Linkers and coupling drugs using them, antibody-drug conjugates and their applications

Abstract

The present invention provides linkers, coupling drugs using them, antibody-drug conjugates, and their applications. Specifically, it provides a conjugate as shown in formula (II), or its stereoisomer, or a pharmaceutically acceptable salt thereof, where L is a linker (-GGFL-) as shown in formula (I). A conjugate having a linker as shown in formula (I) has high stability at non-target sites and can be rapidly activated at target sites. Formula (II): R1-L-R2

Description

[Technical Field]

[0001] This invention relates to the field of pharmaceuticals, and more specifically to linkers, coupling drugs using the same, antibody-drug conjugates, and their applications. [Background technology]

[0002] Coupling technology has a wide range of applications in tumor treatment, disease diagnosis, and efficient screening. Here, small molecule coupling drugs (SMDCs) and antibody-drug conjugates (ADCs) consist of a target molecule or antibody, a coupling arm, and an effector molecule (e.g., a cytotoxic drug). The mechanisms of action of SMDCs and ADCs are similar. They are usually administered into the bloodstream by intravenous injection and enter tumor tissue via the bloodstream. Thereafter, they bind to metastatic receptors or related receptors on the surface of tumor cells. Under the mediation of these receptors, they form endosomes via endocytosis. The endosomes then fuse with lysosomes and release cytotoxic molecules into the cytoplasm. These cytotoxic molecules bind to DNA, tubulin, etc., affecting their replication or mitosis, ultimately leading to apoptosis of the cell. In summary, there is an urgent need in this field for the development of novel coupling drugs with improved stability and enhanced therapeutic efficacy. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] The object of the present invention is to provide linkers for application with coupling drugs, tumor-targeting coupling drugs, antibody-drug conjugates, pharmaceutical compositions, and applications thereof. A first aspect of the present invention provides a conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the conjugate is as shown in formula (II), Formula (II): R1-L-R2 Here, L is a linker (-GGFL-) as shown in equation (I), TIFF2026510850000001.tif37141R1 is a group having one or more functions selected from the group consisting of activating the tumor microenvironment, improving the water solubility of tumor-targeted drugs, and providing a coupling site with antibody elements. R2 is a group derived from a cytotoxic drug or a cytotoxic-carrying drug.

[0004] In another preferred example, the antibody element is an antibody or the antigen-binding domain of the antibody.

[0005] In another preferred example, the conjugate is a tumor-targeting conjugate.

[0006] In another preferred example, the molecule of the cytotoxic drug or cytotoxic-carrying drug contains at least one reactive group (e.g., an amino group or imino group (e.g.) or a hydroxyl group).

[0007] In another preferred example, the group derived from the cytotoxic drug or cytotoxic carrier drug is the reactive group (e.g., an amino group, an imino group, or a hydroxyl group) and, for example, -OC(O)C 1-6 This refers to a monovalent group formed by a reaction with groups such as alkyl groups, -COOH groups, and amino groups.

[0008] In another preferred example, the group derived from the cytotoxic drug or cytotoxic carrier drug means a monovalent group formed by the loss of one hydrogen atom from the amino group or imino group, or by the loss of a hydrogen atom on the hydroxyl group.

[0009] In another preferred example, the cytotoxic drug or cytotoxic carrier drug is selected from the group consisting of doxorubicin, daunorubicin, epirubicin, taxanes, paclitaxel, docetaxel, topoisomerase inhibitors, camptothecins, irinotecan, extecan and its derivative DXd, TLR7 / 8 agonists, reximod, imiquimod, rosolibine, 1-(4-aminobutyl)-2-butyl-1H-imidazo[4,5-C]quinoline-4-amine (T785) or its analogues, cisplatin, carboplatin, rapamycin, everolimus, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitomycin, mitoxantrone, MMAE, MMAF, and MMAD.

[0010] In another preferred example, R2 is -D1 or -Am-D2, where D1 is a monovalent group from a drug having -NH2 or -NH- in the molecule, D2 is a monovalent group from a drug having -OH in the molecule, and Am is -NH-C 1-6 It is an alkylene-O-, and the NH terminus of Am is bound to L.

[0011] In another preferred example, D1 is selected from the groups shown in Table E1.

[0012] In another preferred example, if R2 is D1, the conjugate is selected from Table B1 below.

[0013] TIFF2026510850000002.tif228166TIFF2026510850000003.tif138165Table B1 In another preferred example, -Am- is, The filename is TIFF2026510850000004.tif14142.

[0014] In another preferred example, D2 is selected from the groups shown in Table E2.

[0015] In another preferred example, when R2 is -Am-D2, the conjugate is selected from Table B2 below.

[0016] TIFF2026510850000005.tif191166In another preferred example, R1 comprises one or more groups selected from the group consisting of an activating group in the tumor microenvironment, a modifying group that improves the water solubility of the tumor targeting drug, and a reactive group capable of coupling with an antibody.

[0017] In another preferred example, R1 comprises a reactive group capable of coupling with an antibody.

[0018] In another preferred example, R1 comprises a modifying group that improves the water solubility of the tumor targeting drug.

[0019] In another preferred example, the modifying group that improves the water solubility of the tumor targeting drug is a PEG chain.

[0020] In another preferred example, improving the water solubility of the tumor targeting drug means that R1 contains a PEG chain structural unit.

[0021] In another preferred example, the PEG chain is a linear or branched PEG chain.

[0022] In another preferred example, the PEG chain has 1 to 40 (preferably 1 to 20, more preferably 1 to 10) -CH2CH2O- structural units.

[0023] In another preferred example, providing a coupling position with an antibody means that R1 contains a reactive group capable of coupling with an antibody.

[0024] In another preferred example, the reactive group capable of coupling with the antibody is TIFF2026510850000006.tif20141selected from the group consisting of.

[0025] In another preferred example, the activating group in the tumor microenvironment is The selection is made from the group consisting of TIFF2026510850000007.tif81164.

[0026] In another preferred example, R1 is selected from the following tables C1-A and C1-B: In TIFF2026510850000008.tif255162TIFF2026510850000009.tif246165TIFF2026510850000010.tif150157C1-B, o is an integer between 0 and 20, preferably between 0 and 1 and 10, and more preferably between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). m and n are each independently an integer between 0 and 20, preferably m and n are each independently an integer between 0 and 1 and 10, and more preferably m and n are each independently an integer between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0027] In another preferred example, R1 is a group formed by a click reaction between Ra and Rb.

[0028] In another preferred example, Ra is The filename is TIFF2026510850000011.tif12126, and Rb is, The filename is TIFF2026510850000012.tif12125.

[0029] In another preferred example, Ra is Ra'-N3, and Rb is The filename is TIFF2026510850000013.tif11124.

[0030] In another preferred example, Rb' is concatenated to L.

[0031] In another preferred example, the group formed by the click reaction between Ra and Rb is The filename is TIFF2026510850000014.tif14125.

[0032] In another preferred example, Ra is selected from the following tables D1-A and D1-B: In Table D1-B of TIFF2026510850000015.tif255162, o is an integer between 0 and 20, preferably between 0 and 1 and 10, and more preferably between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). m and n are each independently an integer between 0 and 20, preferably between 0 and 1 and 10, and more preferably between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0033] In another preferred example, Rb is selected from the following tables D2-A and D2-B: TIFF2026510850000016.tif180166 Here, n is an integer between 0 and 20, preferably between 0 and 1 and 10, and more preferably between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0034] In another preferred example, R1 is selected from the following tables C2-A and C2-B: TIFF2026510850000017.tif244165TIFF2026510850000018.tif214166 Here, m and n are each independently 0 or an integer from 1 to 20, preferably 0 or an integer from 1 to 10, and more preferably an integer from 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0035] In another preferred example, the conjugate is selected from Tables A1, A2, and A3.

[0036] In another preferred example, the conjugate is selected from Table A1.

[0037] In another preferred example, the conjugate can release a cytotoxic substance or a cytotoxic carrier in the tumor environment.

[0038] In another preferred example, the tumor environment contains cathepsin L.

[0039] In another preferred example, the cytotoxic substance or cytotoxic carrier is the cytotoxic drug or cytotoxic carrier or a derivative thereof (wherein a derivative is a derivative modified by linking to L).

[0040] In another preferred example, in a tumor environment, the release rate of the conjugate that releases a cytotoxic substance or cytotoxic carrier within 60 hours (preferably within 40 hours, more preferably within 20 hours) based on the total molar amount of the conjugate is >80%, preferably >85%, more preferably >90%.

[0041] In another preferred example, the conjugate substantially does not release cytotoxic substances or cytotoxic carriers in a non-tumor environment (e.g., the environment of blood, plasma, heart, or liver).

[0042] In another preferred example, in a non-tumor environment, the release rate of the conjugate that releases a cytotoxic substance or cytotoxic carrier in 2 hours (preferably within 10 hours, more preferably 20 hours) based on the total molar amount of the conjugate is <10%.

[0043] In another preferred example, the ratio of the release rates of the conjugate over 20 hours in a tumor environment and a non-tumor environment (i.e., release rate in the presence of cathepsin L or in a tumor environment / release rate in the absence of cathepsin L or in a non-tumor environment) is >10, preferably >15.

