Furan-fused substituted glutarimide compounds

Abstract

The application discloses a series of furan-fused ring-substituted glutarimide compounds and application of the compounds in preparation of drugs for treating related diseases, and specifically discloses a compound shown in formula (II) and pharmaceutically acceptable salts of the compound.

Description

[0001] This application claims the following priority:

[0002] CN202110286500.6, Application Date: 2021-03-17;

[0003] CN202110712765.8, application date: 2021-06-25;

[0004] CN202111314330.4, Application Date: 2021-11-08;

[0005] CN202210187905.9, application date: 2022-02-28. Technical Field

[0006] This invention relates to a series of furan-fused-ring substituted glutarimide compounds and their use in the preparation of medicaments for treating related diseases, particularly to the compound shown in formula (II) and its pharmaceutically acceptable salt. Background Technology

[0007] Androgen receptor signaling is known to play a crucial role in the pathogenesis of prostate cancer and is involved in the development of other androgen receptor-positive cancers. Anti-androgens, which antagonize androgen receptors, have been used or are planned to be used in the treatment of prostate cancer to inhibit androgen receptor signaling.

[0008] The AR gene encodes the androgen receptor protein, whose ligands are primarily testosterone and dihydrotestosterone. This receptor is widely distributed in most organs and tissues of the human body, mediating the biological effects of androgens. The androgen receptor binds to heat shock proteins (Hsp) in the cytoplasm. When an androgen binds to the androgen receptor, the receptor is activated, the heat shock protein dissociates, and the androgen receptor forms a dimer that enters the nucleus. It then binds to the androgen response element (ARE) on DNA, initiating the transcriptional expression of a series of downstream genes, including prostate-specific antigen (PSA), prostate acid phosphatase (PAP), and the cyclin-dependent kinase (CDK) inhibitor p21WAF1 / CIPI. Ultimately, this leads to cell differentiation and promotes tissue and organ development. Androgens play a crucial role in maintaining prostate growth and development. In the absence of androgens, prostate cells spontaneously undergo apoptosis, while in an environment with normal androgen levels, prostate cells continue to grow and differentiate. Therefore, some studies suggest that excessive androgen secretion and overly responsive androgen receptors can lead to uninhibited prostate cell growth, which is a risk factor for the development and progression of prostate cancer.

[0009] Prostate cancer (PCa) is one of the most frequently diagnosed non-skin cancers among men in the United States and the second leading cause of cancer death, with over 200,000 new cases and over 30,000 deaths annually in the US. Androgen deprivation therapy (ADT) is the standard treatment for advanced PCa. Patients with advanced PCa receive ADT via luteinizing hormone-releasing hormone (LHRH) agonists, LHRH antagonists, or bilateral orchiectomy. Despite an initial response to ADT, disease progression is inevitable, and the cancer develops into castration-resistant prostate cancer (CRPC). Up to 30% of prostate cancer patients who receive primary treatment with radiation or surgery will develop metastatic disease within 10 years of primary treatment. Approximately 50,000 patients will develop metastatic disease each year, known as metastatic CRPC (mCRPC).

[0010] Protein degradation targeting chimera (PROTAC) is a technique that uses the ubiquitin-proteasome system to target specific proteins and induce their degradation within cells. The ubiquitin-proteasome system is the main pathway for protein degradation in cells; its normal physiological function is primarily to remove denatured, mutated, or harmful proteins from the cell. More than 80% of intracellular protein degradation relies on the ubiquitin-proteasome system. PROTAC utilizes the cell's own protein destruction mechanisms to remove specific target proteins within the cell.

[0011] This disclosure describes compounds, including compositions with the same composition, that function to recruit endogenous proteins to E3 ubiquitin ligases, such as cereblon (CRBN) E3 ubiquitin ligase, for ubiquitination and subsequent degradation, and using the same methods. In particular, this disclosure provides bifunctional or proteolytically targeted chimeric (PROTAC) compounds found to act as regulators of targeted ubiquitination and androgen receptor (AR) degradation. Summary of the Invention

[0012] This invention provides compounds of formula (II) or pharmaceutically acceptable salts thereof.

[0013]

[0014] in,

[0015] PTM is selected from drugs or their derivatives that target and bind to AR proteins;

[0016] L1 is selected from -(CH2) n - Each CH2 may be optionally replaced by R1;

[0017] R1 is selected from C 3-7 Cycloalkyl, 6-membered heterocycloalkyl, -NR a -、-CRb R c -, -CH2CH2O- and -NHC(=O)-;

[0018] R a Selected from H and C 1-3 alkyl;

[0019] R b R c Selected from H, D, and F;

[0020] E1 is selected from single bonds and O;

[0021] Ring A does not exist;

[0022] Alternatively, ring A may be selected from phenyl and 5-membered heteroaryl groups;

[0023] n is selected from 1, 2, 3, 4, 5, and 6;

[0024] The 6-membered heterocyclic alkyl and 5-membered heteroaryl groups each contain 1, 2 or 3 heteroatoms or heterogroups independently selected from -NH-, -O-, -S- and N;

[0025] The condition is that the compound is not selected from:

[0026]

[0027]

[0028] In some embodiments of the present invention, the PTM is selected from... Other variables are as defined in this invention.

[0029] In some embodiments of the present invention, the above-mentioned R a Selected from H and CH3, other variables are as defined in this invention.

[0030] In some embodiments of the present invention, R1 is selected from cyclopropyl, cyclohexyl, piperazinyl, piperidinyl, and -NR. a -, -CH2CH2O-, -NHC(=O)- and -C(=O)NH-, and other variables as defined in this invention.

[0031] In some embodiments of the present invention, R1 is selected from... -NH-, -N(CH3)-, -CH2CH2O-, -NHC(=O)- and -C(=O)NH-, and other variables as defined in this invention.

[0032] In some embodiments of the present invention, R1 is selected from... -N(CH3)-, -CH2CH2O- and -NHC(=O)-, and other variables as defined in this invention.

[0033] In some embodiments of the present invention, L1 is selected from... Other variables are as defined in this invention.

[0034] In some embodiments of the present invention, the above-mentioned structural unit -E1-L1- is selected from... Other variables are as defined in this invention.

[0035] In some embodiments of the present invention, the above-described structural unit Selected from Other variables are as defined in this invention.

[0036] In some embodiments of the present invention, the above-mentioned compounds are selected from the structures shown in formulas (I-1), (I-2), (II-1), (II-2), (III-1), and (IV-1):

[0037]

[0038] in,

[0039] L1 is as defined in this invention.

[0040] This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0041]

[0042] in,

[0043] E1 is selected from single bonds, O, and NH;

[0044] E2 is selected from single bonds and C. 1-3 alkyl;

[0045] E3 is selected from C 1-3 Alkyl and cyclopropyl;

[0046] E4 is selected from single bonds and -C(=O)NH-;

[0047] Ring A does not exist;

[0048] Alternatively, ring A may be selected from phenyl;

[0049] ABM is selected from drugs or derivatives thereof that bind to AR-targeting proteins.

[0050] In some embodiments of the present invention, the above structural units -E1-E2- are selected from single bonds and -OCH2CH2-, and other variables are as defined in the present invention.

[0051] In some embodiments of the present invention, the above structural units -E3-E4- are selected from C. 1-3 alkyl, Other variables are as defined in this invention.

[0052] In some embodiments of the present invention, the above-described structural unit Selected from Other variables are as defined in this invention.

[0053] In some embodiments of the present invention, the above-mentioned ABM is selected from the structure of formula (ABM-1):

[0054]

[0055] in,

[0056] R1 and R2 are selected from methyl groups;

[0057] Alternatively, R1 and R2 form a carbon atom with the carbon atom they are bonded to. 4-6 cycloalkyl;

[0058] Y1 and Y2 are independently selected from O and S, respectively;

[0059] Ring B is selected from phenyl and pyridyl groups, wherein the phenyl and pyridyl groups are optionally surrounded by 1, 2, or 3 R groups. a replace;

[0060] The ring C is selected from a single bond or a phenyl group, wherein the phenyl group is optionally surrounded by 1, 2, or 3 R groups. b replace;

[0061] R a Selected from F, Cl, Br, I, CN, CH3, CF3, and NO2;

[0062] R b Selected from F and Cl.

[0063] In some embodiments of the present invention, the above-mentioned ABM is selected from the structures of formulas (ABM-1a) and (ABM-1b):

[0064]

[0065] in,

[0066] T1 is selected from CH and N;

[0067] R b1 Selected from H and F;

[0068] n is selected from 1, 2, and 3;

[0069] Y1, Y2, R a R1 and R2 are as defined in this invention.

[0070] In some embodiments of the present invention, the above-mentioned ABM is selected from...

[0071] In some embodiments of the present invention, the above-mentioned compounds are selected from the structures shown in formulas (I-1a) and (I-2a):

[0072]

[0073] in,

[0074] T1, R1, R2, R a R b1 E1, E2, E3 and E4 are as defined in any one of the present invention.

[0075] Some solutions in this invention are derived from arbitrary combinations of the above-mentioned variables.

[0076] This invention also provides the following compounds or pharmaceutically acceptable salts thereof:

[0077]

[0078]

[0079] This invention also provides the following compounds or pharmaceutically acceptable salts thereof:

[0080]

[0081]

[0082]

[0083] The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating prostate cancer.

[0084] Definitions and Explanations

[0085] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0086] The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0087] The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0088] The term "drug or its derivative" includes drugs or their derivatives that have been developed to bind to target proteins.

[0089] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0090] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0091] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0092] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0093] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0094] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration representing a solid center; in the absence of a solid center in the structure, the wedge-shaped solid key. and wedge-shaped dashed key Simultaneous appearance indicates relative configurations, for example, using It represents trans-1,4-dimethylcyclohexane.

[0095] Unless otherwise specified, when a group has one or more connectable sites, any one or more of these sites can be connected to other groups by chemical bonds. The chemical bonds connecting these sites to other groups can be straight solid lines. Straight dashed key or wavy line For example, a straight solid line bond in -OCH3 indicates that the oxygen atom in that group is connected to other groups; The straight dashed bond in the diagram indicates that the group is connected to other groups through both ends of the nitrogen atom in the group; The wavy lines in the diagram indicate that the carbon atoms at positions 1 and 2 of the phenyl group are linked to other groups.

[0096] The compounds of this invention can exist in specific forms. Unless otherwise stated, the terms "tautomer" or "tautomer form" refer to isomers of different functional groups in dynamic equilibrium at room temperature, capable of rapidly interconverting into each other. If tautomerization is possible (e.g., in solution), chemical equilibrium of the tautomer can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions involving the rearrangement of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0097] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0098] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0099] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase, optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0100] The compounds of this invention may contain non-natural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or C-14 (14C). As another example, deuterium may be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared to undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged biological half-life. All variations in the isotopic composition of the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0101] The terms “optional” or “optionally” refer to events or conditions that may occur but are not required to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0102] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, unless otherwise specified, and the type and number of substituents can be arbitrary on a chemically feasible basis.

[0103] The term "substituted" refers to the fact that a specific atom or group can be replaced with another specified atom or group. For example, the CH2 in CH3CH2CH3 can be substituted by O, S, or NH to obtain CH3OCH3, CH3SCH3, and CH3NHCH3.

[0104] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0105] When the number of a linking group is 0, such as -(CRR)0-, it indicates that the linking group is a single bond.

[0106] When a substituent is vacant, it means that the substituent does not exist. For example, if X is vacant in AX, the structure is actually A. When the listed substituents do not specify which atom they are attached to the substituted group through, such substituents can be bonded to any of their atoms. For example, a pyridinium group as a substituent can be attached to the substituted group through any carbon atom on the pyridine ring.

[0107] Unless otherwise specified, the term "C1-3 alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C1-3 alkyl group includes C1-2 and C2-3 alkyl groups, etc.; it can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0108] Unless otherwise specified, the number of atoms in a ring is usually defined as the elemental number of the ring. For example, a “5-7 elemental ring” refers to a “ring” with 5-7 atoms arranged around it.

[0109] Unless otherwise specified, "C3-7 cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms, including monocyclic and bicyclic systems, wherein bicyclic systems include spirocyclic, fused, and bridged rings. The C3-7 cycloalkyl group includes C3-6, C3-5, C3-4, C4-7, C4-6, C4-5, C5-7, or C5-6 cycloalkyl groups; it can be monovalent, divalent, or polyvalent. Examples of C3-7 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0110] Unless otherwise specified, the term "6-membered heterocyclic alkyl" on its own or in combination with other terms refers to a saturated cyclic group consisting of six ring atoms, wherein one, two, three, or four ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S(O)p, where p is 1 or 2). It includes monocyclic and bicyclic systems, wherein bicyclic systems include spirocyclic, fused, and bridged rings. Examples of 6-membered heterocyclic alkyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), or morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.).

[0111] Unless otherwise specified, the terms "5-membered heteroaryl" and "5-membered heteroaryl" are used interchangeably in this invention. The term "5-membered heteroaryl" refers to a monocyclic group consisting of one, two, or three ring atoms with a conjugated π-electron system, where one, two, or three ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms. The nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S(O)p, where p is 1 or 2). The 5-membered heteroaryl group can be attached to the rest of the molecule via a heteroatom or a carbon atom. Examples of the 5-membered heteroaryl group include, but are not limited to, pyrrole (including N-pyrrole, 2-pyrrole, and 3-pyrrole), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl), imidazole (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, and 5-imidazolyl), oxazolyl (including 2-oxazolyl, 4-oxazolyl, and 5-oxazolyl), and triazolyl (1H-1,2,3-triazolyl, 2H-2H-3H-4H-3H-4H-3H-4H-5 ... H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isooxazolyl, 4-isooxazolyl and 5-isooxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furanyl (including 2-furanyl and 3-furanyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.).

[0112] Unless otherwise specified, Cn-n+m or Cn-Cn+m includes any specific case of n to n+m carbons, such as C1-12 including C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, and C12, and also any range from n to n+m, such as C1-12 including C1-3, C1-6, C1-9, C3-6, C3-9, C3-12, C6-9, C6-12, and... C9-12, etc.; similarly, n-membered to n+m-membered indicates that the number of atoms on the ring is n to n+m. For example, 3-12-membered rings include 3-membered rings, 4-membered rings, 5-membered rings, 6-membered rings, 7-membered rings, 8-membered rings, 9-membered rings, 10-membered rings, 11-membered rings, and 12-membered rings. It also includes any range from n to n+m. For example, 3-12-membered rings include 3-6-membered rings, 3-9-membered rings, 5-6-membered rings, 5-7-membered rings, 6-7-membered rings, 6-8-membered rings, and 6-10-membered rings, etc.

[0113] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be connected to other groups by chemical bonds. When the chemical bond connection is non-directional and the connectable site contains H atoms, the number of H atoms at that site will decrease accordingly with the number of chemical bonds connected, resulting in a group with a corresponding valence. The chemical bonds connecting the site to other groups can be straight solid line bonds. Straight dashed key or wavy line For example, a straight solid line bond in -OCH3 indicates that the oxygen atom in that group is connected to other groups; The straight dashed bond in the diagram indicates that the group is connected to other groups through both ends of the nitrogen atom in the group; The wavy lines in the text indicate that the phenyl group is connected to other groups through the carbon atoms at positions 1 and 2 of the phenyl group. This indicates that any connectable site on the naphthalene ring can be linked to other groups by a single chemical bond, including at least... These are the 6 connection methods.

[0114] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0115] The structures of the compounds of this invention can be confirmed by conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, that absolute configuration can be confirmed by conventional techniques in the art. For example, single-crystal X-ray diffraction (SXRD) is used, where the cultured single crystal is used to collect diffraction intensity data using a Bruker D8 venture diffractometer with CuKα radiation as the light source. The scanning method is as follows: After scanning and collecting relevant data, the crystal structure can be further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0116] The solvents used in this invention are commercially available. The following abbreviations are used in this invention: aq represents water; eq represents equivalent; CDI represents carbonyl diimidazole; DCM represents dichloromethane; PE represents petroleum ether; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; Boc represents tert-butyloxycarbonyl, which is an amine protecting group; rt represents room temperature; O / N represents overnight; THF represents tetrahydrofuran; Boc2O represents di-tert-butyldicarbonate; M represents mol / L; HPLC represents high-performance liquid chromatography.

[0117] Compounds are named according to conventional naming principles in the field or using Software naming conventions are used; commercially available compounds adopt supplier catalog names. Technical effectiveness.

[0118] The compounds of this invention exhibit excellent AR protein degradation activity, cell proliferation inhibition activity, and significant tumor suppression activity. Attached Figure Description

[0119] Figure 1 Enzalutamide and WX001 on the degradation activity of AR protein in the LNCaP cell line.

