Valine casein protein VCP / p97 inhibitor, pharmaceutical composition and application of valine casein protein VCP / p97 inhibitor and pharmaceutical composition

Abstract

The invention provides a valine casein protein VCP / p97 inhibitor, a pharmaceutical composition and application, and relates to the technical field of medicines. The valine and casein protein VCP / p97 inhibitor is a compound with a structure as shown in a formula (I), and the compound comprises a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug of the compound; the invention provides a pharmaceutical composition which comprises a compound as shown in a formula (I) or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof, and one or more pharmaceutically acceptable carriers, diluents and excipients. The invention provides application of the compound in preparation of drugs for preventing and / or treating VCP / p97 abnormal diseases. The compound has a good inhibition effect on malignant proliferation of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a valine peptide VCP / p97 inhibitor, a pharmaceutical composition, and its application. Background Technology

[0002] Malignant transformation of cells is accompanied by the overactivation of stress management pathways. These pathways, while not driving tumorigenesis themselves, are essential for disease progression; this process is known as non-oncogene addiction. These stress phenotypes include DNA damage and replication stress, protein toxicity stress, and mitotic stress. The signal transduction cascade leading to non-oncogene addiction provides malignant cells with a superior survival and proliferation advantage; therefore, inhibiting this pathway is an effective strategy for cancer treatment.

[0003] Valine-containing proteins (VCP / p97) belong to the type II AAA+ ATPase family and participate in regulating non-oncogene addiction pathways such as proteasome degradation, autophagy, and DNA damage repair, which are crucial for maintaining cellular homeostasis, especially in cancer cells. p97 is known to be essential for ERAD (endoplasmic reticulum-associated protein degradation), and recent studies have shown that cancer cells are particularly dependent on ERAD. When this pathway is impaired, cancer cells may be unable to maintain endoplasmic reticulum homeostasis, potentially activating programmed cell death. Inhibition of p97 / VCP can trigger immunogenic cell death and inhibit proteasome degradation, autophagy, and DNA damage repair, thus becoming an emerging therapeutic target for cancer, viral infections, and neurodegenerative diseases. In particular, compared to proteasome inhibitors, p97 / VCP inhibitors have high specificity and potentially good safety profiles, making them a promising approach for monotherapy or adjuvant cancer therapy.

[0004] Chinese patent CN 116234804 A discloses a crystalline form and formulation of a VCP / p97 inhibitor, specifically 1-(4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxamide or a pharmaceutically acceptable salt or solvate thereof.

[0005] Chinese Patent CN 106458996 A relates to certain fused pyrimidines having a homo- or heterocyclic pentyl ring, cyclohexyl ring, or cycloheptyl ring as a pyrimidine fusion partner; having an aminobenzyl or substituted aminobenzyl group at the 4 position of the pyrimidine ring; and having a 5:6 heterobicycle with at least one N, O, or S at the 2 position of the pyrimidine ring. These compounds can be used to treat cancer by inhibiting the p97 complex. Fused pyrimidine compounds are shown in Formula I below:

[0006]

[0007] Small molecule inhibitors of VCP / p97 protein under investigation include D1-D2 allosteric site inhibitors, D2 covalent inhibitors, and D2 non-covalent inhibitors. Among these, allosteric inhibitors have unclear mechanisms of action, poor pharmacokinetic properties, and require further confirmation of efficacy in animal models; covalent inhibitors suffer from low activity and off-target effects; and while D2 non-covalent inhibitors have been studied more extensively, their molecular structures are limited, and some compounds exhibit low selectivity and high toxicity. Therefore, discovering VCP / p97 inhibitors with novel structures and better drug-like properties has significant research value and social implications.

[0008] Based on this, the present invention provides a valine peptide protein VCP / p97 inhibitor and its application, which has a good inhibitory effect on the malignant proliferation of tumor cells. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a compound that targets and inhibits the valine protein VCP / p97, its optical isomer, and its pharmaceutically acceptable salt, which exhibits good inhibitory effects on the malignant proliferation of tumor cells.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] On one hand, the present invention provides a compound having the structure of Formula I, the compound comprising its stereoisomers, geometric isomers, tautomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs;

[0012]

[0013] in,

[0014] Ring A is selected from benzene rings and 3-6 membered heterocycles; heteroatoms are selected from O, S, and N;

[0015] R1 and R2 are each independently selected from H, substituted or unsubstituted alkyl, cycloalkyl, aryl, heterocyclic, and halogen groups;

[0016] L1 is selected from

[0017] Ring B is independently selected from benzene rings and 3-6 membered heterocycles; heteroatoms are selected from O, S, and N;

[0018] R3 and R4 are each independently selected from H, substituted or unsubstituted C. 1-5 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl, halogen, cyano; the substituents are selected from C 1-4 At least one of alkyl, halogen, cyano, and amino groups; R3 and R4 can form a 5- or 6-membered heterocyclic alkyl group together with the attached atom;

[0019] R5 and R6 are each independently selected from H and C. 1-5 Alkyl groups; R5 and R6 can form 3-6 membered heterocyclic alkyl groups together with the atoms they are attached to;

[0020] R7 is independently selected from H, C 1-5 alkyl;

[0021] m is selected from 0, 1, 2, 3, and 4;

[0022] n is selected from 1, 2, and 3.

[0023] Preferably,

[0024] Ring A is selected from benzene rings and 3-6 membered aromatic heterocycles; heteroatoms are selected from S and N;

[0025] R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-5 Alkyl, C 3-5 cycloalkyl, C 6-10 Aryl, C 3-10 Heterocyclic groups, halogens;

[0026] L1 is selected from

[0027] Ring B is independently selected from benzene rings and 3-6 membered aromatic heterocycles; heteroatoms are selected from S and N;

[0028] R3 and R4 are each independently selected from H, substituted or unsubstituted C. 1-5 Alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkyl, halogen, cyano; the substituents are selected from C 1-3 At least one of alkyl and amino groups; R3 and R4 can form a 5- or 6-membered heterocyclic alkyl group together with the attached atom;

[0029] R5 and R6 are each independently selected from H and C. 1-3 Alkyl groups; R5 and R6 can form 3-5 membered heterocyclic alkyl groups together with the atoms they are attached to;

[0030] R7 is independently selected from H, C 1-3 alkyl;

[0031] m is selected from 0, 1, 2, and 3;

[0032] n is selected from 1, 2, and 3.

[0033] Preferably, ring A is selected from benzene rings or 5-membered aromatic heterocycles; heteroatoms are selected from S and N;

[0034] R1 and R2 are each independently selected from H, substituted or unsubstituted C. 1-3 Alkyl groups, halogens;

[0035] L1 is selected from

[0036] Ring B is independently selected from benzene rings and 5-membered aromatic heterocycles; heteroatoms are selected from S and N;

[0037] R3 and R4 are each independently selected from H, substituted or unsubstituted C. 1-3 Alkyl, C 1-3 Alkoxy, halogen; the substituents are selected from C 1-3 At least one of alkyl and amino groups; R3 and R4 can form a 5- or 6-membered heterocyclic alkyl group together with the attached atom;

[0038] R5 and R6 are each independently selected from H and C. 1-3 Alkyl groups; R5 and R6 can form 3-5 membered heterocyclic alkyl groups together with the atoms they are attached to;

[0039] R7 is independently selected from H, C 1-3 alkyl;

[0040] m is selected from 0, 1, and 2;

[0041] n is selected from 1 or 2.

[0042] Preferably, the compound is a compound represented by formula (II), or a stereoisomer, geometric isomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof; wherein ring A, R1, R2, ring B, R3, R4, and n are as described above;

[0043]

[0044] Preferably, the compound is a compound represented by formula (III), or a stereoisomer, geometric isomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof; wherein rings A, R1, R2, and n are as described above; and p is independently selected from 1 or 2.

[0045]

[0046] Preferably, the compound is a compound of formula (Ⅳ), or a stereoisomer, geometric isomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof; wherein rings A, R1, R2, and n are as described above;

[0047]

[0048] Preferably, the compound is a compound of formula (V), or a stereoisomer, geometric isomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof; wherein rings A, R1, R2, R5, R6, m, and n are as described above;

[0049]

[0050] Preferably, the structural unit Selected from

[0051] Preferably, the structural unit Selected from

[0052] Preferably, the structural unit Selected from

[0053] Preferably, the compound is selected from any of the following compounds:

[0054]

[0055]

[0056] More preferably, the compound is selected from any of the following compounds:

[0057]

[0058]

[0059] More preferably, the compound is selected from any of the following compounds:

[0060]

[0061] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer, geometric isomer, tautomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers, diluents, and excipients.

[0062] In another aspect, the present invention provides the use of the compound of formula (I) or its stereoisomers, geometric isomers, tautomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, in the preparation of pharmaceutical compositions for the prevention and / or treatment of VCP / p97 abnormalities.

[0063] Preferably, the disease is selected from cancer, metabolic diseases, inflammatory diseases, or autoimmune diseases.

[0064] Preferably, the cancer is a solid tumor or a hematoma.

[0065] Preferably, the solid tumor is cholangiocarcinoma, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, or ovarian cancer.

[0066] Preferably, the hematologic malignancy is lymphocytic leukemia or chronic myeloid leukemia.

[0067] 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.

[0068] It should be understood that substitutions and combinations of substitutions as described herein, whether explicitly stated or not, refer to substitutions that conform to the valence of the substituted member. For example, substitution applied to carbon members refers to the tetravalence of C; when applied to nitrogen members, it refers to the trivalence of N; and when generally indicating a positive charge, it refers to the quadratic bond of the nitrogen member. The permissible options for valence are part of the art.

[0069] The term "pharmaceutical acceptable" 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.

[0070] 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, hydrogen 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.

[0071] 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.

[0072] The term "isomer" refers to the fact that 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.

[0073] Unless otherwise stated, "(D)" or "(+)" indicates right-handed rotation, "(L)" or "(-)" indicates left-handed rotation, and "(DL)" or "(±)" indicates racemic rotation.

[0074] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key The relative configuration of the center of a solid is indicated by a wavy line. Indicates wedge-shaped solid line key or wedge-shaped dashed key Or use wavy lines Indicates a straight solid line key and straight dashed key

[0075] The term "substituted" refers to the substitution of one or more hydrogen atoms on a particular atom by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical feasibility.

[0076] 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.

[0077] When the listed substituents do not specify which atom they are attached to the substituted group, such substituents can be bonded to any of their atoms. For example, a phenyl group as a substituent can be attached to the substituted group via any carbon atom on the benzene ring. A ring system formed by a substituent being bonded to a central ring (e.g., (As shown) represents the substitution of one of the substituents at any of the substituted positions in the ring system.

[0078] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group, which may be monosubstituted (e.g., -CH2F) or polysubstituted (e.g., -CF3), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc.

[0079] Unless otherwise specified, the term "cycloalkyl" includes any stable cyclic or polycyclic hydrocarbon group in which all carbon atoms are saturated, and which may be monosubstituted or polysubstituted, and may be monovalent, divalent or polyvalent. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, norbornene, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, etc.

[0080] Unless otherwise specified, the term "heterocyclic alkyl" includes any stable cyclic hydrocarbon group in which at least one carbon atom is substituted by an N, S, or O atom, and can be saturated or unsaturated, monosubstituted or polysubstituted, and monovalent, divalent, or polyvalent.

[0081] Unless otherwise specified, the term "halogen" itself or as part of another substituent means a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.

[0082] Unless otherwise specified, a number range represents all integers including the numbers at both ends of the range. Unless otherwise specified, integers from 0 to 10 represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; integers from 1 to 5 represent 1, 2, 3, 4, 5; integers from 1 to 3 represent 1, 2, 3; C 1-3 Alkyl groups represent C1, C2, and C3 alkyl groups; C 1-6 Alkyl groups represent C1, C2, C3, C4, C5, and C6 alkyl groups; C 3-6 Cycloalkyl refers to C3, C4, C5, C6 cycloalkyl, and so on.

[0083] The term "alkoxy" refers to straight-chain and branched alkyl groups with a specified number of carbon atoms connected by oxygen bridges.

