Application of amide-substituted 2-oxopyridine compound

Abstract

The invention relates to an application of an amide substituted 2-oxopyridine compound. Specifically, a compound of formula (I), or a pharmaceutically acceptable salt, a solvate, an optically pure isomer, a stereoisomer or a mixture thereof, is used for preparing a medicine or a preparation, and the medicine or the preparation is used for one or more of the following applications: (a) a medicine for treating and / or preventing cachexia-related diseases, (b) a medicine for treating and / or preventing cachexia-related diseases, (c) a medicine for treating and / or preventing cachexia-related diseases, and (c) a medicine for treating and / or preventing cachexia-related diseases; (b) inhibiting the browning of fat cells; and / or (c) promoting the degradation of the PRDM16 protein; wherein the compound shown in the formula (I) has a structure shown in the specification,

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to the use of amide-substituted 2-oxopyridine compounds in the preparation of medicaments for the treatment and / or prevention of cachexia-related diseases. Background Technology

[0002] Cachexia is a devastating and often irreversible metabolic syndrome caused by multiple factors, characterized by anorexia and significant weight loss due to a large reduction in skeletal muscle and fat.

[0003] Cachexia is associated with diseases such as cancer and chronic obstructive pulmonary disease. Cancer-related cachexia is particularly dangerous. Statistics show that approximately 40% of diagnosed cancers and 70% of advanced cancers are accompanied by cachexia. Cachexia is a common cause of death from malignant tumors, significantly reducing patient survival rates; about 20% of patients ultimately die from cachexia.

[0004] The 2020 American Society of Clinical Oncology (ASCO) guidelines for cancer cachexia recommend the use of progesterone analogs and corticosteroids to improve appetite and increase weight. However, progesterone analogs and corticosteroids primarily alleviate cachexia symptoms by regulating the patient's overall metabolic state and hormone levels, rather than directly treating the pathophysiological processes of cachexia; therefore, their therapeutic effects are limited.

[0005] Molecular glues are novel small-molecule protein degraders that interact with E3 ubiquitin ligases and target proteins to form ternary complexes, thereby initiating ubiquitination and proteasome degradation of the target protein. Compared to traditional small-molecule inhibitors and receptor antagonists, molecular glues possess a unique mechanism: they do not require specific binding pockets on the target protein during degradation, thus enabling them to degrade targets that are difficult for traditional small molecules to reach. Furthermore, the small molecular weight of molecular glues allows for good cell permeability and oral absorption, resulting in significant advantages in pharmacokinetics. In summary, due to their drug similarity, ability to reveal novel mechanisms of action and targets, and small molecular weight, molecular glues demonstrate enormous potential and application prospects in drug discovery and disease treatment.

[0006] In summary, there is a lack of satisfactory targeted drugs for the effective treatment of cachexia in this field. Therefore, there is an urgent need in this field to develop effective, safe, and targeted molecular colloid degrading agents for the treatment and / or prevention of cachexia-related diseases. Summary of the Invention

[0007] The purpose of this invention is to provide effective, safe and targeted molecular colloid degrading agents for the treatment and / or prevention of cachexia-related diseases and their applications.

[0008] A first aspect of the invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer, or mixture thereof, for the preparation of a medicament or formulation for use in one or more uses selected from the group consisting of:

[0009] (a) Medications for the treatment and / or prevention of cachexia-related diseases;

[0010] (b) Inhibits adipocyte browning; and / or

[0011] (c) Promotes the degradation of PRDM16 protein;

[0012] The compound of formula (I) has the following structure:

[0013]

[0014] in,

[0015] R1 is a C1-C3 alkyl or a C1-C3 haloalkyl;

[0016] R2 either does not exist or is -SR5 or -OR5;

[0017] R5 is a substituted or unsubstituted group of the following: C1-C6 alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclic, C6-C10 aryl, or 5-7 membered heteroaryl; wherein the substitution refers to being independently substituted by 1-3 substituents selected from the group consisting of: halogen, cyano, hydroxyl, C1-C3 alkyl, C1-C3 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -NR 10 R 11 -C(=O)R 12 C1-C4 alkanoyloxy groups, or two adjacent substituents together with the carbon atom attached to them, form a 5-7 membered carbon ring or a 5-7 membered heterocycle.

[0018] R3 is selected from: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, wherein the substitution refers to being independently substituted by 1 to 3 substituents selected from the group consisting of: halogen, OH, C1-C3 alkyl;

[0019] R4 is a substituted or unsubstituted group selected from the group consisting of: C1-C6 straight-chain or branched alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclic, C6-C10 aryl, or 5-7 membered heteroaryl; wherein the substituent is selected from halogen, hydroxyl, nitro, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylcarbonyl-, and C1-C4 carboxyl. The phenyl group is a 5-7-membered heteroaryl group substituted or substituted by 1-3 substituents selected from group A (for example, the heteroaryl group is a heteroaryl group containing 1-4 N, preferably a 5-membered heteroaryl group containing 3 or 4 N, more preferably a tetrazolium group), and an unsubstituted or substituted phenyl group substituted by 1-3 substituents selected from group A, wherein the substituents in group A are selected from the group consisting of: halogens, C1-C3 alkyl groups, OH groups, C1-C3 haloalkyl groups, and C1-C3 alkoxy groups.

[0020] Alternatively, R3 and R4 together with the attached N atom form Where n is 1 to 4;

[0021] R6 is selected from halogen, cyano, hydroxyl, substituted or unsubstituted C1-C3 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted 4-7 membered heterocyclic group, substituted or unsubstituted benzyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted 5-7 membered heteroaryl; wherein the substitution refers to being independently substituted by 1-3 substituents selected from the following group: halogen, hydroxyl, C1-C4 straight-chain or branched alkoxy, C1-C4 straight-chain or branched alkyl, C1-C4 straight-chain or branched haloalkyl, C1-C4 straight-chain or branched haloalkoxy, cyano, nitro, amino, carboxyl.

[0022] In another preferred embodiment, R1 is a C1-C3 fluoroalkyl group, more preferably a trifluoromethyl group.

[0023] In another preferred example, R2 does not exist.

[0024] In another preferred embodiment, R2 is -SR5.

[0025] In another preferred embodiment, R5 is selected from groups substituted with 1-3 substituents: C1-C6 alkyl, phenyl, C4-C6 cycloalkyl, 4-6 heterocyclic, 5-6 heteroaryl; each substituent is independently selected from halogens.

[0026] In another preferred embodiment, R5 is selected from an aryl group that has been substituted with a halogen.

[0027] In another preferred embodiment, R3 and R4 are each independently selected from hydrogen or from the following groups substituted with 1 to 3 substituents: C1-C6 alkyl, phenyl, C4-C6 cycloalkyl, 4-6 membered heterocyclic, 5-6 membered heteroaryl; and each substituent is independently selected from tetrazolium, cyano, nitro, amino, and carboxyl.

[0028] In another preferred embodiment, R3 is selected from hydrogen, and R4 is selected from the following groups substituted with 1 to 3 substituents: C1-C6 alkyl, phenyl, C4-C6 cycloalkyl, 4-6 membered heterocyclic, 5-6 membered heteroaryl; each substituent is independently selected from tetrazolium, cyano, nitro, amino, and carboxyl.

[0029] In another preferred embodiment, the 4-7 membered heterocyclic group contains 1-3 heteroatoms selected from N, O and S.

[0030] In another preferred embodiment, the 5-7 membered heterocycle contains 1-3 heteroatoms selected from N, O and S.

[0031] In another preferred embodiment, the 5-7 membered heteroaryl contains 1-3 heteroatoms selected from N, O and S.

[0032] In another preferred embodiment, the 5-7 membered heteroaryl is a 6 membered heteroaryl containing 1, 2 or 3 nitrogen atoms.