[0120] Figure 2 WX002 activity in the degradation of LNCaP AR protein in cell line. Detailed Implementation

[0121] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

[0122] Reference Example 1

[0123]

[0124] Synthesis route:

[0125]

[0126] Step 1: Synthesis of intermediate BB-1-2

[0127] Compound BB-1-1 (1 g, 3.53 mmol) and tert-butyl carbamate (2.07 g, 17.66 mmol) were added to toluene (15 mL) and water (1.5 mL). Tris(dibenzylacetone)dipalladium (226.41 mg, 247.25 μmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropylbiphenyl (209.98 mg, 494.49 μmol), and potassium phosphate (3.00 g, 14.13 mmol) were added to the reaction mixture sequentially and slowly. The mixture was purged with nitrogen three times and then heated to 100 °C and stirred for 12 hours. The reaction solution was cooled to room temperature, and most of the organic solvent was removed by rotary evaporation under reduced pressure. The solution was diluted with ethyl acetate (30 mL), washed twice with water (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-3 / 2, volume ratio) to obtain intermediate BB-1-2. 1 H NMR(400MHz, CDCl3)δ:7.83-7.73(m,1H),7.57(s,1H),7.47-7.40(m,1H),7.14-7.05(m,1H),6.81(br d,J=1.0Hz,1H),4.19(q,J=7.0Hz,2H),3.67(d,J=1.3Hz,2H),1.49-1.42(m,9H),1.32-1.23(m,3H).

[0128] Step 2: Synthesis of intermediate BB-1-3

[0129] At 0 °C, compound BB-1-2 (300 mg, 939.40 μmol) and acrylamide (73.45 mg, 1.03 mmol, 71.31 μL) were added to tetrahydrofuran (5 mL). Potassium tert-butoxide (158.12 mg, 1.41 mmol) was slowly added to the reaction mixture. After purging the mixture with nitrogen three times, the mixture was stirred at 0 °C for 1 hour. The reaction mixture was slowly added to a saturated ammonium chloride aqueous solution (20 mL), extracted twice with ethyl acetate (20 mL × 2), washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-3 / 1, v / v) to give intermediate BB-1-3.

[0130] Step 3: Synthesis of the hydrochloride of intermediate BB-1

[0131] Compound BB-1-3 (40 mg, 116.16 μmol) was added to ethyl acetate (2 mL), and hydrochloric acid / dioxane (4 M, 1 mL) was slowly added dropwise to the reaction mixture. The mixture was purged with nitrogen three times and then stirred at 25 °C for 12 hours. The reaction solution was directly evaporated to dryness to give the hydrochloride salt of intermediate BB-1.

[0132] See Example 2

[0133]

[0134] Synthesis route:

[0135]

[0136] Step 1: Synthesis of intermediate BB-2-2

[0137] Under nitrogen protection, intermediate BB-2-1 (4 g, 14.13 mmol) and tert-butyl carbamate (4.97 g, 42.39 mmol) were added to a mixture of toluene (70 mL) and water (10 mL), followed by the sequential addition of potassium phosphate (12.00 g, 56.51 mmol), tris(dibenzylacetone)palladium (905.63 mg, 988.99 μmol), and 2-di-tert-butylphosphine-2′,4′,6′-triisopropylbiphenyl (839.93 mg, 1.98 mmol). The reaction mixture was stirred at 110 °C under nitrogen protection for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered through diatomaceous earth, and the filtrate was extracted with ethyl acetate (500 mL × 2) after adding water (100 mL). Combine the organic phases, wash with saturated brine (300 mL × 2), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to remove the solvent, and separate the residue by column chromatography (eluent: petroleum ether / ethyl acetate = 0 / 1-10 / 1, volume ratio) to obtain intermediate BB-2-2. 1 H NMR (400MHz, CDCl3) δ: 7.71 (br s, 1H), 7.63 (s, 1H), 7.37 (d, J = 8.8Hz, 1H), 7.14 (dd, J = 2.0, 8.8Hz, 1H), 6.59 (br s, 1H), 4.20 (q, J = 7.2Hz, 2H), 3.67 (d, J = 0.8Hz, 2H), 1.53 (s, 9H), 1.29 (t, J = 7.2Hz, 3H).

[0138] Step 2: Synthesis of intermediate BB-2-3

[0139] Compound BB-2-2 (0.75 g, 1.69 mmol) and potassium tert-butoxide (284.42 mg, 2.53 mmol) were added to tetrahydrofuran (8 mL). Acrylamide (312.60 mg, 2.03 mmol) was slowly added to the reaction mixture. The mixture was purged with nitrogen three times and stirred at 0–5 °C for 2 hours. After the reaction was complete, the reaction mixture was slowly added to a saturated ammonium chloride aqueous solution (20 mL), extracted twice with ethyl acetate (20 mL × 2), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–1 / 1, v / v) to give intermediate BB-2-3. MS-ESI m / z: 245.0 [M+H] + . 1 H NMR (400MHz, CDCl3) δ: 8.13 (br s, 1H), 7.65 (br s, 1H), 7.46 (s, 1H), 7.32 (d, J = 8.8Hz, 1H), 7.04 (dd, J = 2.1, 8.8Hz, 1H), 6.57 (br s,1H),3.91(t,J=7.6Hz,1H),2.80-2.53(m,2H),2.35-2.20(m,2H),1.45(s,9H).

[0140] Step 3: Synthesis of the hydrochloride salt of intermediate BB-2

[0141] At room temperature, intermediate BB-2-3 (300 mg, 871.18 μmol) was dissolved in hydrochloric acid / ethyl acetate solution (4 M, 5 mL), and the reaction mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, yielding the hydrochloride salt of intermediate BB-2.

[0142] 1 H NMR(400MHz,DMSO-d6)δ:10.97(s,1H),10.34(br s,2H),8.05(s,1H),7.72(d,J=8.8Hz,1H),7.58(d,J=2.0Hz,1H),7.35(dd,J=2.1,8.7Hz,1H),4.20(dd,J =4.8,12.3Hz,1H),2.88-2.73(m,1H),2.68-2.58(m,1H),2.30(dq,J=4.4,12.6Hz,1H),2.20-2.10(m,1H).

[0143] See Example 3

[0144]

[0145] Synthesis route:

[0146]

[0147] Step 1: Synthesis of intermediate BB-3-2

[0148] Under nitrogen protection at room temperature, concentrated sulfuric acid (220.80 g, 2.21 mol, 120 mL, purity: 98%) was added dropwise to ice water (40 mL), followed by compound BB-3-1 (10 g, 44.83 mmol). The mixture was cooled to 5–10 °C, and ethyl 4-chloroacetoacetate (7.38 g, 44.83 mmol) was slowly added dropwise. The reaction mixture was heated to room temperature and stirred for 16 hours, then heated to 50 °C and stirred for another 16 hours. After the reaction was complete, the mixture was cooled to room temperature and poured into ice water (1 L). A large amount of solid precipitated out. The solid was filtered, and the filter cake was collected. Toluene (400 mL) was added to the obtained solid, and the solvent was removed under reduced pressure. Toluene (400 mL) was added again, and the solvent was removed under reduced pressure again to give intermediate BB-3-2.

[0149] Step 2: Synthesis of intermediate BB-3-3

[0150] Under nitrogen protection at room temperature, intermediate BB-3-2 (14.5 g, 44.81 mmol) was dissolved in a 150 mL solution of sodium hydroxide (8.70 g, 217.52 mmol) in water. The reaction mixture was heated to 80 °C and stirred for 5 hours. After the reaction was complete, the mixture was cooled to room temperature and diluted with 150 mL of dichloromethane. The organic phase was collected after separation, and the aqueous phase was extracted with 150 mL × 3 of dichloromethane. The pH of the aqueous phase was adjusted to 4 with 2 M dilute hydrochloric acid and extracted with 200 mL × 3 of ethyl acetate. The combined organic phases were washed with 50 mL × 2 of saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure to obtain intermediate BB-3-3.

[0151] Step 3: Synthesis of intermediate BB-3-4

[0152] Under nitrogen protection at room temperature, intermediate BB-3-3 (11.3 g, 37.03 mmol) was dissolved in ethanol (300 mL), followed by the addition of concentrated sulfuric acid (2.08 g, 20.78 mmol, 1.13 mL, purity: 98%). The reaction mixture was heated to 80 °C and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and water (150 mL) was added. The mixture was extracted with ethyl acetate (150 mL × 1, 100 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0-97 / 3, v / v) to give intermediate BB-3-4.1 H NMR(400MHz,DMSO-d6)δ:8.35(d,J=2.0Hz,1H),8.09(t,J=4.4Hz,2H),7.86( s, 2H), 7.73 (dd, J = 2.0, 8.8Hz, 1H), 4.18-4.09 (m, 4H), 1.18 (t, J = 7.2Hz, 3H).

[0153] Step 4: Synthesis of intermediate BB-3-5

[0154] At room temperature, intermediate BB-3-4 (2.00 g, 6.00 mmol) was dissolved in toluene (60 mL) and water (6 mL) under stirring. Then, tert-butylcarbamate (3.52 g, 30.01 mmol), tris(dibenzylacetone)palladium (384.78 mg, 420.20 μmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropylbiphenyl (356.87 mg, 840.40 μmol), and potassium phosphate (5.10 g, 20.01 mmol) were added sequentially. The reaction mixture was purged with nitrogen three times, and the temperature was slowly raised to 105 °C. The reaction mixture was then reacted for 12 hours under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature, and water (50 mL) was added to the resulting reaction solution. The solution was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed successively with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The resulting residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1-5 / 1, v / v) to give intermediate BB-3-5.

[0155] Step 5: Synthesis of intermediate BB-3-6

[0156] Under nitrogen protection at 0°C, BB-3-5 (4.00 g, 10.83 mmol) was dissolved in N,N-dimethylformamide (60 mL). Potassium tert-butoxide (1.22 g, 10.83 mmol) and acrylamide (1.67 g, 10.83 mmol) were added to the solution, and the reaction mixture was stirred at 0°C for 3 hours. After the reaction was complete, water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed successively with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The resulting residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1-1 / 1, v / v) to give intermediate BB-3-6. MS-ESI m / z: 338.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 10.95 (s, 1H), 9.55 (s, 1H), 8.25 (s, 1H), 7.96 (d, J = 4.0Hz, 2H), 7.72 (s, 2H), 2.89 (s, 3H), 2.74 (s, 2H), 1.52 (s, 9H).

[0157] Step 6: Synthesis of the hydrochloride of intermediate BB-3

[0158] At 0 °C, BB-3-6 (1.0 g, 2.54 mmol) was dissolved in ethyl acetate (20 mL), and ethyl acetate hydrochloride (2.54 mL) was slowly added. The reaction mixture was stirred for 4 hours under a nitrogen atmosphere. After the reaction was complete, the solution was concentrated under reduced pressure to obtain the hydrochloride salt of BB-3. MS-ESI m / z: 294.31 [M+H] + .

[0159] See Example 4

[0160]

[0161] Synthesis route:

[0162]

[0163] Step 1: Synthesis of intermediate BB-4-2

[0164] Compound BB-4-1 (10 g, 78.67 mmol) was dissolved in dichloromethane (120 mL) and acetone (60 mL) under nitrogen protection at 0–5 °C. Cyanotrimethylsilane (12.45 g, 125.50 mmol, 15.70 mL) and trimethylsilyltrifluoromethanesulfonate (820.00 mg, 3.69 mmol, 666.67 μL) were added dropwise sequentially. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the mixture was cooled to 0–5 °C, diluted with water (200 mL), and extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1–5 / 1, v / v) to give compound BB-4-2. H NMR (400MHz, CDCl3) δ: 6.83 (t, J=9.0Hz, 1H), 6.74 (dd, J=2.7, 12.1Hz, 1H), 6.66-6.59 (m, 1H), 1.65 (s, 6H).

[0165] Step 2: Synthesis of compound BB-4-3

[0166] Compound BB-4-2 (8 g, 45.40 mmol) was dissolved in N,N-dimethylacetamide (150 mL) at room temperature and under nitrogen protection. 4-Thioisocyanate-2-(trifluoromethyl)benzonitrile (10.36 g, 45.40 mmol) was added in portions to the reaction mixture. The reaction mixture was stirred at 25 °C for 12 hours. Methanol (60 mL) and dilute hydrochloric acid (2 M, 60 mL) were added, and the reaction mixture was stirred at 70 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with water (500 mL), and extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1-1 / 1, v / v) to give compound BB-4-3. 1 HNMR (400MHz, CDCl3) δ: 8.03-7.94(m,1H),8.02-7.94(m,1H),8.05-7.92(m,1H),8.04-7.92(m,1H),8.1 5-7.89(m,1H),7.86(d,J=1.5Hz,1H),7.82-7.79(m,1H),7.19-7.10(m,2H),6.99-6.90(m,2H),6.06(br s,1H),1.58(s,6H).

[0167] Step 3: Synthesis of compound BB-4-4

[0168] Compound BB-4-3 (2 g, 4.72 mmol) was dissolved in N,N-dimethylformamide (50 mL) at room temperature and under nitrogen protection. Tert-butyl 4-(2-bromoethyl)piperazine-1-carboxylate (1.66 g, 5.67 mmol), potassium carbonate (1.31 g, 9.45 mmol), and potassium iodide (784.17 mg, 4.72 mmol) were added. The reaction mixture was heated to 80 °C and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1-1 / 1, v / v) to give compound BB-4-4. MS-ESI m / z: 636.3 [M+H] + .

[0169] Step 4: Synthesis of the hydrochloride salt of compound BB-4-5

[0170] Compound BB-4-4 (2.0 g, 3.15 mmol) was dissolved in ethyl acetate (50 mL) at room temperature and under nitrogen protection. Ethyl acetate hydrochloride (4 M, 3.93 mL) was slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 25 °C for 12 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain the hydrochloride salt of compound BB-4-5. 1 H NMR (400MHz, CD3OD) δ:8.23-8.13(m,2H),8.00(dd,J=1.8,8.3Hz,1H),7.40(t,J=8.9Hz,1H),7.32(dd,J=2.4,11.4Hz,1H),7.24(br d,J=8.8Hz,1H),4.69-4.60(m,2H),3.88-3.79(m,6H),3.75-3.65(m,4H),1.58(s,6H).

[0171] Step 5: Synthesis of compound BB-4-6

[0172] Compound BB-4-5 (1.5 g, 2.62 mmol, hydrochloride) was dissolved in acetonitrile (50 mL) at room temperature and under nitrogen protection. Ethyl bromoacetate (875.85 mg, 5.24 mmol, 580.04 μL) and potassium carbonate (724.86 mg, 5.24 mmol) were added sequentially. The reaction mixture was heated to 80 °C and stirred for 5 hours. After the reaction was complete, it was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1-1 / 1, v / v) to give compound BB-4-6. MS-ESI m / z: 622.2 [M+H] + .

[0173] Step 6: Synthesis of compound BB-4

[0174] Compound BB-4-6 (1.5 g, 2.42 mmol) was dissolved in ethanol (50 mL) at room temperature under nitrogen protection. Lithium hydroxide (1 M, 7.25 mL) was slowly added dropwise to the reaction mixture, which was stirred at 25 °C for 12 hours. After the reaction was complete, the pH of the reaction mixture was adjusted to 2-3 with 2 M hydrochloric acid, and the mixture was concentrated under reduced pressure to give compound BB-4. MS-ESI m / z: 594.1 [M+H] + .

[0175] See Example 5

[0176]

[0177] Synthesis route:

[0178]

[0179] Compound BB-1-1 (3 g, 10.60 mmol) and allyl alcohol (1.34 g, 23.07 mmol, 1.57 mL) were added to dioxane (30 mL). N-cyclohexyl-N-methyl-cyclohexylamine (2.48 g, 12.72 mmol, 2.70 mL), tri-tert-butylphosphine (4.29 g, 2.12 mmol, 4.97 mL, 10% purity) and tris(dibenzylacetone)palladium (970.32 mg, 1.06 mmol) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and stirred at 30 °C for 12 hours. The reaction solution was slowly added to water (50 mL), extracted three times with ethyl acetate (50 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 0 / 1-3 / 1, volume ratio) to give compound BB-5. 1 HNMR(400MHz, CDCl3)δ:9.84(s,1H),7.60(s,1H),7.50(d,J=8.0Hz,1H),7.33(s,1H),7.12(dd,J=1.0,8.0Hz ,1H),4.24-4.16(m,2H),3.69(d,J=0.8Hz,2H),3.14-3.03(m,2H),2.90-2.78(m,2H),1.29(t,J=7.2Hz,3H).