[0084] The term "halogen" (or "halogenated") refers to fluorine, chlorine, bromine, and iodine (or fluorinated (F), chlorinated (Cl), brominated (Br), and iodinated (I)).

[0085] The term "aryl" refers to aromatic monocyclic and polycyclic ring systems, in which the carbon rings are fused together or linked together by single bonds. Common aryl groups include phenyl, naphthyl, and biphenylene.

[0086] The term "heterocycle" refers to a ring structure composed of carbon atoms and non-carbon atoms. Examples of non-carbon atoms in the ring include nitrogen, oxygen, and sulfur. Common heterocyclic groups include pyridine, quinoline, tropane, phenothiazine, benzodiazepine, furan, pyrazolone, and pyrimidine.

[0087] The term "aromatic heterocycle" refers to a 5- or 6-membered monocyclic aromatic ring or a 7- to 12-membered bicyclic ring, which consists of a carbon atom and one or more heteroatoms selected from N, O, and S. Examples of aromatic heterocycles include pyridyl, pyrroloyl, pyrazinyl, pyrimidinyl, pyridazinyl, thiophene (or thiophenyl), thiazolyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, azole, isozolyl, diazolyl, thiazolyl, isothiazolyl and thiadiazolyl, benzotriazolyl, indolyl, isoindolyl, indolyl, dihydroindolyl, isodihydroindolyl, quinoxalinyl, quinazolinyl, zolinyl, chromanyl, isochoryl, tetrahydroquinolinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo-1,4-dienyl, imidazo(2,1-b)(1,3)thiazole and benzo-1,3-m-dioxanepentenyl.

[0088] The aryl group in the term "substituted aryl" is as defined above. When no substituent is specified for the substituted aryl group, the substituent may be selected from the following groups, including but not limited to: halogens, C1-C... 20 Alkyl, CF3, NH2, N(C1-C6 alkyl)2, NO2, oxo, CN, N3, -OH, -O(C1-C6 alkyl), C3-C 10 Cycloalkyl, C2-C6 alkenyl, C2-C6 ynyl, (C0-C6 alkyl)S(O) 0-2 -, Aryl-S(O) 0-2 -、(C0-C6 alkyl)S(O) 0-2 (C0-C6 alkyl)-, (C0-C6 alkyl)C(O)NH-, H2N-C(NH)-, -O(C1-C6 alkyl)CF3, (C0-C6 alkyl)C(O)-, (C0-C6 alkyl)OC(O)-, (C0-C6 alkyl)2NC(O)-(C0-C6 alkyl)O(C1-C6 alkyl)-, (C0-C6 alkyl)C(O) 1-2 (C0-C6 alkyl)-, (C0-C6 alkyl)OC(O)NH-, aryl, aralkyl, heteroaryl, heterocyclic alkyl, halogen-aryl, halogen-aralkyl, halogen-heterocyclic, halogen-heterocyclic alkyl, cyano-aryl, cyano-aralkyl, cyano-heterocyclic, and cyano-heterocyclic alkyl. The term "substituted phenyl" has a similar definition.

[0089] Unless otherwise stated, all ranges listed in this article are inclusive. For example, "n is an integer between 0 and 2" means that n can be 0, 1, or 2.

[0090] The term "solvate" refers to a variable stoichiometric complex formed by a solute (i.e., a compound of formula I) or a pharmaceutically acceptable salt thereof and a solvent that does not interfere with the biological activity of the solute. Examples of solvents include, but are not limited to, water, ethanol, and acetic acid. When the solvent is water, the solvate is called a hydrate. Hydrates include, but are not limited to, hemihydrates, monohydrates, sesquihydrates, dihydrates, and trihydrates.

[0091] The term "prodrug" refers to a functional derivative of the compound of this invention that is readily converted into the desired compound in vivo.

[0092] Compared with the prior art, the present invention has the following beneficial effects:

[0093] This invention provides a novel valine peptide VCP / p97 inhibitor that can achieve potent inhibition of p97 enzyme activity under low concentration conditions, exhibit significant inhibitory activity against the proliferation of cholangiocarcinoma and colorectal cancer cells, has no significant inhibitory effect on PDE6C, and may avoid ocular toxicity caused by PDE6C inhibition. It also has good pharmacokinetic absorption and pharmacokinetic advantages. Detailed Implementation

[0094] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0095] Example 1. Synthesis of intermediate B1-5

[0096]

[0097] Step 1: Add urea (12.7 g, 145 mmol) to compound 2-aminonicotinic acid (5 g, 36.2 mmol), and stir the mixture at 180 °C for 15 minutes, then at 200 °C for 15 minutes, and finally at 210 °C for 1 hour. After the reaction is complete, cool to room temperature. Add 2 M sodium hydroxide solution (50 mL) to the resulting solid and stir at 50 °C until all the solid dissolves. Cool the solution again, adjust the pH to weakly alkaline with acetic acid, filter the resulting creamy flocculent precipitate to obtain a white solid 1-1, and dry it. The crude product is used directly in the next step.

[0098] Step 2: Add DMAP (0.18 g, 1.5 mmol), POCl3 (200 mL, excess), and PCl5 (3 g, 14.4 mmol) to compound 1-1 (5 g, 30.6 mmol). Reflux the mixture at 130 °C for 48 hours. After complete reaction, distill to recover POCl3, yielding a brownish-black solid. Dissolve the solid in ethyl acetate and quench the reaction dropwise in ice water. Filter the mixture with diatomaceous earth, extract with ethyl acetate, combine the organic layers, wash with saturated NaHCO3 solution and saturated brine, dry on anhydrous Na2SO4, and concentrate. Purify the crude product by silica gel column chromatography (eluent: CH2Cl2) to obtain the desired compound 1-2 as a pale yellow solid. Yield: 21.9%; ESI-MS: m / z = 201 [M+H] + .

[0099] Step 3: Add triethylamine (1.52 g, 15.0 mmol) and benzylamine (0.8 g, 7.5 mmol) to a mixture of compounds 1-2 (1 g, 5.0 mmol) in tetrahydrofuran (25 mL). Stir the mixture at room temperature for 1.5 hours. After complete reaction, evaporate the solvent. Purify the crude product by silica gel column chromatography (eluent: EA) to obtain the desired compounds 1-3 as pale yellow solids. Yield: 93.3%; ESI-MS: m / z = 271 [M+H] + .

[0100] Step 4: Under hydrogen protection, PtO2 (0.106 g, 0.47 mmol) was added to a mixture of compound B1-3 (1.26 g, 4.65 mmol) and EtOH / EtOAc in a 4:1 (50 mL, v:v) solution. The mixture was stirred at room temperature for 6 hours. After complete reaction, the mixture was filtered through diatomaceous earth, and the solid was washed with ethyl acetate. The filtrates were mixed and concentrated to give the desired compound B1-4, a pale yellow solid. Yield: 97%; ESI-MS: m / z = 275 [M+H] + .

[0101] Step 5: Add DMAP (0.25 g, 2.0 mmol) and (Boc)₂O (5.46 g, 25 mmol) to a mixture of compound B1-4 (1.24 g, 5.0 mmol) and acetonitrile (70 mL). Stir the mixture at room temperature for 30 hours. After complete reaction, pour the mixture into 50 mL of H₂O, then extract with ethyl acetate. Wash the combined organic layers with saturated brine, dry on anhydrous Na₂SO₄, and concentrate. Purify the crude product by silica gel column chromatography (eluent: PE / EA = 3:1) to obtain the desired compound B1-5 as a yellow oily liquid. Yield = 83.5%.

[0102] Example 2. Synthesis of Comparative Compound A

[0103]

[0104] Step 1: Under nitrogen protection, Pd2(dba)3 (0.137 g, 0.15 mmol), Cs2CO3 (0.489 g, 1.5 mmol), and 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl (0.072 g, 0.15 mmol) were added to a mixture of compounds B1-5 (0.475 g, 1 mmol) and 1-9 (0.156 g, 1 mmol) in anhydrous dioxane (10 mL). The mixture was stirred and refluxed at 100 °C for 15 hours. After complete reaction, the mixture was dissolved in CH2Cl2, washed with H2O and saturated brine, dried on anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: PE / EA = 10:1) to give the desired compound B1-6 as a yellow solid. Yield: 87.4%; 1 H NMR(400MHz,Chloroform-d)δ8.78(d,J=8.4Hz,1H),7.48(d,J=7.5Hz,1H),7.29(d,J=3.5Hz ,7H),6.61(s,1H),5.04(s,2H),3.79(s,2H),2.76(s,3H),2.47(s,2H),1.64-1.49(m,18H).

[0105] Step 2: Acetaldehyde oxime (0.04 g, 0.67 mmol), PPh3 (0.01 g, 0.04 mmol), and Pd(OAc)2 (0.007 g, 0.03 mmol) were added to a mixture of compound B1-6 (0.2 g, 0.34 mmol) in an EtOH / H2O solution of 8:1 (5.1 mL, v:v). The mixture was stirred and refluxed at 90 °C for 48 hours. After complete reaction, the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: PE / EA = 2:1) to obtain the desired compound B1-7 as a pale yellow solid. Yield: 97.7%.

[0106] Step 3: Add trifluoroacetic acid (2 mL, excess) to a mixture of compound B1-7 (0.1954 g, 0.319 mmol) and CH2Cl2 (2 mL). Stir the mixture at room temperature for 1.5 hours. After complete reaction, evaporate the solvent, dissolve in a small amount of water, adjust the pH to 6-8 with saturated NaHCO3 solution, extract with CH2Cl2, combine the organic layers, wash with saturated brine, dry on anhydrous Na2SO4, and concentrate. Purify the crude product by silica gel column chromatography (eluent: CH2Cl2 / MeOH = 30:1) to obtain the desired compound AIT010001 as a brown solid. Yield: 65.3%; ESI-MS: m / z = 413 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J=8.2Hz,1H),7.48(d,J=7.4Hz,1H),7.35(d,J=6.6Hz,5H),7.05(t,J=7.9Hz,1H),6.74(s,1H), 6.09(s,2H),5.59(s,1H),4.71(s,3H),3.22(s,2H),2.59(s,3H),2.38(t,J=6.4Hz,2H),1.95(t,J=5.9Hz,2H),1.27(d,J=11.7Hz,2H).

[0107] Example 3. Synthesis of compound A1

[0108]

[0109] Step 1: Add B1-5 (250 mg, 0.527 mmol), 6-amino-3,4-dihydro-2(1H)-quinoline (85.4 mg, 0.527 mmol), tris(dibenzylacetone)palladium (72.34 mg, 0.079 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (37.66 mg, 0.079 mmol), and cesium carbonate (257.4 mg, 0.79 mmol) to a two-necked reaction flask. After three gas changes, under nitrogen protection, inject dioxane (10 mL) into the system. The system is then reacted overnight at 100 °C. After the reaction was completed, the reaction solution was cooled to room temperature, filtered with diatomaceous earth, and the diatomaceous earth was washed with ethyl acetate. The filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:3-1:1) to obtain the desired compound B2-6, which is a yellowish-brown oil. Yield: 41.1%.

[0110] Step 2: Dissolve B1-6 (120 mg, 0.20 mmol) in 1.5 mL of dichloromethane solution. Add trifluoroacetic acid (1.5 mL, 20.17 mmol) dropwise to the solution. Stop the reaction after 3 h at room temperature. After evaporating the reaction mixture to dryness, dissolve it in water. If the water cannot completely dissolve it, add a small amount of dichloromethane. Adjust the pH of the mixture to 7 with saturated sodium bicarbonate solution. Extract the mixture with dichloromethane solution (10 mL × 4). Collect the organic phase, wash it with saturated sodium chloride solution, and finally dry it with anhydrous sodium sulfate. Evaporate the excess organic phase to obtain the desired compound AIT010011, a yellowish-brown solid. (Extraction only, no column chromatography). Yield: 62.5%, ESI-MS: m / z = 401.3 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.14(s,1H),7.43(s,2H),7.36-7.27(m,6H),7.03(s,1H),5.10(s,1H),4.59(t,J=5.7H z,1H),4.68(s,2H),3.36-3.29(m,2H),2.80(s,2H),2.53(d,J=4.6Hz,2H),2.32(t,J=6.4Hz,2H),1.99-1.90(m,2H).

