Degradation agents targeting interleukin-1 receptor-associated kinase 4 and therapeutic uses thereof

Abstract

This application belongs to the field of pharmaceutical technology and discloses a class of degrading agents targeting interleukin-1 receptor-associated kinase 4 and their therapeutic uses. It relates to a bifunctional compound having the structure of formula (I), or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or isotopic derivative thereof, having one end containing a cereblon (CRBN) ligand binding to E3 ubiquitin ligase, and the other end containing a target protein binding moiety, namely a small molecule binding to interleukin-1 receptor-associated kinase 4 (IRAK4), such that the target protein is degraded after ubiquitination labeling by the ubiquitin ligase. The bifunctional compounds disclosed herein exhibit broad pharmacological activities associated with target protein degradation. Diseases or disorders caused by IRAK4-mediated cell signal transduction can be treated or prevented using the bifunctional compounds or compositions of this invention.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically to compounds that target the degradation of IRAK4 protein, pharmaceutical compositions thereof, methods for their preparation, and their use in the preparation of medicaments for the treatment and / or prevention of IRAK4-mediated diseases or conditions. Background Technology

[0002] Interleukin-1 receptor-associated kinase 4 (IRAK4) belongs to the serine-threonine kinase family, which includes four members: IRAK1, IRAK2, IRAKM (IRAK3), and IRAK4. IRAK4, as the master kinase among the four, plays a crucial role in the interleukin-1 receptor (IL-1R) / Toll-like receptor (TLR) signaling pathway. TLRs are transmembrane pattern recognition receptors (PRRs) that primarily mediate innate immunity, mainly recognizing pathogen-associated molecular patterns (PAMPs). IL-1R shares the same signaling pathway as TLRs due to the same structural domain, primarily recognizing damage-associated molecular patterns (DAMPs). Upon activation of IL-1R / TLRs, the downstream protein MyD88 is recruited, followed by the recruitment of IRAK4 from the IRAK family. IRAK4 undergoes autophosphorylation, recruiting IRAK1 and IRAK2, ultimately forming the myddosome complex. This complex sequentially activates downstream proteins, ultimately activating the NF-κB, JNK / p38, and IRF3 / 7 pathways. IRAK4 is primarily expressed in various immune cells. Its deficiency may cause recurrent infections in childhood, but this gradually decreases with age, and no significant infections are observed after age 14. However, overexpression and activation of IRAK4 can lead to abnormal signaling pathways, which are closely related to the development and progression of various autoinflammatory / immune diseases and tumors.

[0003] In signal transduction, IRAK4 plays a dual role: kinase activity and scaffolding function. IRAK4 possesses kinase activity, forming the basis for the activation of the entire downstream signaling pathway. IRAK4 itself folds into a unique "pocket-like" structure, where ATP binds to enable protein phosphorylation. Small molecule inhibitors are designed to target this active pocket, competing with ATP for the active site to exert their effects. In recent years, small molecule inhibitor development has dominated, with Pfizer's PF-06650833 and Curis' CA-4948 already in Phase II clinical trials. However, research indicates that the IL-1R / TLR signaling pathway can also be activated through the scaffolding effect of IRAK4. Small molecule inhibitors, by simply inhibiting kinase activity, may not be able to completely inhibit the IL-1R / TLR pathway; therefore, further development of therapeutics targeting IRAK4 is needed.

[0004] To achieve complete inhibition of the IL-1R / TLR signaling pathway, proteolysis-targeting chimeras (PROTACs) are considered a novel drug research strategy for treating autoimmune diseases, inflammatory diseases, and oncology. For example, Kymera's KT-474, which simultaneously blocks kinase activity and scaffold function to achieve complete inhibition of the IL-1R / TLR signaling pathway, is currently in Phase 2 clinical trials for the treatment of atopic dermatitis and hidradenitis suppurativa. KT-474 is the first degradation agent to enter the clinical stage. The development of novel IRAK4 degradation agents aims to achieve comprehensive inhibition of the TLR / IL-1R signaling pathway through effective degradation of IRAK4. This innovative strategy holds promise for providing new avenues for the treatment of various inflammatory and immune-related diseases.

[0005] Parthenolide (PTL) is a natural organic compound extracted from the plant *Tanacetum parthenium*, belonging to the sesquiterpene lactone class. Parthenolide possesses various biological activities, including anti-inflammatory, antitumor, antimicrobial, and anti-angiogenic pharmacological effects. Studies have shown that parthenolide exhibits good inhibitory activity against IRAK4. Designing and synthesizing heterobifunctional molecular degraders targeting IRAK4 using parthenolide represents a novel drug design strategy. Summary of the Invention

[0006] This invention describes a class of bifunctional compounds for recruiting endogenous proteins to E3 ubiquitin ligases for ubiquitination and degradation, and their applications. Specifically, the bifunctional or proteolytically targeted chimeras provided by this invention can hijack E3 ubiquitin ligases to ubiquitinate and label peptides and proteins, ultimately leading to their degradation or inhibition. Furthermore, this invention provides methods for treating or improving diseases, such as autoinflammatory / immune diseases and cancer, using the described effective compounds.

[0007] This invention relates to bifunctional compounds having the structure of formula (I):

[0008]

[0009] Or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or isotopic derivative thereof, wherein parthenolide is the protein / peptide targeting binding moiety, ring A is the E3 ubiquitin ligase binding moiety, i.e., the E3 ubiquitin ligase ligand, and LNK is the linker, which is the chemical bond or chemical group that connects parthenolide to the E3 ubiquitin ligase ligand. Parthenolide is responsible for binding to the target protein IRAK4 and is chemically linked to the E3 ligase ligand directly or through the linker moiety LNK.

[0010] in,

[0011] LNK is selected independently from:

[0012] R1 is independently selected from straight-chain C atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0013] R2 is independently selected from straight-chain carbon atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0014] R3 is independently selected from straight-chain carbon atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0015] in:

[0016] Ring A is independently selected from equations (IIa) and (IIb):

[0017]

[0018] Among them, X1, X2, and X3 are independently selected from C and N, respectively; For LNK connection points;

[0019] R4 and R5 are independently selected from unsubstituted and halogenated, -OH, -NH2, -CN, or -CF3 substituted straight-chain or branched C atoms, respectively. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0020] In another preferred embodiment, formula (Ia) is a bifunctional compound, a solvate, an enantiomer, a diastereomer, or an isotopic derivative thereof, or a pharmaceutically acceptable salt thereof.