[0180] See Example 6

[0181]

[0182] Synthesis route:

[0183]

[0184] Step 1: Synthesis of intermediate BB-6-2

[0185] At 25°C, 1.0 g (6.04 mmol) of ethyl 1-aminocyclopropane-1-carboxylate hydrochloride was added to 10 mL of ethanol, and stirring was started. Then, N-benzyl-2-chloro-N-(2-chloroethyl)ethylamine hydrochloride (1.78 g, 6.64 mmol) and N,N-diisopropylethylamine (7.88 g, 60.98 mmol, 10.62 mL) were added sequentially. The mixture was heated to 80°C under nitrogen protection and stirred for 12 hours. After the reaction was complete, the reaction solution was cooled to room temperature and then evaporated to dryness. The crude product was extracted with water (20 mL) and ethyl acetate (20 mL × 2). The combined organic phases were washed with semi-saturated brine (10 mL × 2), and the organic phases were dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1–1 / 1, volume ratio) to obtain intermediate BB-6-2. 1 H NMR (400MHz, CDCl3) δ: 7.40-7.27 (m, 5H), 4.18 (q, J = 7.20Hz, 2H), 3.55 (s, 2H), 3.02 (s, 4H), 2.40 (s, 4H), 1.28-1.34 (m, 5H), 0.94 (q, J = 3.6Hz, 2H).

[0186] Step 2: Synthesis of intermediate BB-6-3

[0187] Intermediate BB-6-2 (0.3 g, 1.04 mmol) was added to ethanol (20 mL) at 25 °C with stirring. Potassium hydroxide (583.66 mg, 10.40 mmol) was added sequentially, and the mixture was heated to 120 °C and stirred for 12 hours. After the reaction was complete, the reaction solution was cooled to room temperature and evaporated to dryness. Water (40 mL) was then added, followed by extraction with ethyl acetate (40 mL × 2). The pH of the aqueous phase was adjusted to 3 with 4 M dilute hydrochloric acid, and then extracted with ethyl acetate (40 mL). The product remained in the aqueous phase, which was collected and lyophilized to obtain compound BB-6-3. 1 H NMR (400MHz, DMSO-d6) δ: 10.70 (s, 1H), 7.59 (s, 2H), 7.45 (s, 3H), 4.26 (s, 2H), 3.46 (t, J = 12.4 Hz, 2H), 3.21 (d, J = 11.6 Hz, 2H), 2.94 (d, J = 12.8 Hz, 2H), 2.83 (s, 2H), 1.16 (s, 2H), 0.94 (s, 2H).

[0188] Step 3: Synthesis of compound BB-6-4

[0189] At 25°C, intermediate BB-6-3 was added to N,N-dimethylformamide (20 mL), and stirring was started. Compound BB-2 (938.21 mg, 3.84 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.19 g, 5.76 mmol), and triethylamine (1.55 g, 15.37 mmol, 2.14 mL) were added sequentially. Stirring continued for 12 hours under nitrogen protection. After the reaction was complete, the reaction mixture was extracted with water (50 mL) and ethyl acetate (50 mL × 2). The organic phases were combined, washed with semi-saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1–0 / 1, v / v) to obtain intermediate BB-6-4. MS-ESIm / z::487.2[M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.92 (s, 1H), 7.95 (s, 1H), 7.88 (s, 1H), 7.53 (d, J = 8.8Hz, 1H), 7.41 (dd, J = 8.8, 1.6Hz, 1H), 7.35-7.28 (m ,4H),7.27-7.22(m,1H),4.12(dd,J=12.0,4.8Hz,1H),3.50(s,2H),3.4 0-3.30(m,2H),2.89(s,2H),2.83-2.74(m,1H),2.73(s,2H),2.61-2.60 (m,1H),2.56-2.55(m,2H),2.34-2.20(m,1H),2.14-2.10(m,1H),1.08-1.06(m,4H).

[0190] Step 4: Synthesis of compound BB-6

[0191] Compound BB-6-4 (50 mg, 97.91 μmol) was dissolved in tetrahydrofuran (10 mL) at 25 °C. Under argon protection, wet palladium on carbon (5 mg, 10.28 μL) was added, and the mixture was purged with hydrogen three times. The pressure was maintained at 40 Psi. The reaction solution was heated to 30 °C and stirred for 12 hours under a hydrogen atmosphere. After the reaction was complete, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding compound BB-6. MS-ESI m / z: 397.1 [M+H] + .

[0192] See Example 7

[0193]

[0194] Synthesis route:

[0195]

[0196] Step 1: Synthesis of compound BB-7-2

[0197] Triphenyl phosphite (48.42 g, 156.06 mmol, 41.04 mL) was dissolved in dichloromethane (250 mL) at 25 °C. Under a nitrogen atmosphere, the solution was cooled to -70 °C, and liquid bromine (27.21 g, 170.25 mmol, 8.78 mL) was added dropwise. After the addition was complete, triethylamine (18.66 g, 184.44 mmol, 25.67 mL) and a dichloromethane solution of BB-7-1 (25.00 g, 141.88 mmol) were added dropwise sequentially. The solution was then slowly heated to 25 °C and stirred for 12 hours. After the reaction was complete, a saturated aqueous solution of sodium sulfite (400 mL) was slowly poured into the reaction solution, and the mixture was stirred for 10 minutes. The solution was extracted with dichloromethane (200 mL × 3), and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography (eluent: pure petroleum ether) to obtain compound BB-7-2. 1 H NMR (400MHz, CDCl3) δ: 7.48 (d, J = 8.78Hz, 1H), 6.75 (dd, J = 8.53, 2.51Hz, 1H), 6.68 (d, J = 2.76Hz, 1H), 6.30 (t, J = 4.77Hz, 1H), 3.82 (s, 3H), 2.82 (t, J = 8.03Hz, 2H), 2.35 (td, J = 8.03, 5.02Hz, 2H).

[0198] Step 2: Synthesis of compound BB-7-3

[0199] Compound BB-7-2 (10.00 g, 41.82 mmol) was dissolved in toluene (100 mL) at 25 °C. The mixture was cooled to 0 °C under nitrogen protection, and dichlorocyanobenzoquinone (10.44 g, 46.00 mmol) was added in portions. After the addition was complete, the reaction mixture was reacted at 25 °C for 15 hours. The reaction was quenched by adding 200 mL of saturated sodium sulfite solution dropwise. After stirring for 10 minutes, 100 mL of 1 N sodium hydroxide solution was added. The mixture was extracted with ethyl acetate (50 mL × 3), and the organic phase was washed with 100 mL of saturated brine. The phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was slurried in petroleum ether (50 mL) for 10 minutes, filtered, and the filter cake was washed with petroleum ether (25 mL × 2). The filtrate was evaporated to dryness, and the crude product was purified by column chromatography (eluent: pure petroleum ether) to obtain compound BB-7-3. 1H NMR (400MHz, CDCl3) δ: 8.13 (d, J = 9.29Hz, 1H), 7.67 (d, J = 8.28Hz, 1H), 7.61 (d, J = 7.28Hz, 1H), 7.21–7.26 (m, 2H), 7.11 (d, J = 2.51Hz, 1H), 3.92 (s, 3H).

[0200] Step 3: Synthesis of compound BB-7-4

[0201] Compound BB-7-3 (4.90 g, 20.67 mmol) was dissolved in dichloromethane (50 mL) at 25 °C. The solution was cooled to 0 °C, and boron tribromide (6.21 g, 24.80 mmol, 2.39 mL) was slowly added dropwise. After the addition was complete, the mixture was stirred at 25 °C for 3 hours. The reaction mixture was quenched in ice water (250 mL), extracted with dichloromethane (100 mL), and the organic phase was washed with saturated brine (100 mL × 1). The solution was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound BB-7-4. 1 H NMR (400MHz, CDCl3) δ: 8.19 (d, J = 9.03Hz, 1H), 7.63–7.73 (m, 2H), 7.26–7.32 (m, 1H), 7.23 (dd, J = 9.03, 2.51Hz, 1H), 7.19 (d, J = 2.51Hz, 1H).

[0202] Step 4: Synthesis of compound BB-7-5

[0203] Compound BB-7-4 (2.50 g, 11.21 mmol) was dissolved in methanesulfonic acid (25 mL) at 20 °C, and ethyl 4-chloroacetoacetate (2.77 g, 16.81 mmol, 2.27 mL) was added dropwise. The mixture was stirred at 20 °C for 15 hours. The reaction solution was quenched in ice water (200 mL), stirred for 10 minutes, filtered, and the filter cake was washed with water (30 mL × 3) and then evaporated to dryness to obtain compound BB-7-5. 1 H NMR(400MHz,DMSO-d6)δ:8.56(d,J=8.78Hz,1H),8.46(d,J=9.29Hz,1H),7.7 1(d,J=9.29Hz,1H),7.63(dd,J=8.66,7.65Hz,1H),6.92(s,1H),5.39(s,2H).

[0204] Step 5: Synthesis of compound BB-7-6

[0205] Compound BB-7-5 (0.10 g, 309.05 μmol) was added to an aqueous solution of sodium hydroxide (2 M, 1.03 mL) at 25 °C, and stirred at 80 °C for 3 hours. The reaction solution was cooled to 25 °C, diluted with water (10 mL), and the pH was adjusted to 3 with dilute hydrochloric acid (2 M, aqueous solution). The solution was extracted with ethyl acetate (10 mL × 3), the organic phase was washed with saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain compound BB-7-6. 1 H NMR(400MHz,DMSO-d6)δ:12.59(br s,1H),8.24(d,J=8.38Hz,1H),7.96(d,J=9.26Hz,1H),7.90(d,J=7.50Hz,1H),7.54(dd,J=2.8Hz,1H),7.53(s,1H),7.41(s,1H),4.08(s,2H).

[0206] Step 6: Synthesis of compound BB-7

[0207] Compound BB-7-6 (1.40 g, 4.59 mmol) was dissolved in anhydrous ethanol (14 mL) at 25 °C. Sulfuric acid (413.28 mg, 4.13 mmol, 224.61 μL, 98% concentration) was slowly added dropwise, and the mixture was heated to 80 °C and stirred for 12 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, diluted with ethyl acetate (60 mL), extracted with saturated sodium bicarbonate aqueous solution (60 mL), and then extracted with ethyl acetate (60 mL × 2). The organic phase was washed with saturated brine (60 mL × 1), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Petroleum ether (5 mL) was added, and the mixture was stirred at room temperature for 10 minutes, then filtered. The filter cake was washed with petroleum ether (5 mL × 2), collected, and evaporated to dryness. Compound BB-7 was obtained. 1 H NMR(400MHz, CDCl3)δ:8.22(t,J=8.41Hz,2H),7.78–7.85(m,2H),7.75(d,J=9.29Hz,1H ), 7.41 (t, J = 7.91Hz, 1H), 4.23 (q, J = 7.03Hz, 2H), 4.06 (s, 2H), 1.26 (t, J = 7.15Hz, 3H).

[0208] See Example 8

[0209]

[0210] Synthesis route:

[0211]

[0212] Step 1: Synthesis of Compound 8-1

[0213] Compound BB-7 (5 g, 15.01 mmol) was dissolved in a mixed solvent of toluene (50 mL) and water (10 mL). Tert-butyl carbamate (2.64 g, 22.51 mmol), potassium phosphate (12.74 g, 60.03 mmol), tris(dibenzylacetone)palladium (961.96 mg, 1.05 mmol), and 2-di-tert-butylphosphine-2,4,6-triisopropylbiphenyl (892.16 mg, 2.10 mmol) were added. The mixture was purged with nitrogen three times, heated to 100 °C, and stirred for 15 hours. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate (30 mL × 3). The filtrate was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Add 50 mL of methyl tert-butyl ether to the crude product, stir for 10 minutes, filter, rinse the filter cake with 10 mL × 2 of methyl tert-butyl ether, and collect the filter cake to obtain compound BB-8-1.

[0214] Step 2: Synthesis of compound BB-8-2

[0215] Compound BB-8-1 (4.2 g, 11.37 mmol) and acrylamide (888.93 mg, 12.51 mmol, 863.04 μL) were dissolved in N,N-dimethylformamide (40 mL). Under nitrogen protection, the mixture was lowered to 0 °C, and a solution of potassium tert-butoxide (2.55 g, 22.74 mmol) in N,N-dimethylformamide (10 mL) was added dropwise. The mixture was heated to 20 °C and stirred for 2 hours. The reaction solution was poured into 0.2 N dilute hydrochloric acid (200 mL) and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Dichloromethane (20 mL) was added to the crude product, and the mixture was stirred for 10 minutes. The mixture was filtered, and the filter cake was washed with dichloromethane (10 mL). The filter cake was collected to obtain compound BB-8-2. 1 H NMR (400MHz, DMSO-d6) δ: 10.94 (s, 1H), 9.28 (s, 1H), 8.04-7.96 (m, 3H), 7.79 (d, J = 9.6Hz, 1H), 7.55-7.51 (m, 2H), 4.6 7(dd,J=4.4Hz,12.0Hz,1H),2.93-2.84(m,1H),2.66-2.60(m,1H),2.45-2.36(m,1H),2.33-2.26(m,1H),1.49(s,9H).

[0216] Step 3: Synthesis of the hydrochloride salt of compound BB-8

[0217] Compound BB-8-2 (1 g, 2.54 mmol) was dissolved in ethyl acetate (10 mL), and hydrochloric acid / ethyl acetate (4 M, 50.00 mL) was added dropwise.

[0218] The mixture was stirred at 20°C for 15 hours. The reaction solution was then concentrated under reduced pressure to obtain the hydrochloride salt of compound BB-8.

[0219] See Example 9

[0220]

[0221] Synthesis route:

[0222]

[0223] Step 1: Synthesis of compound BB-9-1

[0224] Compound BB-4-3 (13 g, 30.71 mmol) and potassium carbonate (8.49 g, 61.41 mmol) were dissolved in N,N-dimethylformamide (300 mL), and 1,2-dibromoethane (28.84 g, 153.53 mmol, 11.58 mL) was added. The mixture was heated to 80 °C and stirred for 12 hours. The reaction solution was cooled to room temperature, water (1 L) was added, and the mixture was extracted with ethyl acetate (300 mL × 3). The organic phase was washed with saturated brine (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to obtain compound BB-9-1. 1 H NMR (400MHz, CDCl3) δ: 8.00-7.96 (m, 2H), 7.85-7.83 (m, 1H), 7.14-7.04 (m, 3H), 4.44 (t, J = 6.0Hz, 2H), 3.71 (t, J = 6.4Hz, 2H), 1.60 (s, 6H).

[0225] Step 2: Synthesis of compound BB-9

[0226] Compound BB-9-1 (3 g, 5.66 mmol) and methylamine hydrochloride (1.91 g, 28.28 mmol) were placed in N,N-dimethylformamide (30 mL), and potassium carbonate (11.73 g, 84.85 mmol) was added. The mixture was heated to 50 °C and stirred for 12 hours. The reaction mixture was cooled to room temperature, and water (70 mL) was added. The mixture was extracted with ethyl acetate (50 mL × 4). The organic phase was washed with saturated brine (70 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: dichloromethane / methanol = 50 / 1–10 / 1) to obtain compound BB-9.

[0227] Reference Example 10: Fragment BB-10

[0228]

[0229] Synthesis route:

[0230]

[0231] Step 1: Synthesis of compound BB-10-2

[0232] Compound BB-10-1 (5 g, 18.03 mmol) was dissolved in dichloromethane (50 mL) and acetone (25 mL) under nitrogen protection at 0 °C. Cyanotrimethylsilane (2.68 g, 27.04 mmol, 3.38 mL) and trimethylsilyltrifluoromethanesulfonate (400.67 mg, 1.80 mmol, 325.74 μL) were then slowly added dropwise. The reaction mixture was stirred at 20 °C for 2 hours. After the reaction was complete, the solvent was removed from the reaction solution under reduced pressure. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–4 / 1, v / v) to give compound BB-10-2.

[0233] Step 2: Synthesis of compound BB-10

[0234] Compound BB-10-2 (10 g, 29.03 mmol) was dissolved in N,N-dimethylacetamide (100 mL) under nitrogen protection at room temperature. 4-Thioisocyanate-2-(trifluoromethyl)benzonitrile (6.62 g, 29.03 mmol) was added in portions to the reaction mixture. The reaction mixture was stirred at 20 °C for 3 hours. Methanol (100 mL) and dilute hydrochloric acid (2 M, 56.32 mL) were added, and the reaction mixture was stirred at 70 °C for 2 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with water (100 mL), and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by column chromatography (eluent: dichloromethane / methanol = 20 / 1–10 / 1, v / v) to give compound BB-10. 1 HNMR(400MHz, CDCl3)δ:7.99(d,J=8.8Hz,1H),7.96(d,J=1.6Hz,1H),7.84(dd,J=2.0,8.4Hz,1H) ,7.22(d,J=9.2Hz,2H),7.04(d,J=8.8Hz,2H),3.64–3.57(m,4H),3.45–3.38(m,4H),1.58(s,6H).

