Drug delivery system for CRBN-mediated protein degraders

Abstract

Provided in the present invention is a drug delivery system for CRBN-mediated protein degraders. Specifically provided in the present invention is a conjugate as represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein each group is as defined in the text.

Description

A class of drug delivery systems for CRBN-mediated protein degraders Technical Field

[0001] This application belongs to the pharmaceutical field and specifically relates to a drug delivery system for a protein degrader mediated by CRBN. Background Technology

[0002] Protein degraders (including PROTACs and molecular colloids) are an emerging drug intervention approach in recent years. Due to their unique catalytic mechanisms, they possess significant advantages in efficacy. However, because protein degraders themselves lack tissue and cell targeting, they offer no advantage in terms of safety. Antibody-drug conjugate (ADC) delivery systems often improve drug safety, thereby broadening the therapeutic window. Currently, there is a lack of universally applicable, stably linked, and highly efficient protein degradation drug release systems.

[0003] In summary, there is an urgent need in this field for a new type of protein degradation drug release system. Summary of the Invention

[0004] The purpose of this invention is to provide a class of linkers that can be used in targeted drug delivery systems, as well as coupling agents or degrading agents containing the same, and their applications.

[0005] In a first aspect of the invention, a protein degrading agent conjugate or a pharmaceutically acceptable salt thereof is provided, wherein the conjugate is a conjugate of formula (I).

[0006] in,

[0007] The portion shown in (Ib) is derived from a protein degrader based on CRBN E3 ligase;

[0008] in,

[0009] M POI This is the target protein binding region;

[0010] For CRBN bonding portion;

[0011] M Linker For use with or without a connector to link the target protein binding portion and the CRBN binding portion;

[0012] X is CH or N;

[0013] L 1 This is the antibody linker portion;

[0014] L 2 This is a bridging portion used to connect antibody connectors;

[0015] Y is either NH or O;

[0016] R T Selected from the following groups: cathepsin substrate fraction, β-glucuronidase substrate fraction, β-galactosidase substrate fraction;

[0017] L 3 For H, COR N Or containing one or more PEG unit structures and / or polysarcosine unit structures; wherein, R N For H or C 1-6 alkyl

[0018] L 4 Groups that are absent or contain one or more PEG unit structures and / or polysarcosine unit structures;

[0019] a is a value between 1 and 8;

[0020] Bm is the binding moiety that can specifically bind to the target protein.

[0021] In another preferred embodiment, the protein degrading agent is used to degrade the target protein.

[0022] In another preferred embodiment, the target protein is a target protein to be degraded.

[0023] In another preferred embodiment, the target proteins include: GSPT1, IKZF1 / 3, VAV1, DCAF15, CK1α, BET family proteins, BTK, AR, ER, BRD9, STAT3, STAT6, EGFR, TRK, ALK, BRAF, KRAS, SOS1, pan-RAS, IRAK4, BCL2 family proteins, USP7, CDK family proteins, RIPK1, RIPK3, MLKL, EZH2, HDAC family proteins, SMARCA2, SMARCA4, MDM2, PARP1, NAMPT, CBP / P300, and Tau.

[0024] In another preferred embodiment, the target protein is a cell membrane receptor or a cell surface antigen.

[0025] In another preferred embodiment, Bm is an antibody or its antigen-binding portion.

[0026] In another preferred embodiment, the target proteins include 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9, CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, and CD31. CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, CD41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, condensation factor, cKit, closure protein 3, closure protein 18.2, CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1. Growth factors, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2) ETS fusion gene), ETBR, ETV6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24. HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including α4, α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podin, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, MLIAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, copper *Pseudomonas aeruginosa*, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1, or combinations thereof.

[0027] In another preferred embodiment, the target protein includes: HER2, TROP-2, B7H3, EGFR, CD3, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD52, CD79b, CD138, BCMA, PSMA, DLL3, CTLA-4, CA125, Nectin-4, TF, CEGF, folate receptor α, or combinations thereof.

[0028] In another preferred embodiment, Bm is an antibody or an antibody fragment or an antigen-binding fragment thereof as defined below.

[0029] In another preferred embodiment, the antibody is selected from the group consisting of: rituximab, trastuzumab, depatuxizumab, dicetuzumab, gemtuzumab, vobramitamab, pertuzumab, obbituzumab, olfamumab, olatozumab, ixartuzumab, denintuzumab, inelizumab, ifinatamab, moxetumomab, mirvetuximab, verbentuzumab, datopotamab, sacituzumab, rosopatamab, u3-1784, daratumumab, STI-6129, lintuzumab, naltuzumab, oxintuzumab, belantan, polotuzumab, iladatuzumab, indatuzumab, cetuximab, anti-CD38. a2 antibody, alemtuzumab, teimozumab, belantuzumab, atorxizumab, indatuximab, faucituzumab, enroximateb, teximumab, tosimomumab, bevacizumab, panitumumab, trimemumab, teximumab, caputsuzumab, ogovanimab, vepoxetuzumab, and vetozumab.

[0030] In another preferred embodiment, the antibody is selected from the group consisting of: trastuzumab, pertuzumab, dicetuzumab, datopotamab, sacituzumab, datopotamab, rosopatamab, vobramitamab, ifinatamab, depatuxizumab, cetuximab, panitumumab, and vepotuzumab.

[0031] In another preferred embodiment, the antibody is selected from the group consisting of trastuzumab, ifinatamab, cetuximab, sacituzumab, datopotamab, rosopatamab, and vepotuzumab.

[0032] In another preferred embodiment, L 1 -L 11 -M Bm -;in,

[0033] M Bm Selected from the following group: and This is the connection point with Bm;

[0034] L 11 It is a chain-like linker group.

[0035] In another preferred embodiment, M Bm for

[0036] In another preferred embodiment, L 11 The chain length is 5 to 30 chain atoms; more preferably, 10 to 25 chain atoms; even more preferably, 12 to 20 chain atoms; and most preferably, 13, 14, 15, 16, 17, 18 or 19 chain atoms.

[0037] In another preferred embodiment, L 11 -M 11b -(M 11a ) p -M 11c -

[0038] In another preferred embodiment, M 11a Selected from the following groups: -CH2-, -CH2CH2O-; preferably, M 1a It is -CH2CH2O-.

[0039] In another preferred embodiment, p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, p is 2, 3 or 4.

[0040] In another preferred embodiment, M 11b Selected from the group consisting of: none, -CO-, C1-2 alkylene, -C1-2 alkylene-CO-; preferably none.

[0041] In another preferred embodiment, M 11c Selected from the following group: none, C1-2 alkylene, -C≡C-, -C≡C-C1-2 alkylene-, -C1-2 alkylene-CONH-, -C1-2 alkylene-CONH-C1-2 alkylene-.

[0042] In another preferred embodiment, M 11c With M Bm connect.

[0043] In another preferred embodiment, L 2 -M 22 -M 21 -; where M 21 M is a divalent group formed by a click chemical reaction; 22 It is a C1-C4 alkylene group or -(CH2CH2O). p1 -, p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, p1 is 2, 3 or 4.

[0044] In another preferred embodiment, M 22 It is a C1-C4 alkylene group.

[0045] In another preferred embodiment, M 21 Does not exist or is

[0046] In another preferred embodiment, when Y is NH, R T It is the substrate portion of cathepsins; when Y is O, R T It is either the substrate portion of β-glucuronidase or the substrate portion of β-galactosidase.

[0047] In another preferred embodiment, when Y is NH, -R T -L 3 Selected from the following group:

[0048] in,

[0049] Z 1 and Z 2 Each can be independently identified as absent (non-existent) or an amino acid residue;

[0050] Z 3 and Z 4 Each is an amino acid residue;

[0051] R a and R b Each is independently H or C1-4 alkyl; or R a and R b Together with the carbon atoms attached to them, they form C4-6 cycloalkyl groups.

[0052] In another preferred embodiment, Z 1 For the absence of glycine residues; and / or, Z 2 The following amino acid residues are absent or selected: L-glutamine residue, D-glutamine residue, L-glutamate residue, D-glutamate residue, L-aspartic acid residue, D-aspartic acid residue, L-alanine residue, D-alanine residue, and glycine residue; and / or, Z 3 Selected from the group consisting of: L-valine residues, D-valine residues, L-alanine residues, D-alanine residues, L-phenylalanine residues, D-phenylalanine residues, and glycine residues; and / or, Z 4 Selected from the following group: L-alanine residue, D-alanine residue, L-citrulline residue, D-citrulline residue, L-asparagine residue, D-asparagine residue, L-lysine residue, D-lysine residue, L-phenylalanine residue, D-phenylalanine residue, and glycine residue.

[0053] In another preferred embodiment, when Y is NH, -R T -L 3 Selected from the table below

[0054] In another preferred embodiment, when Y is 0, -R T -L 3 Selected from the following group:

[0055] In another preferred embodiment, Y is NH, and R T It is the substrate portion of cathepsins.

[0056] In another preferred embodiment, the group containing one or more PEG unit structures and / or polysarcosine unit structures is -M. 33 -M 31 -(CH2CH2O) q -M 32 or -M 33 -M 31 -[COCH2N(Me)] q -M 32 ;in,

[0057] M 31 Selected from the following group: none, -CO-, -C1-4 alkylene-, -CO-C1-4 alkylene-;

[0058] M 32 Selected from the group consisting of: none, C1-2 alkyl, C1-2 alkylene-acid group, -CO-C1-2 alkyl; and

[0059] M 33 Selected from the following group: none, -NHCO-, -N(C1-4 alkyl)CO-, linking group formed by click chemistry, -C1-6 alkylene-NHCO-, -C1-6 alkylene-N(C1-4 alkyl)CO-, -C1-6 alkylene-linking group formed by click chemistry, -C1-6 heteroalkylene-NHCO-, -C1-6 heteroalkylene-N(C1-4 alkyl)CO-, -C1-6 heteroalkylene-linking group formed by click chemistry; and

[0060] q is an integer between 1 and 50.

[0061] In another preferred embodiment, q is an integer from 5 to 30; more preferably, q is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0062] In another preferred embodiment, L 3 In this context, the group containing one or more PEG unit structures and / or polysarcosine unit structures is -M. 31 -(CH2CH2O) q -M 32 or -M 31-[COCH2N(Me)] q -M 32 Preferably, M 31 It is either none or -CO-.

[0063] In another preferred embodiment, L 3 It is a group containing one or more PEG unit structures and / or polysarcosine unit structures.

[0064] In another preferred embodiment, L 3 -M 31 -(CH2CH2O) q -M 32 or -M 31 -[COCH2N(Me)] q -M 32 .

[0065] In another preferred embodiment, L 4 In this context, the group containing one or more PEG unit structures and / or polysarcosine unit structures is -M. 33 -(CH2CH2O) q -M 32 or -M 33 -[COCH2N(Me)] q -M 32 Preferably, M 33 Selected from the following group: -C1-6 alkylene-NHCO-, -C1-6 alkylene-N(C1-4 alkyl)CO-, -C1-6 alkylene-linking group formed by click chemistry, -C1-6 heteroalkylene-NHCO-, -C1-6 heteroalkylene-N(C1-4 alkyl)CO-, -C1-6 heteroalkylene-linking group formed by click chemistry.

[0066] In another preferred embodiment, L 4 It is none.

[0067] In another preferred embodiment, Selected from Table A1 below, Represents the X-linked junction:

[0068] Table A1

[0069] Wherein, ring B is an optionally substituted C3-20 cycloalkyl or an optionally substituted 4- to 20-membered heterocyclic alkyl; the optional substitution means that the group is unsubstituted or one or more H in the group is replaced by R. 1 The substituted R is selected independently from the group consisting of: C1-4 alkyl, hydroxyl, C1-4 haloalkyl, and cyano.

[0070] In another preferred embodiment, ring B is as shown in equation IB-1, IB-2, or IB-3:

[0071] in,

[0072] X 1 X 2 X 3 and X 4 Each independently as X a X a -X b or X a -X b -X c

[0073] X 5 and X 6 Each independently is C(R) 1 ) or N;

[0074] X 7 None (single key), X a or X a -X b ;and

[0075] X a X b and X c Each is independently selected from the following group: C(R) 1 )2、N(R 1 ), O and S;

[0076] R 1 Each can be H or R independently;

[0077] R is independently selected from the following group: C1-4 alkyl, hydroxyl, C1-4 haloalkyl, cyano.

