VCP / p97 inhibitor containing valine casein protein as well as preparation method and application of VCP / p97 inhibitor

Abstract

The invention belongs to the field of medicines, and provides a compound which is novel in structure type and better in druggability and is used for a VCP / p97 inhibitor, and a raceme, an enantiomer, a diastereoisomer and pharmaceutically acceptable salts thereof. The compound has a structural formula as shown in a formula I. The compound for the VCP / p97 inhibitor, provided by the invention, has a relatively good inhibition effect on malignant proliferation of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a valine peptide protein VCP / p97 inhibitor, its preparation method, 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, making the inhibition of this pathway 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 Degradation) (CarvaIho, P. et al., Cell (2006) 126, 361-373), 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 (BoeLens, J. et al., In Vivo (2007) 21, 215-226). Inhibition of p97 / VCP can trigger immunogenic cell death and inhibit proteasome degradation, autophagy, and DNA damage repair, thus becoming an emerging target for the treatment of 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 / adjuvant cancer therapy.

[0004] Investigational small-molecule inhibitors of the VCP / p97 protein 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. 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. Summary of the Invention

[0005] The purpose of this invention is to provide a compound with a novel structure and better drug-like properties for use as a VCP / p97 inhibitor.

[0006] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0007] According to a first aspect of the present invention, a compound of Formula I, its racemic form, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof are provided; Formula I is shown below:

[0008]

[0009] Where X is selected from N or O;

[0010] When X is 0, one of R1 and R2 does not exist, and the other is not selected from hydrogen.

[0011] When X is N, R1 and R2 are not both selected from hydrogen;

[0012] R1 and R2 are selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, 5-13 heterocyclic alkyl, 5-13 heteroaryl, or R1 and R2 are bonded together to form a 5-13 heterocyclic alkyl or 5-13 heteroaryl; wherein the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, 5-13 heterocyclic alkyl, or 5-13 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino groups, etc. Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

[0013] In one aspect of the invention, the invention provides a compound of Formula I, its racemic form, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof; Formula I is shown below:

[0014]

[0015] X is selected from N or O;

[0016] When X is 0, one of R1 and R2 does not exist, and the other is not selected from hydrogen.

[0017] When X is N, R1 and R2 are not both selected from hydrogen;

[0018] R1 and R2 are selected from hydrogen, C1-5 alkyl, C1-5 alkoxy, 5-7 membered heterocyclic alkyl, 5-7 membered heteroaryl, or R1 and R2 are bonded together to form 5-13 membered heterocyclic alkyl or 5-13 membered heteroaryl; wherein the C1-10 alkyl, C2-10 alkenyl, C2-10 alkoxy, 5-13 membered heterocyclic alkyl, or 5-13 membered heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, or amino groups. Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

[0019] In one aspect of the invention, the compound has a structure selected from any one of the structures shown in Formulas II to V:

[0020]

[0021]

[0022] Wherein, R1 and R2 are selected from C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl; wherein the C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino, Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl;

[0023] n1 is selected from 0, 1, or 2; n2 is selected from 0, 1, 2, or 3, and the n2 R7s may be the same or different from each other; R7 is selected from C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl; the C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino, Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

[0024] In one aspect of the invention, the compound has a structure selected from any one of the structures shown in Formula III-1, Formula IV-1, or Formula V-1:

[0025]

[0026]

[0027] Wherein, R9 is selected from C1-5 alkyl groups; R 10 Selected from hydrogen, amino, hydroxyl,

[0028] R 11 Selected from hydrogen, amino, hydroxyl, C1-5 alkyl, R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

[0029] In one aspect of the invention, the compound is selected from any of the following:

[0030]

[0031]

[0032] According to a second aspect of the present invention, a method is provided for preparing the aforementioned compounds having formula III-1 or V-1, their racemic mixtures, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof, the method comprising the following steps:

[0033]

[0034] Among them, R' 11 For R 11 Or R with a protecting group 11 ;R' 10 For R 10 Or R with a protecting group 10 R9, R 10 and R 11 The definition is as described above.