[0033] In another preferred embodiment, the heteroaryl group is a 5-6 membered heteroaryl group containing 1-4 N atoms, more preferably a 5 membered heteroaryl group containing 3 or 4 N atoms, and even more preferably a tetrazolium group.

[0034] In another preferred example, n is 1 or 2.

[0035] In another preferred embodiment, R3 and R4 together form with the attached N atom. Where n is 1 or 2.

[0036] In another preferred embodiment, R6 is selected from hydrogen, cyano, alkoxy, and hydroxyl.

[0037] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0038]

[0039]

[0040] In another preferred embodiment, the cachexia-related diseases are selected from the group consisting of diseases associated with fat browning, skeletal muscle loss, or combinations thereof.

[0041] In another preferred embodiment, the cachexia-related disease is selected from the group consisting of: chronic obstructive pulmonary disease, chronic heart failure, chronic kidney disease, severe trauma, sepsis, AIDS, inflammation, cancer, or combinations thereof.

[0042] In another preferred embodiment, the cancer is a solid tumor or a hematoma.

[0043] In another preferred embodiment, the disease is selected from the group consisting of: myelodysplastic syndromes, multiple myeloma, mantle cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, chronic myelomonocytic leukemia, myelofibrosis, Burkitt lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, ciliary body and chronic melanoma, iris melanoma, recurrent interocular melanoma, T-cell lymphoma, erythroid lymphoma, monocytoblasts and mononuclear cells. Leukemia, myeloid leukemia, central nervous system lymphoma, meningioma, spinal cord tumor, lung cancer, ovarian cancer, skin cancer, renal cell carcinoma, astrocytoma, amyloidosis, type I complex regional pain syndrome, malignant melanoma, radiculopathy, glioblastoma, glioma, malignant glioma, refractory plasmacytoma, extraocular melanoma, papillary and follicular thyroid carcinoma, breast cancer, pancreatic cancer, colon cancer, prostate cancer, hepatocellular carcinoma, or primary macroglobulinemia.

[0044] In another preferred embodiment, the cachexia is cachexia caused by cancer selected from the group consisting of lung cancer, breast cancer, pancreatic cancer, colon cancer, or a combination thereof.

[0045] In another preferred embodiment, the compound or pharmaceutical composition is used for:

[0046] (1) Promotes the binding of PRDM16 to E3 ubiquitin ligase;

[0047] (2) Degradation of PRDM16 protein;

[0048] (3) Reduces fat browning in patients;

[0049] (4) Promotes the increase of skeletal muscle in patients;

[0050] (5) Reduce patients' cachexia.

[0051] In another preferred embodiment, the compound or pharmaceutical composition is used to degrade the PRDM16 protein in adipocytes.

[0052] In another preferred embodiment, the E3 ubiquitin ligase is selected from SCF. β-TRCP .

[0053] In another preferred embodiment, the compound is administered via a method selected from the group consisting of oral, rectal, and parenteral administration.

[0054] In another preferred embodiment, the parenteral administration method is selected from the group consisting of intravenous, intramuscular, or subcutaneous administration.

[0055] In another preferred embodiment, the oral formulation is selected from the group consisting of capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups, and tinctures.

[0056] In a second aspect of the invention, there is provided the use of a pharmaceutical composition for preparing a medicament for treating and / or preventing cachexia-related diseases;

[0057] And the pharmaceutical composition comprises:

[0058] (i) A first active ingredient, wherein the first active ingredient is selected from the group consisting of compounds of formula (I), or pharmaceutically acceptable salts, solvates, optically pure isomers, and stereoisomers thereof;

[0059] (ii) Optionally, a second active ingredient selected from the group consisting of: dexamethasone, rituximab, trastuzumab, PD-1 inhibitors, PDL-1 inhibitors, pemetrexed, topotecan, doxorubicin, gemcitabine, dacarbazine, clarithromycin, vincristine, cytarabine, prednisone, docetaxel, clofarabine injection, HDAC inhibitors, androgen receptor inhibitors, androgen biosynthesis inhibitors, BTK inhibitors, erythrocyte growth hormone, minocycline, elotuzumab, palbociclib, nivolumab, pembrolizumab, panobinostat, ubliximab, romidepsin, eltrombopag, CAR-T, melphalan, or combinations thereof;

[0060] (iii) A pharmaceutically acceptable carrier;

[0061] The compound of formula (I) is as defined in the first aspect of the present invention.

[0062] In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

[0063]

[0064]

[0065] In another preferred embodiment, the content of the first active ingredient in the pharmaceutical composition is 0.01-99 wt%, more preferably 0.1-90 wt%, based on the total weight of the pharmaceutical composition.

[0066] In another preferred embodiment, the compound of formula (I) is used as a single component in combination with other effective treatments for cachexia-related diseases (surgical treatment, radiotherapy, etc.) in the comprehensive treatment of cachexia-related diseases.

[0067] In a third aspect of the invention, a method for treating and / or preventing cachexia-related diseases is provided, comprising administering to a subject in need a medically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer, or mixture thereof;

[0068] The compound of formula (I) is as defined in the first aspect of the present invention.

[0069] In another preferred embodiment, the object is a primate mammal, such as a human.

[0070] In another preferred embodiment, a medicament for treating and / or preventing multiple sclerosis is prepared using an amide-substituted 2-oxopyridine compound of the general formula (I) or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, prodrug, or mixture thereof as the active ingredient.

[0071] In another preferred embodiment, the drug may be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, by means of injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it may be introduced into the body by being mixed with or encapsulated by other substances.

[0072] In another preferred embodiment, the drug further includes one or more pharmaceutically acceptable carriers.

[0073] In another preferred embodiment, the carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, lubricants, etc., which are commonly used in the pharmaceutical field.

[0074] In another preferred embodiment, an amide-substituted 2-oxopyridine compound of the general formula (I) or its pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, or prodrug is used as an active ingredient, either alone or in combination, or formulated with other drugs or excipients into various dosage forms.

[0075] In another preferred embodiment, the dosage form includes, but is not limited to, tablets, powders, pills, injections, capsules, films, suppositories, ointments, and granules.

[0076] In another preferred embodiment, the drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0077] In a fourth aspect of the invention, a method for inhibiting PRDM16 protein levels in adipocytes in vitro is provided, comprising the steps of:

[0078] Adipocytes were cultured in the presence of a compound of formula (I) or a pharmaceutically acceptable salt thereof, thereby inhibiting the level of PRDM16 protein in the adipocytes.

[0079] In another preferred embodiment, the adipocytes are adipocytes co-cultured with tumor cells or adipocytes pretreated with tumor cells.

[0080] In another preferred embodiment, the tumor cells are cachectic tumor cells.

[0081] In another preferred embodiment, the tumor cells are selected from the group consisting of lung cancer cells, breast cancer cells, pancreatic cancer cells, colon cancer cells, or combinations thereof.

[0082] In another preferred embodiment, the method further includes the step of: (b) detecting the protein level or ubiquitination level of PRDM16 in the adipocytes.

[0083] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0084] In a fifth aspect of the invention, a method for inhibiting adipocyte browning in vitro is provided, comprising the steps of:

[0085] (a) In the presence of a compound of formula I or a pharmaceutically acceptable salt thereof, adipocytes are cultured to inhibit adipocyte browning.

[0086] In another preferred embodiment, the method further includes the step of: (b) detecting the degree of browning of the fat cells.