[0078] In another preferred embodiment, the conjugate is capable of releasing a CRBN-based protein degrader, as shown in (Ib1), within cells or in the presence of cathepsins, β-glucuronidase, or β-galactosidase (β-gal).

[0079] In the formula, M POI M Linker X and As stated above.

[0080] In another preferred embodiment, the CRBN-based protein degrader is a CRBN-based protein degrader that can be released from the specific compounds shown in Table B (i.e., the specific compounds shown in Table B containing CRBN-based protein degraders). form The protein degrading agent that is subsequently formed and released.

[0081] In another preferred embodiment, L 1 L 2 Y, R T L 3 and L 4 Each is an independent group corresponding to the coupling compound described in Table A.

[0082] In another preferred embodiment, the coupling agent is selected from Table A below;

[0083] Table A

[0084] In another preferred embodiment, the portion shown in (Ib) (and M in formula (Ib)) POI M Linker , and X), L 1 L 2 Y, R T L 3 and L 4 Each of these is an independent group corresponding to the compound shown in Table B.

[0085] In another preferred embodiment, the conjugate is a conjugate formed by the compound shown in Table B of the fourth aspect and the antibody.

[0086] In a second aspect of the invention, a pharmaceutical composition is provided comprising the conjugate as described in the first aspect and optionally a pharmaceutically acceptable carrier.

[0087] In a third aspect of the invention, a conjugate as described in the first aspect or a pharmaceutically acceptable salt thereof is provided for the preparation of a treatment or prevention of a disease associated with or caused by excessive expression of an intracellular target protein.

[0088] In another preferred embodiment, the disease is a tumor.

[0089] In a fourth aspect of the invention, a compound or a salt thereof is provided, wherein the compound is as shown in II.

[0090] in,

[0091] L i 1 For antibody linkers to be coupled with antibodies;

[0092] Furthermore, as shown in (Ib), L 2 L 3 L 4 R T X, Y, M POI M Linker,and As defined in claim 1;

[0093] In another preferred embodiment, L i 1 -L 11 -M i Bm ;in,

[0094] M i Bm Selected from the following group:

[0095] L 11 It is a chain-like linking group;

[0096] In another preferred embodiment, L 11 As defined above.

[0097] In another preferred embodiment, M i Bm for

[0098] In another preferred embodiment, the compound is selected from Table B below;

[0099] Table B

[0100] In another preferred embodiment, the compound is used to prepare the coupling compound as described in the first aspect.

[0101] In a fifth aspect of the invention, an intermediate is provided, said intermediate as shown in III.

[0102] in,

[0103] L ii 2 The portion containing reactive groups;

[0104] Furthermore, as shown in (Ib), L 3 L 4 R T X, Y, MPOI M Linker ,and As defined in the first aspect;

[0105] In another preferred embodiment, the reactive group is a group capable of undergoing a click chemistry reaction.

[0106] In another preferred embodiment, the reactive group is Or azide group.

[0107] In another preferred embodiment, the intermediate is used to prepare a compound as shown in Formula II.

[0108] In another preferred embodiment, the intermediate is used to prepare the conjugate as described in the first aspect.

[0109] In a sixth aspect of the invention, a method for degrading a target protein is provided, comprising the steps of: contacting a conjugate as described in the first aspect with a target, thereby degrading the target protein in the target.

[0110] In another preferred embodiment, the object highly expresses one or more target proteins.

[0111] In another preferred embodiment, the conjugate is capable of selectively targeting a target and degrading the target protein therein.

[0112] In another preferred embodiment, the object is a cell; more preferably, a tumor cell.

[0113] In another preferred embodiment, the method is non-therapeutic in vitro.

[0114] 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 or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0115] none Detailed Implementation

[0116] Through long-term and in-depth research, the inventors unexpectedly discovered a specific structure (such as -YR) T -L 3 A class of conjugates possesses high DAR values ​​and can efficiently release protein degrading agents in specific environments. Based on this, the inventors completed this invention.

[0117] the term

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0119] In this document, unless otherwise defined, groups written from left to right are also intended to include those written from right to left, i.e., -CH2-O- includes both -CH2-O- and -O-CH2-.

[0120] As used herein, unless otherwise defined, the term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (i.e., C64 ... 1-6 (Indicates 1-6 carbons). Preferably, the alkyl group specifically has 1-4 carbons, i.e., C1-C2. 1-4 Alkyl groups. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc.

[0121] As used herein, unless otherwise stated, the term "heteroalkyl" itself or as part of another substituent refers to a group formed by the substitution of one or more carbon atoms in an alkyl group with a specified number of carbon atoms as defined above, by one to three heteroatoms selected from O, N, and S. The total number of carbon atoms and heteroatoms in a heteroalkyl group is expressed as a subset (e.g., 3-4-membered heteroalkyl) in which nitrogen and sulfur atoms may optionally be oxidized, and nitrogen heteroatoms may optionally be quaternized. Heteroatoms O, N, and S may be located at any internal position of the heteroalkyl group. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. At most two heteroatoms can be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0122] As used herein, the term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkane as defined above. Alkylenes typically have 1-4 carbon atoms (i.e., C1-4 alkylenes) or 1-2 carbon atoms (C1-2 alkylenes). Examples of alkylenes include, but are not limited to, methylene (-CH2-) and ethylene (e.g., -CH2CH2-).

[0123] As used herein, the term “heteroalkylene” itself or as part of another substituent refers to a saturated or unsaturated or polyunsaturated divalent group derived from a heteroalkylene group, such as -CH2-CH2-S-CH2CH2- and -CH2-O-CH2-CH2-CH2-.

[0124] As used herein, the term "cycloalkyl" or "cycloalkane" refers to a hydrocarbon ring having a specified number of ring atoms (e.g., C3-20 cycloalkyl (cycloalkane), C4-6 cycloalkyl (cycloalkane)) and being fully saturated or having no more than one double bond between the ring apexes (preferably a fully saturated hydrocarbon ring). The term also includes bicyclic and polycyclic hydrocarbon rings, such as bridged rings, fused rings, spirocyclic rings, etc. Non-limiting examples of cycloalkyl or cycloalkane include cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc. Similarly, the term "heterocyclic alkyl" or "heterocyclic alkane" refers to a cycloalkyl group having a specified number of ring atoms (e.g., 4-20 membered heterocyclic alkyl (heterocyclic alkane)) and containing one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Heterocyclic alkyl groups can be monocyclic, bicyclic, or polycyclic systems (e.g., bridged rings, fused rings, spirocyclic rings, etc.). Non-limiting examples of heterocyclic alkyl groups or heterocyclic alkanes include pyrrolidine, imidazoline, pyrazolidine, butyrolactam, valproic acid, imidazolidinone, hydantoin, dioxolane, benzodiimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrrolidine, thiaran, pyranone, tetrahydrofuran, tetrahydrothiophene, quinine ring, etc. Heterocyclic alkyl groups may be attached to the rest of the molecule via a cyclic carbon or heteroatom.

[0125] Unless otherwise stated, the term "aryl" or "aromatic ring" refers to a polyunsaturated (usually aromatic) hydrocarbon group or hydrocarbon ring, which may be a monocyclic or fused together or covalently linked polycyclic (up to three rings). The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule by a carbon atom or a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups or heteroaromatic rings include pyridinyl, pyrazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, terpineyl, phthalazinyl, benzotriazinyl, purine, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzoisoxazolyl, and isobenzuranyl. zofuryl), isoindolyl, indoleyl, benzotriazinyl, thienopyridyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothiaphenyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazoleyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrroleyl, thiazolyl, furanyl, thiophenyl, etc.

[0126] In some embodiments, the terms above (such as "alkyl", "aryl", or "heteroaryl") will include substituted and unsubstituted forms of the specified group. Preferred substituents for each type of group are provided below. For brevity, the terms aryl and heteroaryl will refer to the substituted or unsubstituted forms as provided below, while the term "alkyl" and the associated aliphatic group refer to the unsubstituted form unless specified otherwise.

[0127] The substituents of alkyl groups (including those commonly referred to as alkylene, alkenyl, ynyl, and cycloalkyl) can be a variety of groups selected from the group consisting of: -halogen, -OR', -NR'R”, -SR', -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -CONR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O)NR”R”', -NR” C(O)₂R', -NH-C(NH₂)=NH, -NR'C(NH₂)=NH, -NH-C(NH₂)=NR', -S(O)R', -S(O)₂R', -S(O)₂NR'R”, -NR'S(O)₂R”, -CN, and -NO₂, in quantities ranging from zero to (2M'+1), where M' is the total number of carbon atoms in this group. R', R”, and R”’ each independently represent hydrogen, and unsubstituted C 1-8 Alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl, C1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4 Alkyl groups. When R' and R” are attached to the same nitrogen atom, they can combine with the nitrogen atom to form 3-, 4-, 5-, 6-, or 7-membered rings. For example, -NR'R” refers to a group comprising 1-pyrrolidinyl and 4-morpholinyl. The term “acyl”, used alone or as part of another group, refers to a group in which the substituents on the carbon closest to the connection point of the group are substituted with =O (e.g., -C(O)CH3, -C(O)CH2CH2OR', etc.).

[0128] Similarly, the substituents of aryl and heteroaryl groups are diverse and are usually selected from: -halogen, -OR', -OC(O)R', -NR'R”, -SR', -R', -CN, -NO2, -CO2R', -CONR'R”, -C(O)R', -OC(O)NR'R”, -NR”C(O)R', -NR”C(O)2R', -NR'-C(O)NR”R”', -NH- C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R”, -NR'S(O)2R”, -N3, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in numbers ranging from zero to the total number of open valences on the aromatic ring system; wherein R', R” and R”' are independently selected from hydrogen, C 1-8 Alkyl, C 3-6 cycloalkyl, C 2-8 alkenyl, C 2-8 Alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C 1-4 Alkyl and unsubstituted aryloxy-C 1-4 Alkyl groups. Other suitable substituents include each of the above aryl substituents attached to the ring atom via an alkylene chain of 1-4 carbon atoms.

[0129] As used herein, the term "amino acid residue" refers to the group formed by removing an H atom from the N-terminal -NH2 group and the -OH atom from the C-terminal -COOH group of an amino acid. Generally, the segment of an amino acid (residue) including both the N-terminus and the C-terminus is called the main chain, while the portion that determines the specific type of amino acid is called the side chain. Unless otherwise defined, in this document, amino acids include both native and non-native amino acids, including D-type and / or L-type amino acids. Examples of amino acids include, but are not limited to, Ala (A), Arg I, Asn (N), Asp (D), Cys (C), Gln (Q), GI (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V). Preferably, in this document, the amino acid is selected from the group consisting of: L-glycine (L-Gly), L-alanine (L-Ala), β-alanine (β-Ala), L-glutamic acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L-Lys), L-valine (L-Val), L-serine (L-Ser), and L-threonine (L-Thr); furthermore, when the amino acid has two or more amino groups and / or two or more carboxyl groups, the term also includes groups formed by removing one H from -NH2 and -OH from -COOH on different carbon atoms, such as the divalent group -C(O)-(CH2)2-C(COOH)-NH- formed by removing one H from -NH2 and non-α-COOH of glutamic acid respectively.

[0130] In this document, unless otherwise defined, bonds indicated by dashed lines or marked with wavy lines represent positions that connect to other parts of the molecule.

[0131] For the compounds presented herein, a bond from a substituent (typically an R group) to the center of the ring will be understood as a bond that provides a connection at any available vertex of the aromatic ring.

[0132] In this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0133] In this invention, the term "effective amount" or "therapeutic effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health condition, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is useless to pre-specify an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0134] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0135] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but 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.

[0136] Unless otherwise specified, the term “amino acid” as used in this article is intended to include any common amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine, tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, and arginine.