[0021]

[0022] in:

[0023] The ring C is selected from the following groups: substituted or unsubstituted 5-6 membered heterocyclic groups, substituted or unsubstituted 7-9 membered bridged heterocyclic groups, substituted or unsubstituted 6-10 membered fused heterocyclic groups, and substituted or unsubstituted 3-6 membered cycloalkyl groups;

[0024] Y is selected from -(D1)n1;

[0025] D1 is selected from saturated or unsaturated C. 1-3 The methylene chain, wherein any methylene unit is optionally surrounded by -O-, -C(O)-, or -C(R)-. a (R) b )-、-N(R c - Replaced; n1 is selected from 0 and 1;

[0026] R a R b R c They are independently selected from H and halogens, respectively;

[0027] R6 is independently selected from unsubstituted and halogenated, -OH, NH2, -CN, or -CF3 substituted straight-chain or branched C atoms. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0028] In another preferred embodiment, formula (Ib) is a bifunctional compound, a solvate, an enantiomer, a diastereomer, or an isotopic derivative thereof, or a pharmaceutically acceptable salt thereof.

[0029]

[0030] in:

[0031] The ring E is selected from the following groups: substituted or unsubstituted 5-6 membered heterocyclic groups, substituted or unsubstituted 7-9 membered bridged heterocyclic groups, substituted or unsubstituted 6-10 membered fused heterocyclic groups, and substituted or unsubstituted 3-6 membered cycloalkyl groups;

[0032] Z is selected from -(D2)n2;

[0033] D2 is selected from saturated or unsaturated C. 1-3 A hydrocarbon chain, wherein any methylene unit is optionally surrounded by -O-, -C(O)-, or -C(R)-. a (R) b )-、-N(R c - Replaced; n2 is selected from 0 and 1;

[0034] R a R b R c They are independently selected from H and halogens, respectively;

[0035] R7 is independently selected from unsubstituted and halogenated, -OH, NH2, -CN, or -CF3 substituted straight-chain or branched C atoms. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkynyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl;

[0036] In another preferred embodiment, LNK is selected from...

[0037] One end of the LNK is linked to the E3 ubiquitin ligase ligand, and the other end is linked to ternolactone.

[0038] In another preferred embodiment, the bifunctional compound, solvate, enantiomer, diastereomer, or isotope derivative or a pharmaceutically acceptable salt thereof is selected from the following compounds:

[0039]

[0040]

[0041]

[0042] In a second aspect, this disclosure also provides a pharmaceutical composition comprising the bifunctional compound, solvate, enantiomer, diastereomer, or isotope derivative or a pharmaceutically acceptable salt thereof described in the first aspect of this disclosure, and one or more pharmaceutically acceptable excipients, said pharmaceutical composition being in any pharmaceutically acceptable dosage form. According to this disclosure, a pharmaceutically acceptable excipient is a substance that is non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in a living organism. The selection of a specific excipient will depend on the route of administration or the type and state of disease for treating a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, antioxidants, etc., conventional in the pharmaceutical field. Where necessary, flavoring agents, preservatives, and sweeteners may also be added to the pharmaceutical composition.

[0043] In another preferred embodiment, the content of the bifunctional compound, solvate, enantiomer, diastereomer or isotope derivative or pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1%-95%.

[0044] A third aspect of this disclosure also provides the use of a bifunctional compound, solvate, enantiomer, diastereomer or isotope derivative or pharmaceutically acceptable salt thereof, as described in any of the embodiments of the first aspect of this disclosure, in the preparation of a medicament for the prevention and / or treatment of IRAK4-mediated related diseases.

[0045] In another preferred embodiment, the IRAK4-mediated related diseases are selected from autoinflammatory / immune diseases.

[0046] In another preferred embodiment, the IRAK4-mediated related diseases are selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis, atopic dermatitis, hidradenitis suppurativa, alcoholic hepatitis, inflammatory bowel disease, multiple sclerosis, rash, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, alcoholic hepatitis, Hashimoto's thyroiditis, toxic diffuse goiter, type 1 diabetes, and Crohn's disease.

[0047] Detailed description of the invention

[0048] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0049] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc., and their various branched isomers. The present invention preferably uses methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, and hydroxyl-substituted alkyl.

[0050] The term "alkylene" refers to an alkyl group in which one hydrogen atom is further substituted, for example: "methylene" refers to -CH2-, "ethylene" refers to -(CH2)2, "propylene" refers to -(CH2)3-, "butylene" refers to -(CH2)4-, etc.

[0051] The term "alkenyl" refers to an alkyl group as defined above, consisting of at least two carbon atoms and at least one carbon-carbon double bond, such as vinyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc. Alkenyl groups can be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0052] The term "alkenyl" refers to the further substitution of one hydrogen atom of an alkenyl group, for example, "vinylene" refers to -C=CH2-.

[0053] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups, and non-limiting examples include...

[0054] The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylazyl, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0055] The term "cycloalkylene" refers to a cycloalkyl group in which one hydrogen atom is further substituted, with non-limiting examples including:

[0056]

[0057] The term "heterocyclic group" refers to a substituent in a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, C(O), S(O) (=NH), or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 12 ring atoms; most preferably, it contains 3 to 8 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include oxetane, thiohepane, azahepane, tetrahydropyranyl, azaheptanyl, pyrrolidinyl, imidazoyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, etc., preferably oxetane, thiohepane, azahepane, tetrahydrofuranyl, tetrahydropyranyl, 1-aminoimylide-1-oxothiran, azaheptanyl, piperidinyl, and piperazinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups, non-limiting examples of which include:

[0058] The heterocyclic group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from hydrogen, alkyl, hydroxyalkyl, amino, imino, cyano, oxo, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0059] The term "heterocyclic group" refers to a heterocyclic group in which one hydrogen atom is further substituted, and non-limiting examples include: piperidinyl, piperazine, pyrrolopyrroleyl, diazaspiro[5.5]undecyl, diazaspiro[5.5]undecyl, benzopiperidinyl, diazaspirobutane, diazaspirobutane, pyrroleyl, diazaspiro[3.5]nonyl, diazaspiro[3.5]nonyl, diazabicyclo[3.1.1]heptyl, diazaspiro[2.5]octyl,

[0060] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. Phenyl is more preferred. 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.

[0061] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, benzoyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0062] The term "aryl" refers to the further substitution of one hydrogen atom of an aryl group, with non-limiting examples including:

[0063]

[0064] The term "heteroaryl" refers to a heteroaryl system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl is preferably a 5-8 member mono-heteroaryl or an 8-14 member original di-heteroaryl, more preferably a 5-membered mono-heteroaryl, 6-membered mono-heteroaryl, or 9-membered di-heteroaryl, such as imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, pyrroleyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, pyridinium-imidazolyl, pyrimidium-imidazolyl, etc., with pyridinium-imidazolyl and pyrimidium-imidazolyl being preferred.