[0035] According to a third aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising a therapeutically effective amount of the aforementioned compound, its racemic form, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof; the pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0036] In one aspect of the invention, the pharmaceutical composition is an inhibitor of a VA-type peptide-containing protein, a member of the AAA family.

[0037] In one aspect of the invention, the valine-containing protein is present in human cells.

[0038] According to a fourth aspect of the invention, a method is provided for reducing the activity of valine-containing proteins or reducing the degradation of proteasome system substrates, the method comprising administering an effective amount of the aforementioned compound, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof to a patient.

[0039] According to a fifth aspect of the invention, a method for reducing the activity of valine-containing proteins or reducing the degradation of proteasome system substrates is provided, the method comprising administering an effective amount of the aforementioned pharmaceutical composition to a patient.

[0040] According to a sixth aspect of the invention, the use of the aforementioned compounds, their racemic mixtures, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof, or the aforementioned pharmaceutical compositions, in the prevention and / or treatment of VCP / p97 abnormalities is provided.

[0041] In one aspect of the invention, the abnormal disease is selected from cancer, metabolic diseases, inflammatory diseases, or autoimmune diseases.

[0042] In one aspect of the invention, the cancer is selected from solid tumors or hematologic malignancies.

[0043] In one aspect of the invention, the solid tumor is selected from bile duct cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, and ovarian cancer; the hematologic tumor is selected from lymphocytic leukemia and chronic myeloid leukemia.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention provides a compound that targets and inhibits the valine protein VCP / p97, which has a good inhibitory effect on the malignant proliferation of tumor cells. Detailed Implementation

[0046] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Unless otherwise stated, "(D)" indicates right-handed, "(L)" indicates left-handed, and "(DL)" indicates racemic.

[0054] 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

[0055] 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.

[0056] 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.

[0057] 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 substituent can be attached to the substituted group through any carbon atom on the benzene ring. A ring system formed by drawing a bond between a substituent and the central ring (as shown) represents the selective substitution of the substituent at all substituted positions on the ring system.

[0058] In this invention, the term "alkyl" refers to an aliphatic hydrocarbon group, which can be straight-chain or branched. Branched refers to one or more lower alkyl groups, such as methyl, ethyl, or propyl, that are linked together by a linear alkyl chain. "Lower alkyl" refers to a group containing about 1 to about 6 carbon atoms in the chain, which can be straight-chain or branched.

[0059] In this invention, the term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, which can be straight-chain or branched. Branched refers to one or more lower alkyl groups, such as methyl, ethyl, or propyl, attached to a linear alkenyl chain. "Lower alkenyl" refers to a group containing about 2 to about 6 carbon atoms in the chain, which can be straight-chain or branched.

[0060] In this invention, the term "alkynyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, which can be straight-chain or branched. Branching refers to one or more lower alkyl groups, such as methyl, ethyl, or propyl, attached to a linear alkynyl chain. "Lower alkynyl" refers to a chain containing about 2 to about 6 carbon atoms, which can be straight-chain or branched. Non-limiting examples of alkynyl groups include ethynyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, and decynyl.

[0061] In this invention, the term "aryl" refers to an aromatic monocyclic or polycyclic ring system. An aryl group may optionally be substituted with one or more "cyclic substituents," which may be the same or different, as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

[0062] In this invention, the term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system, wherein one or more ring atoms are elements other than carbon, such as nitrogen, oxygen, or sulfur, either individually or in combination. Preferably, a heteroaryl contains about 5 to about 6 ring atoms. A "heteroaryl" may optionally be substituted with one or more "cyclic substituents," which may be the same or different, as defined herein. The prefixes azido, oxa, or thiado preceding the heteroaryl root name indicate that at least one nitrogen, oxygen, or sulfur atom is present as a ring atom, respectively. The nitrogen atom of a heteroaryl may optionally be oxidized to the corresponding N-oxide. Suitable, non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl, furanyl, phenylthio, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrroleyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, 2,3-diazanaphthyl, imidazo[1,2-a]pyridyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indoleyl, azaindoleyl, benzimidazolyl, benzothiopheneyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindoleyl, 1,2,4-triazinyl, benzothiazolyl, etc.