[0235] See Example 11: Fragment BB-11

[0236]

[0237] Synthesis route:

[0238]

[0239] Step 1: Synthesis of compound BB-11-2

[0240] Compound BB-11-1 (0.87 g, 5.57 mmol) and compound [(1R,4R)-4-hydroxycyclohexyl] tert-butyl carbamate (1.00 g, 4.64 mmol) were dissolved in N,N-dimethylformamide (20 mL) under nitrogen protection at 0 °C. Sodium hydroxide (278.68 mg, 6.97 mmol, 60%) was then added, and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed successively with water (50 mL × 3) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–6 / 1, v / v) to obtain compound BB-11-2. 1 H NMR(400MHz, CDCl3)δ:8.00(s,1H),7.56-7.47(m,1H),6.97-6.92(m,1H),6.84-6.72(m,1H),4 .29-4.17(m,1H),4.13-4.08(m,1H),2.14-2.03(m,4H),1.47-1.38(m,9H),1.26-1.20(m,4H).

[0241] Step 2: Synthesis of the hydrochloride salt of compound BB-11-3

[0242] Compound BB-11-2 (2.70 g, 7.70 mmol) was dissolved in methanol (5 mL) at room temperature, followed by the addition of 4 M hydrogen chloride methanol solution (25 mL). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure to obtain the hydrochloride salt of compound BB-11-3, which was used directly in the next reaction.

[0243] Step 3: Synthesis of compound BB-11-6

[0244] At room temperature, compound BB-11-5 (10.00 g, 57.95 mmol) and 4-piperidinemethanol (6.67 g, 57.95 mmol) were dissolved in dimethyl sulfoxide (100 mL), followed by the addition of triethylamine (11.73 g, 115.90 mmol, 16.13 mL). The reaction mixture was heated to 90 °C and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, and water (200 mL) was added to the reaction solution. The mixture was extracted with dichloromethane (100 mL × 4). The organic phases were combined and washed successively with water (100 mL × 2) and saturated brine (100 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: dichloromethane / methanol = 1 / 0–20 / 1, v / v) to give compound BB-11-6. 1 H NMR(400MHz, DMSO-d6)δ:7.79(d,J=9.2Hz,1H),7.27(d,J=9.6Hz,1H),4.57-4.53(m,1H),4.53-4.48(m, 2H), 3.86 (s, 3H), 3.30-3.24 (m, 2H), 2.99 (td, J = 1.9, 12.7Hz, 2H), 1.81-1.66 (m, 3H), 1.21-1.06 (m, 2H).

[0245] Step 4: Synthesis of compound BB-11-7

[0246] At room temperature, BB-11-6 (2.00 g, 7.96 mmol) was dissolved in tetrahydrofuran (15 mL), followed by the addition of 2M sodium hydroxide aqueous solution (15 mL). The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the pH was adjusted to 4-5 with 4M hydrochloric acid, and the solvent was removed by concentration under reduced pressure to obtain crude compound BB-11-7, which was directly used in the next reaction.

[0247] Step 5: Synthesis of compound BB-11-4

[0248] Under nitrogen protection, the hydrochloride salt (7.65 mmol) of compound BB-11-3 and the crude compound BB-11-7 (7.65 mmol) were dissolved in N,N-dimethylformamide (46 mL), followed by the addition of diisopropylethylamine (1.03 g, 7.96 mmol, 1.39 mL) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.03 g, 7.96 mmol), and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (70 mL × 3). The organic phases were combined, washed successively with water (100 mL × 2) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: dichloromethane / methanol = 1 / 0–20 / 1, v / v) to give compound BB-11-4. MS–ESI m / z: 470.2 [M+H] + . 1 H NMR(400MHz, DMSO-d6)δ:8.58(d,J=8.4Hz,1H),7.85(d,J=8.4Hz,1H),7.79(d,J=9.6Hz,1 H),7.38(d,J=2.4Hz,1H),7.33(d,J=9.6Hz,1H),7.13(dd,J=2.4,8.8Hz,1H),4.60-4.44( m,4H),3.92-3.79(m,1H),3.32-3.27(m,2H),3.07-2.95(m,2H),2.18-2.05(m,2H),1.95- 1.84(m,2H),1.81-1.68(m,3H),1.68-1.51(m,2H),1.51-1.48(m,2H),1.20-1.06(m,2H).

[0249] Step 6: Synthesis of compound BB-11

[0250] Compound BB-11-4 (100.02 mg, 205.06 μmol) was dissolved in dichloromethane (6 mL) at room temperature, followed by the addition of Dys-Martin oxidant (130.46 mg, 307.59 μmol), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–0 / 1, v / v) to give compound BB-11. MS–ESI m / z: 468.2 [M+H] + .

[0251] Reference Example 12: Fragment BB–12

[0252]

[0253] Synthesis route:

[0254]

[0255] Step 1: Synthesis of compound BB-12-1

[0256] Compound BB-3-4 (10.00 g, 30.01 mmol) and 1-tert-butyloxycarbonylpiperazine (8.39 g, 45.02 mmol) were added to toluene (150 mL) and water (20 mL) at 25 °C. Tris(dibenzylacetone)dipalladium (1.92 g, 2.10 mmol), potassium phosphate (19.11 g, 90.04 mmol), and 2-di-tert-butylphosphine-2',4',6'-triisopropylbiphenyl (1.78 g, 4.20 mmol) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and then heated to 100 °C and stirred for 12 hours. The reaction solution was cooled to 25 °C, filtered through diatomaceous earth, and the filter cake was washed with 20 mL of ethyl acetate. The filtrate was added to 300 mL of ethyl acetate and 300 mL of water. The aqueous phase was extracted with ethyl acetate (200 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (eluent: petroleum ether: ethyl acetate = 50 / 1 to 5 / 1) to obtain compound BB-12-1.

[0257] MS–ESI m / z: 439.0 [M+H] + . 1 H NMR (400MHz, CDCl3) δ: 8.15 (d, J = 9.0Hz, 1H), 7.74 (s, 1H), 7.60 (s, 2H), 7.36 (dd, J = 2.5, 9.0Hz, 1H), 7.31 (d, J = 2.5Hz ,1H),4.23(q,J=7.1Hz,2H),4.04(s,2H),3.71-3.62(m,4H),3.29-3.20(m,4H),1.53(s,9H),1.28(dt,J=7.2Hz,3H).

[0258] Step 2: Synthesis of compound BB-12-2

[0259] Compound BB-12-1 (8.6 g, 15.86 mmol) and acrylamide (1.13 g, 15.86 mmol, 1.09 mL) were added to N,N-dimethylformamide (80 mL) at 0 °C. Stirring was started, and potassium tert-butoxide (2.67 g, 23.80 mmol) was added to the reaction mixture in portions. The reaction mixture was purged with nitrogen three times and then stirred at 0 °C for 1 hour. After the reaction was complete, the reaction mixture was slowly added dropwise to a saturated ammonium chloride aqueous solution (480 mL), resulting in the precipitation of a large amount of solid. The solid was filtered, and the filter cake was washed twice with water (20 mL × 2). The filter cake was collected and evaporated under reduced pressure. The crude product was added to methanol (10 mL) and stirred for 30 minutes. The mixture was filtered, and the filter cake was washed with methanol (5 mL). The filter cake was collected and evaporated under reduced pressure to obtain compound BB-12-2. 1 H NMR (400MHz, DMSO-d6) δ: 10.96 (br dd, J=2.9, 6.1Hz, 1H), 8.02 (br d, J=8.8Hz, 1H), 7.97-7.91 (m, 1H), 7.72-7.65 (m, 2H), 7.44-7.37 (m, 2H), 4.62 (br dd,J=4.3,11.8Hz,1H),3.53(br s,4H),3.22-3.20(m,4H),2.93-2.83(m,1H),2.68-2.57(m,1H),2.39(dq,J=4.3,12.5Hz,1H),2.26(br dd,J=3.9,8.7Hz,1H),1.44(s,9H).

[0260] Step 3: Synthesis of the hydrochloride salt of compound BB-12

[0261] Compound BB-12-2 (3.4 g, 7.34 mmol) was added to ethyl acetate (20 mL), and stirring was started. Ethyl hydrochloric acid solution (4 M, 30 mL) was slowly added. The reaction mixture was purged with nitrogen three times and then stirred at 25 °C for 12 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed with ethyl acetate (5 mL × 2). The filter cake was collected and evaporated under reduced pressure to obtain the hydrochloride salt of compound BB-12. 1H NMR(400MHz,DMSO-d6)δ:10.96(s,1H),9.65(br s,2H),8.07(br d,J=9.3Hz,1H),7.96(s,1H),7.75-7.69(m,2H),7.53(d,J=2.3Hz,2H),7.45(dd,J=2.4,9.2Hz,1H),4.64(br dd,J=4.4,11.9Hz,1H),3.54(br d,J=5.0Hz,4H),3.28(br s,4H),2.94-2.82(m,1H),2.67-2.58(m,1H),2.39(br dd,J=4.0,12.5Hz,1H),2.30-2.17(m,1H).

[0262] Reference Example 13: Fragment BB-13

[0263]

[0264] Synthesis route:

[0265]

[0266] Step 1: Synthesis of compound BB-13-1

[0267] Compound BB-7 (1.4 g, 4.20 mmol) and 1-tert-butyloxycarbonylpiperazine (1.17 g, 6.30 mmol) were added to dioxane (20 mL) and water (4 mL) at 25 °C. Tris(dibenzylacetone)dipalladium (269.35 mg, 294.14 μmol), potassium phosphate (2.68 g, 12.61 mmol), and 2-di-tert-butylphosphine-2',4',6'-triisopropylbiphenyl (249.81 mg, 588.28 μmol) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and then heated to 100 °C and stirred for 12 hours. The reaction solution was cooled to 25 °C, filtered through diatomaceous earth, and the filter cake was washed with 20 mL of ethyl acetate. The filtrate was added to ethyl acetate (100 mL) and water (100 mL). The aqueous phase was extracted with ethyl acetate (100 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give compound BB-13-1. MS: ESI m / z: 439.0 [M+H] + . 1H NMR(400MHz, DMSO-d6)δ:8.17(d,J=9.3Hz,1H),8.04(s,1H),7.86(d,J=8.3Hz,1H),7.77(d,J=9.3Hz,1H),7.53( t,J=8.0Hz,1H),7.19(d,J=7.3Hz,1H),4.20-4.09(m,4H),3.36-2.59(m,8H),1.45(s,9H),1.20(t,J=7.0Hz,3H).

[0268] Step 2: Synthesis of compound BB-13-2

[0269] Compound BB-13-1 (1.5 g, 3.26 mmol) and acrylamide (231.68 mg, 3.26 mmol, 224.93 μL) were added to N,N-dimethylformamide (20 mL) at 0 °C. Stirring was started, and potassium tert-butoxide (438.91 mg, 3.91 mmol) was slowly added in portions to the reaction mixture. The reaction mixture was purged with nitrogen three times and then stirred at 0 °C for 1 hour. The mixture was slowly added dropwise to a saturated ammonium chloride aqueous solution (75 mL), resulting in the precipitation of a solid, which was filtered. The filter cake was washed with water (5 mL × 2), and the solid was collected and evaporated to dryness under reduced pressure. The crude product was added to 5 mL of methanol and stirred for 15 minutes. The mixture was filtered, washed with methanol (3 mL × 2), and the solid was collected and evaporated to dryness under reduced pressure. Compound BB-13-2 was obtained. MS–ESI m / z: 464.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 8.19 (d, J = 9.3Hz, 1H), 7.97 (d, J = 16.8Hz, 2H), 7.78 (d, J = 9.0Hz, 1H), 7.51 (br t, J = 8.0Hz, 1H), 7.18 (br d,J=7.5Hz,1H),4.66(br d,J=7.8Hz,1H),3.34(m,4H),3.17(m,4H),2.89-2.81(m,1H),2.68-2.55(m,1H),2.40(dq,J=3.8,12.4Hz,1H),2.26(br s,1H),1.44(s,9H).

[0270] Step 3: Synthesis of the hydrochloride salt of compound BB-13

[0271] Compound BB-13-2 (0.6 g, 1.16 mmol) was added to ethyl acetate (6 mL), and stirring was started. Ethyl hydrochloride (4 M, 10 mL) was slowly added to the reaction mixture. The reaction was purged with nitrogen three times and then stirred at 25 °C for 12 hours. The mixture was evaporated to dryness under reduced pressure to give the hydrochloride salt of compound BB-13. MS–ESI m / z: 364.0 [M+H] + .

[0272] Example 1

[0273]

[0274] Synthesis route:

[0275]

[0276] Under nitrogen protection at room temperature, compound BB-4 (0.3 g, 505.40 μmol) was dissolved in N,N-dimethylformamide (10 mL), and BB-2 (123.44 mg, 505.40 μmol, hydrochloride), triethylamine (255.70 mg, 2.53 mmol, 351.72 μL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (288.25 mg, 758.10 μmol) were added. The reaction mixture was stirred at 20 °C for 2 hours. After the reaction was complete, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was separated by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% hydrochloric acid), lyophilized, and the target compound WX001 was obtained. MS-ESI m / z: 820.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 10.94 (s, 1H), 10.61 (s, 1H), 8.40 (d, J = 8.0Hz, 1H ),8.29(d,J=1.6Hz,1H),8.08(m,1H),7.93-7.92(m,2H),7.57(d,J=8.8Hz, 1H),7.49-7.43(m,3H),7.40(d,J=2.4Hz,1H),4.59(s,2H),4.15-4.11(m, 1H),3.62-3.54(m,6H),2.90-2.51(m,8H),2.25-2.15(m,2H),1.52(s,6H).

[0277] Example 2

[0278]

[0279] Synthesis route:

[0280]

[0281] The hydrochloride salts of compound BB-4 (100 mg, 168.47 μmol) and intermediate BB-1 (47.29 mg, 168.47 μmol) were added to N,N-dimethylformamide (2 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (96.08 mg, 252.70 μmol) and triethylamine (34.09 mg, 336.93 μmol, 46.90 μL) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and then stirred at 25 °C for 12 hours. The reaction solution was poured into 20 mL of water and 30 mL of ethyl acetate. The organic phase was separated and washed twice with water (20 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% hydrochloric acid) to obtain compound WX002. MS-ESI m / z: 820.2 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ:10.90(s,1H),10.47(br s,1H),8.40(d,J=8.3Hz,1H),8.28(d,J=1.5Hz,1H),8.13-7.97(m,2H),7.87 (s,1H),7.54(d,J=8.5Hz,1H),7.47-7.29(m,3H),7.26-7.13(m,1H),4.54(br s,2H),4.12(dd,J=4.8,11.8Hz,1H),3.67-3.45(m,2H),2.83-2.67(m,2H ),2.63-2.43(m,10H),2.39-2.25(m,1H),2.19-2.03(m,1H),1.52(s,6H).

[0282] Example 3

[0283]

[0284] Synthesis route:

[0285]

[0286] Under nitrogen protection and at room temperature, BB-3 hydrochloride (378 mg, 1.08 mmol) and BB-4 (767.86 mg, 1.29 mmol) were dissolved in N,N-dimethylformamide (5 mL). The reaction mixtures of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (614.82 mg, 1.62 mmol) and triethylamine (272.70 mg, 2.69 mmol) were added separately, and the mixture was stirred at 25 °C for 3 hours. After the reaction was complete, water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 2). The combined organic phases were washed successively with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% hydrochloric acid) to obtain the target compound WX003. MS-ESI m / z: 870.1 [M+H] + . 1 H NMR(400MHz, DMSO-d6)δ:10.96(s,1H),10.73(s,1H),8.41-8.40(m,2H),8.39(d,J=1.6Hz ,1H),8.08(d,J=8.8Hz,1H),8.07(dd,J=1.6,8.4Hz,1H),8.01(s,1H),7.78(s,3H),7.75( d,J=2.0Hz,1H),7.46-7.37(m,2H),7.23(d,J=8.8Hz,1H),4.68(dd,J=4.0,11.2Hz,1H),4 .58(s,2H),3.98(m,1H),3.47(s,9H),2.95-2.58(m,3H),2.49-2.21(m,3H),1.52(s,6H).

[0287] Example 4

[0288]

[0289] Synthesis route:

[0290]

[0291] Step 1: Synthesis of WX004-1

[0292] Compounds BB-5 (102.38 mg, 393.35 μmol) and BB-4-5 (150 mg, 262.23 μmol, 145.82 μL, hydrochloride) were added to 10 mL of dichloromethane. Triethylamine (53.07 mg, 524.46 μmol, 73.00 μL) was slowly added to the reaction mixture. The mixture was stirred at 25 °C for 0.5 h. Sodium borohydride acetate (138.94 mg, 655.58 μmol) was slowly added to the reaction mixture. After purging the mixture with nitrogen three times, it was stirred at 25 °C for 11.5 h. The reaction mixture was then slowly added to 50 mL of water and extracted three times with dichloromethane (50 mL × 3). The combined organic phases were evaporated to dryness under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane / methanol = 1 / 0 - 5.7 / 1, v / v) to give compound WX004-1. MS-ESI m / z: 780.1 [M+H] + .