[0087] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0088] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new technical solutions or in another preferred embodiment. The various features disclosed in the specification can be replaced by any alternative features that provide the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0089] Figure 1The results show the experimental results verifying that β-TRCP (expressed by the Btrc gene) is the ubiquitinase of PRDM16. Figure A shows the effect of siBtrc knockdown on PRDM16 protein by immunoblotting analysis in 3T3-L1 cells. Figure B shows the results of immunoprecipitation analysis of the degree of PRDM16 polyubiquitination after overexpression of exogenous PRDM16 (PRDM16-HA), β-TRCP (Myc-β-TRCP), and Ubiquitin (His-Ub) plasmids in 293T cells and treatment with 50 μM MG132 for 9 h. β-ACTIN represents the internal reference.

[0090] Figure 2 The diagram shows the results of NRX series drugs in this invention promoting the binding of PRDM16 to the E3 ubiquitin ligase β-TRCP, leading to PRDM16 polyubiquitination and degradation. Figures A and D represent the results of immunoblotting analysis of PRDM16 protein changes after treating 3T3-L1 cell lines with different concentrations of NRX drugs (NRX-252262, NRX-1933, NRX-103094, and NRX-252114) for 24 hours. Figure E shows a schematic diagram of the PRDM16 protein structure, where the blue area represents the PR / SET domain, the pink area represents the zinc finger structure (Zinc-finger (ZF)), and the green area represents PLDLS. The top image in the F-plot shows the binding of NRX-252262 and PRD peptide at different concentrations detected by surface plasmon resonance. Changes in the response units reflect binding and dissociation events between biomolecules; an increase in the response unit indicates binding, and a decrease to zero indicates dissociation. The bottom image shows the relationship between the equilibrium response unit and the compound concentration plotted using Graphs. The dissociation constant (K0) of RX-252262 and PRD peptide is derived from this relationship. D The concentration was 2.77 μM; Figure G shows the results of immunoprecipitation analysis of the interaction between β-TRCP and PRDM16 proteins after treating 3T3-L1 cells with 12 nM NRX-252262 for 24 h and then treating them with 50 μM G132 for 16 h. Figure H shows the results of immunoprecipitation analysis of the effect of NRX-252262 on PRDM16 polyubiquitination after treating 3T3-L1 cells with 12 nM NRX-252262 for 24 h and then treating them with 50 μM G132 for 16 h.

[0091] Figure 3The diagrams show the results of the NRX series drugs in this invention promoting the degradation of PRDM16 and inhibiting cachexia. Figure A shows the changes in PRDM16 protein after treating differentiated 3T3-L1 cells with different tumor cell culture supernatants (including MC38 colon cancer cells and LLC lung cancer cells) for 48 hours, followed by treatment with 12 nM NRX-252262 for 24 hours. Figure B shows the changes in PRDM16 protein after treating differentiated 3T3-L1 cells with LLC lung cancer cell culture supernatants for 48 hours, followed by treatment with different concentrations of NRX series drugs for 24 hours. The concentrations are: NRX-252262 (12 nM), NRX-252114 (80 nM), NRX-103094 (400 nM), and NRX-1933 (160 nM). Figures C and D show the changes in PRDM16 protein after treating differentiated 3T3-L1 cells with different tumor cell culture supernatants for 48 hours, followed by treatment with 12 nM NRX-252262 for 24 hours. The results of immunoblotting analysis of PRDM16 protein changes after 24 hours of NRX-252262 treatment are shown in Figure C, which represents the supernatant of breast cancer cell line culture medium (4T1-CM); Figure D represents the supernatant of colorectal cancer cell line culture medium (C26-CM); Figure E represents the supernatant of pancreatic duct adenocarcinoma cell line culture medium (Panc02-CM); Figures F and G represent the changes in UCP1 protein in inguinal adipose tissue of mice after tumor removal and weight measurement 28 days after subcutaneous injection of LLC cell line or PBS (NTB) (Figure G); Figure HI represents the changes in PRDM16 and UCP1 protein in subcutaneous adipose tissue of mice after treatment with DMSO or NRX-252262 (10 mg / kg) on ​​day 7 after subcutaneous injection of LLC cell line (before treatment), tumor removal and weight measurement 35 days later (post-treatment) (Figure I); and NS represent no significant difference. Detailed Implementation

[0092] Through extensive and in-depth research and numerous screenings, the inventors have unexpectedly developed, for the first time, a class of active ingredients that can effectively inhibit cachexia-related diseases, namely compounds represented by general formula (I) or their pharmaceutically acceptable salts, enantiomers, diastereomers, or racemates. Experiments show that the active ingredients of this invention can efficiently promote the binding of PRDM16 protein to the E3 ubiquitin ligase β-TRCP via molecular gelation, thereby efficiently leading to the degradation of PRDM16 protein, thus effectively and specifically treating and / or preventing cachexia-related diseases. This invention was completed based on this.

[0093] the term

[0094] When a substituent is described using a conventional chemical formula written from left to right, it also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0095] Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible. Substitution in the relevant structure in this invention includes both substitution and non-substitution; for example, "optionally" or "optionally" substituted by a substituent means both substitution and non-substitution.

[0096] The statement in this invention that when the number of substituents is greater than 1, the substituents R can be the same or different substituents means that when there are multiple substituents in a certain structure, the combination of substituents R can be selected from a variety of different types of substituents.

[0097] As used in this article, the term "SCF" β-TRCP "" and "β-TRCP" are used interchangeably, both referring to the E3 ubiquitin ligase SCF. β-TRCP .

[0098] The term "substitution" applies only to sites that can be replaced by a substituent and does not include substitutions that are not achievable with existing chemical knowledge. The term "substitution" refers to the replacement of one or more hydrogen atoms on a specific group by a specific substituent. A specific substituent is a substituent described above or that appears in the examples. Unless otherwise specified, an arbitrarily substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Cyclic substituents, such as heterocyclic alkyl groups, may be attached to another ring, such as a cycloalkyl group, thereby forming a spirobicyclic system, for example, where the two rings share a single carbon atom.

[0099] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0100] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group, which typically has 1-10, 1-8, 1-6, or 1-4 carbon atoms. For example, "C1-8 alkyl" refers to straight-chain alkyl and branched alkyl groups comprising 1 to 8 carbon atoms, including but not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, etc.

[0101] The term "cycloalkyl group" refers to a cyclic hydrocarbon substituent that is saturated or partially unsaturated, either monocyclic or polycyclic, typically having 3-12, 3-10, 3-8, or 4-6 carbon atoms. For example, "C3-8 cycloalkyl" refers to a cycloalkyl group containing 3 to 8 carbon atoms, and is classified into monocyclic and polycyclic cycloalkyl groups. Monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl. Polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0102] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein one or more ring atoms, in addition to the ring carbon atom, are heteroatoms selected from N, O, or S.

[0103] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring that shares adjacent carbon atom pairs) group, a polycyclic (i.e., a ring with adjacent carbon atom pairs) group with a conjugated π-electron system, including but not limited to phenyl and naphthyl.

[0104] The term "halogen-substituted aryl" refers to a 6-14 membered all-carbon monocyclic or fused polycyclic group with a conjugated p-electron system, substituted by one or more halogens, preferably a 6- to 10-membered ring (i.e., C6-C10 aryl), more preferably phenyl and naphthyl, and most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring.

[0105] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, including nitrogen, oxygen, and S. For example, 5-7-membered heteroaryl refers to a heteroaromatic system containing 5-7 ring atoms, and 5-10-membered heteroaryl refers to a heteroaromatic system containing 5-10 ring atoms, including but not limited to furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc.

[0106] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. For example, "C1-8 alkoxy" refers to alkyloxy groups containing 1-8 carbons, including but not limited to methoxy, ethoxy, propoxy, butoxy, etc. The present invention is further illustrated below with specific embodiments.

