A compound with STING protein degradation effect, its preparation method and uses

Abstract

The present invention provides a compound having the effect of degrading STING protein, a preparation method thereof, and uses thereof. The compound having the effect of degrading STING protein according to the present invention has a structural formula as shown in Formula I. The compound provided by the present invention can effectively degrade STING protein in cells through the ubiquitin-proteasome pathway. The experimental results of human retinal pigment epithelial cells and human immune cells show that the cytotoxicity of the compound of the present invention is much lower than that of the only currently marketed STING degrader, SP23, while the degradation effect on STING is almost the same. The present invention provides more options for the treatment of STING-related autoimmune and inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and particularly relates to a compound having the effect of degrading STING protein, and a preparation method and use thereof. Background Art

[0002] Stimulator of Interferon Genes (STING) is a protein that plays a key role in the innate immune system of cells and is widely expressed in various tissues. STING protein is a transmembrane protein mainly located on the endoplasmic reticulum of cells. By binding to intracellular DNA, it activates downstream signaling pathways, triggering immune cells to produce interferons and other immune regulatory molecules, thereby participating in antiviral immune responses and autoimmune regulation.

[0003] Since STING protein plays a core role in the innate immune response of cells, its abnormal activation is related to a variety of diseases, especially diseases related to the immune system. For example, abnormal STING signals may cause the immune system to attack its own tissues, thereby triggering autoimmune diseases; the over-activation of STING protein promotes the production of inflammatory cytokines, which may be related to chronic inflammatory diseases. Based on the double-edged sword nature of STING in immune responses and its potential role in various diseases, small molecules targeting STING have become a new treatment strategy for treating these diseases.

[0004] Ubiquitin is a small molecule protein consisting of 76 amino acids with a highly conserved sequence present in eukaryotic cells. The main function of ubiquitin is to label target proteins, and the labeled target proteins can be recognized and degraded by the proteasome, and this process is called the ubiquitin / proteasome system. Among them, the E3 ubiquitin-protein ligase directly binds to the protein, determining the specificity of degradation. The degradation process of the ubiquitin / proteasome system mainly includes the following four steps: 1) Ubiquitin activation: The carboxyl residue of ubiquitin binds to the sulfhydryl group of ubiquitin-activating enzyme E1; 2) Ubiquitin cross-linking: E1 transfers the activated ubiquitin to ubiquitin-conjugating enzyme E2 through a transesterification process; 3) E3 binds the ubiquitin complex to the target protein: The ubiquitin-protein ligase E3 links the ubiquitin bound to E2 to the target protein. If ubiquitin already exists on the target protein, the ubiquitin bound to E2 can be directly linked to the target protein; 4) Proteasome degradation: The proteasome recognizes the labeled target protein, thereby hydrolyzing the target protein into peptide chains with a length of 7-8 amino acids, completing the degradation of the target protein.

[0005] A proteolysis targeting chimeric molecule is a bifunctional molecule that can bind to both an E3 ubiquitin ligase and a target protein simultaneously, ubiquitinate the target protein that could not originally bind to the E3, and undergo selective degradation through the ubiquitin / proteasome system, thereby controlling the level of the target protein in the cell. Currently, the main E3 ubiquitin ligases include CRBN, VHL, MDM2, cIAP1, etc. PROTAC has three essential components: a warhead, an E3 ligase ligand, and a linker connecting them. The selection of the warhead, the composition, length, and attachment site of the linker are important factors in constructing PROTAC.

[0006] The currently marketed STING degradation inhibitor is SP23. Through experiments, it was found that although SP23 has good degradation efficiency, it has relatively high cytotoxicity, which greatly limits its subsequent use. Summary of the Invention

[0007] The purpose of the first aspect of the present invention is to provide a compound with the effect of degrading STING protein. The experimental results of human retinal pigment epithelial cells and human immune cells show that the cytotoxicity of the small molecule PROTAC drugs of the present invention is much lower than that of the currently marketed STING degrader (SP23), while the degradation effect on STING is almost the same.

[0008] The purpose of the second aspect of the present invention is to provide a pharmaceutical composition containing the above compound.

[0009] The purpose of the third aspect of the present invention is to provide a preparation method of the above compound.

[0010] The purpose of the fourth aspect of the present invention is to provide the use of the above compound in the preparation of drugs related to diseases associated with STING activity, or in the preparation of drugs related to inflammatory diseases and / or autoimmune diseases.

[0011] The purpose of the fifth aspect of the present invention is to provide a method for preventing and / or treating diseases related to the function of STING protein.

[0012] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0013] According to the first aspect of the present invention, there is first provided a compound represented by Formula I, or its deuterated compound, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, prodrug, or its pharmaceutically acceptable salt:

[0014] X - Y - Z

[0015] Formula I

[0016] wherein,

[0017] X is selected from

[0018]

[0019] wherein,

[0020] R 1 is selected from hydrogen, halogen, cyano, nitro, hydroxy, -C 1～6 alkyl, -C 2～6 alkenyl, -C 2～6 alkynyl, halogen-substituted -C 1～6 alkyl, halogen-substituted -C 2～6 alkenyl, halogen-substituted -C 2～6 alkynyl, -O(C 1～6 alkyl), -NH2, -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl);

[0021] R 2 is selected from hydrogen, halogen, cyano, nitro, hydroxy, -C 1～6 alkyl, -C 2～6 alkenyl, -C 2～6 alkynyl, halogen-substituted -C 1～6 alkyl, halogen-substituted -C 2～6 alkenyl, halogen-substituted -C 2～6 alkynyl, -O(C 1～6 alkyl), -NH2, -NH(C 1～6 alkyl), -N(C 1～6 alkyl)(C 1～6 alkyl);

[0022] Y is a linking group;

[0023] Z is a group that binds to the E3 ubiquitin ligase.

[0024] In some embodiments of the present invention, the X is selected from

[0025]

[0026] R 1 is selected from hydrogen, fluorine, chlorine, bromine, iodine, cyano, hydroxy, methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, -NH2, -NH(methyl), -N(methyl)(methyl); more preferably, R 1 is selected from fluorine.

[0027] R 2 is selected from hydrogen, fluorine, chlorine, bromine, iodine, cyano, hydroxy, methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, -NH2, -NH(methyl), -N(methyl)(methyl); more preferably, R 2 is selected from hydroxy.