[0137] The conjugates of the present invention, pharmaceutical compositions containing them, and their applications

[0138] This invention provides a conjugate or a pharmaceutically acceptable salt thereof, and conducts systematic characterization and biological evaluation of it, demonstrating that the conjugate of this invention can target and deliver protein degraders of multiple targets to the target site and release the active protein degraders, indicating that it has good efficacy, safety and versatility.

[0139] In some aspects, this document provides conjugates or pharmaceutically acceptable salts thereof for use with the drug delivery system of the present invention, said conjugates being shown in formula (I).

[0140] The variables are defined as in the first aspect.

[0141] In the field of protein degrading agents, CRBN is one of the most widely used E3 ligases. The conjugate of this invention can release an active protein degrading agent in a specific environment. The initiating group (i.e., R) in the conjugate of this invention... T It can be recognized and cleaved by cathepsins, β-glucuronidase or β-galactosidase (β-gal), ultimately triggering autolysis to release active protein degraders such as Ib1 (e.g., PROTAC protein degradation targeting consortium, molecular glue).

[0142] As used in this article, "protein degraders" include protein degradation targeting chimeras (PROTACs) and molecular glues. PROTAC-based heterobifunctional molecules have a group or ligand binding to a target protein (POI) at one end and an E3 ligase (such as CRBN) ligand at the other end, linked by a suitable linker. PROTACs degrade target proteins through the ubiquitin-proteasome system (UPS). The general process is as follows: the PROTAC molecule binds to the target protein (POI) and the E3 ligase, forming a ternary complex that ubiquitinates the target protein. The ubiquitinated protein is then recognized and degraded by the intracellular proteasome 26S. Molecular glues have a similar catalytic degradation mechanism, but their structure is often more streamlined than that of PROTACs.

[0143] Considering that the conjugates of the present invention can release the complete CRBN-mediated protein degrader upon reaching the target environment (such as intracellular or an environment containing cathepsins, β-glucuronidase, or β-galactosidase (β-gal)), the drug delivery system of the present invention can be used with various existing, developing, or pending CRBN-mediated protein degraders, provided that the E3 ubiquitinase ligand moiety of the CRBN-mediated protein degrader contains the following groups:

[0144] As used herein, “cell membrane receptors” are a class of proteins embedded in the cell membrane that play a crucial role in transmitting information between the cell and its external environment. Some of them are overexpressed in cells associated with specific tissues or diseases and are often used for targeted drug therapy. For example, antibody-drug conjugates (ADCs) specifically recognize and bind to specific antigens (usually cell membrane receptors) on the surface of cancer cells through their antibody portions. This specific binding ensures that the ADC primarily acts on target cells, rather than normal cells, thereby reducing non-specific toxicity. For the purposes of this application, preferred cell membrane receptors are those that are overexpressed on the cell membrane of cells where the target protein to be degraded is overexpressed (especially receptors that are overexpressed relative to normal cells), such as: 5T4, ACE, ADRB3, AKAP-4, ALK, androgen receptor, AOC3, APP, axonin 1, AXL, ASGR1, ASGR2, B7H3, B7-H4, BCL2, BCMA, bcr-abl, BORIS, BST2, C242, C4.4a, CA 125, CA6, CA9, CAIX, CCL11, CCR5, CD123, CD133, CD138, CD142, CD15, CD15-3, CD171, CD179a, CD18, CD19, CD19-9 , CD2, CD20, CD22, CD23, CD24, CD25, CD27L, CD28, CD3, CD30, CD31, CD300LF, CD33, CD352, CD37, CD38, CD4, CD40, C D41, CD44, CD44v6, CD5, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD71, CD72, CD74, CD79a, CD79b, CD80, CD90, CD97, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM5, CFTR, condensation factor, cKit, closure protein 3, closure protein 18.2. CLDN6, CLEC12A, CLL-1, cll3, c-MET, Crypto 1 growth factor, CS1, CTLA-4, CXCR2, CXORF61, cyclin B1, CYP1B1, cadherin-3, cadherin-6, DLL3, E7, EDNRB, EFNA4, EGFR, EGFRvIII, ELF2M, EMR2, ENPP3, EPCAM, EphA2, liver glycoside A4, liver glycoside B2, EPHB4, ERBB2 (Her2 / neu), ErbB3, ERG (TMPRSS2ETS fusion gene), ETBR, ET V6-AML, FAP, FCAR, FCRL5, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, folate receptor α, folate receptor β, FOLR1, Fos-associated antigen 1, fucose GM1, GCC, GD2, GD3, GloboH, GM3, GPC1, GPC2, GPC3, gplOO, GPNMB, GPR20, GPRC5D, GUCY2C, HAVCR1, HER2, HER3, HGF, HMI.24, HMWMAA, HPV E6, hTERT, human telomerase reverse transcriptase, ICAM, ICOS-L, IFN-α, IFN-γ, IGF-I receptor, IGLL1, IL-2 receptor, IL-4 receptor, IL-13Ra2, IL-11Ra, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, integrins (including α4 and α). v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), integrin αV, intestinal carboxyl esterase, KIT, LAGE-1a, LAIR1, LAMP-1, LCK, podin, LewisY, LFA-1 (CD11a), L-selectin (CD62L), LILRA2, LIV-1, LMP2, LRRC15, LY6E, LY6K, LY75, MAD-CT-1, MAD-CT-2, MAGE A1, MelanA / MART1, mesothelin, MLIAP, MSLN, mucin, MUC1, MUC16, mut hsp70-2, MYCN, myostatin, NA17, NaPi2b, NCA-90, NCAM, connexin-4, NGF, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NY-BR-1, NY-ESO-1, o-acetyl-GD2, OR51E2, OY-TES1, p53, p53 mutant, PANX3, PAP, PAX3, PAX5, p-CAD, PCTA-1 / galactagoguerin 8, PD-L1, PD-L2, PDGFR, PDGFR-β, phosphatidylserine, PIK3CA, PLAC1, polysialic acid, prostate enzymes, prostate cancer cells, prostaglandins, copper *Pseudomonas aeruginosa*, rabies virus, survivin and telomerase, PRSS21, PSCA, PSMA, PTK7, RAGE-1, RANKL, Ras mutant, respiratory syncytial virus, rhesus monkey factor, RhoC, RON, ROR1, ROR2, RU1, RU2, sarcoma translocation breakpoint, SART3, SLAMF7, SLC44A4, sLe, SLITRK6, spermin 17, sphingosine 1-phosphate, SSEA-4, SSX2, STEAP1, TAG72, TARP, TCRβ, TEM1 / CD248, TEM7R, tendinin C, TF, TGF-1, TGF-β2, TNF-α, TGS5, Tie 2. TIM-1, TnAg, TRAC, TRAIL-R1, TRAIL-R2, TROP-2, TRP-2, TRPV1, TSHR, tumor antigen CTAA16.88, tyrosinase, UPK2, VEGF, VEGFR1, VEGFR2, vimentin, WT1, XAGE1, or combinations thereof.

[0145] In some respects, this article also provides a pharmaceutical composition comprising the said conjugate or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0146] Pharmaceutical Compositions and Administration

[0147] Because the conjugates provided by this invention possess excellent ability to target and deliver protein degrading agents to target sites (such as inside diseased cells like cancer cells) and release the active protein degrading agents, conjugates using the drug delivery system of this invention can be used to treat or prevent various diseases related to or caused by protein overexpression, such as tumors or cancer. In some embodiments, the cancer includes breast cancer, lung cancer, gastric cancer, hepatocellular carcinoma, lymphoma, leukemia, pancreatic cancer, head and neck cancer, squamous cell carcinoma, urethral cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, plasmacytoma, myeloma, or sarcoma.

[0148] The aforementioned conjugates may be administered to subjects (e.g., humans) in a therapeutically effective amount via a suitable route. The therapeutically effective amount of the conjugates described in this invention may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the antibody-drug conjugate, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Given that the conjugates of this invention employ a special drug delivery system, the conjugates of this invention can be administered at lower doses and / or frequencies compared to inhibitors or PROTACs targeting the corresponding target, for example, at a dose of about 0.01 mg to 50 mg / kg animal body weight (preferably 0.1 mg to 20 mg / kg animal body weight).

[0149] The conjugates or pharmaceutical compositions of the present invention can be administered by conventional methods, depending on the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, such as parenteral administration, oral administration, via inhalation spray, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, or via implantation. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, it can be administered via injectable reservoir routes, such as using injectable or biodegradable materials and methods with 1-, 3-, or 6-month reservoirs.

[0150] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered via infusion, thereby delivering a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile formulations of a suitable soluble salt form of the antibody, may dissolve and administer pharmaceutical excipients such as water-based injections, 0.9% saline, or 5% glucose solutions.

[0151] When using the conjugates of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump. Other methods include various transport and carrier systems using conjugates and biodegradable polymers.

[0152] The pharmaceutical compositions of the present invention contain a safe and effective amount of the conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated as solutions, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0153] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0154] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents or adjuvants.

[0155] The main advantages of this invention include

[0156] 1. The antibody-drug conjugate of this application has the characteristics of high DAR (drug / antibody ratio) and low aggregation, and has the characteristics of an ideal antibody-drug conjugate.

[0157] 2. The conjugates of the drug delivery system of the present invention can efficiently release protein degrading agents in the target environment. In particular, further attaching hydrophilic groups (such as PEG chains) to the initiating group can further improve the release efficiency of the protein degrading agent.

[0158] 3. The target environment includes, but is not limited to, environments containing cathepsins, β-glucuronidase, or β-galactosidase, and has good compatibility.

[0159] 4. The drug delivery system of this application can be further linked or coupled with functional groups such as ligands of cell membrane receptors and modifying groups without affecting the release capability of the active protein degrader.

[0160] 5. The drug delivery system (or conjugate) and its preparation method of this application are compatible with a variety of different types of CRBN-related protein degraders, exhibiting strong versatility. Specifically, as can be seen from Examples 1-3, the preparation method of the conjugate of this application has good versatility and is adaptable to degraders targeting different sites. See Example 4, for example, the drug delivery system of this invention can efficiently release a variety of different types of CRBN-related protein degraders.

[0161] 6. Compared with traditional small molecules, the antibody-drug conjugate of this application exhibits significant receptor expression correlation, suggesting that it has good safety.

[0162] 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.

[0163] Example 1:

[0164] Compound I-2: Compound I-1 (1.0 equivalent), 1-imidazolium sulfonyl azide hydrochloride (1.05 equivalent), potassium carbonate (1.3 equivalent), and copper sulfate (0.1 equivalent) were stirred overnight in methanol. The mixture was filtered and evaporated to dryness. The residue was purified by column chromatography to obtain the title compound. 1H NMR (600MHz, CDCl3) δ9.15(s,1H),7.43(d,J=8.1Hz,2H),7.38(d,J=7.6Hz,1H),7.20(d,J=8.1Hz,2H),4.80-4.73(m,1H),4.64-4.56( m,2H),3.76(d,J=4.9Hz,1H),2.39-2.31(m,1H),1.50(d,J=7.0Hz,3H),1.07(d,J=6.9Hz,3H),0.96(d,J=6.7Hz,3H).LC-MS(ESI):m / z 320.1[M+H] + .

[0165] Compound I-4a: Compound I-2 (1.0 equivalent), bis(p-nitrobenzene) carbonate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 3 hours. Then, I-3a (1.8 equivalent), HOBt (1.8 equivalent), and DIEA (2.5 equivalent) were added sequentially to the reaction solution, and the mixture was stirred overnight at room temperature. After the reaction was monitored by LC-MS to be complete, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ10.13 (s, 1H), 8.51 (d, J = 6.9Hz, 1H), 7.70-7.63 (m, 1H ),7.63-7.53(m,2H),7.30(d,J=8.2Hz,2H),4.97(s,2H),4.52-4.40(m,1H),3 .78(dd,J=5.8,2.5Hz,2H),3.50(d,J=8.1Hz,1H),3.13-3.08(m,1H),2.11-2. 00(m,1H),1.34(d,J=7.1Hz,3H),0.94(dd,J=9.8,6.7Hz,6H).LC-MS(ESI):m / z 423.2[M+Na] + .