[0044] A second aspect of the present invention provides an antibody conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the antibody conjugate is as shown in formula (III), Formula (III): R3-(R1'-L-R2) x Here, R3 represents the antibody portion. R1'-L-R2 represents a group formed by coupling a conjugate as shown in formula (II) to an antibody element via the R1 group, where R1, L, and R2 are as defined in claim 1, and R1' is a group formed by coupling R1 to an antibody element, and x is between 1 and 8.

[0045] In another preferred example, R1 is TIFF2026510850000019.tif18133 is a base that exists and R1' is in R1. TIFF2026510850000020.tif19134 is coupled This is the base that forms TIFF2026510850000021.tif20134.

[0046] In another preferred example, R1 is TIFF2026510850000022.tif22133 is a base that exists and R1' is in R1. TIFF2026510850000023.tif22135 is coupled This is the group that forms TIFF2026510850000024.tif19134.

[0047] In another preferred example, R1 is TIFF2026510850000025.tif21135 is a base that exists and R1' is in R1. TIFF2026510850000026.tif20133 is coupled This is the base that forms TIFF2026510850000027.tif27133.

[0048] In another preferred example, R1 is TIFF2026510850000028.tif19134 is a base that exists and R1' is in R1. TIFF2026510850000029.tif19133 are coupled and each To form TIFF2026510850000030.tif38134, or, This is the base that forms TIFF2026510850000031.tif38131.

[0049] In another preferred example, the antibody in the antibody portion may be unmodified, and it can be linked to the R1 group of the conjugate via the side chain group of an amino acid in the antibody (e.g., the -SH of a cysteine ​​residue in the antibody).

[0050] In another preferred example, the antibody in the antibody moiety can be modified with an active linking group (e.g., an alkynyl group) which then links to the R1 group of the conjugate via the modified active group.

[0051] In another preferred example, the antibodies include HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylyl cyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1, and PDL. Selected from the group consisting of 1, HER2, NY-ESO-1, BCMA, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC1, and MAGE-A3, or compositions thereof.

[0052] A third aspect of the present invention provides a pharmaceutical composition comprising (i) a conjugate or stereoisomer thereof or a pharmaceutically acceptable salt thereof as described in the first aspect, or an antibody conjugate or stereoisomer thereof or a pharmaceutically acceptable salt thereof as described in the second aspect, and (ii) a pharmaceutically acceptable carrier thereof.

[0053] A fourth aspect of the present invention provides the use of the conjugate described in the first aspect or the antibody conjugate described in the second aspect in the preparation of a drug for treating or preventing tumors or inflammation.

[0054] In another preferred example, the tumor is selected from the group consisting of bladder cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and hematological cancer, or a combination thereof.

[0055] A fifth aspect of the present invention provides a linker, which is shown in formula (I).

[0056] In another preferred example, the linker is used to couple drugs.

[0057] In another preferred example, the linker is selective.

[0058] In another preferred example, the linker is enzymatically digestible by cathepsin L.

[0059] In another preferred example, the linker is substantially enzymatically digestible by an enzyme other than cathepsin L (e.g., cathepsin B).

[0060] In another preferred example, in the tumor environment, based on the total molar amount of the conjugate, >80%, preferably >85%, more preferably >90% of the conjugate having the linker is enzymatically digested by enzymes within 60 hours (preferably within 40 hours, more preferably within 20 hours).

[0061] In another preferred example, in a non-tumor environment (e.g., blood, plasma, cardiac, or hepatic environment), the conjugate having the linker is substantially not enzymatically digested by enzymes.

[0062] In another preferred example, in a non-tumor environment, based on the total molar amount of the conjugate, <10% of the conjugate having the linker is enzymatically digested by an enzyme within 2 hours (preferably within 10 hours, more preferably 20 hours). [Effects of the Invention]

[0063] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0064] [Figure 1] Example 5 shows a pattern in which L-AM-DXD is not enzymatically digested by cathepsin L. [Figure 2] The enzymatic digestion pattern of EMC-GGFG-AM-DXD by cathepsin L in Example 6 is shown. [Figure 3] The enzymatic digestion pattern of MI-PEG6-GGFL-AM-DXD by cathepsin L in Example 6 is shown. [Figure 4] Example 7 shows a pattern in which MI-PEG6-GGFL-AM-DXD is not enzymatically digested by cathepsin B. [Figure 5] The enzymatic digestion pattern of EMC-GGFG-AM-DXD by cathepsin B in Example 7 is shown. [Figure 6] The stability detection pattern of EMC-GGFG-AM-DXD in human plasma in Example 8 is shown. [Figure 7] The stability detection pattern of MI-PEG6-GGFL-AM-DXD in human plasma in Example 8 is shown. [Figure 8] The stability detection pattern of GGFG-AM-DXD for monkey heart homogenate in Example 9 is shown. [Figure 9] The stability detection pattern of MI-PEG6-GGFL-AM-DXD for monkey heart homogenate in Example 9 is shown. [Figure 10] The stability profile of MI-PEG6-GGFL-AM-DXD in monkey liver homogenate in Example 10 is shown. [Figure 11]The stability profile of the EMC-GGFG-AM-DXD of the monkey liver homogenate in Example 10 is shown. [Figure 12] The stability profile of GGFG-AM-DXD of human gastric cancer homogenate in Example 11 is shown. [Figure 13] The stability profile of MI-PEG6-GGFL-AM-DXD of human gastric cancer homogenate in Example 11 is shown. [Figure 14] The stability profile of GGFG-AM-DXD for CT26 tumor homogenate in Example 12 is shown. [Figure 15] The stability profile of GGFL-AM-DXD for CT26 tumor homogenate in Example 12 is shown. [Figure 16] This example demonstrates the in vivo therapeutic effect of antibody-drug conjugates based on GGFL linker and GGFG linker in mice. [Modes for carrying out the invention]

[0065] As a result of extensive and meticulous research, the inventors unexpectedly discovered a drug that possesses excellent selectivity, has a -GGFL-linker (i.e., the linker shown in formula (I)), is almost completely enzymatically digested under the action of cathepsin L to release cytotoxic substances, is stable in environments where cathepsin L is absent (e.g., liver, heart, plasma, etc.), is therefore extremely safe outside tumors, effectively releases cytotoxic substances within tumors, and thus can effectively treat tumors. Based on this, the inventors completed the present invention.

[0066] term As used herein, the terms “conjugate of the present invention” and “coupling drug of the present invention” are interchangeable and refer to a class of conjugates having a linker represented by formula (I) such as formula (II) (i.e., -GGFL-).

[0067] As used herein, the terms “antibody conjugate of the present invention” and “antibody-drug conjugate of the present invention” are interchangeable and refer to an antibody-conjugate formed by an antibody (Ab) or its active fragment or its antigen-binding domain and the conjugate shown in formula (II) or its pharmaceutically acceptable salt or solvent compound.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs. Where used herein in reference to a specific numerical value, the term “about” means that the value can vary by no more than 1% from the stated value. For example, the term “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0069] As used herein, the term “amino acid residue” refers to the group formed by removing one H from the N-terminal -NH2 of an amino acid and one -OH from the C-terminal -COOH. Generally, the portion of an amino acid (residue) including the N-terminus and C-terminus is called the main chain, and the portion that determines the specific type of amino acid is called the side chain. Unless otherwise specified herein, amino acids include natural amino acids and unnatural amino acids, including D-type and / or L-type amino acids. Examples of amino acids include, but are not limited to, Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(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 specification, an amino acid is an amino acid 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), and further, if the amino acid has two or more amino groups and / or two or more carboxyl groups, the term further includes groups formed by removing one H from -NH2 on different carbon atoms and -OH from -COOH, for example, the divalent group -C(O)-(CH2)2-C(COOH)-NH- formed by removing one H from -NH2 of glutamic acid and one H from -COOH other than the α-position.

[0070] In the present invention, the term "pharmaceutically acceptable" component means a substance that is suitable for use in humans and / or animals without excessive side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit-risk ratio.

[0071] In this invention, the term "effective dose" 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 preventive effect. The exact effective dose for a particular subject varies depending on the subject's size and health status, the nature and severity of the disease, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to pre-determine the exact effective dose. However, in certain situations, the effective dose can be determined by conventional experiments and is left to the clinician's judgment.

[0072] Unless otherwise specified, all compounds appearing in this invention are intended to include all possible optical isomers, such as single chiral compounds or mixtures of various different chiral compounds (i.e., racemic compounds). In all compounds of this invention, each chiral carbon atom may optionally be in an R configuration, an S configuration, or a mixture of R and S configurations.

[0073] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is suitable as a drug when formed with an acid or base of the compound of the present invention. pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is those formed with an acid of the compound of the present invention. Acids suitable for salt formation 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, benzenemethanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0074] Unless otherwise specified, the term "amino acid" as used herein is intended to include all conventional amino acids, 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.

[0075] Where a trade name is used herein, it is intended to include the formulation of the trade name product, its corresponding generic drug, and the active ingredient of the trade name product.

[0076] antibody As used herein, the term “antibody element” includes an antibody or the antigen-binding domain of such antibody. Preferred antibody elements include antibodies (e.g., complete antibodies, single-chain antibodies, nanobodies, antibody fragments), and in particular antibodies that target tumor cell markers (e.g., tumor markers present on the surface of tumor cells).