[0107] PR domain protein 16 (PRDM16)

[0108] PRDM16 is a transcription factor whose main function is to regulate cell differentiation, especially in adipocyte differentiation and the development of brown adipose tissue and muscle tissue. Overactivation of PRDM16 can lead to the loss of muscle and fat, resulting in various metabolic diseases; therefore, inhibiting PRDM16 overactivation is of significant value.

[0109] Studies have shown that inhibition of PRDM16 may have important applications in promoting skeletal muscle growth, inhibiting muscular dystrophy, treating metabolic diseases related to fat browning and skeletal muscle loss, inhibiting the proliferation and metastasis of malignant tumor cells, and treating acute myeloid leukemia, especially in the treatment of cachexia.

[0110] Studies have shown that PRDM16 can inhibit the formation of white adipose tissue, thereby suppressing lipid accumulation. PRDM16 can also promote the differentiation of white adipocyte precursor cells into beige adipocytes, and simultaneously participate in regulating the proliferation and differentiation of brown adipocyte precursor cells, promoting the development of brown adipose tissue and thus promoting the formation of thermogenic adipose tissue. PRDM16 also plays an important role in other cell types; for example, in myoblasts, PRDM16 can inhibit muscle formation and induce differentiation into brown adipocytes.

[0111] PRDM16 acts as a "switch" in adipogenesis, regulating the transcription of various target genes (such as UCP1 and Cidea) involved in the transformation from white adipose tissue to brown adipose tissue and from skeletal muscle to brown adipose tissue. Inhibiting PRDM16 expression in fibroadipocytes can induce their differentiation into muscle cells, thereby promoting the formation of damaged muscle and reducing fat accumulation in muscle, thus inhibiting muscular dystrophy and related metabolic diseases caused by skeletal muscle loss.

[0112] In cancer patients, inhibiting the expression of PRDM16 in adipocytes can suppress tumor-induced adipose browning, thereby inhibiting tumor proliferation and metastasis.

[0113] The study of this invention shows that targeted degradation of PRDM16 by the molecular glue compound of this invention can promote skeletal muscle formation, inhibit muscular dystrophy, treat metabolic diseases related to fat browning and skeletal muscle reduction, and inhibit the proliferation and metastasis of malignant tumor cells, thereby effectively improving or treating cachexia.

[0114] Uncoupling protein (UCP1)

[0115] UCP1 is a specialized mitochondrial protein that promotes mitochondrial respiration, thereby generating energy expenditure. UCP1 was the first uncoupling protein discovered in brown adipocytes; it is located on the inner mitochondrial membrane and can reduce the production of protons (H+) by the electron transport system. + The gradient makes it easier to directly generate heat. UCP1 activity in brown adipocytes is considered a promising strategy to combat obesity and metabolic diseases.

[0116] The active ingredient of the present invention

[0117] As used herein, the terms “compound of the present invention”, “active ingredient of the present invention”, “molecular glue compound of the present invention”, “molecular glue of the present invention”, etc., are used interchangeably to refer to the compound represented by general formula (I) or its pharmaceutically acceptable salt, enantiomer, diastereomer or racemate.

[0118] The compounds of the present invention have the structure of formula (I):

[0119]

[0120] The groups are defined as described above.

[0121] As used herein, “compound of the invention” or “active ingredient” refers to a compound of formula I and also includes a pharmaceutically acceptable salt, tautomer, stereoisomer, or prodrug.

[0122] The compound shown in formula (I) may also exist in different tautomer forms, all of which are included within the scope of this invention.

[0123] The term "tautomer" refers to structural isomers with different energies that interconvert via a low energy barrier. The reaction generally results in the movement of hydrogen atoms or protons, accompanied by the transformation of single bonds and adjacent double bonds.

[0124] The compounds of this invention can exist in specific geometric or stereoisomeric forms. All such compounds contemplated by this invention, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, non-corresponding isomers, racemates, and other mixtures, are within the scope of this invention. Additional asymmetric carbon atoms may be present in alkyl or other substituents. All such isomers and mixtures thereof are included within the scope of this invention.

[0125] The compounds represented by formula (I) may contain one or more asymmetric or chiral centers, and therefore may exist in different stereoisomer forms. The compounds of this invention include all stereoisomer forms, including but not limited to diastereomers, enantiomers, and transisomers, as well as mixtures thereof (such as racemates), all of which are included within the scope of this invention.

[0126] Unless otherwise stated, the term "enantiomer" refers to stereoisomers that are mirror images of each other.

[0127] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0128] A racemic mixture is a stereoisomer that is a mirror image of another, with opposite optical rotations that cancel each other out.

[0129] The compounds in the general formula and examples of this invention contain chiral centers; therefore, the single configuration or racemate of the compounds of this invention is also within the scope of this application. Furthermore, prior art evidence indicates that compounds with a amine structure, in their R configuration, S configuration, and racemate, are converted to a 1:1 racemate in vivo after entering cells from in vitro cell experiments. Therefore, those skilled in the art can expect that the technical solutions corresponding to the enantiomers or racemates of the compounds of this invention can achieve comparable technical effects. To obtain a single configuration of the example compounds in this invention, a single configuration example compound can be obtained through synthesis method 1 in the compound preparation method using the single configuration intermediate 1B; thus, an enantiomer can be obtained. Alternatively, diastereomeric example compounds can be obtained through the preparation of the single configuration intermediate 1B; therefore, diastereomeric compounds can also be obtained in this invention, such as compounds 48 and 54, which can form diastereomeric forms. These different isomer forms all belong to stereoisomers.

[0130] The compounds of this invention may contain non-natural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, the compounds may be radiolabeled with radioactive isotopes such as deuterium (D), tritium (T), 12C, 13C, and 14C. As another example, deuterium may be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared to undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations in the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

[0131] As used herein and unless otherwise specified, the term "prodrug" refers to a derivative of a compound containing a bioreactive functional group, such that, under biological conditions (in vitro or in vivo), the bioreactive functional group can be cleaved from the compound or otherwise reacted to provide said compound. Typically, prodrugs are inactive, or at least less active than the compound itself, such that the compound does not exert its activity until the bioreactive functional group is cleaved from it. The bioreactive functional group can be hydrolyzed or oxidized under biological conditions to provide said compound. For example, a prodrug may contain a biohydrolyzable group. Examples of biohydrolyzable groups include, but are not limited to, biohydrolyzable phosphates, biohydrolyzable esters, biohydrolyzable amides, biohydrolyzable carbonates, biohydrolyzable carbamates, and biohydrolyzable ureas. For reviews of prodrugs, see, for example, J. Rautio et al., Nature Reviews Drug Discovery 2008, 7, 255-270 and Prodrugs: Challenges and Rewards (V. Stella et al. ed., Springer, 2007).

[0132] When the compounds of this invention contain a basic group, they can be prepared into pharmaceutically acceptable salts, including inorganic acid salts and organic acid salts. Suitable acids for salt formation are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0133] Pharmaceutical Compositions and Administration

[0134] In this application, "pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally accepted in the art for delivering bioactive compounds to mammals (e.g., humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity.

[0135] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components of the composition can be mixed with compounds of general formula (I) of the present invention, their pharmaceutically acceptable salts or solvates thereof, and with each other, without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0136] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0137] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0138] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0139] There are no particular limitations on the administration of the compound or pharmaceutical composition of formula (I) of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0140] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0141] In this invention, the compound of formula (I) can be used in combination with other known drugs for treating or improving similar symptoms. When administered in combination, the original drug's administration method and dosage can remain unchanged, while the compound of formula I is taken simultaneously or subsequently. When the compound of formula I is taken concurrently with one or more other drugs, a pharmaceutical composition containing one or more known drugs and the compound of formula I is preferably used. Drug combination also includes taking the compound of formula I with one or more other known drugs during overlapping time periods. When the compound of formula I is used in combination with one or more other drugs, the dosage of the compound of formula I or the known drug may be lower than the dosage of either drug alone.

