Halogen-substituted pyridazinone compounds and their use

Abstract

Disclosed are halogen-substituted pyridazinone compounds and uses thereof, specifically compounds represented by formula (I) or pharma- ceutically acceptable salts thereof. [Formula 1] TIFF2024533411000120.tif43170

Description

Detailed Description of the Invention

[0001] The present invention claims the following priorities: CN202111064358.7, with a filing date of September 10, 2021, CN202210119219.8, with a filing date of February 8, 2022.

[0002] [Technical Field] The present invention relates to halogen-substituted pyridazinone compounds and their uses, specifically to compounds represented by formula (I) or pharmaceutically acceptable salts thereof.

[0003] [Background Art] In 2017, the number of patients with chronic kidney disease (CKD) worldwide reached nearly 700 million, accounting for 9.1% of the world's population. Among them, China had approximately 132 million. In 2017, a total of 2.6 million people died due to CKD and related cardiovascular diseases, accounting for 4.6% of the total deaths worldwide. In the past nearly 30 years, the mortality rates of other non-communicable diseases such as cancer (14.9%), chronic obstructive pulmonary disease (41.3%), and cardiovascular diseases (30.4%) have decreased significantly, but CKD has not shown a similar decrease, and the mortality rate has changed to +2.8%. By 2040, CKD is expected to cause 2.2 to 4 million deaths.

[0004] Proteinuria is one of the common symptoms of kidney diseases. Due to damage to kidney tissue, most patients with chronic kidney disease show symptoms of urinary protein in the initial stage. The formation of proteinuria is closely related to the barrier function of the glomerulus. Due to the filtration effect of the glomerular filtration membrane and the reabsorption effect of the renal tubules, the protein content in the urine of healthy people (mainly referring to proteins with a small molecular weight) is very low (the excretion amount per day is less than 150 mg). When the filtration effect of the glomerular filtration membrane is impaired and the reabsorption effect of the renal tubules is inhibited, a large amount of protein is excreted in the urine. When the protein content in the urine is 3.5 g / 24 h or more, it is called massive proteinuria. If the leakage of protein continues for a long time, it may lead to renal insufficiency. Therefore, a more effective method is needed to treat or reduce proteinuria and reduce the risk of developing kidney diseases.

[0005] The TRP superfamily of ion channels is very important in renal physiology. TRP channels are usually non-selective cation channels with high Ca2 + permeability. Based on sequence homology, the TRP superfamily is further divided into six subfamilies with functions of TRPC, TRPV, TRPA1, TRPM, TRPML, and TRPP. It has been reported that several TRP proteins: TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPV1, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4, TRPM6, and PKD2 are expressed in various cells of the kidney. Podocytes, that is, glomerular epithelial cells of the kidney, adhere to the outside of the glomerular basement membrane (GBM) and together with vascular endothelial cells and the glomerular basement membrane, form the glomerular blood filtration barrier. TRPC5 expressed in podocytes mainly mediates the influx of Ca 2+ and the activation of the Ras-related protein 1 (Rac1) signal causes TRPC5 to move to the cell membrane surface of podocytes, so that the ion channel is activated by the angiotensin II type 1 receptor (AT1R), and the activated ion channel causes a transient influx of Ca 2+ into podocytes, further activates Rac1 in podocytes, induces remodeling of the actin cytoskeleton structure, dissociates damaged podocytes from the glomerular basement membrane, and the detached podocytes affect the filtration rate of the glomerulus and cause proteinuria. Inhibition by TRPC5 knockout or inhibitors such as ML204 and AC1903 significantly reduces LPS-induced urinary protein, inhibits protamine sulfate-induced podocyte damage, and protects the remodeling of the podocyte cytoskeleton. Therefore, TRPC5 is a potential therapeutic target for kidney diseases.

Summary of the Invention

[0006] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0007]

Chemical formula

[0008] However, X1 and X2 are each independently selected from C and N, L is -O-, -S-, -S(=O)-, -C(=O)-, -CF2-, -CH(OCH3)-, -N(OCH3)-,

[0009]

Chemical formula

[0010]

Chemical formula

[0011] R1 is selected from F, Cl, Br and I, R2 is selected from H, F, Cl, Br, I, OH, NH2, CN, COOH, CONH2, C 1-3 alkyl, C 1-3 alkoxy and C 1-3 alkyl-C 1-3 alkoxy, and said C 1-3 alkyl, C 1-3 alkoxy and C 1-3 alkyl-C 1-3 alkoxy are each independently optionally substituted by 1, 2 or 3 R b groups, the structural unit

[0012]

Chemical formula

[0013]

Chemical formula

[0014] Ring B is selected from phenyl and 5- to 6-membered heteroaryl, and the phenyl and 5- to 6-membered heteroaryl are each independently substituted by 1, 2, or 3 R c s Ring C is selected from 6-membered heteroaryl R a is selected from F, Cl, Br, I, and C 1-3 alkyl R b is selected from F, Cl, Br, I, OH, NH2, CN, COOH, and CONH2 R c is selected from F, Cl, Br, I, CONH2, C 1-3 alkyl, C 1-3 alkoxy, and C 2-3 alkenyl, and the C 1-3 alkyl, C 1-3 alkoxy, and C 2-3 alkenyl are each independently optionally substituted by 1, 2, or 3 halogens The conditions are as follows: 1) When R1 is Cl, ring A is

[0015]

Chemical formula

[0016] 2) When R1 is Cl and ring A is

[0017]

Chemical formula

[0018] In some embodiments of the present invention, the "hetero" of the above 5- or 6-membered heteroaryl or 6-membered heteroaryl independently represents 1, 2 or 3 heteroatoms or heteroatomic groups independently selected from N, NH, O and S.

[0019] In some embodiments of the present invention, the above R c is selected from F, Cl, Br, I, CONH2, CH3, CH2CH3, OCH3 and -CH=CH2, and the CH3, CH2CH3, OCH3 and -CH=CH2 are each independently optionally substituted by 1, 2 or 3 halogens, and the other variables are as defined in the present invention.

[0020] In some embodiments of the present invention, the above R c is selected from F, Cl, Br, I, CONH2, CH3, CH2F, CHF2, CF3, CH2CH3, CF2CH3, OCH3, OCF3 and -CF=CH2, and the other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, the above L is -O-, -S-, -S(=O)-, -C(=O)-, -CF2-, -CH(OCH3)-, -N(OCH3)-,

[0022]

Chemical formula

[0023]

Chemical formula

[0024] In some embodiments of the present invention, the above L is -O-, -S-, -S(=O)-, -C(=O)-, -CF2-, -CH(OCH3)-, -N(OCH3)-,

[0025]

Chemical formula

[0026] In some embodiments of the present invention, the above L is selected from -O-, -S-, -S(=O)- and -C(=O)-, and the other variables are as defined in the present invention. In some embodiments of the present invention, the above L is selected from -O- and -S-, and the other variables are as defined in the present invention.

[0027] In some embodiments of the present invention, the above L is

[0028]

Chemical formula

[0029] In some embodiments of the present invention, the above L is -CF2-, -CH(OCH3)-, -N(OCH3)- and

[0030]

Chemical formula

[0031] In some embodiments of the present invention, the above R2 is selected from H, F, Cl, Br, I, OH, NH2, CN, COOH, CONH2, CH3, CH2CH3, OCH3 and CH2OCH3, and the CH3, CH2CH3, OCH3 and CH2OCH3 are each independently, optionally 1, 2 or 3 R bis replaced, and other variables are as defined in the present invention.

[0032] In some embodiments of the present invention, the above R2 is selected from H, F, Cl, Br, I, OH, NH2, CN, COOH, CONH2, CH3, CH2CN, CH2COOH, CH2CONH2, CH2OH, CH2CH3, CH(OH)CH3, CH(OH)CH2OH, OCH3 and CH2OCH3, and other variables are as defined in the present invention.

[0033] In some embodiments of the present invention, the above R2 is selected from CN, COOH, CONH2, CH3, CH2CN, CH2COOH, CH2CONH2 and CH2OCH3, and other variables are as defined in the present invention.

[0034] In some embodiments of the present invention, the above R2 is selected from CH3, CH2CN, CH2COOH, CH2CONH2 and CH2OCH3, and other variables are as defined in the present invention.

[0035] In some embodiments of the present invention, the above R2 is selected from CN, COOH and CONH2, and other variables are as defined in the present invention. In some embodiments of the present invention, the above R2 is selected from H, and other variables are as defined in the present invention.

[0036] In some embodiments of the present invention, the above R2 is selected from CH2OCH3, and other variables are as defined in the present invention. In some embodiments of the present invention, the above ring A is

[0037]

Chemical formula

[0038] In some embodiments of the present invention, the above ring A is

[0039] [Chemical formula] selected from, and other variables are as defined in the present invention.

[0040] In some embodiments of the present invention, the above ring B is selected from phenyl, thienyl and pyridyl, and the phenyl, thienyl and pyridyl are each independently optionally substituted with 1, 2 or 3 R c and other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, the above ring B is

[0042] [Chemical formula] selected from, and other variables are as defined in the present invention.

[0043] In some embodiments of the present invention, the above ring B is

[0044] [Chemical formula] selected from, and other variables are as defined in the present invention.

[0045] In some embodiments of the present invention, the above ring B is

[0046] [Chemical formula] selected from, and other variables are as defined in the present invention.

[0047] In some embodiments of the present invention, the above ring B is

[0048] [Chemical formula] selected from, and other variables are as defined in the present invention.

[0049] In some embodiments of the present invention, the above ring B is

[0050]

Chemical formula

[0051] In some embodiments of the present invention, the above ring B is

[0052]

Chemical formula

[0053] In some embodiments of the present invention, the above ring B is

[0054]

Chemical formula

[0055] In some embodiments of the present invention, the above ring C is selected from pyridyl and pyrimidinyl, and the other variables are as defined in the present invention. In some embodiments of the present invention, the above ring C is selected from pyrimidinyl, and the other variables are as defined in the present invention.

[0056] In some embodiments of the present invention, the above compound or its pharmaceutically acceptable salt is selected from the following formulas.

[0057]

Chemical formula

[0058] However, n is selected from 0, 1, 2, and 3, R1, R3, Rc L and ring A are as defined in the present invention.

[0059] In some embodiments of the present invention, the above compound or its pharmaceutically acceptable salt is selected from the following formulae.

[0060]

Chemical formula

[0061] Provided that, R1, R3 and R c are as defined in the present invention. The present invention further provides a compound represented by formula (I) or its pharmaceutically acceptable salt.

[0062]

Chemical formula

[0063] Provided that, X1 and X2 are each independently selected from CH and N, L is selected from -O-, -S-, -S(=O)-, -C(=O)-, -CF2-, -CH(OCH3)-, -N(OCH3)-,

[0064]

Chemical formula

[0065]

Chemical formula

[0066] R1 is selected from F, Cl, Br and I, R2 is selected from H, F, Cl, Br, I, OH, NH2, CN, COOH, CONH2, C1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkyl-C 1-3 Selected from alkoxy, said C 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 Alkyl-C 1-3 Alkoxy are each independently optionally substituted by 1, 2 or 3 R b and Ring A is

[0067]

Chemical formula

[0068] Ring B is selected from phenyl and 5- to 6-membered heteroaryl, said phenyl and 5- to 6-membered heteroaryl are each independently substituted by 1, 2 or 3 R c and Ring C is selected from 6-membered heteroaryl, R a is selected from F, Cl, Br, I and C 1-3 alkyl, R b is selected from F, Cl, Br, I, OH, NH2, CN, COOH and CONH2, R c is selected from F, Cl, Br, I, CONH2, C 1-3 alkyl, C 1-3 alkoxy and C 2-3 alkenyl, said C 1-3 alkyl, C 1-3 alkoxy and C 2-3 alkenyl are each independently optionally substituted by 1, 2 or 3 halogens, The conditions are as follows: 1) R1 is Cl and ring A is

[0069]

Chemical formula

[0070] wherein R1 is Cl and ring A is

[0071] [Chemical formula] and when L is O and ring B is 2-trifluoromethyl-4-fluorophenyl, R2 is CN, COOH, CONH2, CH3, CH2CN, CH2COOH, CH2CONH2 or CH2OCH3.

[0072] Some further aspects of the present invention are formed by any combination of the above variables. The present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof.

[0073] [Chemical formula] TIFF0007701559000031.tif233170TIFF0007701559000032.tif221170 In some aspects of the present invention, the above compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0074] [Chemical formula] The present invention further provides the use of the above compound or a pharmaceutically acceptable salt thereof in drugs related to TRPC5 inhibitors.

[0075] [Advantages of the Invention] The compounds of the present invention have a significant inhibitory effect on TRPC5, exhibit good pharmacokinetic properties in rats, and the compounds of the present invention have a high distribution in the kidneys and liver and a low distribution in the brain. In addition, it can significantly reduce the level of urinary albumin in a rat model of hypertensive renal insufficiency and can dose-dependently reduce the expression of Rac1 in the urine of a rat model of hypertensive renal insufficiency.

[0076] Definitions and Explanations Unless otherwise specified, the following terms and phrases used in this specification are intended to have the following meanings. Specific terms and phrases should not be considered uncertain or unclear when not specifically defined, but should be understood according to their ordinary meanings. When a trade name appears in this specification, it is intended to refer to the corresponding trade name or its active ingredient.

[0077] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, and have little excessive toxicity, irritation, allergic reaction or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0078] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention prepared from a relatively non-toxic acid or base with a specific substituent found in the present invention. When a relatively acidic functional group is included in the compound of the present invention, a base addition salt can be obtained by contacting such a compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. When a relatively basic functional group is included in the compound of the present invention, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Some specific compounds of the present invention contain both basic and acidic functional groups and can therefore be converted into either a base addition salt or an acid addition salt.

[0079] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acidic or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

[0080] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers, and tautomers.

[0081] The compounds of the present invention can exist in the form of specific geometric or stereoisomers. All such compounds contemplated by the present invention include cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present invention. Substituents such as alkyl may have other asymmetric carbon atoms. All of these isomers and their mixtures are included within the scope of the present invention.

[0082] Unless otherwise specified, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other. Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" are due to the inability of double bonds or single bonds of ring-forming carbon atoms to rotate freely.

[0083] Unless otherwise specified, the term "diastereomer" refers to stereoisomers in which the molecules have two or more chiral centers and the molecules are non-mirror images of each other. Unless otherwise specified, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

[0084] Unless otherwise specified, the wedge solid line bond (

[0085] [Chem.] ) and a wedge-dotted line bond (

[0086] [Chem.] ) to represent the absolute configuration of a stereocenter by a straight solid line bond (

[0087] [Chem.] ) and a straight dotted line bond (

[0088] [Chem.] ) to represent the relative configuration of a stereocenter by a wavy line (

[0089] [Chem.] ) to represent a wedge solid line bond (

[0090] [Chem.] ) or a wedge-dotted line bond (

[0091] [Chem.] ) or by a wavy line (

[0092] [Chem.] ) to represent a straight solid line bond (

[0093] [Chem.] ) or a straight dotted line bond (

[0094] [Chemistry] represents

[0095] Unless otherwise specified, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer", or "enriched in enantiomers" mean that the content of one isomer or enantiomer is less than 100%, and the content of this isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0096] Unless otherwise specified, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, when the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.