[0293] Step 2: Synthesis of WX004

[0294] Compound WX004-1 (280 mg, 309.14 μmol) and acrylamide (26.37 mg, 370.97 μmol, 25.60 μL) were added to N,N-dimethylformamide (5 mL) at 0 °C. Potassium tert-butoxide (173.45 mg, 1.55 mmol) was slowly added to the reaction mixture in portions. After purging the mixture with nitrogen three times, the mixture was stirred at 0 °C for 1 hour. The reaction mixture was then slowly added to a saturated ammonium chloride solution and extracted twice with ethyl acetate (20 mL × 2). The combined organic phases were washed twice with saturated brine (20 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The residue was separated by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain the target compound WX004. MS-ESI m / z: 805.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 10.89 (s, 1H), 8.40 (d, J = 8.3Hz, 1H), 8.28 (d, J = 1.0Hz, 1H), 8.07 (dd, J =1.4,8.2Hz,1H),7.85(d,J=6.8Hz,1H),7.53(dd,J=4.0,7.8Hz,1H),7.49-7.32(m,3H),7.21(br d,J=8.8Hz,1H),7.15(dd,J=5.5,7.3Hz,1H),4.53(br d,J=1.0Hz,2H),4.12(dd,J=4.9,11.9Hz,1H),3.83-3.62(m,5H),3.27-3.06(m,2H),2.84-2.65(m,3H),2.57(br dd,J=4.1,17.2Hz,4H),2.49-2.42(m,2H),2.40-2.26(m,1H),2.19-2.01(m,3H),1.52(s,6H).

[0295] Example 5

[0296]

[0297] Synthesis route:

[0298]

[0299] Step 1: Synthesis of intermediate WX005-1

[0300] At room temperature, BB-4-3 (1.00 g, 2.36 mmol) and bromoacetaldehyde diethanolate (512.01 mg, 2.60 mmol) were dissolved in N,N-dimethylformamide (10 mL), and potassium carbonate (391.73 mg, 2.83 mmol) was added. The reaction mixture was stirred at 80 °C under nitrogen protection for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, and water (50 mL) was added to the mixture. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed successively with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–3 / 1, v / v) to give intermediate WX005-1. 1H NMR(400MHz, CDCl3)δ:8.01-7.94(m,2H),7.84(dd,J=2.0,8.4Hz,1H),7.16-7.10(m,1H),7.09-7.00(m,2H),4.88 (t,J=5.2Hz,1H),4.13(d,J=5.2Hz,2H),3.86-3.76(m,2H),3.72-3.62(m,2H),1.59(s,6H),1.27(t,J=7.0Hz,6H).

[0301] Step 2: Synthesis of intermediate WX005-2

[0302] At room temperature, WX005-1 (500.00 mg, 926.71 μmol) was dissolved in ethanol (20 mL), and acetic acid (5.25 g, 87.42 mmol) and hydrochloric acid (1 M, 2.5 mL) were added. The reaction mixture was stirred at 80 °C under nitrogen protection for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent (ethanol) was removed by concentration under reduced pressure. Water (30 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined and washed successively with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 0–1 / 1, v / v) to give intermediate WX005-2. 1 H NMR (400MHz, CDCl3) δ: 9.92 (s, 1H), 8.02-7.92 (m, 2H), 7.84 (dd, J = 2.0, 8.0Hz, 1H), 7.19-7.10 (m, 1H), 7.10-7.01 (m, 2H), 4.74 (s, 2H), 1.60 (s, 6H).

[0303] Step 3: Synthesis of compound WX005

[0304] Compound WX005-2 (40 mg, 85.94 μmol) was added to dichloromethane (10 mL) at 25 °C with stirring. Then, compound BB-6 (36.80 mg, 77.35 μmol) and sodium triacetoxyborohydride (54.65 mg, 257.83 μmol) were added. Stirring continued for 12 hours under nitrogen protection. After the reaction was complete, the reaction mixture was extracted with water (50 mL) and ethyl acetate (50 mL × 2). The combined organic phases were washed with semi-saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was separated by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain the target compound WX005. ESI m / z: 846.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 10.89 (s, 2H), 9.77 (s, 1H), 8.39 (d, J = 8.4Hz, 1H), 8.27 (d, J = 1.25Hz, 1H), 8.07 (d, J = 8.4Hz, 1H) ), 7.96 (d, J = 1.2Hz, 1H), 7.88 (s, 1H), 7.51-7.56 (m, 1H), 7.44-7.50 (m, 1H), 7.35-7.44 (m, 2H), 7.21-7.23 (m, 1H), 4.61 ( s,2H), 4.11(dd,J=12.0,4.8Hz,1H), 3.5-3.60(m,4H), 3.50-3.51(m,2H), 2.93-3.02(m,2H), 2.80-2.90(m,2H), 2.70-2 .79(m,1H), 2.55-2.68(m,1H), 2.22-2.35(m,1H), 2.06-2.19(m,1H), 1.51(s,6H), 1.14-1.21(m,2H), 1.08-1.10(m,2H).

[0305] Example 6

[0306]

[0307] Synthesis route:

[0308]

[0309] Step 1: Synthesis of compound WX006-1

[0310] Compound BB-9 (71.00 mg, 499.50 μmol, 66.36 μL) and compound ethyl 2-formyl-1-cyclopropanecarboxylate (0.2 g, 416.25 μmol) were dissolved in a mixed solvent of dichloromethane (4 mL) and glacial acetic acid (0.1 mL). Sodium triacetoxyborohydride (132.33 mg, 624.38 μmol) was added at 20 °C, and the mixture was stirred at 20 °C for 15 hours. Water (2 mL) was added to the reaction solution, and the mixture was stirred for 10 minutes. Saturated sodium bicarbonate aqueous solution (5 mL) was added, and the mixture was extracted with dichloromethane (5 mL × 4). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The mixture was purified by plate chromatography (developing solvent: dichloromethane / methanol = 10 / 1) to give compound WX006-1.

[0311] Step 2: Synthesis of compound WX006-2

[0312] Compound WX006-1 (0.11 g, 181.33 μmol) was dissolved in a mixed solvent of tetrahydrofuran (2 mL) and water (0.4 mL), and lithium hydroxide monohydrate (38.05 mg, 906.65 μmol) was added. The mixture was stirred at 20 °C for 15 hours. Ethyl acetate (5 mL) was added to the reaction solution, and the mixture was washed with 1 N lithium hydroxide aqueous solution (2 mL × 5). The pH of the aqueous phase was adjusted to 4-5 with 1 N dilute hydrochloric acid, and the phase was extracted with dichloromethane (5 mL × 3). The organic phase was dried directly with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound WX006-2.

[0313] Step 3: Synthesis of compound WX006

[0314] Compound WX006-2 (90 mg, 155.55 μmol) was dissolved in N,N-dimethylformamide (2 mL), followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (88.72 mg, 233.33 μmol) and N,N-diisopropylethylamine (100.52 mg, 777.77 μmol, 135.47 μL). After stirring for 10 minutes, the hydrochloride salt of compound BB-8 (54.94 mg, 186.66 μmol) was added, and the mixture was stirred at 20 °C for 60 hours. The reaction solution was purified by preparative HPLC (mobile phase: acetonitrile / water; neutral system) to obtain compound WX006. 1H NMR (400MHz, CD3OD) δ: 8.15-8.13 (m, 2H), 8.06-8.05 (m, 1H), 7.98 (t, J = 10Hz, 2H), 7.84 (s, 1H), 7.69 (d, J=9.2Hz,1H),7.58-7.55(m,2H),7.29-7.19(m,2H),7.10-7.08(m,1H),4.65-4.61(m,1H),4.33(t,J=5.2 Hz,2H),3.07(t,J=4.8Hz,2H),2.92-2.84(m,1H),2.77-2.70(m,2H),2.61-2.58(t,1H),2.55(s,3H)2.4 6-2.42(m,2H),1.94-1.91(m,1H),1.67-1.65(m,1H),1.52(s,6H),1.35-1.31(m,1H),0.95-0.90(m,1H).

[0315] Example 7

[0316]

[0317] Synthesis route:

[0318]

[0319] Step 1: Synthesis of compound WX007-1

[0320] Compound WX007-2 (0.5 g, 3.47 mmol) was dissolved in dichloromethane (5 mL). The reaction solution was cooled to 0 °C, and Dys-Martin oxidant (2.94 g, 6.94 mmol, 2.15 mL) was added in portions. The mixture was then slowly heated to 20 °C and stirred for 2 hours. The reaction solution was poured into a saturated sodium bicarbonate aqueous solution (10 mL) and a 10% sodium thiosulfate aqueous solution (30 mL) at 0-10 °C. The mixture was extracted with dichloromethane (10 mL × 3), and the organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered to obtain a solution of compound WX007-3. A solution of compound WX007-3 and compound BB-9 (1.4 g, 2.91 mmol) were dissolved in dichloromethane (5 mL) and glacial acetic acid (0.1 mL). Sodium triacetoxyborohydride (1.24 g, 5.83 mmol) was added at 20 °C and the mixture was stirred for 12 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phase was washed with saturated brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give compound WX007-1. 1H NMR(400MHz, CDCl3)δ:8.00-7.96(m,2H),7.85(dd,J=1.6Hz,8.4Hz,1H),7.14-7.02(m,3H),4.21-4.18(m,2H),4.1 3(q,J=7.2Hz,2H),2.98-2.90(m,2H),2.78(s,2H),2.41(s,3H),1.59(s,6H),1.29-1.23(m,5H),0.88-0.85(m,2H).

[0321] Step 2: Synthesis of compound WX007-4

[0322] Compound WX007-1 (0.53 g, 873.68 μmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL), and lithium hydroxide monohydrate (183.30 mg, 4.37 mmol) was added. The mixture was stirred at 20 °C for 24 hours. The temperature was then raised to 40 °C and stirred for another 24 hours. The pH of the reaction mixture was adjusted to 6–7 with 1 N hydrochloric acid. Most of the tetrahydrofuran was removed by rotary evaporation. Methyl tert-butyl ether (30 mL) and 10% potassium carbonate aqueous solution (20 mL × 3) were added. The mixture was separated, and the pH of the aqueous phase was adjusted to 5–6 with 1 N hydrochloric acid. The phase was extracted with dichloromethane (50 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and evaporated to dryness under reduced pressure to obtain compound WX007-4.

[0323] Step 3: Synthesis of compound WX007

[0324] Compound WX007-4 (50 mg, 86.42 μmol) and N,N-diisopropylethylamine (44.68 mg, 345.68 μmol, 60.21 μL) were dissolved in N,N-dimethylformamide (2 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (39.43 mg, 103.70 μmol) was added at 20 °C and stirred for 5 minutes. Then, the hydrochloride salt of compound BB-8 (34.30 mg, 103.70 μmol) was added, and the mixture was stirred at 20 °C for 12 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, yielding a crude product. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain compound WX007. 1H NMR (400MHz, CD3OD) δ: 8.17-8.15 (m, 3H), 7.98 (d, J = 8.4Hz, 1H), 7.87-7.84 (m, 2H), 7.74 (d, J = 9.6Hz, 1H), 7.62 (t, J = 7. 6Hz, 1H), 7.45 (d, J = 7.2Hz, 1H), 7.28-7.25 (m, 2H), 7.12 (d, J = 8.8Hz, 1H), 4.65 (dd, J = 5.6Hz, 11.2Hz, 1H), 4.52 (t, J = 4. 4Hz,2H),3.90(dd,J=4.0Hz,13.2Hz,1H),3.83-3.79(m,1H),3.65-3.61(m,1H),3.32-3.29(m,1H),3.08(s,3H),2.94-2 .85(m,1H),2.77-2.72(m,1H),2.53-2.43(m,2H),1.92-1.84(m,2H),1.53(s,6H),1.48-1.45(m,1H),1.39-1.35(m,1H).

[0325] Example 8

[0326]

[0327] Synthesis route:

[0328]

[0329] Step 1: Synthesis of compound WX008-2

[0330] Compound WX008-1 (100 g, 460.70 mmol) and acetyl chloride (54.25 g, 691.05 mmol, 49.31 mL) were dissolved in dichloromethane (1000 mL), purged with nitrogen for protection, and aluminum trichloride (92.15 g, 691.05 mmol, 37.76 mL) was added in portions at 20 °C. The reaction mixture was stirred for 2 hours. The reaction mixture was poured into ice-cold 6N hydrochloric acid (400 mL), extracted with dichloromethane (700 mL × 3), washed with saturated brine (700 mL × 3), dried over anhydrous sodium sulfate, filtered, and the crude product obtained by rotary evaporation was stirred with methanol (500 mL) for 15 minutes, filtered, and the filter cake was washed with methanol (100 mL × 2). The filter cake was collected to obtain compound WX008-2.

[0331] Step 2: Synthesis of compound WX008-3

[0332] WX008-2 (20 g, 77.19 mmol) was dissolved in dichloromethane (200 mL), cooled to -70 °C, and boron tribromide (17.40 g, 69.47 mmol, 6.69 mL) was added dropwise. The mixture was then slowly heated to 20 °C and stirred for 1 hour. The reaction solution was poured into 4N hydrochloric acid (100 mL), extracted with ethyl acetate (100 mL × 3), and the organic phase was washed with saturated brine (100 mL × 3). The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 7 / 1) to obtain compound WX008-3.

[0333] Step 3: Synthesis of compound WX008-4

[0334] Compound WX008-3 (13.84 g, 56.47 mmol) was dissolved in chloroform (70 mL) and ethyl acetate (70 mL), and copper bromide (25.23 g, 112.95 mmol, 5.29 mL) was added. The mixture was heated to 90 °C and stirred for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with dichloromethane (110 mL) to obtain a dichloromethane solution of compound WX008-4.

[0335] Step 4: Synthesis of compound WX008-5

[0336] At 0°C, triethylamine (11.43 g, 112.97 mmol, 15.72 mL) was slowly added dropwise to a dichloromethane solution of compound WX008-4. After the addition was complete, the temperature was raised to 20°C and stirred for 2 hours. The solution was washed with saturated ammonium chloride aqueous solution (100 mL × 3), dried over anhydrous sodium sulfate, and filtered to obtain a dichloromethane solution of compound WX008-5.

[0337] Step 5: Synthesis of compound WX008-6

[0338] Toluene (150 mL) and ethoxyformylmethylenetriphenylphosphine (23.62 g, 67.79 mmol) were added to a dichloromethane solution of WX008-5. The mixture was heated to 130 °C, and the low-boiling solvent was separated using a water separator. The mixture was stirred for 12 hours. After cooling to room temperature, the solution was concentrated directly under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain compound WX008-6. ¹H NMR (400 MHz, CDCl₃) δ: 7.70 (s, 1H), 7.60 (s, 1H), 7.05 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.67 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H).

[0339] Step 6: Synthesis of compound WX008-7

[0340] Compound WX008-6 (4 g, 12.77 mmol) was dissolved in dichloromethane (40 mL), cooled to -70 °C, and boron tribromide (3.36 g, 13.41 mmol, 1.29 mL) was added dropwise. The mixture was kept at this temperature and stirred for 0.5 hours, then slowly heated to 0 °C and stirred for 1 hour. The temperature was then lowered to -70 °C, and boron tribromide (3.36 g) was added again. The mixture was slowly heated to 0 °C and stirred for 1 hour. The reaction solution was poured into 1 N hydrochloric acid (50 mL), extracted with dichloromethane (50 mL × 3), and the organic phase was washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1) to obtain compound WX008-7.

[0341] Step 7: Synthesis of compound WX008-8

[0342] Compound WX008-7 (1.8 g, 6.02 mmol) and potassium carbonate (1.83 g, 13.24 mmol) were placed in N,N-dimethylformamide (10 mL), and 2-bromo-1,1-dimethoxyethane (1.22 g, 7.22 mmol, 847.58 μL) was added. The mixture was heated to 100 °C and stirred for 12 hours. The reaction solution was cooled to room temperature, and water (50 mL) was added. The mixture was extracted with ethyl acetate (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain compound WX008-8.

[0343] Step 8: Synthesis of compound WX008-9

[0344] Compound WX008-8 (1.84 g, 4.75 mmol) was placed in toluene (20 mL), and polyphosphoric acid (1.84 g, 8.74 mmol) was added. The mixture was heated to 100 °C and stirred for 12 hours. The reaction solution was cooled to room temperature, and water (30 mL) was added. The mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated sodium bicarbonate aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain compound WX008-9. 1 H NMR (400MHz, CDCl3) δ: 7.81 (d, J = 2.4Hz, 1H), 7.71 (m, 1H), 7.66 (s, 1H), 7.13 (d, J =2.0Hz, 1H), 4.20 (q, J = 7.2Hz, 2H), 3.85 (d, J = 0.8Hz, 2H), 1.25 (t, J = 7.2Hz, 3H).