[0077] As used herein, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the constant region of the light chain facing the first constant region of the heavy chain, and the variable region of the light chain facing the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0078] As used herein, the terms “single-domain antibody” and “nanobody” have the same meaning and refer to a single-domain antibody constructed by cloning the variable region of the antibody heavy chain, which consists of only one heavy chain variable region, and is the smallest antigen-binding fragment that functions fully. Typically, an antibody spontaneously lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0079] As used herein, the term “variable” refers to a difference in the arrangement of specific portions of the variable region in an antibody, which shapes the binding and specificity of different particular antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folding configuration, connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folding structure. The CDRs of each chain are closely adjacent by the FR regions and, together with the CDRs of another chain, form the antigen-binding site of the antibody. While the constant regions are directly involved in the binding of the antibody to the antigen, they also exhibit various effector functions, such as involvement in the antibody-dependent cytotoxicity of the antibody.

[0080] The "light chain" of vertebrate antibodies (immunoglobulins) can be assigned to one of two different classes (called κ and λ) according to the amino acid sequence of its constant region. The immunoglobulin can also be assigned to a different class according to the amino acid sequence of its heavy chain's constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0081] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located within the variable regions of the heavy and light chains, called variable regions (CDRs). These sections are divided into four framework regions (FRs), and the amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-folds formed by the FRs between them are spatially close. The CDRs of the heavy chain and their corresponding CDRs of the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of similar antibodies, the amino acids that make up the FR or CDR region can be determined.

[0082] In the present invention, polypeptide elements include not only complete antibodies but also antibody fragments having immunoactivity (e.g., Fab or (Fab')2 fragments, antibody heavy chains, or antibody light chains), or fusion proteins formed by antibodies with other sequences. Accordingly, the present invention further includes the antibody fragments, derivatives, and analogs.

[0083] Cathepsin In mammals, papain-like cysteine ​​proteases are classified as cathepsins, which are a type of intracellular protease primarily found in lysosomes. They are readily activated in weakly acidic environments and unstable glycoproteins (except cathepsins D, E, and S) in basic and neutral solutions. All cathepsins are hydrolyzed from inactive preprocathepsins, and their in vivo synthesis pathway is as follows: First, they are synthesized as preprocathepsins on the ribosome-binding membrane, transported to the endoplasmic reticulum via transferrin, then enter the Golgi apparatus, where they simultaneously undergo glycosylation and phosphorylation to form mannose-6-phosphate proteins. Finally, they are indirectly transported to lysosomes via recognition by mannose-6-phosphate specific receptors on lysosomes. Recent studies have shown that cathepsins are increasingly recognized for their roles in lesions, tumor invasion, immune system-related diseases, and various parasitic infections. The main cause of death in tumor patients is the degradation of the extracellular matrix and basement membrane, which allows tumor cells to escape from the tumor tissue and metastasize. The proteases necessary for this degradation process include various cathepsins, which promote tumor spatial expansion, tumor angiogenesis, and metastasis of tumor cells both inside and outside blood vessels through protein hydrolysis. Currently, it is known that the expression levels of cathepsins are increased in various human tumors, with cathepsin B being particularly prominent.

[0084] Most cathepsins are intracellular enzymes found in lysosomes, playing a crucial role in human protein hydrolysis. They are closely associated with various tumor diseases such as human tumors, osteoporosis, and arthritis, and have recently attracted significant attention as targeted proteases. Based on their substrate specificity, they can be classified into endopeptidases, exopeptidases, aminopeptidases, and carboxypeptidases. Based on their protein hydrolysis mechanism, they can be classified into serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, and metalloproteases. Currently, the most studied enzyme is cathepsin B. Because this enzyme is highly expressed in many tumor cells and is a specific intracellular enzyme, it is being considered as a Linker enzyme digestor in the development of major ADC drugs, thus enabling specific and efficient intracellular enzyme digestion. Cathepsin L is a key member of the lysosomal cysteine ​​protease family. Its preprocathepsin peptide, which has a very unique synthesis and transport method, contains the spatial structure of cathepsin L with ERF / WNIN and GNFD motifs, and is mainly composed of an α-helical L domain and a β-sheet R domain. Extensive research has shown that cathepsin L plays extremely important roles in physiological and pathological processes in vivo, as well as in parasites. Its physiological functions include protein hydrolysis, antigen presentation, T cell sorting, cell apoptosis, and embryonic development. Cathepsin L is closely associated with various types of tumorigenesis, cardiovascular diseases, and renal diseases.

[0085] Shyam S et al. studied the expression of cathepsin L in human tumor cells and found that cancer generally showed higher levels of cathepsin L expression than normal tissue, kidney tumors, and testicular tumors. Non-small cell lung cancer showed the next highest levels, and other cancers such as breast cancer, ovarian cancer, colon cancer, adrenal cancer, bladder cancer, prostate cancer, and thyroid cancer also showed higher expression. Cathepsin L may be a useful diagnostic agent or biomarker for human malignancies.

[0086] Dhhivya R et al. investigated targeted therapy with cathepsin L in cancer and revealed that cathepsin L is a conventional phenomenon in human cancer and is strongly associated with metastasis, invasiveness, and poor patient prognosis. They also suggested that cathepsin L contributes to cancer-related bone resorption. Furthermore, they elucidated the mechanism by which cathepsin L promotes advanced tumors and their spread, and described the therapeutic usefulness of cathepsin L intervention strategies aimed at preventing metastatic progression and bone resorption.

[0087] drugs As used herein, “drug” means a compound having a desired biological activity and possessing a reactive functional group (e.g., an amino group (-NH2), a hydroxyl group (-OH), etc.) for preparing the conjugate described in the present invention or for forming a covalent bond with the linker described in the present invention. The desired biological activity includes the diagnosis, cure, alleviation, treatment, and prevention of diseases in humans or other animals. Thus, as long as the necessary reactive functional group is present, the compound referred to in the term “drug” refers to a drug identified in the official National Pharmacopoeia of China, as well as, for example, the official Homeopathic Pharmacopoeia of the United States, the official National Prescription Collection, or any supplement thereof. Typical drugs are listed in the Physician’s Reference Book of Drugs (PDR) and the U.S. Food and Drug Administration (FDA) Orange Book. As new drugs are discovered and developed, it should be understood that these drugs will also be included in the “drug” in the coupling drugs described in the present invention.

[0088] Preferably, the drugs that can be used in the configuration of the present invention include, but are not limited to, cytotoxic drugs or cytotoxic-carrying drugs.

[0089] The term "cytotoxic drug or cytotoxic-carrying drug" refers to a substance that inhibits or blocks the expression activity, function, and / or causes cell destruction of a cell. This term includes radioisotopes, chemotherapeutic agents, and toxins (including fragments and / or variants thereof) of low molecular weight toxins or enzymatic toxins derived from, for example, bacteria, fungi, plants, or animals. Preferably, the cytotoxic drug or cytotoxic-carrying drug includes, but is not limited to, doxorubicin, daunorubicin, epirubicin, taxanes, paclitaxel, docetaxel, topoisomerase inhibitors, camptothecins, irinotecan, extecan and its derivatives DXd, TLR7 / 8 agonists, reximod, imiquimod, rosolibine, 1-(4-aminobutyl)-2-butyl-1H-imidazo[4,5-C]quinoline-4-amine (T785) or analogues, cisplatin, carboplatin, rapamycin, everolimus, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitomycin, mitoxantrone, MMAE, MMAF, and MMAD.

[0090] Linker As used herein, the term “linker” refers to a divalent linking group used to link two different functional parts. One embodiment of this application provides a linker such as that shown in formula (I) (i.e., -GGFL-).

[0091] TIFF2026510850000033.tif41134 The linker is efficiently enzymatically digested by cathepsin L in a tumor environment but is stable in a non-tumor environment. As a result, the conjugate (or coupling drug) and antibody conjugate (or antibody conjugate) using the linker of the present invention are stable until they reach the target site (e.g., tumor site), and once they reach the target site (e.g., tumor site), they are rapidly activated, thereby treating the tumor while avoiding excessive side effects (e.g., off-target toxicity).

[0092] Coupling drugs (also called conjugates) One embodiment of the present invention provides a conjugate (or coupling drug) using the linker described in formula (I).

[0093] Preferably, the present invention provides a tumor-targeting coupling drug as shown in formula (II), Formula (II): R1-L-R2 Here, the structure of L is as shown in equation (I), and R1 and R2 are as defined in the first embodiment.

[0094] In another embodiment, R1 is a modifying group that improves the water solubility of an activating group or tumor-targeting drug in the tumor microenvironment, and / or R2 is doxorubicin, daunorubicin, epirubicin, taxanes, paclitaxel, docetaxel, topoisomerase inhibitors, camptothecins, irinotecan, extecan and its derivatives DXd, TLR7 / 8 agonists, reximod, imiquimod, rosolibine, 1-(4-aminobutyric acid) The cytotoxic drug or cytotoxic-carrying drug is selected from 2-butyl-1H-imidazo[4,5-C]quinoline-4-amine (T785) or its analogues, cisplatin, carboplatin, rapamycin, everolimus, methotrexate, fludarabine, gemcitabine, cytarabine, melphalan, nimustine, mitomycin, mitoxantrone, drastatin 10 derivatives, MMAE, MMAF, and MMAD.

[0095] Preferably, R2 includes -D1 or -Am-D2.