[0111] Example 4. Synthesis of compound A2

[0112]

[0113] Step 1: Solid reactants 7-amino-3,4-dihydroquinazolin-2-one (70 mg, 0.42 mmol), Pd2(dba)3 (70 mg, 0.063 mmol), X-Phos (30 mg, 0.063 mmol), and cesium carbonate (205 mg, 0.63 mmol) were added to the reaction system under nitrogen atmosphere. B1-5 (200 mg, 0.42 mmol) was dissolved in 1,4-dioxane and injected into the reaction system. The mixture was stirred overnight at 100°C until the reactants were completely reacted. The reaction system was filtered, washed with EA, and evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:20) to obtain the desired compound B3-6 as a pale yellow oil (53.3 mg). Yield: 25%. 1H NMR(400MHz,Chloroform-d)δ7.90(s,1H),7.29(s,6H),6.99(d,J=8.0Hz,1H),6.77(d,J=8.0Hz,1H),4.92( s,2H),3.69(s,2H),2.87(t,J=7.5Hz,2H),2.63-2.53(m,2H),2.40-2.17(m,2H),1.56(s,9H),1.39(s,9H).

[0114] Step 2: Dissolve B3-6 (55 mg, 0.092 mmol) in 1.5 mL of dichloromethane, then add excess TFA (1.5 mL, 0.020 mol) and stir at room temperature for 3 hours. After the reaction, evaporate the solvent to dryness, wash with saturated NaHCO3 solution and saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 1:1) to obtain the desired compound A2, a pale yellow oil (25.9 mg). ESI-MS: m / z = 401.3 [M+H] + Yield: 70.4% 1 H NMR(400MHz,Chloroform-d)δ8.18(s,1H),7.40-7.28(m,5H),7.20(d,J=2.1Hz,1H),7.14(s,1H),6.93-6.80(m,2H),5.37(d,J=20.9Hz,1 H), 4.68 (d, J = 5.6Hz, 2H), 3.30 (t, J = 5.5Hz, 2H), 2.83-2.74 (m, 2H), 2.51 (dd, J = 8.5, 6.4Hz, 2H), 2.32 (t, J = 6.4Hz, 2H), 1.99-1.89 (m, 2H).

[0115] Example 5. Synthesis of compound A3

[0116]

[0117] Step 1: Add B1-5 (250 mg, 0.527 mmol), 7-amino-3,4-dihydroisoquinoline-1(2H)-one (85.4 mg, 0.527 mmol), tris(dibenzylideneacetone)palladium (72.34 mg, 0.079 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (37.66 mg, 0.079 mmol), and cesium carbonate (257.4 mg, 0.79 mmol) to a two-necked reaction flask. After three gas changes, under nitrogen protection, inject dioxane (10 mL) into the system. The system is then reacted overnight at 100 °C. After the reaction was completed, the reaction solution was cooled to room temperature, filtered with diatomaceous earth, and the diatomaceous earth was washed with ethyl acetate. The filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:3-1:1) to obtain the desired compound B4-6, which is a yellowish-brown oil. Yield: 41%.

[0118] Step 2: Dissolve B4-6 (120 mg, 0.20 mmol) in 1.5 mL of dichloromethane solution. Add trifluoroacetic acid (1.5 mL, 20.17 mmol) dropwise to the solution. Stop the reaction after 3 h at room temperature. After evaporating the reaction mixture, dissolve it in water. If the water cannot completely dissolve it, add a small amount of dichloromethane. Adjust the pH of the mixture to 7 with saturated sodium bicarbonate solution. Extract the mixture with 10 mL × 4 dichloromethane solutions. Collect the organic phase, wash it with saturated sodium chloride solution, and finally dry it with anhydrous sodium sulfate. Evaporate the excess organic phase to obtain the crude product. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 10:1 - MeOH / DCM = 1:20) to obtain the desired compound A3 as a pale yellow solid. Yield: 50%, ESI-MS: m / z = 401.3 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.23(s,1H),7.85-7.68(m,1H),7.42-7.26(m,6H),7.04(s,1H),6.29(s,1H),5.20(s,1H), 4.72(s,2H),4.55(s,1H),4.27-4.05(m,1H),3.48(s,2H),3.32(s,2H),2.89(s,2H),2.31(s,2H),2.06(d,J=12.5Hz,1H).

[0119] Example 6. Synthesis of compound A4

[0120]

[0121] Step 1: The solid reactants 6-aminoindol-1-one (129 mg, 0.87 mmol), Pd2(dba)3 (108.9 mg, 0.119 mmol), X-Phos (56.7 mg, 0.119 mmol), and cesium carbonate (387.7 mg, 1.19 mmol) were added to the reaction system. The system was placed under nitrogen atmosphere. B1-5 (375 mg, 0.42 mmol) was dissolved in 1,4-dioxane and injected into the reaction system. The mixture was stirred overnight at 100 °C until the reactants were completely reacted. The reaction system was filtered, washed with EA, and evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:50) to obtain the desired compound B5-6, a pale yellow oil. ESI-MS: m / z = 587 [M+H] + .

[0122] Step 2: Dissolve B5-6 in 1.5 mL of dichloromethane, then add excess TFA (1.5 mL, 0.020 mol) and stir at room temperature for 3 hours. After the reaction, evaporate the solvent to dryness, wash with saturated NaHCO3 solution and saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 1:2) to obtain the desired compound A4 as a white solid (21.5 mg). ESI-MS: m / z = 387.2 [M+H] + ; 1 H NMR (400MHz, Methanol-d4) δ7.37-7.34(m,2H),7.32-7.12(m,5H),7.08(d,J=2.8Hz,1H),6.98(dd,J=8 .1,2.2Hz,1H),4.71(s,2H),4.65(s,2H),3.35-3.32(m,2H),2.40(t,J=6.4Hz,2H),1.96-1.90(m,2H).

[0123] Example 7. Synthesis of compound A5

[0124]

[0125] Step 1: Add the starting materials 4-aminophthalimide (350 mg, 0.74 mmol), Pd2(dba)3 (101.4 mg, 0.11 mmol), X-Phos (52.8 mg, 0.11 mmol), and cesium carbonate (721.7 mg, 2.22 mmol) to the reaction system. Under nitrogen atmosphere, dissolve B1-5 in 1,4-dioxane and inject it into the reaction system. Stir overnight at 100°C until the starting materials are completely reacted. Extract the reaction system with EA, wash with saturated brine, dry with anhydrous sodium sulfate, and then evaporate to dryness. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 1:2) to obtain the desired compound B6-6 as a pale yellow solid (225 mg). ESI-MS: 601; Yield: 50.7%;

[0126] Step 2: Dissolve B6-6 (225 mg, 0.37 mmol) in 5 mL of dichloromethane, then add excess TFA (2.5 mL) and stir the reaction at room temperature for 3 hours. After the reaction, evaporate the solvent to dryness, adjust the pH to 6 with saturated NaHCO3 solution, extract with DCM, wash with saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH = 30:1) to obtain the desired compound A5 as a white solid. ESI-MS: m / z = 401 [M+H] + Yield: 30% 1 H NMR(400MHz,DMSO-d6)δ10.93(s,1H),9.24(s,1H),8.24(d,J=1.9Hz,1H),8.00 (dd,J=8.4,2.0Hz,1H),7.47(d,J=8.4Hz,1H),7.40-7.34(m,2H),7.28(t,J=7.6 Hz,2H),7.21-7.14(m,1H),6.77(t,J=6.2Hz,1H),6.25(d,J=2.6Hz,1H),4.61(d ,J=6.1Hz,2H),3.24-3.17(m,2H),2.38(t,J=6.4Hz,2H),1.82(t,J=5.8Hz,2H).

[0127] Example 8. Synthesis of compound A6

[0128]

[0129] The synthesis method was the same as in Example 7, except that the 4-aminophthalimide raw material was replaced with 5-amino-2,3-dihydroisoindole-1-one to prepare compound A6.

[0130] Yield: 50%, ESI-MS: m / z = 401.3 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ10.51(s,1H),8.02(s,1H),7.72-7.66(m,3H),7.39(d,J=7.5Hz,2H),7.34(d,J=7.1Hz,1H),7 .30(d,J=7.5Hz,2H),6.49(s,1H),5.29(s,1H),4.76(d,J=5.4Hz,2H),4.32(s,2H),2.39(s,2H),2.03(s,2H),1.25(s,2H).

[0131] Example 9. Synthesis of Compound A7

[0132]

[0133] The synthesis method was the same as in Example 7, except that the raw material 4-aminophthalimide was replaced with 4-amino-isoindoline-1-one to prepare compound A7.

[0134] Yield: 50%, ESI-MS: m / z = 401.3 [M+H] + ;1H NMR (400MHz, DMSO-d6) δ8.55(s,1H),8.35(t,J=6.1Hz,1H),7.47(d,J=7.5Hz,2H),7.34(t,J=7.5Hz,2H),7.27(q,J=8.1Hz,2H),7.04(d,J= 7.5Hz, 1H), 6.94 (d, J = 7.9Hz, 1H), 4.79 (s, 2H), 4.72 (d, J = 5.9Hz, 2H), 3.33 (d, J = 5.7Hz, 2H), 2.41 (t, J = 6.3Hz, 2H), 1.84 (p, J = 6.0Hz, 2H).

[0135] Example 10. Synthesis of Compound A8

[0136]

[0137] The synthesis method was the same as in Example 7, except that the 4-aminophthalimide raw material was replaced with 5-amino-1,3-dihydroindole-2-one to prepare compound A8.

[0138] Yield: 50%, ESI-MS: m / z = 387.2 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.35(s,1H),9.86(s,1H),8.01(t,J=5.7Hz,1H),7.36(d,J=20.3Hz,1H),7.33-7.21(m,6H),7.12(d,J=7.6H z, 1H), 6.69 (d, J = 8.3Hz, 1H), 4.56 (d, J = 5.9Hz, 2H), 3.35 (s, 2H), 3.27 (d, J = 5.9Hz, 2H), 2.38 (t, J = 6.3Hz, 2H), 1.83 (t, J = 6.1Hz, 2H).

[0139] Example 11. Synthesis of compound A9

[0140]

[0141] The synthesis method was the same as in Example 7, except that the 4-aminophthalimide raw material was replaced with 6-amino-indole ketone to prepare compound A9.

[0142] Yield: 50%, ESI-MS: m / z = 387.2 [M+H] + ; 1 H NMR(400MHz, Acetone-d6)δ7.42-7.26(m,5H),7.23(dddd,J=6.3,4.3,2.8,1.4Hz,2H),7.09(d,J=8.1Hz,1H),4.77 (d,J=4.7Hz,2H),3.54(s,4H),3.49-3.42(m,2H),3.39(s,2H),2.50(t,J=6.3Hz,2H),1.98(dt,J=11.6,6.3Hz,2H).

[0143] Example 12. Synthesis of intermediate B2-5

[0144]

[0145] The synthesis method was the same as in Example 1, except that the starting material benzylamine was replaced with 3-fluorobenzylamine to prepare compound B2-5. Yield: 82%, ESI-MS: 493.1 [M+H] + .

[0146] Example 13. Synthesis of compound A10

[0147]

[0148] The synthesis method is the same as in Example 7, except that the raw material 4-aminophthalimide is replaced with 5-amino-isoindoline-1-one and B1-5 is replaced with B2-5 to prepare compound A10.

[0149] Yield: 50%, ESI-MS: m / z = 405.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.35(s,2H),8.57(s,1H),8.40(s,1H),7.37(dd,J=8.0,6.5Hz,2H),7.28-7.20(m,2H),7.07(td,J=8. 4,2.3Hz,1H),7.03-6.93(m,2H),4.76(s,2H),4.66(d,J=6.0Hz,2H),2.43(t,J=6.3Hz,2H),1.84(t,J=5.7Hz,2H),1.15(s,2H).