[0065] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from leucyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0066] The term "hybrid aryl" refers to a heteroaryl group in which one hydrogen atom is further substituted, and non-limiting examples include:

[0067] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0068] "Hydroxy" refers to the -OH group. "Halogen" refers to fluorine, chlorine, bromine, or iodine. "Amino" refers to -NH2. "Cyano" refers to -CN. "Nitro" refers to -NO2.

[0069] “Carboxyl group” refers to -C(O)OH. “THF” refers to tetrahydrofuran. “DCM” refers to dichloromethane. “EtOAc” refers to ethyl acetate. “MeOH” refers to methanol. “DMF” refers to N,N-dimethylformamide. “TFA” refers to trifluoroacetic acid. “TEA” refers to triethylamine. “MeCN” refers to acetonitrile. “Et2O” refers to diethyl ether. “DIPEA” refers to N,N-diisopropylethylamine. “Pd2(dba)3” refers to tris(dibenzylacetone)dipalladium. “Dppf” refers to 1,1'-bis(diphenylphosphine)ferrocene. “HATU” refers to 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate. “DMAP” refers to 4-dimethylaminopyridine. “EDCI” refers to carbodiimide. “Pd(PPh3)2Cl2” refers to di(triphenylphosphine)palladium dichloride. “TBHP” refers to tert-butyl hydroperoxide. “DMSO” refers to dimethyl sulfoxide. “K2CO3” refers to potassium carbonate. “PBS” represents phosphate buffer.

[0070] The chiral carbon in the compounds of this invention can be either R-configuration or S-configuration.

[0071] All hydrogen atoms described in this invention can be replaced by their isotope deuterium, and any hydrogen atom in the compounds of the embodiments of this invention can also be replaced by deuterium atoms.

[0072] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0073] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0074] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0075] "Medicinal salts" refer to the salts of the compounds of this invention, which are safe and effective when used in mammals and have the appropriate biological activity. Detailed Implementation

[0076] The preparation methods of some compounds in this disclosure reference the preparation methods of the aforementioned similar compounds. Those skilled in the art should understand that when using or referring to the referenced preparation methods, the reactant ratios, reaction solvents, reaction temperatures, etc., can be appropriately adjusted according to the different reactants.

[0077] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0078] Unless otherwise stated, all reactions in this disclosure are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, using a dry solvent, wherein: (i) temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature, generally 15-35°C, preferably 20-30°C, more preferably 20-25°C; (ii) solvent removal is performed by rotary evaporator distillation under reduced pressure; (iii) the reaction process is monitored by LC-MS or thin-layer chromatography (TLC); and (iv) the final product has nuclear magnetic resonance (NMR) and / or high-resolution mass spectrometry (HRMS) data. NMR chemical shifts (δ) are given in parts per million (ppm). NMR determinations are performed using a QOne Quantum-400MHz NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) and deuterated chloroform (CDCl3) as solvents and tetramethylsilane (TMS) as an internal standard.

[0079] The purification methods used in this disclosure include, but are not limited to, preparative high-performance liquid chromatography, thin-layer chromatography, or column chromatography. The purification reagents used are conventional solvents in the art, such as dichloromethane, methanol, ethyl acetate, petroleum ether, acetonitrile, and water. The specific types and proportions used can be determined by conventional methods in the art.

[0080] The raw materials or intermediate compounds used in this disclosure are commercially available or synthesized in-house. The structures and preparation methods of the in-house synthesized intermediates are described in this disclosure. The chirality of the chiral intermediates or chiral products in this disclosure can be determined by the chiral raw materials used. When the reaction site is an achiral site, the chirality of the raw material is usually the same as that of the product.

[0081] Synthesis of Example 1

[0082] Synthetic route of Example 1:

[0083]

[0084] Synthesis of (1aR,7aS,10bS,E)-5-(hydroxymethyl)-1a-methyl-8-methylene-2,3,6,7,7a,8,10a,10b-octahydrooxocyclopentene[2',3':9,10]cyclodeca[1,2-b]furan-9(1aH)-one (intermediate A-1):

[0085] At room temperature, SeO2 (447 mg, 4.03 mmol, 0.5 equiv) and 70% t-buOOH (TBHP) (1.11 mL, 1 equiv) were added to a 30 mL DCM solution of the starting material PTL (2 g, 8.05 mmol, 1 equiv) in a 100 mL reaction flask. The mixture was stirred overnight at room temperature. After the reaction of the starting material PTL was complete as monitored by TLC, DCM (20 mL) was added to the system. The mixture was then transferred to a separatory funnel, and Na2S2O3 (30 mL) was added to remove excess TBHP. The upper organic phase was collected, washed with saturated sodium chloride solution (200 mL), and dried over anhydrous sodium sulfate (200 mL). The DCM phase was concentrated under reduced pressure and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white crystalline solid A-1 (1.41 g, 66.2%).

[0086] LC-MS:(ESI,m / z):265.1[M+H] + .

[0087] Synthesis of methyl 5-bromopentanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyeno[2',3':9,10]cyclodeca[1,2-b]furan-5-yl) (intermediates 1-2):

[0088] At room temperature, intermediate A-1 (100 mg, 0.38 mmol, 1 equiv), starting material 1-1 (104 mg, 0.57 mmol, 1.5 equiv), EDCI (110 mg, 0.57 mmol, 1.5 equiv), and DMAP (2.5 mg, 0.02 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 100:1) to obtain a white crystalline solid 1-2 (53 mg, 32.7%).

[0089] LC-MS (ESI, m / z): 427.1 [M+H] + .

[0090] Synthesis of methyl 5-((2-(2,6-dioxopiridine-3-yl)-1-oxoisoindol-4-yl)amino)valerate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)5-((2-(2,6-dioxopiridine-3-yl)-1-oxoisoindol-4-yl)amino)valerate (Example 1):

[0091] At room temperature, intermediate 1-2 (53 mg, 0.12 mmol, 1.2 equiv), starting material 1-3 (27 mg, 0.10 mmol, 1 equiv), and anhydrous K2CO3 (21 mg, 0.15 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to give white solid 1 (12 mg, 20.0%).

[0092] HRMS:(ESI,m / z):606.2814[M+H] + .

[0093] Synthesis of Example 2

[0094] Synthetic route of Example 2:

[0095]

[0096] Synthesis of methyl 4-bromobutyrate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl) (intermediate 2-2)

[0097] At room temperature, intermediate A-1 (150 mg, 0.57 mmol, 1 equiv), starting material 2-1 (143 mg, 0.85 mmol, 1.5 equiv), EDCI (163 mg, 0.85 mmol, 1.5 equiv), and DMAP (3.5 mg, 0.03 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (3 mL) was added as a solvent. After stirring at room temperature for 6 h, the reaction was monitored by TLC to ensure complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, mixed with silica gel, and purified by column chromatography (DCM:CH3OH (100:1)) to obtain a white crystalline solid intermediate 2-2 (101 mg, 42.9%).