[0096] Preferably, when the drug (D) of R2 is a drug having a -NH2 free end or an -NH- free end, and R2 is D1, the structure of the tumor-targeting coupling drug is as shown in Table B1.

[0097] Preferably, when the drug (D) of R2 is a drug having a free -OH end, R2 is -AM-D2, where the structure of -AM- is If the product code is TIFF2026510850000034.tif15133, the structure of the tumor-targeting coupling drug is as shown in Table B2.

[0098] Preferably, R1 is selected from the structures shown in Table C1.

[0099] Preferably, R1 is Formed by a click reaction with TIFF2026510850000035.tif11159 Selected from the TIFF2026510850000036.tif16156 structure.

[0100] Preferably, when Ra is selected from the structures shown in Table D1, Rb is selected from compounds containing the groups described in Table D2.

[0101] Preferably, the coupling drug is selected from structures shown in Table A2 or Table A3.

[0102] Preferably, the coupling drug is selected from structures as shown in Table A1.

[0103] Antibody-drug conjugates One embodiment of the present invention further provides an antibody conjugate (or antibody-drug conjugate) using the linker described in formula (I), as well as stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0104] Preferably, the antibody-drug conjugate is as shown in formula (III), Formula (III): R3-(R1'-L-R2) x R3 represents the antibody portion, R1'-L-R2 represents a group formed by coupling a conjugate as shown in formula (II) to the antibody element via the R1 group, where R1, L, and R2 are as defined in claim 1, R1' is the group formed by coupling R1 to the antibody element, and x is 1 to 8.

[0105] Preferably, the antibody-drug conjugate is as shown in formula (IV), Formula (IV): R4-cys-S-(R1'-L-R2) x In another embodiment, (R1'-L-R2) is any structure described in the first embodiment or the coupling drug described above. R3 is an antibody having one or more cysteine ​​residue mutations, cys is a cysteine ​​residue contained in R3, and S is the sulfur atom in cysteine. X is the number of (R1-L-R2) molecules bound to the antibody, and x = 1 to 8.

[0106] Preferably, the antibody is HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylyl cyclase. C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK 6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1, PDL1, HER2, NY-ESO-1, BCMA, WT1, MU The antigen-binding domains can be selected from (but are not limited to) C1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC-1, and MAGE-A3, or from any of the antigen-binding domains of any of the antibodies referred to in this invention.

[0107] Pharmaceutical composition and administration method The linker used in the coupling drug (conjugate) or antibody-drug conjugate provided by the present invention remains stable outside the tumor environment and, after entering the tumor environment, is efficiently enzymatically digested by cathepsin L, thereby releasing a specific drug (e.g., a cytotoxic drug) within the tumor site or other target sites. Therefore, the coupling drug or antibody-drug conjugate of the present invention can be used to treat tumors or inflammation (e.g., bladder cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and hematological cancers).

[0108] The coupling drug or antibody-drug conjugate described above can be administered to a subject (e.g., a human) in a therapeutically effective dose via an appropriate route. Subjects requiring treatment may be patients at risk of or suspected of having a disease associated with the activity or expression level of a particular antigen. Such patients can be identified by conventional physical examination.

[0109] Depending on the type of disease or site of the disease requiring treatment, the pharmaceutical composition may be administered to a subject using conventional methods known to those skilled in the medical field. The composition may also be administered via other conventional routes, such as orally, parenterally, inhalation spray, topically, rectally, nasally, orally, vaginally, or via implant. As used herein, the term "parenterally" includes techniques of injection or infusion subcutaneously, intradermally, intravenously, intramuscularly, intra-articularly, intra-arterially, intra-bursally, intrasternally, intra-spinally, intra-lesionally, and intracranially. Furthermore, it may be administered by injectable depot preparations, such as depot injections lasting for 1 month, 3 months, or 6 months, or by biodegradable materials and methods.

[0110] The injectable composition includes, but is not limited to, various carriers such as vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby injecting a drug 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 formulations can be administered by dissolving a sterile formulation of a suitable soluble salt form of the antibody in a pharmaceutical excipient such as an injection solution, 0.9% saline, or 5% glucose solution.

[0111] When the coupling drug or antibody-drug conjugate of the present invention is used for therapeutic purposes, it can be delivered by conventional methods 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 infusion pump. Other methods include various delivery and carrier systems using the conjugate and biodegradable polymers.

[0112] The pharmaceutical composition of the present invention comprises a safe and effective amount of the coupling drug or antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Typically, drug formulations need to be adapted to the route of administration, and the pharmaceutical composition of the present invention can be prepared in solution form by conventional methods, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective dose.

[0113] The effective dose of the coupling drug or antibody-drug conjugate described in the present invention can vary depending on the method of administration and the severity of the disease being treated. A person skilled in the art can determine the preferred effective dose based on various factors (e.g., clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the antibody conjugate (e.g., bioavailability, metabolism, half-life), the severity of the disease in the patient requiring treatment, the patient's body weight, the patient's immune status, and the route of administration. Typically, the antibody-drug conjugate of the present invention can be administered daily at a dose of approximately 0.0001 mg to 50 mg (preferably 0.001 mg to 10 mg per kg of animal body weight) to obtain a good effect. For example, depending on the urgency of the treatment situation, it may be administered in several divided doses per day, or the dose may be proportionally reduced.

[0114] The dosage forms of the compounds of the present invention used for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0115] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0116] When the pharmaceutical composition is used, the antibody conjugate of the present invention, which is safe and available in a sufficient dose, is applied to mammals (e.g., humans) in need of treatment, where the dose at the time of administration is the effective dose to be considered, and for a person weighing 60 kg, the daily dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dose must also take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of the skills of a skilled physician.

[0117] The main advantages of this invention are as follows: (1) The linker, tumor-targeting coupling drug, antibody-drug conjugate, pharmaceutical composition, and applications of the present invention utilize a linker that is selectively enzymatically digested by cathepsin, thereby enabling GGFL to be effectively identified and enzymatically digested by cathepsin L. The drug enters tumor cells as a target and is then enzymatically digested by cathepsin present in the tumor cells, releasing drug molecules with therapeutic effects.

[0118] (2) The conjugate or antibody conjugate having the linker (-GGFL-) of the present invention exhibits excellent stability in non-tumor environments such as plasma, cardiac homogenate, and liver homogenate, and significantly surpasses the stability of conjugates using other linkers (-GGFG-) (see Examples 8 to 10).

[0119] (3) The conjugate or antibody conjugate having the linker (-GGFL-) of the present invention can be efficiently activated in the tumor environment and release specific drugs (see, for example, Examples 11 and 12).

[0120] (4) The conjugate or antibody conjugate having the linker (-GGFL-) of the present invention exhibits a superior cure rate compared to the linker (-GGFG-) of the prior art.

[0121] (5) The conjugate or antibody conjugate of the present invention exhibits excellent quality and efficacy in vivo (see Examples 13 and 14).

[0122] The present invention will be further described below in conjunction with specific examples. These examples are used solely to illustrate the present invention and should not be used to limit its scope. In the following examples, experimental methods that do not specify conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: An Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer. Unless otherwise specified, percentages and quantities are calculated as weight percentages and weight quantities.

[0123] Example 1. Linker applied to coupling drugs The linker provided by the present invention has the following structure.

[0124] The linker of formula (I) can be applied to drug coupling drugs, can be applied to antibody-drug conjugates, and can be further applied to pharmaceutical compositions containing the above drug coupling drugs or antibody-drug conjugates.

[0125] Example 2. Tumor-targeted coupling drug The drug has the structure of formula (II) below, Formula (II): R1-L-R2 Here, the structure of L is as follows: TIFF2026510850000038.tif40132 Here, R1 is an activating group or a modifying group that improves the water solubility of tumor-targeting drugs in the tumor microenvironment. R2 is a cytotoxic drug or a cytotoxic-carrying drug.

[0126] In a preferred embodiment of this example, R2 here includes -D1 or -Am-D2.

[0127] Preferably, drug (D) is a drug whose free end has -NH2 or -NHR', where R' is C1-6 It is an alkyl group (for example, a methyl group), and R2 is D1, and D1 is a structure selected from Table E1 below. TIFF2026510850000039.tif253165TIFF2026510850000040.tif76166 Preferably, if the free end of D is a drug having -OH, then R2 is -Am-D2, Here, the structure of -Am- is, The filename is TIFF2026510850000041.tif15133, and D2 is a structure selected from Table E2 below.

[0128] TIFF2026510850000042.tif221165 In a preferred embodiment of this example, R1 is selected from the structures shown in Table C1, or R1 is Formed by a click reaction with TIFF2026510850000043.tif10132 Selected from the TIFF2026510850000044.tif13132 structure, Here, if Ra is selected from the structures shown in Table D1 (see above), Rb is selected from compounds containing the groups shown in Table D2 (see above).

[0129] In a specific embodiment, R1 is selected from Table B2 (see above).

[0130] In a preferred embodiment, the drug is selected from Table A1 below.