[0150] Example 14. Synthesis of intermediate B3-5

[0151]

[0152] The synthesis method was the same as in Example 1, except that the starting material benzylamine was replaced with 2-fluorobenzylamine to prepare compound B3-5. Yield: 75.5%, ESI-MS: 493 [M+H] + .

[0153] Example 15. Synthesis of compound A11

[0154]

[0155] The synthesis method was the same as in Example 7, except that the raw material 4-aminophthalimide was replaced with 5-amino-isoindoline-1-one and B1-5 was replaced with B3-5 to prepare compound A11.

[0156] Yield: 89.6%; ESI-MS: m / z = 405 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ8.44(s,1H),8.18(s,1H),7.44(t,J=7.7Hz,1H),7.36(d,J=8.3Hz,1H),7.31(t,J=6.1Hz,1H),7.25-7.14(m,2H),6.9 9(dd,J=8.2,2.2Hz,1H),6.95(d,J=2.1Hz,1H),4.74(s,2H),4.71(d,J=5.8Hz,2H),3.37(s,4H),2.42(t,J=6.2Hz,2H),1.85(q,J=5.8Hz,2H).

[0157] Example 16. Synthesis of compound A12

[0158]

[0159] Step 1: Under nitrogen protection, Pd2(dba)3 (0.137 g, 0.15 mmol), Cs2CO3 (0.489 g, 1.5 mmol), and 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl (0.072 g, 0.15 mmol) were added to a mixture of compound B1-5 (0.475 g, 1 mmol) and m-aminobenzonitrile (0.118 g, 1 mmol) in anhydrous dioxane (10 mL). The mixture was stirred and refluxed at 100 °C for 15 hours. After complete reaction, the mixture was dissolved in CH2Cl2, washed with H2O and saturated brine, dried on anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: PE / EA = 8:1) to give the desired compound B13-6 as a yellow solid. Yield: 95.3%. 1 H NMR(400MHz,Chloroform-d)δ8.47(s,1H),7.62(d,J=8.2Hz,1H),7.31(t,J=7.9Hz,1H),7.26 -7.20(m,7H),4.92(s,2H),3.70(s,2H),2.25(s,2H),1.81(s,2H),1.56(s,9H),1.42(s,9H).

[0160] Step 2: Acetaldehyde oxime (0.042 g, 0.72 mmol), PPh3 (0.0113 g, 0.043 mmol), and Pd(OAc)2 (0.008 g, 0.036 mmol) were added to a mixture of compound B13-6 (0.2 g, 0.36 mmol) in an EtOH / H2O solution of 8:1 (5.4 mL, v:v). The mixture was stirred and refluxed at 90 °C for 48 hours. After complete reaction, the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: PE / EA = 2:1) to give the desired compound B13-7 as a white solid. Yield: 93.7%.

[0161] Step 3: Add trifluoroacetic acid (2 mL, excess) to a mixture of compound B13-7 (0.1775 g, 0.345 mmol) and CH2Cl2 (2 mL). Stir the mixture at room temperature for 2 hours. After complete reaction, evaporate the solvent, dissolve in a small amount of water, adjust the pH to 6-8 with saturated NaHCO3 solution, and extract with CH2Cl2. A white solid precipitates. Filter, combine the organic layers, wash with saturated brine, dry on anhydrous Na2SO4, concentrate, and combine with the filtered solid to obtain the desired compound A12 as a white solid. Yield: 95.4%; ESI-MS: m / z = 375 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ9.97(s,1H),8.04(d,J=2.0Hz,1H),7.95(s,2H),7.57(d,J=7.4Hz,1H),7.54(s,1H),7.46(s,1H),7.39(s,1 H),7.33-7.24(m,5H),7.22-7.16(m,1H),4.61(d,J=5.9Hz,2H),3.29(t,J=5.7Hz,2H),2.39(t,J=6.2Hz,2H),1.84(t,J=6.0Hz,2H).

[0162] Example 17. Synthesis of compound A13

[0163]

[0164] The synthesis method was the same as in Example 16, except that the raw material m-aminobenzonitrile was replaced with 3-amino-4-methylbenzonitrile to prepare compound A13.

[0165] Yield: 95%; ESI-MS: m / z = 389.3 [M+H] + ; 1 H NMR (400MHz, Methanol-d4) δ8.03(d,J=1.9Hz,1H),7.71(dd,J=7.9,1.9Hz,1H),7.36(d,J=8.0Hz,1H),7.24-7.12(m,3H ),7.09-6.98(m,2H),4.46(s,2H),3.38-3.32(m,2H),2.39(t,J=6.3Hz,2H),2.22(s,3H),1.93(dd,J=11.4,6.4Hz,2H).

[0166] Example 18. Synthesis of intermediate C15-3

[0167]

[0168] Step 1: Compound C15-1 (1 g, 8.8 mmol) was dissolved in an appropriate amount of THF, and triethylamine (2.5 mL, 17.6 mmol) and N,N-dimethylformyl chloride (1.14 g, 10.6 mmol) were added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed by TLC monitoring, the solvent was evaporated to dryness, and the crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:5) to obtain the desired compound C15-2 as a white solid (1.03 g). ESI-MS: 185; Yield: 62.3%;

[0169] Step 2: Dissolve C15-2 (1.03 g, 5.5 mmol) in sufficient methanol solution, then add palladium on carbon (100 mg), and react overnight at 40 °C. After the reaction is complete, monitor by TLC (PE:EA = 1:1, v:v) and LC-MS, and filter to obtain a pale yellow solid, which can be used for the next experiment without purification.

[0170] Example 19. Synthesis of compound A14

[0171]

[0172] Step 1: Solid reactants B1-3 (200 mg, 0.74 mmol), Pd2(dba)3 (220 mg, 0.24 mmol), X-Phos (115 mg, 0.24 mmol), and cesium carbonate (723 mg, 2.2 mmol) were added to the reaction system. The system was placed under nitrogen atmosphere. C15-3 was dissolved in acetonitrile and injected into the reaction system. The mixture was stirred at 100 °C for 3 h until the reactants were completely reacted. The reaction system was extracted with EA, washed with saturated brine, dried with anhydrous sodium sulfate, and then evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: DCM / MeOH = 20:1) to obtain the desired compound B15-4 as a red solid (160 mg). ESI-MS: 389; Yield: 55.5%;

[0173] Step 2: Dissolve B15-4 (140 mg, 0.36 mmol) in a solvent of ethanol:ethyl acetate = 4:1 (v:v), then add 10% of the starting material of platinum dioxide (8.19 mg, 0.036 mmol), and react overnight under hydrogen atmosphere. After the reaction is complete, monitor the reaction by TLC (DCM / MeOH = 20:1) and LC-MS. Filter with diatomaceous earth, wash the diatomaceous earth with DCM, and evaporate the filtrate to dryness. Purify the crude product by silica gel column chromatography (eluent: DCM:MeOH = 50:1) to obtain the desired compound A14 (70 mg). ESI-MS: m / z = 393 [M+H] + Yield: 49.6%; 1H NMR (400MHz, Methanol-d4) δ7.78(d,J=2.9Hz,1H),7.25(s,5H),7.19(q,J=5.1,4.6Hz,1H),6.71(d,J=3.0 Hz, 1H), 4.59 (s, 2H), 3.39 (s, 4H), 3.22 (q, J = 5.5, 4.8Hz, 2H), 2.24 (t, J = 6.4Hz, 2H), 1.86 (t, J = 5.9Hz, 2H).

[0174] Example 20. Synthesis of compound A15

[0175]

[0176] The synthesis method was the same as in Example 19, except that starting material C15-3 was replaced with 3-aminopyrazole to prepare compound A15. Yield: 30%; ESI-MS: m / z = 322 [M+H] + .

[0177] Example 21. Synthesis of intermediate C17-2

[0178]

[0179] C17-1 (500 mg, 2.7 mmol) was dissolved in sufficient methanol, and then palladium on carbon (50 mg) was added. The mixture was reacted overnight at 40 °C. After the reaction was complete, as monitored by TLC (PE:EA = 1:1, v:v) and LC-MS, the mixture was filtered and concentrated under vacuum to give C17-2 (400 mg) as a pale yellow solid. Yield: 95.6%; ESI-MS: m / z = 155 [M+H] + .

[0180] Example 22. Synthesis of compound A16

[0181]

[0182] Step 1: Solid reactants B1-3 (400 mg, 1.476 mmol), Pd2(dba)3 (202.7 mg, 0.221 mmol), X-Phos (105.5 mg, 0.221 mmol), and cesium carbonate (1.44 g, 4.43 mmol) were added to a double-necked flask. The system was placed under nitrogen atmosphere. C17-2 was dissolved in 1,4-dioxane and injected into the reaction system. The mixture was stirred at 100 °C for 3 h until the reactants were completely reacted. The reaction system was extracted with EA, washed with saturated brine, dried with anhydrous sodium sulfate, and then evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: pure EA) to obtain the desired compound B17-4 as a red solid (490 mg). Yield: 85.6%; ESI-MS: m / z = 389 [M+H] + .

[0183] Step 2: Dissolve B17-4 (490 mg, 1.26 mmol) in a solvent with an ethanol:ethyl acetate ratio of 4:1 (v:v), add 10% of the starting material of platinum dioxide (28.7 mg, 0.126 mmol), and react overnight under hydrogen atmosphere. After the reaction is complete as monitored by TLC (pure EA) and LC-MS, filter with diatomaceous earth, wash the diatomaceous earth with EA, and evaporate the filtrate to dryness to obtain crude white solid B17-5 (486 mg), which is directly used in the next step of the reaction. Yield: 97.9%; ESI-MS: m / z = 393 [M+H] + .

[0184] Step 3: Dissolve B17-5 (250 mg, 0.637 mmol) in 5 mL of methanol, then add 30% NaOH aqueous solution (3 mL) and reflux at 100 °C for 2 hours. After the reaction is complete, extract with DCM, wash with saturated brine, dry with anhydrous sodium sulfate, and then evaporate to dryness. The desired compound B17-6 is obtained as a yellow oil (228 mg). Yield: 94.2%; ESI-MS: m / z = 379 [M+H] + .

[0185] Step 4: Add reactants B17-6 (228 mg, 0.608 mmol), EDCI (174.8 mg, 0.912 mmol), HOBT (123.2 mg, 0.912 mmol), and DIPEA (235.7 mg, 1.824 mmol) to the reaction system. Incubate the system at 0°C for 30 min. Add NH4Cl (64.5 mg, 1.206 mmol) to the reaction system and stir overnight at room temperature until the reactants have reacted completely. Extract the reaction system with EA, wash with saturated brine, dry with anhydrous sodium sulfate, and then evaporate to dryness. Purify the crude product by silica gel column chromatography (eluent: DCM:MeOH = 100:1) to obtain the desired compound A16 (45 mg). Yield: 19.8%; ESI-MS: m / z = 378 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.23(s,1H),7.34-7.26(m,5H),7.23-7.16(m,2H),6.99(s,1H),6.66(s,2H),6.58(s,1H) ,6.24(s,1H),4.60(d,J=6.0Hz,2H),3.65(s,3H),3.24-3.15(m,2H),2.34(t,J=6.3Hz,2H),1.80(p,J=5.8Hz,2H).

[0186] Example 23. Synthesis of intermediate B4-5

[0187]

[0188] The synthesis method is the same as in Example 1, except that the raw material benzylamine is replaced with 3-aminomethylthiophene to prepare intermediate B4-5.

[0189] Yield = 54.7%; ESI-MS: m / z = 481 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ7.48(dd,J=5.0,2.9Hz,1H),7.30(d,J=1.7Hz,1H),7.04(dd,J=5.0,1.3 Hz, 1H), 4.79 (s, 2H), 3.65 (s, 2H), 2.24 (d, J = 6.3Hz, 2H), 1.67 (s, 2H), 1.49 (s, 9H), 1.38 (s, 9H).