[0063] In this invention, the term "amino" refers to the -NR'R" group. The amino group may optionally be substituted. In an unsubstituted amino group, R' and R" are hydrogen. In a substituted amino group, R' and R" can each independently be, but are not limited to, hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, alkylheteroalkyl, alkoxy, sulfonyl, alkenyl, alkanoyl, aryl, arylalkyl, or heteroaryl, provided that R' and R" are not both hydrogen. In a substituted amino group, R' and R" can be cyclized to form a cyclic amino group, such as pyrrolidinyl or piperidinyl. Such cyclic amino groups may incorporate other heteroatoms, for example, to form piperazine or morpholine groups. Such cyclic amino groups may optionally be substituted, for example, by an amino, hydroxyl, or oxo group.

[0064] In this invention, the term "alkoxy" refers to -O-alkyl. Alkoxy can refer to a straight-chain, branched, or cyclic, saturated or unsaturated oxy-hydrocarbon chain, including, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, and pentoxy. Alkoxy groups may optionally be substituted with one or more alkoxy substituents ("substituted alkoxy").

[0065] In this invention, the term "cycloalkyl" refers to a non-aromatic mono- or polycyclic ring system, preferably containing about 5 to about 7 ring atoms. The cycloalkyl group may optionally be substituted with one or more "cyclic substituents," which may be the same or different, as defined above. Suitable monocyclic cycloalkyl groups, without limitation, include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Suitable polycyclic cycloalkyl groups, without limitation, include 1-decahydronaphthyl, norcamphenyl, adamantyl, etc. In this invention, the term "cycloalkoxy" refers to a group in which one or more carbon atoms in the mono- or polycyclic ring system of the "cycloalkyl" group are substituted with oxygen atoms.

[0066] In this invention, the term "heterocyclic group" refers to a non-aromatic saturated monocyclic or polycyclic ring system, wherein one or more ring atoms in the ring system are elements other than carbon, such as nitrogen, oxygen, or sulfur, either individually or in combination. Adjacent oxygen and / or sulfur atoms are absent in the ring system, and the preferred heterocycle contains about 5 to about 6 ring atoms. The prefixes aza, oxa, or thioa preceding the name of the heterocyclic group indicate that at least one nitrogen, oxygen, or sulfur atom is present as a ring atom, respectively. The heterocyclic group may optionally be substituted with one or more "cyclic substituents," which may be the same or different, as defined herein. The nitrogen or sulfur atom of the heterocyclic group may optionally be oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclic rings include piperidinyl, pyrrolyl, piperazine, morpholinyl, thiomorpholinyl, thiazolyl, 1,3-dioxolanecycloyl, 1,4-dioxacyclohexyl, tetrahydrofuranyl, tetrahydrophenylthio, tetrahydrothiopyranyl, etc.

[0067] Unless otherwise specified, a range of numbers represents all integers including the numbers at both ends of the range. Unless otherwise specified, 1-5 represents 1, 2, 3, 4, 5; 5-7 represents 5, 6, 7; 1-3 represents 1, 2, 3; C1-3 alkyl represents C1, C2, C3 alkyl; C1-5 alkyl represents C1, C2, C3, C4, C5 alkyl; 5-7 heterocyclic alkyl represents 5, 6, 7 heterocyclic alkyl, and so on.

[0068] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0069] Example 1. Synthesis of intermediate A5

[0070]

[0071] Step 1: Dissolve A2 (1.47 g, 10.3 mmol) in anhydrous tetrahydrofuran. Add 1.6N n-butyllithium (8.13 mL, 13 mmol) dropwise under ice bath conditions. Stir at 0°C for 1 h. After the reaction is complete, add the resulting reaction solution dropwise to a solution of A1 dissolved in anhydrous tetrahydrofuran under ice bath conditions. Slowly restore to room temperature and stir for 2 h. After the reaction is complete, quench the reaction solution with saturated ammonium chloride. Extract three times with ethyl acetate, separate and combine the organic phases, wash the organic phase with an appropriate amount of saturated brine, dry the organic phase with anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 0 / 1 to 1 / 10) to obtain product A3 (550 mg).