[0097] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. To obtain one enantiomer of a certain compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomers are resolved by conventional methods known in the art and recovered to obtain the pure enantiomer. Also, the separation of enantiomers and diastereomers is usually carried out using chromatography with a chiral stationary phase and optionally in combination with a chemical derivatization method (e.g., generating a carbamate from an amine).

[0098] The compounds of the present invention may contain unnatural proportions of atomic isotopes in one or more atoms constituting the compounds. For example, the compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). Or for example, deuterium can be substituted for hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by normal hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reducing toxic side effects, enhancing drug stability, enhancing efficacy, and extending the biological half-life of the drug. The conversion of the isotope composition of the compounds of the present invention is included within the scope of the present invention regardless of whether it is radioactive or not.

[0099] The term "optionally" or "optionally" means that the event or situation described thereafter may occur, but does not necessarily occur, and the description includes both the case where the event or situation occurs and the case where the event or situation does not occur.

[0100] The term "substituted" means that any one or more hydrogen atoms at a particular atom are substituted with substituents. As long as the valence of the particular atom is normal and the substituted compound is stable, the substituents may include deuterium and variants of hydrogen. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur in aromatic groups. The term "optionally substituted" means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents are chemically feasible and arbitrary.

[0101] If any of the variables (e.g., R) appears one or more times in the composition or structure of the compound, its definition is independent in each case. Thus, for example, if one group is substituted by 0 to 2 R's, the said group may optionally be substituted by up to 2 R's, and in each case R has independent options. Also, combinations of substituents and / or their variants are only permitted if such combinations result in stable compounds.

[0102] When the number of linking groups is 0, for example, -(CRR)0- means that the said linking group is a single bond. When the number of substituents is 0, it indicates the absence of the said substituent. For example, -A-(R)0 represents that the structure is actually -A.

[0103] When the substituent is empty, it means the absence of the said substituent. For example, when X in A-X is empty, the structure actually means A. When one of the variables is a single bond, it means that the two groups linked to it are directly linked. For example, when L in A-L-Z represents a single bond, the structure actually means A-Z.

[0104] When the bond of a substituent can cross-link to two or more atoms on one ring, this substituent can be bonded to any atom on this ring. For example, the structural unit

[0105]

Chemical formula

[0106] When the listed linking group does not indicate its linking direction, the linking direction is arbitrary. For example,

[0107]

Chemical formula

[0108]

Chemical formula

[0109]

Chemical formula

[0110] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups via a chemical bond. When the bonding mode of the chemical bond is delocalized and there is an H atom at the bondable site, when the chemical bond is formed, the number of H atoms at the site decreases to a group with a corresponding valence according to the number of formed chemical bonds. The chemical bond by which the site is bonded to another group is a solid straight-line bond (

[0111]

Chemical formula

[0112]

Chemical formula

[0113]

Chemical formula

[0114]

Chemical formula

[0115]

Chemical formula

[0116]

Chemical formula

[0117]

Chemical formula

[0118]

Chemical formula

[0119]

Chemical formula

[0120] Unless otherwise specified, the number of atoms in the ring is generally defined as the number of ring members. For example, "5- to 7-membered ring" refers to a "ring" with 5 to 7 atoms arranged around it. Unless otherwise indicated, the term "halo" or "halogen" means a fluorine, chlorine, bromine, or iodine atom, either by itself or as part of another substituent.

[0121] Unless otherwise indicated, the term "C 1-3 alkyl" is used to represent a straight-chain or branched-chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C 1-3 alkyl includes C 1-2 and C 2-3 alkyl, etc., which may be monovalent (e.g., methyl), divalent (e.g., methylene), and polyvalent (e.g., methine). Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0122] Unless otherwise indicated, the term "C 1-3 alkoxy" means an alkyl group containing 1 to 3 carbon atoms bonded to the remainder of the molecule through one oxygen atom. The C 1-3 alkoxy includes C 1-2 , C 2-3 , C3, and C2 alkoxy, etc. Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy or isopropoxy), etc.

[0123] Unless otherwise indicated, the term "C 2-3 alkenyl" is used to represent a straight-chain or branched-chain hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, and the carbon-carbon double bond may be at any position in the group. The C 2-3 alkenyl includes C3 and C2 alkenyl, and the C 2-3 alkenyl may be monovalent, divalent, or polyvalent. Examples of C 2-3 alkenyl include, but are not limited to, ethenyl, propenyl, etc.

[0124] Unless otherwise indicated, the terms "5- to 6-membered heteroaryl ring" and "5- to 6-membered heteroaryl" can be used interchangeably in the present invention. The term "5- to 6-membered heteroaryl" is a monocyclic group having a conjugated π-electron system consisting of 5 to 6 ring atoms, 1, 2, 3, or 4 of which are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms. Here, the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O) p , where p is 1 or 2). The 5- to 6-membered heteroaryl can be attached to the remainder of the molecule via a heteroatom or a carbon atom. The 5- to 6-membered heteroaryl includes 5-membered and 6-membered heteroaryls. Examples of the 5- to 6-membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (including 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, etc.), furanyl (including 2-furanyl, 3-furanyl, etc.), thienyl (including 2-thienyl, 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, 4-pyridyl, etc.), pyrazinyl, or pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, etc.).

[0125] The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxyl protecting group" or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino acid protecting groups include, but are not limited to, acyl such as formyl; alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl such as benzyl (Bn), triphenylmethyl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; silyl such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS). The term "hydroxyl protecting group" refers to a protecting group suitable for preventing side reactions of hydroxyl. Representative hydroxyl protecting groups include, but are not limited to, alkyl such as methyl, ethyl and tert-butyl, acyl such as alkanoyl (e.g., acetyl), arylmethyl such as benzyl (Bn), p-formyloxybenzyl (PMB), 9-fluorenylthyl (Fm) and diphenylmethyl (diphenylmethyl, DPM), silyl such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS).

[0126] The structure of the compounds of the present invention can be confirmed by ordinary methods well-known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by ordinary technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the diffraction intensity data of the cultured single crystal is collected with a Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure using the direct method (Shelxs97).

[0127] The compounds of the present invention can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combination with other chemical synthesis methods, and equivalent alternative methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0128] The following abbreviations are used in the present invention: aq represents water. eq represents equivalent. DCM represents dichloromethane. PE represents petroleum ether. DMSO represents dimethyl sulfoxide. EA or EtOAc represents ethyl acetate. EtOH represents ethanol. MeOH represents methanol. DMF represents N,N-dimethylformamide. Cbz represents benzyloxycarbonyl, an amine protecting group. Boc represents tert-butoxycarbonyl, an amine protecting group. r.t. represents room temperature. O / N represents overnight. THF represents tetrahydrofuran. Boc2O represents di-tert-butyl dicarbonate. TFA represents trifluoroacetic acid. HCl represents hydrochloric acid. iPrOH represents 2-propanol. mp represents melting point. Pd(PPh3)4 represents tetrakis(triphenylphosphine)palladium(0). Pd(dppf)Cl2 represents [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II). DIBAL-H represents diisobutylaluminum hydride. FA represents formic acid. ACN represents acetonitrile. DEA represents diethylamine. THP represents 2-tetrahydropyranyl ether. prep-TLC represents separation by preparative thin-layer chromatography. DIEA represents N,N-diisopropylethylamine. DMA represents N,N-dimethylacetamide. DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0129] The solvents used in the present invention can be obtained from commercially available products. Compounds are named according to the usual naming principles in the art or using ChemDraw® software, and commercially available compounds are named in the supplier's catalog.

Brief Description of the Drawings

[0130]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0131] Hereinafter, the present invention will be described in detail with reference to Examples, which should not be construed as limiting the invention in any way. The present invention has been described in detail herein, and its specific embodiments have also been disclosed. It will be apparent to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0132] Reference Example 1: Fragment A-1

[0133]

Chemical Formula

[0134] Synthesis Route:

[0135]

Chemical Formula

[0136] Step 1: Synthesis of Compound A-1-2 Sodium ethoxide (3.2 g, 47.02 mmol, 2.55 eq) was dissolved in ethanol (100 mL). Under nitrogen gas protection, compound A-1-1 (5 g, 18.43 mmol, 1 eq) and formamidine acetate (2.9 g, 27.86 mmol, 1.51 eq) were added, and the reaction system was stirred at 100 °C for 5 h. After the reaction system was cooled, 250 mL of water was added, and the mixture was extracted with dichloromethane (3 × 250 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. Ethyl acetate (75 mL) was added to the crude product, and the mixture was stirred for 2 h and filtered. The cake was washed with 5 mL of ethyl acetate and then dried to obtain compound A-1-2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.42 - 1.56 (m, 9 H), 2.64 (br s, 2 H), 3.65 (t, J=5.77 Hz, 2 H), 4.43 (br s, 2 H), 8.07 (s, 1 H), 12.62 (br s, 1 H).

[0137] Step 2: Synthesis of compound A-1 Compound A-1-2 (18 g, 71.63 mmol, 1 eq) and trichloroacetonitrile (15.55 g, 107.71 mmol, 1.5 eq) were suspended in toluene (250 mL), triphenylphosphine (56.00 g, 213.51 mmol, 2.98 eq) was added, and the mixture was stirred at 120 °C for 1.5 h under nitrogen gas protection. The reaction solution was concentrated under reduced pressure, 250 mL of water was added to the residue, and the mixture was extracted with dichloromethane (3 × 150 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain compound A-1-3. 1 H NMR (400 MHz, CDCl3) δ ppm 1.50 (s, 9 H), 2.88 (t, J=5.52 Hz, 2 H), 3.75 (t, J=5.77 Hz, 2 H), 4.65 (s, 2 H), 8.80 (s, 1 H).

[0138] Reference Example 2: Fragment A-2

[0139]

Chem.

[0140] Synthesis Route:

[0141]

Chem.

[0142] A-2-1 (186 g, 1.13 mol, 1 eq) was added to a reaction flask containing tetrahydrofuran (1116 mL), pyridinium p-toluenesulfonate (56.66 g, 225.49 mmol, 0.2 eq), and 3,4-dihydro-2H-pyran (142.25 g, 1.69 mol, 154.62 mL, 1.5 eq) were added, and the reaction was carried out at 100 °C for 5 hours. Next, 3,4-dihydro-2H-pyran (94.83 g, 1.13 mol, 103.08 mL, 1 eq) was added, and the reaction was carried out at 100 °C for 12 hours. After concentrating the reaction solution, ethyl acetate was added to dissolve it, and the organic phase was washed successively with a 2M sodium hydroxide solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was first purified by column chromatography (petroleum ether:ethyl acetate = 1:0~15:1~10:1~5:1~2.5:1), and then slurried with petroleum ether to obtain Compound A-2. LCMS: m / z [M+23] + = 271.

[0143] Reference Example 3: Fragment A-3

[0144]

Chem.

[0145] Synthesis Route:

[0146]

Chem.

[0147] A-3-1 (35 g, 115.07 mmol, 1 eq) was added to a reaction flask containing acetonitrile (300 mL), potassium carbonate (31.81 g, 230.13 mmol, 2 eq) was added, and then a solution of 4-fluoro-2-(trifluoromethyl)phenol (24.87 g, 138.08 mmol, 1.2 eq) in acetonitrile (240 mL) was added. The mixture was reacted at 50 °C for 16 hours. 300 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered by suction, and concentrated to obtain a crude product. A mixed solvent (petroleum ether:ethyl acetate = 9:1) was added to the crude product and stirred, and then filtered by suction. The cake was washed with the mixed solvent (petroleum ether:ethyl acetate = 9:1) and then concentrated to obtain a cake. The cake was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 - 9:1 - 4:1 - 0:1) to obtain Compound A-3. 1 H NMR (400 MHz, CDCl3) δ ppm 1.51 (s, 9 H), 2.86 (br t, J = 5.02 Hz, 2 H), 3.76 (t, J = 5.77 Hz, 2 H), 4.63 (s, 2 H), 7.31 - 7.38 (m, 2 H), 7.40 - 7.48 (m, 1 H); LCMS: m / z [M - 55] + = 391.9.

[0148] Example 1

[0149]

Chemical formula

[0150] Synthesis route:

[0151]

Chemical formula

[0152] Step 1: Synthesis of Compound WX001-2 Under nitrogen gas protection, WX001-1 (5 g, 19.04 mmol, 1 eq) was added to a reaction flask containing 1,4-dioxane (30 mL). Then, water (7.5 mL) and tris(2-carboxyethyl)phosphine hydrochloride (21.83 g, 76.14 mmol, 4 eq) were added, and the mixture was reacted at 120 °C for 2 hours. After cooling to room temperature, 75 mL of water was added, and the mixture was extracted with dichloromethane (3 × 100 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by vacuum column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain Compound WX001-2. 1 H NMR (400 MHz, DMSO-d6) δ ppm 6.13 (br s, 1 H), 7.33 - 7.97 (m, 3 H).

[0153] Step 2: Synthesis of Compound WX001-3 A-1 (2.47 g, 9.16 mmol, 1 eq) and WX001-2 (1.89 g, 9.62 mmol, 1.05 eq) were added to a reaction flask containing N,N-dimethylformamide (27 mL). 1,8-Diazabicyclo[5.4.0]undec-7-ene (2.79 g, 18.31 mmol, 2.76 mL, 2 eq) was added, and the mixture was reacted at 100 °C for 0.5 hour. After cooling to room temperature, 300 mL of ethyl acetate was added, and the organic phase was washed successively with semi-saturated brine (5 × 30 mL) and brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 4:1) to obtain Compound WX001-3. LCMS: m / z [M+1] + = 430.

[0154] Step 3: Synthesis of Compound WX001-4 WX001-3 (1.64 g, 3.82 mmol, 1 eq) was added to a reaction flask containing dichloromethane (76 mL), trifluoroacetic acid (13.06 g, 114.57 mmol, 8.48 mL, 30 eq) was added, and the mixture was reacted at 20 °C for 0.5 h. 70 mL of water was added to the reaction solution, sodium carbonate was added to adjust the pH to 8, and the mixture was extracted with dichloromethane (3 × 70 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain WX001-4. LCMS: m / z [M+1] + = 330.

[0155] Step 4: Synthesis of compound WX001-5 WX001-4 (2.87 g, 8.72 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (64.75 g, 500.96 mmol, 87.26 mL, 57.48 eq), A-2 (2.39 g, 9.59 mmol, 1.1 eq) was added, and the mixture was reacted at 100 °C for 16 h. The reaction solution was directly concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 1:1) to obtain compound WX001-5. LCMS: m / z [M+1] + = 542.