[0190] Example 24. Synthesis of compound A17

[0191]

[0192] The synthesis method is the same as in Example 2, except that the raw material B1-5 is replaced with B4-5 to prepare compound A17.

[0193] Yield: 51.8%; ESI-MS: m / z = 419 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.16(d,J=8.2Hz,1H),7.51(s,1H),7.43-7.36(m,1 H),7.24(s,1H),7.11(d,J=4.9Hz,1H),7.01(t,J=7.9Hz,1H),6.89(s,1H),6.28(t ,J=5.4Hz,1H),4.72(d,J=5.8Hz,2H),3.56(q,J=6.9Hz,1H),3.38(t,J=5.5Hz,2H) ,2.61(s,3H),2.54(t,J=6.4Hz,2H),1.97(p,J=6.0Hz,2H),1.12(t,J=7.0Hz,2H).

[0194] Example 25. Synthesis of intermediate B5-5

[0195]

[0196] The synthesis method was the same as in Example 1, except that the starting material benzylamine was replaced with 4-(aminomethyl)-1-methylpyrazole to prepare intermediate B5-5. Yield: 82%.

[0197] Example 26. Synthesis of compound A18

[0198]

[0199] The synthesis method is the same as in Example 2, except that the raw material B1-5 is replaced with B5-5 to prepare compound A18.

[0200] Yield: 50%, ESI-MS: m / z = 417.3 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ8.10(d,J=8.3Hz,1H),7.73(s,1H),7.50(s,1H),7.46(d,J=7.4Hz,1H),7.30(s,1H),7.21(s,1H),7.03(t,J=7.8Hz,1H) ,6.82(s,1H),6.79(s,1H),6.70(t,J=5.9Hz,1H),4.40(d,J=5.7Hz,2H) ,3.76(s,3H),2.58(s,3H),2.39(t,J=6.4Hz,2H),1.83(d,J=7.4Hz,2H).

[0201] Example 27. Synthesis of compound A19

[0202]

[0203] Step 1: Triethylamine (161.21 mg, 0.75 mmol) was added to a tetrahydrofuran solution (20 mL) of compounds 1-2 (50 mg, 0.25 mmol) and 2-thiophene methylamine (28.3 mg, 0.25 mmol). The system was incubated at room temperature for 5 hours. After the reaction was complete, the mixture was extracted with water and ethyl acetate (10 mL × 3), washed with saturated sodium chloride solution, and finally dried over anhydrous sodium sulfate. The solution was not purified by column chromatography to give a pale yellow solid 053-3. Yield: 87%, ESI-MS: m / z = 277.0 [M+H] + .

[0204] Step 2: Add B20-3 (30 mg, 0.109 mmol), 1-9 (17 mg, 0.109 mmol), tris(dibenzylacetone)palladium (30 mg, 0.0327 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (15.6 mg, 0.0327 mmol), and cesium carbonate (106.54 mg, 0.327 mmol) to a two-necked reaction flask. After three gas changes, under nitrogen protection, inject dioxane (10 mL) into the system. React overnight at 100 °C. After the reaction is complete, cool the reaction solution to room temperature, filter with diatomaceous earth, wash the diatomaceous earth with ethyl acetate, and evaporate the filtrate to dryness to obtain the crude product. Purify the crude product by silica gel column chromatography (eluent: DCM:MeOH = 50:1) to obtain the desired compound B20-4, which is a yellow solid. Yield: 60%, ESI-MS: 397.1 [M+H] + .

[0205] Step 3: Platinum dioxide (14.33 mg, 0.0631 mmol) was added to compound B20-4 (250 mg, 0.631 mmol), followed by the addition of a mixed solvent of ethanol / ethyl acetate (4 mL / 1 mL). The system was purged three times and reacted overnight under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the diatomaceous earth was washed with ethyl acetate. The crude product was purified by silica gel column chromatography (eluent: DCM:MeOH = 100:1) to obtain the desired compound B20-5 as a pale yellow solid. Yield: 98%, ESI-MS: m / z = 401.2 [M+H] + .

[0206] Step 4: Dissolve B20-5 (200 mg, 0.5 mmol), palladium acetate (11.23 mg, 0.05 mmol), triphenylphosphine (15.74 mg, 0.06 mmol), and acetaldehyde oxime (59.07 mg, 1 mmol) in a 0.25 mL ethanol / water mixture and reflux overnight. After the reaction, extract with water and ethyl acetate (20 mL × 3), wash with saturated sodium chloride solution, and finally dry with anhydrous sodium sulfate. Reduce the excess organic phase by rotary evaporation to obtain the crude product. Purify the crude product by silica gel column chromatography (eluent: PE:EA = 1:1) to obtain the desired compound A19 as a yellow solid. Yield: 50%, ESI-MS: m / z = 419.2 [M+H] + ;1H NMR(400MHz,DMSO-d6)δ8.03(dt,J=8.2,0.9Hz,1H),7.73(s,1H),7.45(dd,J=7.5,1.0Hz ,1H),7.35-7.31(m,1H),7.21(s,1H),7.10(t,J=6.1Hz,1H),7.00(dd,J=8.3,7.4Hz,1H), 6.97-6.93(m,2H),6.87(d,J=2.6Hz,1H),6.85-6.79(m,1H),4.73(d,J=5.9Hz,2H),3.25( q, J=4.4, 2.6Hz, 2H), 2.56 (d, J=1.0Hz, 3H), 2.42 (t, J=6.4Hz, 2H), 1.86 (t, J=5.8Hz, 2H).

[0207] Example 28. Synthesis of intermediate C21-2

[0208]

[0209] At 0 °C, a borane dimethyl sulfide complex (2 mol / L, 10 mL) was added dropwise to a mixture of compound 3-thiopheneacetonitrile (1.232 g, 10 mmol) and tetrahydrofuran (10 mL). The mixture was stirred and refluxed at 80 °C for 6 hours. After complete reaction, MeOH was added dropwise to quench the reaction until no bubbles were produced, and the solvent was evaporated to dryness. An EA / HCl solution (2 mol / L, 20 mL) was added to the above mixture, and the mixture was stirred at room temperature for 5 hours. After complete reaction, the mixture was filtered and dried to give the desired compound 5-1 as a white solid. Yield: 67.2%; ESI-MS: m / z = 128 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ8.17(s,3H),7.51(dd,J=4.9,2.9Hz,1H),7.32(s,1H) ,7.06(dd,J=4.9,1.3Hz,1H),3.08-2.96(m,2H),2.91(dd,J=9.5,6.3Hz,2H).

[0210] Example 29. Synthesis of intermediate B6-5

[0211]

[0212] The synthesis method was the same as in Example 1, except that the starting material benzylamine was replaced with C21-2 to prepare intermediate B6-5. Yield = 16.8%; ESI-MS: m / z = 495 [M+H].

[0213] Example 30. Synthesis of compound A20

[0214]

[0215] The synthesis method is the same as in Example 2, except that the raw material B1-5 is replaced with B6-5 to prepare compound A20.

[0216] Yield: 18.7%; ESI-MS: m / z = 433 [M+H] + ; 1H NMR(400MHz,DMSO-d6)δ8.22(d,J=8.3Hz,1H),7.74(s,1H),7.51-7.44(m,2H),7 .26-7.16(m,2H),7.11-7.03(m,1H),6.99(dd,J=4.9,1.3Hz,1H),6.88-6.82(m, 1H),6.79(s,1H),6.49(t,J=5.7Hz,1H),3.65-3.53(m,2H),3.25(d,J=5.3Hz,2H ), 2.94-2.83 (m, 2H), 2.64 (s, 3H), 2.36 (t, J = 6.2Hz, 2H), 1.84 (t, J = 5.7Hz, 2H).

[0217] Example 31. Synthesis of compound A21

[0218]

[0219] The synthesis method was the same as in Example 27, except that the starting material 2-thiophene methylamine was replaced with 2-aminomethyl-5-methylthiophene to prepare compound A21. Yield: 21.3%; ESI-MS: m / z = 433 [M+H] + .

[0220] Example 32. Synthesis of compound A22

[0221]

[0222] Step 1: Solid reactants 3-acetylindole (36.6 mg, 0.23 mmol), Pd2(dba)3 (27 mg, 0.03 mmol), X-Phos (14.3 mg, 0.03 mmol), and cesium carbonate (97.7 mg, 0.3 mmol) were added to the reaction system. The system was placed under nitrogen atmosphere. B1-5 (100 mg, 0.21 mmol) was dissolved in 1,4-dioxane and injected into the reaction system. The mixture was stirred overnight at 100 °C until the reactants were completely reacted. The reaction system was filtered, washed with EA, and evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:10) to obtain the desired compound B23-6 as a colorless oil. ESI-MS: m / z = 598.3 [M+H] +

[0223] Step 2: Dissolve B23-6 in 1.5 mL of dichloromethane, then add excess TFA (1.5 mL, 0.020 mol) and stir the reaction at room temperature for 3 hours. After the reaction is complete, evaporate the solvent to dryness, wash with saturated NaHCO3 solution and saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 1:2) to obtain the desired compound A22 as a pale yellow solid (18 mg). ESI-MS: m / z = 398.2 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.77(s,1H),8.60(dt,J=8.0,0.9Hz,1H),8.42-8.36(m,1H),7.44-7.35(m,4H),7.33-7.26(m,2H),7.26-7.21(m,1H) ,5.15(s,1H),4.79(d,J=5.3Hz,3H),3.41(t,J=5.5Hz,2H),2.57(s,3H),2 .41(t,J=6.4Hz,2H), 2.03(dd,J=10.9,5.1Hz,2H), 1.26(d,J=1.5Hz,2H).

[0224] Example 33. Synthesis of compound A23

[0225]

[0226] The synthesis method was the same as in Example 2, except that raw materials 1-9 were replaced with 3-cyanoindole to prepare compound A23.

[0227] ESI-MS: m / z = 399.2 [M+H] + ; 1 H NMR(400MHz,DMSO-d6)δ8.81(s,1H),

[0228] 8.57(dd,J=7.5,2.1Hz,1H),8.17(dd,J=7.1,2.3Hz,1H),7.76(s,1H),7.44-7.37(m,2H),7.31(t,J=7.6Hz,2H),7.2 4-6.93(m,5H),6.82(s,1H),4.73(d,J=5.8Hz,2H),3.30-3.24(m,2H),2.45(t,J=6.3Hz,2H),1.87(q,J=5.9Hz,2H).

[0229] Example 34. Synthesis of compound A24

[0230]

[0231] B24-6 was dissolved in 1.5 mL of dichloromethane, and then excess TFA (1.5 mL, 0.020 mol) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was washed with saturated NaHCO3 solution and saturated brine. The resulting organic layer was dried over anhydrous sodium sulfate and then evaporated to dryness before being passed through a column chromatography column. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:2) to obtain the desired compound A24 as a pale yellow solid (22 mg). ESI-MS: m / z = 381.2 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.66-8.59(m,2H),7.74-7.68(m,1H),7.40-7.27(m,7H),5.01(s,1H),4 .82-4.73(m,3H),3.42-3.37(m,2H),2.40(t,J=6.4Hz,2H),2.04-1.98(m,2H),1.26(d,J=1.5Hz,2H).

[0232] Example 35. Synthesis of intermediate C25-2

[0233]

[0234] C25-1 (500 mg, 3.12 mmol) was dissolved in sufficient acetonitrile solution, and then di-tert-butyl dicarbonate ((Boc)₂O, 749 mg, 3.43 mmol) was added. The mixture was reacted at room temperature for 1 h. After the reaction was complete, it was monitored by TLC (PE:EA = 5:1, v:v) and LC-MS. The reaction solution was extracted with saturated brine and EA. The organic layer was evaporated to dryness and passed through a column to obtain a white oily liquid C25-2 (550 mg). ESI-MS: 205; Yield: 67.6%; ¹H NMR (400MHz, DMSO-d⁶) δ 10.78 (s, 1H), 7.51 (d, J = 7.9Hz, 1H), 7.32 (d, J = 6.2Hz, 1H), 7.12 (d, J = 2.3Hz, 1H), 7.07–7.02 (m, 1H), 6.95 (ddd, J = 7.9, 7.0, 1.1Hz, 1H), 2.84–2.77 (m, 2H), 2.77–2.71 (m, 2H).