[0131] TIFF2026510850000045.tif230165TIFF2026510850000046.tif255158TIFF2026510850000047.tif255160TIFF2026510850000048.tif249166TIFF2026510850000049.tif237164TIFF2026510850000050.tif118165 Example 3. Preparation method for tumor-targeting drugs 1. Synthesis of compound R1-Gly-Gly-Phe-Leu-N(H)-D A typical preparation method is as shown in synthesis scheme 1. TIFF2026510850000051.tif11146 synthesis method: Compound R1-Gly-Gly-Phe-Leu-OH (1.0 eq) is dissolved in an appropriate amount of DMF solvent, then D(NH2 / NH) (i.e., a drug having an -NH2 or -NH- group, 1.0 eq) and the condensing agent HBTU (1.2 eq) are added sequentially, DIPEA (2.0 eq) is added to the reaction mixture under an ice bath, then the mixture is slowly returned to room temperature and stirred continuously for 3 to 5 hours. Most of the solvent is removed from the resulting reaction mixture under high vacuum, then diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to obtain the target product. The compounds shown in Table A2 below can be obtained according to the above method.

[0132] TIFF2026510850000052.tif247165TIFF2026510850000053.tif253166TIFF2026510850000054.tif252165TIFF2026510850000055.tif671662. Synthesis of compound R1-Gly-Gly-Phe-Leu-AM-D The synthesis route is as shown in Synthesis Scheme 2.

[0133] Synthesis scheme 2: Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-Gly-OH (TIFF2026510850000056.tif41161) Dissolve Fmoc-Gly-Gly-Phe-Leu-Gly-OH (1.0 eq) in DMF, cool to 0°C, and under nitrogen gas protection, add H-Gly-OtBu (1.0 eq), HBTU (1.0 eq), and DIPEA (2.0 eq), and stir at room temperature for 2-3 hours. Monitor the completion of the reaction by TLC. Dilute the reaction mixture with dichloromethane, wash sequentially with water, separate the aqueous phase with dichloromethane, combine with the organic phase, dry over anhydrous sodium sulfate, spin dry, and perform column chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-Gly-OtBu.

[0134] The crude product is dissolved in DCM, TFA / DCM (20%) is added, and the mixture is stirred at room temperature for 2-3 hours. The completion of the reaction is monitored by TLC. The reaction mixture is concentrated and separated by preparative chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-Gly-OH.

[0135] 1) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-AM-OAc (where -AM- is replaced by -NH-CH2-) Dissolve Fmoc-Gly-Gly-Phe-Leu-Gly-OH (1.0 eq) in dichloromethane / tetrahydrofuran (15 / 5 mL), cool to 0°C, add lead acetate (1.2 eq) and copper acetate (0.2 eq) under nitrogen gas protection, and stir at 40-50°C for 2-3 hours under nitrogen gas protection. Monitor the completion of the reaction by TLC. Dilute the reaction solution with dichloromethane, wash sequentially with water, separate the aqueous phase with dichloromethane, combine with the organic phase, dry over anhydrous sodium sulfate, spin dry, and perform column chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-AM-Oac.

[0136] 2) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-AM-O-Da (where -AM-O-Da is -AM-D2) TIFF2026510850000059.tif20153 Compound Fmoc-Gly-Gly-Phe-Leu-AM-OAc (1.0 eq) is added to a three-necked bottle, compound D(OH) (i.e., a drug with an -OH group, 2.0 eq) is added, and TFA (0.3 eq) is added under nitrogen gas protection and in an ice bath, and the mixture is stirred at room temperature for 2-3 hours. After the reaction is complete, the reaction mixture is concentrated and separated by high-pressure preparative chromatography to obtain a white solid Fmoc-Gly-Gly-Phe-Leu-AM-O-Da.

[0137] 3) Synthesis of compound H-Gly-Gly-Phe-Leu-AM-O-Da Compound Fmoc-Gly-Gly-Phe-Leu-AM-O-Da (1.0 eq) is dissolved in DMF, piperidine (2.0 eq) is added, and the mixture is stirred at room temperature for 2-3 hours under nitrogen gas protection. The completion of the reaction is monitored by TLC. The reaction mixture is redissolved, the solution is filtered, and then prepared by high-pressure liquid chromatography to obtain compound H-Gly-Gly-Phe-Leu-AM-O-Da as a solid product.

[0138] 4) Synthesis of compound R1-Gly-Gly-Phe-Leu-AM-O-Da TIFF2026510850000061.tif18163 Compound R1-OH (1.0 eq) is dissolved in DMF (9 / 1 10 mL), H-Gly-Gly-Phe-Leu-AM-D2 (1.0 eq) is added, and HBTU (1.2 eq) and DIPEA (2.0 eq) are added under nitrogen gas protection, and the mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound R1-Gly-Gly-Phe-Leu-AM-D2 (e.g., shown in Table A3) as a solid product.

[0139] TIFF2026510850000062.tif250165TIFF2026510850000063.tif255163TIFF2026510850000064.tif311653. Synthesis of compounds S1, S2, S3, S4 and S5 3.1. Synthesis of compound S1: (TIFF2026510850000065.tif441491) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-OH TIFF2026510850000066.tif35161 Add H-Phe-Leu-OH (1.0 eq) and NaHCO3 (5.0 eq) to a three-necked bottle, suspend in THF / H2O (4 / 1), and stir for 30 minutes under ice bath and nitrogen gas protection. Then add compound Fmoc-Gly-Gly-OSu (1.0 eq) in batches and stir at room temperature for 5-6 hours. Acidify the reaction mixture with dilute hydrochloric acid, extract the resulting crude product with the organic solvent siRNA, separate, dry, remove most of the organic solvent using a rotary evaporator, and slurry the residue with the organic solvent MTBE to obtain compound Fmoc-Gly-Gly-Phe-Leu-OH as a white solid.

[0140] 2) Synthesis of compound H-Gly-Gly-Phe-Leu-OH TIFF2026510850000067.tif37155Fmoc-Gly-Gly-Phe-Leu-OH (1.0 eq) is dissolved in DMF, piperidine (5.0 eq) is added, and the mixture is stirred at room temperature for 2 hours. The solvent is removed under reduced pressure using an oil pump, the residue is poured into water, filtered, and the solid is dried. The resulting crude product is slurryed with the organic solvent MTBE to obtain H-Gly-Gly-Phe-Leu-OH as an off-white solid.

[0141] 3) Synthesis of compound EMC-Gly-Gly-Phe-Leu-OH TIFF2026510850000068.tif30165 Add H-Gly-Gly-Phe-Leu-OH (1.0 eq), NaHCO3 (5.0 eq), and solvent THF / H2O (4 / 1) to a three-necked bottle and stir for 30 minutes under ice bath and nitrogen gas protection. Then add compound EMC-OSu (1.0 eq) in batches and stir at room temperature for 10-13 hours. Acidify the reaction mixture with dilute hydrochloric acid, extract the resulting crude product with the organic solvent siRNA, separate the solution, dry it, remove most of the organic solvent using a rotary evaporator, and slurry the residue with the organic solvent MTBE to obtain compound EMC-Gly-Gly-Phe-Leu-OH as a white solid.

[0142] 4) Synthesis of compound S1 TIFF2026510850000069.tif24150 Add EMC-Gly-Gly-Phe-Leu-OH (1.0 eq) and DEPBT (1.2 eq) to a three-necked bottle, dissolve in DMF solvent, and stir for 30 minutes under ice bath and nitrogen gas protection. Then, at 0°C, add the compounds doxorubicin (1.0 eq) and DIPEA (2.0 eq), respectively, and stir at room temperature for 10-13 hours. Remove most of the organic solvent using a rotary evaporator, and separate and purify the resulting crude product by high-pressure preparative chromatography to obtain a red solid powder.

[0143] 3.2. When the obtained toxin molecule is replaced with extecan or its derivatives DXd-NH2, T785, melphalan, or MMAE, compounds S2, S3, S4, and S5 can be prepared in the same manner.

[0144] 4. Synthesis of compounds S6, S13, S14, S15, and S16 4.1. Synthesis of compound S6: (TIFF2026510850000070.tif821651) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-Gly-OH Dissolve Fmoc-Gly-Gly-Phe-Leu-OH (1.0 eq) in DMF, cool to 0°C, and under nitrogen gas protection, add H-Gly-OtBu (1.0 eq), HBTU (1.0 eq), and DIPEA (2.0 eq), and stir at room temperature for 2-3 hours. Monitor the completion of the reaction by TLC. Dilute the reaction mixture with dichloromethane, wash sequentially with water, separate the aqueous phase with dichloromethane, combine with the organic phase, dry over anhydrous sodium sulfate, spin dry, and perform column chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-Gly-OtBu.

[0145] The crude product is dissolved in DCM, TFA / DCM (20%) is added, and the mixture is stirred at room temperature for 2-3 hours. The completion of the reaction is monitored by TLC. The reaction mixture is concentrated and separated by preparative chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-Gly-OH.

[0146] 2) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-OAc Dissolve Fmoc-Gly-Gly-Phe-Leu-Gly-OH (1.0 eq) in dichloromethane / tetrahydrofuran (15 / 5 mL), cool to 0°C, add lead acetate (1.2 eq) and copper acetate (0.2 eq) under nitrogen gas protection, and stir at 40-50°C for 2-3 hours under nitrogen gas protection. Monitor the completion of the reaction by TLC. Dilute the reaction solution with dichloromethane, wash sequentially with water, separate the aqueous phase with dichloromethane, combine with the organic phase, dry over anhydrous sodium sulfate, spin dry, and perform column chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-OAc.