[0166] Compound I-4b: The title compound can be obtained by replacing I-3a with I-3b using the above method. 1H NMR (400MHz, DMSO-d6) δ10.12 (s, 1H), 8.51 (d, J = 7.0Hz, 1H), 7.61-7.54 (m, 2H), 7.33-7.26(m,2H),7.24(t,J=5.8Hz,1H),4.95(s,2H),4.52-4.40(m,1H),4.14( d,J=2.4Hz,2H),3.59-3.47(m,5H),3.45-3.37(m,3H),3.18-3.10(m,2H),2.12- 2.02(m,1H),1.34(d,J=7.0Hz,3H),0.94(dd,J=9.9,6.6Hz,6H).LC-MS(ESI):m / z 511.2[M+Na] + .

[0167] Compound I-6a: Compound I-4a (1.0 equivalent), TMSCl (6.0 equivalent), and paraformaldehyde (10.0 equivalent) were stirred overnight in dichloromethane. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure. The crude product I-5a was dissolved in DMF and used directly in the next step. CC-885 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. The DMF solution of the above compound I-5a (1.5 equivalent) was added dropwise under ice bath, and then reacted at room temperature for 30 minutes. After the reaction was complete as monitored by LC-MS, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the intermediate. This compound was dissolved in THF, water (20 equivalent) and trimethylphosphine (1.5 equivalent) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the title compound. LC-MS(ESI): m / z 827.3 [M+H] + .

[0168] Compound I-6b: The title compound was obtained by replacing I-4a with I-4b using the method described above. LC-MS (ESI): m / z 915.4 [M+H] + .

[0169] The general method for compound I-9A-C: Compound I-6a (1.0 equivalent), carboxylic acids I-7a / b / c (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 30 minutes. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure. Compound I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were added in a DMSO / tert-butanol / water mixed solvent (10 / 5 / 2, v / v) and reacted for 2 hours. The mixture was then separated by reverse-phase reaction to obtain the corresponding compounds.

[0170] Compound I-9A: The title compound can be obtained from carboxylic acid I-7a using the general method described above. 1 H NMR (600MHz, DMSO-d6) δ9.91 (s, 1H), 8.74 (s, 1H), 8.20 (d, J = 7.0Hz, 1H), 7.99 (t, J = 5 .7Hz,1H),7.93-7.70(m,3H),7.67(d,J=2.1Hz,1H),7.57(d,J=8.2Hz,2H),7.52(s,1 H),7.45(d,J=8.0Hz,1H),7.39-7.23(m,2H),7.20-7.10(m,2H),6.99(s,2H),6.80(t ,J=6.0Hz,1H),5.41(d,J=13.2Hz,1H),5.29(d,J=13.1Hz,1H),5.17-5.07(m,1H),5. 03(s,2H),4.56-4.34(m,8H),4.30-4.20(m,1H),4.17(dd,J=8.5,6.8Hz,1H),3.85-3 .72(m,2H),3.59(t,J=7.3Hz,2H),3.42-3.28(m,10H),3.16-3.09(m,2H),3.01-2.82 (m,1H),2.76-2.65(m,1H),2.32(t,J=7.3Hz,2H),2.29-2.17(m,4H),2.03-1.92(m,2 H),1.88(s,3H),1.31(d,J=7.1Hz,3H),0.86(dd,J=21.3,6.7Hz,6H).LC-MS(ESI):m / z 1238.4[M+H] + .

[0171] Compound I-9B: The title compound can be obtained from carboxylic acid I-7b using the general method described above. 1H NMR(600MHz,DMSO-d6)δ9.93(s,1H),8.74(s,1H),8.18(d,J=7.0Hz,1H),7.99(t,J=5 .7Hz,1H),7.91-7.64(m,4H),7.57(d,J=8.3Hz,2H),7.52(s,1H),7.45(d,J=7.8Hz,1H ),7.38-7.22(m,2H),7.20-7.11(m,2H),6.99(s,2H),6.79(t,J=6.0Hz,1H),5.41(d, J=13.2Hz,1H),5.29(d,J=12.9Hz,1H),5.16-5.07(m,1H),5.03(s,2H),4.56-4.46(m, 2H),4.44-4.35(m,6H),4.29-4.18(m,2H),3.85-3.74(m,2H),3.64-3.54(m,12H),3. 51-3.37(m,36H),3.36-3.31(m,2H),3.24(s,3H),3.16-3.09(m,2H),3.00-2.84(m,1H ),2.77-2.65(m,1H),2.49-2.36(m,2H),2.32(t,J=7.3Hz,2H),2.28-2.19(m,4H),2. 04-1.92(m,2H),1.30(d,J=7.1Hz,3H),0.85(dd,J=23.3,6.8Hz,6H).LC-MS(ESI):m / z 884.4[(M+2H) / 2] + .

[0172] Compound I-9C: The title compound was obtained from carboxylic acid I-7c using the general method described above. LC-MS (ESI): m / z 975.4 [(M+2H) / 2] + .

[0173] Compound I-11: Compound I-6a (1.0 equivalent), compound I-10 (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was monitored by LC-MS until complete, the mixture was separated by reverse-phase reaction to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ9.93 (s, 1H), 8.70 (s, 1H), 8.17 (d, J = 7.0Hz, 1H), 7.8 5(d,J=8.6Hz,1H),7.72(d,J=7.8Hz,1H),7.58(d,J=8.2Hz,2H),7.54(s,1H) ,7.46(d,J=7.9Hz,1H),7.41(d,J=2.5Hz,1H),7.37-7.30(m,2H),7.29-7.21 (m,2H),7.01(s,2H),6.79(t,J=6.1Hz,1H),5.39(d,J=13.1Hz,1H),5.27-5. 20(m,2H),5.04(s,2H),4.48-4.36(m,4H),4.27(d,J=17.2Hz,1H),4.19(dd, J=8.6,6.7Hz,1H),4.02-3.94(m,2H),3.49-3.44(m,5H),3.23(s,1H),3.06- 2.96(m,1H),2.85-2.76(m,1H),2.47-2.30(m,4H),2.27(s,3H),2.07-1.92( m,2H),1.30(d,J=7.1Hz,3H),0.84(dd,J=24.6,6.7Hz,6H).LC-MS(ESI):m / z 1022.3[M+H] + .

[0174] Compound I-13: Compounds I-11 (1.0 equivalent), I-12 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a THF / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was completed as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ9.92(s,1H),8.70(s,1H),8.16(d,J=7.0Hz,1H),7.91-7.69(m,3H),7.57(d,J=8.2Hz,2H),7.52(s,1H),7.45(d, J=7.9Hz,1H),7.41(d,J=2.5Hz,1H),7.37-7.21(m,4H),7.01(s,2H),6.78(t,J=6.1Hz,1H),5.47-5.24(m,2H),5.17-4.98(m,3H),4.55- 4.34(m,8H),4.29-4.15(m,2H),3.84-3.73(m,2H),3.64-3.52(m,14H),3.51-3.33(m,84H),3.24(s,3H),3.01-2.87(m,1H),2.77-2.67( m,1H),2.46-2.38(m,1H),2.36-2.30(m,1H),2.27(s,3H),2.04-1.90(m,3H),1.30(d,J=7.1Hz,3H),0.87-0.80(m,6H).LC-MS(ESI):m / z 1068.6[(M+2H) / 2] + .

[0175] Compound I-15: Compound I-14 (1.0 equivalent), bis(p-nitrobenzene) carbonate (1.05 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 3 hours. Then, I-3a (1.2 equivalent), HOBt (1.2 equivalent), and DIEA (2.5 equivalent) were added sequentially to the reaction solution, and the mixture was stirred overnight at room temperature. After the reaction was monitored by LC-MS to be complete, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1 H NMR (400MHz, CDCl3) δ7.32 (d, J = 8.2Hz, 2H), 7.04-6.96 (m, 2H), 5.54-5.44 (m, 2H), 5.16-5.02 (m, 4H), 4.98 (s, 1H), 4.27-4.16 (m,2H),4.10-4.04(m,1H),4.03-3.97(m,2H),2.26(t,J=2.5Hz,1H),2.20(s,3H),2.08(s,6H),2.03(s,3H).LC-MS(ESI):m / z 558.2[M+Na] + .

[0176] Compound I-20: The title compound is obtained by replacing I-14 with compound I-19 using the above method. LC-MS (ESI): m / z 567.1 [M+H] + .

[0177] Compound I-17: Compound I-15 (1.0 equivalent), TMSCl (6.0 equivalent), and paraformaldehyde (10.0 equivalent) were stirred overnight in dichloromethane. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure. The crude product I-16 was dissolved in DMF and used directly in the next step. CC-885 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. The DMF solution of the above compound I-16 (1.5 equivalent) was added dropwise under ice bath, and then the reaction was carried out at room temperature for 2 hours. After the reaction was complete as monitored by LC-MS, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 988.3 [M+H] + .

[0178] Compound I-22: The title compound was obtained by replacing I-15 with compound I-20 using the method described above. LC-MS (ESI): m / z 1019.3 [M+H] + .

[0179] Compound I-18: Compound I-17 was dissolved in a tetrahydrofuran-water mixture, and scandium trifluoromethanesulfonate (20 equivalents) was added. The mixture was reacted overnight at room temperature. After the reaction was complete as monitored by LC-MS, the intermediate was purified by reverse-phase chromatography. This intermediate was reacted with I-8 (2.0 equivalents), copper sulfate pentahydrate (0.2 equivalents), and sodium ascorbate (0.4 equivalents) in a THF / tert-butanol / water mixture (4 / 4 / 1, v / v) for 2 hours. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1189.4 [M+H] + .

[0180] Compound I-23: The title compound is obtained by replacing I-17 with compound I-22 using the above method. LC-MS (ESI): m / z 1248.4 [M+H] + .

[0181] Compound I-25: I-24 (1.0 equivalent), allyl chloroformate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 2 hours. After the reaction was completed as monitored by LC-MS, the DCM in the reaction solution was evaporated to dryness, slurried with water, filtered and dried to obtain the title compound.1 H NMR (600MHz, DMSO-d6) δ10.99(s,1H),7.89(t,J=6.2Hz,1H),7.68(d,J=7.9Hz,1H),7.4 7(s,1H),7.39(d,J=7.8Hz,1H),5.97-5.87(m,1H),5.33-5.26(m,1H),5.21-5.15(m,1H) ,5.13-5.06(m,1H),4.51-4.48(m,2H),4.44(d,J=17.2Hz,1H),4.35-4.23(m,3H),2.99 -2.82(m,1H),2.62-2.56(m,1H),2.43-2.34(m,1H),2.04-1.95(m,1H).LC-MS(ESI):m / z 358.1[M+H] + .

[0182] Compound I-26: I-25 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 2 hours. After the reaction was monitored by LC-MS to be complete, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1H NMR(600MHz,DMSO-d6)δ11.00(s,1H),10.14(s,1H),8.52(t,J=6.0Hz,1H),7.9 6(s,1H),7.91(d,J=7.0Hz,1H),7.73-7.67(m,1H),7.57(d,J=8.2Hz,2H),7.50 (dd,J=14.1,8.9Hz,2H),7.41(t,J=7.0Hz,1H),7.34(s,1H),5.97-5.88(m,1H) ,5.33-5.27(m,1H),5.27-5.21(m,1H),5.19(d,J=10.4Hz,1H),5.14-5.09(m,1 H),5.05(s,1H),4.84-4.70(m,1H),4.53-4.50(m,2H),4.48-4.42(m,2H),4.33 -4.31(m,2H),4.01-3.92(m,1H),3.49(d,J=8.2Hz,1H),3.01-2.91(m,1H),2.8 3-2.77(m,1H),2.65-2.56(m,1H),2.55-2.52(m,1H),2.44-2.34(m,1H),2.08- 2.01(m,2H),1.36-1.31(m,3H),0.93(dd,J=17.0,6.7Hz,6H).LC-MS(ESI):m / z 770.4 [M+H] + .