[0098] LC-MS:(ESI,m / z):413.1[M+H] + .

[0099] Synthesis of methyl ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)butyrate (compound 2):

[0100] At room temperature, intermediate 2-2 (80 mg, 0.19 mmol, 1.2 equiv), starting material 1-3 (42 mg, 0.16 mmol, 1 equiv), and anhydrous K2CO3 (36 mg, 0.26 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to give white solid 2 (22 mg, 18.0%).

[0101] HRMS:(ESI,m / z):592.2660[M+H] + .

[0102] Synthesis of Example 3

[0103] Synthetic route of Example 3:

[0104]

[0105] Synthesis of methyl 6-bromohexanoate (intermediate 3-2) of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)

[0106] At room temperature, intermediate A-1 (150 mg, 0.57 mmol, 1 equiv), starting material 3-1 (166 mg, 0.85 mmol, 1.5 equiv), EDCI (163 mg, 0.85 mmol, 1.5 equiv), and DMAP (3.5 mg, 0.03 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (3 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 100:1) to obtain a white crystalline solid 3-2 (120 mg, 47.7%).

[0107] LC-MS:(ESI,m / z):441.1[M+H] + .

[0108] Synthesis of methyl hexanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)6-((2-(2,6-dioxoperidin-3-yl)-1-oxoisoindol-4-yl)amino)hexanoate (Example 3):

[0109] At room temperature, intermediate 3-2 (80 mg, 0.18 mmol, 1.2 equiv), starting material 1-3 (40 mg, 0.15 mmol, 1 equiv), and anhydrous K2CO3 (32 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was then monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to give a white solid 3 (16 mg, 14.3%).

[0110] HRMS:(ESI,m / z):620.2966[M+H] + .

[0111] Synthesis of Example 4

[0112] Synthetic route of Example 4:

[0113]

[0114] Synthesis of methyl 7-bromoheptanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl) (intermediate 4-2)

[0115] At room temperature, intermediate A-1 (150 mg, 0.57 mmol, 1 equiv), starting material 4-1 (178 mg, 0.85 mmol, 1.5 equiv), EDCI (163 mg, 0.85 mmol, 1.5 equiv), and DMAP (3.5 mg, 0.03 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (3 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 100:1) to obtain a white crystalline solid intermediate 4-2 (115 mg, 44.3%).

[0116] LC-MS (ESI, m / z): 455.1 [M+H] + .

[0117] Synthesis of methyl heptanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)7-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)heptanoate (Example 4):

[0118] At room temperature, intermediate 4-2 (80 mg, 0.18 mmol, 1.2 equiv), starting material 1-3 (40 mg, 0.15 mmol, 1 equiv), and anhydrous K2CO3 (32 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to give a white solid 4 (18 mg, 15.8%).

[0119] HRMS:(ESI,m / z):634.3123[M+H] + .

[0120] Synthesis of Example 5

[0121] Synthetic route of Example 5:

[0122]

[0123] Synthesis of methyl 5-((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)5-((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)valerate (Example 5):

[0124] At room temperature, intermediate 1-2 (80 mg, 0.19 mmol, 1.2 equiv), starting material 5-1 (43 mg, 0.16 mmol, 1 equiv), and anhydrous K2CO3 (33 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to obtain a pale yellow solid powder 5 (26 mg, 26.3%).

[0125] HRMS:(ESI,m / z):620.2607[M+H] + .

[0126] Synthesis of Example 6

[0127] Synthetic route of Example 6:

[0128]

[0129] Synthesis of methyl ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)butyrate (Example 6):

[0130] At room temperature, intermediate 2-2 (80 mg, 0.19 mmol, 1.2 equiv), starting material 5-1 (43 mg, 0.16 mmol, 1 equiv), and anhydrous K2CO3 (33 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to obtain a pale yellow solid powder 6 (23 mg, 20.0%).

[0131] HRMS:(ESI,m / z):606.2458[M+H] + .

[0132] Synthesis of Example 7

[0133] Synthetic route of Example 7:

[0134]

[0135] Synthesis of methyl hexanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyeno[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)6-((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)hexanoate (Example 7):

[0136] At room temperature, intermediate 2-2 (80 mg, 0.19 mmol, 1.2 equiv), starting material 5-1 (43 mg, 0.16 mmol, 1 equiv), and anhydrous K2CO3 (33 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to obtain a pale yellow solid powder 7 (23 mg, 20.0%).

[0137] HRMS:(ESI,m / z):634.2770[M+H] + .

[0138] Synthesis of Example 8

[0139] Synthetic route of Example 8:

[0140]

[0141] Synthesis of methyl heptanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)7-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)heptanoate (Example 8):

[0142] At room temperature, intermediate 2-2 (60 mg, 0.19 mmol, 1.2 equiv), starting material 5-1 (43 mg, 0.16 mmol, 1 equiv), and anhydrous K2CO3 (33 mg, 0.23 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and then purified by reversed-phase preparative liquid chromatography (CH3CN:H2O) (5:95 to 95:5) to obtain a pale yellow solid powder 7 (23 mg, 20.0%).

[0143] HRMS:(ESI,m / z):648.2935[M+H] + .

[0144] Synthesis of Example 9

[0145] Synthetic route of Example 9:

[0146]

[0147] Synthesis of methyl 8-bromooctanoate ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)8-bromooctanoate (intermediate 9-2):

[0148] At room temperature, intermediate A-1 (150 mg, 0.57 mmol, 1 equiv), starting material 9-1 (190 mg, 0.85 mmol, 1.5 equiv), EDCI (163 mg, 0.85 mmol, 1.5 equiv), and DMAP (3.5 mg, 0.03 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (3 mL) was added as a solvent. After stirring at room temperature for 6 h, the reaction was monitored by TLC to ensure complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, mixed with silica gel, and purified by column chromatography (DCM:CH3OH = 100:1) to obtain a white crystalline solid 9-2 (118 mg, 44.2%).

[0149] LC-MS (ESI, m / z): 469.1 [M+H] + .

[0150] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)8-((2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindole-4-yl)amino)methyl octanoate (Example 9):

[0151] At room temperature, intermediate 9-2 (80 mg, 0.17 mmol, 1.2 equiv), starting material 1-3 (37 mg, 0.14 mmol, 1 equiv), and anhydrous K2CO3 (30 mg, 0.21 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 30:1) to obtain a white solid powder 9 (18 mg, 15.8%).

[0152] HRMS:(ESI,m / z):648.3276[M+H] + .