[0142] Drugs or active ingredients that can be used in combination with compounds of general formula (I) include, but are not limited to, dexamethasone, rituximab, trastuzumab, PD-1 inhibitors, PDL-1 inhibitors, pemetrexed, topotecan, doxorubicin, bortezomib, gemcitabine, dacarbazine, clarithromycin, vincristine, cytarabine, prednisone, docetaxel, clofarabine injection, HDAC inhibitors, kinase-targeting inhibitors, androgen receptor inhibitors, androgen biosynthesis inhibitors, erythrocyte growth hormone, minocycline, elotuzumab, palbociclib, nivolumab, pembrolizumab, panobinostat, ubliximab, romidepsin, eltrombopag, CAR-T, and melphalan.

[0143] Typical formulations are prepared by mixing compounds of general formula (I) of the present invention with a carrier, diluent, or excipient. Suitable carriers, diluents, or excipients are well known to those skilled in the art and include substances such as carbohydrates, waxes, water-soluble and / or expandable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, and water. The specific carrier, diluent, or excipient used will depend on the manner and purpose of use of the compound according to the present invention. Solvents are generally selected based on solvents that are considered safe and effective for administration to mammals by those skilled in the art. Generally, safe solvents are non-toxic aqueous solvents such as pharmaceutical water, as well as other non-toxic solvents that are soluble in or miscible with water. Suitable aqueous solvents include one or more of water, ethanol, propylene glycol, polyethylene glycol (such as PEG400, PEG300), etc. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavorings, seasonings or other known additives, so that the compound represented by formula (I) is manufactured or used in an acceptable form.

[0144] When the compounds of formula (I) of the present invention are used in combination with at least one other drug, the two or more drugs may be used separately or in combination, preferably in the form of a pharmaceutical composition. The compounds or pharmaceutical compositions of formula (I) of the present invention can be administered to the subject separately or together in any known oral, intravenous, rectal, vaginal, transdermal, or other local or systemic forms of administration.

[0145] These pharmaceutical compositions may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, flow aids, processing aids, colorants, sweeteners, flavorings, or other known additives to make or use the pharmaceutical composition in an acceptable form.

[0146] The preferred route of administration for the pharmaceutical products of this invention is oral administration. Solid dosage forms for oral administration may include capsules, tablets, powders, or granules. In solid dosage forms, the compounds or pharmaceutical compositions of this invention are mixed with at least one inert excipient, diluent, or carrier. Suitable excipients, diluents, or carriers include substances such as sodium citrate or dicalcium phosphate, or starch, lactose, sucrose, mannitol, silicic acid, etc.; binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic, etc.; humectants such as glycerin, etc.; disintegrants such as agar, calcium carbonate, potato or cassava starch, alginate, specific complex silicates, sodium carbonate, etc.; solution blocking agents such as paraffin, etc.; absorption enhancers such as quaternary ammonium compounds, etc.; adsorbents such as kaolin, bentonite, etc.; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, etc. In the case of capsules and tablets, the dosage form may also include a buffer. Similar solid compositions can also be used as fillers in soft and hard filled gelatin capsules, using lactose and high molecular weight polyethylene glycol as excipients.

[0147] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the compounds or compositions thereof of the present invention, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide; oils (such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, etc.); glycerin; tetrahydrofurfuryl alcohol; fatty acid esters of polyethylene glycol and sorbitol; or mixtures of several of these substances.

[0148] In addition to these inert diluents, the composition may also include one or more excipients, such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, and fragrances.

[0149] In the case of suspensions, in addition to the compounds or combinations of the present invention, they may further contain carriers such as suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, dehydrated sorbitol ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar and astragalus gum, or mixtures of several of these substances.

[0150] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds or combinations of the present invention with suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, or suppository wax, which are solid at normal room temperature and liquid at body temperature, and can melt in the rectum or vagina to release the active compound.

[0151] The compounds or pharmaceutical compositions of this invention can be administered in other topical dosage forms, including ointments, powders, sprays, and inhalers. The drug can be mixed under sterile conditions with pharmaceutically acceptable excipients, diluents, or carriers, and any desired preservatives, buffers, or propellants. Ophthalmic formulations, ophthalmic ointments, powders, and solutions are also intended to be covered within the scope of this invention.

[0152] As used herein, the terms “administration,” “application,” “dosage,” etc., refer to methods that deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, oral administration, duodenal administration, parenteral administration (including intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial injection or infusion), local administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used with the compounds and methods described herein, such as those discussed in Goodman and Gilman, *The Pharmacological Basis of Therapeutics*, current ed.; Pergamon; and Remington's, *Pharmaceutical Sciences* (current edition), Mack Publishing Co., Easton, Pa. In a preferred embodiment, the compounds and compositions discussed herein are administered orally.

[0153] The terms "drug combination," "drug co-administration," "combined drug therapy," "administration of other treatments," and "administration of other therapeutic agents" used in this invention refer to drug therapy obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to the simultaneous administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity or single dosage form. The term "non-fixed combination" refers to the simultaneous, combined, or sequential administration to a patient of at least one compound described herein and at least one synergistic agent in the form of a single entity at variable intervals. These also apply to cocktail therapies, such as the administration of three or more active ingredients.

[0154] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0155] Indications

[0156] The present invention also provides the use of the compounds or pharmaceutical compositions of the present invention in the treatment or prevention of cachexia or cachexia-related diseases.

[0157] As used herein, the term "cachexia" refers to a devastating and often irreversible metabolic syndrome induced by multiple factors, characterized primarily by anorexia and significant weight loss due to a substantial reduction in skeletal muscle and fat. Cachexia is associated with diseases such as cancer, chronic obstructive pulmonary disease, chronic heart failure, chronic kidney disease, severe trauma, sepsis, infectious diseases such as HIV / AIDS, and inflammation. Preferably, the cachexia described is cancer-induced cachexia.

[0158] Preferably, the cachexia-related diseases include (but are not limited to): chronic obstructive pulmonary disease, chronic heart failure, chronic kidney disease, severe trauma, sepsis, AIDS, inflammation, cancer, or combinations thereof.

[0159] Preferably, the cancer is a solid tumor or a hematoma.

[0160] Preferably, the diseases include (but are not limited to): myelodysplastic syndromes, multiple myeloma, mantle cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, chronic myelomonocytic leukemia, myelofibrosis, Burkitt lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, ciliary body and chronic melanoma, iris melanoma, recurrent interocular melanoma, T-cell lymphoma, erythroid lymphoma, monocytic and... Monocytic leukemia, myeloid leukemia, central nervous system lymphoma, meningioma, spinal cord tumor, lung cancer, ovarian cancer, skin cancer, renal cell carcinoma, astrocytoma, amyloidosis, type I complex regional pain syndrome, malignant melanoma, radiculopathy, glioblastoma, glioma, malignant glioma, refractory plasmacytoma, extraocular melanoma, papillary and follicular thyroid carcinoma, breast cancer, prostate cancer, hepatocellular carcinoma, or primary macroglobulinemia.

[0161] The main advantages of this invention include:

[0162] (a) The compounds in this invention promote the interaction between PRDM16 and SCF via a molecular glue mechanism. β-TRCP Binding of E3 ubiquitin ligase.

[0163] (b) The compounds in this invention have excellent pharmacokinetics and efficacy, and can significantly promote the degradation of PRDM16 protein, with effective concentrations reaching the nM level.

[0164] (c) The compounds in this invention can significantly inhibit tumor-induced fat browning, fat consumption and cachexia.