[0345] Step 9: Synthesis of compound WX008-10

[0346] Compound WX008-9 (700 mg, 2.17 mmol), 2-di-tert-butylphosphine-2',4',6'-triisopropylbiphenyl (128.78 mg, 303.28 μmol), tert-butyl carbamate (304.53 mg, 2.60 mmol), potassium phosphate (1.84 g, 8.67 mmol), and tris(dibenzylacetone)palladium (138.86 mg, 151.64 μmol) were placed in toluene (10 mL) and water (2 mL), purged with nitrogen for protection, and heated to 100 °C with stirring for 6 hours. The reaction mixture was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Add n-heptane (10 mL) to the crude product and stir for 15 minutes. Filter the mixture and wash the filter cake with n-heptane (3 mL × 3). Collect the filter cake to obtain compound WX008-10.

[0347] Step 10: Synthesis of compound WX008-11

[0348] Compound WX008-10 (760 mg, 2.11 mmol) and acrylamide (165.35 mg, 2.33 mmol, 160.53 μL) were dissolved in N,N-dimethylformamide (20 mL). Potassium tert-butoxide (498.34 mg, 4.44 mmol) was added at 20 °C, and the mixture was stirred at 20 °C for 1 hour. The reaction solution was poured into 1N hydrochloric acid (20 mL) at 0-10 °C, extracted with ethyl acetate (20 mL × 3), and the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was separated by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound WX008-11.

[0349] Step 11: Synthesis of the hydrochloride salt of compound WX008-12

[0350] Compound WX008-11 (240 mg, 624.38 μmol) was placed in ethyl acetate (1 mL), and hydrochloric acid / ethyl acetate (4 M, 10 mL) was added. The mixture was stirred at 20 °C for 3 hours. The reaction solution was then concentrated under reduced pressure to obtain the hydrochloride salt of compound WX008-12. 1HNMR(400MHz,DMSO-d6)δ:10.94(s,1H),8.16(d,J=2.0Hz,1H),7.87(s,1H),7.26(s,1H),7.09(d,J=2.0Hz,1H ), 4.29 (dd, J = 4.8Hz, 12.0Hz, 1H), 2.86-2.77 (m, 1H), 2.68-2.58 (m, 1H), 2.34-2.22 (m, 1H), 2.19-2.14 (m, 1H).

[0351] Step 12: Synthesis of compound WX008

[0352] Compound BB-4 (125.29 mg, 211.07 μmol) and N,N-diisopropylethylamine (90.93 mg, 703.57 μmol) were dissolved in N,N-dimethylformamide (2 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (86.94 mg, 228.66 μmol) was added at 20 °C and the mixture was stirred for 5 minutes. Then, the hydrochloride salt of compound WX008-12 (50 mg, 175.89 μmol) was added at 20 °C and the mixture was stirred for 12 hours. The reaction solution was directly concentrated under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain compound WX008. 1 H NMR (400MHz, CD3OD) δ: 8.19 (s, 1H), 8.17-8.15 (m, 2H), 7.98 (dd, J = 1.6Hz, 8.0Hz, 1H), 7.91 (d, J =2.0Hz,1H),7.81(s,1H),7.40-7.35(m,1H),7.32(dd,J=2.4Hz,11.6Hz,1H),7.25-7.22(m,1H) ,7.02(d,J=2.0Hz,1H),4.60-4.58(m,2H),4.29(dd,J=5.2Hz,11.6Hz,1H),3.92(s,2H),3.77-3 .69(m,6H),3.42(m,4H),2.92-2.83(m,1H),2.79-2.73(m,1H),2.44-2.30(m,2H),1.57(s,6H).

[0353] Example 9

[0354]

[0355] Synthesis route:

[0356]

[0357] Synthesis of compound WX009

[0358] Compounds BB-4 (200 mg, 336.93 μmol) and the hydrochloride salt of BB-8 (133.73 mg, 404.32 μmol) were added to N,N-dimethylformamide (10 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (256.22 mg, 673.86 μmol) and N,N-diisopropylethylamine (130.64 mg, 1.01 mmol) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and stirred at 20 °C for 12 hours. The reaction solution was poured into 10 mL of water and 10 mL of ethyl acetate. The organic phase was separated and washed three times with water (10 mL × 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain compound WX009. 1 H NMR (400MHz, DMSO-d6) δ: 10.95 (s, 1H), 10.69-10.37 (m, 1H), 8.39 (d, J = 8.0Hz, 1H), 8.27 (d, J = 1. 5Hz,1H),8.17-7.97(m,4H),7.86(d,J=9.3Hz,1H),7.68-7.54(m,2H),7.49-7.33(m,2H),7.22(br d,J=8.0Hz,1H),4.70(br dd,J=4.3,11.8Hz,1H),4.66-4.46(m,2H),4.28-3.98(m,2H),3.81-3.61(m,10H),3.0 0-2.81(m,1H),2.72-2.60(m,1H),2.47-2.37(m,1H),2.35-2.21(m,1H),1.51(s,6H).

[0359] Example 10

[0360]

[0361] Synthesis route:

[0362]

[0363] Step 1: Synthesis of compound WX010-1

[0364] Compound BB-3-4 (2 g, 6.00 mmol) was added to a solvent of dioxane (20 mL) at room temperature. Stirring was started, and then compound pinacol diboronate (2.29 g, 9.00 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (439.23 mg, 600.28 μmol), and potassium acetate (1.18 g, 12.01 mmol) were added. The reaction mixture was heated to 100 °C and stirred for 12 hours under nitrogen protection. After the reaction was complete, the reaction mixture was cooled to room temperature and extracted with water (30 mL) and ethyl acetate (30 mL × 2). The combined organic phases were washed with semi-saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1 / 0 to 4 / 1) to obtain compound WX010-1. MS–ESI m / z: 380.9 [M+H] + . 1 H NMR(400MHz, CDCl3)δ:8.47(s,1H),8.19(d,J=8.5Hz,1H),7.94(d,J=8.3Hz,1H),7.83-7.75(m,2H),7.65 (d, J = 9.0Hz, 1H), 4.23 (q, J = 7.1Hz, 2H), 4.09-4.09 (m, 1H), 4.08 (s, 1H), 1.41 (s, 12H), 1.30-1.25 (m, 3H).

[0365] Step 2: Synthesis of compound WX010-2

[0366] Compound WX010-1 (2.3 g, 6.05 mmol) was added to a mixed solvent of tetrahydrofuran (40 mL) and water (20 mL) at room temperature. Stirring was started, and sodium bicarbonate (1.02 g, 12.10 mmol, 470.52 μL) was added. The reaction solution was cooled to 0 °C, and hydrogen peroxide (5.49 g, 48.39 mmol, 4.65 mL, 30% concentration) was slowly added dropwise to the reaction solution. Stirring continued for 2 hours under nitrogen protection. A saturated sodium sulfite aqueous solution (50 mL) was slowly added dropwise to the reaction solution under an ice bath. After the addition was complete, stirring continued for 10 minutes. Ethyl acetate (100 mL × 2) was added for extraction. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 8 / 1 to 4 / 1) to obtain compound WX010-2. 1H NMR (400MHz, CDCl3) δ: 8.13 (d, J = 8.9Hz, 1H), 7.74 (s, 1H), 7.64-7.51 (m, 2H), 7.29 (d, J = 2.6Hz, 1 H),7.18(dd,J=2.6,8.9Hz,1H),4.23(q,J=7.1Hz,2H),4.05(d,J=0.9Hz,2H),1.31-1.22(m,3H).

[0367] Step 3: Synthesis of compound WX010-3

[0368] Compound WX010-2 (400 mg, 1.33 mmol) and 1,5-dibromopentane (1.53 g, 6.64 mmol, 897.79 μL) were added to N,N-dimethylformamide (10 mL). Stirring was started, and potassium carbonate (550.41 mg, 3.98 mmol) and sodium iodide (198.99 mg, 1.33 mmol) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and stirred at 80 °C for 10 hours. The reaction solution was cooled to 25 °C, and ethyl acetate (30 mL) and water (30 mL) were added. The organic phase was separated and washed twice with saturated brine (30 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to obtain compound WX010-3. MS–ESI m / z: 418.9 [M+H] + . 1 H NMR (400MHz, CDCl3) δ: 8.06 (d, J = 9.0 Hz, 1H), 7.66 (s, 1H), 7.53 (s, 2H), 7.25-7.10 (m, 3H), 4.17-4.08 (m, 3H), 4.03 (t, J = 6.3 Hz,2H),3.96(s,2H),3.39(t,J=6.8Hz,2H),1.97-1.86(m,2H),1.85-1.77(m,2H),1.69-1.57(m,3H),1.18(t,J=7.2Hz,3H).

[0369] Step 4: Synthesis of compound WX010-4

[0370] Compound BB-10 (200 mg, 422.37 μmol) and WX010-3 (177.11 mg, 422.37 μmol, 72.61 μL) were added to N,N-dimethylformamide (5 mL). Stirring was started, and potassium carbonate (175.12 mg, 1.27 mmol) and potassium iodide (70.12 mg, 422.37 μmol) were slowly added to the reaction mixture. The reaction solution was purged with nitrogen three times and stirred at 80 °C for 12 hours. The reaction solution was cooled to room temperature and diluted with ethyl acetate (30 mL). The organic phase was washed twice with water (20 mL × 2), collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 10 / 1) to obtain compound WX010-4.

[0371] MS–ESI m / z: 812.3 [M+H] + .

[0372] Step 5: Synthesis of compound WX010

[0373] Compound WX010-4 (300 mg, 369.50 μmol) and acrylamide (26.26 mg, 369.50 μmol, 25.50 μL) were added to N,N-dimethylformamide (10 mL) at 0 °C. Potassium tert-butoxide (82.92 mg, 739.00 μmol) was slowly added to the reaction mixture in portions. After purging the mixture with nitrogen three times, the mixture was stirred at 0 °C for 1 hour. The reaction mixture was then slowly added to saturated ammonium chloride (50 mL), and extracted twice with ethyl acetate (50 mL × 2). The combined organic phases were washed three times with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain compound WX010. MS–ESI m / z: 837.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ: 11.23 (br s,1H),10.95(s,1H),8.39(d,J=8.3Hz,1H),8.30(d,J=1.3Hz,1H),8.15-8.04(m,2H),7.98(d,J=6.8H z,1H),7.82-7.68(m,2H),7.53(d,J=2.3Hz,1H),7.34-7.20(m,3H),7.14(brd,J=9.0Hz,2H),4.65(br dd,J=4.1,11.9Hz,1H),4.18-4.09(m,2H),4.03(s,1H),3.93(br s,2H),3.60(br d,J=11.3Hz,2H),3.38-3.23(m,2H),3.22-3.04(m,4H),2.98-2.79(m,1H),2.71-2.53(m,1H),2.41(br dd,J=3.9,12.7Hz,1H),2.31-2.17(m,1H),1.95-1.76(m,4H),1.64-1.43(m,8H).

[0374] Example 11

[0375]

[0376] Synthesis route:

[0377]

[0378] Step 1: Synthesis of compound WX011-1

[0379] Compound BB-4-3 (1 g, 2.36 mmol) was dissolved in N,N-dimethylformamide (30 mL), and potassium carbonate (652.86 mg, 4.72 mmol) and 2,2'-dibromodiethyl ether (2.74 g, 11.81 mmol, 1.48 mL) were added. The mixture was heated to 80 °C and stirred for 30 hours. Water (150 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Compound WX011-1 was obtained by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1).

[0380] Step 2: Synthesis of compound WX011-2

[0381] Ethyl 1-aminocyclopropanecarboxylate hydrochloride (346.01 mg, 2.09 mmol) and potassium carbonate (577.48 mg, 4.18 mmol) were dissolved in N,N-dimethylformamide (10 mL). Compound WX011-1 (0.6 g, 1.04 mmol) was added, and the mixture was heated to 75 °C and stirred for 15 hours. Water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL × 2). The organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1) to give compound WX011-2.

[0382] Step 3: Synthesis of compound WX011-3

[0383] Compound WX011-2 (0.2 g, 321.22 μmol) was dissolved in a mixed solvent of tetrahydrofuran (4 mL) and water (1 mL). Lithium hydroxide monohydrate (67.40 mg, 1.61 mmol) was added at 20 °C, and the mixture was stirred for 30 hours. Water (5 mL) was added to the reaction solution, and the pH was adjusted to 5 with 1 N dilute hydrochloric acid. The mixture was extracted with ethyl acetate (10 mL × 4). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound WX011-3.

[0384] Step 4: Synthesis of compound WX011

[0385] Compound WX011-3 (80 mg, 134.55 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (76.74 mg, 201.82 μmol) and N,N-diisopropylethylamine (69.56 mg, 538.20 μmol, 93.74 μL) were added. The mixture was stirred for 10 minutes, and then the hydrochloride salt of compound BB-1 (49.10 mg, 174.91 μmol) was added. The mixture was stirred at 20 °C for 15 hours. The reaction solution was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain compound WX011. 1H NMR(400MHz, CD3OD)δ:8.16-8.14(m,2H),7.95(dd,J=1.6Hz,8.4Hz,1H),7.91(d,J=1.6Hz,1H),7 .71(s,1H),7.50(d,J=8.8Hz,1H),7.30-7.22(m,3H),7.16-7.14(m,1H),4.35(t,J=4.0Hz,2H),4 .10(dd,J=4.2Hz,11.6Hz,1H),3.99-3.97(m,2H),3.89(t,J=2.8Hz,2H),3.47(s,2H),2.83-2.66 (m,2H),2.42-2.32(m,1H),2.29-2.22(m,1H),1.81-1.78(m,2H),1.65-1.63(m,2H),1.54(s,9H).

[0386] Example 12

[0387]

[0388] Synthesis route:

[0389]

[0390] Step 1: Synthesis of compound WX012-1

[0391] Compound BB-10 and ethyl 5-bromopentanoate (662.33 mg, 3.17 mmol, 505.59 μL) were added to N,N-dimethylformamide (5 mL). Stirring was started, and potassium carbonate (262.69 mg, 1.90 mmol) and sodium iodide (94.97 mg, 633.56 μmol) were slowly added to the reaction mixture. The reaction solution was purged with nitrogen three times and stirred at 80 °C for 12 hours. After the reaction was complete, the mixture was cooled to 25 °C, diluted with ethyl acetate (20 mL), washed twice with water (20 mL × 2), and once with saturated brine (20 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 10 / 1) to obtain compound WX012-1. MS–ESI m / z: 602.1 [M+H] + .

[0392] Step 2: Synthesis of compound WX012-2

[0393] Compound WX012-1 (170 mg, 282.54 μmol) was added to anhydrous ethanol (2.5 mL) and water (0.5 mL). Stirring was started, and lithium hydroxide monohydrate (29.64 mg, 706.35 μmol) was slowly added to the reaction mixture. The reaction solution was purged with nitrogen three times and then stirred at 25 °C for 12 hours. After the reaction was complete, water (10 mL) was added to the reaction solution, and most of the organic solvent was removed by rotary evaporation under reduced pressure. The aqueous phase was adjusted to pH 3 with 2N hydrochloric acid and then lyophilized directly to obtain compound WX012-2. MS–ESI m / z: 574.0 [M+H] + .

[0394] Step 3: Synthesis of the hydrochloride salt of compound WX012

[0395] The hydrochloride salt of compound BB-3 (54.30 mg, 139.46 μmol) was added to N,N-dimethylformamide (5 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (165.71 mg, 435.82 μmol) and triethylamine (52.92 mg, 522.99 μmol, 72.79 μL) were slowly added to the reaction mixture. After purging the mixture three times with nitrogen, the mixture was stirred at 25 °C for 3 hours. The reaction mixture was diluted with ethyl acetate (30 mL), and the organic phase was washed twice with water (20 mL × 2). The collected organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product was evaporated under reduced pressure. The hydrochloride salt of compound WX012 was obtained by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl). MS–ESI m / z: 850.4 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ:10.95(s,1H),10.54(br d,J=4.5Hz,1H),10.29(s,1H),8.48-8.35(m,2H),8.29(d,J=1.3Hz,1H),8.18-8.04(m, 2H),7.99(s,1H),7.79-7.69(m,3H),7.28-7.21(m,2H),7.14(d,J=9.0Hz,2H),4.65(br dd,J=4.4,12.2Hz,1H),3.94(br d,J=11.3Hz,2H),3.62(br d,J=10.8Hz,2H),3.28-3.07(m,6H),2.95-2.80(m,1H),2.69-2.59(m,1H),2.49-2 .36(m,3H),2.33-2.22(m,1H),1.92-1.77(m,2H),1.76-1.65(m,2H),1.49(s,6H).