[0028] In some embodiments of the present invention, the X is selected from

[0029]

[0030] In some embodiments of the present invention,

[0031] Y is selected from -(L Y ) q -;

[0032] q is an integer from 1 to 30;

[0033] Each L Y is independently selected from C(R)2, C(O), O, S, S(O), S(O)2, NR, -CR=CR-, -C≡C-, 3- to 10-membered cycloalkane, 3- to 10-membered heterocycloalkane, 6- to 10-membered aromatic ring, 5- to 10-membered heteroaromatic ring, 5- to 12-membered spiro ring, 5- to 12-membered spiroheterocyclic ring, 5- to 12-membered bridged ring, 5- to 12-membered bridged heterocyclic ring; wherein the cycloalkane, heterocycloalkane, aromatic ring, heteroaromatic ring, spiro ring, spiroheterocyclic ring, bridged ring, bridged heterocyclic ring may be further substituted by one, two or three R YL substituents;

[0034] Each R YL is independently selected from hydrogen, halogen, cyano, nitro, -C 1～6 alkyl, halogen-substituted C 1～6 alkyl, -OR, -N(R)(R);

[0035] Each R is independently selected from hydrogen, halogen, -C 1～6 alkyl, halogen-substituted C 1～6 alkyl.

[0036] In some embodiments of the present invention,

[0037] Y is selected from wherein the aa end indicates the direction in which the group is connected to X;

[0038] n1 is an integer from 0 to 10; preferably, n1 is selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0039] In some embodiments of the present invention, the Y is selected from

[0040]

[0041] Among them, the aa end represents the direction in which the group is connected to X.

[0042] In some embodiments of the present invention, the E3 ubiquitin ligase is selected from CRBN, von Hippel-Lindau (VHL), XIAP, MDM2 or cIAP-1; preferably, the E3 ubiquitin ligase is selected from CRBN.

[0043] The ubiquitin ligase, also known as the E3 ubiquitin ligase, is an enzyme that can connect a ubiquitin molecule to a certain lysine of the target protein.

[0044] Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex and is the target of immunomodulatory drugs such as thalidomide.

[0045] Von Hippel-Lindau or VHL is an E3 ligase.

[0046] X-linked inhibitor of apoptosis protein (XIAP) is a major member of the newly discovered IAP family. Experiments have confirmed that phosphorylated XIAP can exert the function of ubiquitin ligase.

[0047] MDM2 (Murine double minute 2) is one of the important members of the RING (Really interesting new gene) type ubiquitin protein ligase.

[0048] cIAP-1 (Cellular inhibitor of apoptosis protein-1) is the cellular inhibitor of apoptosis protein-1 and also has ubiquitin ligase activity.

[0049] In some embodiments of the present invention, Z is selected from

[0050]

[0051] In some preferred embodiments of the present invention, the compound represented by formula I is selected from the following compounds:

[0052]

[0053]

[0054] Generally speaking, PROTACs have three essential components: a warhead, an E3 ligase ligand, and a linker connecting them. The selection of the warhead, the composition, length, and attachment site of the linker are important factors in constructing PROTACs.

[0055] The compounds A1 (ZOC6), A2 (ZOC1), A3 (ZOC2), A4 (ZOC3), A5 (ZOC4), A6 (ZOC5), B2 (ZOC8), B3 (ZOC9), and B4 (ZOC10) described in the present invention all have a degrading effect on STING.

[0056] The currently marketed STING degrading inhibitor is SP23. Through experiments, it was found that although SP23 has good degradation efficiency, its cytotoxicity is relatively high, which has great limitations for subsequent use. The applicant obtained the compounds in the present invention by changing the warhead and the linker. In ARPE cells, the degradation efficiency of B2 (ZOC8) and SP23 on STING protein is close: the maximum degradation rate of B2 is 72.2%, and the maximum degradation rate of SP23 is 71.8%; in THP1 cells, the degradation efficiency of A1 (ZOC6) and SP23 on STING protein is close: the maximum degradation rate of A1 is 76.6%, and the maximum degradation rate of SP23 is 87.8%. However, under the same treatment conditions, the cytotoxicity of A1 (ZOC6) and B2 (ZOC8) among the compounds is 3 to 7 times lower than that of SP23. In in vivo experiments, after injection of SP23, the visual function of mice decreased, while the compound A1 (ZOC6) in the present invention has no effect on visual function.

[0057] According to the second aspect of the present invention, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned compounds, or their deuterated compounds, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, prodrugs, or their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier.

[0058] Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, etc.

[0059] The dosage forms of the composition include tablets, capsules, emulsions, suspensions, dispersions, or solutions.

[0060] According to the third aspect of the present invention, the present invention provides a method for synthesizing a compound, comprising the following steps:

[0061] Mix compound M1, compound M2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), and N,N-dimethylformamide (DMF), and react at room temperature for 10 to 20 hours to prepare compound S1; the reaction equation is as follows:

[0062]

[0063] Among them, n1 is selected from integers of 0 to 10;

[0064] R 1 is selected from hydrogen, halogen, cyano, nitro, hydroxyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, halogen-substituted -C 1-6 alkyl, halogen-substituted -C 2-6 alkenyl, halogen-substituted -C 2-6 alkynyl, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)(C 1-6 alkyl).

[0065] Preferably, stirring is required during the reaction. 1 Preferably, after the reaction, compound S1 is obtained through extraction, purification, and concentration under reduced pressure. 1 is preferably selected from fluorine.

[0066] Preferably, stirring is required during the reaction.

[0067] Preferably, after the reaction, compound S1 is obtained through extraction, purification, and concentration under reduced pressure.

[0068] The present invention also provides a method for synthesizing a compound, comprising the following steps:

[0069] Mix compound M2, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and pyridine and react for 1 to 10 minutes, then add compound M3 and react at room temperature for 1 to 4 hours to prepare compound S2; the reaction equation is as follows:

[0070]

[0071] Among them, n1 is selected from integers of 0 to 10;

[0072] R 1Selected from hydrogen, halogen, cyano, nitro, hydroxy, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, halogen-substituted -C 1-6 alkyl, halogen-substituted -C 2-6 alkenyl, halogen-substituted -C 2-6 alkynyl, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)(C 1-6 alkyl).

[0073] Preferably, R 1 is selected from hydrogen, fluorine, chlorine, bromine, iodine, cyano, hydroxy, methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, -NH2, -NH(methyl), -N(methyl)(methyl); more preferably, R 1 is selected from fluorine.

[0074] R 2 is selected from hydrogen, halogen, cyano, nitro, hydroxy, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, halogen-substituted -C 1-6 alkyl, halogen-substituted -C 2-6 alkenyl, halogen-substituted -C 2-6 alkynyl, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)(C 1-6 alkyl).

[0075] Preferably, R 2 is selected from hydrogen, fluorine, chlorine, bromine, iodine, cyano, hydroxy, methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, -NH2, -NH(methyl), -N(methyl)(methyl); more preferably, R 2 is selected from hydroxy.

[0076] Preferably, stirring is required during the reaction.

[0077] Preferably, after the reaction is completed, the compound S2 is obtained through extraction, purification, and concentration under reduced pressure.