[0183] Compound I-27: I-26 was dissolved in THF, water (20 equivalents) and trimethylphosphine (5.0 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by reverse phase preparation to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ10.18(s,1H),8.72(d,J=7.0Hz,1H),8.07(d,J=5.5H z,3H),7.91(t,J=6.3Hz,1H),7.71(d,J=7.8Hz,1H),7.57(d,J=8.2Hz,2H),7 .50(s,1H),7.42(d,J=7.8Hz,1H),7.35(s,2H),6.00-5.87(m,1H),5.39(d,J =13.2Hz,1H),5.33-5.28(m,1H),5.26-5.24(m,1H),5.21-5.16(m,1H),5.05 (s,2H),4.52-4.48(m,3H),4.46(d,J=17.1Hz,1H),4.32(d,J=6.1Hz,2H),4. 27(d,J=17.0Hz,1H),4.03-3.94(m,2H),3.62(t,J=5.6Hz,1H),3.24(s,1H), 3.01(s,1H),2.80(d,J=17.3Hz,1H),2.53-2.52(m,1H),2.40-2.35(m,1H),2 .12-2.07(m,1H),1.36(d,J=7.1Hz,3H),1.00-0.92(m,6H).LC-MS(ESI):m / z 744.4[M+H] + .

[0184] Compound I-28: I-27 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was monitored by LC-MS until complete, the mixture was separated by reverse-phase reaction to obtain the title compound. 1HNMR(600MHz,DMSO-d6)δ9.94(s,1H),8.19(d,J=7.0Hz,1H),7.89(d,J=8.7Hz,2H) ,7.71(d,J=7.8Hz,1H),7.58(d,J=8.2Hz,2H),7.50(s,1H),7.41(d,J=8.1Hz,1H),7 .33(s,2H),5.93(s,1H),5.39(d,J=13.2Hz,1H),5.32-5.16(m,3H),5.04(s,2H),4 .53-4.48(m,2H),4.45(d,J=17.2Hz,1H),4.41-4.36(m,1H),4.32(d,J=6.2Hz,2H), 4.27(d,J=17.1Hz,1H),4.24-4.19(m,1H),3.99(s,2H),3.60(td,J=6.5,2.7Hz,2H ),3.53-3.48(m,40H),3.44-3.40(m,2H),3.24(s,4H),3.01(s,1H),2.80(d,J=17.2 Hz,1H),2.54-2.52(m,3H),2.46(t,J=6.9Hz,1H),2.42-2.32(m,2H),1.99-1.92(m ,1H),1.30(d,J=7.0Hz,3H),1.27-1.21(m,1H),0.90-0.80(m,6H).LC-MS(ESI):m / z 1336.6[M+Na] + .

[0185] Compound I-29: I-28 (1.0 equivalent), 1,3-dimethylbarbituric acid (3.0 equivalent), and tetra-triphenylphosphine palladium (0.1 equivalent) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by reverse phase preparation to obtain the title compound. 1H NMR(600MHz,DMSO-d6)δ9.94(s,1H),8.19(d,J=6.9Hz,1H),7.89(d,J=8.5Hz,1 H),7.82(d,J=7.8Hz,1H),7.69(s,1H),7.63-7.52(m,3H),7.33(s,2H),5.39(d, J=13.2Hz,1H),5.29-5.20(m,2H),5.03(s,2H),4.49(d,J=17.3Hz,1H),4.38(p ,J=7.1Hz,1H),4.29(d,J=17.4Hz,1H),4.22-4.15(m,3H),3.98(s,2H),3.64-3. 55(m,2H),3.54-3.46(m,41H),3.44-3.41(m,2H),3.23(s,4H),3.10(d,J=18.7 Hz,1H),3.04(d,J=2.9Hz,1H),3.00(d,J=9.6Hz,2H),2.81(d,J=17.3Hz,1H),2. 46(t,J=6.9Hz,1H),2.42-2.31(m,2H),2.10-2.01(m,1H),1.99-1.91(m,1H),1. 30(d,J=7.0Hz,3H),1.24(d,J=14.1Hz,1H),0.93-0.78(m,6H).LC-MS(ESI):m / z 1230.6 [M+H] + .

[0186] Compound I-31A: I-30A (1.0 equivalent), triphosgene (0.4 equivalent), and triethylamine (4.0 equivalent) were dissolved in DCM and reacted for 1 hour. I-24 (1.0 equivalent) was dissolved in DCM and added dropwise to the above reaction solution, and the reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and purified by reverse-phase synthesis to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ10.99(s,1H),9.46(s,1H),7.93(d,J=2.1Hz,1H),7.79(d,J=8.7H z,1H),7.70(d,J=7.8Hz,1H),7.55-7.49(m,1H),7.45(d,J=7.8Hz,1H),7.41-7.37(m,1H) ,7.14(t,J=6.1Hz,1H),5.14-5.07(m,1H),4.48-4.40(m,3H),4.32(d,J=17.2Hz,1H),2.9 6-2.86(m,1H),2.63-2.59(m,1H),2.40-2.36(m,1H),2.04-1.97(m,1H).LC-MS(ESI):m / z 452.1[M+H] + .

[0187] Compound I-31B: The title compound can be obtained by replacing I-30A with I-30B using the above method. 1 H NMR (600MHz, DMSO-d6) δ10.99(s,1H),9.55(s,1H),8.06(d,J=9.1Hz,1H),7.92(d,J= 2.3Hz,1H),7.70(d,J=7.8Hz,1H),7.54-7.50(m,1H),7.47-7.41(m,2H),7.17(t,J=6 .1Hz,1H),5.14-5.06(m,1H),4.49-4.41(m,3H),4.31(d,J=17.2Hz,1H),2.96-2.86( m,1H),2.63-2.58(m,1H),2.37(d,J=4.6Hz,1H),2.03-1.96(m,1H).LC-MS(ESI):m / z 472.1[M+H] + .

[0188] Compound I-32A: The title compound can be obtained by replacing I-24 with I-29 using the above method. 1H NMR (600MHz, DMSO-d6) δ9.95 (s, 1H), 9.47 (s, 1H), 8.20 (d, J = 7.0Hz, 1H), 7.93 (d, J = 2.0Hz, 1H),7.90(d,J=8.6Hz,1H),7.79(dd,J=8.7,2.7Hz,1H),7.72(d,J=7.8Hz,1H),7.58(d,J=8. 1Hz,2H),7.54(s,1H),7.46(d,J=7.9Hz,1H),7.40(dd,J=8.8,2.1Hz,1H),7.33(s,2H),7.1 5(t,J=6.0Hz,1H),5.39(d,J=13.2Hz,1H),5.29-5.18(m,2H),5.04(s,2H),4.44(d,J=5.8Hz ,2H),4.39(t,J=7.1Hz,1H),4.27(d,J=17.2Hz,1H),4.23-4.17(m,1H),3.98(s,1H),3.64- 3.56(m,2H),3.55-3.45(m,40H),3.44-3.42(m,2H),3.24(s,3H),3.07-2.94(m,1H),2.80(d ,J=14.1Hz,1H),2.62(p,J=1.9Hz,2H),2.41-2.31(m,4H),2.07-1.89(m,1H),1.30(d,J=7. 0Hz, 3H), 1.24 (s, 1H), 1.10 (t, J=7.0Hz, 2H), 0.85 (dd, J=24.0, 6.8Hz, 6H). LC-MS (ESI): m / z 1430.6[M+Na] + .

[0189] Compound I-32B: The title compound was obtained by replacing I-24 with I-29 and I-30A with I-30B using the method described above. LC-MS (ESI): m / z 1450.6 [M+Na] + .

[0190] Compound I-33A: I-32A (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was completed as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ9.94(s,1H),9.47(s,1H),8.19(d,J=6.9Hz,1H),7.99(s,1H),7.92(d,J=2.0Hz, 1H),7.88(d,J=8.6Hz,1H),7.78(d,J=8.7Hz,1H),7.75-7.68(m,1H),7.56(d,J=8.4Hz,2H),7.52(s,1H) ,7.45(d,J=7.9Hz,1H),7.40(dd,J=8.7,2.1Hz,1H),7.33(d,J=8.2Hz,1H),7.25(d,J=7.7Hz,1H),7.15( t,J=6.0Hz,1H),6.98(s,2H),5.40(d,J=13.1Hz,1H),5.27(d,J=13.3Hz,1H),5.10(s,1H),5.02(s,2H), 4.50(d,J=22.0Hz,2H),4.45-4.39(m,4H),4.37(d,J=7.2Hz,1H),4.28-4.17(m,2H),3.78(d,J=17.8Hz, 2H),3.64-3.54(m,4H),3.50(d,J=1.9Hz,40H),3.45-3.41(m,6H),3.23(s,3H),3.19-3.07(m,2H),2.98 -2.82(m,1H),2.75-2.58(m,1H),2.45(t,J=7.0Hz,1H),2.42-2.37(m,1H),2.31(t,J=7.3Hz,2H),2.01- 1.93(m,2H),1.29(d,J=7.0Hz,3H),1.23(d,J=4.3Hz,3H),0.84(dd,J=24.1,6.6Hz,6H).LC-MS(ESI):m / z 889.5[(M+2H) / 2] + .

[0191] Compound I-33B: The title compound can be obtained by replacing I-32A with I-32B using the above method. 1H NMR(600MHz,DMSO-d6)δ9.94(s,1H),9.58(s,1H),8.19(d,J=6.8Hz,1H),8.06(d,J=9.1Hz,1H) ,7.99(s,1H),7.92(d,J=2.3Hz,1H),7.88(d,J=8.5Hz,1H),7.76-7.65(m,1H),7.60-7.48(m,3H ),7.48-7.40(m,2H),7.33(d,J=8.3Hz,1H),7.25(d,J=8.5Hz,1H),7.19(t,J=5.9Hz,1H),6.98( s,2H),5.40(d,J=13.1Hz,1H),5.28(d,J=13.2Hz,1H),5.11(s,1H),5.02(s,2H),4.49(d,J=29. 2Hz,2H),4.46-4.41(m,4H),4.40-4.32(m,2H),4.28-4.15(m,2H),3.78(d,J=18.4Hz,2H),3.62 -3.54(m,4H),3.51-3.41(m,52H),3.23(s,3H),3.18-3.06(m,2H),2.97-2.84(m,1H),2.69(t,J =18.1Hz,1H),2.47-2.43(m,1H),2.41-2.34(m,1H),2.31(t,J=7.3Hz,2H),2.24(t,J=7.5Hz,1H ),1.95(dq,J=14.0,7.2,6.7Hz,2H),1.30(d,J=7.0Hz,3H),0.92-0.77(m,6H).LC-MS(ESI):m / z 899.5[(M+2H) / 2] + .

[0192] Compound I-35: Compound I-34 (1.0 equivalent), bromopropylene (1.2 equivalent), and cesium carbonate (3.0 equivalent) were dissolved in DMF and reacted at room temperature for 4 hours. After the reaction was completed as monitored by LC-MS, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1H NMR (600MHz, CDCl3) δ8.32(d,J=2.2Hz,1H),8.16(dd,J=8.6,2.2Hz,1H),7.99(d,J=8.5Hz,1H) ,6.11-5.95(m,1H),5.50-5.41(m,1H),5.41-5.32(m,1H),4.95-4.84(m,2H).LC-MS(ESI):m / z 242.0[M+H] + .

[0193] Compound I-36: I-35 (1.0 equivalent), iron powder (3.0 equivalent), and ammonium chloride (3.0 equivalent) were dissolved in methanol / water (4 / 1, volume ratio), and the mixture was heated to 80°C and reacted for 3 hours. After the reaction was monitored by LC-MS until complete, the reaction solution was filtered, concentrated under reduced pressure, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ7.67(d,J=8.6Hz,1H),6.64(d,J=2.2Hz,1H),6.57-6.48(m,1H),6.20(s, 2H),6.07-5.93(m,1H),5.43-5.31(m,1H),5.27-5.20(m,1H),4.72-4.64(m,2H).LC-MS(ESI):m / z 212.0[M+H] + .