[0147] 3) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000073.tif34165 Compound Fmoc-Gly-Gly-Phe-Leu-AM-OAc (1.0 eq) is added to a three-necked bottle, and DXd-OH (1.0 eq), a derivative of the compound extecan, is added and dissolved in dichloromethane solution. TFA (0.3 eq) is added under nitrogen gas protection and in an ice bath, and the mixture is stirred at room temperature for 2-3 hours. After the reaction is complete, the reaction mixture is concentrated and separated by high-pressure preparative chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-AM-DXd as a pale yellow solid.

[0148] Synthesis of compound H-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000074.tif38165 Compound Fmoc-Gly-Gly-Phe-Leu-AM-D (1.0 eq) is dissolved in DMF, piperidine (2.0 eq) is added, and the mixture is stirred at room temperature for 2-3 hours under nitrogen gas protection. The completion of the reaction is monitored by TLC. The reaction mixture is redissolved, the solution is filtered, and then subjected to high-pressure liquid chromatography to obtain compound H-Gly-Gly-Phe-Leu-AM-DXd as a solid product.

[0149] 4) Synthesis of compound S6 TIFF2026510850000075.tif31165 Compound EMC-OSu (1.0 eq) is dissolved in DMF (10 mL), H-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is added, and DIPEA (2.0 eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound S6 as a pale yellow solid product.

[0150] 4.2. When the obtained toxin molecules are replaced with paclitaxel, SN38, and gemcitabine, respectively, compounds S13, S14, S15, and S16 can be prepared in the same manner.

[0151] 5. Synthesis of compounds S7, S8, S9, and S10: 5.1. Synthesis of compound S7 (MI-PEG6-GGFL-AM-DXD): TIFF2026510850000076.tif63162 Compound MI-PEG6-OSu (1.0 eq) is dissolved in DMF (10 mL), H-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is added, and DIPEA (2.0 eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound S7 as a pale yellow solid product.

[0152] 5.2. When MI-PEG6-OSu is replaced with the corresponding branched chain structure, compounds S8, S9, and S10 can be prepared in a similar manner.

[0153] 6. Synthesis of compounds S11 and S12 6.1. Synthesis of compound S11: (TIFF2026510850000077.tif881651) Synthesis of compound Fmoc-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000078.tif40160 Compound Fmoc-Gly-Gly-Phe-Leu-AM-OAc (1.0 eq) is added to a three-necked bottle, compound paclitaxel (1.0 eq) is added and dissolved in dichloromethane solution, and TFA (0.3 eq) is added under nitrogen gas protection and ice bath, and the mixture is stirred at room temperature for 2-3 hours. After the reaction is complete, the reaction mixture is concentrated and separated by high-pressure preparative chromatography to obtain Fmoc-Gly-Gly-Phe-Leu-AM-Taxol as a white solid.

[0154] 2) Synthesis of compound H-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000079.tif39162 Add compound Fmoc-Gly-Gly-Phe-Leu-AM-Taxol (1.0 eq) to a three-necked bottle, dissolve in DMF, add piperidine (0.2 eq), and stir at room temperature for 2-3 hours under nitrogen gas protection. After the reaction is complete, concentrate the reaction mixture and separate by high-pressure preparative chromatography to obtain H-Gly-Gly-Phe-Leu-AM-Taxol as a white solid.

[0155] 3) Synthesis of compound H-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000080.tif75161 Compound Azid-PEG4-Diglycolic acid (1.0 eq) is dissolved in DMF (10 mL), H-Gly-Gly-Phe-Leu-AM-Taxol (1.0 eq) is added, and HBTU (1.2 eq) and DIPEA (2.0 eq) are added under ice bath and nitrogen gas protection, and the mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The reaction mixture is redissolved, the solution is filtered, and then subjected to high-pressure liquid chromatography to obtain the product of compound Azid-PEG4-Diglycolic-Gly-Gly-Phe-Leu-AM-Taxol as a white solid.

[0156] 4) Synthesis of compound S11 TIFF2026510850000081.tif63166 Compound Azid-PEG4-Diglycolic-Gly-Gly-Phe-Leu-AM-Taxol (1.0 eq) is dissolved in DMSO / H2O (9 / 1 10 mL), EMC-Lys(PEG5)-Yne (1.0 eq) is added, and CuBr (1.2 eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain the product of compound S11 as a white solid.

[0157] 6.2. When EMC-Lys(PEG5)-Yne is replaced with EMC-Lys(PEG8)-Yne, compound S12 can be prepared in a similar manner.

[0158] 7. Synthesis of compounds S17, S18, S30, and S34 7.1. Synthesis of compound S17: TIFF2026510850000082.tif34165 Compound H-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is dissolved in DMF (10 mL), lactobionic acid (2.0 eq) is added, and HBTU (1.2 eq) and DIPEA (2.0 eq) are added under ice bath and nitrogen gas protection, and the mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The reaction mixture is redissolved, the solution is filtered, and then subjected to high-pressure liquid chromatography to obtain compound S17 as a pale yellow solid product.

[0159] 7.2. When lactobionic acid LA is replaced with galactosamine GalNAc, S18 can be prepared in a similar manner.

[0160] 7.3. When lactobionic acid LA is replaced with BCN or DBCO, S30 and S34 can be prepared in the same manner.

[0161] 8. Synthesis of compounds S19 and S20 8.1. Synthesis of compound S19: (TIFF2026510850000083.tif1071651) Synthesis of compound Yne-PEG5-Diglycolic-Gly-Gly-Phe-Leu-AM-DXd TIFF2026510850000084.tif70166 Compound Yne-PEG5-Diglycolic acid (1.0 eq) is dissolved in DMF (10 mL), H-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is added, and HBTU (1.2 eq) and DIPEA (2.0 eq) are added under nitrogen gas protection, and the mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound Yne-PEG5-Diglycolic-Gly-Gly-Phe-Leu-AM-DXd as a pale yellow solid product.

[0162] 2) Synthesis of compound S19 TIFF2026510850000085.tif76165 Compound CyRGDfK(N3)(1.0eq) is dissolved in DMSO / H2O (9 / 1 10 mL), Yne-PEG5-Diglycolic-Gly-Gly-Phe-Leu-AM-DXd(1.0eq) is added, and CuBr(1.2eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound S19 as a pale yellow solid product.

[0163] 8.2. When replacing CyRGDfK(N3) with CyRGDyK(N3), compound S20 can be prepared in a similar manner.

[0164] 9. Synthesis of compounds S21 and S22 9.1. Synthesis of compound S21: Synthesis of compound Yne-PEG5-Gly-Gly-Phe-Leu-AM-DXd (TIFF2026510850000086.tif1041601) TIFF2026510850000087.tif74159 Compound Yne-PEG5-OH (1.0 eq) is dissolved in DMF (10 mL), H-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is added, and HBTU (1.2 eq) and DIPEA (2.0 eq) are added under nitrogen gas protection, and the mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound Yne-PEG5-Gly-Gly-Phe-Leu-AM-D as a pale yellow solid product.

[0165] 2) Synthesis of compound S21 TIFF2026510850000088.tif68165 Compound CyRGDfK(N3)(1.0eq) is dissolved in DMSO / H2O (9 / 1 10mL), Yne-PEG5-Gly-Gly-Phe-Leu-AM-DXd(1.0eq) is added, and CuBr(1.2eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The crude product of the reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound S21 as a pale yellow solid product.

[0166] 9.2. When replacing CyRGDfK(N3) with CyRGDyK(N3), compound S22 can be prepared in a similar manner.

[0167] 10. Synthesis of compounds S23, S24, and S25 10.1. Synthesis of Compound S23 (TIFF2026510850000089.tif781651) Synthesis of Compound 3 Compound 1 (10.0 g, 1.0 eq) was dissolved in DCM, BTC (0.33 eq) was added at -78°C, and after 2 hours, Compound 2 (1.1 eq) and triethylamine (3.4 eq) were added. The mixture was stirred at room temperature for 16 hours, water was added to quench the mixture, and the mixture was extracted with ethyl acetate. The organic solvent was removed using a rotary evaporator, the sample was stirred, and the mixture was subjected to column chromatography (PE / siRNA = 60:40) to obtain Compound 3 (10 g) as a white solid.

[0168] 2) Synthesis of Compound 4 Compound 3 (10 g, 1.0 eq) was dissolved in DCM, Pd / C (2 g) was added at room temperature, the mixture was stirred overnight at room temperature, the reaction mixture was filtered to remove Pd / C, the sample was stirred, and the mixture was subjected to column chromatography (DCM / MeOH = 92 / 8) to obtain Compound 4 (6.5 g) as an off-white solid.

[0169] 3) Synthesis of Compound 5 Compound 4 (720 mg, 1.0 eq) and azidoacetic acid (1.2 eq) were dissolved in DMF, DIPEA (1.2 eq) and HBTU (1.2 eq) were added at room temperature, and the mixture was stirred overnight at room temperature. The reaction mixture was quenched with sat.NaHCO3 solution, extracted with ethyl acetate, the sample was stirred, and subjected to column chromatography (PE / SiO=1 / 1) to obtain (870 mg) of a colorless liquid.