[0078] The compounds of formula I of the present invention can all be prepared by the above-mentioned or similar preparation methods, and the corresponding starting materials can be selected according to the differences in substituents and the positions of substituents. Those skilled in the art should recognize that the above route helps to understand the present invention, but does not limit the content of the present invention.

[0079] According to the fourth aspect of the present invention, the present invention provides the use of any of the above-mentioned compounds, or their deuterated compounds, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, prodrugs, or their pharmaceutically acceptable salts, or pharmaceutical compositions in the preparation of drugs for preventing and / or treating diseases related to STING protein function.

[0080] Preferably, the diseases related to STING protein function include STING-associated vasculopathy with onset in infancy SAVI, Aicardi-Goutières syndrome AGS, COPA syndrome caused by mutations in the α subunit gene of the coatomer protein complex, systemic lupus erythematosus SLE, familial chilblain lupus erythematosus FCL, Parkinson's disease PD, amyotrophic lateral sclerosis ALS, Huntington's disease, non-alcoholic steatohepatitis NASH, alcoholic liver disease, nerve injury, rheumatoid arthritis, renal fibrosis, systemic sclerosis, intervertebral disc degeneration, pulmonary fibrosis, aging, scleroderma, psoriasis, inflammatory bowel disease, autoimmune colitis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, uveitis, mucositis, diabetes, cardiovascular disease or neurodegenerative disease.

[0081] The present invention also provides the use of any of the above-mentioned compounds, or their deuterated compounds, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotope-labeled compounds, metabolites, prodrugs, or their pharmaceutically acceptable salts, or pharmaceutical compositions in the preparation of drugs for preventing and / or treating inflammatory diseases and / or autoimmune diseases.

[0082] Preferably, the inflammatory diseases and autoimmune diseases include STING-associated vasculopathy with onset in infancy SAVI, Aicardi-Goutières syndrome AGS, COPA syndrome caused by mutations in the α subunit gene of the coatomer protein complex, systemic lupus erythematosus SLE, familial chilblain lupus erythematosus FCL, Parkinson's disease PD, amyotrophic lateral sclerosis ALS, Huntington's disease, non-alcoholic steatohepatitis NASH, alcoholic liver disease, nerve injury, rheumatoid arthritis, renal fibrosis, systemic sclerosis, intervertebral disc degeneration, pulmonary fibrosis, aging, scleroderma, psoriasis, inflammatory bowel disease, autoimmune colitis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, uveitis, mucositis, diabetes, cardiovascular disease or neurodegenerative disease.

[0083] According to a fifth aspect of the present invention, the present invention also provides a method for preventing and / or treating a disease, an inflammatory disease or an autoimmune disease related to the function of STING protein, which comprises administering to a subject in need thereof an effective dose of any of the above-mentioned compounds, or a deuterated compound, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, prodrug, pharmaceutically acceptable salt, or pharmaceutical composition thereof.

[0084] The compounds or pharmaceutical compositions of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir. The compounds or pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Oral compositions can be any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, and suspensions, dispersions and solutions.

[0085] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0086] Definition of terms used in the present invention: Unless otherwise specified, the initial definitions provided for groups or terms herein apply to such groups or terms throughout the specification; for terms not specifically defined herein, meanings that can be given to them by those skilled in the art should be provided according to the disclosure and context.

[0087] "Substituted" means that a hydrogen atom in a molecule is replaced by another different atom or group; or a lone pair of electrons of an atom in the molecule is replaced by another atom or group.

[0088] "Optionally substituted" means that "substituted" may or may not occur, and this description includes the cases of occurrence or non-occurrence.

[0089] The minimum and maximum carbon atom contents in a hydrocarbon group are indicated by a prefix. For example, the prefix C a～b alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C 1～6 alkyl refers to an alkyl group containing 1 to 6 carbon atoms.

[0090] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. The alkyl group can be straight-chain or branched-chain. Representative branched-chain alkyl groups have one, two, or three branches. The alkyl group can optionally be substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl. The alkyl group can also be part of other groups, such as -O(C 1～6 alkyl).

[0091] As used herein, "cycloalkane" refers to a saturated or non-aromatic partially saturated divalent cyclic alkane having a single ring or multiple rings (fused) with multiple carbon atoms and no ring heteroatoms. The term "cycloalkane" includes cycloalkenes, such as cyclohexene. Examples of monocyclic carbon-based groups include, for example, cyclopropane, cyclobutane, cyclohexane, cyclopentane, cyclooctane, cyclopentene, and cyclohexene, etc. Examples of fused cycloalkane systems include bicyclohexane, bicyclopentane, bicyclooctane, etc. As used herein, "cycloalkane" is implemented to include, but is not limited to etc.

[0092] As used herein, "heterocycloalkane" refers to a saturated ring or non-aromatic partially saturated divalent ring having a single ring or multiple rings (fused) containing at least one heteroatom; where the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. Examples of heterocycloalkanes in monocyclic heterocycloalkane systems are oxetane, azetidine, pyrrolidine, 2-oxo-pyrrolidine, tetrahydrofuran, tetrahydro-thiophene, pyrazolidine, imidazolidine, thiazolidine, piperidine, tetrahydropyran, tetrahydrothiopyran, piperazine, morpholine, thiomorpholine, 1,1-dioxo-thiomorpholine, azepane, diazepane, etc. Examples of fused heterocycloalkane systems include 8-aza-bicyclo[3.2.1]octane, quinuclidine, 8-oxa-3-aza-bicyclo[3.2.1]octane, 9-aza-bicyclo[3.3.1]nonane, etc. The term "heterocycloalkane" also includes cases where a partially saturated ring is formed by the fusion of an aromatic ring containing at least one heteroatom and a non-aromatic ring, and the connection site can be located at a non-aromatic carbon atom, an aromatic carbon atom, or a heteroatom.

[0093] As used herein, "spirocycle" refers to a saturated or non-aromatic partially saturated divalent ring formed by the spiro-fusion of two or more rings having multiple carbon atoms and no ring heteroatoms. As used herein, "spiroheterocycle" refers to a saturated ring or non-aromatic partially saturated divalent ring formed by the spiro-fusion of two or more rings containing at least one heteroatom.

[0094] As used herein, the term "bridged ring" refers to a saturated or partially saturated divalent ring formed by bridging multiple rings having multiple carbon atoms and no ring heteroatoms. The term "bridged heterocycle" as used herein refers to a saturated ring or a non-aromatic partially saturated divalent ring formed by bridging multiple rings containing at least one heteroatom.

[0095] As used herein, the term "unsaturated" means that a group or molecule contains a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-oxygen double bond, a carbon-sulfur double bond, a carbon-nitrogen triple bond, etc.