[0235] Example 36. Synthesis of compound A25

[0236]

[0237] Step 1: Solid reactants B1-3 (200 mg, 0.74 mmol), Pd2(dba)3 (223.6 mg, 0.24 mmol), X-Phos (116 mg, 0.24 mmol), and cesium carbonate (723 mg, 2.22 mmol) were added to the reaction system. The system was placed under nitrogen atmosphere. C25-2 was dissolved in acetonitrile and injected into the reaction system. The mixture was stirred at 100 °C for 3 h until the reactants were completely reacted. The reaction system was extracted with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and then evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 5:1) to obtain the desired compound B26-3 as a pale yellow oil (280 mg). ESI-MS: 495; Yield: 77%.

[0238] Step 2: Dissolve B26-3 (280 mg, 0.567 mmol) in a solvent with an ethanol:ethyl acetate ratio of 4:1 (v:v), then add 10% of the starting material's amount of platinum dioxide (12.6 mg, 0.0567 mmol). React overnight under hydrogen atmosphere. After the reaction is complete, monitor the reaction by TLC (PE:EA = 1:1, v:v) and LC-MS. Filter with diatomaceous earth, wash the diatomaceous earth with EA, and evaporate the filtrate to dryness to obtain crude white solid B26-4 (250 mg), which is directly used in the next step of the reaction. ESI-MS: m / z = 499 [M+H] + .

[0239] Step 3: Dissolve B26-4 (250 mg, 0.501 mmol) in 5 mL of dichloromethane, then add excess TFA (2.5 mL) and stir the reaction at room temperature for 3 hours. After the reaction is complete, evaporate the solvent, adjust the pH to 6 with saturated NaHCO3 solution, extract with DCM, wash with saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH = 20:1) to obtain the desired compound A25 as a white solid (106 mg). Yield: 53%; ESI-MS: m / z = 399 M+H + . 1H NMR (400MHz, DMSO-d6) δ8.51(dd,J=6.2,3.3Hz,1H),7.95(s,1H),7.55(dd,J=6.1,3 .1Hz,1H),7.38(d,J=7.5Hz,2H),7.31(t,J=7.6Hz,2H),7.18(t,J=7.3Hz,1H),7.08( dt,J=6.1,3.5Hz,2H),7.01(t,J=6.1Hz,1H),6.81(s,1H),4.66(d,J=5.8Hz,3H),3. 31-3.19(m,3H),3.02(d,J=6.9Hz,5H),2.43(t,J=6.3Hz,2H),1.85(q,J=6.1Hz,2H).

[0240] Example 37. Synthesis of compound A26

[0241]

[0242] Step 1: The solid reactants 6-fluoroindole-3-acetonitrile (144 mg, 0.83 mmol), Pd2(dba)3 (99 mg, 0.11 mmol), X-Phos (51.5 mg, 0.11 mmol), and cesium carbonate (351.9 mg, 1.1 mmol) were added to the reaction system. The system was placed under nitrogen atmosphere. B1-5 (360 mg, 0.76 mmol) was dissolved in 1,4-dioxane and injected into the reaction system. The mixture was stirred overnight at 100 °C until the reactants were completely reacted. The reaction system was filtered, washed with EA, and evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: EA / PE = 1:10) to obtain compound B27-6 498 mg, a yellow oily substance. ESI-MS: m / z = 613.3 [M+H] + .

[0243] Step 2: Dissolve B27-6 in 2 mL of tetrahydrofuran, add BH3-THF (3 mL) dropwise under ice bath, and stir the reaction at room temperature for 3 hours. After the reaction is complete, quench the reaction with saturated NaHCO3 solution, extract with EA, dry the resulting organic layer with anhydrous sodium sulfate, and then evaporate to dryness and pass through a column. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH = 100:1) to obtain the desired compound B27-7 as a pale yellow solid (100 mg).

[0244] Step 3: Dissolve B27-7 in 1.5 mL of dichloromethane, then add excess TFA (1.5 mL, 0.020 mol) and stir the reaction at room temperature for 3 hours. After the reaction is complete, evaporate the solvent to dryness, wash with saturated NaHCO3 solution and saturated brine, dry the resulting organic layer with anhydrous sodium sulfate, evaporate to dryness, and pass through a column. Purify the crude product by silica gel column chromatography (eluent: EA / PE = 1:2) to obtain compound A26 as a yellow oil (30 mg). ESI-MS: m / z = 417 [M+H] + ; 1 H NMR(400MHz, Methanol-d4)δ8.30(dd,J=11.1,2.4Hz,1H),7.96(s,1H),7.50(dd,J=8.7,5.4Hz,1H),7.41-7.26(m,4H),7.22-7.15(m,1H),6.93 (td,J=9.0,2.5Hz,1H),4.76(s,2H),3.41-3.34(m,2H),3.24(t,J=7.4Hz,2H),3.07(t,J=7.3Hz,2H),2.49(t,J=6.4Hz,2H),2.02-1.96(m,2H).

[0245] Example 38. Synthesis of compound A27

[0246]

[0247] Step 1: Add aminoacetaldehyde dimethyl acetal (3.680 g, 35 mmol) to a mixture of compound 2,4-dipiperidinone-1-carboxylic acid tert-butyl ester (7.463 g, 35 mmol) and ethanol (150 mL). Stir the mixture at 95 °C for 8 hours. After complete reaction, evaporate the solvent. Purify the crude product by silica gel column chromatography (eluent: PE:EA = 1:2) to obtain the desired compound 6-1 as a milky white solid. Yield: 84.4%; ESI-MS: m / z = 301 [M+H] + ;

[0248] Step 2: At 0°C, trifluoroacetic acid (17.103 g, 11.14 mL, 150 mmol) was added dropwise to a mixture of compound 6-1 (4.505 g, 15 mmol) and CH2Cl2 (40 mL). The mixture was then stirred at room temperature for 24 hours. After complete reaction, the solvent was concentrated by rotary evaporation, diluted with water, and the pH was adjusted to 7 by adding saturated NaHCO3 solution. The solvent was then evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: CH2Cl2:MeOH = 40:1) to obtain the desired compound 6-2 as a colored solid. Yield: 65.2%; ESI-MS: m / z = 137 [M+H]+ ; 1 H NMR (400MHz, DMSO-d6) δ 11.14 (s, 1H), 6.86 (s, 1H), 6.64 (s, 1H), 6.20 (s, 1H), 3.34 (dt, J = 6.9, 3.5Hz, 2H), 2.72 (t, J = 6.9Hz, 2H).

[0249] Step 3: Under nitrogen protection, Cs₂CO₃ (0.977 g, 3 mmol), 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl (0.143 g, 0.3 mmol), and Pd₂(dba)₃ (0.275 g, 0.3 mmol) were added to a mixture of compounds B₁-5 (0.950 g, 2 mmol) and 6-2 (0.272 g, 2 mmol) in 1,4-dioxane (20 mL). The mixture was stirred and refluxed at 105 °C for 5 hours. After complete reaction, the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: CH₂Cl₂:EA = 4:1) to give the desired compound 6-4 as a yellow oily liquid. Yield: 28.7%; ESI-MS: m / z = 575 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ7.56(d,J=3.3Hz,1H),7.34-7.24(m,5H),7.22-7.18(m,1H),6.44(d,J=3.3Hz,1H),4.90(s,2H),3 .69(s,2H),3.29(td,J=7.0,2.4Hz,2H),3.15(d,J=5.0Hz,2H),2.41-2.30(m,2H),1.71(s,2H),1.49(s,9H),1.36(s,9H).

[0250] Step 4: Add HCl / EA solution (2 mol / L, 30 mL) to compound 6-4 (0.33 g, 0.574 mmol). Stir the mixture at room temperature for 4 hours. After complete reaction, filter, dissolve the resulting brown solid in a small amount of water, adjust the pH to 6-8 with saturated NaHCO3 solution, extract with CH2Cl2, and concentrate to obtain a brown oily liquid. Purify the crude product by silica gel column chromatography (eluent CH2Cl2:MeOH = 100:1) to obtain the desired compound A27 as a white solid. Yield: 65.1%; ESI-MS: m / z = 375 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ7.40(d,J=3.3Hz,1H),7.29(d,J=4.4Hz,4H),7.23-7.14(m,1H),7.07(d,J=6.0Hz,1H),7.04(t,J=2.6Hz,1H),6.82-6.75(m ,1H),6.29(d,J=3.3Hz,1H),4.56(d,J=6.0Hz,2H),3.21(td,J=7.0,2.5Hz ,4H), 3.02(t,J=6.9Hz,2H), 2.42(t,J=6.3Hz,2H), 1.84(q,J=5.6Hz,2H).

[0251] Example 39. Synthesis of compound A28

[0252]

[0253] Step 1: 1,5,6,7-Tetrahydro-4H-indol-4-one (500 mg, 3.7 mmol) and hydroxylamine hydrochloride (308 mg, 4.44 mmol) were dissolved in anhydrous methanol (5 mL), and sodium acetate (607 mg, 7.4 mmol) was added. The reaction was carried out at 70 °C for 2 h. After the reaction was completed, the mixture was concentrated under reduced pressure, extracted with EA and water, and the organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give compound 2-1 (500 mg, off-white solid). ESI-MS: m / z = 151.1 [M+H] + ;

[0254] Step 2: Compound 2-1 (500 mg, 3.3 mmol) was dissolved in THF (10 mL), and thionyl chloride (435 mg, 3.6 mmol) was added dropwise. The mixture was stirred at room temperature for 2 h. After the reaction was complete, the mixture was quenched with saturated NaHCO3 solution (3 mL), concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: DCM / Me = 30:1) to obtain compound 2-2 (355 mg, powdery white solid). ESI-MS: m / z = 151.2 [M+H] + , 1 H NMR (400MHz, DMSO-d6) δ10.91(s,1H),7.25(t,J=5.1Hz,1H),6.61(t,J=2.7Hz,1H), 6.29(t,J=2.8Hz,1H),3.17-3.10(m,2H),2.86(t,J=6.6Hz,2H),1.92-1.84(m,2H).

[0255] Step 3: Compound 2-2 (221 mg, 1.47 mmol), Pd2(dba)3 (202 mg, 0.22 mmol), x-phos (105 mg, 0.22 mmol), and cesium carbonate (720 mg, 2.2 mmol) were dissolved in 1,4-dioxane (20 mL), N2 was replaced, and compound B1-5 (700 mg, 1.47 mmol) was added. The mixture was reacted at 105 °C for 3 h. After the reaction was complete, the mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: DCM / Me = 50:1) to obtain compound 4 (534 mg, yellow liquid). ESI-MS: m / z = 589.3 [M+H] + .

[0256] Step 4: Compound 4 (100 mg, 0.17 mmol) was dissolved in DCM (2 mL), and HCl / EA solution (2 M, 1 mL, 0.17 mmol) was added. The mixture was stirred at room temperature for 6 h. After the reaction was complete, the pH was adjusted to 7-8 with saturated NaHCO3 solution, and the mixture was extracted with DCM. The organic phases were combined, dried over anhydrous Na2SO4, and the crude product was purified by thin-layer chromatography (developing solvent: DCM / MeOH = 20:1) to give compound A28 (45 mg, pale yellow solid). ESI-MS: m / z = 389.2 [M+H] + , 1 H NMR (400MHz, DMSO-d6) δ7.46(t,J=5.1Hz,1H),7.29(d,J=6.6Hz,5H),7.19(d dd,J=8.6,5.6,2.3Hz,1H),7.04(t,J=6.1Hz,1H),6.76(d,J=2.7Hz,1H),6.39 (d,J=3.2Hz,1H),4.55(d,J=5.9Hz,2H),3.26-3.19(m,2H),3.11-2.99(m,4H ), 2.42 (t, J = 6.4Hz, 2H), 1.83 (p, J = 6.1Hz, 2H), 1.73 (dt, J = 11.1, 6.5Hz, 2H).