[0170] 4) Synthesis of compound 6 Compound 6 (150 mg) was dissolved in formic acid (1 mL), stirred at room temperature for 2 hours, and the reaction was confirmed to be complete by LC-MS. The solvent was spin-dried, and the compound was separated by direct high-pressure preparative chromatography to obtain compound 6 (70 mg) as the product.

[0171] 5) Synthesis of compound S23 TIFF2026510850000094.tif64165 Compound 6 (1.0 eq) is dissolved in DMSO / H2O (9 / 1 10 mL), Yne-PEG5-Gly-Gly-Phe-Leu-AM-DXd (1.0 eq) is added, and CuBr (1.2 eq) is added under nitrogen gas protection. The mixture is stirred at room temperature for 1-2 hours. The completion of the reaction is monitored by TLC. The reaction mixture is filtered and then subjected to high-pressure liquid chromatography to obtain compound S23 as a pale yellow solid product.

[0172] 10.2. When the drug toxin molecule exetecan derivative (DXd-OH) in the above compounds is replaced with paclitaxel and docetaxel, respectively, compounds S24 and S25 can be prepared in the same manner.

[0173] 11. Synthesis of compound S26: Synthesis of the compound Br-MI-Gly-Gly-Phe-Leu-OH (TIFF2026510850000095.tif701631) TIFF2026510850000096.tif30155 Add H-Gly-Gly-Phe-Leu-OH (1.0 eq), NaHCO3 (5.0 eq), and solvent THF / H2O (4 / 1) to a three-necked bottle and stir for 30 minutes under ice bath and nitrogen gas protection. Then add compound Br-MI-OSu (1.0 eq) in batches and stir at room temperature for 10-13 hours. Acidify the reaction mixture with dilute hydrochloric acid, extract the resulting crude product with the organic solvent ELISA, separate the solution, dry it, remove most of the organic solvent using a rotary evaporator, and slurry the residue with the organic solvent MTBE to obtain compound Br-MI-Gly-Gly-Phe-Leu-OH as a white solid.

[0174] 2) Synthesis of compound S26 TIFF2026510850000097.tif33157 Add Br-MI-Gly-Gly-Phe-Leu-OH (1.0 eq) and DEPBT (1.2 eq) to a three-necked bottle, dissolve in DMF solvent, and stir for 30 minutes under ice bath and nitrogen gas protection. Then, at 0°C, add compound extecan (1.0 eq) and DIPEA (3.0 eq), respectively, and stir at room temperature for 10-13 hours. Remove most of the organic solvent using a rotary evaporator, and separate and purify the resulting crude product by high-pressure preparative chromatography to obtain a pale yellow solid powder.

[0175] 12. Synthesis of compounds S27, S28, and S29: 12.1. Synthesis of compound S27: Synthesis of compound BCN-PEG3-Gly-Gly-Phe-Leu-OH (TIFF2026510850000098.tif561651) TIFF2026510850000099.tif28166 Add H-Gly-Gly-Phe-Leu-OH (1.0 eq), NaHCO3 (5.0 eq), and solvent THF / H2O (4 / 1) to a three-necked bottle and stir for 30 minutes under ice bath and nitrogen gas protection. Then add compound BCN-PEG3-OSu (1.0 eq) in batches and stir at room temperature for 10-13 hours. Acidify the reaction mixture with dilute hydrochloric acid, extract the resulting crude product with the organic solvent ELISA, separate the solution, dry it, remove most of the organic solvent using a rotary evaporator, and slurry the residue with the organic solvent MTBE to obtain compound BCN-PEG3-Gly-Gly-Phe-Leu-OH as a white solid.

[0176] 2) Synthesis of compound S27 TIFF2026510850000100.tif28165 Add BCN-PEG3-Gly-Gly-Phe-Leu-OH (1.0 eq) and DEPBT (1.2 eq) to a three-necked bottle, dissolve in DMF solvent, and stir for 30 minutes under ice bath and nitrogen gas protection. Then, at 0°C, add compound T785 (1.0 eq) and DIPEA (2.0 eq), respectively, and stir at room temperature for 10-13 hours. Remove most of the organic solvent using a rotary evaporator, and separate and purify the resulting crude product by high-pressure preparative chromatography to obtain a white solid powder.

[0177] 12.2.When T785 is replaced with the extecan derivatives DXd-NH2 and MMAE, compounds S28 and S29 can be prepared in the same manner.

[0178] 13. Synthesis of compounds S31, S32, and S33: 13.1. Synthesis of Compound S31 Synthesis of compound DBCO-PEG3-Gly-Gly-Phe-Leu-OH (TIFF2026510850000101.tif501651) TIFF2026510850000102.tif24165 Add H-Gly-Gly-Phe-Leu-OH (1.0 eq), NaHCO3 (5.0 eq), and solvent THF / H2O (4 / 1) to a three-necked bottle and stir for 30 minutes under ice bath and nitrogen gas protection. Then add compound DBCO-PEG3-OSu (1.0 eq) in batches and stir at room temperature for 10-13 hours. Acidify the reaction mixture with dilute hydrochloric acid, extract the resulting crude product with the organic solvent ELISA, separate the solution, dry it, remove most of the organic solvent using a rotary evaporator, and slurry the residue with the organic solvent MTBE to obtain compound DBCO-PEG3-Gly-Gly-Phe-Leu-OH as a white solid.

[0179] 2) Synthesis of compound S31 TIFF2026510850000103.tif25166 Add EMC-Gly-Gly-Phe-Leu-OH (1.0 eq) and DEPBT (1.2 eq) to a three-necked bottle, dissolve in DMF solvent, and stir for 30 minutes under ice bath and nitrogen gas protection. Then, at 0°C, add compound T785 (1.0 eq) and DIPEA (2.0 eq), respectively, and stir at room temperature for 10-13 hours. Remove most of the organic solvent using a rotary evaporator, and separate and purify the resulting crude product by high-pressure preparative chromatography to obtain a white solid powder.

[0180] 13.2. When T785 is replaced with the extecan derivatives DXd-NH2 and MMAE, compounds S32 and S33 can be prepared in the same manner.

[0181] 14. Synthesis of Leu-AM-DXd: TIFF2026510850000104.tif84166 Step 1: Synthesis of compound Fmoc-Leu-OAc Dissolve Fmoc-Leu-Gly-OH (1.0 eq) in dichloromethane / tetrahydrofuran (15 / 5 mL), cool to 0°C, add lead acetate (1.2 eq) and copper acetate (0.2 eq) under nitrogen gas protection, and stir at 40-50°C for 2-3 hours under nitrogen gas protection. Monitor the completion of the reaction by TLC. Dilute the reaction solution with dichloromethane, wash sequentially with water, separate the aqueous phase with dichloromethane, combine with the organic phase, dry over anhydrous sodium sulfate, spin dry, and perform column chromatography to obtain Fmoc-Leu-OAc.

[0182] Step 2: Synthesis of compound Fmoc-Leu-AM-DXd TIFF2026510850000106.tif43147 Add compound Fmoc-Leu-AM-OAc (1.0 eq) to a three-necked bottle, add compound DXd (1.0 eq), dissolve in dichloromethane solution, add TFA (0.3 eq) under nitrogen gas protection and ice bath, and stir at room temperature for 2-3 hours. After the reaction is complete, concentrate the reaction mixture and separate by high-pressure preparative chromatography to obtain Fmoc-Leu-AM-DXd as a pale yellow solid.

[0183] Step 3: Synthesis of compound Leu-AM-DXd TIFF2026510850000107.tif44151 Compound Fmoc-Leu-AM-D (1.0 eq) is dissolved in tetrahydrofuran solution, DBU (1.0 eq) is added, and the mixture is stirred at room temperature for 2-3 hours under nitrogen gas protection. The completion of the reaction is monitored by TLC. The reaction mixture is redissolved in methanol, the solution is filtered, and then subjected to high-pressure liquid chromatography to obtain compound Leu-AM-DXd as a yellow solid product.

[0184] Example 4. Determination of DXD One method for testing whether a coupling drug is enzymatically digested by Cathepsin is to add Cathepsin to the coupling drug, initiate the enzymatic digestion reaction, and simultaneously detect the content of each substance in the mixture to determine the rate and progress of the enzymatic digestion reaction.

[0185] Test conditions 1.1. Preparation of control solution: Precisely weigh approximately 25 mg of DXD control, place it in a 5 mL volumetric flask, add DMSO to the mark, mix uniformly to prepare a 10 mM solution, mark it as the standard stock solution, take the standard stock solution, and prepare standard solutions at concentrations of 20 nmol / mL, 10 nmol / mL, 5 nmol / mL, 2 nmol / mL, 1 nmol / mL, and 0.2 nmol / mL. Inject 10 μL of each sample into an HPLC, record the peak area, plot the peak area on the vertical axis and the concentration on the horizontal axis, and calculate the linear equation.

[0186] 1.2. Chromatography conditions: Chromatography column: Agilent Eclipse Plus C18 5um, 4.6 × 250 mm Fluorescence detector: Em=380nm, Ex=460nm Mobile phase A:0.1%TFA / H2OB:ACN Flow rate: 1mL / min Injection volume: 10uL TIFF2026510850000108.tif88165 Example 5. L-AM-DXD that is not enzymatically digested by cathepsin L 1. Preparation of experimental solutions: Detection buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w / v) Brij-35, pH 6.0.