[0096] As used herein, the term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group having at least 1 vinyl unsaturation site (>C═C<). For example, C a-b An alkenyl group refers to an alkenyl group having from a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0097] As used herein, the term "alkynyl" refers to a straight-chain monovalent hydrocarbon group or a branched-chain monovalent hydrocarbon group containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, C 2-6 Alkynyl is intended to include ethynyl, propynyl, etc.

[0098] As used herein, the term "aromatic ring" refers to an aromatic hydrocarbon group having multiple carbon atoms. An aryl group is typically a monocyclic, bicyclic or tricyclic aryl group having multiple carbon atoms. In addition, the term "aryl" as used herein refers to an aromatic substituent that can be a single aromatic ring or multiple aromatic rings fused together. Non-limiting examples include phenyl, naphthyl or tetrahydronaphthyl.

[0099] As used herein, the term "heteroaromatic ring" refers to an aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. It is typically an aromatic monocyclic or bicyclic hydrocarbon containing multiple ring atoms, one or more of which are heteroatoms selected from O, N, S. Preferably, there are one to three heteroatoms. Heteroaryl groups, for example, represent: pyridyl, indolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, benzothienyl, benzopyranyl, benzothiopyranyl, furanyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl.

[0100] As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0101] As used herein, the term "halogen-substituted alkyl" means that one or more hydrogen atoms in an alkyl group are substituted by a halogen; for example, halogen-substituted C 1～4The alkyl group refers to an alkyl group containing 1 to 4 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms; for example, monofluoromethyl, difluoromethyl, and trifluoromethyl.

[0102] In the present invention, the "halogen-substituted alkenyl" means that one or more hydrogen atoms in the alkenyl group are replaced by halogen atoms; for example, halogen-substituted C 2-6 The alkenyl group refers to an alkenyl group containing 2 to 6 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms; for example, monofluoroethenyl, difluoroethenyl, and trifluoropropenyl.

[0103] In the present invention, the "halogen-substituted alkynyl" means that one or more hydrogen atoms in the alkynyl group are replaced by halogen atoms; for example, halogen-substituted C 2-6 The alkynyl group refers to an alkynyl group containing 2 to 6 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms; for example, monofluoroethynyl, difluoroethynyl, and trifluoropropynyl.

[0104] In the present invention, "-OR", "-N(R)2", etc. mean that the R group is connected to an oxygen atom or a nitrogen atom by a single bond.

[0105] In the present invention, "=O" means that an oxygen atom replaces two hydrogen atoms in the molecule through a double bond.

[0106] In the present invention, the oxygen atom in "-C(O)R", "-S(O)2R", etc. is connected to a carbon atom or a sulfur atom by a double bond, and the R group is connected to the oxygen atom or the sulfur atom by a single bond; for example, "-S(O)(NH)R" means that an oxygen atom and a nitrogen atom are connected to a sulfur atom by a double bond, and the R group is connected to the sulfur atom by a single bond.

[0107] In the description of the groups in the present invention is used to describe the position of group substitution.

[0108] The "deuterated compound" of the present invention means that one or more hydrogen atoms in a molecule or a group are replaced by deuterium atoms, and the proportion of deuterium atoms is greater than the natural abundance of deuterium.

[0109] The term "pharmaceutically acceptable" means that a certain carrier, vehicle, diluent, excipient, and / or the formed salt is usually chemically or physically compatible with other components constituting a pharmaceutical dosage form and is physiologically compatible with a receptor.

[0110] The terms "salt" and "pharmaceutically acceptable salt" refer to acid addition and / or base salts formed by the above-mentioned compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (inner salts), and also including quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained during the final separation and purification of the compound. They can also be obtained by appropriately (e.g., equimolarly) mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base. These salts may form precipitates in solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by lyophilization after reaction in an aqueous medium.

[0111] The term "prevention" includes inhibiting and delaying the onset of a disease, and includes not only prevention before the development of the disease, but also prevention of recurrence of the disease after treatment.

[0112] The term "treatment" means reversing, alleviating or eliminating the condition or disorder to which such term applies or the progression of one or more symptoms of such condition or disorder.

[0113] In certain embodiments, one or more compounds of the present invention can be used in combination with each other. Optionally, the compounds of the present invention can be combined with any other active agent for preparing a drug or pharmaceutical composition for regulating cell function or treating a disease. If a group of compounds is used, these compounds can be administered to a subject simultaneously, separately or sequentially. Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0114] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0115] The beneficial effects of the present invention are:

[0116] In vitro cell experiments prove that the novel STING small molecule PROTAC drugs with the structure of the present invention can effectively degrade STING protein in cells through the ubiquitin-proteasome pathway.

[0117] The experimental results of human retinal pigment epithelial cells and human immune cells show that the cytotoxicity of the small molecule PROTAC drugs of the present invention is much lower than that of the only currently marketed STING degrader (SP23), while the degradation effect on STING is almost the same. The present invention provides a better choice for treating STING-related autoimmune diseases and inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 1H NMR spectrum of compound A1 (ZOC6) in the embodiment of the present invention 1 1H NMR spectrum;

[0119] Figure 2 1H NMR spectrum of compound B2 (ZOC8) in the embodiment of the present invention 1 1H NMR spectrum;

[0120] Figure 3 Western blot analysis of the degradation effect of the compound in human retinal pigment epithelial cell line ARPE

[0121] Figure 4 SDS-PAGE analysis of the degradation effect of the compound in human immune cell line THP1

[0122] Figure 5 Results of live and dead cell staining experiment

[0123] Figure 6 Results of mouse visual function ERG experiment Detailed implementation mode

[0124] The known starting materials of the present invention can be used or synthesized according to methods known in the art, or can be purchased from companies such as Energy Chemical, Chengdu Kelong Chemical Industry, Shanghai Yuanye Bio-Technology, and J&K Scientific Limited

[0125] Unless otherwise specified in the examples, the reactions are carried out under a nitrogen atmosphere. Unless otherwise specified in the examples, the solution refers to an aqueous solution. Unless otherwise specified in the examples, the reaction temperature is room temperature. Room temperature is the most suitable reaction temperature, which is 20°C to 30°C. Unless otherwise specified in the examples, M is mole per liter

[0126] The structure of the compound was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR chemical shift (δ) is in ppm -6Given in the unit of (ppm). The NMR measurements were performed using (Bruker AvanceIII 400 and Bruker Avance 600) nuclear magnetic resonance spectrometers. The solvents for the measurements were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (Methanol-d4), and the internal standard was tetramethylsilane (TMS). The LC-MS measurements were carried out using a Shimadzu liquid chromatography-mass spectrometer (Shimadzu LC-MS2020 (ESI)). The HPLC measurements were performed using a Shimadzu high-performance liquid chromatograph (Shimadzu LC-20A). MPLC (medium-pressure preparative chromatography) used a Gilson GX-281 reverse-phase preparative chromatograph. The thin-layer chromatography silica gel plates were Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates, and the specifications for separating and purifying products by thin-layer chromatography were 0.4 mm to 0.5 mm. Column chromatography generally used Yantai Huanghai silica gel with 200 to 300 mesh as the carrier.