[0072] Step 2: Dissolve A3 (550 mg, 1.83 mmol) in tetrahydrofuran, then add sodium bicarbonate (462 mg, 5.49 mmol) and A4 (107 mg, 1.65 mmol) sequentially, and react at room temperature for 2 h. After the reaction is complete, filter the reaction solution and collect the filter cake. Wash the filter cake twice with dichloromethane to obtain product A5 (425 mg). ESI-MS: m / z = 361 [M+H]+.

[0073] Example 2: Synthesis of Compound 1

[0074]

[0075] Step 1: Dissolve A6 (102 mg, 1.2 mmol) and potassium carbonate (525 g, 3.8 mmol) in 30 mL of acetonitrile, add A5 (425 mg, 1.2 mmol), and react at room temperature for 16 h. After the reaction is complete, filter the reaction solution and collect the filtrate. Concentrate the filtrate under reduced pressure and purify it by TLC (PE; EA = 1 / 5) to obtain product A7 (300 mg). ESI-MS: m / z = 412 [M+H]+.

[0076] Step 2: A7 (100 mg, 0.23 mmol), acetaldehyde oxime (30 mg, 0.5 mmol), Pd(OAc)2 (7 mg, 0.02 mmol), and triphenylphosphine (8 mg, 0.04 mmol) were dissolved in an appropriate amount of 1,4-dioxane and reacted by microwave heating to 100 °C for 1 h under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by TLC (PE:EA = 0 / 1) to obtain compound 1 (40 mg). ESI-MS: m / z = 430 [M+H]+. 1H NMR (400MHz, DMSO-d6) δ8.98–8.92(m,1H),8.25–8.19(m,1H),8.15–8.05(m,1H),7.86(s,1 H),7.55(t,J=8.2Hz,1H),7.35(d,J=9.2Hz,2H),7.32–7.27(m,3H),7.19(dd,J=13.5,8.2H z,1H),7.13(dd,J=8.3,3.7Hz,1H),5.01–4.95(m,1H),4.56(d,J=6.3Hz,1H),4.50(d,J=6. 3Hz,1H),4.34(d,J=11.2Hz,1H),3.77–3.41(m,4H),2.03–1.92(m,1H),1.90–1.81(m,1H).

[0077] Example 3: Synthesis of Compound 2

[0078]

[0079] The steps in Example 3 are the same as in Example 2, except that A6 is replaced with A6-1 to obtain compound 2. ESI-MS: m / z = 430 [M+H]+ 1 H NMR(400MHz, DMSO-d6)δ9.09–8.66(m,1H),8.26(q,J=3.8,3.1Hz,1H),8.19–8.08(m,1H),7.91(s,1H),7.65–7.55(m,1H),7.44– 7.13(m,8H),5.02(dt,J=6.1,3.2Hz,1H),4.57(dd,J=24.5,6.4Hz,2H),4.43–4.31(m,1H),3.81–3.44(m,4H),2.08–1.81(m,2H).

[0080] Example 4: Synthesis of Compound 3

[0081]

[0082] The procedure for Example 4 was the same as that for Example 2, except that A6 was replaced with A6-2, yielding compound 3. ESI-MS: m / z = 430.2 [M+H] + , 1 H NMR(400MHz, DMSO-d6)δ9.00–8.67(m,1H),8.22(q,J=3.9,3.3Hz,1H),8.09(dt,J=8.8,5.9Hz,1H),7.86(s,1H),7.61–7.51(m,1H),7 .38–7.11(m,8H),4.98(dt,J=6.0,3.0Hz,1H),4.53(dd,J=24.3,6.4Hz,2H),4.40–4.29(m,1H),3.76–3.42(m,4H),2.04–1.78(m,2H).

[0083] Example 5: Synthesis of Compound 4

[0084]

[0085] The steps in Example 5 were the same as in Example 2, except that A6 was replaced with A6-3, resulting in compound 4. ESI-MS: m / z = 457.3 [M+H]+ 1 H NMR (400MHz, DMSO-d6) δ8.34–8.09(m,2H),7.90(s,1H),7.67–7.54(m,1H),7.41–7.14(m,8H),4.56(d d,J=25.7,6.1Hz,2H),3.96–3.12(m,6H),2.73(dt,J=15.1,7.8Hz,1H),2.19(dd,J=12.4,2.8Hz,6H).