[0153] Synthesis of Example 10

[0154] Synthetic route of Example 10:

[0155]

[0156] Synthesis of methyl 4-(bromomethyl)benzoate (intermediate 10-2): ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)

[0157] At room temperature, intermediate A-1 (150 mg, 0.57 mmol, 1 equivalent), starting material 10-1 (183 mg, 0.85 mmol, 1.5 equivalent), EDCI (163 mg, 0.85 mmol, 1.5 equivalent), and DMAP (3.5 mg, 0.03 mmol, 0.05 equivalent) were added to a 50 mL reaction flask. DCM (3 mL) was added as a solvent. After stirring at room temperature for 6 h, the reaction was monitored by TLC to ensure complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, mixed with silica gel, and purified by silica gel column chromatography (DCM:CH3OH (100:1)) to obtain a white crystalline solid powder intermediate 10-2 (152 mg, 57.8%).

[0158] LC-MS (ESI, m / z): 461.1 [M+H] + .

[0159] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindole-4-yl)amino)methyl)benzoate (Example 10):

[0160] At room temperature, intermediate 10⁻² (80 mg, 0.17 mmol, 1.2 equiv), starting material 1⁻³ (38 mg, 0.14 mmol, 1 equiv), and anhydrous K₂CO₃ (30 mg, 0.22 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography using DCM:CH₃OH (30:1) as the mobile phase, yielding a white solid powder 10 (53 mg, 57.6%).

[0161] HRMS:(ESI,m / z):640.2647[M+H] + Synthesis of Example 11

[0162] Synthetic route of Example 11:

[0163]

[0164] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)amino)methyl octanoate (Example 11):

[0165] At room temperature, intermediate 9-2 (80 mg, 0.17 mmol, 1.2 equiv), starting material 5-1 (39 mg, 0.14 mmol, 1 equiv), and anhydrous K2CO3 (30 mg, 0.22 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 5 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 30:1) to obtain a white solid powder 11 (37 mg, 39.4%).

[0166] HRMS:(ESI,m / z):662.3038[M+H] + .

[0167] Synthesis of Example 12

[0168] Synthetic route of Example 12:

[0169]

[0170] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)4-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindole-4-yl)amino)methyl)benzoate (Example 12):

[0171] At room temperature, intermediate 10⁻² (60 mg, 0.13 mmol, 1.2 equiv), starting material 5⁻¹ (30 mg, 0.11 mmol, 1 equiv), and anhydrous K₂CO₃ (24 mg, 0.17 mmol, 1.5 equiv) were added to a 50 mL reaction flask. DMF (2 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC to indicate complete reaction. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH₃OH = 30:1) to obtain 12 (28 mg, 39.8%) of yellow solid powder.

[0172] HRMS:(ESI,m / z):654.2471[M+H] + .

[0173] Synthesis of Example 13

[0174] Synthetic route of Example 13:

[0175]

[0176] Synthesis of 3-(4-(6-hydroxyhex-1-en-1-yl)-1-oxoisoindol-2-yl)piperidine-2,6-dione (intermediate 13-3):

[0177] At room temperature, 13-1 (235 mg, 0.73 mmol, 1 equiv), 13-2 (230 mg, 2.34 mmol, 3.2 equiv), CuI (15 mg, 0.079 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (57 mg, 0.05 mmol, 0.68 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (5 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 1 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three more times and reacted at 80 °C for 32 h. TLC monitoring showed that the reactant 13-1 had completely reacted. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 13-3 (152 mg, 61.3%).

[0178] LC-MS (ESI, m / z): 341.1 [M+H] + .

[0179] Synthesis of 6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)hex-5-alkynic acid (intermediate 13-4):

[0180] (1) Preparation of Jones' reagent:

[0181] Under ice bath conditions, 240 μL of water was added to a small sample vial, followed by 64 mg of CrO3. After dissolution, the solution turned orange-red. Then, 80 μL of concentrated sulfuric acid was added dropwise to prepare 2M Jones' reagent.

[0182] (2) Synthesis of intermediate 13-4:

[0183] Under ice bath conditions, intermediate 13-3 (100 mg, 0.29 mmol) was added to a 100 mL reaction flask and dissolved in 30 mL of acetone. The solution changed from clear to turbid, and the color of the solution changed from yellow to red and finally to green. After stirring for 30 min under ice bath conditions, the reaction was monitored by TLC to be complete. The reaction was stopped, and after vacuum distillation, 2 mL of water was added. After sonication, the mixture was filtered and washed with water to obtain a white solid powder 13-4 (85 mg, 81.7%).

[0184] LC-MS (ESI, m / z): 355.1 [M+H] + .

[0185] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyeno[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)hex-5-acetylacetic acid methyl ester (Example 13):

[0186] At room temperature, intermediate 13-4 (80 mg, 0.23 mmol, 1 equiv), starting material A-1 (72 mg, 0.27 mmol, 1.2 equiv), EDCI (67 mg, 0.35 mmol, 1.5 equiv), and DMAP (1.5 mg, 0.012 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 13 (57 mg, 39.8%).

[0187] HRMS:(ESI,m / z):601.2556[M+H] + .

[0188] Synthesis of Example 14

[0189] Synthetic route of Example 14:

[0190]

[0191] Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(6-hydroxyhex-1-yn-1-yl)isoindole-1,3-dione (intermediate 14-2):

[0192] At room temperature, 14-1 (300 mg, 0.89 mmol, 1 equiv), 13-2 (175 mg, 1.78 mmol, 2 equiv), CuI (18 mg, 0.089 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (52 mg, 0.045 mmol, 0.05 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (10 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 2 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three more times and reacted at 80 °C for 32 h. TLC monitoring showed that the reactant 14-1 had completely reacted. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 14-2 (165 mg, 52.4%).

[0193] LC-MS (ESI, m / z): 355.1 [M+H] + .

[0194] Synthesis of 6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hex-5-alkynic acid (intermediate 14-3):

[0195] Under ice bath conditions, intermediate 14-2 (120 mg, 0.34 mmol) was added to a 100 mL reaction flask and dissolved in 30 mL of acetone. The solution changed from clear to turbid, and the color of the solution changed from yellow to red and finally to green. After stirring for 30 min under ice bath conditions, the reaction was monitored by TLC to be complete. The reaction was stopped, and after vacuum distillation, 2 mL of water was added. After sonication, the mixture was filtered and washed with water to obtain a white solid powder 14-3 (116 mg, 93.0%).

[0196] LC-MS (ESI, m / z): 369.1 [M+H] + .

[0197] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)6-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxaindol-4-yl)hex-5-acetylacetic acid methyl ester (Example 14):

[0198] At room temperature, intermediate 13-4 (100 mg, 0.28 mmol, 1 equiv), starting material A-1 (90 mg, 0.34 mmol, 1.2 equiv), EDCI (81 mg, 0.42 mmol, 1.5 equiv), and DMAP (2.0 mg, 0.014 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 14 (87 mg, 50.6%).