[0156] Step 5: Synthesis of compound WX001-6 WX001-5 (100 mg, 184.52 μmol, 1 eq) was added to a reaction flask containing dichloromethane (4.4 mL), and a solution of 3-chloroperoxybenzoic acid (44.95 mg, 221.42 μmol, purity: 85%, 1.2 eq) in dichloromethane (2.2 mL) was added at 0 °C, and the mixture was reacted at 0 °C for 3 h. A saturated sodium thiosulfate solution was added to the reaction solution to quench the reaction system (the potassium iodide-starch test paper did not change to blue), then sodium carbonate was added to adjust the pH to 8, and the mixture was extracted with dichloromethane (3 × 4 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 2:3) to obtain compound WX001-6.1 1H NMR (400 MHz, CDCl3) δ ppm 1.60 - 1.82 (m, 4 H), 2.04 - 2.21 (m, 2 H), 3.33 - 3.51 (m, 1 H), 3.54 - 3.68 (m, 1 H), 3.77 (br t, J = 11.54 Hz, 1 H), 3.81 - 3.95 (m, 2 H), 4.12 - 4.17 (m, 1 H), 4.71 (d, J = 4.52 Hz, 2 H), 6.01 - 6.15 (m, 1 H), 7.48 (dd, J = 8.03, 2.51 Hz, 1 H), 7.53 - 7.63 (m, 1 H), 7.79 (s, 1 H), 8.38 - 8.45 (m, 1 H), 8.94 (s, 1 H); LCMS: m / z [M+1] + = 558。

[0157] Step 6: Synthesis of Compound WX001 WX001-6 (1.23 g, 2.20 mmol, 1 eq) was added to a reaction flask containing dichloromethane (22 mL), trifluoroacetic acid (7.54 g, 66.14 mmol, 4.90 mL, 30 eq) was added, and the reaction was carried out at 20 °C for 0.5 h. 22 mL of water was added to the reaction solution, sodium carbonate was added to adjust the pH to 8, and the mixture was extracted with dichloromethane (3 × 22 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. 5 mL of dichloromethane was added to the crude product, and it was sonicated for 2 minutes. It was suction filtered, 3 mL of methanol was added to the cake, and it was sonicated for 2 minutes. It was suction filtered, and the cake was dried to obtain Compound WX001. LCMS: m / z [M+1] + = 474。

[0158] Step 7: Synthesis of Compounds WX001A and WX001B WX001-7 was separated by SFC (chromatographic column: DAICEL CHIRALPAK IG (250 mm × 30 mm, 10 μm); mobile phase: A is CO2, B is [0.1% NH3H2O - EtOH]; gradient B%: 60% - 60%) to obtain WX001A and WX001B.

[0159] WX001A: (Retention time: 3.558 minutes, ee = 100%). 1 H NMR (400 MHz, CD3CN) δ ppm 3.24 - 3.36 (m, 1 H), 3.40 - 3.51 (m, 1 H), 3.80 (t, J = 5.77 Hz, 2 H), 4.70 (s, 2H), 7.59 - 7.71 (m, 2 H), 7.80 (s, 1 H), 8.29 (dd, J = 8.78, 5.27 Hz, 1 H), 8.88 (s, 1 H), 10.92 (br s, 1 H); LCMS: m / z [M+1] + = 474.

[0160] WX001B: (Retention time: 1.738 minutes, ee = 100%). 1 H NMR (400 MHz, CD3CN) δ ppm 3.19 - 3.38 (m, 1 H), 3.39 - 3.53 (m, 1 H), 3.80 (t, J = 5.77 Hz, 2 H), 4.70 (s, 2 H), 7.54 - 7.73 (m, 2 H), 7.80 (s, 1 H), 8.29 (dd, J = 8.78, 5.27 Hz, 1 H), 8.88 (s, 1 H), 10.93 (br s, 1 H); LCMS: m / z [M+1] + = 474.

[0161] Detection conditions for SFC analysis: chromatographic column: Chiralpak IG-3 50 × 4.6 mm I.D., 3 μm; mobile phase: phase A is CO2; phase B is ethanol (0.05% DEA); gradient B%: 40%.

[0162] Example 2

[0163]

Chem.

[0164] Synthesis route:

[0165]

Chem.

[0166] WX001-5 (16 g, 29.52 mmol, 1 eq) was added to a reaction flask containing dichloromethane (160 mL), trifluoroacetic acid (100 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. It was cooled to 0 °C, saturated sodium carbonate solution was added dropwise to the reaction solution to adjust the pH to 8, and suction filtration was carried out to obtain a cake. The cake was washed with dichloromethane and then dried to obtain compound WX002. 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.90 (br t, J = 5.27 Hz, 2 H), 3.80 (t, J = 5.52 Hz, 2 H), 4.63 (s, 2 H), 7.71 (td, J = 8.47, 2.89 Hz, 1 H), 7.84 - 7.93 (m, 2 H), 8.00 (s, 1 H), 8.61 (s, 1 H), 13.00 (br s, 1 H); LCMS: m / z [M+1] + = 458.

[0167] Example 3

[0168]

Chem.

[0169] Synthesis route:

[0170]

Chem.

[0171] Step 1: Synthesis of Compound WX003-2 WX003-1 (8 g, 38.81 mmol, 1 eq) was added to a reaction flask containing ethanol (160 mL), replaced with nitrogen gas, p-toluenesulfonyl hydrazide (7.23 g, 38.81 mmol, 1 eq) was added in one batch, and the mixture was reacted at 90 °C for 12 hours. It was concentrated to obtain a crude product. 10 mL of methanol was added to the crude product, stirred for 5 minutes, suction filtered, and the cake was dried to obtain Compound WX003-2. LCMS: m / z [M+1] + = 375.

[0172] Step 2: Synthesis of Compound WX003-3 Under the protection of nitrogen gas, WX003-2 (3.74 g, 10.00 mmol, 1 eq), tris(dibenzylideneacetone)dipalladium (915.72 mg, 1.00 mmol, 0.1 eq), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (953.43 mg, 2.00 mmol, 0.2 eq) were added to a reaction flask containing 1,4-dioxane (60 mL), lithium tert-butoxide (1.76 g, 22.00 mmol, 1.98 mL, 2.2 eq) was added, and the mixture was stirred for 1 minute. A-1 (2.70 g, 10 mmol, 1 eq) was added, and the mixture was reacted at 100 °C for 12 hours. It was cooled to room temperature, ethyl acetate was added, filtered through diatomaceous earth, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0~4:1) to obtain Compound WX003-3. LCMS: m / z [M+1] + = 424.

[0173] Step 3: Synthesis of Compound WX003-4 WX003-3 (1.82 g, 4.30 mmol, 1 eq) was added to a reaction flask containing dichloromethane (6.5 mL), trifluoroacetic acid (2.60 g, 22.78 mmol, 1.69 mL, 5.3 eq) was added, and the reaction was carried out at 20 °C for 12 h. 10 mL of water was added, the pH was adjusted to 8 with sodium carbonate, extracted with dichloromethane (3 × 10 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 1:0~9:1) to obtain compound WX003-4. LCMS: m / z [M+1] + = 324.

[0174] Step 4: Synthesis of compound WX003-5 WX003-4 (986.1 mg, 2.81 mmol, purity: 92.07%, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (25.25 g, 195.34 mmol, 34.02 mL, 69.56 eq), A-2 (839.45 mg, 3.37 mmol, 1.2 eq) was added, and the reaction was carried out at 100 °C for 16 h. It was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0~0:1) to obtain compound WX003-5. LCMS: m / z [M+1] + = 536.

[0175] Step 5: Synthesis of compound WX003 WX003-5 (0.3 g, 559.78 μmol, purity: 100%, 1 eq) was added to a reaction flask containing dichloromethane (10 mL), trifluoroacetic acid (1.91 g, 16.79 mmol, 1.24 mL, 30 eq) was added, and the reaction was carried out at 20 °C for 0.5 h. 10 mL of water was added, the pH was adjusted to 8 with sodium carbonate, at this point a solid precipitated in the organic phase, the aqueous phase was extracted with a mixed solvent (DCM:MeOH = 20:1), the organic phases were combined, washed with saturated brine, and concentrated to obtain a crude product. 5 mL of dichloromethane and 5 mL of methanol were added to the crude product, stirred for 10 min, suction filtered, and the cake was dried to obtain compound WX003. 11H NMR (400 MHz, DMSO-d6) δ ppm 3.07 (broad singlet, 2 H), 3.70 (broad singlet, 2 H), 4.73 (singlet, 2 H), 5.94 (singlet, 1 H), 6.12 (singlet, 1 H), 7.52 - 7.73 (multiplet, 3 H), 8.00 (singlet, 1 H), 8.87 (singlet, 1 H), 12.97 (broad singlet, 1 H); LCMS: m / z [M+1] + = 452.

[0176] Example 4

[0177]

Chemical Structure

[0178] Synthesis Route:

[0179]

Chemical Structure

[0180] Step 1: Synthesis of Compound WX004-2 Add A-1 (34 g, 126.05 mmol, 1 eq) to a reaction flask containing N,N-dimethylformamide (328 mL). After complete dissolution, add WX004-1 (25.28 g, 132.36 mmol, 1.05 eq), and then add 1,8-diazabicyclo[5.4.0]undec-7-ene (38.38 g, 252.11 mmol, 38.00 mL, 2 eq). React at 100 °C for 1.5 hours. Slowly add the reaction solution to 3500 mL of water while stirring. At this point, a solid precipitates. Filter by suction through diatomaceous earth, wash the cake with water, dissolve it in dichloromethane, then filter by suction again, and concentrate the filtrate to obtain the crude product. Purify the crude product by chromatography column (petroleum ether:ethyl acetate = 1:1) to obtain Compound WX004-2. LCMS: m / z [M+1] + = 424, 426.

[0181] Step 2: Synthesis of Compound WX004-3 WX004-2 (51 g, 120.21 mmol, 1 eq) was added to a reaction flask, followed by dichloromethane (500 mL) and trifluoroacetic acid (255.64 g, 2.24 mol, 166.00 mL, 18.65 eq). The reaction was carried out at 20 °C for 0.5 h. 500 mL of water was added to the reaction solution, and then sodium carbonate was added to adjust the pH to 7. The mixture was extracted with dichloromethane (3 × 500 mL). The combined organic phases were washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound WX004-3. LCMS: m / z [M+1] + = 324, 326.

[0182] Step 3: Synthesis of compound WX004-4 Under nitrogen gas protection, WX004-3 (20 g, 61.70 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (371.00 g, 2.87 mol, 500 mL, 46.52 eq), and A-2 (16.91 g, 67.87 mmol, 1.1 eq) was added. The reaction was carried out at 100 °C for 16 h. It was directly concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:4) to obtain compound WX004-4. LCMS: m / z [M+1] + = 536, 538.

[0183] Step 4: Synthesis of compound WX004 WX004-4 (24 g, 44.71 mmol, 1 eq) was added to a reaction flask containing dichloromethane (238 mL), followed by trifluoroacetic acid (244.86 g, 2.15 mol, 159 mL, 48.03 eq). The reaction was carried out at 20 °C for 0.5 h. 500 mL of water was added to the reaction solution, and sodium carbonate was added to adjust the pH to 7. The mixture was extracted with a mixed solvent (dichloromethane:methanol = 20:1) (2 × 500 mL). After concentration under reduced pressure until about 500 mL of the solvent remained, suction filtration was carried out, and the cake was dried to obtain compound WX004. 11H NMR (400 MHz, DMSO-d6) δ ppm 3.01 (broad singlet, 2 H), 3.73 - 3.82 (multiplet, 2 H), 4.66 (singlet, 2 H), 7.37 (transverse doublet, J=8.53, 2.51 Hz, 1 H), 7.43 - 7.53 (multiplet, 1 H), 7.74 (broad doublet of doublets, J=8.03, 2.51 Hz, 1 H), 8.02 (singlet, 1 H), 8.53 (singlet, 1 H), 11.21 - 13.86 (multiplet, 1 H); LCMS: m / z [M+1] + = 452,454。

[0184] Example 5

[0185]

Chemical Structure

[0186] Synthesis Route:

[0187]

Chemical Structure

[0188] Step 1: Synthesis of Compound WX005-2 A-1 (1.5 g, 5.56 mmol, 1 eq) and WX005-1 (812.81 mg, 5.56 mmol, 630.08 μL, 1 eq) were added to N,N-dimethylformamide (15 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.69 g, 11.12 mmol, 1.68 mL, 2 eq) was added. The mixture was reacted at 100 °C for 1.5 hours. 150 mL of ethyl acetate was added to the reaction solution, and it was washed successively with semi-saturated brine (6 × 50 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain Compound WX005-2. LCMS: m / z [M+1] + = 380。

[0189] Step 2: Synthesis of Compound WX005-3 WX005-2 (1 g, 2.64 mmol, 1 eq) was added to a reaction flask containing dichloromethane (50 mL), trifluoroacetic acid (11.59 g, 101.63 mmol, 7.52 mL, 38.56 eq) was added, and the mixture was reacted at 20 °C for 0.5 h. 50 mL of water was added to the reaction solution, the pH was adjusted to 9 with sodium carbonate, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Compound WX005-3. LCMS: m / z [M+1] + = 280.

[0190] Step 3: Synthesis of Compound WX005-4 WX005-3 (570 mg, 2.04 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (18.35 g, 141.96 mmol, 24.73 mL, 69.56 eq), A-2 (508.34 mg, 2.04 mmol, 1 eq) was added, and the mixture was reacted at 100 °C for 16 h. After the reaction solution was concentrated, 30 mL of water was added, and the mixture was extracted with dichloromethane (3 × 30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain Compound WX005-4. LCMS: m / z [M+1] + = 492.

[0191] Step 4: Synthesis of Compound WX005 WX005-4 (0.43 g, 874.09 μmol, 1 eq) was added to a reaction flask containing dichloromethane (10 mL), trifluoroacetic acid (2.99 g, 26.22 mmol, 1.94 mL, 30 eq) was added, and the mixture was reacted at 20 °C for 0.5 h. 20 mL of water was added to the reaction solution, sodium carbonate was added to adjust the pH to 9, and the mixture was extracted with dichloromethane (3 × 20 mL). After concentration, first 7 mL of dimethyl sulfoxide was added to dissolve it, then 7 mL of water was added, a solid precipitated, and it was suction filtered to obtain a cake. 1 mL of methanol and 1 mL of ethyl acetate were added to the cake, stirred, suction filtered, and the cake was dried to obtain compound WX005. 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.91 (br t, J = 5.40 Hz, 2 H), 3.81 (br t, J = 5.65 Hz, 2 H), 4.63 (s, 2 H), 7.26 (td, J = 8.34, 2.38 Hz, 1 H), 7.52 (td, J = 9.22, 2.64 Hz, 1 H), 7.66 - 7.80 (m, 1 H), 8.00 (s, 1 H), 8.64 (s, 1 H), 12.98 (s, 1 H); LCMS: m / z [M+1] + = 408.