[0396] Example 13

[0397]

[0398] Synthesis route:

[0399]

[0400] Step 1: Synthesis of compound WX013-1

[0401] Compound BB-10 (0.15 g, 284.79 μmol) and ethyl bromide propionate (103.11 mg, 569.57 μmol, 72.61 μL) were added to acetonitrile (5 mL), and stirring was started. Potassium carbonate (78.72 mg, 569.57 μmol) and potassium iodide (47.28 mg, 284.79 μmol) were slowly added to the reaction mixture. The reaction solution was purged with nitrogen three times and stirred at 80 °C for 2 hours. The reaction solution was cooled to room temperature, and ethyl acetate (50 mL) and water (50 mL) were added. The organic phase was separated and washed twice with saturated brine (30 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 50 / 1–10 / 1, v / v) to obtain compound WX013-1. MS–ESI m / z: 574.0 [M+H] + .

[0402] Step 2: Synthesis of compound WX013-2

[0403] Compound WX013-1 (0.2 g, 348.66 μmol) was added to anhydrous ethanol (4 mL) and water (1 mL). Stirring was started, and lithium hydroxide monohydrate (43.89 mg, 1.05 mmol) was slowly added to the reaction mixture. The reaction solution was purged with nitrogen three times and then stirred at 25 °C for 12 hours. Most of the ethanol was removed under reduced pressure, and 20 mL of water was added. The aqueous phase was adjusted to pH 3-4 with 2N hydrochloric acid and then lyophilized directly to obtain compound WX013-2. MS–ESI m / z: 546.0 [M+H] + .

[0404] Step 3: Synthesis of the hydrochloride salt of compound WX013

[0405] Compound WX013-2 (60 mg, 109.98 μmol) and the hydrochloride salt of BB-3 were added to N,N-dimethylformamide (3 mL). 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (83.63 mg, 219.95 μmol) and N,N-diisopropylethylamine (42.64 mg, 329.93 μmol, 57.47 μL) were slowly added to the reaction mixture. The mixture was purged with nitrogen three times and then stirred at 20 °C for 12 hours. After the reaction was complete, the reaction solution was poured into a mixed solvent of water and ethyl acetate (10 mL:10 mL). The organic phase was separated and washed three times with water (10 mL × 3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the crude product was evaporated under reduced pressure. The crude product was then purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain the hydrochloride salt of compound WX013. MS–ESI m / z: 822.3 [M+H] + .

[0406] 1 H NMR(400MHz,DMSO-d6)δ:10.95(s,1H),10.54(br s,1H),10.46(br s,1H),8.47-8.35(m,2H),8.29(br s,1H),8.16(br s,1H),8.08(br d,J=8.5Hz,1H),8.00(s,1H),7.80-7.67(m,3H),7.34-7.20(m,2H),7.16(br d,J=8.8Hz,2H),4.68(br s,1H),3.98(m,2H),3.64(m,2H),3.55(m,4H),3.23(m,2H),3.05(br s,2H),2.86(m,1H),2.67(m,1H),2.63-2.62(m,1H),2.41-2.33(m,1H),1.49(br s,6H).

[0407] Example 14

[0408]

[0409] Synthesis route:

[0410]

[0411] Synthesis of hydrochloride of compound WX014

[0412] Compound BB-11 (400 mg, 854.80 μmol) and the hydrochloride salt of compound BB-12 (403.83 mg, 1.11 mmol) were dissolved in dichloromethane (30 mL), and sodium borohydride acetate (543.50 mg, 2.56 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours under nitrogen protection. After the reaction was complete, the reaction solution was diluted with dichloromethane (100 mL) and water (100 mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to obtain the hydrochloride salt of compound WX014. MS–ESI m / z: 815.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 10.97 (s, 1H), 10.83 (br s, 1H), 8.63 (d, J = 8.0Hz, 1H), 8.05 (br d,J=8.5Hz,1H),7.96(s,1H),7.91-7.84(m,2H),7.73(s,2H),7.54-7.43(m,3H),7.41-7.38(m,1H),7.16-7.11(m,1H),4.63(br dd,J=4.0,12.0Hz,1H),4.58-4.46(m,3H),3.98-3.81(m,3H),3.66(br d,J=11.0Hz,2H),3.43(br t,J=12.2Hz,2H),3.28-3.05(m,6H),2.96-2.83(m,1H),2.76-2.58(m,2H),2.56-2.53(m,1H),2.46-2.37(m,1H),2.31-2.21(m,2H),2.11(br d,J=9.0Hz,2H),2.02(br d,J=12.0Hz,2H),1.90(br d,J=10.5Hz,2H),1.72-1.58(m,2H),1.58-1.45(m,2H).

[0413] Example 15

[0414]

[0415] Synthesis route:

[0416]

[0417] Synthesis of hydrochloride of compound WX015

[0418] Compound BB-11 (400 mg, 854.80 μmol) and the hydrochloride salt of compound BB-13 (403.83 mg, 1.11 mmol) were dissolved in dichloromethane (30 mL), and sodium borohydride acetate (181.17 mg, 854.80 μmol) was added. The reaction mixture was stirred at room temperature for 12 hours under nitrogen protection. After the reaction was complete, the reaction solution was diluted with dichloromethane (100 mL) and water (100 mL), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.05% HCl) to give the hydrochloride salt of compound WX015. MS–ESI m / z: 815.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ: 10.96 (s, 1H), 10.82 (br s, 1H), 8.63 (d, J = 8.3Hz, 1H), 8.17 (d, J = 9.3Hz, 1H), 8.03 (s, 1H), 7.96 (br d,J=5.8Hz,1H),7.86(dt,J=9.4,15.6Hz,3H),7.60-7.51(m,2H),7.39(d,J=2.5Hz,1H),7.27-7.22(m,1H),7.14(dd,J=2.3,8.8Hz,1H),4.68(br dd,J=3.9,11.9Hz,1H),4.58-4.47(m,3H),3.92-3.81(m,1H),3.69(br s,3H),3.50-3.38(m,3H),3.23-3.06(m,4H),2.98-2.81(m,1H),2.70-2.58(m,1H),2.45-2.20(m,4H),2.16-1.99(m,5H),1.91(br d,J=10.3Hz,2H),1.71-1.58(m,2H),1.57-1.42(m,2H),1.40-1.27(m,2H).

[0419] Examples 16 and 17

[0420]

[0421] Synthesis route:

[0422]

[0423] Synthesis of Compounds 16 and 17

[0424] Compound BB-11 (7 g, 14.96 mmol) and the hydrochloride salt of compound BB-13 (7.18 g, 17.95 mmol) were dissolved in dichloromethane (500 mL) and stirred at room temperature for 12 hours. Sodium borohydride acetate (4.76 g, 22.44 mmol) was added, and the reaction mixture was stirred at room temperature for 12 hours under nitrogen protection. After the reaction was complete, water (200 mL) was added to dilute the reaction solution, the organic phase was separated, washed with saturated brine (100 mL), dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was separated by column chromatography (eluent: dichloromethane:methanol = 10 / 1) to obtain compound WX015. Compound WX015 was then separated by chiral HPLC (column type: DAICL CHIRALPAK IE (250 mm * 30 mm, 10 μm); mobile phase: A (IPA), B (ACN), B%: 50%–100%; flow rate: 80 mL / min) to obtain compounds WX016 and WX017.

[0425] Analytical methods: Column: Chiralpak IA 100*4.6mm, 3μm; Mobile phase: A: n-hexane (0.1% diethylamine); B: isopropanol:acetonitrile = 2:1, A:B = 40:60; Column temperature: 35℃; Wavelength: 220nm.

[0426] WX016: Retention time 4.679 min, ee% 98.77%; MS–ESI m / z 815.4 [M+H] + . 1 H NMR(400MHz, DMSO-d6)δ:10.97(s,1H),8.60(d,J=8.3Hz,1H),8.16(d,J=9.3Hz,1H),7.99(s, 1H),7.91-7.74(m,4H),7.51(t,J=7.9Hz,1H),7.41-7.31(m,2H),7.21-7.10(m,2H),4.66(br dd,J=4.1,11.7Hz,1H),4.58-4.45(m,3H),3.94-3.79(m,1H),3.223.00(m,7H),2 .89(ddd,J=5.3,12.2,17.4Hz,2H),2.69-2.56(m,2H),2.47-2.33(m,2H),2.29(br d,J=7.0Hz,3H),2.11(br d,J=9.3Hz,2H),2.00-1.81(m,5H),1.72-1.58(m,2H),1.58-1.44(m,2H),1.26-1.09(m,2H).

[0427] WX017: Retention time 5.797 min, ee% 99.86%; MS–ESI m / z 815.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ = 11.00 (br s, 1H), 8.60 (br d,J=8.3Hz,1H),8.22-8.13(m,1H),8.04-7.95(m,1H),7.94-7.71(m,4H),7.57-7.47(m,1H),7.41-7.31(m,2H),7.16(br dd,J=8.0,19.1Hz,2H),4.66(br d,J=7.3Hz,1H),4.58-4.40(m,3H),3.87(br d,J=7.8Hz,1H),3.28-2.97(m,7H),2.88(br t,J=12.3Hz,2H),2.69-2.56(m,2H),2.49-2.35(m,2H),2.28(br d,J=6.0Hz,3H),2.17-2.00(m,2H),2.00-1.79(m,5H),1.71-1.58(m,2H),1.51(q,J=10.6Hz,2H),1.26-1.01(m,2H).

[0428] Biological data

[0429] Test Example 1: In vitro assay of AR protein levels and downstream effector prostate-specific antigen (PSA) protein regulation in human prostate cancer LNCaP cells.

[0430] Experimental objective: To investigate the regulation of AR protein levels and downstream effector prostate-specific antigen (PSA) protein in human prostate cancer LNCaP cells under different concentration conditions using Western blotting (WB).

[0431] Experimental plan:

[0432] 1) Thaw and passage LNCaP cells at least twice;

[0433] 2) LNCaP cells were seeded at 3 × 10⁵ cells per well in 6-well plates, and after overnight adhesion, they were treated with a certain concentration of the test compound.

[0434] 3) After 24 hours of treatment, discard the supernatant of the cultured cell sample, wash twice with DPBS (Dubor's phosphate buffer), then lyse the cells with a certain amount of 2% SDS lysis buffer preheated to 100°C, collect the cells and denature them at 100°C for 15 minutes.

[0435] 4) After the above lysis buffer is denatured and cooled, protein quantification test is performed (Pierce BCA protein assay kit, Thermo). Then, the volume is adjusted to the same protein concentration with 5 times loading buffer (containing dithiothreitol (DDT), Beyotime), and then reduced and denatured at 100℃ for 10 minutes.

[0436] 5) Separate the above samples (10-20 μg protein) by SDS-PAGE and transfer them onto a PVDF membrane (Biorad);

[0437] 6) Cut the bands according to the molecular weight of the target protein, block with blocking buffer (3% bovine serum albumin TBS-T solution, where TBS-T solution is Tris-HCl buffer containing 0.2% Tween-20) for 1 hour, and then incubate with primary antibodies (anti-AR (#5153, CST), anti-PSA / KLK3 (#5365, CST) and anti-β-actin (#4970, CST), which are prepared by diluting with blocking buffer at 1:1000, 1:1000 and 1:2000 respectively) at 4°C overnight;

[0438] 7) Finally, incubate the membrane with HRP-linked secondary antibody (anti-rabbit IgG (#7074, CST, prepared by diluting blocking buffer at 1:2000) at room temperature for 1 hour, and then detect the bands on the membrane with chemiluminescent substrate (Clarity ECL, Biorad).

[0439] Experimental results:

[0440] Test results are as follows Figure 1 , Figure 2 As shown.

[0441] Conclusion: The compounds of this invention exhibit good degradation activity against AR proteins, and their degradation ability shows a good concentration-dependent effect. The compounds of this invention can significantly affect the corresponding downstream PSA effect, and the degree of influence shows a good concentration-dependent effect.

[0442] Test Example 2: Evaluation of Antiproliferative Effect in Human Prostate Cancer LNCaP Cells

[0443] Experimental Objective

[0444] This experiment investigated the inhibitory effect of the test compound on cell proliferation in human prostate cancer LNCaP cells.

[0445] Experimental materials:

[0446] 1. Cell lines and culture methods

[0447] Table 1. Cell lines and culture methods

[0448] LNCaP Human prostate cancer Adhesive Phenol Red 1640 + 10% FBS

[0449] 2. Culture media and reagents

[0450] Table 2. Culture media and reagents

[0451] Phenol Red-Free RPMI 1640 GIBCO 11835030 Dulbecco's PBS CORNING 21-031-CVC FBS ExCell Bio FSP500 Penicillin-Streptomycin Solution HyClone SV30010 0.25% Trypsin GIBCO 25200072

[0452] 3. Perforated plate

[0453] Greiner 96-Perforated plate, flat-bottomed blackboard (with cover and transparent bottom), #655090.

[0454] 4. Reagents and instruments used in cell viability experiments

[0455] (1) Promega CellTiter-Glo luminescent cell viability assay kit (Promega-G7573).

[0456] (2)2104 PerkinElmer, a board reader.

[0457] Experimental protocol

[0458] 1. Cell Culture

[0459] The tumor cell lines were cultured under the conditions described above in an incubator at 37°C and 5% CO2. Cells were passaged periodically, and cells in the logarithmic growth phase were used for plating.

[0460] 2. Cell plating

[0461] (1) Stain cells with trypan blue and count viable cells;

[0462] (2) Adjust the cell concentration to a suitable level;

[0463] Table 3. Cell Plating Number

[0464] LNCaP 4500

[0465] (3). Add 90 μL of cell suspension to each well of the culture plate as shown in the table above, and add cell-free culture medium to the blank control well;

[0466] (4) Incubate the culture plate overnight in an incubator at 37°C, 5% CO2 and 100% relative humidity.

[0467] 3. Preparation of compound storage plates

[0468] To prepare a stock solution storage plate with an initial concentration 400 times that of the compound: dilute the compound with DMSO from the highest concentration to the lowest concentration. Prepare fresh solution each time you need to use it.

[0469] Preparation of working solution with a starting concentration of 10 times the compound concentration and cell treatment with the compound.

[0470] (1) Add 78 μL of cell culture medium to a 96-well plate with a V-bottom, and take 2 μL of the stock solution from a storage plate containing 400 times the initial concentration of the compound.

[0471] Add μL of the compound to the cell culture medium in a 96-well plate. Add 2 μL of LDMSO to the solvent control and blank control. Add the compound or DMSO.

[0472] Then use a blower to mix it thoroughly.

[0473] (2) Drug addition: Add 10 μL of the working solution (10 times the initial concentration of the compound) to the cell culture plate. (Compare the solvent control and blank control.)

[0474] Add 10 μL of DMSO-cell culture medium mixture to the control group.

[0475] (3) Place the 96-well cell plate back into the incubator and incubate for 6 days.

[0476] 5. CellTiter–Glo luminescence assay for cell viability detection

[0477] The following steps were performed in accordance with the instructions for the Promega CellTiter–Glo luminescence assay kit (Promega–G7573).

[0478] (1). Melt the CellTiter–Glo buffer and let it reach room temperature;

[0479] (2). Allow the CellTiter–Glo substrate to reach room temperature;

[0480] (3) Add 100 mL of CellTiter–Glo buffer to a bottle of CellTiter–Glo substrate to dissolve the substrate, thereby preparing CellTiter–Glo working solution;

[0481] (4) Slow vortexing ensures complete dissolution;

[0482] (5) Remove the cell culture plate and let it equilibrate to room temperature for 30 minutes;

[0483] (6) Add 50 μL of CellTiter-Glo working solution (equivalent to half the volume of cell culture medium in each well) to each well. Wrap the cell plate with aluminum foil to protect it from light;

[0484] (7) Shake the culture plate on a track shaker for 2 minutes to induce cell lysis;

[0485] (8) The culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal;

[0486] (9) Detect the light emission signal on the 2104EnVision reader.

[0487] 6. Data Analysis

[0488] The inhibition rate (IR) of the detected compound is calculated using the following formula: IR (%) = (RLU solvent control – RLU compound) / (RLU solvent control – RLU blank control) * 100%. The inhibition rates of different concentrations of the compound are calculated in Excel, and then GraphPad Prism software is used to create inhibition curves and calculate relevant parameters, including minimum inhibition rate, maximum inhibition rate, and IC50.

[0489] Experimental results:

[0490] The test results are shown in Table 4.

[0491] Table 4. Inhibitory effect of the compounds of the present invention on cell proliferation in LNCaP cell line.

[0492] compound LNCaP IC50(nM)

[0493] WX002 46 WX003 230 WX004 25 WX005 190 WX015 hydrochloride 45 WX016 31 WX017 44

[0494] Conclusion: The compounds of this invention exhibit excellent inhibitory effects on cell proliferation in human prostate cancer cells LNCaP.