[0086] Example 6: Synthesis of Compound 5

[0087]

[0088] The procedure for Example 6 was the same as that for Example 2, except that A6 was replaced with A6-4, yielding compound 5. ESI-MS: m / z = 457.3 [M+H] + , 1H NMR(400MHz,DMSO-d6)δ8.86(dt,J=107.1,9.1Hz,1H),8.31–8.22(m,1H),8.21–8.11(m ,1H),7.90(d,J=5.8Hz,1H),7.59(dd,J=9.9,7.5Hz,1H),7.45–7.14(m,8H),4.65–4.46 (m,2H),3.96–3.66(m,2H),3.58–3.41(m,1H),3.32–3.13(m,1H),2.74(dq,J=15.5,7.7 Hz,1H),2.19(dd,J=12.3,2.8Hz,6H),2.15–2.04(m,1H),1.78(dq,J=12.5,9.4Hz,1H).

[0089] Example 7: Synthesis of Compound 6

[0090]

[0091] Step 1: Step 1 is the same as in Example 2, except that A6 is replaced with A6-5 to obtain intermediate A8. ESI-MS: m / z = 529.0 [M+H]+.

[0092] Step 2: Place A8 (53 mg, 0.1 mmol) in a single-necked flask, add 2 ml of DCM, then add 1 ml of trifluoroacetic acid, and stir at room temperature for 2 hours. Add saturated Na2CO3 solution dropwise to adjust the pH to approximately 7.0, evaporate to dryness, and perform preparative silica gel plate purification (DCM:MeOH = 15:1) to obtain 23 mg of white solid. ESI-MS: m / z = 429.0 [M+H]+. 1 H NMR (400MHz, DMSO-D6) δ8.24(q,J=4.1Hz,1H),8.18–8.06(m,1H),7.88(s,1H),7.58(t,J=8.4Hz,1H),7.46–7.26(m,6H),7.26–7.13(m,2H) ,4.56(dd,J=22.5,6.2Hz,2H),3.79–3.63(m,2H),3.52(d,J=17.8Hz,2H),2.09–1.94(m,1H),1.74(d,J=6.2Hz,1H),0.85(t,J=6.5Hz,1H).

[0093] Example 8: Synthesis of Compound 7

[0094]

[0095] The steps in Example 8 were the same as in Example 7, except that A6-5 was replaced with A6-6, resulting in compound 7. ESI-MS: m / z = 429.0 [M+H]+. 1 H NMR (400MHz, DMSO-D6) δ8.25(q,J=4.4Hz,1H),8.17(s,1H),7.90(s,1H),7.59(t,J=8.4Hz,1H),7.34(dt,J=18.6,6.4Hz,6H) ,7.26–7.15(m,2H),4.57(dd,J=23.1,6.3Hz,2H),3.86–3.63(m,4H),2.14(s,1H),1.84(s,1H),0.85(dd,J=9.5,6.4Hz,1H).

[0096] Example 9: Synthesis of Compound 8

[0097]

[0098] The steps in Example 9 were the same as in Example 2, except that A6 was replaced with A6-7, yielding compound 8. ESI-MS: m / z = 444.3 [M+H] + , 1 H NMR(400MHz,DMSO-d6)δ8.99(d,J=8.3Hz,1H),8.77(dt,J=51.6,6.3Hz,1H),8.23( dd,J=5.4,3.7Hz,1H),7.93(d,J=7.6Hz,1H),7.63(ddd,J=12.8,7.6,0.9Hz,1H),7. 41–7.29(m,6H),7.28–7.19(m,2H),5.47–5.37(m,1H),4.61(dd,J=29.5,6.2Hz,2H ),2.91–2.67(m,3H),2.47–2.35(m,1H),2.30(d,J=13.7Hz,4H),1.95–1.82(m,1H).