[0192] Example 6

[0193]

Chemical formula

[0194] Synthesis route:

[0195]

Chemical formula

[0196] Step 1: Synthesis of compound WX006-2 A solution of (diethylamino)sulfur trifluoride (1.19 g, 7.39 mmol, 976.23 μL, 1.5 eq) in dichloromethane (5 mL) was added dropwise to a solution of WX006-1 (1 g, 4.93 mmol, 1 eq) in dichloromethane (10 mL) at 0 °C. The reaction solution was allowed to warm to 25 °C naturally and stirred for 18 hours. The reaction solution was added dropwise to saturated sodium bicarbonate solution (40 mL), stirred for 10 minutes, dichloromethane (20 mL) was added, and the organic phase was washed with water (2 × 20 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound WX006-2. 1 H NMR (400 MHz, CDCl3) δ ppm 6.72 - 7.04 (m, 1 H), 7.11 (br dd, J=2.01, 1.00 Hz, 1 H), 7.41 (dd, J=8.66, 2.89 Hz, 1 H), 7.60 (dd, J=8.78, 5.02 Hz, 1 H).

[0197] Step 2: Synthesis of compound WX006-3 To a solution of WX006-2 (2 g, 8.89 mmol, 1 eq) and N,N-diisopropylethylamine (3.45 g, 26.67 mmol, 4.64 mL, 3 eq) in 1,4-dioxane (20 mL) was added 4-methoxybenzyl mercaptan (1.51 g, 9.78 mmol, 1.36 mL, 1.1 eq). After purging with nitrogen gas, tris(dibenzylideneacetone)dipalladium (244.19 mg, 266.66 μmol, 0.03 eq) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (360.02 mg, 622.21 μmol, 0.07 eq) were added. After purging with nitrogen gas again, the reaction was carried out at 90 °C for 2 hours. After concentrating the reaction solution, it was dissolved in ethyl acetate (20 mL), washed with water (3 × 10 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0~19:1) to obtain compound WX006-3. 11H NMR (400 MHz, DMSO-d6) δ ppm 3.71 (s, 3 H), 4.14 (s, 2 H), 6.82 - 6.88 (m, 3 H), 7.14 - 7.19 (m, 2 H), 7.25 (d, J=8.53 Hz, 1 H), 7.41 (br d, J=9.29 Hz, 1 H), 7.63 (br dd, J=8.16, 5.40 Hz, 1 H).

[0198] Step 3: Synthesis of Compound WX006-4 WX006-3 (2.5 g, 8.38 mmol, 1 eq) was dissolved in trifluoroacetic acid (10 mL) and anisole (5 mL), and stirred at 80 °C for 1 hour. The reaction solution was cooled to 25 °C, added to ice water, extracted with ethyl acetate (50 mL), the organic phase was extracted with 5 M aqueous sodium hydroxide solution (3 × 30 mL), the aqueous phases were combined, the pH was adjusted to 2 with 2 M hydrochloric acid, extracted with dichloromethane (2 × 50 mL), the organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain Compound WX006-4. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 3.75 (s, 1 H), 6.79 - 6.98 (m, 1 H), 7.26 - 7.37 (m, 1 H), 7.39 - 7.45 (m, 1 H), 7.55 - 7.67 (m, 1 H).

[0199] Step 4: Synthesis of Compound WX006-5 WX006-4 (450.84 mg, 2.53 mmol, 1.05 eq), 1,8-diazabicyclo[5.4.0]undec-7-ene (733.74 mg, 4.82 mmol, 726.47 μL, 2 eq), and A-1 (650 mg, 2.41 mmol, 1 eq) were added to N,N-dimethylformamide (6.5 mL), and the mixture was stirred at 100 °C for 0.5 h. The reaction solution was cooled to 25 °C, 10 mL of ethyl acetate was added, and the organic phase was washed with water (3 × 5 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 5:1) to obtain compound WX006-5. LCMS: m / z [M+1] + = 412.

[0200] Step 5: Synthesis of compound WX006-6 WX006-5 (550 mg, 1.34 mmol, 1 eq) was added to a reaction flask containing trifluoroacetic acid (2 mL) and dichloromethane (5 mL), and the mixture was reacted at 25 °C for 1 h. Saturated sodium carbonate solution was added to adjust the pH to 8, 5 mL of dichloromethane was added, and the mixture was washed with water (3 × 3 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound WX006-6. LCMS: m / z [M+1] + = 312.

[0201] Step 6: Synthesis of compound WX006-7 WX006-6 (330 mg, 739.02 μmol, purity: 69.72%, 1 eq) and A-2 (202.49 mg, 812.92 μmol, 1.1 eq) were added to a reaction flask containing N,N-diisopropylethylamine (6 mL), and the mixture was reacted at 100 °C for 16 h. The reaction solution was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:1 to 1:3) to obtain compound WX006-7. LCMS: m / z [M+1] + = 524.

[0202] Step 7: Synthesis of compound WX006 WX006-7 (357 mg, 681.35 μmol, 1 eq) was added to a reaction flask containing dichloromethane (3.5 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. It was cooled to 0 °C, saturated sodium carbonate solution was added dropwise to the reaction solution to adjust the pH to 8, and the mixture was suction filtered. The cake was washed with water and dichloromethane and then dried to obtain compound WX006. 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.92 (br t, J = 5.40 Hz, 2 H), 3.77 (t, J = 5.65 Hz, 1 H), 3.83 - 3.88 (m, 1 H), 4.59 (s, 2 H), 7.12 (s, 1 H), 7.53 - 7.62 (m, 1 H), 7.64 - 7.71 (m, 1 H), 7.73 - 7.84 (m, 1 H), 7.99 (s, 1 H), 8.61 (s, 1 H), 12.95 (br s, 1 H); LCMS: m / z [M+1] + = 440.

[0203] Example 7

[0204]

Chemical formula

[0205] Synthesis route:

[0206]

Chemical formula

[0207] Step 1: Synthesis of compound WX007-2 Under nitrogen gas protection, WX007-1 (2 g, 9.75 mmol, 1 eq) and sulfur powder (469.19 mg, 14.63 mmol, 1.5 eq) were added to a reaction flask containing tetrahydrofuran (50 mL). The reaction flask was stirred in a dry ice - acetone bath for 10 minutes, then tert - butyllithium (1.3 M, 16.51 mL, 2.2 eq) was slowly added dropwise, and the reaction was carried out in a dry ice - acetone bath for 1 hour. After the reaction was completed, the reaction solution was added dropwise to 80 mL of saturated ammonium chloride solution, extracted with ethyl acetate (2×80 mL), the organic phases were combined, the organic phase was washed with 2 M hydrochloric acid (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 - 10:1) to obtain compound WX007 - 2. 1 H NMR (400 MHz, DMSO - d6) δ ppm 3.83 (s, 3 H), 4.83 (s, 1 H), 6.71 (td, J = 8.41, 2.76 Hz, 1 H), 6.90 - 6.97 (m, 1 H), 7.32 (dd, J = 8.53, 6.53 Hz, 1 H).

[0208] Step 2: Synthesis of compound WX007 - 3 A - 1 (2.65 g, 9.82 mmol, 1 eq) was dissolved in N,N - dimethylformamide (20 mL), 1,8 - diazabicyclo[5.4.0]undec - 7 - ene (2.99 g, 19.65 mmol, 2.96 mL, 2 eq) was added dropwise, WX007 - 2 (1.55 g, 9.82 mmol, 1 eq) was added, and the reaction was carried out at 100 °C for 1.5 hours. 200 mL of ethyl acetate was added, the organic phase was washed with semi - saturated brine (6×50 mL) and 1 M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 1:0 - 4:1) to obtain compound WX007 - 3. LCMS: m / z [M + 1] + = 392.

[0209] Step 3: Synthesis of compound WX007 - 4 WX007-3 (1.55 g, 3.96 mmol, 1 eq) was dissolved in a reaction flask containing dichloromethane (20 mL), and trifluoroacetic acid (17.41 g, 152.68 mmol, 11.30 mL, 38.56 eq) was added dropwise. The mixture was reacted at 20 °C for 0.5 h. Under an ice bath, saturated sodium carbonate solution was added dropwise to the reaction solution to adjust the pH to 8. The mixture was extracted with dichloromethane (3 × 50 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound WX007-4. LCMS: m / z [M+1] + = 292.

[0210] Step 4: Synthesis of compound WX007-5 WX007-4 (1.1 g, 3.78 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (46 mL), and A-2 (1.13 g, 4.53 mmol, 1.2 eq) was added. The mixture was reacted at 100 °C for 16 h. After concentrating the reaction solution, 50 mL of water was added, and the mixture was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:2) to obtain compound WX007-5. LCMS: m / z [M+1] + = 504.

[0211] Step 5: Synthesis of compound WX007 WX007-5 (1 g, 1.98 mmol, 1 eq) was added to a reaction flask containing dichloromethane (30 mL), and trifluoroacetic acid (6.79 g, 59.53 mmol, 4.41 mL, 30 eq) was added dropwise. The mixture was reacted at 20 °C for 0.5 h. Under an ice bath, the pH was adjusted to 9 with saturated brine, and the mixture was extracted with dichloromethane (3 × 40 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Phenomenex C18 80×40 mm×3 μm; mobile phase: [water (NH3H2O)-ACN]; gradient (ACN%): 31% - 61%) to obtain compound WX007. 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.88 (br t, J=5.14 Hz, 2 H), 3.76 (s, 3 H), 3.79 (br t, J=5.65 Hz, 2 H), 4.60 (s, 2 H), 6.90 (td, J=8.41, 2.26 Hz, 1 H), 7.11 (dd, J=11.17, 2.38 Hz, 1 H), 7.47 - 7.65 (m, 1 H), 8.00 (s, 1 H), 8.58 (s, 1 H), 12.96 (br s, 1 H); LCMS: m / z [M+1] + =420.

[0212] Example 8

[0213]

Chemical formula

[0214] Synthesis route:

[0215]

Chemical formula

[0216] Step 1: Synthesis of compound WX008-2 A-1 (5 g, 18.54 mmol, 1 eq) was dissolved in N,N-dimethylformamide (500 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (5.64 g, 37.07 mmol, 5.59 mL, 2 eq) was added, WX008-1 (5.29 g, 22.24 mmol, 1.2 eq) was added, and the mixture was reacted at 100 °C for 1.5 h. 1000 mL of ethyl acetate was added to the reaction solution, and it was washed with semi-saturated brine (6 × 100 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 4:1) to obtain compound WX008-2. LCMS: m / z [M+1] + = 472.

[0217] Step 2: Synthesis of compound WX008-3 Under nitrogen gas protection, WX008-2 (5 g, 10.61 mmol, 1 eq) was dissolved in a mixed solution of N,N-dimethylformamide (50 mL) and H2O (10 mL), vinylboronic acid pinacol ester (5.72 g, 37.13 mmol, 6.30 mL, 3.5 eq), tetrakis(triphenylphosphine)palladium(0) (1.23 g, 1.06 mmol, 0.1 eq) and sodium carbonate (6.75 g, 63.66 mmol, 6 eq) were added, and the mixture was reacted at 80 °C for 1 h. 1000 mL of ethyl acetate was added to the filtrate, and it was washed with semi-saturated brine (6 × 100 mL) and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 6:1) to obtain compound WX008-3. LCMS: m / z [M+1] + = 372.

[0218] Step 3: Synthesis of compound WX008-4 At 0 °C, WX008-3 (3.4 g, 9.15 mmol, 1 eq) was dissolved in dichloromethane (60 mL), N-bromosuccinimide (1.79 g, 10.07 mmol, 1.1 eq) was added dropwise, and then triethylamine hydrofluoride (2.95 g, 18.31 mmol, 2.98 mL, 2 eq) was added. The temperature was slowly raised to 20 °C and the reaction was carried out for 16 hours. The reaction solution was poured into a mixed solution of ice water (50 mL) and aqueous ammonia (10 mL), extracted with dichloromethane (3 × 60 mL), the organic phases were combined, washed with 1 M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 5:1) to obtain compound WX008-4. LCMS: m / z [M+1] + = 470.

[0219] Step 4: Synthesis of compound WX008-5 WX008-4 (2.54 g, 5.40 mmol, 1 eq) was dissolved in a reaction flask containing dichloromethane (30 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (1.15 g, 7.56 mmol, 1.14 mL, 1.4 eq) was added dropwise, and the reaction was carried out at 60 °C for 16 hours. The reaction solution was directly concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain compound WX008-5. LCMS: m / z [M+1] + = 390.

[0220] Step 5: Synthesis of compound WX008-6 WX008-5 (1.71 g, 4.39 mmol, 1 eq) was dissolved in a reaction flask containing dichloromethane (20 mL), trifluoroacetic acid (19.31 g, 169.33 mmol, 12.54 mL, 38.56 eq) was added dropwise, and the reaction was carried out at 20 °C for 0.5 hour. Under an ice bath, saturated sodium carbonate solution was added dropwise to the reaction solution to adjust the pH to 8, extracted with dichloromethane (3 × 20 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound WX008-6. LCMS: m / z [M+1]+ = 290。

[0221] Step 6: Synthesis of Compound WX008-7 WX008-6 (1.14 g, 3.94 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (48 mL), A-2 (1.18 g, 4.73 mmol, 1.2 eq) was added, and the reaction was carried out at 100 °C for 16 h. After concentrating the reaction solution, 50 mL of water was added, and the mixture was extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:2) to obtain Compound WX008-7. LCMS: m / z [M+1] + = 502。

[0222] Step 7: Synthesis of Compound WX008 WX008-7 (1 g, 1.99 mmol, 1 eq) was added to a reaction flask containing dichloromethane (15 mL), trifluoroacetic acid (6.82 g, 59.77 mmol, 4.43 mL, 30 eq) was added dropwise, and the reaction was carried out at 20 °C for 0.5 h. Under an ice bath, the pH was adjusted to 9 with saturated sodium carbonate solution, and the mixture was extracted with dichloromethane (3 × 15 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (chromatography column: Phenomenex C18 80×40 mm×3 μm; mobile phase: [water (NH3H2O)-ACN]; gradient (ACN%): 33% - 63%) to obtain Compound WX008. 11H NMR (400 MHz, DMSO-d6) δ ppm 3.00 (broad t, J = 5.27 Hz, 2 H), 3.79 (broad t, J = 5.52 Hz, 2 H), 4.66 (s, 2 H), 5.06 - 5.11 (m, 1 H), 5.11 - 5.24 (m, 1 H), 7.39 - 7.43 (m, 2 H), 7.50 (dd, J = 9.16, 2.38 Hz, 1 H), 8.03 (s, 1 H), 8.53 (s, 1 H), 12.56 - 13.07 (m, 1 H); LCMS: m / z [M+1] + = 418。

[0223] Example 9

[0224]

Chem.

[0225] Synthesis route:

[0226]

Chem.