[0495] Test Example 3: In vivo pharmacodynamic study of a human prostate cancer LNCaP cell subcutaneous xenograft tumor CB-17 SCID model

[0496] Cell culture

[0497] Human prostate cancer LNcap cells (ECACC-89110211) were cultured in vitro as a monolayer under the following conditions: RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested, counted, and seeded.

[0498] laboratory animals

[0499] CB-17 SCID mice, 6-8 weeks old, male, weighing 18-22 grams.

[0500] Experimental protocol

[0501] 0.2 mL (1×10⁷ cells) of LNcap cells (with matrix gel, volume ratio 1:1) were subcutaneously inoculated into the right posterior back of each mouse. When the average tumor volume reached approximately 80 mm³, the mice were castrated. When the tumor volume reached 166 mm³, grouping and drug administration began. The experimental animal grouping and drug administration regimens are shown in Table 5.

[0502] Table 5. Grouping and Dosing Regimens of Experimental Animals

[0503]

[0504] Note:

[0505] 1. N: Number of mice in each group

[0506] 2. Dosage volume: 10 μL / g based on mouse body weight. If body weight decreases by more than 15%, the dosing regimen should be adjusted accordingly.

[0507] 3. The BID time interval is 8 hours.

[0508] Tumor measurements and experimental indicators

[0509] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5a × b², where a and b represent the major and minor diameters of the tumor, respectively.

[0510] The antitumor efficacy of the compound was evaluated using TGI (%). TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0511] Experimental results

[0512] The test results are shown in Table 6.

[0513] Table 6. Antitumor effects of compounds on LNCaP xenograft tumor models

[0514]

[0515] Conclusion: The compounds of this invention exhibit significant tumor-suppressive effects in a human prostate cancer LNCaP xenograft model.

[0516] Test Example 4: In vitro test of the regulation of AR protein levels in human prostate cancer LNCaP cells

[0517] Experimental objective: To test the degradation effect of a compound on the androgen receptor AR in LNCap cells using the ICW method.

[0518] Experimental methods:

[0519] Day 1

[0520] 1. Preheat the cell culture medium, 0.025% trypsin and phosphate buffer in a 37°C water bath.

[0521] 2. After tryingptin digestion, centrifuge the cells at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in fresh culture medium.

[0522] 3. Seed cells at a density of 4kJ / 36μL / well in COL-Coated CellCarrier-384 plates.

[0523] 4. Place the 384 cell plate inoculated with cells in a clean bench and let it stand for 10 minutes.

[0524] 5. Place the cell culture plate in a carbon dioxide cell culture incubator and incubate overnight.

[0525] the next day

[0526] 1. Dilute the 10 mM compound to 3 mM (3 μL + 7 μL dimethyl sulfoxide) before serial dilution.

[0527] 2. The compound was serially diluted 3-fold (4 μL + 8 μL) in Echo plates using a Bravo instrument.

[0528] 3. Set up ZPE and HPE control wells, with dimethyl sulfoxide as the ZPE control well.

[0529] 4. After centrifuging the Echo plate at 1000 rpm for 1 minute, transfer the diluted compound from the Echo plate to the intermediate plate using the Echo, 200 nL / well.

[0530] 5. Add cell culture medium to the intermediate plate using Multidrop combi, 20 μL / well.

[0531] 6. Place the intermediate plate on a horizontal shaker and shake for 1 minute to ensure the compound is thoroughly mixed with the culture medium.

[0532] 7. Use Bravo to transfer the compound from the intermediate dilution plate to the cell plate, 4 μL / well.

[0533] 8. Place the cell plate on a horizontal shaker and shake for 1 minute, then centrifuge at 800 rpm for 30 seconds.

[0534] 9. Place the cell plate back into the CO2 incubator and continue culturing for 24 hours.

[0535] Day 3

[0536] 1. Remove 4% of paraformaldehyde from the refrigerator and let it reach room temperature.

[0537] 2. Remove the cell plate from the incubator.

[0538] 3. Add 4% paraformaldehyde directly to the cell plate, 40 μL / well.

[0539] 4. Incubate at room temperature for 20 minutes.

[0540] 5. Manually shake off the culture medium from the cell plate, invert it into a centrifuge, and centrifuge at 800 rpm for 7 seconds to thoroughly remove any residual culture medium.

[0541] 6. Add 4% paraformaldehyde to the cell plate, 25 μL / well.

[0542] 7. Incubate at room temperature for 30 minutes.

[0543] 8. Manually remove the paraformaldehyde solution from the cell plate, invert it into a centrifuge, and centrifuge at 800 rpm for 7 seconds.

[0544] 9. Add 0.1% Tritium, 25 μL / well.

[0545] 10. Incubate at room temperature for 10 minutes.

[0546] 11. Manually remove the 0.1% Tritium solution from the cell plate, invert it into a centrifuge, and centrifuge at 800 rpm for 7 seconds.

[0547] 12. Add (PBS)Blocking Buffer, 25μL / well.

[0548] 13. Incubate at room temperature for 1 hour.

[0549] 14. Manually shake off the cells from the cell plate. (PBS) Blocking Buffer solution, inverted in a centrifuge, centrifuged at 800 rpm for 7 seconds.

[0550] 15. Add primary antibody solution, 20 μL / well. Place the cell plate in a centrifuge and centrifuge at 800 rpm for 7 seconds.

[0551] 16. After sealing the cell plate, incubate it overnight at 4°C.

[0552] Day 4

[0553] 1. Manually remove the primary antibody solution from the cell plate, invert it into a centrifuge, and centrifuge at 800 rpm for 7 seconds.

[0554] 2. Wash 3 times with 110 μL PBS.

[0555] 3. Invert the cell plate in a centrifuge and centrifuge at 800 rpm for 7 seconds.

[0556] 4. Add secondary antibody and DAPI solution, 20 μL / well. Place the cell plate in a centrifuge and centrifuge at 800 rpm for 7 seconds.

[0557] 5. Incubate at room temperature for 1 hour.

[0558] 6. Manually remove the secondary antibody and DAPI solution from the cell plate, invert it into a centrifuge, and centrifuge at 800 rpm for 7 seconds.

[0559] 7. After washing three times with 110 μL PBS, 30 μL of PBS remained in the cell plate.

[0560] 8. Place the cell plate in a centrifuge and centrifuge at 800 rpm for 7 seconds.

[0561] 9. The Operetta instrument scans the cell plate.

[0562] Experimental results

[0563] The test results are shown in Table 4.

[0564] Table 7. Degradation of androgen receptor AR in LNCap cells by compounds

[0565] 1 WX002 8.9 72.01 2 WX009 10.1 64.66 3 WX014 hydrochloride 22.4 76.22 4 WX015 hydrochloride 13.7 102.09 5 WX016 14.0 95.66 6 WX017 15.1 100.32

[0566] Conclusion: The compounds of this invention exhibit good degradation activity against AR.

[0567] Test Example 5: In-cell Western blotting analysis of AR protein expression levels in 293T AR F876L cells

[0568] Experimental Objective: This experiment used In Cell Western blotting to detect the effects of compounds WX015, WX016, and WX017 on the expression level of point-mutated AR protein in cell line 293T AR F876L, and to evaluate the degradation effect of the compounds on AR F876L point-mutated protein.

[0569] Experimental materials:

[0570] 1. Cell lines and culture methods

[0571] Table 8. Cell lines and culture methods

[0572] 293T AR F876L Embryo kidney cell Adhesive growth DMEM + 10% FBS + 1% AA

[0573] 2. Culture media and reagents

[0574] Table 9. Culture media and reagents

[0575] DMEM GIBCO 11995-065 PBS Cytiva SH30256.01 FBS Hyclone SH30084.03 Antibiotic-antimycotic GIBCO 15240-062 0.25% Trypsin GIBCO 25200072

[0576] DMSO SIGMA D2650

[0577] 3. Instruments

[0578] Table 10. Instruments

[0579] Odyssey Gel Imaging System LI-COR CLx CO2 incubator Thermo Scientific Heracell 2401 Automated cell counter Countstar IC-1000 Inverted microscope Nikon ECLIPSE Ts2 Biosafety cabinet Su Jing An Tai BSC-1600 A3 Bleaching Shaker Hm-Kylin TS-1

[0580] 4. Antibodies

[0581] Table 11. Antibodies

[0582]

[0583] Experimental methods:

[0584] 1. Cell culture:

[0585] The cell lines were cultured in an incubator at 37°C and 5% CO2. Cells were passaged periodically, and cells in the logarithmic growth phase were used for plating.

[0586] 2. Cell plating:

[0587] 1) Use trypan blue to stain cells and count live cells.

[0588] 2) Adjust the cell concentration to a suitable level, with a density of 20,000 cells / well.

[0589] 3) Add 90 μL of cell suspension to each well of a 96-well culture plate, and add cell-free culture medium to the blank control well.

[0590] 4) Incubate the culture plates overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0591] 3. Compound storage plate preparation and In-cell Western spectroscopy:

[0592] 1) Preparation of 400x compound storage plates: The compound was serially diluted with DMSO from the highest concentration to the lowest concentration.

[0593] 2) Preparation of 10-fold compound working solution: Add 78 μL of cell culture medium to a 96-well plate with a V-bottom. Add 2 μL of the compound from a 400X compound storage plate to the cell culture medium in the 96-well plate. Add 2 μL of DMSO to the solvent control and blank control. After adding the compound or DMSO, mix thoroughly by pipetting.

[0594] 3) Drug addition: Add 10 μL of the 10-fold working solution of the compound to the cell culture plate. Add 10 μL of DMSO-cell culture medium mixture to the solvent control and blank control. The final concentration of DMSO is 0.25%.

[0595] 4) Place the 96-well cell plate back into the incubator and incubate for 48 hours.

[0596] 5) Remove the culture medium and wash once with PBS;

[0597] 6) Add 8% formalin solution (diluted with PBS) and incubate overnight at 4°C.

[0598] 7) Remove the 8% formalin solution, add 150 μL of Licor Blocking buffer to each well, and place in a shaker at room temperature for 1.5 h to seal;

[0599] 8) Discard the blocking solution, add 50 μL of primary antibody diluted with Licor Blocking buffer to each well (do not add primary antibody to DMSO wells), incubate overnight at 4°C, and then wash 5 times with PBST (containing 0.1% Triton-X) for 5 min each time;

[0600] 9) Add 50 μL of secondary antibody diluted with Licor Blocking buffer to each well, incubate at room temperature for 1 h, wash 3 times with PBST (containing 0.1% Triton-X), wash 2 times with ddH2O, and then add 100 μL of PBS to each well for detection.

[0601] 10) Detection: Pour off the PBS from the well plate and use a Li-COR Odyssey dual-color near-infrared laser imager to detect the fluorescence signal at 700 nm in each well.

[0602] 4. Data Analysis

[0603] The density and intensity of the immunoblot chemiluminescence bands were relatively quantified using Image Studio Ver 5.2 software.

[0604] Experimental results

[0605] The test results are shown in Table 12.

[0606] Table 12. Degradation of androgen receptor AR by compounds in 293T AR F876L cells

[0607] 1 WX015 hydrochloride 43 85 2 WX016 18 86 3 WX017 97 95

[0608] Conclusion: The compounds of this invention have a strong ability to degrade the F876L point mutant AR protein.

[0609] Test Example 6: Pharmacokinetic Evaluation of Compounds in Mice

[0610] Experimental objective:

[0611] In this study, CB-17 SCID male mice were used as test compounds. LCMS / MS was employed to quantitatively determine the plasma drug concentrations of the test and reference compounds at different time points after intravenous or oral administration to evaluate the pharmacokinetic characteristics of the test drugs in mice. Experimental materials:

[0612] CB-17 SCID mice (male, 20-30g, 7-10 weeks old, from Vital Rivers Beijing or Slack Shanghai).

[0613] Experimental procedure:

[0614] The clear or suspension solution of the test compound was injected into mice via the tail vein (without fasting) or administered via gavage (without fasting). For intravenous administration, blood was collected by jugular vein puncture at 0 h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration. The blood was placed in anticoagulant tubes supplemented with EDTA-K2, and the mixture was thoroughly vortexed and centrifuged at 13,000 rpm for 10 minutes at 4°C. For oral gavage administration, blood was collected by jugular vein puncture at 0 h (before administration) and 0.5, 1, 2, 4, 6, 8, and 24 h after administration. The blood was placed in anticoagulant tubes supplemented with EDTA-K2, and the mixture was thoroughly vortexed and centrifuged at 13,000 rpm for 10 minutes. Blood drug concentrations were determined by LC-MS / MS. Relevant pharmacokinetic parameters were calculated using the WinNonlin™ Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software with the non-compartmental linear logarithmic trapezoidal method.

[0615] Experimental results: The test results are shown in Table 13.

[0616] Table 13. Pharmacokinetic parameters of the compounds of the present invention in mice.

[0617]

[0618] Conclusion: The compounds of this invention exhibit good drug-like properties in mice.

[0619] Test Example 7: Rat Pharmacokinetic Evaluation of Compounds

[0620] Experimental objective:

[0621] In this study, male SD rats were used as test compounds. The plasma drug concentrations of the test and reference compounds administered intravenously or orally to the rats at different time points were quantitatively determined using LCMS / MS to evaluate the pharmacokinetic characteristics of the test drugs in rats.

[0622] Experimental materials:

[0623] SD rats (male, Vital Rivers, Beijing).

[0624] Experimental procedure:

[0625] A clear or suspension solution of the test compound was injected into rats via the tail vein (without fasting) or administered via gavage (without fasting). For intravenous administration, blood was collected via jugular vein at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after administration. The blood samples were placed in anticoagulant tubes containing heparin sodium. After collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6000 g, 3 min, 2-8 °C). For oral gavage administration, blood was collected via jugular vein at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after administration. The blood samples were placed in anticoagulant tubes containing heparin sodium. After collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6000 g, 3 min, 2-8 °C). Blood drug concentrations were determined by LC-MS / MS. Relevant pharmacokinetic parameters were calculated using the WinNonlin™ Version 8.2.0 (Pharsight, Mountain View, CA) pharmacokinetic software and the non-compartmental linear logarithmic trapezoidal method.

[0626] Experimental results: The test results are shown in Table 14.

[0627] Table 14. Pharmacokinetic parameters of the compounds of the present invention in rats

[0628]

[0629] Conclusion: The compounds of this invention exhibit good drug-like properties in rats.

[0630] Test Example 8: Pharmacokinetic Evaluation of Compounds in Beagle Dogs

[0631] Experimental objective:

[0632] In this study, beagle dogs were used as test compounds. The plasma drug concentrations of the test and reference compounds administered intravenously or orally to beagle dogs at different time points were quantified using LCMS / MS to evaluate the pharmacokinetic characteristics of the test drugs in beagle dogs.

[0633] Experimental materials:

[0634] Beagle (male, Jiangsu Yadong Experimental Animal Research Institute Co., Ltd.)

[0635] Experimental procedure:

[0636] The clear or suspension solution of the test compound was injected into the beagle via the tail vein (with normal feeding) or administered via gavage (with normal feeding). For intravenous administration, blood was collected via forelimb vein puncture at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after administration. The blood samples were placed in anticoagulant tubes supplemented with EDTA-2K. After collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6000g, 3 minutes, 2-8℃). For oral gavage administration, blood was collected via forelimb vein puncture at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h after administration. The blood samples were placed in anticoagulant tubes supplemented with EDTA-2K. After collection, the blood samples were placed on ice and centrifuged within 1 hour to separate the plasma (centrifugation conditions: 6000g, 3 minutes, 2-8℃). Blood drug concentrations were determined by LC-MS / MS. Relevant pharmacokinetic parameters were calculated using the WinNonlin™ Version 8.2.0 (Pharsight, Mountain View, CA) pharmacokinetic software and the non-compartmental linear logarithmic trapezoidal method.

[0637] Experimental results: The test results are shown in Table 15.

[0638] Table 15. Pharmacokinetic parameters of the compounds of the present invention in beagle dogs

[0639]

[0640] Conclusion: The compounds of this invention exhibit good drug-like properties in beagle dogs.

Claims

1. The compound represented by formula (II) or a pharmaceutically acceptable salt thereof, ， in, PTM is selected from , and ; L1 is selected from , , , , , , , , and ; E1 is selected from single bonds and O; Structural unit Selected from , and .

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, Structural unit - E1 - L1 - selected from , , , , , , , and .

3. The compound according to any one of claims 1-2 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the structures shown in formulas (I-1), (I-2), (II-1), (II-2), (III-1), and (IV-1): 、 、 、 、 、 ， in, L1 is defined as in any one of claims 1-2.

4. The compound shown in the following formula or a pharmaceutically acceptable salt thereof, selected from: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 6. The use of the compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating prostate cancer.

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