[0199] HRMS:(ESI,m / z):615.2327[M+H] + .

[0200] Synthesis of Example 15

[0201] Synthetic route of Example 15:

[0202]

[0203] Synthesis of 7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hepta-6-alkynic acid (intermediate 15-2):

[0204] At room temperature, 14-1 (300 mg, 0.89 mmol, 1 equiv), 15-1 (175 mg, 1.78 mmol, 2 equiv), CuI (17 mg, 0.089 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (52 mg, 0.045 mmol, 0.05 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (10 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 2 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three more times and reacted at 80 °C for 32 h. TLC monitoring showed that the reactant 14-1 had completely reacted. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 15-2 (225 mg, 66.2%).

[0205] LC-MS (ESI, m / z): 383.1 [M+H] + .

[0206] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)hept-6-acetylacetic acid methyl ester (Example 15):

[0207] At room temperature, intermediate 15-2 (81 mg, 0.22 mmol, 1 equiv), starting material A-1 (70 mg, 0.25 mmol, 1.2 equiv), EDCI (64 mg, 0.33 mmol, 1.5 equiv), and DMAP (2.0 mg, 0.010 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 14 (84 mg, 60.8%).

[0208] HRMS:(ESI,m / z):629.2495[M+H] + .

[0209] Synthesis of Example 16

[0210] Synthetic route of Example 16:

[0211]

[0212] Synthesis of 8-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oct-7-alkynic acid (intermediate 16-2):

[0213] At room temperature, 14-1 (300 mg, 0.89 mmol, 1 equiv), 16-1 (250 mg, 1.78 mmol, 2 equiv), CuI (17 mg, 0.089 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (52 mg, 0.045 mmol, 0.05 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (10 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 2 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three more times and reacted at 80 °C for 32 h. TLC monitoring showed that the reactant 14-1 had completely reacted. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 16-2 (194 mg, 55.0%).

[0214] LC-MS (ESI, m / z): 397.1 [M+H] + .

[0215] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)8-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oct-7-ynyl methyl ester (Example 16):

[0216] At room temperature, intermediate 16-2 (81 mg, 0.21 mmol, 1 equiv), starting material A-1 (65 mg, 0.25 mmol, 1.2 equiv), EDCI (61 mg, 0.33 mmol, 1.5 equiv), and DMAP (2.0 mg, 0.010 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 16 (101 mg, 74.8%).

[0217] HRMS:(ESI,m / z):643.2646[M+H] + .

[0218] Synthesis of Example 17

[0219] Synthetic route of Example 17:

[0220]

[0221] Synthesis of 7-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)hepta-6-alkynic acid (intermediate 17-1):

[0222] At room temperature, 13-1 (85 mg, 0.26 mmol, 1 equiv), 15-1 (67 mg, 0.53 mmol, 2 equiv), CuI (5 mg, 0.027 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (17 mg, 0.014 mmol, 0.05 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (5 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 1 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three times again, and the reaction was carried out at 80 °C for 32 h. The reaction mixture was monitored by TLC to indicate that the starting material 13-1 had reacted completely. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 17-1 (57 mg, 57.6%).

[0223] LC-MS (ESI, m / z): 369.1 [M+H] + .

[0224] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)7-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)hept-6-acetylginate methyl ester (Example 17):

[0225] At room temperature, intermediate 17-1 (50 mg, 0.14 mmol, 1 equiv), starting material A-1 (43 mg, 0.16 mmol, 1.2 equiv), EDCI (41 mg, 0.21 mmol, 1.5 equiv), and DMAP (2.0 mg, 0.010 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 17 (26 mg, 30.6%).

[0226] HRMS:(ESI,m / z):615.2664[M+H] + .

[0227] Synthesis of Example 18

[0228] Synthetic route of Example 18:

[0229]

[0230] Synthesis of 8-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)oct-7-alkynic acid (intermediate 18-1):

[0231] At room temperature, 13-1 (85 mg, 0.26 mmol, 1 equiv), 16-1 (74 mg, 0.53 mmol, 2 equiv), CuI (5 mg, 0.027 mmol, 0.1 equiv), and Pd(PPh3)2Cl2 (16 mg, 0.014 mmol, 0.05 equiv) were rapidly added to a 50 mL two-necked reaction flask. DMF (5 mL) was added as a solvent, and the mixture was purged with argon three times. After reacting at 80 °C for 5 min, 1 mL of triethylamine was added under argon atmosphere. The mixture was then purged with argon three times again, and the reaction was carried out at 80 °C for 32 h. The reaction mixture was monitored by TLC to indicate that the starting material 13-1 had reacted completely. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 20:1) to obtain a milky white solid powder 18-1 (71 mg, 71.7%).

[0232] LC-MS (ESI, m / z): 383.1 [M+H] + .

[0233] Synthesis of ((1aR,7aS,10bS,E)-1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b decahydroxyenono[2',3':9,10]cyclodeca[1,2-b]furan-5-yl)8-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)oct-7-acetylacetic acid methyl ester (Example 18):

[0234] At room temperature, intermediate 18-1 (60 mg, 0.16 mmol, 1 equiv), starting material A-1 (51 mg, 0.19 mmol, 1.2 equiv), EDCI (46 mg, 0.24 mmol, 1.5 equiv), and DMAP (2.0 mg, 0.010 mmol, 0.05 equiv) were added to a 50 mL reaction flask. DCM (5 mL) was added as a solvent, and the mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until complete. The reaction solution was extracted three times with water, and the organic phase was collected, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography (DCM:CH3OH = 50:1) to obtain a white solid powder 18 (37 mg, 37.0%).

[0235] HRMS:(ESI,m / z):629.2818[M+H] + .

[0236] Bioactivity testing examples

[0237] The control molecule used in this test example is KT-474, which was prepared according to the preparation method of compound I-417 in patent WO2020113233A1, and its structure is as follows:

[0238]

[0239] Test Example 1: Degradation of IRAK4 in THP-1 Cells by Compounds

[0240] (1) Experimental Objective

[0241] Flow cytometry was used to detect the degradation level of IRAK4 protein in THP-1 cells by the compound.

[0242] (2) Experimental Principle

[0243] The target protein is identified by using specific fluorescently labeled antibodies. When the fluorescently labeled target protein is irradiated by a laser of a specific wavelength, it will emit light of different wavelengths.

[0244] (3) Experimental steps

[0245] Step 1: Cell culture: The THP-1 cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% triple antibodies.