[0227] Step 1: Synthesis of compound WX009-2 Add A-1 (3 g, 11.12 mmol, 1 eq) to a reaction flask containing N,N-dimethylformamide (30 mL), add 1,8-diazabicyclo[5.4.0]undec-7-ene (3.39 g, 22.24 mmol, 3.35 mL, 2 eq), then add WX009-1 (2.20 g, 12.23 mmol, 1.1 eq), and react at 100 °C for 0.5 h. Next, add 800 mL of ethyl acetate to the reaction solution, wash the organic phase with semi-saturated brine (5 × 250 mL), then wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain compound WX009-2. LCMS: m / z [M+1] + = 414。

[0228] Step 2: Synthesis of compound WX009-3 WX009-2 (2 g, 4.84 mmol, 1 eq) was added to a reaction flask, followed by dichloromethane (20 mL). Then, trifluoroacetic acid (10.29 g, 90.23 mmol, 6.68 mL, 18.65 eq) was added, and the mixture was reacted at 20 °C for 0.5 h. 20 mL of water was added to the reaction solution, and then sodium carbonate was added to adjust the pH to 7. The mixture was extracted with dichloromethane (2 × 20 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound WX009-3. LCMS: m / z [M+1] + = 314.

[0229] Step 3: Synthesis of compound WX009 Under nitrogen gas protection, WX009-3 (1 g, 3.19 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (19.19 g, 148.51 mmol, 25.87 mL, 46.52 eq). Then, 4,5-dibromopyridazin-3-one (891.52 mg, 3.51 mmol, 1.1 eq) was added, and the mixture was reacted at 100 °C for 24 h. The reaction solution was directly concentrated, and then 20 mL of a mixed solvent (dichloromethane:methanol = 20:1) was added. The organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated by preparative high-performance liquid chromatography (chromatography column: Xtimate C18 150 × 40 mm × 10 μm; mobile phase: [water (FA)-ACN]; ACN%: 35% - 65%) to obtain a crude product. 2 mL of DCM was added, and the mixture was stirred at 20 °C for 16 h. Then, it was suction filtered, and the cake was washed with 1 mL of DCM and then dried to obtain compound WX009. 1 H NMR (400 MHz, CDCl3) δ ppm 3.12 (t, J = 5.77 Hz, 2 H), 3.88 (t, J = 5.77 Hz, 2 H), 4.66 (s, 2 H), 7.32 - 7.38 (m, 2 H), 7.45 (br d, J = 7.53 Hz, 1 H), 7.77 (s, 1 H), 8.55 (s, 1 H); LCMS: m / z [M+1] + = 486.

[0230] Example 10

[0231]

Chem.

[0232] Synthesis route:

[0233]

Chem.

[0234] Step 1: Synthesis of compound WX010-2 A-1 (5 g, 18.54 mmol, 1 eq) and WX010-1 (4.29 g, 27.81 mmol, 1.5 eq) were dissolved in N,N-dimethylformamide (40 mL), then potassium carbonate (7.69 g, 55.61 mmol, 3 eq) was added. After the reaction system was replaced with nitrogen gas three times, it was stirred at 60 °C for 20 h. 100 mL of ethyl acetate was added, the organic phase was washed with water (100 mL), the aqueous phase was extracted with dichloromethane (3 × 50 mL), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 - 15:1) to obtain compound WX010-2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.52 (s, 9 H), 2.47 (s, 3 H), 2.92 (br t, J = 5.4 Hz, 2 H), 3.79 (t, J = 5.8 Hz, 2 H), 4.65 (s, 2 H), 7.14 (dd, J = 8.8, 4.5 Hz, 1 H), 7.28 - 7.35 (m, 1 H), 7.56 (dd, J = 8.7, 3.1 Hz, 1 H), 8.49 (s, 1 H); LCMS: m / z [M+1] + = 388.

[0235] Step 2: Synthesis of compound WX010-3 WX010-2 (2.3 g, 5.94 mmol, 1 eq) was dissolved in dichloromethane (100 mL), and then trifluoroacetic acid (25.72 g, 225.60 mmol, 16.70 mL, 38 eq) was added. The reaction system was stirred at 20 °C for 1 hour. 100 mL of water was added to the reaction solution, sodium carbonate was added to adjust the pH to 9, and the aqueous phase was extracted with dichloromethane (3 × 50 mL). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound WX010-3. LCMS: m / z [M+1] + = 288.

[0236] Step 3: Synthesis of compound WX010-4 WX010-3 (2.25 g, 7.83 mmol, 1 eq) was dissolved in N,N-diisopropylethylamine (70.85 g, 548.23 mmol, 95.49 mL, 70 eq), A-1 (2.34 g, 9.40 mmol, 1.2 eq) was added, and the reaction system was stirred at 100 °C for 16 hours. It was distilled under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 5:1) to obtain compound WX010-4. LCMS: m / z [M+1] + = 500.

[0237] Step 4: Synthesis of compound WX010-5 WX010-4 (0.5 g, 1.00 mmol, 1 eq) was dissolved in dichloromethane (20 mL), and then bis(2-methoxyethyl)aminosulfur trifluoride (2.21 g, 10.00 mmol, 2.19 mL, 10 eq) was slowly added. The reaction system was stirred at 40 °C for 20 hours. The reaction solution was slowly added to 50 mL of saturated sodium bicarbonate solution, stirred for 5 minutes, and the aqueous phase was extracted with dichloromethane (2 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product. The crude product was separated by preparative high-performance liquid chromatography (chromatography column: Phenomenex C18 150×40 mm×5 μm; mobile phase: [water (HCl)-ACN]; gradient (ACN%): 50% - 60%) to obtain compound WX010-5.1 1H NMR (400 MHz, CDCl3) δ ppm 1.55 - 1.62 (m, 1 H), 1.70 - 1.82 (m, 3 H), 2.00 - 2.07 (m, 1 H), 2.09 - 2.20 (m, 1 H), 2.49 (s, 3 H), 3.11 (br t, J = 5.5 Hz, 2 H), 3.70 - 3.94 (m, 3 H), 4.09 - 4.20 (m, 1 H), 4.62 (s, 2 H), 6.08 (dd, J = 10.7, 1.6 Hz, 1 H), 7.17 (dd, J = 9.0, 4.5 Hz, 1 H), 7.28 - 7.36 (m, 1 H), 7.57 (dd, J = 8.5, 3.0 Hz, 1 H), 7.81 (s, 1 H), 8.50 (s, 1 H); LCMS: m / z [M+23] + = 544。

[0238] Step 5: Synthesis of Compound WX010 WX010-5 (0.088 g, 168.61 μmol, 1 eq) was dissolved in ethyl acetate (1 mL), and then hydrogen chloride - 4M ethyl acetate solution (2 mL) was added. The reaction system was stirred at 20 °C for 1 hour. The reaction solution was concentrated to obtain a crude product. The crude product was dissolved in 50 mL of water, and saturated sodium carbonate solution was slowly added to adjust the pH to 9. It was filtered and dried. Next, 2 mL of dichloromethane was added and stirred for 0.5 hour, then filtered, and the cake was washed with 0.5 mL of dichloromethane. The cake was dried to obtain Compound WX010. 1 1H NMR (400 MHz, DMSO-d6) δ ppm 1.97 (br t, J = 19.1 Hz, 3 H), 3.00 (br s, 2 H), 3.79 (br s, 2 H), 4.66 (br s, 2 H), 7.47 (br s, 3 H), 8.03 (s, 1 H), 8.54 (s, 1 H), 13.00 (br s, 1 H); LCMS: m / z [M+1] + = 438。

[0239] Example 11

[0240] [Chemistry]

[0241] Synthesis route:

[0242] [Chemistry]

[0243] Step 1: Synthesis of compound WX011-1 A-3 (10 g, 22.33 mmol, 1 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (326.79 mg, 446.62 μmol, 0.02 eq) and triethylamine (44.66 mmol, 6.22 mL, 2 eq) were added to an autoclave containing methanol (400 mL), and the reaction was carried out under a nitric oxide (150 Psi) atmosphere at 100 °C for 18 hours. The reaction solution was concentrated to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 1:0 - 9:1) to obtain compound WX011-1. LCMS: m / z [M+1] + = 472.

[0244] Step 2: Synthesis of compound WX011-2 WX011-1 (0.20 g, 424.27 μmol, 1 eq) and tert-butanol (5 mL) were added to a reaction flask, and sodium borohydride (50 mg, 1.32 mmol, 3.12 eq) was added at room temperature. The temperature was slowly raised to 70 °C and stirred at 70 °C for 3 hours. A saturated ammonium chloride solution (10 mL) was added dropwise to the reaction solution to quench the reaction system, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound WX011-2. LCMS: m / z [M+1] + = 444.

[0245] Step 3: Synthesis of Compound WX011-3 WX011-2 (60 mg, 135.32 μmol, 1 eq), anhydrous tetrahydrofuran (1 mL) were added to a reaction flask, and phosphorus tribromide (270.64 μmol, 25.44 μL, 2 eq) was added at 0 °C. The reaction system was stirred at 0 °C for 1 hour. Saturated sodium carbonate solution (5 mL) was added to the reaction solution to quench the reaction system, and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 3:1) to obtain Compound WX011-3. 1 H NMR (400 MHz, CDCl3) δ ppm 1.51 (s, 9 H), 2.89 (br s, 2 H), 3.77 (br t, J = 5.77 Hz, 2 H), 4.32 (s, 2 H), 4.64 (s, 2 H), 7.30 - 7.40 (m, 2 H), 7.43 (dd, J = 8.16, 2.64 Hz, 1 H); LCMS: m / z [M+1] + = 506.

[0246] Step 4: Synthesis of Compound WX011-4 WX011-3 (40 mg, 79.01 μmol, 1 eq), anhydrous methanol (0.5 mL) were added to a reaction flask, and sodium methoxide (42.68 mg, 790.06 μmol, 10 eq) was added. The mixture was stirred at room temperature for 1 hour. Citric acid solution (1 M) was added dropwise to the reaction solution to adjust the pH of the reaction solution to about 7, and the mixture was concentrated to remove methanol. Water (5 mL) was added, and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Compound WX011-4. LCMS: m / z [M+1] + = 458.

[0247] Step 5: Synthesis of Compound WX011-5 WX011-4 (60 mg, 131.17 μmol, 1 eq), anhydrous dichloromethane (1 mL) were added to a reaction flask, trifluoroacetic acid (4.05 mmol, 0.3 mL, 30.89 eq) was added, and the mixture was stirred at room temperature for 1 hour. Saturated sodium bicarbonate solution was added dropwise to the reaction solution to adjust the pH of the reaction solution to about 7, and then separated. The aqueous phase was extracted with dichloromethane (2 × 5 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound WX011-5. LCMS: m / z [M+1] + = 358.

[0248] Step 6: Synthesis of compound WX011-6 WX011-5 (60 mg, 167.93 μmol, 1 eq), A-2 (50 mg, 200.73 μmol, 1.20 eq), N,N-diisopropylethylamine (5.74 mmol, 1 mL, 34.19 eq) were added to a reaction flask, and the mixture was stirred at 90 °C for 16 hours. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound WX011-6. LCMS: m / z [M+1] + = 570.

[0249] Step 7: Synthesis of compound WX011 WX011-6 (30 mg, 52.64 μmol, 1 eq), anhydrous dichloromethane (0.5 mL) were added to a reaction flask, trifluoroacetic acid (2.70 mmol, 0.20 mL, 51.32 eq) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated to obtain a crude product. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150×25mm×5μm; mobile phase: [water (NH4HCO3)-ACN]; gradient) (ACN%): 23% - 53%) to obtain compound WX011. 11H NMR (400 MHz, CDCl3) δ ppm 3.09 (s, 2H), 3.45 (s, 3 H), 3.87 (t, J = 5.65 Hz, 2 H), 4.43 (s, 2 H), 4.69 (s, 2 H), 7.32 - 7.39 (m, 2 H), 7.42 - 7.46 (m, 1 H), 7.73 (s, 1 H), 10.33 (br s, 1 H); LCMS: m / z [M+1] + = 486。

[0250] Example 12

[0251]

Chem.

[0252] Synthesis route:

[0253]

Chem.

[0254] Step 1: Synthesis of compound WX012-2 WX012-1 (478.35 mmol, 35.56 mL, 1 eq), MeOH (170 mL), sodium methoxide (3.40 g, 62.94 mmol, 0.13 eq) were added to the reaction flask, and the reaction system was stirred at room temperature (20 °C) for 1 hour. Next, ammonium chloride (25.84 g, 483.07 mmol, 1.01 eq) was added, and the reaction system was stirred at 40 °C for 3 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude hydrochloride product of WX012-2, which was directly used in the next step. 1 1HNMR (400 MHz, DMSO-d6) δ ppm 3.35 (s, 3H), 4.24 (s, 2H), 8.86 (br s, 4 H).

[0255] Step 2: Synthesis of compound WX012-3 Ethyl 1-BOC-3-oxo-4-piperidinecarboxylate (2.00 g, 7.37 mmol, 1 eq), WX012-2 (2.20 g, crude hydrochloride product), sodium ethoxide (1.11 g, 16.33 mmol, 2.21 eq), and absolute ethanol (30 mL) were added to a reaction flask, and the reaction system was stirred at 100 °C for 4 hours. The reaction solution was concentrated, water (20 mL) was added, the pH was adjusted to about 6 with hydrochloric acid (1 M), and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. Petroleum ether:ethyl acetate (20:1, 20 mL) was added to the crude product, and the mixture was stirred at room temperature (20 °C) for 0.2 hour, filtered, and the cake was dried to obtain WX012-3. 1 H NMR (400 MHz, CDCl3) δ ppm 1.49 (s, 9H), 2.62 (br s, 2H), 3.52 (s, 3H), 3.62 (br t, J=5.57 Hz, 2H), 4.35 (br s, 2H), 4.38 (s, 2H), 9.69 (br s, 1 H);LCMS: m / z [M+1] + =295.9.

[0256] Step 3: Synthesis of Compound WX012-4 WX012-3 (1.60 g, 5.42 mmol, 1 eq), triphenylphosphine (2.85 g, 10.87 mmol, 2.01 eq), and anhydrous toluene (20 mL) were added to a reaction flask. Under a nitrogen gas atmosphere, 2,2,2-trichloroacetonitrile (1.18 g, 8.18 mmol, 0.82 mL, 1.51 eq) was added, and the reaction system was stirred at 100 °C for 1.5 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. A mixed solvent (petroleum ether:ethyl acetate = 10:1, 30 mL) was added to the crude product, and the mixture was stirred at room temperature (20 °C) for 0.2 hour, filtered, and the filtrate was concentrated. The crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 5:1) to obtain WX012-4. LCMS: m / z [M+1] + =313.9.