[0127] TCFH: N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate; NMI: N-methylimidazole; TEA: triethylamine; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP: 4-(dimethylamino)pyridine; Pd / C: palladium on carbon; (Boc)2O: di-tert-butyl dicarbonate; MeCN: acetonitrile; MeOH: methanol; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; Found: the MS value shown when the product was measured by LC-MS.

[0128] SP23 was used as a positive control and is a latest STING PROTAC drug with the patent application publication number CN1140852215A. The said SP23 was purchased from MCE (#HY-150608). Its structural formula is shown as follows:

[0129]

[0130] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited.

[0131] Example 1. Preparation of Compound A1 (ZOC6)

[0132] The synthetic route is shown as follows:

[0133]

[0134] Step 1. Preparation of Compound 3

[0135]

[0136] Under ice bath conditions, TCFH (1.55 g, 5.55 mmol), NMI (1.06 g, 12.61 mmol) and MeCN (20 mL) were successively added to a 100 mL reaction flask. After the reaction mixture was stirred for 5 minutes, compound 1 (1.00 g, 5.04 mmol) and compound 2 (848.31 mg, 5.04 mmol) were added. After stirring the reaction at room temperature for 2 hours, the reaction was quenched (monitored by LC-MS). The reaction mixture was filtered, and the solid was washed with MeCN (3 × 20 mL) to obtain compound 3 (1.75 g, crude product).

[0137] Step 2: Preparation of compound 4

[0138]

[0139] Compound 3 (1.25 g, crude product), NH4Cl (2.67 g, 49.95 mmol), zinc powder (3.25 g, 49.95 mmol) and methanol (30 mL) were successively added to a 100 mL reaction flask. After stirring the reaction at 60 °C for 2 hours, the reaction was quenched (monitored by LC-MS). The zinc powder was filtered off, washed with methanol (2 × 20 mL), the filtrate was collected, and the solvent was removed by concentration under reduced pressure to obtain compound 4 (1.25 g, crude product).

[0140] Step 3: Preparation of compound 6

[0141]

[0142] Compound 4 (1.25 g, 3.93 mmol), compound 5 (916.91 mg, 4.71 mmol), TEA (595.95 mg, 5.89 mmol, 821.43 μL) and DMF (20 mL) were added to a 100 mL reaction flask. After stirring the reaction at room temperature for 3 hours, water was added to quench the reaction (monitored by LC-MS). The mixture was extracted with saturated NaCl solution (25 mL) and ethyl acetate (3 × 25 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, and separated by column chromatography. The volume ratio of the eluent used was dichloromethane / methanol = 100:1 to 10:1. After removing the solvent by concentration under reduced pressure, compound 6 (650.00 mg, 1.36 mmol, 34.74% yield) was obtained.

[0143] Step 4: Preparation of compound 7

[0144]

[0145] In a 100 mL reaction flask, add compound 6 (650.00 mg, 1.36 mmol), MeCN (20 mL), Al (184.02 mg, 6.82 mmol), and I2 (380.83 mg, 1.50 mmol). Stir the reaction at 80 °C for 16 hours, then quench the reaction by adding water (monitored by LC-MS). Extract with saturated NaCl solution (25 mL) and ethyl acetate (3 × 25 mL). Combine the organic phases, dry the organic phase with anhydrous sodium sulfate, evaporate the solvent, and separate by column chromatography. The volume ratio of the eluent used is dichloromethane / methanol = 100:1 to 10:1. After concentrating under reduced pressure to remove the solvent, compound 7 (450.00 mg, 972.99 μmol, 71.33% yield) is obtained.

[0146] Step 5. Preparation of compound A1 (ZOC6)

[0147]

[0148] Add compound 7 (46.00 mg, 99.46 μmol), compound 8 (37.78 mg, 109.41 μmol), EDCI (28.50 mg, 149.19 μmol), DMAP (14.58 mg, 119.35 μmol), and DMF (5 mL) to a 50 mL reaction flask. Stir the reaction at room temperature for 16 hours, then quench the reaction (monitored by LC-MS). Extract with saturated NaCl solution (10.0 mL) and ethyl acetate (3 × 10 mL). Combine the organic phases, dry the organic phase with anhydrous sodium sulfate, evaporate the solvent, and purify by prep-HPLC. After concentrating under reduced pressure to remove the solvent, compound A1 (22.7 mg, 28.08 μmol, 28.23% yield, 97.7% purity) is obtained. LC-MS m / z: [M+H] + calcd.for C 41 H 33 FN5O9S + : 790.19; found: 790.1. 11H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.41 (s, 1H), 10.03 (s, 1H), 8.10 - 8.02 (m, 2H), 7.97 (d, J = 2.4 Hz, 1H), 7.88 - 7.83 (m, 2H), 7.83 - 7.79 (m, 2H), 7.79 - 7.74 (m, 2H), 7.68 - 7.60 (m, 2H), 7.52 (t, J = 7.8 Hz, 2H), 7.47 - 7.41 (m, 1H), 7.40 - 7.35 (m, 2H), 7.22 - 7.16 (m, 1H), 7.09 (d, J = 6.9 Hz, 1H), 7.01 (d, J = 8.7 Hz, 1H), 6.78 (t, J = 6.3 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 3.62 (q, J = 6.6 Hz, 2H), 2.94 - 2.85 (m, 1H), 2.84 (t, J = 6.9 Hz, 2H), 2.64 - 2.51 (m, 1H), 2.07 - 1.98 (m, 1H).

[0149] For compound A1 (ZOC6) 1 The 1H NMR spectrum is shown in Figure 1 .

[0150] Referring to the synthesis method of compound A1 (ZOC6), by replacing compound 8 with the raw materials in the following list 1 and keeping other raw materials and operation methods unchanged, compounds A2 - A6 (ZOC1 - 5) can be obtained.

[0151] Table 1. Compounds A2 - A6

[0152]

[0153]

[0154]

[0155]

[0156] Example 2. Preparation of compound B1 (ZOC7)

[0157]

[0158] Step 1. Preparation of compound 15

[0159]

[0160] To a 100 mL reaction flask, compound 14 (1.22 g, 5.02 mmol), methanol (20 mL), and palladium on carbon (609.21 mg, 5.02 mmol) were added successively. The reaction system was purged with hydrogen, maintained under a hydrogen atmosphere of one atmosphere, and stirred at room temperature for 3 hours (monitored by LC-MS). Palladium on carbon was removed by filtration, washed with methanol (2×20 mL), the filtrate was collected, and the solvent was removed by concentration under reduced pressure to obtain compound 15 (1.10 g, crude product).