[0099] Example 10: Synthesis of Compound 9

[0100]

[0101] The steps in Example 10 are the same as in Example 2, except that A6 is replaced with A6-8, yielding compound 9. ESI-MS: m / z = 444.2 [M+H]+; 1H NMR(400MHz,DMSO-d6)δ8.99(d,J=8.3Hz,1H),8.82(t,J=6.2Hz,1H),8.75–8 .64(m,1H),8.23(t,J=4.4Hz,1H),7.94(d,J=7.6Hz,1H),7.63(dd,J=12.9,7 .5Hz,1H),7.47–7.17(m,8H),5.49–5.32(m,1H),4.61(dd,J=29.3,6.2Hz,2H ),2.86–2.58(m,3H),2.29(dd,J=28.9,14.8Hz,5H),1.86(t,J=15.8Hz,1H).

[0102] Example 11: Synthesis of Compound 10

[0103]

[0104] The procedure in Example 11 was the same as in Example 7, except that A6-5 was replaced with A6-9, yielding compound 10. ESI-MS: m / z = 430.3 [M+H] + , 1 H NMR (400MHz, DMSO-d6) δ9.01–8.91(m,1H),8.83–8.61(m,1H),8.23(t,J=4.3Hz, 1H),7.95(d,J=8.0Hz,1H),7.64(dd,J=13.5,7.5Hz,1H),7.43–7.19(m,8H),5.6 5(dt,J=6.8,3.2Hz,1H),4.62(dd,J=31.7,6.2Hz,2H),3.54–3.42(m,2H),3.37( t,J=12.2Hz,1H),3.30–3.22(m,2H),2.21(dtd,J=25.5,13.8,11.7,6.4Hz,2H).

[0105] Example 12: Synthesis of Compound 11

[0106]

[0107] The steps in Example 12 are the same as in Example 7, except that A6-5 is replaced with A6-10, yielding compound 11. ESI-MS: m / z = 430.2 [M+H]+; 1H NMR (400MHz, DMSO-d6) δ9.32(s,2H),9.06–8.92(m,1H),8.86–8.60(m,1H),8.23(t,J=4.2Hz,1H),7.96(d,J=8.1Hz,1H),7.65(dd,J=13.5,7.5Hz,1H ),7.46–7.19(m,9H),5.67(td,J=4.9,2.5Hz,1H),4.63(dd,J=32.4,6.2Hz ,2H),3.63–3.43(m,2H),3.37–3.27(m,2H),2.28(th,J=19.8,7.1Hz,2H).

[0108] Example 13: Synthesis of Compound 12

[0109]

[0110] The steps in Example 13 are the same as in Example 2, except that A6 is replaced with A6-11 to obtain compound 12. ESI-MS: m / z = 431 [M+H]+ 1 H NMR (400MHz, DMSO-d6) δ9.14–8.67(m,1H),8.29–8.03(m,2H),7.93(d,J=19.5Hz,1H),7.64–7.50(m,1H),7.44–7.14(m,9H),4.57( dd,J=18.1,6.3Hz,2H),3.45(ddt,J=32.4,12.8,6.9Hz,2H),2.53(d,J=7.6Hz,1H),2.41(t,J=6.6Hz,1H),2.20(d,J=38.0Hz,6H).

[0111] Example 14: Synthesis of Compound 13

[0112]

[0113] The procedure for Example 14 is the same as that for Example 2, except that A6 is replaced with A6-12, yielding compound 13. ESI-MS: m / z = 432.3 [M+H] + , 1H NMR(400MHz,DMSO-d6)δ9.15–8.65(m,1H),8.31–8.05(m,2H),7.96–7.85(m,1H),7.63–7.56(m,1H),7.42–7.15(m,9H),4 .65–4.55(m,2H),4.53(d,J=7.4Hz,1H),3.41(d,J=6.2Hz,1H),3.33(t,J=6.8Hz,1H),1.15(d,J=2.2Hz,4H),1.05(s,2H).