[0257] Step 4: Synthesis of Compound WX012-5 WX012-5 (0.65 g, 2.07 mmol, 1 eq), WX001-2 (0.45 g, 2.29 mmol, 1.11 eq), potassium carbonate (0.56 g, 4.05 mmol, 1.96 eq), and anhydrous DMF (10 mL) were added to a reaction flask, and the reaction system was stirred at 100 °C for 1 hour. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography (PE:EA = 1:0~2:1) to obtain WX012-5. LCMS: m / z [M+1] + = 473.9

[0258] Step 5: Synthesis of Compound WX012-6 WX012-5 (0.30 g, 633.60 μmol, 1 eq) and anhydrous DCM (3 mL) were added to a reaction flask, and trifluoroacetic acid (20.26 mmol, 1.50 mL, 31.98 eq) was added. The reaction system was stirred at room temperature (20 °C) for 2 hours. Saturated sodium bicarbonate solution was added dropwise to the reaction solution to adjust the pH of the reaction solution to about 7. The layers were separated, and the aqueous phase was extracted with DCM (2 × 10 mL). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product WX012-6, which was directly used in the next step reaction. LCMS: m / z [M+1] + = 373.9

[0259] Step 6: Synthesis of Compound WX012-7 WX012-6 (0.15 g, 401.75 μmol, 1 eq), A-2 (0.10 g, 401.46 μmol, 1 eq), N,N-diisopropylethylamine (1.44 mmol, 0.25 mL, 3.57 eq), and DMA (1.5 mL) were added to a reaction flask, and the reaction system was stirred at 100 °C for 3 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by prep-TLC (PE:EA = 1:1) to obtain WX012-7. LCMS: m / z [M+23] + = 607.8.

[0260] Step 7: Synthesis of Compound WX012 WX012-7 (0.12 g, 204.78 μmol, 1 eq) and anhydrous DCM (2 mL) were added to a reaction flask, and boron tribromide (830.26 μmol, 80 μL, 4.05 eq) was added. The reaction system was stirred at room temperature (20 °C) for 1 hour. A saturated sodium carbonate solution was added dropwise to the reaction solution to quench the reaction system, and the pH of the solution was adjusted to about 7. The mixture was concentrated to obtain a crude product. A mixed solvent (DCM:MeOH = 10:1, 5 mL) was added to the crude product and stirred uniformly, then filtered, and the filtrate was concentrated. The crude product was separated and purified by preparative plate (DCM:MeOH = 20:1), and further separated and purified by preparative high performance liquid chromatography (chromatography column: Phenomenex C18 80×40 mm×3 μm; mobile phase: [water (ammonia water)-ACN]; gradient (ACN)%: 35% - 65%) to obtain WX012. 11H NMR (400 MHz, CDCl3) δ ppm 2.96 - 3.15 (m, 3H), 3.89 (br t, J = 5.40 Hz, 2H), 4.52 (br d, J = 4.02 Hz, 2H), 4.61 (s, 2H), 7.37 (br t, J = 6.90 Hz, 1H), 7.59 (dd, J = 8.66, 2.38 Hz, 1H), 7.69 (br dd, J = 8.41, 5.40 Hz, 1H), 7.76 (s, 1H), 11.38 (br s, 1 H); LCMS: m / z [M+1] + = 488.0。

[0261] Example 13

[0262]

Chem.

[0263] Synthesis route:

[0264]

Chem.

[0265] Step 1: Synthesis of Compound WX013-1 A-3-1 (18 g, 59.18 mmol, 0.95 eq) and WX001-2 (15.27 g, 62.29 mmol, purity: 80%, 1 eq) were dissolved in ACN (200 mL), potassium carbonate (25.83 g, 186.88 mmol, 3 eq) was added, and the reaction system was stirred at 50 °C for 4 h. It was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to column chromatography (petroleum ether:ethyl acetate = 20:1~10:1) to obtain WX013-1. LCMS: m / z [M+1] + = 464.0。

[0266] Step 2: Synthesis of Compound WX013-2 WX013-1 (13 g, 28.02 mmol, 1 eq), ACN (100 mL), and 2,4-dimethoxybenzylamine (23.43 g, 140.12 mmol, 21.11 mL, 5 eq) were added to a reaction flask and stirred at 80 °C under nitrogen gas for 12 h. The reaction solution was cooled to room temperature, filtered, and the cake was taken out separately. After the filtrate was concentrated under reduced pressure, it was separated and purified by column chromatography (PE:EA = 3:1) to obtain a crude product. After combining the crude product with the cake, it was added to 15 mL of MeOH and stirred at room temperature (20 °C) for 1 h, filtered, and the cake was collected to obtain WX013-2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.49 (s, 9H), 2.64 (br s, 2H), 3.70 (br s, 2H), 3.79 (s, 6H), 4.04 (br s, 2H), 4.41 (br s, 2H), 6.29 (br d, J = 8.78 Hz, 1H), 6.34 - 6.57 (m, 2H), 7.18 (br s, 1H), 7.45 (br d, J = 8.03 Hz, 1H), 7.59 (dd, J = 8.41, 5.65 Hz, 1H); LCMS: m / z [M+1] + = 595.

[0267] Step 3: Synthesis of compound WX013-3 WX013-2 (10 g, 16.82 mmol, 1 eq) and a dioxane solution of hydrogen chloride (4 M, 100 mL) were added to a reaction flask, and the reaction system was stirred at room temperature (20 °C) for 3 h. Saturated sodium bicarbonate solution was added to the reaction solution to adjust the pH to about 8, and DCM (100 mL × 3) was added for extraction. The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain WX013-3, which was directly used in the next step. 11H NMR (400 MHz, CDCl3) δ ppm 2.63 (broad t, J = 5.65 Hz, 2H), 3.21 (t, J = 5.77 Hz, 2H), 3.79 (d, J = 4.27 Hz, 6H), 3.83 (singlet, 2H), 4.04 (broad singlet, 2H), 6.29 (broad d, J = 8.28 Hz, 1H), 6.41 (d, J = 2.26 Hz, 1H), 6.59 (broad singlet, 1H), 7.15 (broad t, J = 8.03 Hz, 1H), 7.43 (doublet of doublets, J = 9.16, 2.89 Hz, 1H), 7.59 (doublet of doublets, J = 8.53, 5.52 Hz, 1H); LCMS: m / z [M+1] + = 494.9。

[0268] Step 4: Synthesis of Compound WX013-4 WX013-3 (11 g, 22.24 mmol, 1 eq), A-2 (4.99 g, 20.02 mmol, 0.9 eq), and N,N-diisopropylethylamine (110 mL) were mixed, heated to 100 °C, and stirred for 12 hours. After concentrating the reaction solution under reduced pressure, the crude product was separated and purified by column chromatography (PE:EA, EA ratio = 0 - 60%) to obtain WX013-4. 1 1H NMR (400 MHz, CDCl3) δ ppm 1.72 (broad singlet, 6H), 2.81 (broad t, J = 5.52 Hz, 2H), 3.79 (d, J = 1.76 Hz, 8H), 3.92 - 4.20 (multiplet, 4H), 4.37 (singlet, 2H), 6.04 - 6.14 (multiplet, 1H), 6.30 (broad d, J = 7.78 Hz, 1H), 6.36 - 6.53 (multiplet, 2H), 7.15 - 7.22 (multiplet, 1H), 7.46 (doublet of doublets, J = 8.78, 2.76 Hz, 1H), 7.60 (doublet of doublets, J = 8.41, 5.40 Hz, 1H), 7.75 (singlet, 1H), ; LCMS: m / z [M+1] + = 707。

[0269] Step 4: Synthesis of Compound WX013 WX013-4 (5 g, 7.07 mmol, 1 eq), DCM (50 mL) were added to a reaction flask, trifluoroacetic acid (16.12 g, 141.42 mmol, 10.47 mL, 20 eq) was added dropwise, and the reaction system was stirred at 35 °C for 12 h. The reaction solution was concentrated, MeOH (50 mL) was added, and then concentrated again to obtain the crude product. 50 mL of DCM was added to the crude product, stirred at room temperature for 1 h, filtered, ethyl acetate (15 mL) was added to the cake, stirred at room temperature for 3 h, filtered, and the cake was dried to obtain WX013. 1 H NMR (400 MHz, CDCl3) δ ppm 2.88 (t, J = 5.65 Hz, 2H), 3.86 (t, J = 5.77 Hz, 2H), 4.58 (s, 2H), 7.53 (td, J = 8.22, 2.64 Hz, 1H), 7.70 (dd, J = 9.03, 2.76 Hz, 1H), 7.81 (dd, J = 8.53, 5.27 Hz, 1H), 7.97 (s, 1H); LCMS: m / z [M+1] + = 472.9.

[0270] Example 14

[0271]

Chemical formula

[0272] Synthesis route:

[0273]

Chemical formula

[0274] Step 1: Synthesis of compound WX014-2 WX014-1 (30 g, 332.89 mmol, 35.21 mL, 1 eq) and trimethylsilyl cyanide (33.02 g, 332.89 mmol, 41.64 mL, 1 eq) were added to a reaction flask. Boron trifluoride diethyl ether solution (3.45 g, 24.31 mmol, 3.00 mL, 0.073 eq) was slowly added dropwise at 0 °C. After the addition was complete, the mixture was stirred at 20 °C for 16 h. 20 mL of saturated sodium bicarbonate solution was added to the reaction solution to quench the reaction system, and then it was extracted with DCM (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and then concentrated under reduced pressure at room temperature (25 °C) to obtain WX014-2, which was directly used in the next step. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.45 (s, 3H), 3.37 (s, 3H), 4.44 (q, J = 6.78 Hz, 1H).

[0275] Step 2: Synthesis of compound WX014-3 WX014-2 (20 g, 235.01 mmol, 1 eq) was added to a reaction flask containing ethanol (100 mL). Ammonia gas was bubbled through the reaction system at -5 °C for 15 min, and then the reaction mixture was stirred at 25 °C for 16 h. The reaction solution was concentrated under reduced pressure to remove part of the ammonia gas, and an ethanol solution of WX014-3 was obtained, which was directly charged into the next step.

[0276] Step 3: Synthesis of compound WX014-4 To the ethanol solution of WX014-3 obtained in Step 2, sodium ethoxide (3.45 g, 50.68 mmol, 2.5 eq) was added. Next, ethyl 1-Boc-3-oxo-4-piperidinecarboxylate (5.5 g, 20.27 mmol, 1 eq) was added, and the reaction was carried out with stirring at 100 °C for 6 hours under nitrogen gas protection. After the reaction solution was concentrated under reduced pressure, 100 mL of water was added to dissolve it, and the pH was adjusted to 6 with 1 M hydrochloric acid aqueous solution. Then, it was extracted with ethyl acetate (2 × 100 mL). After combining the organic phases, they were washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and then the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (MeOH / DCM, MeOH ratio = 0% - 6%) to obtain WX014-4. LCMS: m / z [M+1] + = 309.9.

[0277] Step 4: Synthesis of compound WX014-5 WX014-4 (4.16 g, 13.45 mmol, 1 eq) and triphenylphosphine (10.58 g, 40.34 mmol, 3 eq) were added to anhydrous toluene (50 mL). Under nitrogen gas protection, 2,2,2-trichloroacetonitrile (2.91 g, 20.17 mmol, 2.02 mL, 1.5 eq) was added, and the reaction system was reacted at 100 °C for 1 hour. After the reaction solution was cooled, 80 mL of saturated sodium bicarbonate solution was added to quench the reaction system. The aqueous phase was extracted with ethyl acetate (2 × 80 mL). The organic phases were combined, washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. 100 mL of a mixed solvent (PE:EA = 3:1) was added to the crude product and stirred for 1 hour. Then, it was filtered, and the cake was washed with 50 mL of the mixed solvent (PE:EA = 3:1). The filtrates were combined and concentrated to obtain a crude product. The crude product was purified by column chromatography (EA / PE, EA ratio = 0% - 20%) to obtain WX014-5. LCMS: m / z [M+1] + = 327.9.

[0278] Step 5: Synthesis of compound WX014-6 Under nitrogen gas protection, WX014-5 (1.62 g, 4.94 mmol, 1 eq) and WX001-2 (1.51 g, 4.94 mmol, purity: 64%, 1 eq) were added to a reaction flask containing DMF (16 mL). Next, 1,8-diazabicyclo[5.4.0]undec-7-ene (1.50 g, 9.88 mmol, 1.49 mL, 2 eq) was added, and the mixture was stirred at 120 °C for 1 hour. After the reaction solution was cooled to room temperature, 30 mL of ethyl acetate was added, and the mixture was washed with 30 mL of saturated brine. Then, the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain a crude product. The crude product was purified by column chromatography (EA / PE, EA ratio = 0% - 25%) to obtain WX014-6. LCMS: m / z [M+1] + = 488.1.

[0279] Step 6: Synthesis of compound WX014-7 WX014-6 (1.79 g, 3.67 mmol, 1 eq) was added to a reaction flask containing DCM (18 mL). Next, trifluoroacetic acid (5 mL) was added, and the reaction system was reacted at 25 °C for 1 hour. 20 mL of water was added to the reaction solution, and then sodium carbonate was added to adjust the pH to 8. The aqueous phase was extracted with DCM (2 × 20 mL). The combined organic phases were washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to obtain the crude product WX014-7, which was directly used in the next step. LCMS: m / z [M+1] + = 388.0.

[0280] Step 7: Synthesis of compound WX014-8 WX014-7 (1.5 g, 3.87 mmol, 1 eq) was added to a reaction flask containing N,N-diisopropylethylamine (30 mL). Next, A-2 (1.06 g, 4.26 mmol, 1.1 eq) was added, and the reaction system was stirred at 100 °C for 16 hours. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (MeOH / DCM, MeOH ratio = 0% - 8%) to obtain WX014-8. LCMS: m / z [M+1] + = 600.1.

[0281] Step 8: Synthesis of Compound WX014 WX014-8 (400 mg, 666.64 μmol, 1 eq) was added to a reaction flask containing anhydrous DCM (10 mL). After cooling to 0 °C, a solution of boron tribromide (1.67 g, 6.67 mmol, 642.33 μL, 10 eq) in anhydrous DCM (5 mL) was slowly added dropwise. After the addition was complete, the reaction was carried out at 25 °C for 16 h. The reaction solution was added to an aqueous sodium carbonate solution (10 mL) at 0 °C to quench the reaction system. The aqueous phase was extracted with DCM (2 × 10 mL). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography (chromatography column: Phenomenex C18 150×40 mm×5 μm; mobile phase: water (HCl)-ACN]; gradient (ACN)%: 35% - 55%) to obtain WX014. 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.08 (d, J=6.53 Hz, 3H), 2.87 (br t, J=5.27 Hz, 2H), 3.79 (br t, J=5.52 Hz, 2H), 4.41 (q, J=6.53 Hz, 1H), 4.62 (s, 2H), 7.69 (td, J=8.47, 2.89 Hz, 1H), 7.82 - 7.91 (m, 2H), 7.98 (s, 1H), 12.96 (s, 1 H);LCMS: m / z [M+1] + = 502.0。

[0282] Step 9: Synthesis of Compounds WX014A and WX014B WX014 (230 mg) was separated by chiral preparative chromatography (chromatography column: DAICEL CHIRALPAK AD (250 mm×30 mm, 10 μm); mobile phase: A: CO2, B: MeOH (0.1% aqueous ammonia); gradient (B%): 35% - 35%) to obtain WX014A and WX014B.