[0161] Step 2: Preparation of compound 16

[0162]

[0163] To a 100 mL reaction flask, compound 15 (1.10 g, 5.16 mmol), (Boc)2O (1.12 g, 5.16 mmol), NaHCO3 (866.98 mg, 10.32 mmol), and methanol (20 mL) were added successively. The reaction was stirred at room temperature for 16 hours and then quenched (monitored by LC-MS). The solvent was evaporated, and extraction was performed with saturated NaCl solution (20 mL) and ethyl acetate (3×20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain compound 16 (1.56 g, crude product).

[0164] Step 3: Preparation of compound 17

[0165]

[0166] Under ice bath conditions, to a 100 mL reaction flask, TCFH (1.67 g, 5.98 mmol), NMI (1.23 g, 14.94 mmol), and MeCN (5 mL) were added successively. The reaction mixture was stirred for 5 minutes, and then compound 16 (1.56 g, 4.98 mmol) and compound 2 (837.13 mg, 4.98 mmol) were added. The reaction was stirred at room temperature for 2 hours and then quenched (monitored by LC-MS). Extraction was performed with saturated NaCl solution (20.0 mL) and dichloromethane (3×20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by MPLC. After removing the solvent by concentration under reduced pressure, compound 17 (2.00 g, 4.32 mmol, 86.68% yield) was obtained.

[0167] Step 4: Preparation of compound 18

[0168]

[0169] To a 100 mL reaction flask, compound 17 (2.00 g, 4.32 mmol), methanol (15 mL), THF (5 mL), and palladium on carbon (524.08 mg, 4.32 mmol) were added successively. The reaction system was purged with hydrogen, maintained under a hydrogen atmosphere at one atmosphere, and stirred at room temperature for 3 hours (monitored by LC-MS). The palladium on carbon was filtered off and washed with methanol (2×20 mL). The filtrate was collected, and the solvent was removed by concentration under reduced pressure to obtain compound 18 (1.80 g, crude product).

[0170] Step 5: Preparation of compound 19

[0171]

[0172] To a 50 mL reaction flask, compound 18 (1.80 g, crude product), compound 5 (808.08 mg, 4.15 mmol), NaHCO3 (697.58 mg, 8.30 mmol), THF (10 mL), and H2O (5 mL) were added. The reaction was stirred at room temperature for 16 hours and then quenched by adding water (monitored by LC-MS). The mixture was extracted with saturated NaCl solution (25 mL) and ethyl acetate (3×25 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by MPLC. After concentration under reduced pressure to remove the solvent, compound 19 (2.10 g, 3.55 mmol, 85.48% yield) was obtained.

[0173] Step 6: Preparation of compound 20

[0174]

[0175] To a 50 mL reaction flask, compound 19 (1.00 g, 1.69 mmol), MeCN (10 mL), Al (228.01 mg, 8.45 mmol), and I2 (471.89 mg, 1.86 mmol) were added. The reaction was stirred at 80 °C for 16 hours and then quenched by adding water (monitored by LC-MS). The mixture was extracted with saturated NaCl solution (25 mL) and ethyl acetate (3×25 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by MPLC. After concentration under reduced pressure to remove the solvent, compound 20 (650.00 mg, 1.36 mmol, 80.54% yield) was obtained.

[0176] Step 7: Preparation of compound B1 (ZOC7)

[0177]

[0178] Under ice bath conditions, compound 8 (34.5 mg, 99.91 μmol), EDCI (38.17 mg, 199.82 μmol) and pyridine (2 mL) were successively added to a 20 mL reaction flask. After stirring the reaction mixture for 5 minutes, compound 20 (47.71 mg, 99.91 μmol) was added. After stirring the reaction at room temperature for 2 hours, the reaction was quenched. Extraction was completed with saturated NaCl solution (20.0 mL) and dichloromethane (3 × 15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, purified by MPLC, and the solvent was removed under reduced pressure to obtain compound B1 (20.7 mg, 25.72 μmol, 25.74% yield, 99.1% purity). LC-MS m / z: [M+H] + calcd.for C 41 H 34 FN6O9S + : 805.20; found: 805.1. 1 1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 10.15 (d, J = 21.2 Hz, 2H), 9.38 (d, J = 38.8 Hz, 2H), 8.09 - 8.00 (m, 2H), 7.98 (d, J = 2.2 Hz, 1H), 7.87 - 7.79 (m, 2H), 7.78 - 7.69 (m, 3H), 7.66 - 7.58 (m, 2H), 7.46 - 7.33 (m, 5H), 7.22 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.78 (t, J = 6.2 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 5.04 (dd, J = 12.8, 5.4 Hz, 1H), 3.66 (q, J = 6.4 Hz, 2H), 2.94 - 2.97 (m, 1H), 2.75 - 2.63 (m, 2H), 2.63 - 2.51 (m, 2H), 2.09 - 1.95 (m, 1H).

[0179] of B1(ZOC7) 1 1H NMR spectrum is shown in Figure 2 .

[0180] Referring to the synthesis method of compound B1(ZOC7), using the raw materials in Table 2 below to replace compound 8, with other raw materials and operation methods unchanged, compounds B2 - B4(ZOC8 - 10) can be obtained.

[0181] Table 2. Compounds B2 - B4

[0182]

[0183]

[0184] The effects of the present invention are illustrated by the following experimental examples:

[0185] Experimental Example 1. Evaluation of the degradation of STING protein in human retinal pigment epithelial cell line ARPE-19 by PROTAC through Western blot method

[0186] 1. Experimental instruments and reagents:

[0187] SDS-PAGE gel (10%), Cocktail 100× (Selleck), P / S (Gibco), FBS (Nobimpex), 5× loading buffer (EpiZyme), Tween-20, Anti-α-actinin (Proteintech), RIPA, skim milk powder (Biofroxx), Anti-STING (CST), 24-well plate (LABSELECT), Gel Imaging System (Tanon), -PSQ PVDF membrane (Millipore), 1.5 mL centrifuge tube (AXYGEN), CriterionTM Blotter transfer tank (BIO-RAD), DMEM / F12 culture medium (Gibco), Super Signal ECL Chemiluminescent Kit (NCM).

[0188] 2. Experimental method:

[0189] Protein immunoblot analysis: Cells were evenly seeded into 24-well plates and treated with the indicated compounds at corresponding concentrations for 12 hours. Whole cell lysates were collected using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentrations were quantified by BCA analysis. Equal amounts of protein were electrophoresed on 10% SDS-PAGE and transferred to a polyvinylidene difluoride transfer membrane (PVDF, 0.45 μm), and incubated overnight with different target antibodies at 4°C. The primary antibodies included α-actinin antibody (proteintech, #11313-2-AP, 1:5000) and anti-STING antibody (CST, #50494, 1:1000). The secondary antibody (1:5000) was incubated at room temperature for 1 hour. The expression level of STING protein was normalized to the expression level of α-actinin protein. The target bands were exposed and the images were recorded. Gray scale analysis of the bands was performed using imagej.