[0246] Step 2: Plating and Drug Administration: On Day 1, seed cells at a rate of 2 x 10⁵ cells / well (500 μL) in each well of a 24-well plate. Dissolve the compound in DMSO to a final concentration of 10 mM. For a partial final concentration, dilute the compound 100-fold with complete culture medium and add 55 μL / well to achieve a final concentration of 10 μM. The control group consisted of DMSO diluted with complete culture medium. After drug administration, incubate at 37°C in a 5% CO₂ incubator for 24 hours.

[0247] Step 3: Cell collection: Transfer the cell groups in the 24-well plate to centrifuge tubes in sequence, centrifuge (2000 rpm, 4℃, 5 min), aspirate the supernatant, add 500 μL PBS to wash the cells, centrifuge (2000 rpm, 4℃, 5 min), and aspirate the supernatant.

[0248] Step 4: Membrane disruption: Add 200 μL of 60% methanol per tube and incubate on ice for 30 min. The 60% methanol should be prepared in advance and stored at -20°C.

[0249] Step 5: Termination of membrane disruption: After incubation on ice for 30 min, add 800 μL of PBS per tube to terminate membrane disruption. Centrifuge (600 x g, 4 °C, 5 min), aspirate the supernatant, and resuspend.

[0250] Step 6: Stain the cells with fluorescently labeled anti-Human IRAK4 Antibody and incubate at 4°C for 30 min.

[0251] Step 7: Add 600 μL of PBS per tube to stop staining, centrifuge (600 x g, 4 °C, 5 min), and aspirate the supernatant. Add PBS and perform flow cytometry analysis.

[0252] (4) Data processing

[0253] Streaming data were analyzed using Flowjo software. Data display and inhibition rate calculation were performed using Prism 10 (GraphPad).

[0254] (5) Experimental Results

[0255] The degradation activity results of the compounds in the embodiments of this disclosure on IRAK4 are shown in Table 1.

[0256] The results show that some of the compounds in the embodiments of this disclosure have good degradation activity against IRAK4 in THP-1 cells.

[0257] Table 1

[0258]

[0259]

[0260] Test Example 2: Degradation of IRAK4 by the Compound in THP-1 and RAW264.7 Cells

[0261] (1) Experimental Objective

[0262] Western blot analysis was performed to assess the degradation level of the compound on IRAK4 protein in THP-1 cells (RAW264.7 cells).

[0263] (2) Experimental Principle

[0264] Cellular or tissue proteins are transferred from a gel to a solid support NC or PVDF membrane by electrophoresis. Then, a specific antigen (i.e., protein) is stained with a specific antibody. Finally, the expression of the protein in the analyzed cells or tissues is obtained by analyzing the location and depth of the staining.

[0265] (3) Experimental methods

[0266] Step 1: Cell culture: THP-1 cell line and RAW264.7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% triple antibodies.

[0267] Step 2: Plating and Drug Administration: On Day 1, 500 μL of cells were seeded into 24-well plates at a density of 2 x 10⁵ cells per well. The compound was dissolved in DMSO to a final concentration of 10 mM, with some compounds reaching a final concentration of 3 mM. This was then diluted 100-fold with complete culture medium and added to each well (55 μL / well) to achieve final concentrations of 10 μM and 3 μM in RAW264.7 cells, and 3 μM in THP-1 cells. The control group consisted of DMSO diluted with complete culture medium. After drug administration, the cells were incubated at 37°C in a 5% CO₂ incubator for 24 hours.

[0268] Step 3: Cell collection: Transfer cells to centrifuge tubes according to their corresponding groups, centrifuge (2500 rpm, 4℃, 5 min), discard the supernatant, add 500 μL PBS / tube to wash the cells, centrifuge (2500 rpm, 4℃, 5 min), discard the supernatant.

[0269] Step 4: Lyse cells: Add RIPA lysis buffer (RIPA + 10% protease inhibitor + 10% phosphatase inhibitor) and incubate on ice for 30 min to lyse cells.

[0270] Step 5: Protein extraction and sample preparation: After lysis for 30 min, centrifuge (12000 rpm, 4℃, 15 min) and collect a fixed amount of supernatant. Add 5× loading buffer, mix well, boil at 100℃ for 10 min to prepare the sample, and store at -20℃.

[0271] Step 6: Western blotting was used to detect IRAK4 protein levels.

[0272] (4) Data processing

[0273] Band data were analyzed using ImageJ software. Data display and relative IRAK4 expression level calculation were performed using Prism 10 (GraphPad).

[0274] (5) Experimental Results

[0275] The degradation activity results of the compounds in the embodiments of this disclosure on IRAK4 are shown in Table 2.

[0276] The results show that some of the compounds in the embodiments of this disclosure have good degradation activity against IRAK4 in THP-1 cells. The degradation effects of P10 and P12 are more significant.

[0277] Table 2

[0278]

[0279]

[0280] Test Example 3: Anti-inflammatory activity of the compound against LPS-induced IL-6 in RAW264.7 cells

[0281] (1) Experimental Objective

[0282] The anti-inflammatory activity of the compound against LPS-induced IL-6 in RAW264.7 cells was tested by ELISA.

[0283] (2) Experimental Principle

[0284] A certain concentration of antigen or antibody is fixed on the surface of a polystyrene microplate by physical adsorption. The sample to be tested is added, and the presence or amount of the antigen or antibody to be tested is indirectly reflected by the color intensity of the enzyme label.

[0285] (3) Experimental methods

[0286] Step 1: Cell culture: RAW264.7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% triple antibodies.

[0287] Step 2: Plating: Seed 100 μL of cells at a density of 2 x 10⁵ cells per well in a 96-well plate. After 24 hours, once the cells have adhered, replace the complete medium containing 10% FBS with medium containing 2% FBS to starve the cells. Four hours later, LPS was added for stimulation, followed by drug administration. The control group received no stimulation after starvation, the model group received LPS after starvation but no drug administration, and the drug administration group received LPS after starvation and drug administration. The final concentrations of the compounds were 3 μM, 1 μM, and 0.3 μM. Some compounds had final concentrations of 3 μM, 1 μM, 0.3 μM, and 0.1 μM.

[0288] Step 2: IL-6 primary antibody coating, overnight at 4°C.

[0289] Step 3: After washing three times with PBST, block with 1×Diluent for 1 hour.

[0290] Step 4: After blocking, wash three times with PBST, prepare the standard curve and load the sample. Dilute the sample 10-fold. Incubate at room temperature for 2 hours or at 4°C overnight.

[0291] Step 5: Wash three times with PBST and incubate with 1×Diluent secondary antibody for 1 hour.

[0292] Step 6: After washing three times with PBST, add Avidin-HRP for 30 minutes.

[0293] Step 7: After washing five times with PBST, add TMB and develop the color.

[0294] Step 8: Add stop solution, then use an ELISA reader to detect the enzyme.