[0194] Compound I-37: I-36 (1.0 equivalent), triphosgene (0.4 equivalent), and triethylamine (4.0 equivalent) were dissolved in DCM and reacted for 1 hour. I-24 (1.0 equivalent) was dissolved in DCM and added dropwise to the above reaction solution, and the reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and purified by reverse-phase synthesis to obtain the title compound. 1H NMR (600MHz, DMSO-d6) δ10.99(s,1H),9.27(s,1H),7.84-7.79(m,2H),7.70(d,J=7.8Hz,1H),7.52(s,1 H),7.47-7.42(m,1H),7.37-7.31(m,1H),7.02(t,J=6.1Hz,1H),6.07-5.97(m,1H),5.43-5.36(m,1H), 5.30-5.24(m,1H),5.13-5.07(m,1H),4.80-4.71(m,2H),4.48-4.40(m,3H),4.31(d,J=17.3Hz,1H),2. 95-2.86(m,1H),2.63-2.57(m,1H),2.54(s,1H),2.41-2.34(m,1H),2.03-1.96(m,1H).LC-MS(ESI):m / z 511.2[M+H] + .

[0195] Compound I-39: The title compound was obtained by replacing I-24 with I-29 using the method described above. LC-MS (ESI): m / z 1489.5 [M+Na] + .

[0196] Compound I-38: I-37, 1,3-dimethylbarbituric acid (3.0 equivalents), and tetra-triphenylphosphine palladium (0.1 equivalents) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by reverse phase preparation to obtain the title compound. 1 H NMR(600MHz,DMSO-d6)δ11.00(s,1H),9.20(s,1H),7.81-7.75(m,2H),7.70(d,J =7.8Hz,1H),7.53(s,1H),7.45(d,J=7.9Hz,1H),7.36-7.28(m,1H),7.00(t,J=6 .0Hz,1H),5.15-5.08(m,1H),4.50-4.40(m,3H),4.32(d,J=17.2Hz,1H),2.95-2 .88(m,1H),2.59(s,2H),2.44-2.34(m,2H),2.04-1.96(m,1H).LC-MS(ESI):m / z 471.1[M+H] + .

[0197] Compound I-40: The title compound was obtained by replacing I-37 with I-39 using the method described above. LC-MS (ESI): m / z 1449.6 [M+Na] + .

[0198] Compound I-41: I-40 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was completed as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. 1 H NMR (600MHz, DMSO-d6) δ9.95 (s, 1H), 9.31 (s, 1H), 8.21 (s, 1H), 8.01 (d, J = 5.7Hz, 1H),7.94-7.84(m,1H),7.78-7.63(m,3H),7.57(d,J=8.3Hz,2H),7.51(d,J=6.5H z,1H),7.45(d,J=7.9Hz,1H),7.38-7.19(m,3H),7.13(s,1H),6.98(s,2H),5.40( d,J=12.9Hz,1H),5.28(d,J=13.2Hz,1H),5.10(s,1H),5.02(s,1H),4.55-4.35(m, 7H),4.27-4.16(m,2H),3.78(d,J=20.6Hz,2H),3.63-3.55(m,4H),3.55-3.39(m, 48H),3.23(s,3H),3.19-3.08(m,2H),2.93(d,J=30.7Hz,1H),2.69(t,J=18.1Hz, 1H),2.45(s,1H),2.41-2.34(m,1H),2.34-2.16(m,3H),2.03-1.87(m,2H),1.30( d,J=6.9Hz,3H),1.26-1.19(m,2H),0.84(dd,J=24.0,6.7Hz,6H).LC-MS(ESI):m / z 899.0[(M+2H) / 2] + .

[0199] Compound I-43: I-42 (1.0 equivalent), bromopropylene (1.2 equivalent), and cesium carbonate (3.0 equivalent) were dissolved in ACN and reacted at 80°C for 4 hours. After the reaction was completed by LC-MS monitoring, the reaction solution was poured into water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1H NMR (400MHz, CDCl3) δ8.30 (d, J = 2.7Hz, 1H), 8.20-8.01 (m, 1H), 7.07-6.90 (m, 1H), 6.20 -5.97(m,1H),5.57-5.45(m,1H),5.43-5.34(m,1H),4.81-4.64(m,2H).LC-MS(ESI):m / z 237.1[M+Na] + .

[0200] Compound I-44: I-43 (1.0 equivalent), iron powder (3.0 equivalent), and ammonium chloride (3.0 equivalent) were dissolved in methanol / water (4 / 1, volume ratio), and the mixture was heated to 80°C and reacted for 3 hours. After the reaction was monitored by LC-MS until complete, the reaction solution was filtered, concentrated under reduced pressure, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound. 1 H NMR (400MHz, DMSO-d6) δ6.85 (d, J = 8.7Hz, 1H), 6.64 (t, J = 2.2Hz, 1H), 6.50-6.42 (m, 1H), 6.10-5. 94(m,1H),5.47-5.32(m,1H),5.30-5.17(m,1H),4.91(s,2H),4.53-4.42(m,2H).LC-MS(ESI):m / z 184.0[M+H] + .

[0201] Compound I-45: I-44 (1.0 equivalent), triphosgene (0.4 equivalent), and triethylamine (4.0 equivalent) were dissolved in DCM and reacted for 1 hour. I-24 (1.0 equivalent) was dissolved in DCM and added dropwise to the above reaction solution, and the reaction was continued for 1 hour. The reaction solution was concentrated under reduced pressure and purified by reverse-phase synthesis to obtain the title compound. 1H NMR (400MHz, DMSO-d6) δ10.98(s,1H),8.65(s,1H),7.77-7.61(m,2H),7.51(s,1H),7.43(d,J=7.9Hz,1H), 7.23-7.13(m,1H),7.03(d,J=8.9Hz,1H),6.77(t,J=6.1Hz,1H),6.10-5.94(m,1H),5.40(d,J=17.2Hz,1H) ,5.25(d,J=10.6Hz,1H),5.15-5.02(m,1H),4.57(d,J=5.1Hz,2H),4.50-4.37(m,3H),4.31(d,J=17.2Hz,1 H),3.00-2.83(m,1H),2.67-2.57(m,1H),2.38(dd,J=13.3,4.5Hz,1H),2.05-1.94(m,1H).LC-MS(ESI):m / z 483.1[M+H] + .

[0202] Compound I-47: The title compound was obtained by replacing I-24 with I-29 using the method described above. LC-MS (ESI): m / z 1461.6 [M+Na] + .

[0203] Compound I-46: I-45, 1,3-dimethylbarbituric acid (3.0 equivalents), and tetra-triphenylphosphine palladium (0.1 equivalents) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by reverse phase preparation to obtain the title compound. 1 H NMR (600MHz, DMSO-d6) δ11.00(s,1H),9.66(s,1H),8.49(s,1H),7.69(d,J=7.8Hz,1H),7.54(d,J =2.6Hz,1H),7.51(s,1H),7.44(d,J=7.9Hz,1H),7.04-7.01(m,1H),6.84(d,J=8.8Hz,1H),6.69( t,J=6.1Hz,1H),5.14-5.07(m,1H),4.45(d,J=17.2Hz,1H),4.39(d,J=6.0Hz,2H),4.31(d,J=17. 2Hz,1H),2.96-2.85(m,1H),2.55(s,2H),2.39-2.35(m,1H),2.04-1.97(m,1H).LC-MS(ESI):m / z 443.1[M+H] + .

[0204] Compound I-48: The title compound was obtained by replacing I-45 with I-47 using the method described above. LC-MS (ESI): m / z 1449.6 [M+Na] + .

[0205] Compound I-49: I-48 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was completed as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. 1 H NMR (600MHz, DMSO-d6) δ8.49(s,1H),8.20(d,J=6.9Hz,1H),8.02(d,J=15.7Hz,1H),7.89(d,J=8.5Hz,1H),7 .77-7.69(m,1H),7.57(d,J=8.5Hz,2H),7.54(d,J=2.6Hz,1H),7.51(s,1H),7.44(d,J=7.9Hz,1H),7.34(d, J=8.2Hz,1H),7.29-7.16(m,1H),7.03(dd,J=8.8,2.6Hz,1H),6.99(s,1H),6.84(d,J=8.8Hz,1H),6.69(t,J =6.1Hz,1H),5.41(d,J=13.2Hz,1H),5.36-5.31(m,1H),5.29(d,J=13.1Hz,1H),5.11(s,1H),5.03(s,1H),4. 51(d,J=21.7Hz,2H),4.44-4.37(m,4H),4.30-4.16(m,2H),3.79(d,J=17.5Hz,1H),3.64-3.56(m,3H),3.54 -3.47(m,37H),3.47-3.37(m,8H),3.28(s,1H),3.24(s,2H),3.13(q,J=10.1Hz,2H),2.99-2.87(m,1H),2.72 (d,J=17.8Hz,1H),2.46(s,1H),2.39(d,J=14.6Hz,1H),2.35-2.29(m,1H),1.98(ddt,J=22.5,16.7,7.1Hz, 3H),1.46(t,J=7.1Hz,1H),1.30(d,J=7.0Hz,3H),1.25(d,J=6.3Hz,8H),0.92-0.78(m,6H).LC-MS(ESI):m / z 899.0[(M+2H) / 2] + .

[0206] Compound I-51: I-50 (1.0 equivalent), 2-[2-(2-azidoethoxy)ethoxy]ethylamine (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 30 minutes. The reaction solution was poured into water, extracted with DCM, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. 1 H NMR (600MHz, DMSO-d6) δ9.11(s,2H),7.93(t,J=5.8Hz,1H),3.58(t,J=4.9Hz,2H),3.45(t,J=5.9Hz,2H),3.41(s,3H ),3.39(t,2H),3.26-3.18(m,2H),2.55(t,J=7.1Hz,2H),2.26(t,J=7.4Hz,2H),1.85-1.76(m,2H).LC-MS(ESI):m / z 381.1[M+H] + .

[0207] Compound I-52: I-29 (1.0 equivalent), 3-chloro-4-methylphenyl isocyanate (1.2 equivalent), and triethylamine (4.0 equivalent) were dissolved in DCM and reacted for 1 hour. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1419.6 [M+Na] + .

[0208] Compound I-53: I-52 (1.0 equivalent), I-51 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was completed as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. 1H NMR(600MHz,DMSO-d6)δ9.94(s,1H),9.09(s,2H),8.78(s,1H),8.19(d,J=6.8Hz,1H),7.88( d,J=8.3Hz,3H),7.70(d,J=7.8Hz,1H),7.66(d,J=2.2Hz,1H),7.56(d,J=8.1Hz,2H),7.51(s ,1H),7.44(d,J=7.9Hz,1H),7.36-7.21(m,2H),7.21-7.10(m,2H),6.82(t,J=6.1Hz,1H),5. 39(d,J=13.1Hz,1H),5.27(d,J=13.2Hz,1H),5.10(s,1H),5.01(s,2H),4.58-4.33(m,8H),4. 27-4.16(m,2H),3.77(s,2H),3.61-3.53(m,3H),3.49(d,J=7.4Hz,40H),3.44-3.35(m,8H), 3.23(s,3H),3.16(s,2H),3.06-2.82(m,1H),2.69(s,1H),2.45(s,1H),2.40-2.34(m,1H),2 .23(d,J=12.0Hz,6H),2.02-1.91(m,J=7.1,6.6Hz,2H),1.78(p,J=8.6,7.9Hz,2H),1.54-1. 39(m,1H),1.29(d,J=7.0Hz,3H),1.24(d,J=12.5Hz,2H),0.92-0.77(m,6H).LC-MS(ESI):m / z 889.5[(M+2H) / 2] + .

[0209] Compound I-55: I-54 (1.0 equivalent), allyl chloroformate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 2 hours. After the reaction was complete as monitored by LC-MS, the DCM in the reaction solution was evaporated to dryness, slurried with water, filtered, and dried to obtain the compound. LC-MS (ESI): m / z 612.2 [M+H] + .

[0210] Compound I-56: I-55 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 1024.4 [M+H] + .

[0211] Compound I-57: I-56 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound. LC-MS (ESI): m / z 998.5 [M+H] + .