[0190] 3. Data analysis:

[0191] Analysis was performed using gray-scale analysis software. After correcting the amount of STING protein by the amount of α-actinin, the degradation of STING protein by 10 μM compound was analyzed. The data presentation form was mean + standard deviation, and one-way ANOVA and Tukey's test were used for data analysis. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0192] The experimental results are shown in Table 3 and Figure 3 .

[0193] Table 3. Degradation of STING protein in ARPE cells by 10 μM compound (average of three replicates, P < 0.05)

[0194]

[0195] Figure 3 Part A in the figure shows the results of immunoblotting of STING protein in ARPE cells. It can be seen from the figure that compound ZOC1-10 has different degrees of degradation effects on STING protein in ARPE cells. Among them, the degradation effects of ZOC6, ZOC8, and ZOC9 are more obvious, and the degradation rates are 56.5%, 72.2%, and 67.7% respectively. SP23 is a reported effective STING degrader and is used as a positive control. Part B is the statistical result of gray-scale analysis. The results show that compared with DMSO, the degradation effects of compounds ZOC6, ZOC8, and ZOC9 are statistically significant.

[0196] Test Example 2. Evaluation of the degradation of STING protein in human immune cell line THP1 by PROTAC through microscale protein electrophoresis (WES) experimental method

[0197] 1. Experimental instruments and reagents:

[0198] RIPA, Cocktail 100× (Selleck), Anti-α-actinin (Proteintech), Anti-STING (CST), 24-well plate (LABSELECT), P / S (Gibco), FBS (Nobimpex), RPMI1640 culture medium (Gibco), microscale protein electrophoresis kit (Protein Simple).

[0199] 2. Experimental method:

[0200] Cells were evenly seeded into 24-well plates and treated with the corresponding concentration of the indicated compound for 12 hours, after which the cells were collected. Whole cell lysates were collected using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentrations were quantified by BCA analysis. The protein supernatant was assayed for concentration using a BCA protein concentration assay kit, and protein samples with a final concentration of 0.2 μg / μl were taken for microprotein electrophoresis experiments. The detailed steps of the microprotein electrophoresis experiment were carried out according to the instructions provided by the manufacturer (Protein Simple). Primary antibodies were α-actinin antibody (proteintech, #11313-2-AP, 1:10000) and anti-STING antibody (CST, #50494, 1:100). The STING protein expression level was normalized to the α-actinin protein expression level.

[0201] 3. Data analysis: The degradation rate was calculated based on the STING protein level. The experimental results are shown in Table 4 and Figure 4 .

[0202] Table 4. Degradation rate of STING protein in THP1 cells by 10 μM compound (average of three replicates, P<0.05)

[0203]

[0204]

[0205] Figure 4 The results of immunoblotting of STING protein in THP1 cells are presented. It can be seen from the figure that compound ZOC1-10 has different degrees of degradation effects on STING protein in THP1 cells. Among them, compound A1 (ZOC6) has the most obvious degradation effect, with a degradation rate of 76.6%.

[0206] Experimental Example 3. Cytotoxicity experiment

[0207] 1. Experimental instruments and reagents: ARPE (human retinal pigment epithelial cells), 48-well plates / 96-well plates (costar), Calcein / PI Cell Viability and Cytotoxicity Detection Kit (Beyotime, C2015S), Cell Counting Kit-8 (Beyotime, C0037), high-content cell imaging system (ImageXpress Micro 4, PerkinElmer), microplate reader (Synergy HTX, Biotek).

[0208] 2. Experimental method:

[0209] 2.1 Live and dead cell staining

[0210] a. Inoculation and culture. Inoculate the cells into a 48-well plate and treat them with the corresponding drug for 6 hours.

[0211] b. Washing. Aspirate the culture medium and wash the cells once with PBS.

[0212] c. Staining. Prepare the Calcein AM / PI detection working solution (Calcein AM 1:1000, PI 1:1000), add 150 μl of the Calcein AM / PI detection working solution to each well, and incubate at 37 °C for 1 hour.

[0213] d. Detection. After the incubation, observe and take pictures under a high-content cell imaging system.

[0214] 2.2 CCK8 cell viability assay

[0215] a. Inoculation and culture. Inoculate the cells into a 96-well plate and treat them with the corresponding drug for 12 hours.

[0216] b. Washing. Aspirate the culture medium and wash the cells once with PBS.

[0217] c. Incubation. Prepare the CCK8 detection working solution (CCK8: medium = 10:100), add 100 μl of the CCK8 detection working solution to each well, and incubate at 37 °C for 2 hours.

[0218] d. Detection. After the incubation, measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0219] 3. Data analysis: Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%, where As is the absorbance of the experimental group (containing cells, medium, CCK-8 solution, and drug solution); Ac is the absorbance of the control group (containing cells, medium, CCK-8 solution, without drug); Ab is the absorbance of the blank group (containing medium, CCK-8 solution, without cells and drug). The data is presented in the form of mean + standard deviation, and two-way ANOVA and Tukey's test are used for data analysis.

[0220] Among them, SP23 is a positive control, which is a latest STING PROTAC drug with the patent application publication number CN1140852215A. The SP23 used as the positive control is purchased from MCE (#HY-150608).

[0221] Figure 5 This is a live and dead cell staining experiment. In the figure, green represents live cells and red represents dead cells. SP23 is the positive control. Figure 5The results showed that there were no significant differences in the number of dead cells and cell morphology between the DMSO group and the groups treated with ZOC6 and ZOC8 at different concentrations (0.1, 1, 10 μM). However, at a concentration of 1 μM of SP23, the number of dead cells increased significantly, and at a concentration of 10 μM of SP23, the toxicity to ARPE cells was greater, with a large number of cells dying and the cell morphology changing and aggregating.

[0222] The specific data of the live and dead cell staining experiment are shown in Table 5.

[0223] Table 5. Statistical results of live and dead cell staining (average of three replicates)

[0224]

[0225] Table 6 shows the data of the CCK8 cell viability experiment. This data indicates that under the condition of a concentration of 10 μM and a treatment time of 12 hours, the cytotoxicity of the compounds described in the table is more than 3 times lower than that of SP23. After treatment with SP23 at a concentration of 10 μM, the cell viability was 28.375%, indicating that SP23 has relatively high cytotoxicity. While for A1 (ZOC6), B2 (ZOC8) and B3 (ZOC9) at a concentration of 10 μM, the cell viabilities were 92.3%, 94.34% and 92% respectively (p < 0.05), indicating that even under the treatment of high concentrations of A1 (ZOC6), B2 (ZOC8) and B3 (ZOC9), the cell activity was not affected and the compounds had relatively low cytotoxicity to the cells.