[0257] Example 40. Synthesis of compound A29

[0258]

[0259] Step 1: At 0°C, (flask A) potassium tert-butoxide (4.040 g, 36 mmol, 1 M in THF) was slowly added dropwise to a mixture of compound mono-tert-butyl malonate (5.766 g, 36 mmol) and anhydrous MgCl2 (3.428 g, 36 mmol) in anhydrous tetrahydrofuran (60 mL), keeping the temperature below 10°C. The mixture was then stirred at room temperature for 3 hours. Under nitrogen protection, (flask B) N,N'-carbonyldiimidazole (4.865 g, 30 mmol) was added in portions to a mixture of compound Boc-D-alanine (5.676 g, 30 mmol) in anhydrous tetrahydrofuran (50 mL), and the mixture was stirred at room temperature for 3 hours. The reaction solution from flask B was added to flask A and stirred at room temperature for 17 hours. After the reaction was complete, 10% HCl solution was added dropwise to gradually clarify the solution, keeping the temperature below 20°C. The mixture was then extracted with EA, and the combined organic layers were washed with a small amount of saturated NaHCO3 solution and saturated brine, dried on anhydrous Na2SO4, and concentrated. The desired compound C7-1 was obtained as a yellow oily liquid.

[0260] Step 2: Chloroacetaldehyde (8.621 g, 30 mmol) was slowly added dropwise to a mixture of compound C7-1 (8.621 g, 30 mmol), ammonium acetate (23.124 g, 300 mmol), and amine-methanol solution (43 mL, 300 mmol, 7 mol / L) in 40 mL of methanol, keeping the temperature below 38 °C. The mixture was stirred at 50 °C for 3 hours. After complete reaction, the mixture was diluted with CH2Cl2, and a small amount of solid NaHCO3 and saturated NaHCO3 solution were added until no more bubbles were produced. The mixture was extracted with CH2Cl2, and the combined organic layers were washed with saturated brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: CH2Cl2:MeOH = 10:1) to obtain the desired compound C7-2, a brownish-yellow oily liquid. Yield: 17.9%; ESI-MS: m / z = 311 [M+H] + ;

[0261] Step 3: At 0°C, sodium hydride (0.075 g, 1.885 mmol, 60%) and p-toluenesulfonyl chloride (0.252 g, 1.319 mmol) were added sequentially to a mixture of compound C7-2 (0.390 g, 1.256 mmol) and anhydrous tetrahydrofuran (10 mL). The mixture was stirred at room temperature for 3 hours. After complete reaction, the reaction was quenched with water, extracted with EA, and the combined organic layers were washed with saturated brine, dried on anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: PE:EA = 15:1) to give the desired compound C7-3 as a yellow oily liquid. Yield = 37.2%; ESI-MS: m / z = 465 [M+H]+ ;

[0262] Step 4: Trifluoroacetic acid (2 mL) was slowly added dropwise to a mixture of compound C7-3 (0.24 g, 0.517 mmol) and CH2Cl2 (2 mL), and the mixture was stirred at room temperature for 5 min. The mixture was then refluxed at 50 °C with stirring for 5 h under drying conditions. After complete reaction, the solvent was evaporated and the mixture was dried under vacuum to obtain the desired compound C7-4 as a black solid, a trifluoroacetate. Yield = 87.1%; ESI-MS: m / z = 309 [M+H] + ;

[0263] Step 5: At 0°C, DIPEA (0.156 g, 1.207 mmol) was added to a mixture of compound C7-4 (0.17 g, 0.402 mmol) and ethyl acetate (12 mL). After stirring for 10 min, DPPA (0.122 g, 0.443 mmol) was added, and the mixture was stirred for another 10 min. The mixture was then transferred to room temperature and stirred for 24 hours. After complete reaction, the pH was adjusted with saturated NaHCO3 solution, and the mixture was extracted with EA. The combined organic layers were washed with saturated brine, dried on anhydrous Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography (eluent: PE:EA = 2:1) to obtain the desired compound C7-5 as a yellow oily liquid. Yield: 51.4%; ESI-MS: m / z = 291 [M+H] + ;

[0264] Step Six: Add K₂CO₃ (0.057 g, 0.413 mmol) to a mixture of compound C7-5 (0.060 g, 0.207 mmol) and methanol (3 mL). Stir the mixture at room temperature for 24 hours. After complete reaction, evaporate the solvent to obtain the desired compound C7-6 as a yellow-brown solid. ESI-MS: m / z = 137 [M+H] + ;

[0265] Step 7: Under nitrogen protection, Cs₂CO₃ (0.135 g, 0.414 mmol), 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl (0.020 g, 0.041 mmol), and Pd₂(dba)₃ (0.038 g, 0.041 mmol) were added to a mixture of compounds B₁-5 (0.098 g, 0.207 mmol) and C₇-6 (0.028 g, 0.207 mmol) in 1,4-dioxane (3 mL). The mixture was stirred and refluxed at 105 °C for 4 hours. After complete reaction, the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: DCM:EA = 9:1) to give the desired compound C₇-7 as a yellow oily liquid. Yield: 50.4%; ESI-MS: m / z = 575 [M+H] + ;

[0266] Step 8: Add HCl / EA solution (2 mol / L, 10 mL) to compound C7-7 (0.060 g, 0.104 mmol). Stir the mixture at room temperature for 24 hours. After complete reaction, filter, dissolve the resulting brown solid in a small amount of water, adjust the pH to 6-8 with saturated NaHCO3 solution, filter again, and slurry the solid with EA / PE to obtain the desired compound AIT010078, a brownish-gray solid. Yield: 74.6%; ESI-MS: m / z = 375 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ7.81(s,1H),7.55(d,J=3.1Hz,1H),7.31(s,2H),7.30(d,J=2.6Hz,2H),7.19(tt,J=5.8,3.0Hz,1H),6.23(d,J=3.1Hz ,1H),4.64(s,1H),4.61(s,2H),4.33(s,1H),3.24(t,J=5.5Hz,2H),2.42(t,J=6.3Hz,2H),1.84(q,J=5.8,4.7Hz,2H),1.27(d,J=6.4Hz,3H).

[0267] Example 41. Synthesis of compound A30

[0268]

[0269] Step 1: Under nitrogen protection, Cs₂CO₃ (0.679 g, 2.084 mmol), 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl (0.099 g, 0.208 mmol), and Pd₂(dba)₃ (0.191 g, 0.208 mmol) were added to a mixture of compounds B1-5 (0.660 g, 1.389 mmol) and D₇-6 (0.206 g, 1.389 mmol) in 20 mL of 1,4-dioxane. The mixture was stirred and refluxed at 105 °C for 5 hours. After complete reaction, the solvent was evaporated to dryness. The crude product was purified by silica gel column chromatography (eluent: DCM:EA = 4:1) to give the desired compound D₇-7 as a yellow solid. Yield: 85.8%; ESI-MS: m / z = 587 [M+H] + ;

[0270] Step 2: Add HCl / EA solution (2 mol / L, 30 mL) to compound D7-7 (0.7 g, 1.193 mmol). Stir the mixture at room temperature for 15 hours. After complete reaction, filter, dissolve the resulting brown solid in a small amount of water, adjust the pH to 6-8 with saturated NaHCO3 solution, filter, and slurry with EA / PE to obtain the desired compound AIT010079, a white solid. Yield: 82.4%; ESI-MS: m / z = 387 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ7.88(s,1H),7.58(d,J=3.2Hz,1H),7.34-7.27(m,4H),7.22-7.16(m,1H),7.07(s,1H),6.24(d,J=3.2Hz ,1H),4.57(s,2H),3.24-3.18(m,2H),2.38(t,J=6.3Hz,2H),2.09(q,J=4.0Hz,2H),1.82(d,J=6.1Hz,2H),1.20(q,J=4.2Hz,2H).

[0271] Example 38. Evaluation of bioactivity

[0272] I. In vitro p97 enzyme activity assay

[0273] The p97 enzyme activity assay is an initial screening assay used in this invention to determine the inhibitory activity of compounds against the p97 complex. Inhibition of the p97 proteasome complex activity can lead to the accumulation of pro-apoptotic transcription factors in cancer cells, ultimately resulting in apoptosis of cancer cells.

[0274] Reagents used for p97 enzyme activity assay include:

[0275] The assay buffer consisted of a mixture of 50 mM Tris HCl pH 7.5, 20 mM MgCl2, 0.02% Triton TX-100, 1 mM DTT, and 0.2% (v / v) glycerol. The plate type was Corning 3674, 384-well plate. The identification kit was the ADP-G1o kit (manufacturer: Promega, catalog number: V9102).

[0276] The ADP-Glo ​​kit assay is a luminescent assay used to determine ADP formed by an enzymatic reaction. In this specific study, the enzymatic reaction was completed by the hydrolysis of ATP by the p97 protein. In the second step, the enzymatic reaction terminated, and all remaining ATP was depleted. In the final step, ADP was converted to ATP, and the newly synthesized ATP was measured using a luciferase / luciferin reaction. The resulting light was measured using an Envision plate reader, where the obtained luminescent signal was positively correlated with kinase activity.

[0277] The measurement procedure is as follows:

[0278] 1. Add buffer, p97 protein (final concentration 50 nM), compound / DMSO (5 μL), and ATP (20 μL, final concentration 20 μM) to a 384-well plate and mix well to form a 50 μL reaction system. Centrifuge at 1000 rpm for 10 s and incubate at room temperature for 15 min.

[0279] 2. Take 5 μL of reaction solution, add 5 μL of ADP-Glo ​​reagent, centrifuge at 1000 rpm for 10 s, and incubate at room temperature for 40 min;

[0280] 3. Add 10 μL of kinase detection reagent, centrifuge at 1000 rpm for 10 s, incubate at room temperature for 30 min, and detect luminescence using an Envision plate reader;

[0281] Data processing:

[0282] The inhibition rate is calculated as follows:

[0283] Inhibition rate (%) = [1 - (RLU)] 样品 -RLU 空白对照组 ) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0284] Data Analysis:

[0285] Using the concentration logarithmic value as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using the log(Inhibitor) vs. response-variable slope (four parameters) analysis software GraphPad Prism9 to obtain the IC50 values ​​of each compound on enzyme activity. 50 value.

[0286] Table 1. In vitro p97 enzyme activity inhibition rate under the action of 500 nM compound.

[0287]

[0288] As shown in Table 1, the compounds of the present invention exhibit good p97 enzyme activity inhibition rates under the action of 500 nM compound, especially A11, A18, A19 and A25, which show good p97 enzyme activity inhibition rates under the action of 20 nM compound.

[0289] Table 2. IC50 of the compounds on p97 enzyme activity inhibition in vitro 50 value

[0290]

[0291] As shown in Table 2, the compounds of the present invention can achieve strong inhibition of p97 enzyme activity under low concentration conditions, and the inhibitory effects of compounds A4, A5, A8, A9 and A19 on p97 enzyme activity are significantly better than those of comparative compound A.

[0292] II. Detection of Cell Proliferation Inhibition Activity

[0293] The specific measurement procedure is as follows:

[0294] (1) Take colorectal cancer cells (HCT116) or bile duct cancer cells (RBE, HuCCT1, HCCC9810) in logarithmic growth phase and seed them in 96-well plates at 3000 to 5000 per well, with 100 μL of culture medium per well, and incubate at 37°C and 5% CO2 for 24 hours.

[0295] (2) Add 10 μL of different concentrations of the compound to each well, with at least two replicates for each concentration. Set up a blank control group and a DMSO solvent control group, and use compound A as a positive control.

[0296] (3) After incubating at 37°C for 72 hours, collect the plate and perform SRB testing.