[0212] Compound I-58: I-57 (1.0 equivalent), I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1568.7 [M+Na] + .

[0213] Compound I-59: I-58 (1.0 equivalent), 1,3-dimethylbarbituric acid (3.0 equivalent), and tetrakis(triphenylphosphine)palladium (0.1 equivalent) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1484.6 [M+H] + .

[0214] Compound I-60: I-59 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1853.9 [M+H] + .

[0215] Compound I-62: I-61 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 954.4 [M+H] +

[0216] Compound I-63: I-62 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound. LC-MS (ESI): m / z 928.5 [M+H] + .

[0217] Compound I-64: I-63 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1498.7 [M+H] + .

[0218] Compound I-65: I-64 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1867.9 [M+H] + .

[0219] Compound I-67: I-66 (1.0 equivalent), allyl chloroformate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 2 hours. After the reaction was complete as monitored by LC-MS, the DCM in the reaction solution was evaporated to dryness, slurried with water, filtered, and dried to obtain the compound. LC-MS (ESI): m / z 652.3 [M+H] + .

[0220] Compound I-68: I-67 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 1064.4 [M+H] + .

[0221] Compound I-69: I-68 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound. LC-MS (ESI): m / z 1038.5 [M+H] + .

[0222] Compound I-70: I-69 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1608.8 [M+H] + .

[0223] Compound I-71: I-70 (1.0 equivalent), 1,3-dimethylbarbituric acid (3.0 equivalent), and tetrakis(triphenylphosphine)palladium (0.1 equivalent) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1524.8 [M+H] + .

[0224] Compound I-72: I-71 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1893.9 [M+H] + .

[0225] Compound I-74: I-73 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 966.4 [M+H] + .

[0226] Compound I-75: I-74 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound. LC-MS (ESI): m / z 940.4 [M+H] + .

[0227] Compound I-76: I-75 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1510.7 [M+H] + .

[0228] Compound I-77: I-76 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1879.9 [M+H] + .

[0229] Compound I-79: I-76 (1.0 equivalent), allyl chloroformate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 2 hours. After the reaction was complete as monitored by LC-MS, the DCM in the reaction solution was evaporated to dryness, slurried with water, filtered, and dried to obtain the compound. LC-MS (ESI): m / z 638.2 [M+H] + .

[0230] Compound I-80: I-79 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 1050.4 [M+H] + .

[0231] Compound I-81: I-80 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the title compound. LC-MS (ESI): m / z 1024.4 [M+H] + .

[0232] Compound I-82: I-81 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1594.8 [M+H] + .

[0233] Compound I-83: ​​I-82 (1.0 equivalent), 1,3-dimethylbarbituric acid (3.0 equivalent), and tetrakis(triphenylphosphine)palladium (0.1 equivalent) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1510.7 [M+H] + .

[0234] Compound I-84: I-83 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1879.9 [M+H] + .

[0235] Compound I-86: I-85 (1.0 equivalent), allyl chloroformate (1.2 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 2 hours. After the reaction was complete as monitored by LC-MS, the DCM in the reaction solution was evaporated to dryness, slurried with water, filtered, and dried to obtain the compound. LC-MS (ESI): m / z 652.2 [M+H] + .

[0236] Compound I-87: I-86 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 1064.4 [M+H] + .

[0237] Compound I-88: I-87 was dissolved in THF, water (20 equivalents) and trimethylphosphine (1.5 equivalents) were added, and the reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure to give the title compound. LC-MS (ESI): m / z 1038.4 [M+H] + .

[0238] Compound I-89: I-88 (1.0 equivalent), compound I-7b (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 30 minutes. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1608.8 [M+H] + .

[0239] Compound I-90: I-89 (1.0 equivalent), 1,3-dimethylbarbituric acid (3.0 equivalent), and tetrakis(triphenylphosphine)palladium (0.1 equivalent) were dissolved in DCM and reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1524.7 [M+H] + .

[0240] Compound I-91: I-90 (1.0 equivalent), I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a DMSO / tert-butanol / water mixed solvent (4 / 4 / 1, v / v) for 1 hour. After the reaction was complete as monitored by LC-MS, the mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1893.9 [M+H] + .

[0241] Example 2:

[0242] Compound II-2: Compound II-1 (1.0 equivalent), N-Boc glycine (1.05 equivalent), HATU (1.08 equivalent), and DIEA (3.0 equivalent) were stirred overnight in DCM. The mixture was filtered and evaporated to dryness. The residue was purified by column chromatography to give the title compound. LC-MS (ESI): m / z 431.2 [M+H] + .

[0243] Compound II-3: Compound II-2 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 843.4 [M+H] + .

[0244] Compound II-4: Compound II-3 and TFA were stirred in DCM for 2 hours. After the reaction was complete as monitored by LC-MS, triethylamine was added dropwise to neutralize the reaction solution, followed by concentration under reduced pressure. The residue was purified by column chromatography to obtain the title compound. LC-MS (ESI): m / z 743.4 [M+H] + .

[0245] Compound II-6: Compounds II-4 (1.0 equivalent), II-5 (1.0 equivalent), HATU (1.05 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 1 hour. The mixture was filtered and evaporated to dryness. The residue was dissolved in THF, and water (20 equivalent) and trimethylphosphine (5.0 equivalent) were added. The mixture was reacted under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and isolated by reverse phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1199.4 [M+H] + .

[0246] The general method for compounds II-7A-C: Compound II-6 (1.0 equivalent), carboxylic acids I-7a / b / c (1.05 equivalent), HATU (1.1 equivalent), and DIEA (3.0 equivalent) were stirred in DCM for 30 minutes. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure. Compound I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were added and reacted in a DMSO / tert-butanol / water mixed solvent (10 / 5 / 2, v / v) for 2 hours. The mixture was then separated by reverse-phase reaction to obtain the corresponding compounds.

[0247] Compound II-7A: The title compound was obtained from carboxylic acid I-7a using the general method described above. LC-MS (ESI): m / z 1610.6 [M+H] + .

[0248] Compound II-7B: The title compound was obtained from carboxylic acid I-7b using the general method described above. LC-MS (ESI): m / z 1070.1 [(M+2H) / 2] + .

[0249] Compound II-7C: The title compound was obtained from carboxylic acid I-7c using the general method described above. LC-MS (ESI): m / z 1161.2 [(M+2H) / 2] + .

[0250] Compound II-8: Compounds II-6 (1.0 equivalent), I-10 (1.05 equivalent), HATU (1.08 equivalent), and DIEA (3.0 equivalent) were stirred in DMSO for 1 hour. After the reaction was completed as monitored by LC-MS, the crude product was isolated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1394.5 [M+H] + .

[0251] Compound II-9: Compound II-8 (1.0 equivalent), I-12 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) were reacted in a mixed solvent of DMSO / tert-butanol / water (10 / 5 / 2, v / v) for 2 hours. The mixture was then separated by reverse-phase chromatography to obtain the corresponding compound. LC-MS (ESI): m / z 1255.1 [(M+2H) / 2] + .

[0252] Example 3:

[0253] Compound III-2: Compound III-1 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, dissolved in THF, and then water (20 equivalent) and trimethylphosphine (5.0 equivalent) were added. The reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1106.5 [M+H] + .

[0254] Compound III-3A: Compound III-2 (1.0 equivalent), carboxylic acid I-7b (1.05 equivalent), DIEA (3.0 equivalent), and HATU (1.1 equivalent) were dissolved in DCM and stirred at room temperature for 30 minutes. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure and reacted with compound I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) in a DMSO / tert-butanol / water mixed solvent (10 / 5 / 2, v / v) for 2 hours. The mixture was separated by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1023.6 [(M+2H) / 2] + .

[0255] Compound III-3B: The title compound was obtained by replacing carboxylic acid I-7b with carboxylic acid I-7c using the above synthetic method. LC-MS (ESI): m / z 1114.7 [(M+2H) / 2] + .

[0256] Compound IV-2: Compound IV-1 (1.0 equivalent) and potassium tert-butoxide (1.2 equivalent) were stirred in DMF for 5 minutes. A DMF solution of compound I-5a (1.5 equivalent) was added dropwise under ice bath conditions, followed by reaction at room temperature for 30 minutes. After the reaction was monitored by LC-MS until complete, the reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, dissolved in THF, and then water (20 equivalent) and trimethylphosphine (5.0 equivalent) were added. The reaction was carried out under nitrogen protection for 2 hours. The reaction solution was concentrated under reduced pressure and purified by reverse-phase chromatography to obtain the title compound. LC-MS (ESI): m / z 1198.5 [M+H] + .

[0257] Compound IV-3A: Compound IV-2 (1.0 equivalent), carboxylic acid I-7b (1.05 equivalent), DIEA (3.0 equivalent), and HATU (1.1 equivalent) were dissolved in DCM and stirred at room temperature for 30 minutes. After the reaction was complete as monitored by LC-MS, the reaction solution was concentrated under reduced pressure and reacted with compound I-8 (2.0 equivalent), copper sulfate pentahydrate (0.2 equivalent), and sodium ascorbate (0.4 equivalent) in a DMSO / tert-butanol / water mixed solvent (10 / 5 / 2, v / v) for 2 hours. The mixture was separated by reverse-phase chromatography to obtain the corresponding compound. LC-MS (ESI): m / z 1069.6 [(M+2H) / 2] + .

[0258] Compound IV-3B: The title compound was obtained by replacing carboxylic acid I-7b with carboxylic acid I-7c using the above synthetic method. LC-MS (ESI): m / z 1160.7 [(M+2H) / 2] + .

[0259] Example 4: Enzymatic hydrolysis experiment of representative compounds

[0260] Protocol 1: Cathepsin B (CTB)-triggered digestion. Dissolve 10 units of CTB in 1 mL of buffer containing 25 mM sodium acetate (pH 5.0) and 1 mM EDTA. Dilute 5 μL of the stock solution to 184 μL using buffer containing 25 mM sodium acetate (pH 5.0) and 1 mM EDTA. Add 10 μL of 200 mM dithiothreitol (DTT) solution and incubate at 37 °C for 20 min to activate the enzyme, resulting in a final CTB concentration of 0.25 units / mL. Then, add 1 μL of DMSO solution containing the specified compound (10 mM) to the CTB solution and incubate at 37 °C for 24 h. Next, collect 200 μL of sample and rapidly quench with 200 μL of acetonitrile. Centrifuge at 10,000 rpm for 10 min and collect the supernatant. Quantitatively analyze the sheared substrate and corresponding products using HPLC. Repeat the above experimental procedure three times.

[0261] Option 2: β-Galactosidase (β-Gal)-triggered enzymatic digestion. β-Gal and the shearing substrate solution were added to phosphate-buffered saline (PBS), with 20 units (5 μL) of β-Gal and 0.5 μL of the shearing substrate solution (10 mM in DMSO). The PBS buffer volume was 994.5 μL, pH 6.0, and the mixture was incubated at 37°C for 24 hours. Then, 150 μL of the sample was collected and rapidly quenched with 350 μL of acetonitrile. The mixture was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. HPLC was used for quantitative analysis of the shearing substrate and corresponding products. The above experimental procedure was repeated three times.

[0262] Option 3: β-glucosidase-triggered enzymatic digestion. β-glucosidase and the cleavage substrate solution were added to sodium acetate buffer (PBS), with 500 units (1 μL) of glucosidase and 1 μL of the cleavage substrate solution (10 mM in DMSO). The PBS buffer volume was 998 μL, pH 5.0, and the mixture was incubated at 37°C for 24 hours. Then, 150 μL of the sample was collected and rapidly quenched with 350 μL of acetonitrile. The mixture was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. HPLC was used for quantitative analysis of the cleavage substrate and corresponding products. The above experimental procedure was repeated three times.

[0263] The test results are shown in the table below.

[0264] Table 1. Results of enzymatic hydrolysis experiments for representative compounds [a] A: ≥95% converted to enzymatic hydrolysis products; B: <95% converted to enzymatic hydrolysis products; [b] Add excess acetylcysteine ​​and pre-incubate for 30 minutes; [c] The enzymatic hydrolysis conditions were the same as the corresponding enzymatic hydrolysis protocol, except that the final CTB concentration was reduced to 0.10 units / mL and the incubation was carried out at 37°C for 2 hours.