[0295] Step 9: Add the 96-well plate sample with the supernatant removed to CCK-8 and incubate for 1 hour.

[0296] (4) Data processing

[0297] A microplate reader was used to analyze 96-well plates and obtain data. This included cell viability, IL-6 inhibition rate, and IC50 values ​​of some compounds. 50 The calculations were performed using Prism 10 (GraphPad).

[0298] (5) Experimental Results

[0299] The results of the anti-inflammatory activity of the compounds in the embodiments of this disclosure against IL-6 in LPS-induced RAW264.7 cells are shown in Table 3.

[0300] The results show that some of the compounds in the embodiments of this disclosure have good inhibitory activity against IL-6 in RAW264.7 cells.

[0301] Table 3

[0302]

[0303]

[0304] Test Example 4: Test of the kinase activity of the compound against IRAK4

[0305] (1) Experimental Objective

[0306] HTRF assay was used to assess the inhibitory effect of the compound on IRAK4 kinase.

[0307] (2) Experimental Principle

[0308] HTRF (homogeneous time-resolved fluorescence) is one of the most commonly used methods for detecting analytes in homogeneous systems. This technique combines fluorescence resonance energy transfer (FRET) and time-resolved fluorescence (TR). In TR-FRET experiments, when the donor and acceptor are very close, fluorescence resonance energy transfer occurs between them, generating a signal. Using dual-wavelength detection significantly reduces interference from buffer and culture medium, and the final signal is proportional to the amount of product formed.

[0309] (3) Experimental methods

[0310] Step 1: Compound preparation: Prepare the compound to 25 μM using KBA buffer. (KBA buffer preparation: sterile water + 1 mM DTT + 1 mM MgCl2). Prepare a concentration gradient by sequentially diluting P10 and P12 by 3-fold.

[0311] Step 2: Add 4 μL of the compound to a 384-well plate. Reserve blank and control groups.

[0312] Step 3: Add 2 μL of IRAK4 kinase solution prepared with KBA buffer.

[0313] Step 4: Add 2 μL of substrate solution prepared using KBA buffer.

[0314] Step 5: Add 2 μL of ATP solution prepared with KBA buffer. The blank solution contains no compound and no ATP, while the control solution contains no compound. The unadded portion is supplemented with KBA buffer. After addition, mix well and incubate at 37°C for 1 hour.

[0315] Step 6: After incubation, add 5 μL of antibody prepared with detection buffer and 5 μL of XL665 prepared with detection buffer. Incubate for 1 hour.

[0316] Step 7: After incubation, the data is detected by a microplate reader (Synergy H1, BioTek, USA) according to the HTRF method.

[0317] (4) Data processing

[0318] IRAK4 inhibition rate and IC50 values ​​for P10 and P12 50 The calculations were performed using Prism 10 (GraphPad).

[0319] (5) Experimental Results

[0320] The inhibition results of the compounds in the embodiments of this disclosure on IRAK4 kinase are shown in Table 4.

[0321] The results show that some of the compounds in the embodiments of this disclosure have good inhibitory activity against IRAK4 kinase.

[0322] Table 4

[0323]

[0324]

Claims

1. A bifunctional compound having the structure of formula (I), or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer or isotopic derivative thereof; in, LNK is selected independently from: R1 is independently selected from straight-chain C atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkyne group, NH-C 1-10 Alkyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl; R2 is independently selected from straight-chain carbon atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkyne group, NH-C 1-10 Alkyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl; R3 is independently selected from straight-chain carbon atoms substituted with unsubstituted or halogenated, -OH, -NH2, -CN, or -CF3. 1-10 Alkyl, C 2-10 Alkyne group, NH-C 1-10 Alkyl, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl; in: Ring A is independently selected from equations (IIa) and (IIb): Among them, X1, X2, and X3 are independently selected from C and N, respectively; For LNK connection points; R4 and R5 are independently selected from unsubstituted and halogenated, -OH, -NH2, -CN, or -CF3 substituted straight-chain or branched C atoms. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkyne, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is shown in formula (Ia). in: The ring C is selected from the following groups: substituted or unsubstituted 5-6 membered heterocyclic groups, substituted or unsubstituted 7-9 membered bridged heterocyclic groups, substituted or unsubstituted 6-10 membered fused heterocyclic groups, and substituted or unsubstituted 3-6 membered cycloalkyl groups; Y is selected from -(D1)n1; D1 is selected from saturated or unsaturated C. 1-3 The methylene chain, wherein any methylene unit is optionally surrounded by -O-, -C(O)-, or -C(R)-. a (R) b )-、-N(R c - Replaced; n1 is selected from 0 and 1; R a R b R c They are independently selected from H and halogens, respectively; R6 is independently selected from unsubstituted and halogenated, -OH, -NH2, -CN, or -CF3 substituted straight-chain or branched C atoms. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkyne, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is shown in formula (Ib). in: The ring E is selected from the following groups: substituted or unsubstituted 5-6 membered heterocyclic groups, substituted or unsubstituted 7-9 membered bridged heterocyclic groups, substituted or unsubstituted 6-10 membered fused heterocyclic groups, and substituted or unsubstituted 3-6 membered cycloalkyl groups; Z is selected from -(D2)n2; D2 is selected from saturated or unsaturated C. 1-3 The methylene chain, wherein any methylene unit is optionally surrounded by -O-, -C(O)-, or -C(R)-. a (R) b )-、-N(R c - Replaced; n2 is selected from 0 and 1; R a R b R c They are independently selected from H and halogens, respectively; R7 is independently selected from unsubstituted and halogenated, -OH, -NH2, -CN, or -CF3 substituted straight-chain or branched C atoms. 1-10 Alkyl, OC 1-10 Alkyl, NH-C 1-10 Alkyl, C 2-10 Alkyne, 5-8 membered cycloalkyl, 5-8 membered heterocyclic, phenyl or 5-8 membered heteroaryl.

4. The bifunctional compound of claim 1, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or isotopic derivative thereof, selected from the following compounds:

5. A pharmaceutical composition comprising the bifunctional compound of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or isotope derivative thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

6. Use of the bifunctional compound of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or isotope derivative thereof, or the pharmaceutical composition of claim 5, in the preparation of a medicament for the prevention and / or treatment of autoinflammatory / immune diseases.

7. The use as described in claim 6, wherein, The autoinflammatory / immune diseases mentioned are selected from rheumatoid arthritis, systemic lupus erythematosus, psoriasis, atopic dermatitis, hidradenitis suppurativa, alcoholic hepatitis, inflammatory bowel disease, multiple sclerosis, rash, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, alcoholic hepatitis, Hashimoto's thyroiditis, toxic diffuse goiter, type 1 diabetes, and Crohn's disease.

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