[0165] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0166] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0167] Experimental methods:

[0168] 1. siRNA transfection experiment:

[0169] The siRNA transfection process is as follows: Taking a 6-well culture dish as an example, take two 1.5mL EP tubes (A and B) containing RNase- / DNase-free enzymes and add 75μL of Opti-MEM to each. Add 25μM siRNA to tube A and 9μL of Lipofectamine RNAIMAX (Thermo Fisher 13778150) to tube B. After shaking, let stand for about 4 minutes. Then, mix solutions A and B dropwise until homogeneous and let stand for 8 minutes. At this point, discard the culture medium in the culture dish and replace it with preheated new cell culture medium. After standing, add the AB mixture dropwise to the cell culture dish. After 24 hours, replace the medium with new cell culture medium, or choose not to replace the medium.

[0170] 2. Immunoblotting:

[0171] Prepare a Western blot gel of the corresponding concentration according to the size of the target protein. Add approximately 25 μg of protein to the wells of the gel and incubate at 80V for 30 min. Then, change the voltage to 120V for 60 min to stop electrophoresis. Perform wet transfer using the sandwich method, adding pre-cooled transfer buffer and incubating at a constant voltage of 105V for 120 min. After the experiment, cut the target protein band according to the position of the protein marker, then block with 5% BSA blocking buffer at room temperature for 1 hour. Then, incubate overnight at 4°C with anti-protein antibodies (Anti-β-Actin Sigam A2228, Anti-PRDM16 R&D AF6295, Anti-β-TRCP CST 4394, Anti-Myc-Tag CST 2276, Anti-HA-Tag CST 3724, Anti-His-Tag Proteintech 10001, Anti-UCP1 ThermoFisher PA124894). Remove the incubated protein bands, wash the membrane with TBST buffer at room temperature (5 min / wash, 3 washes), then add HRP-labeled anti-primary antibody (Anti-Rabbit IgG Merck401315, Anti-Mouse IgG Bio-RAD 170-6516), incubate on a shaker at room temperature for 1 hour, and wash the membrane again with TBST buffer (5 min / wash, 3 washes). Finally, develop using an ECL development kit (Millipore WBKLS0500).

[0172] 3. Immunoprecipitation:

[0173] Antibody incubation: Prepare cell lysis buffer (50mM NaCl, 20mM HEPES pH 7.4, 1% Triton X-100, 1.5mM MgCl2, 12.5mM β-glycerophosphate, 2mM EGTA, 10mM NaF, 1mM PMSF, 1mM Na3VO4, and protease inhibitor). Collect cultured cells, discard the supernatant, add an appropriate amount of sterile PBS solution, scrape cells with a scraper and transfer to a 1.5mL EP tube, centrifuge at 3000rpm, 4℃ for 4min, discard the supernatant, add an appropriate amount of lysis buffer according to the cell number, and lyse by rotating in a Ferris wheel at 4℃ for 45min. After lysis, centrifuge at 12000rpm, 4℃ for 15min. Transfer the supernatant to a new 1.5 mL EP tube. Measure the protein concentration of multiple samples using BCA. Then prepare protein lysis buffers of the same concentration and volume. Add the anti-immunoprotein antibody according to the protein concentration and incubate overnight in a Ferris wheel at 4°C.

[0174] Magnetic bead incubation (Protein A / G Magnetic Beads (MCE HY-K0202)): Depending on the protein content, take 30μL-50μL of beads, resuspend and wash the beads with 0.5% PBST solution, place them on a Ferris wheel and rotate for 2 minutes, then precipitate the beads on a magnetic rack. Discard the supernatant and repeat the washing process 3 times. Add the protein lysate incubated with antibody to the washed beads and incubate at 4 degrees Celsius with a Ferris wheel for 3-4 hours. Take the incubated lysate, precipitate the beads on a magnetic rack, discard the supernatant, and resuspend and wash the beads again with 0.5% PBST solution. Place them on a Ferris wheel and rotate for 2 minutes, then precipitate the beads on a magnetic rack. Discard the supernatant and repeat the washing process 4 times.

[0175] Elution of proteins: Add SDS-PAGE protein loading buffer (1X) to a test tube, heat at 95°C for 15 min to denature, shake once during the process, centrifuge at 12000 rpm at room temperature for 5 min after denaturation, precipitate the beads with a magnetic rack, collect the supernatant, use it for immunoblotting experiments or store at -30°C.

[0176] 4. Adipocyte differentiation experiment

[0177] Adipocytes were cultured in a cell culture medium containing fetal bovine serum (FeBSS) (penicillin and streptomycin) for 2 days after confluence with the cells. Then, differentiation induction solution A (0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (MCE HY-12318), 1 mM dexamethasone (MCE HY-14648), and 10 μg / mL insulin (Roche 11376497001) added to the FBS cell culture medium) was added for 2 days of induction. The culture medium was then discarded, and differentiation induction solution B (10 μg / mL insulin added to the FBS cell culture medium) was added for another 2 days. After 2 days, the culture medium was discarded again, and the medium was replaced with FBS cell culture medium (penicillin and streptomycin) for another 2 days. Stimulation was then performed according to experimental requirements, and cells were subsequently collected.

[0178] 5. Subcutaneous tumor transplantation experiment:

[0179] 1.5 (or 2.5) × 10⁶ LLC cells were subcutaneously inoculated into one side of 8-10 week old male C57BL / 6J mice. The control group was injected subcutaneously with sterile PBS. Depending on the experimental requirements, cells could be transplanted into one or both sides of the mice. Tumor growth was observed, and mouse weight was measured weekly. When significant weight loss was observed compared to the control group, the mice were anesthetized, and inguinal adipose tissue was extracted and cryopreserved at -80°C.

[0180] 6. SPR Experiment

[0181] SPR experiments were performed using a BIAcore 8K instrument (Cytiva). Briefly, the protein PRD was dissolved at a concentration of 40 μg / mL in 10 mM sodium acetate at pH 3.6 and coupled to a CM5 chip according to the manufacturer's instructions. The run buffer was 1x PBST plus 0.1% DMSO. Small molecules were serially diluted and injected at a flow rate of 30 μL / min for 60 seconds for the binding step, followed by an additional 60 seconds for dissociation. Affinity was calibrated using a steady-state affinity 1:1 binding model using the BIAcore 8K evaluation software.

[0182] Example 1. Detection of β-TRCP as a ubiquitinating enzyme of PRDM16

[0183] The 3T3-L1 adipocyte density was approximately 80%. siRNA (siNC, siBtrc#1, siBtrc#2) (Germazon gene) was transferred into the 3T3-L1 adipocyte line (see Experimental Method 1). After 48 hours, cells were collected, proteins were extracted, and then immunoblotting was performed (see Experimental Method 2) to analyze the knockdown effect of β-TRCP and the protein expression level of PRDM16.

[0184] The 293T cell density was approximately 80%. After overexpressing equal amounts of PRDM16, β-TRCP, and Ubiquitin plasmid in 293T cells for 40 h, the cells were treated with 50 μM MG132 for 9 h. Cells were then collected, and the degree of PRDM16 polyubiquitination was analyzed by immunoprecipitation (see Experimental Method 3).

[0185] 3T3-L1 adipocyte precursor cells were treated with different concentrations of NRX for 24 hours, and the cells were collected and then immunoblot analysis was performed to analyze changes in PRDM16 protein.

[0186] The results showed that after inhibiting the expression of E3 ubiquitin ligase β-TRCP with siRNA in 3T3-L1 adipocytes, the protein level of PRDM16 was significantly increased. Figure 1 A). Further experiments revealed that β-TRCP regulates the degradation of PRDM16 by promoting the ubiquitin ligase of PRDM16. Figure 1 B).

[0187] The above experimental results indicate that β-TRCP is a ubiquitin ligase of PRDM16.