[0297] SRB staining method: Discard the cell supernatant, rinse once with 1xPBS, and fix with 10% trichloroacetic acid for 1 h. Discard the trichloroacetic acid, wash away excess trichloroacetic acid with tap water, and dry the 96-well plate at 60℃. Add 70 μL of SRB staining solution to each well of the dried 96-well plate and stain at room temperature for 30 min. Wash away excess SRB staining solution with 1% glacial acetic acid, and dry the 96-well plate at 60℃. Dissolve the SRB in 100 μL of 10 mM Tris-base solution. Measure the absorbance at 540 nm using a microplate reader.

[0298] Data processing:

[0299] Cell viability is calculated as follows:

[0300] Cell viability (%) = [(RLU)] 样品 -RLU 空白对照组 ) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0301] Data Analysis:

[0302] Using the concentration logarithm as the X-axis and cell viability as the Y-axis, dose-response curves were fitted using the log(Inhibitor) vs. response-variable slope (four parameters) analysis software GraphPad Prism9 to derive the IC50 of each compound on cell proliferation inhibition. 50 value.

[0303] The results are shown in Tables 3 and 4.

[0304] Table 3. Effects of compounds on the proliferation of human colorectal cancer HCT116 cells

[0305] Compound numbering HCT116 cell proliferation inhibition IC50 (nM) A4 200.80 A5 144.00 A8 189.00 A9 195.00 A10 332.80 A11 271.50 A19 215.50 Comparison of compound A 230.90

[0306] Conclusion: The compounds of this invention have significant inhibitory activity against the proliferation of HCT116 cells, indicating that the compounds of this invention have good anti-colorectal cancer effects, and compounds A4, A5, A8, A9, and A19 have better activity than comparative compound A.

[0307] Table 4. Effects of compounds on the proliferation of human cholangiocarcinoma RBE, HuCCT1, and HCCC9810 cells. Conclusion: The compounds of this invention have significant inhibitory activity against the proliferation of RBE, HuCCT1 and RBE cells, indicating that the compounds of this invention have good anti-cholangiocarcinoma effects, and the activities of compounds A4 and A19 are superior to those of comparative compound A.

[0308] III. Pharmacokinetic Testing

[0309] The experiment was conducted at Hangzhou Pioneer Pharmaceutical Technology Co., Ltd.

[0310] Experimental materials: Male SD rats (7-9 weeks old, Shanghai Slack)

[0311] Experimental Procedure: The clear solution obtained after dissolving the test compound was administered to male SD rats by gavage (overnight fasting, 7-9 weeks old). Following administration of the test or control compound, blood was collected from the orbital cavity at 0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, and 24.00 hours, and plasma was obtained after centrifugation. Plasma drug concentrations were determined by LC-MS / MS, and relevant pharmacokinetic parameters were calculated using the non-compartmental linear logarithmic trapezoidal method with WinNonLin 8.0 pharmacokinetic statistical software.

[0312] Preparation and processing of standard curves and quality control samples: Take the mixed stock solution of compounds and dilute it with 50% methanol-water to prepare standard working solutions containing concentrations of 20, 40, 100, 200, 400, 1000, 2000, 4000, and 10000 ng / mL of each compound, and quality control working solutions containing concentrations of 60, 600, and 8000 ng / mL of each compound. 47.5 μL of blank rat plasma was taken and 2.50 μL of standard curve working solution and quality control working solution were added to prepare standard curves containing 1.00, 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, and 500.00 ng / mL of each compound, and quality control samples containing 3.00, 30.00, and 400.00 ng / mL. 200 μL of acetonitrile (containing 2 ng / mL of internal standard verapamil) was added to each sample. After vortexing for 3 min, the samples were centrifuged at 20000 rf and 4 °C for 10 min. The supernatant was then used for LC-MS / MS analysis.

[0313] Unknown sample preparation and processing: Take 50 μL of plasma sample, add 200 μL of acetonitrile (containing internal standard verapamil 2 ng / mL), vortex for 3 min, centrifuge at 20000 rcf, 4℃ for 10 min, and take the supernatant for LC-MS / MS analysis.

[0314] Unknown sample diluted 10-fold: Take 45 μL of blank plasma, add 5 μL of plasma sample, add 200 μL of acetonitrile (containing internal standard verapamil 2 ng / mL), vortex for 3 min, centrifuge at 20000 rcf, 4℃ for 10 min, and take the supernatant for LC-MS / MS analysis.

[0315] The results are shown in Table 5.

[0316] Table 5. Pharmacokinetic properties of the compounds of the present invention in rats

[0317]

[0318] Conclusion: The compounds of this invention are well absorbed in rats and exhibit pharmacokinetic advantages. The Cmax and AUC of A4 are significantly higher than those of the control compound A, while its clearance rate is lower.

[0319] IV. In vitro PDE6C enzyme activity assay

[0320] The previously reported p97 inhibitor CB-5083 was reported to have ocular toxicity due to its inhibition of PDE6C enzyme activity (Leinonen H. et al., J Pharmacol Exp Ther (2021) 378(1):31-41). Therefore, the inhibitory effect of the compound of the present invention on PDE6C enzyme activity was tested to rule out potential ocular toxicity.

[0321] The experiment was conducted at Beijing Aisiyipu Biotechnology Co., Ltd., and the specific operation process is as follows:

[0322] (1) Dilute the compound to 100 times the final concentration of the reaction with 100% DMSO. Transfer 50 μL of the diluted compound solution to the wells of a 384-well plate. For example, if the required maximum inhibitor concentration is 1 μM, prepare a 100 μM compound DMSO solution in this step.

[0323] (2) Transfer all compounds to one well of a 384-well plate and then dilute 100% DMSO three times in the next well, and so on, for a total of 10 concentrations.

[0324] (3) Transfer 0.05 μL of the reference solution to a 384 reaction plate.

[0325] (4) Add 2.5 μL of 2x PDE6C to each well.

[0326] (5) Add 2.5 μL of 2×fam-cycle-3,5-gmp to each well and incubate for 60 min.

[0327] (6) Add 15 μL of binder mixture to each well and incubate for 60 min.

[0328] (7) BMG reads the FP signal.

[0329] Data processing:

[0330] The inhibition rate is calculated as follows:

[0331] Inhibition rate (%) = [1 - (RLU)] 样品 -RLU 空白对照组) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0332] Data Analysis:

[0333] Using the concentration logarithmic value as the X-axis and the percentage inhibition rate as the Y-axis, dose-response curves were fitted using the log(Inhibitor) vs. response-variable slope (four parameters) analysis software GraphPad Prism9 to obtain the IC50 values ​​of each compound on enzyme activity. 50 value.

[0334] The results are shown in Table 6.

[0335] Table 6. IC50 of the compounds of the present invention inhibiting PDE6C enzyme activity in vitro. 50 value

[0336]

[0337] Conclusion: The compounds of this invention have no significant inhibitory effect on PDE6C at low concentrations.

[0338] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A compound, characterized in that, a compound of formula (I), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof; wherein, ring A is selected from a benzene ring, a 3-6 membered heterocyclic ring; heteroatom is selected from O, S, N; R1, R2are each independently selected from H, substituted or unsubstituted alkyl, cycloalkyl, aryl, heterocyclyl, halogen; L1is selected from ring B is independently selected from a benzene ring, a 3-6 membered heterocyclic ring; heteroatom is selected from O, S, N; R3, R4are each independently selected from the group consisting of H, substituted or unsubstituted C 1-5 alkyl, C 1-4 alkoxy, C 1-4 haloalkyl, halogen, cyano; the substituents are selected from the group consisting of C 1-4 at least one of alkyl, halogen, cyano, amino; R3and R4may form together with the connecting atoms a 5- or 6-membered heterocycloalkyl; R5and R6are each independently selected from H, C 1-5 alkyl; R5and R6may together with the atoms to which they are attached form a 3-6 membered heterocycloalkyl; R7is independently selected from H, C 1-5 alkyl; m is selected from 0, 1, 2, 3, 4; n is selected from 1, 2, 3.

2. The compound of claim 1, wherein, ring A is selected from a benzene ring, a 3-6 membered heterocyclic ring; heteroatom is selected from S, N; R1, R2are each independently selected from the group consisting of H, substituted or unsubstituted C 1-5 alkyl, C 3-5 cycloalkyl, C 6-10 aryl, C 3-10 heterocyclyl, halogen; L1is selected from ring B is independently selected from a benzene ring, a 3-6 membered heterocyclic ring; heteroatom is selected from S, N; R3, R4are each independently selected from the group consisting of H, substituted or unsubstituted C 1-5 alkyl, C 1-4 alkoxy, C 1-4 haloalkyl, halogen, cyano; the substituents are selected from the group consisting of C 1-3 alkyl, amino; R3and R4may form together with the connected atoms a 5- or 6-membered heterocycloalkyl; R5and R6are each independently selected from H, C 1-3 alkyl; R5and R6may together with the atoms to which they are attached form a 3-5 membered heterocycloalkyl; R7is independently selected from H, C 1-3 alkyl; m is selected from 0, 1, 2, 3; n is selected from 1, 2, 3.

3. The compound of claim 2, wherein, ring A is selected from a benzene ring, a 5 membered heterocyclic ring; heteroatom is selected from S, N; R1, R2are each independently selected from the group consisting of H, substituted or unsubstituted C 1-3 alkyl, halogen; L1is selected from ring B is independently selected from a benzene ring, a 5 membered heterocyclic ring; heteroatom is selected from S, N; R3, R4are each independently selected from the group consisting of H, substituted or unsubstituted C 1-3 alkyl, C 1-3 alkoxy, halogen; said substituents are selected from the group consisting of C 1-3 alkyl, at least one of amino; R3and R4may form together with the atoms to which they are attached a 5- or 6-membered heterocycloalkyl; R5and R6are each independently selected from H, C 1-3 alkyl; R5and R6may together with the atoms to which they are attached form a 3-5 membered heterocycloalkyl; R7is independently selected from H, C 1-3 alkyl; m is selected from 0, 1, 2; n is selected from 1 or 2.

4. The compound according to any one of claims 1 to 3, characterized in that, said compound is a compound of formula (II), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof; wherein ring A, R1, R2, ring B, R3, R 4、 n as in any one of claims 1-3; 5. The compound according to any one of claims 1 to 3, wherein said compound is a compound of formula (III), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof; wherein ring A, R1, R2, n are as described in any one of claims 1-3; p is independently selected from 1 or 2; 6. The compound of any one of claims 1-3, wherein said compound is a compound of formula (IV), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof; wherein ring A, R1, R2, n are as described in any one of claims 1-3; 7. The compound of any one of claims 1-3, wherein said compound is a compound of formula (V), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof; wherein ring A, R1, R2, R5, R6, m, n are as described in any one of claims 1-3; 8. The compound of claim 1, wherein The structural unit selected from 9. The compound of claim 1, wherein The structural unit selected from 10. The compound of claim 1, wherein The structural unit selected from 11. The compound of any one of claims 1-10, wherein, said compound is selected from any one of the following compounds:

12. The compound of claim 11, wherein, said compound is selected from any one of the following compounds:

13. The compound of claim 12, wherein said compound is selected from any one of the following compounds:

14. A pharmaceutical composition, characterized by, a compound of formula (I), or a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients.

15. Use of a compound, a stereoisomer, a geometric isomer, a tautomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof of any one of claims 1-13, or a pharmaceutical composition of claim 14, in the preparation of a medicament for preventing and / or treating a VCP / p97 abnormality disease.

16. The use according to claim 15, characterized in that, said disease is selected from a cancer, a metabolic disease, an inflammatory or autoimmune disease.

17. Use according to claim 16, characterized in that, said cancer is a solid tumor or a hematological tumor.

18. The use according to claim 17, characterized in that, The solid tumor is cholangiocarcinoma, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, ovarian cancer; the blood tumor is lymphocytic leukemia, chronic myeloid leukemia. The solid tumor is cholangiocarcinoma, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, ovarian cancer; the blood tumor is lymphocytic leukemia, chronic myeloid leukemia. The solid tumor is cholangiocarcinoma, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, ovarian cancer; the blood tumor is lymphocytic leukemia, chronic myeloid leukemia. The solid tumor is cholangiocarcinoma, colorectal cancer, gastric cancer, pancreatic cancer, liver

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