[0283] WX014A: SFC analysis method: Chromatography column: ChiralPak AD-3 150×4.6 mm I.D., 3 μm; Mobile phase: A: CO2, B: MeOH (0.05% DEA); Gradient of B: 40%, Retention time: 3.356 min, ee = 100% 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.09 (d, J = 6.53 Hz, 3H), 2.87 (br t, J = 5.27 Hz, 2H), 3.80 (t, J = 5.77 Hz, 2H), 4.37 - 4.46 (m, 1H), 4.63 (s, 2H), 4.88 (d, J = 5.27 Hz, 1H), 7.70 (td, J = 8.41, 2.76 Hz, 1H), 7.83 - 7.93 (m, 2H), 7.99 (s, 1H), 12.96 (br s, 1 H); LCMS: m / z [M+1] + = 502.0。

[0284] WX014B: SFC analysis method: Chromatography column: ChiralPak AD-3 150×4.6 mm I.D., 3 μm; Mobile phase: A: CO2, B: MeOH (0.05% DEA); Gradient of B: 40%, Retention time: 3.859 min, ee = 97.5% 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.09 (d, J = 6.53 Hz, 3H), 2.87 (br t, J = 5.52 Hz, 2H), 3.80 (br t, J = 5.40 Hz, 2H), 4.42 (quin, J = 6.21 Hz, 1H), 4.63 (s, 2H), 4.87 (d, J = 5.27 Hz, 1H), 7.66 - 7.74 (m, 1H), 7.84 - 7.92 (m, 2H), 7.99 (s, 1H), 12.90 (s, 1 H); LCMS: m / z [M+1] + = 502.0。

[0285] Example 15

[0286]

Chemical formula

[0287] Synthesis route:

[0288]

Chem.

[0289] Step 1: Synthesis of compound WX015-2 WX015-1 (4.58 g, 27.81 mmol, 1.5 eq) and A-1 (5 g, 18.54 mmol, 1 eq) were dissolved in DMA (50 mL), and DBU (5.64 g, 37.07 mmol, 2 eq) was added. The reaction system was reacted at 100 °C for 1.5 h. The reaction system was cooled to room temperature, 500 mL of semi-saturated brine was added, then extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 - 8:1) to obtain WX015-2, which was directly used in the next step. LCMS: m / z [M+1] + = 398.1.

[0290] Step 2: Synthesis of compound WX015-3 WX015-2 (5.4 g, 13.57 mmol, 1 eq) was dissolved in anhydrous DCM (120 mL), and TFA (20 mL) was added. The reaction system was reacted at 20 °C for 1 h. Saturated aqueous sodium carbonate solution was added to the reaction solution to adjust the pH to 9, then extracted with DCM (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product WX015-3. LCMS: m / z [M+1] + = 298.0.

[0291] Step 3: Synthesis of compound WX015-4 WX015-3 (2.0 g, 5.81 mmol, 1 eq) and A-2 (1.59 g, 6.39 mmol, 1.1 eq) were added to a reaction flask, DIEA (229.64 mmol, 40 mL, 39.53 eq) was added, and the reaction system was stirred at 100 °C for 16 hours under nitrogen gas protection. The reaction system was cooled to room temperature and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:2) to obtain WX015-4. LCMS: m / z [M+1] + = 510.0。

[0292] Step 4: Synthesis of Compound WX015 WX015-4 (1.59 g, 3.12 mmol, 1 eq) was dissolved in anhydrous DCM (20 mL), and TFA (9 mL) was added. The reaction system was stirred at 20 °C for 2 hours. A saturated aqueous sodium carbonate solution was added to the reaction system to adjust the pH to about 9, filtered, and the cake was dried to obtain WX015. 1 H NMR (400 MHz, DMSO-d6) δ ppm 3.03 (br t, J=5.27 Hz, 2 H) 3.79 (t, J=5.77 Hz, 2 H) 4.68 (s, 2 H) 7.57 - 7.65 (m, 2 H) 8.01 (s, 1 H) 8.58 (s, 1 H) 12.95 (s, 1 H); LCMS: m / z [M+1] + = 425.9。

[0293] Example 16

[0294]

Chemical Structure

[0295] Synthesis Route:

[0296]

Chemical Structure

[0297] Step 1: Synthesis of Compound WX016-2 WX016-1 (2.93 g, 26.54 mmol, 2.4 eq) and ethyl 1-Boc-3-oxo-4-piperidinecarboxylate (3 g, 11.06 mmol, 1 eq) were dissolved in MeOH (30 mL), and sodium methoxide methanol solution (4.36 mL, 24.33 mmol, 2.2 eq, 30 wt%) was added. The reaction system was stirred at 100 °C for 4 hours. After the reaction system was cooled to room temperature, it was concentrated under reduced pressure. 100 mL of water was added to the residue, and the pH was adjusted to about 6 with 1 M HCl. Ethyl acetate (30 mL × 2) was added for extraction. After combining the organic phases, they were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 2:1~DCM:MeOH = 5:1). Then, petroleum ether:ethyl acetate = 15:1 (30 mL) was added, and the mixture was stirred at room temperature for 30 minutes, followed by filtration. The cake was collected to obtain WX016-2. 1 H NMR (400 MHz, CDCl3) δ ppm 1.50 (s, 9 H) 2.57 (br t, 2 H) 3.60 (br t, J = 5.57 Hz, 2 H) 3.97 (s, 3 H) 4.29 (br s, 2 H) 11.14 (br s, 1 H).

[0298] Step 2: Synthesis of compound WX016-3 WX016-2 (1.5 g, 5.33 mmol, 1 eq) was mixed in anhydrous toluene (35 mL), and triphenylphosphine (2.80 g, 10.66 mmol, 2 eq) and 2,2,2-trichloroacetonitrile (8.00 mmol, 801.99 μL, 1.5 eq) were added sequentially. Under nitrogen gas protection, the reaction system was stirred at 100 °C for 1.5 hours. After the reaction system was cooled to room temperature, it was concentrated under reduced pressure, and the residue was purified by column chromatography (PE:EA = 1:0~5:1) to obtain WX016-3. 11H NMR (400 MHz, CDCl3) δ ppm 1.46 (s, 9 H) 2.74 (br t, J = 5.77 Hz, 2 H) 3.68 (t, J = 5.90 Hz, 2 H) 3.97 (s, 3 H) 4.52 (s, 2 H).

[0299] Step 3: Synthesis of Compound WX016-4 WX016-3 (1.27 g, 4.24 mmol, 1 eq) and WX001-2 (1.30 g, 4.24 mmol, 1 eq) were dissolved in ACN (13 mL), potassium carbonate (1.76 g, 12.71 mmol, 3 eq) was added, and the reaction system was stirred at 50 °C for 4 hours. After the reaction system was cooled to room temperature, it was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to obtain WX016-4. 1 1H NMR (400 MHz, CDCl3) δ ppm 1.48 (s, 9 H) 2.71 (br t, J = 5.63 Hz, 2 H) 3.55 (s, 3 H) 3.74 (br t, J = 5.75 Hz, 2 H) 4.50 (s, 2 H) 7.31 (td, J = 8.13, 2.75 Hz, 1 H) 7.53 (dd, J = 8.82, 2.81 Hz, 1 H) 7.67 (dd, J = 8.63, 5.38 Hz, 1 H).

[0300] Step 4: Synthesis of Compound WX016-5 WX016-4 (1.39 g, 3.03 mmol, 1 eq) was dissolved in DCM (14 mL), TFA (2.50 mL) was added dropwise. After the addition was complete, the reaction system was stirred at 25 °C for 1 hour. Saturated sodium carbonate solution was added to the reaction system to adjust the pH to 9, and it was extracted with DCM (15 mL × 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain the crude product WX016-5, which was directly used in the next step. LCMS: m / z [M+1] + = 360.3.

[0301] Step 5: Synthesis of Compound WX016-6 WX016-5 (1.01 g, 2.81 mmol, 1 eq) and A-2 (700.13 mg, 2.81 mmol, 1 eq) were placed in a 100 mL single-neck flask, DIEA (196.75 mmol, 34.27 mL, 70 eq) was added, and the reaction system was stirred at 100 °C for 16 h under nitrogen gas protection. The reaction system was cooled to room temperature, concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether:ethyl acetate = 4:1~1:1) to obtain WX016-6. 1 H NMR (400 MHz, CDCl3) δ ppm 1.60 - 1.70 (m, 6 H) 2.92 (br t, J = 5.69 Hz, 2 H) 3.60 (s, 3 H) 3.70 - 3.95 (m, 4 H) 4.49 - 4.53 (m, 2 H) 6.06 - 6.12 (m, 1 H) 7.36 (td, J = 8.10, 2.69 Hz, 1 H) 7.57 (dd, J = 8.82, 2.69 Hz, 1 H) 7.72 (dd, J = 8.38, 5.50 Hz, 1 H) 7.77 (s, 1 H); LCMS: m / z [M+1] + = 572.1.

[0302] Step 6: Synthesis of Compound WX016 WX016-6 (400 mg, 699.33 μmol, 1 eq) was dissolved in DCM (3 mL), TFA (570 μL) was added dropwise, and after the addition was complete, the reaction system was reacted at 25 °C for 1 h. Saturated aqueous sodium carbonate solution was added to the reaction system to adjust the pH to about 8 - 9, filtered, the cake was washed with 2 mL of dichloromethane, and dried to obtain WX016. 11H NMR (400 MHz, DMSO-d6) δ ppm 2.75 - 2.86 (m, 2 H) 3.55 (s, 3 H) 3.77 (br t, J=5.57 Hz, 2 H) 4.54 (s, 2 H) 7.71 (td, J=8.38, 2.75 Hz, 1 H) 7.90 (dd, J=9.13, 3.25 Hz, 2 H) 7.97 (s, 1 H) 12.97 (br s, 1 H); LCMS: m / z [M+1] + = 488.1。

[0303] Example 17

[0304]

Chem.

[0305] Synthesis route:

[0306]

Chem.

[0307] Step 1: Synthesis of compound WX017-2 Under nitrogen gas protection, A-1 (2.5 g, 9.27 mmol, 1 eq) and WX017-1 (1.51 g, 9.27 mmol, 1 eq) were added to DMF (25 mL), then DBU (18.54 mmol, 2.79 mL, 2 eq) was added, and the reaction system was stirred at 120 °C for 1 hour. After the reaction system was cooled to room temperature, 50 mL of ethyl acetate was added, and it was washed successively with semi-saturated brine (30 mL × 3) and 30 mL of saturated brine, then dried over anhydrous sodium sulfate. The crude product after concentrating the organic phase under reduced pressure was purified by column chromatography (EA / PE, EA ratio: 0% - 30%) to obtain WX017-2. LCMS: m / z [M+1] + = 396.0 Step 2: Synthesis of compound WX017-3 WX017-2 (2.9 g, 7.33 mmol, 1 eq) was added to DCM (29 mL), then TFA (9.67 mL) was added, and the reaction system was stirred at 25 °C for 1 hour. 30 mL of water was added to the reaction system, then anhydrous sodium carbonate was added to adjust the pH to 8. The aqueous phase was extracted with dichloromethane (30 mL × 2). After combining the organic phases, they were washed with 30 mL of saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the crude product WX017-3, and the next step was carried out directly. LCMS: m / z [M+1] + = 295.9。

[0308] Step 3: Synthesis of compound WX017-4 WX017-3 (1.5 g, 3.87 mmol, 1 eq) was added to DIEA (40 mL), then A-2 (1.95 g, 7.81 mmol, 1.05 eq) was added, and the reaction system was stirred at 100 °C for 3 hours. After cooling the reaction system, it was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (DCM:MeOH, MeOH ratio: 0% - 6%) to obtain WX017-4. LCMS: m / z [M+23] + = 530.0。

[0309] Step 4: Synthesis of compound WX017 WX017-4 (2.8 g, 5.51 mmol, 1 eq) was added to a reaction flask containing DCM (30 mL), then TFA (7.34 mL) was added, and the reaction system was stirred at 25 °C for 1 hour. 30 mL of water was added to the reaction system, then anhydrous sodium carbonate was added to adjust the pH to 8, followed by filtration. The cake was washed successively with 30 mL of DCM and 10 mL of water, dried to obtain a crude product, and the crude product was purified by preparative high-performance liquid chromatography (chromatography column: Phenomenex C18 150×40 mm×5 μm; mobile phase: [water (HCl)-ACN]; gradient (acetonitrile)%: 30% - 60%) to obtain WX017. 11H NMR (400 MHz, DMSO-d6) δ ppm 2.90 (br t, J = 5.13 Hz, 2 H) 3.80 (br t, J = 5.50 Hz, 2 H) 4.62 (s, 2 H) 7.37 (td, J = 8.47, 2.69 Hz, 1 H) 7.72 (dd, J = 8.75, 2.63 Hz, 1 H) 7.80 (dd, J = 8.63, 6.13 Hz, 1 H) 8.00 (s, 1 H) 8.62 (s, 1 H) 13.00 (br s, 1 H); LCMS: m / z [M+1] + = 424.1

[0310] Biological test data Experimental Example 1: hTRPC5-T478C_FLIPR Calcium Flux IC 50 Detection Experimental method: Cell culture: hTRPC5-T478C-HEK cells were cultured at 37 °C in a humidified environment with 5% CO2.

[0311] Components of cell culture medium: DMEM, Supplier: Gibco, Catalog number: 11965-092. FBS, Supplier: Gibco, Catalog number: 10099-141C, Concentration: 10%.

[0312] Pen / Strep, Supplier: Invitrogen, Catalog number: 15140-122, Concentration: 1%. Zeocin, Supplier: Invitrogen, Catalog number: R25005, Concentration: 200 μg / mL.

[0313] Blasticidin S, Supplier: Invitrogen, Catalog number: R21001, Concentration: 5 μg / mL. Cell passage The TRPC5-T478C cell line was usually passaged three times a week, and the dilution factor for each passage was 1:3. When the cells reached approximately 80% confluence in a T-75 flask, the cell line was digested with trypsin for about 1 minute, and then the cell suspension was pipetted out of the flask. Depending on the dilution rate, the removed cell suspension was transferred to another T-75 flask containing cell culture medium. It should be noted that to maintain the logarithmic growth of the cells, it is necessary to maintain the cell confluence at the sub-confluent stage and passage once every 2-3 days according to the growth rate of the cell line.

[0314] Steps of FLIPR calcium flux (1) Preparation of cell plates: Cells were separated as described in the above passage, and the cell density and viability were measured using a cell counter. After adjusting the volume of the cell suspension to the plating density with cell culture medium, 1 μg / mL of tetracycline was added to induce hTRPC5-T478C channel expression. Next, the induced hTRPC5-T478C cells were seeded at a density of 20,000 cells / well (30 μL / well) in a 384-well plate coated with poly-D-lysine (PDL). Then, the cells were cultured overnight in a cell culture incubator.

[0315] (2) Preparation of compound plates: Compounds were usually diluted with DMSO to prepare stock solutions and stored in a -20°C refrigerator. The compound plate program was set up with an ECHO processor, and using ECHO, the compounds were plated according to the set concentration gradient, usually up to 10 μM, diluted three-fold at nine concentrations, and there were two replicate wells. The channel agonist used was englerin A.