[0226] Table 6. Statistical results of CCK8 cell viability (average of three replicates)

[0227]

[0228]

[0229] Experimental Example 4. Detection of the effects of intravitreal injection of compounds ZOC6 and SP23 on mouse retinal function

[0230] Multiple previous studies have shown that high expression of STING caused by retinal damage leads to loss of visual function in mice (PMID: 35347235, PMID: 37217935). In this experimental example, the effect of ZOC6 on visual function was evaluated through an electroretinogram (ERG) experiment and compared with SP23 to clarify its toxic and side effects in the eye.

[0231] 1. Experimental instruments and reagents:

[0232] 5 - 8 - week - old C57BL / 6J mice (Experimental Animal Center of Sun Yat - sen University, Guangzhou), 0.1% sodium pentobarbital, compound tropicamide (Zhuobian), hydroxypropyl methylcellulose eye drops (The Affiliated Ophthalmic Hospital of Sun Yat - sen University), insulin syringe (BD), micro - syringe (Hamilton), ERG measuring instrument (Diagnosys Celeris).

[0233] 2. Experimental methods:

[0234] Intravitreal injection: After anesthetizing the mice with 1% sodium pentobarbital, first use compound tropicamide eye drops to dilate the pupils of the mice, and use hydroxyethyl cellulose gel to lubricate the corneas; during intravitreal injection, first use an insulin syringe to make a small incision at the corneal limbus, preferably without bleeding, and then use a micro - syringe to slowly insert the needle obliquely at about 45 degrees along the opening at the corneal limbus. Do not insert too deep to avoid piercing the retina or scratching the lens. Slowly push the drug, and keep the needle in the vitreous cavity for about 5 seconds after injection to prevent drug leakage, and then slowly withdraw the needle.

[0235] Retinal function analysis: Two days after intravitreal injection, anesthetize the mice with 0.1% sodium pentobarbital. After dilating the pupils with tropicamide, apply hydroxypropyl methylcellulose to keep the corneas of the mice moist. Place the mice on the ERG instrument (Diagnosys Celeris), and let two electrodes contact the two corneas of the mice respectively, and stimulate the mice with flashes of different intensities.

[0236] 3. Data analysis:

[0237] The data is presented in the form of mean + standard deviation, and two - way ANOVA and Tukey's test are used for data analysis. *: P < 0.05.

[0238] The experimental results are shown in Table 7 and Figure 6 .

[0239] The retinal function was analyzed by ERG to observe the toxic effects of intravitreal injection of the compound on the eyes of mice. Figure 6 In part A, it is the representative ERG waveform diagram of mice in each treatment group, and in part B, it is the statistical chart of the amplitudes of the a - wave and b - wave of ERG in mice under flashes of different intensities. As Figure 6 shown, compared with the control DMSO group and the ZOC6 treatment group, the amplitudes of the a - wave and b - wave in the mice injected with the SP23 group decreased under different flash stimulations, while there was no significant change in the amplitudes of the a - wave and b - wave in the ZOC6 treatment group compared with the control DMSO group. Among them, at 0.003 cd.s / m 2Under the intensity, the differences in the amplitudes of the a-wave and b-wave between ZOC6 and SP23 were statistically significant at 0.01, 0.03, and 0.1 cd.s / m 2 Under the intensity, the difference in the amplitude of the b-wave between ZOC6 and SP23 was statistically significant. Generally speaking, the response threshold of mice to flash stimulation increased after injecting SP23, indicating that the visual function of the mice in the SP23 injection group was worse than that in the DMSO group and the ZOC6 group, and the compound SP23 had certain toxicity to the retina.

[0240] Table 7. Results of the mouse ERG experiment

[0241]

[0242]

[0243]

[0244] The results in Table 7 showed that intravitreal injection of ZOC6 had no effect on the function of retinal cells, and it could be subsequently applied to the treatment of ophthalmic diseases by intravitreal injection.

[0245] In summary, the compounds A1 (ZOC6), A2 (ZOC1), A3 (ZOC2), A4 (ZOC3), A5 (ZOC4), A6 (ZOC5), B2 (ZOC8), B3 (ZOC9), and B4 (ZOC10) described in the present invention all have a degrading effect on STING.

[0246] The currently marketed STING degrading inhibitor is SP23. Through experiments, it was found that although SP23 has good degrading efficiency, its cytotoxicity is relatively large, which has great limitations for subsequent use. The applicant obtained the compounds in the present invention by changing the warhead and linker. In ARPE cells, the degrading efficiency of B2 (ZOC8) and SP23 on STING protein was close: the maximum degrading rate of B2 was 72.2%, and the maximum degrading rate of SP23 was 71.8%; in THP1 cells, the degrading efficiency of A1 (ZOC6) and SP23 on STING protein was close: the maximum degrading rate of A1 was 76.6%, and the maximum degrading rate of SP23 was 87.8%. However, under the same treatment conditions, the cytotoxicity of A1 (ZOC6) and B2 (ZOC8) among the compounds was 3 - 7 times lower than that of SP23. In in vivo experiments, the visual function of mice decreased after injecting SP23, while the compound A1 (ZOC6) described in the present invention had no effect on visual function. The present invention provides a better choice for the treatment of STING-related autoimmune diseases and inflammatory diseases.

[0247] The above is a further detailed description of the present invention and should not be construed as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, any simple deduction or substitution without departing from the concept of the present invention falls within the protection scope of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, characterized in that, The compound shown in Formula I is selected from the following compounds: 、 、 、 、 、 、 、 、 、 。 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound shown in Formula I is selected from the following compounds: 、 、 。 3. A pharmaceutical composition comprising the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3, in the preparation of a medicament for preventing and / or treating a disease related to the function of STING protein.

5. The use according to claim 4, wherein The diseases related to the function of STING protein are selected from STING-related angiomatosis of infancy SAVI, Aicardi-Goutières syndrome AGS, COPA syndrome caused by mutations in the gene encoding the alpha subunit of the coatomer protein complex, systemic lupus erythematosus SLE, familial chilblain lupus FCL, Parkinson's disease PD, amyotrophic lateral sclerosis ALS, Huntington's disease, non-alcoholic steatohepatitis NASH, alcoholic liver disease, nerve injury, rheumatoid arthritis, renal fibrosis, systemic sclerosis, intervertebral disc degeneration, pulmonary fibrosis, aging, scleroderma, psoriasis, inflammatory bowel disease, autoimmune colitis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, uveitis, mucositis, diabetes, cardiovascular diseases.

6. The use according to claim 4, wherein, The diseases related to the function of STING protein are selected from neurodegenerative diseases.

7. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3, in the preparation of a medicament for preventing and / or treating inflammatory diseases and / or autoimmune diseases.

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