[0188] Example 2. Detection of the effect of NRX series drugs on the ubiquitination and degradation of PRDM16

[0189] This embodiment further investigates whether NRX series drugs can regulate the degradation of PRDM16. The method is as follows: 3T3-L1 adipocyte progenitor cells were treated with different concentrations of NRX drugs for 24 h, and the cells were collected and then analyzed by immunoblotting to detect changes in PRDM16 protein.

[0190] 2.1 NRX drugs promote PRDM16 degradation

[0191] When the density of 3T3-L1 adipocytes was approximately 80%, 3T3-L1 adipocytes were treated with different concentrations of NRX series drugs (including NRX-252262, NRX-1933, NRX-103094, and NRX-252114) for 24 hours, and the changes in PRDM16 protein were analyzed by Western blotting. The concentrations of each NRX series drug are shown below:

[0192] NRX-252262 (Taoshu T9182) concentrations were 0, 4, 8 and 12 nM;

[0193] NRX-1933 (Taoshu T60031) concentrations were 0, 20, 40, 80 and 160 nM;

[0194] NRX-103094 (MCE HY-141449) concentrations were 0, 25, 50, 100, 200 and 400 nM;

[0195] NRX-252114 (Taoshu T63516) concentrations were 0, 5, 10, 20, 40 and 80 nM.

[0196] The results showed that treatment of 3T3-L1 adipocytes with NRX series drugs (including NRX-252262, NRX-1933, NRX-103094, and NRX-252114) significantly promoted the downregulation of PRDM16 protein levels. Figure 2 Among the various inhibitory agents (AD), NRX-252262 had the lowest effective concentration (8 nM) and the best inhibitory effect on PRDM16. Figure 2 A).

[0197] 2.2 PRD and NRX-252262 are combined

[0198] To further investigate how NRX series drugs regulate the degradation of PRDM16, the inventors analyzed the interaction between PRDM16, β-TRCP, and NRX-252262.

[0199] First, the protein polypeptide containing the conserved PR domain (PRD) of PRDM16 was obtained. Figure 2 E), and then surface plasmon resonance (SPR) experiments were conducted on PRD and NRX-252262. The results show that PRD can interact with NRX-252262, and its dissociation constant K D 2.77 μM ( Figure 2 F).

[0200] 2.3 In the presence of NRX, β-TRCP and PRDM16 proteins undergo immunoprecipitation.

[0201] When the 3T3-L1 adipocyte density was approximately 80%, the 3T3-L1 adipocytes were treated with 12 nM NRX-252262 for 24 h, and the interaction between β-TRCP and PRDM16 proteins was analyzed by immunoprecipitation.

[0202] The results showed that in the presence of NRX-252262, β-TRCP and PRDM16 exhibited significant interaction or binding. Figure 2G), which indicates that the compounds of the present invention (such as NRX-252262) enable β-TRCP to form a complex with PRDM16 via a molecular glue mechanism.

[0203] 2.4NRX-252262 promotes PRDM16 ubiquitination.

[0204] When the density of 3T3-L1 adipocytes was approximately 80%, 3T3-L1 adipocytes were treated with 12 nM NRX-252262 for 24 h and simultaneously treated with 50 μM MG132 (a proteasome inhibitor) for 16 h. Immunoprecipitation was then used to analyze the effect of NRX-252262 on PRDM16 polyubiquitination.

[0205] The results showed that when MG132 was used to inhibit the proteasomal degradation of ubiquitinated PRDM16, thus allowing the ubiquitination level of PRDM16 to be observed, NRX252262 significantly promoted the ubiquitination level of PRDM16. This indicates that NRX-252262 promotes the polyubiquitination of PRDM16 through a molecular gel mechanism, thereby promoting the degradation of PRDM16. Figure 2 H).

[0206] The above data indicate that NRX series drugs promote the polyubiquitination and degradation of PRDM16 through molecular glue interaction.

[0207] Example 3. NRX series drugs promote the degradation of PRDM16 and inhibit fat browning, thereby improving cachexia.

[0208] Tumor cells can induce the expression of PRDM16 in adipocytes, leading to fat browning, fat consumption, and cachexia. This example further investigates the inhibitory effect of NRX on tumor cachexia.

[0209] 3.1 NRX-252262 blocks the increase in PRDM16 caused by cachectic tumor cells.

[0210] Equal numbers of tumor cells (caustic lung cancer cells LLC and non-caustic tumor cells MC38) were seeded in cell culture dishes. When the tumor cells reached 70%-80% growth, the tumor cell culture medium was replaced with serum-free medium (DMEM). After 24 hours, the supernatant from the tumor cell culture medium was collected. Differentiated 3T3-L1 adipocytes (CM, Conditioned Medium) were treated with the tumor culture supernatant from the cachectic lung cancer cells LLC and the non-caustic tumor cells MC38, along with the control medium DMEM, for 48 hours. Simultaneously, they were treated with 12 nM NRX-252262 for 24 hours. Changes in PRDM16 protein were then analyzed by Western blotting.

[0211] The results showed that treatment of 3T3-L1 adipocytes with tumor cell culture supernatant (CM) induced upregulation of PRDM16 protein in cachectic lung cancer cells (LLC), but this effect was not observed in non-cachecinic tumor cells (MC38). Furthermore, the addition of NRX-252262 significantly blocked the upregulation of PRDM16 protein levels by LLC. Figure 3 A).

[0212] 3.2 NRX series drugs block the rise in PRDM16 caused by cachexia tumor cells.

[0213] LLC lung cancer cell culture supernatant and normal culture medium were added to differentiated 3T3-L1 adipocytes at a 1:1 ratio for 48 h. After 24 h, PRDM16 protein changes were analyzed by immunoblotting after treatment with different concentrations of NRX series drugs. The concentrations of NRX series drugs were NRX-252262 (12 nM), NRX-252114 (80 nM), NRX-103094 (400 nM), and NRX-1933 (160 nM).

[0214] The results showed that other NRX series drugs (NRX-252114, NRX-103094, and NRX-1933) also had the same effect as NRX-252262, all of which could block the regulation of PRDM16 protein levels in LLC cells. Figure 3 B).

[0215] 3.3 NRX-252262 blocks the rise in PRDM16 induced by various cachectic tumor cells.

[0216] Equal numbers of tumor cells (4T1 breast cancer cells, C26 colon cancer cells, and Panc02 pancreatic cancer cells) were seeded in cell culture dishes. When the tumor cells grew to 70%-80%, the tumor cell culture medium was replaced with serum-free medium (DMEM). After 24 hours, the supernatants of the various tumor cell culture media were collected. The tumor cell culture supernatants and normal culture medium were added to differentiated 3T3-L1 adipocytes at a ratio of 1:1. At 24 hours, 12 nM NRX-252262 was added. At 48 hours, the cells were collected, proteins were extracted, and the expression of PRDM16 was analyzed by Western blotting.

[0217] The results showed that NRX-252262 could block the 4T1 cell line in breast cancer cells. Figure 3 C), colon cancer cells C26 ( Figure 3 D), Pancreatic cancer cells Panc02 ( Figure 3E) These cachectic tumor cells regulate the level of PRDM16 protein in adipocytes. The above data indicate that NRX series compounds can inhibit tumor cell-induced adipose tissue browning at the cellular level.

[0218] 3.4 NRX-252262 inhibits fat browning induced by cachectic tumor cells.

[0219] (1) To further investigate whether NRX-252262 can inhibit adipose browning in mice, a cachectic mouse model was constructed: C57BL / 6J mice were subcutaneously injected with 2.5 x 10 6 LLC cells were used, while the control group received an equal volume of PBS (NTB) subcutaneously. Mice were weighed before injection, designated as day 0 (0 Days); tumors were removed and mouse weights were recorded again after day 28 (28 Days). Simultaneously, inguinal adipose tissue (subcutaneous fat) was harvested from the mice on day 28, and proteins were extracted. Western blotting analysis was performed to assess the expression of the adipose browning marker protein UCP1.