[0265] The structures of the pyrolysis products are as follows:

[0266] Experimental results show that the linker and its conjugate described in this invention have good versatility for substrates with different enzymatic hydrolysis mechanisms and can achieve high enzymatic hydrolysis efficiency.

[0267] Example 5: Antibody Conjugation

[0268] Antibody reduction: 5 mg / mL monoclonal antibody solution was treated with 10 equivalents of tri-(2-carboxyethyl)phosphine (TCEP) in 10 mM PBS pH 7.4 buffer and incubated at 37°C for 1 hour to reduce interchain disulfide bonds.

[0269] Antibody-drug conjugation: 12 equivalents of the compound in DMSO solution were added to the above-described reduced antibody solution, bringing the final concentration of DMSO to 15% (v / v). After stirring the mixture at room temperature for 1 hour, the resulting conjugate was filtered through a 0.20 μm PES membrane and concentrated using an Amicon Millipore ultracentrifuge with a molecular weight cutoff of 30 kD.

[0270] The concentration of the antibody-drug conjugate was detected by Nanodrop at 280 nm. Conjugate purity and aggregation were determined by size exclusion chromatography (SEC): a 7.8 × 300 mm TSKGel 3000SWXL column with 5 μm particles was used, with isocratic elution at 1.0 mL / min for 15 min using a mobile phase of 50 mM potassium dihydrogen phosphate at pH 6.5 and 15% (v / v) acetonitrile. The purity of the conjugate of the newly degrading agent conjugate was determined based on the retention time and peak area at 280 nm and 254 nm. The drug-antibody ratio (DAR) was analyzed by hydrophobic interaction chromatography (HIC): a 4.6 × 100 mm TSKgel Butyl-NPR column with 2.5 μm particles was used. Mobile phase A was an aqueous solution at pH 7.0 containing 1.5 mM ammonium sulfate and 25 mM disodium hydrogen phosphate, and mobile phase B was an aqueous solution at pH 7.0 containing 20% ​​isopropanol and 25 mM disodium hydrogen phosphate. The analytes were eluted with a linear gradient of 30%–70% B over 30 min at a flow rate of 0.5 mL / min and a column temperature of 25 °C. Detection was performed at 280 nm and 254 nm. Analysis showed that the representative antibody conjugates had high DAR values, and SEC aggregation and peak width analysis indicated good stability and compatibility of the antibody conjugates (see table below). [a] Aggregation test: A: <5%; B: 5%-20%; C: >20%. Note: SMol012 is shown below.

[0271] Experimental results show that the linker described in this disclosure has good compatibility with ligands including cell membrane receptors, such as antibodies.

[0272] Example 6: In vitro antiproliferative activity test

[0273] Cells in the logarithmic growth phase were seeded at an appropriate density into 96-well plates, 100 μL per well. After overnight culture, different concentrations of the drug were added for 3 days of treatment, with each concentration in triplicate. Corresponding solvent controls and cell-free zeroing wells were also included. After treatment, adherent cells were decanted, and 10% (w / v) trichloroacetic acid (100 μL / well) was added for fixation at 4°C for 1 h. Cells were then washed five times with distilled water and dried at room temperature. 100 μL of SRB solution (Sigma, St. Louis, MO, USA) (4 mg / mL, dissolved in 1% glacial acetic acid) was added to each well, and the cells were incubated for staining at room temperature for 15 min. Unbound SRB was washed five times with 1% glacial acetic acid. After drying at room temperature, 150 μL of 10 mM Tris solution was added to each well, and the optical density (OD value) at 560 nm was measured using a SpectraMax 190 microplate reader. The inhibition rate of the compound on cell proliferation was calculated using the following formula: Inhibition rate (%) = [1 - (OD administration wells - OD negative control wells) / (OD positive control wells - OD negative control wells)] × 100%. The IC50 was estimated using the four-parameter method. 50 Each experiment was repeated three times independently, with three replicates for each concentration each time.

[0274] The test results (Table 2) show that, compared with the positive compound, the conjugate of the present invention not only has advantages in terms of activity, but also exhibits good receptor expression-related cell activity (the conjugate of the present invention shows higher inhibitory activity against cells with higher HER2 expression, namely BT-474 or JIMT-1, while exhibiting lower inhibitory activity against cells with lower HER2 expression, namely MDA-MB-231), indicating good safety.

[0275] Table 2. In vitro antiproliferative activity assays of representative compounds [a] A + IC 50 ≤1nM; A: 1nM <IC 50 ≤10nM; B: 10nM <IC 50 ≤50nM; C: 50nM <IC 50 ≤300nM; D: IC 50 >300nM; [b] log2(TPM+1), data source: depmap.org.

[0276] The results (Table 3) show that, compared with the positive compound, the conjugate of the present invention has higher cell inhibitory activity in NCI-N87 and SK-OV-3 cell lines.

[0277] Table 3. In vitro antiproliferative activity assays of representative compounds [a] A+ IC 50 ≤1nM; A: 1nM <IC 50 ≤10nM; B: 10nM <IC 50 ≤50nM; C: 50nM <IC 50 ≤300nM; D: IC 50 >300nM.

[0278] The results (Table 4) show that, compared to the positive compound, the conjugates described in this invention have advantages in activity on both Huh-7 and LnCaP cell lines.

[0279] Table 4. In vitro antiproliferative activity assays of representative compounds [a] A + IC 50 ≤1nM; A: 1nM <IC 50 ≤15nM; B: 15nM <IC 50 ≤50nM; C: 50nM <IC 50 ≤300nM; D: IC 50 >300nM.

[0280] Example 7: Evaluation of the in vivo antitumor activity of representative conjugates

[0281] We used a subcutaneous xenograft model to investigate the in vivo activity of the conjugates, and these new degrading agent conjugates also showed good in vivo antitumor activity.

[0282] Under aseptic conditions, tumor tissue in its vigorous growth phase was cut into 1.5 mm pieces. 3 Subcutaneous xenografts were inoculated into the right axilla of SCID mice. The diameter of the subcutaneous xenografts in SCID mice was measured using calipers. The xenografts were allowed to grow to an average volume of approximately 100-150 mm². 3 Animals were randomly divided into groups. Each treatment group received a single intravenous injection via tail vein on the day of grouping (d0), while the solvent control group received an equal volume of PBS solution. Throughout the experiment, tumor diameter was measured twice weekly, and mouse weight was also measured. Tumor volume (TV) was calculated using the formula: TV = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = V t / V0. Where V0 is the tumor volume measured at the time of administration (i.e., d0), and Vt is the tumor volume at each measurement. The tumor growth inhibition rate (%) is used as the evaluation index of antitumor activity, and the calculation formula is as follows: Tumor growth inhibition rate (TI)(%) = 100 - (T RTV / C RTV )×100%, T RTV Treatment group RTV; C RTV : Negative control group RTV.

[0283] The test results (Table 5) show that the conjugate of the present invention exhibits significant antitumor activity after a single dose.

[0284] Table 5. Efficacy of representative compounds against Huh-7 xenografts in tumor-bearing mice [a] A: TI (%) > 50; B: 30 <TI(%)≤50;C:10<TI(%)≤30;D:TI(%)≤10。

[0285] ARV-766 is a candidate protein degradation drug for the treatment of prostate cancer. The results of the assay (Table 6) show that, compared with ARV-766, the antibody-drug conjugate of the present invention exhibits better in vivo antitumor activity after only a single dose.

[0286] Table 6. Efficacy of representative compounds against 22RV1 xenograft tumors in tumor-bearing mice [a] A: TI (%) > 50; B: 30 <TI(%)≤50;C:10<TI(%)≤30;D:TI(%)≤10。

[0287] 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. A protein degrading agent conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The coupling is as shown in formula (I). in, The portion shown in (Ib) is derived from a protein degrader based on CRBN E3 ligase; in, M POI This is the target protein binding region; This is the CRBN binding portion; M Linker For use with or without a connector to link the target protein binding portion and the CRBN binding portion; X is CH or N; L 1 This is the antibody linker portion; L 2 This is a bridging portion used to connect antibody connectors; Y is either NH or O; R T Selected from the following groups: cathepsin substrate fraction, β-glucuronidase substrate fraction, β-galactosidase substrate fraction; L 3 For H, COR N Or containing one or more PEG unit structures and / or polysarcosine unit structures; wherein, R N For H or C 1-6 alkyl L 4 Groups that are absent or contain one or more PEG unit structures and / or polysarcosine unit structures; a is a value between 1 and 8; Bm is the binding moiety that can specifically bind to the target protein.

2. The coupling as described in claim 1, characterized in that, The target protein is a target protein to be degraded, and / or the target protein is a cell membrane receptor or a cell surface antigen.

3. The coupling as described in claim 1, characterized in that, Bm refers to the antibody or its antigen-binding portion.

4. The coupling as described in claim 1, characterized in that, When Y is NH, -R T -L 3 Selected from the table below Alternatively, when Y is 0, -R T -L 3 Selected from the following group:

5. The coupling as described in claim 1, characterized in that, The group containing one or more PEG unit structures and / or polysarcosine unit structures is -M. 33 -M 31 -(CH2CH2O) q -M 32 or -M 33 -M 31 -[COCH2N(Me)] q -M 32 ; in, M 31 Selected from the following group: none, -CO-, -C1-4 alkylene-, -CO-C1-4 alkylene-; M 32 Selected from the group consisting of: none, C1-2 alkyl, C1-2 alkylene-acid group, -CO-C1-2 alkyl; and M 33 Selected from the following group: none, -NHCO-, -N(C1-4 alkyl)CO-, linking group formed by click chemistry, -C1-6 alkylene-NHCO-, -C1-6 alkylene-N(C1-4 alkyl)CO-, -C1-6 alkylene-linking group formed by click chemistry, -C1-6 heteroalkylene-NHCO-, -C1-6 heteroalkylene-N(C1-4 alkyl)CO-, -C1-6 heteroalkylene-linking group formed by click chemistry; and q is an integer between 1 and 50.

6. The coupling as described in claim 1, characterized in that, L 3 It is a group containing one or more PEG unit structures and / or polysarcosine unit structures.

7. The coupling as described in claim 1, characterized in that, Selected from Table A1 below, Represents the X-linked junction: Table A1 Wherein, ring B is an optionally substituted C3-20 cycloalkyl or an optionally substituted 4- to 20-membered heterocyclic alkyl; the optional substitution means that the group is unsubstituted or one or more H in the group is replaced by R. 1 The substituted R is selected independently from the group consisting of: C1-4 alkyl, hydroxyl, C1-4 haloalkyl, and cyano.

8. The coupling as described in claim 1, characterized in that, The conjugate is capable of releasing a CRBN-based protein degrader, as shown in (Ib1), within cells or in the presence of cathepsins, β-glucuronidase, or β-galactosidase (β-gal). In the formula, M POI M Linker X and As stated above.

9. The coupling as claimed in claim 1, characterized in that, The coupling agents are selected from Table A below; Table A 10. A pharmaceutical composition, characterized in that, This includes the conjugate as described in claim 1 and optionally a pharmaceutically acceptable carrier.

11. The conjugate of claim 1 or a pharmaceutically acceptable salt thereof, in the preparation of a treatment or prevention of diseases associated with or caused by excessive expression of intracellular target proteins.

12. The use as described in claim 11, characterized in that, The disease described is a tumor.

13. A compound or a salt thereof, characterized in that, The compound is shown in II. in, L i 1 The antibody linker to be coupled with the antibody; Furthermore, as shown in (Ib), L 2 L 3 L 4 R T X, Y, M POI M Linker ,and As defined in claim 1; 14. The compound of claim 13, wherein the precursor is characterized in that the compound is selected from Table B below; Table B 15. An intermediate, characterized in that, The intermediate is shown in Figure III. in, L ii 2 The portion containing reactive groups; Furthermore, as shown in (Ib), L 3 L 4 R T X, Y, M POI M Linker ,and As defined in claim 1;

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