[0114] Example 15: Synthesis of Compound 14

[0115]

[0116] The procedure in Example 15 was the same as in Example 2, except that A6 was replaced with A6-13, yielding compound 14. ESI-MS: m / z = 466.2 [M+H] + ,1H NMR (400MHz, DMSO-d6) δ9.07–8.69(m,1H),8.36–8.21(m,1H),8.21–8.03(m,1H),7.90(d,J=6.1Hz,1H),7.77(dt,J=44.3,6.0Hz,1H),7.65–7.55(m ,1H),7.45–7.26(m,6H),7.25–7.15(m,2H),4.65–4.49(m,2H),3.85–3.6 6(m,2H),3.44(t,J=7.1Hz,1H),3.31(t,J=7.0Hz,1H),3.08–2.93(m,3H).

[0117] Example 16: Synthesis of Compound 15

[0118]

[0119] The steps in Example 15 are the same as in Example 2, except that A6 is replaced with A6-14, resulting in compound 15. ESI-MS: m / z = 467.3 [M+H]+; 1H NMR(400MHz, DMSO-d6)δ9.15–8.64(m,1H),8.37–8.01(m,2H),7.90(d,J=5.7Hz,1H),7.76–7.54(m,2H),7.46–7.12 (m,9H),6.99(d,J=17.5Hz,2H),4.58(dd,J=23.2,6.2Hz,2H),3.75(dq,J=29.6,6.8,6.1Hz,2H),3.35–3.23(m,2H).

[0120] Bioactivity assay 1: In vitro p97 enzyme activity determination

[0121] 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.

[0122] Reagents used for p97 enzyme activity assay include:

[0123] 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).

[0124] 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.

[0125] The measurement procedure is as follows:

[0126] (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.

[0127] (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;

[0128] (3) Add 10 μL of kinase detection reagent, centrifuge at 1000 rpm for 10 s, incubate at room temperature for 30 min, and use an Envision plate reader to detect luminescence;

[0129] Data processing:

[0130] The inhibition rate is calculated as follows:

[0131] Inhibition rate (%) = [1 - (RLU)] 样品 -RLU 空白对照组 ) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0132] Data Analysis:

[0133] 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.

[0134] The experimental results are shown in Tables 1 and 2 below:

[0135] Table 1. In vitro p97 enzyme activity inhibition rate under the action of 500 nM compound.

[0136]

[0137]

[0138] Table 2. IC50 of the compounds on p97 enzyme activity inhibition in vitro. 50 value

[0139] Compound numbering <![CDATA[p97 enzyme activity inhibition IC 50 (nM)]]> Compound numbering <![CDATA[p97 enzyme activity inhibition IC 50 (nM)]]> 1 31.70 13 70.31 2 18.88 14 70.61 4 58.62 15 10.58 5 15.21

[0140] As shown in Tables 1 and 2, the compounds of the present invention can achieve strong inhibition of p97 enzyme activity even at low concentrations.

[0141] Bioactivity assay 2: Detection of cell proliferation inhibitory activity

[0142] The specific measurement procedure is as follows:

[0143] (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.

[0144] (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.

[0145] (3) After incubating at 37°C for 72 hours, collect the plate and perform SRB testing.

[0146] 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.

[0147] Data processing:

[0148] Cell viability is calculated as follows:

[0149] Cell viability (%) = [(RLU)] 样品 -RLU 空白对照组 ) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0150] Data Analysis:

[0151] 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.

[0152] Bioactivity assay 3: Pharmacokinetic assay

[0153] Experimental materials: Male SD rats (7-9 weeks old, Shanghai Slack)

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] Bioactivity assay 4: In vitro PDE6C enzyme activity assay

[0159] 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.

[0160] The specific operation procedure of the experiment is as follows:

[0161] (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.

[0162] (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.

[0163] (3) Transfer 0.05 μL of the reference solution to a 384 reaction plate.

[0164] (4) Add 2.5 μL of 2x PDE6C to each well.

[0165] (5) Add 2.5 μL of 2×fam-cycle-3,5-gmp to each well and incubate for 60 min.

[0166] (6) Add 15 μL of binder mixture to each well and incubate for 60 min.

[0167] (7) BMG reads the FP signal.