[0220] The results showed that, compared with mice not inoculated with LLC tumor cells, mice subcutaneously inoculated with LLC tumor cells for 28 days exhibited significant cachexia symptoms, mainly manifested by increased expression of UCP1, a key marker of fat browning, in the inguinal adipose tissue of mice. Figure 3 F) and a significant decrease in mouse body weight after tumor removal (F) Figure 3 G).

[0221] The above data indicate that cachectic tumor cells LLC can induce browning of adipose tissue in mice, thereby inducing weight loss and cachexia.

[0222] (2) Subcutaneous injection of 1.5 x 10⁻⁶ ppm in C57BL / 6J mice 6 LLC cells were used. Mice were weighed on day 7 (before treatment), and then administered 10 mg / kg of NRX-252262 via gavage. Control mice received DMSO. Gavage was administered every two days from day 7 to 20, and daily from day 21 to 35. On day 35, tumors were removed, and mouse weight was recorded (post-treatment). Simultaneously, inguinal adipose tissue (subcutaneous fat) was collected on day 35, and proteins were extracted. Immunoblotting analysis was performed to assess the expression of the adipose browning marker proteins PRDM16 and UCP1.

[0223] The results showed that after subcutaneous injection of LLC cells into C57BL / 6J mice, treatment with NRX-252262 significantly inhibited the expression of PRDM16 and UCP1 in the inguinal fat of mice compared with that before treatment. Figure 3 H), and significantly inhibited the decrease in mouse body weight (H). Figure 3 I).

[0224] The above data indicate that NRX-252262 can promote the degradation of PRDM16, thereby preventing tumor-induced fat browning, and thus inhibiting weight loss and cachexia.

[0225] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer, or mixture thereof, characterized in that, for the manufacture of a medicament or a formulation for one or more uses selected from the group consisting of: (a) a medicament for the treatment and / or prevention of a cachexia-related disease; (b) inhibiting the browning of adipocytes; and / or (c) promoting the degradation of PRDM16 protein; wherein the compound of formula (I) has the following structure: wherein, R1 is C1-C3 alkyl or C1-C3 haloalkyl; R2 is absent or -SR5 or -OR5; R5is substituted or unsubstituted C1-C6alkyl, C3-C7cycloalkyl, 4-7 membered heterocyclyl, C6-C10aryl, or 5-7 membered heteroaryl; wherein the substitution is with from one to three substituents each independently selected from the group consisting of halogen, cyano, hydroxyl, C1-C3alkyl, C1-C3haloalkyl, C1-C4alkoxy, C1-C4haloalkoxy, -NR 10 R 11 , -C(=O)R 12 , C1-C4alkanoyloxy, or two adjacent substituents together with the carbon atoms to which they are attached form a 5-7 membered carbocyclic or 5-7 membered heterocyclic ring; R3 is selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, wherein the substitution is with 1-3 substituents each independently selected from the group consisting of: halo, OH, C1-C3 alkyl; R4 is substituted or unsubstituted selected from the group consisting of: C1-C6 straight or branched alkyl, C3-C7 cycloalkyl, 4-7 membered heterocyclyl, C6-C10 aryl or 5-7 membered heteroaryl; wherein the substituents are selected from the group consisting of: halo, hydroxyl, nitro, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C1-C4 alkylcarbonyl-, C1-C4 carboxyl, 5-7 membered heteroaryl substituted or substituted with 1-3 substituents selected from Group A, phenyl unsubstituted or substituted with 1-3 substituents selected from Group A, wherein Group A substituents are selected from the group consisting of: halo, C1-C3 alkyl, OH, C1-C3 haloalkyl, C1-C3 alkoxy; or R3and R4together with the N atom to which they are attached form wherein n is an integer from 0 to 4; R6 is selected from the group consisting of: halo, cyano, hydroxyl, substituted or unsubstituted C1-C3 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted 4-7 membered heterocyclyl, substituted or unsubstituted benzyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted 5-7 membered heteroaryl; wherein the substitution is with 1-3 substituents each independently selected from the group consisting of: halo, hydroxyl, C1-C4 straight or branched alkoxy, C1-C4 straight or branched alkyl, C1-C4 straight or branched haloalkyl, C1-C4 straight or branched haloalkoxy, cyano, nitro, amino, carboxyl.

2. Use according to claim 1, characterized in that, the compound of formula (I) is selected from the group consisting of:

3. Use according to claim 1, characterized in that, the cachexia-related disease is selected from the group consisting of: Myelodysplastic syndrome, multiple myeloma, mantle cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myelomonocytic leukemia, myelofibrosis, Burkitt's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, ciliary and chronic melanoma, iris melanoma, recurrent binocular melanoma, T-cell lymphoma, erythroid lymphoma, monocytic and mononuclear leukemia, myeloid leukemia, central nervous system lymphoma, meningioma, spinal cord tumor, lung cancer, ovarian cancer, skin cancer, renal cell carcinoma, astrocytoma, amyloidosis, complex regional pain syndrome type I, malignant melanoma, radiculopathy, glioblastoma, gliosarcoma, malignant glioma, refractory plasmacytoma, extraocular extension melanoma, papillary and follicular thyroid cancer, breast cancer, pancreatic cancer, colon cancer, prostate cancer, hepatocellular carcinoma, or primary macroglobulinemia.

4. The use according to claim 1, characterized in that, The drug or preparation is used for: (1) promoting the binding of PRDM16 to E3 ubiquitin ligase; (2) degrading PRDM16 protein; (3) reducing the fat browning of the patient; (4) promoting the increase of skeletal muscle of the patient; (5) reducing the cachexia of the patient.

5. The use of claim 1, wherein the drug or preparation is used for degrading PRDM16 protein in adipocytes.

6. Use of a pharmaceutical composition characterized in that, The pharmaceutical composition is used for preparing a medicament for treating and / or preventing a cachexia-related disease; and the pharmaceutical composition comprises: (i) a first active ingredient selected from the group consisting of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer thereof; (ii) optionally a second active ingredient selected from the group consisting of dexamethasone, rituximab, trastuzumab, a PD-1 inhibitor, a PDL-1 inhibitor, pemetrexed, topotecan, doxorubicin, gemcitabine, dacarbazine, clarithromycin, vincristine, cytarabine, prednisone, docetaxel, clofarabine injection, an HDAC inhibitor, an androgen receptor inhibitor, an androgen biosynthesis inhibitor, a BTK inhibitor, an erythropoietin growth hormone, minocycline, Elotuzumab, Palbociclib, Nivolumab, Pembrolizumab, Panobinostat, Ublituximab, Romidepsin, Eltrombopag, CAR-T, melphalan, or a combination thereof; (iii) a pharmaceutically acceptable carrier; The compound of formula (I) is as defined in claim 1.

7. A method for treating and / or preventing cachexia-related diseases, characterized by, The method comprises administering to a subject in need thereof a medically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, optically pure isomer, stereoisomer thereof; the compound of formula (I) is as defined in claim 1.

8. A method of inhibiting PRDM16 protein levels in adipocytes in vitro, comprising contacting the adipocytes with a compound of any one of claims 1-7. 8 The method comprises the step of culturing an adipocyte in the presence of a compound of formula (I) or a pharmaceutically acceptable salt thereof, thereby inhibiting the level of PRDM16 protein in the adipocyte.

9. A method of inhibiting browning of adipocytes in vitro, characterized in that, The method comprises the steps of: (a) culturing an adipocyte in the presence of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, thereby inhibiting browning of the adipocyte.

10. The method of claim 9, wherein, The method is non-diagnostic and non-therapeutic.

Images