[0316] (3) Computer test: The cell plate and the compound plate were prepared as described in the above method. When the cell confluence of the cell plate reached 80 - 90%, the cells were taken out of the incubator and the test was started. Ca5 dye (Molecular Devices #R8185) was prepared using the test buffer. After aspirating the cell plate medium using Bravo, 25 μL of Ca5 dye was added to each well and cultured for 1 hour. After 1 hour, the cell plate and the compound plate were placed TETRA inside FLIPR (Molecular Devices, USA), and FLIPR detection was performed.

[0317] Data analysis: Data analysis was performed using Excel 2013 (Microsoft, USA) software and GraphPad Prism 6.01 software. The maximum signal was generated by the FLIPR software. Data analysis was performed using Excel (2013) and Prism 6.01. S / B > 2.50 and Z - coefficient > 0.50 were used to control the data quality and data stability. The calculation formulas are as follows: S / B = Avg100% / Avg0%, Z - coefficient = 1 - (3×SD100% + 3×SD0%) / |Avg100% - Avg0%|.

[0318] Experimental results: The experimental results were as shown in Table 1.

[0319]

Table 1

[0320] Conclusion: The compound of the present invention has a significant inhibitory effect on TRPC5. Experimental example 2: hTRPC5 - T478C_patch - clamp IC 50 Detection Experimental method: Cell culture: hTRPC5 - T478C - HEK cells were cultured at 37°C in a humidified environment with 5% CO2.

[0321] Components of the cell medium: DMEM, Supplier: Gibco, Catalog Number: 11965 - 092. FBS, Supplier: Gibco, Catalog Number: 10099 - 141C, Concentration: 10%.

[0322] Pen / Strep, Supplier: Invitrogen, Catalog Number: 15140 - 122, Concentration: 1%. Zeocin, Supplier: Invitrogen, Catalog Number: R25005, Concentration: 200 μg / mL.

[0323] Blasticidin S, Supplier: Invitrogen, Catalog Number: R21001, Concentration: 5 μg / mL. Sub - culture of cells: The TRPC5 - T478C cell line was usually sub - cultured three times a week, and the dilution factor for each sub - culture was 1:3. When the cells reached approximately 80% confluence in a T - 75 flask, the cell line was digested with trypsin for about 1 minute, and then the cell suspension was pipetted out of the flask. According to the dilution ratio, the taken - out cell suspension was transferred to another T - 75 flask containing cell culture medium. It should be noted that to maintain the logarithmic growth of cells, it is necessary to maintain the cell confluence at the sub - confluent stage and sub - culture once every 2 - 3 days according to the growth rate of the cell line.

[0324] Solutions: (1) Extracellular fluid: Sodium chloride (145 mmol / L), Potassium chloride (4 mmol / L), Magnesium chloride (1.5 mmol / L), Glucose (10 mmol / L), 4 - (2 - Hydroxyethyl) - 1 - piperazineethanesulfonic acid (HEPES, 10 mmol / L). The pH of the extracellular fluid was adjusted to 7.4 using NaOH, and the osmotic pressure was adjusted to approximately 305 mOsm / L using pure water or sucrose.

[0325] (2) Intracellular fluid: cesium chloride (140 mmol / L), ethylene glycol diethyl ether diamine tetraacetic acid (EGTA, 1 mmol / L), magnesium chloride (2 mmol / L), glucose (10 mmol / L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mmol / L). The pH of the intracellular fluid was adjusted to 7.2 using HCl, and the osmotic pressure was adjusted to approximately 295 mOsm / L using pure water or sucrose.

[0326] (3) The extracellular fluid needs to be prepared once a week and stored in aliquots at -20 °C after the intracellular fluid is prepared. Preparation of compounds: Using 100% DMSO, the positive reference compounds englerin A and 2-aminoethoxydiphenyl borate (2-APB) were dissolved to prepare and store a 10 mM working solution. The test compounds were dissolved using 100% DMSO to prepare and store a 10 mM working solution. The final concentration of DMSO in the test solution needed to be less than 0.3%.

[0327] Whole-cell voltage clamp recording: The whole-cell patch clamp was clamped at room temperature. The electrical signals recorded using an EPC 10 USB amplifier (HEKA Elektronik, Germany) were low-pass filtered at 3 kHz and finally recorded using PatchMaster 2×90.5 software (HEKA Elektronik, Germany). The quality control criteria for this step were that the high-resistance seal of the cell exceeded 500 MOhms and the detected current exceeded 0.3 nA.

[0328] The recording electrodes were pulled out and polished through a vertical puller (NARISHIGE PC-10, Japan) using borosilicate glass capillaries (GC150tF-10, Harvard Apparatus Co., UK). The quality control standard for this step was an electrode resistance of 2 - 5 MΩ.

[0329] During the whole-cell patch-clamp recording, the extracellular solution was continuously perfused using a continuous perfusion system (BT100-2J, LongerPump, China). The perfusion system was attached to the stage of an upright microscope (FN-S2N, Nikon, Japan), and the perfusion head was manually placed under the microscope.

[0330] The voltage instructions for detecting the current amplitude of hTRPC5-T478C were as follows: The cells were stepped up from a clamp potential of -40 mV to -80 mV over 50 ms. Next, the clamp voltage was increased to +80 mV, and the whole process lasted for 200 ms. Then, the cells were clamped at +80 mV for 50 ms, and finally the voltage was returned to the clamp potential of -40 mV. The voltage detection command was repeated every 5000 ms, and this command was continuously executed during the test of the compound. The quality control criteria for the stability of the compound action were the current amplitudes under three consecutive voltage commands, and the coefficient of variation was <5%. When the compound did not affect the current amplitude of hTRPC5-T478C, continuous monitoring for 5 minutes was required.

[0331] Data analysis: Data analysis was performed using PatchMaster 2×90.5 software (HEKA Elektronik, Germany), Excel 2013 (Microsoft, USA) software, and GraphPad Prism 6.01 software. In the patch-clamp recording of each cell, the hTRPC5-T478C channel current amplitude (I unblock ) stimulated by the hTRPC5 agonist Englerin A, the hTRPC5-T478C channel current amplitude (I block ) inhibited by the hTRPC5 inhibitor 2-APB, and the hTRPC5-T478C channel current amplitude (I test ) corresponding to the concentration of the test compound had to be obtained. The remaining channel current after the action of the compound was the value obtained by subtracting the background current of the cell (Iblock) from I test , and the channel current of the whole cell (I unblock - I block) Dividing by it gives the ratio of the remaining current. Subtracting the ratio of the remaining current from 100% gives the compound inhibition efficiency. That is, compound inhibition efficiency = 100% - (I test - I blocked ) / (I unblocked - I blocked ).

[0332] Experimental results: As shown in Table 1.

[0333]

Table 2

[0334] Experimental conclusion: The compound of the present invention has a significant inhibitory effect on TRPC5. Experimental Example 3: In vivo PK study Experimental purpose: Using male SD rats as test animals, after intravenous injection, measure the blood drug concentration of the compound and evaluate the pharmacokinetic behavior.

[0335] Experimental operation: Select 3 healthy adult male SD rats, mix the test compound with an appropriate amount of intravenous injection group solvent to prepare a clear solution of 0.5 or 1 mg / mL, and filter it through a 0.22 μM microporous membrane for later use. After intravenously administering 0.5 or 1 mg / kg to the rats, collect whole blood at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 hours after administration, put it into an EDTA-K2 anticoagulant tube, vortex mix the mixture thoroughly, and centrifuge at 4°C at 3200 g for 10 minutes to obtain plasma. Use the LC-MS / MS method to measure the drug concentration, and use the Phoenix WinNonlin 6.3 pharmacokinetic software and the linear logarithmic trapezoidal method of the non-compartment model to calculate the relevant pharmacokinetic parameters.

[0336] Solvent 1: 10% DMSO / 10% Kolliphor® HS 15 / 80% (20% sulfobutyl-β-cyclodextrin); Solvent 2: 10% DMSO + 20% polyoxyethylene castor oil + 70% water; Solvent 3: 5% DMSO + 10% Kolliphor® HS 15 + 85% saline; Solvent 4: 10% DMSO + 60% polyethylene glycol 400 + 30% (aqueous solution of 10% hydroxypropyl-β-cyclodextrin).

[0337] The experimental results are as shown in Table 2.

[0338]

Table 3

[0339] Conclusion: The compound of the present invention shows a large apparent volume of distribution, low in vivo clearance, and high exposure in rats, indicating good pharmacokinetic properties. Experimental Example 4: Tissue distribution study in rat groups Experimental purpose: In this study, male Sprague Dawley (SD) rats were used as test animals, and the drug concentrations in plasma, cerebrospinal fluid, brain, liver, and kidney at different time points after oral administration of the test compound to rats were quantitatively measured by LC / MS / MS method to evaluate the tissue distribution of the test drug in rats.

[0340] Experimental method: Experimental materials: Sprague Dawley (SD) rats (male, 200 - 300 g, 7 - 10 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0341] Experimental operation: A transparent or suspension solution of the test compound was administered intragastrically to SD rats (fasted) (solvent: 5% DMSO / 10% Kolliphor® HS 15 / 85% water). Approximately 0.2 mL of blood was collected from the jugular vein at 1, 4, and 24 hours after administration, placed in an EDTA-K2 anticoagulant tube, the mixture was vortexed thoroughly, and centrifuged at 3200 g for 10 minutes at 4°C to obtain plasma. Approximately 0.02 mL of cerebrospinal fluid was collected. The brain, liver, and kidney were collected, washed with pre-cooled physiological saline, suction-dried, and then homogenized at a ratio of 1:4 (1 g of tissue: 4 mL of 15 mM phosphate buffered saline / MeOH (2:1)). The drug concentration was measured using the LC-MS / MS method, and the related pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.3 pharmacokinetic software and the non-compartmental model linear log trapezoidal method.

[0342] The experimental results were as shown in Table 3.

[0343]

Table 4

[0344] Conclusion: The compound of the present invention has a high distribution in the kidney and liver and a low distribution in the brain. Experimental Example 5: Pharmacodynamic Study Experimental Purpose: To study the PD and pharmacodynamics of the compound of the present invention in a male SD rat hypertension model induced by deoxycorticosterone acetate-sodium chloride (DOCA-NaCl).

[0345] Experimental Program: After male Sprague-Dawley rats were acclimated for one week at 7 - 8 weeks of age, surgery was initiated. On the day of surgery, the surgical area table was disinfected with a disinfectant, the rats were anesthetized with 2 - 5% isoflurane, the affected area was fixed, the abdominal hair was removed, the skin at the surgical site was disinfected, the skin and abdominal wall were incised, the right kidney was exposed, the arteries and veins near the renal hilum were ligated, and after confirming no bleeding points, the blood vessels and surrounding ligaments of the kidney were bluntly dissected, the right kidney was excised, after confirming no bleeding points, the abdominal wall muscles and skin were sutured. After the animals woke up, they were returned to the cage and observed. Meloxicam injection was subcutaneously injected once a day for 3 consecutive days as an analgesic.

[0346] One week after unilateral nephrectomy, the animals were randomly divided into a solvent group, a low-dose group, and a high-dose group according to body weight / blood pressure. The drinking water of all animals was changed to 1% sodium chloride + 0.2% potassium chloride water and they were allowed to drink freely for 4 consecutive weeks. 20 mg / kg of DOCA (deoxycorticosterone acetate) was subcutaneously injected twice a week for 4 consecutive weeks. Except for the solvent group, the remaining groups were administered intragastrically once a day for 28 consecutive days according to the dose. 28 days after administration, the animals were placed in a metabolic cage overnight, and urine was collected to detect the levels of urinary albumin and the biomarker Rac1.

[0347] Experimental results: When compound WX002 was administered at doses of 30 mg / kg and 60 mg / kg for 28 days, compared with the solvent control group, the levels of urinary albumin in the rat hypertensive nephropathy model could be significantly decreased at both doses (shown in Figure 1). As a result of further detecting the urinary Rac1 content of the biomarker, compound WX002 could dose-dependently decrease the expression of Rac1 in the urine of the rat hypertensive nephropathy model (shown in Figure 2). *p<0.05, ***p<0.001 vs solvent group, data were shown as mean ± standard error (Mean±SEM).

[0348] Conclusion: The compound of the present invention can significantly decrease the level of urinary albumin in the rat hypertensive nephropathy model and can dose-dependently decrease the expression of Rac1 in the urine of the rat hypertensive nephropathy model.

Claims

Claim 1 A compound represented by formula (P-1) or a pharmaceutically acceptable salt thereof. 【Chemical 1】 However, L is -S-, R １ is selected from F, Cl, Br, and I, R ２ is selected from H, F, Cl, Br, I, OH, NH ２ , C １－３ alkyl, C １－３ alkoxy and C １－３ alkyl-C １－３ alkoxy, and the C １－３ alkyl, C １－３ alkoxy and C １－３ alkyl-C １－３ alkoxy are each independently optionally substituted by 1, 2 or 3 R ｂ s, R ｂ is selected from F, Cl, Br, I, OH and NH2, R ｃ is selected from F, Cl, Br, I, C １－３ alkyl and C １－３ alkoxy, and the C １－３ alkyl and C １－３ alkoxy are each independently optionally substituted by 1, 2 or 3 halogens. Claim 2 R ｃ is selected from F, Cl, Br, I, CH ３ , CH ２ CH ３ and OCH3, and the CH ３ , CH ２ CH ３ and OCH3 are each independently optionally substituted with 1, 2, or 3 halogens, the compound according to claim 1 or a pharmaceutically acceptable salt thereof. Claim 3 R ｃ is selected from F, Cl, Br, I, CH ３ , CH ２ F, CHF ２ , CF ３ , CH ２ CH ３ , CF ２ CH ３ , OCH ３ and OCF3, and is the compound according to claim 2 or a pharmaceutically acceptable salt thereof. Claim 4 R ２ is selected from H, F, Cl, Br, I, OH, NH ２ , CH ３ , CH ２ CH ３ , OCH ３ and CH ２ OCH ３ ; and the CH ３ , CH ２ CH ３ , OCH ３ and CH ２ OCH ３ are each independently optionally substituted with 1, 2, or 3 R ｂ ; a compound according to claim 1 or a pharmaceutically acceptable salt thereof. Claim 5 R ２ is H, F, Cl, Br, I, OH, NH ２ , CH ３ , CH ２ OH, CH ２ CH ３ , CH(OH)CH ３ , CH(OH)CH ２ OH, OCH ３ and CH ２ OCH ３ selected from the compound according to claim 4 or a pharmaceutically acceptable salt thereof.

6. 【Fig. 2】 is 【Chemical Formula 3】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from Claim 7 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the following formulas 【Chemical Formula 4】 Claim 8 The compound according to claim 7 or a pharmaceutically acceptable salt thereof, selected from the following formulas 【Chemical Formula 5】 Claim 9 A TRPC5 inhibitor comprising the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof. Claim 10 A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for treating a disease associated with TRPC5 kinase, wherein the disease associated with TRPC5 kinase is a renal disease.

Images