[0168] Data processing:

[0169] The inhibition rate is calculated as follows:

[0170] Inhibition rate (%) = [1 - (RLU)] 样品 -RLU 空白对照组 ) / (RLU DMSO组 -RLU 空白对照组 )]×100%

[0171] Data Analysis:

[0172] 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.

Claims

1. The compound represented by Formula I, its racemic mixture, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof: in, X is selected from N or O; When X is 0, one of R1 and R2 does not exist, and the other is not selected from hydrogen. When X is N, R1 and R2 are not both selected from hydrogen; R1 and R2 are selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, 5-13 heterocyclic alkyl, 5-13 heteroaryl, or R1 and R2 are bonded together to form a 5-13 heterocyclic alkyl or 5-13 heteroaryl; wherein the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C1-10 alkoxy, 5-13 heterocyclic alkyl, or 5-13 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino groups, etc. Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

2. The compound according to claim 1, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, characterized in that, The compound satisfies the following: X is selected from N or O; When X is 0, one of R1 and R2 does not exist, and the other is not selected from hydrogen. When X is N, R1 and R2 are not both selected from hydrogen; R1 and R2 are selected from hydrogen, C1-5 alkyl, C1-5 alkoxy, 5-7 membered heterocyclic alkyl, 5-7 membered heteroaryl, or R1 and R2 are bonded together to form 5-13 membered heterocyclic alkyl or 5-13 membered heteroaryl; wherein the C1-10 alkyl, C2-10 alkenyl, C2-10 alkoxy, 5-13 membered heterocyclic alkyl, or 5-13 membered heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, or amino groups. Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

3. The compound according to claim 1, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, characterized in that, The compound has a structure selected from any of the structures shown in Formulas II to V: Wherein, R1 and R2 are selected from C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl; wherein the C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino, Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl; n1 is selected from 0, 1, or 2; n2 is selected from 0, 1, 2, or 3, and the n2 R7s may be the same or different from each other; R7 is selected from C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl; the C1-5 alkyl, C1-5 alkoxy, 5-7 heterocyclic alkyl, or 5-7 heteroaryl is optionally surrounded by one or more halogen atoms, C1-3 alkyl, hydroxyl, amino, Substitution; wherein R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

4. The compound according to claim 3, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, characterized in that, The compound has a structure selected from any one of the structures shown in Formula III-1, Formula IV-1, or Formula V-1: Wherein, R9 is selected from C1-5 alkyl groups; R 10 Selected from hydrogen, amino, hydroxyl, R 11 Selected from hydrogen, amino, hydroxyl, C1-5 alkyl, R3 and R4 are each independently selected from hydrogen, amino, hydroxyl or C1-5 alkyl.

5. The compound according to any one of claims 1-4, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from any of the following:

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of the compound according to any one of claims 1-5, its racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof; the pharmaceutical composition further contains one or more pharmaceutically acceptable carriers, diluents, or excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is an inhibitor of a VA-type peptide-containing protein, a member of the AAA family.

8. The pharmaceutical composition according to claim 7, characterized in that, The valine-containing protein is found in human cells.

9. A method for reducing the activity of valine-containing proteins or reducing the degradation of proteasome system substrates, the method comprising administering an effective amount of the compound according to any one of 1-5, its racemic mixture, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof to a patient.

10. A method for reducing the activity of valine-containing proteins or reducing the degradation of proteasome system substrates, the method comprising administering an effective amount of the pharmaceutical composition according to any one of claims 6-8 to a patient.

11. The use of the compound according to any one of claims 1-5, its racemic mixture, enantiomer, diastereomer, and pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 6-8 in the prevention and / or treatment of VCP / p97 abnormalities.

12. The application according to claim 11, characterized in that, The abnormal disease is selected from cancer, metabolic disease, inflammatory disease, or autoimmune disease.

13. The application according to claim 12, characterized in that, The cancer is selected from solid tumors or hematologic malignancies.

14. The application according to claim 13, characterized in that, The solid tumors are selected from bile duct cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, melanoma, breast cancer, prostate cancer, and ovarian cancer; the hematologic tumors are selected from lymphocytic leukemia and chronic myeloid leukemia.

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