[0187] Cathepsin L: Incubate in detection buffer at 100 ug / mL on ice for 15 minutes.

[0188] Preparation of sample solution: Weigh an appropriate amount of L-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL and add it to 80 uL of DMSO to dilute it to 1 umol / mL, and then add purified water in a 1:4 ratio to dilute the sample solution to a concentration of 0.2 umol / mL.

[0189] 2. Enzyme digestion reaction: 50 μL of sample solution was taken, and 150 μL of incubated cathepsin L solution was added in a volume ratio of 1:3 (v:v). Enzymatic digestion was performed at 37°C. Another 50 μL of sample solution was taken, and 150 μL of detection buffer solution was added in a volume ratio of 1:3 (v:v). 30 μL of each solution was transferred to a sample vial and injected at 0, 2, 4, and 20 hours. The HPLC method was as described in 1.2.

[0190] 3. Experimental Results: The experimental results are shown in Figure 1.

[0191] 4. Conclusion: After treatment with cathepsin L for 20 hours, the DXD produced by L-AM-DXD (calculated according to the DXD standard curve external standard method) was only 1.45% of the reaction substrate L-AM-DXD, indicating that it was hardly enzymatically digested, which shows that L-AM-DXD is not enzymatically digested by cathepsin L.

[0192] In Examples 6 to 13, GGFL-AM-DXD and MI-PEG6-GGFL-AM-DXD are used interchangeably, and GGFG-AM-DXD and EMC-GGFG-AM-DXD are used interchangeably.

[0193] Example 6 Linker screening experiments revealed that MI-PEG6-GGFL-AM-DXD(S7) is enzymatically digested by cathepsin L, suggesting that GGF bound to L is recognized and enzymatically digested by cathepsin L. Comparative results showed that its activation efficiency was superior to that of EMC-GGFG-AM-DXD.

[0194] 1. Preparation of experimental solutions: Detection buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w / v) Brij-35, pH 6.0.

[0195] Cathepsin L: Incubate in detection buffer at 100 ug / mL on ice for 15 minutes.

[0196] Preparation of sample solutions: Weigh appropriate amounts of EMC-GGFG-AM-DXD (purchased from Shanghai Chemexpress) and MI-PEG6-GGFL-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL and add it to 80 uL of DMSO to dilute to 1 umol / mL, and further dilute the sample solution mp concentration to 0.2 umol / mL by adding purified water in a 1:4 ratio.

[0197] 2. Enzyme digestion reaction: 50 μL of sample solution was taken, and 150 μL of incubated cathepsin L solution was added in a volume ratio of 1:3 (v:v). Enzymatic digestion was performed at 37°C. Another 50 μL of sample solution was taken, and 150 μL of detection buffer solution was added in a volume ratio of 1:3 (v:v). 30 μL of each solution was transferred to a sample vial and injected at 0, 2, 4, and 20 hours. The HPLC method was as described in 1.2.

[0198] 3. Experimental Results: The results are shown in Figures 2 and 3.

[0199] 4. Conclusion: After treatment with cathepsin L for 20 hours, the DXD produced by MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD (calculated according to the DXD standard curve external standard method) was calculated. The amount of DXD produced by the enzymatic digestion of MI-PEG6-GGFL-AM-DXD accounted for 98.3% of the reaction substrate, i.e., the enzymatic digestion activation efficiency was 98.3%. The amount of DXD produced by the enzymatic digestion of EMC-GGFG-AM-DXD accounted for 27.8% of the reaction substrate, i.e., the enzymatic digestion activation efficiency was 27.8%. This indicates that MI-PEG6-GGFL-AM-DXD is more easily digested by cathepsin L than EMC-GGFG-AM-DXD, and that L-AM-DXD is not enzymatically digested by cathepsin L, but rather is enzymatically digested by cathepsin L after being bound to GGF.

[0200] The results of this enzyme digestion experiment show that compounds S1 to S34 of the present invention all achieved an enzyme digestion efficiency of over 90% within 20 hours, significantly exceeding that of the control compound EMC-GGFG-AM-DXd. Specific data are shown in Table F1.

[0201] TIFF2026510850000109.tif224165 Example 7 Linker screening experiments revealed that MI-PEG6-GGFL-AM-DXD is not enzymatically digested by cathepsin B, while EMC-GGFG-AM-DXD is activated by cathepsin B. This indicates that cathepsin L is the only enzyme that activates MI-PEG6-GGFL-AM-DXD.

[0202] 1. Preparation of experimental solutions: Activation buffer: 25 mM MES, 5 mM DTT, pH 5.0.

[0203] Detection buffer: 25 mM MES, pH 5.0.

[0204] Cathepsin B: Incubate in 100 ug / mL of activation buffer at room temperature for 15 minutes.

[0205] Preparation of sample solutions: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL of each and add it to 80 uL of DMSO to dilute to 1 umol / mL, and then add purified water in a 1:4 ratio to dilute the sample solution to a concentration of 0.2 umol / mL.

[0206] 2. Enzyme digestion reaction: 50 μL of sample solution was taken, and 150 μL of incubated cathepsin B solution was added in a volume ratio of 1:3 (v:v). Enzymatic digestion was performed at 37°C. Another 50 μL of sample solution was taken, and 150 μL of detection buffer solution was added in a volume ratio of 1:3 (v:v). 30 μL of each solution was transferred to a sample vial and injected at 0, 2, 4, and 20 hours. The HPLC method was as described in 1.2 of Example 4.

[0207] 3. Experimental Results: The experimental results are shown in Figures 4 and 5.

[0208] 4. Conclusion: After treating with cathepsin B for 20 hours, the DXD produced by MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD (calculated according to the DXD standard curve external standard method) was compared. The amount of DXD produced by the enzymatic digestion of MI-PEG6-GGFL-AM-DXD accounted for 3.75% of the reaction substrate, while the amount of DXD produced by the enzymatic digestion of EMC-GGFG-AM-DXD accounted for 45.2% of the reaction substrate and also contained some GFG-AM-DXD fragments. This indicates that cathepsin B is more sensitive to EMC-GGFG-AM-DXD and less sensitive to MI-PEG6-GGFL-AM-DXD, and that cathepsin L is the only enzyme that activates MI-PEG6-GGFL-AM-DXD.

[0209] Example 8 Stability experiments in human plasma of MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD showed that MI-PEG6-GGFL-AM-DXD is more stable than EMC-GGFG-AM-DXD. 1. Preparation of experimental solutions: PBS buffer: PH=7.2 Protein precipitant: DMSO / MEOH = 1:1 Preparation of sample solution: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL and add it to 80 uL of DMSO to dilute to 1 umol / mL, and further dilute the sample solution to 0.2 umol / mL by adding purified water in a 1:4 ratio.

[0210] 2. Stability in human plasma: An appropriate amount of sample was weighed and prepared as a 1 mM solution with DMSO. Human plasma was added in a volume ratio of 1:9, and the mixture was incubated in a 37°C incubator. Samples were taken at 0, 2, 4, 6, and 20 hours, and DMSO / MEOH (1:1) was added in a volume ratio of 1:3 to remove proteins. The mixture was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. The HPLC method was as described in 1.2 of Example 4.

[0211] 3. Experimental Results: The experimental results are shown in Figures 6 and 7.

[0212] 4. Conclusion: DXD (calculated according to the DXD standard curve external standard method) generated by incubating MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD in human plasma for 20 hours was calculated. The DXD generated by MI-PEG6-GGFL-AM-DXD(S7) and EMC-GGFG-AM-DXD accounted for 1.75% and 2.88% of the substrate, respectively. That is, the substrate content was 98.3% and 97.1%, respectively, indicating that MI-PEG6-GGFL-AM-DXD has superior stability.

[0213] The results of this plasma stability experiment show that the substrate content of the compound of the present invention can reach 98% or more, or even 100%, within 20 hours (i.e., the generated payload ≤ 98%, or the generated payload is undetectable), indicating good stability in the linkage performance between the linker and the payload. Here, the payload is the base of a cytotoxic drug or cytotoxic-carrying drug such as DXD. Specific data are shown in Table F2.

[0214] TIFF2026510850000110.tif236165 Example 9 MI-PEG6-GGFL-AM-DXD monkey heart homogenate exhibits superior stability compared to GGFG-AM-DXD. 1. Preparation of experimental solutions: Detection buffer: 25 mM MES, pH 5.0.

[0215] Protein precipitant: DMSO / MEOH = 1:1 Preparation of sample solution: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL and add it to 80 uL of DMSO to dilute to 1 umol / mL, and further dilute the sample solution to 0.2 umol / mL by adding purified water in a 1:4 ratio.

[0216] Preparation of cardiac homogenate: A suitable amount of heart rapidly frozen with liquid nitrogen is collected, placed in a 4 mL EP tube, weighed, and detection buffer with pH 5.0 added in a mass-to-volume ratio of 1:5. The tissue is homogenized for 3 minutes using a tissue grinder, then the cardiac homogenate is centrifuged at 12,000 rpm for 5 minutes, the supernatant is collected, and stored in a refrigerator at -40°C.

[0217] 2. Stability of cardiac homogenates: Each sample solution and cardiac homogenate were thoroughly shaken in a volume ratio of 1:9, and another sample solution and buffer (pH 5.0) were also thoroughly shaken in a volume ratio of 1:9 to be used as a blank control solution. These were placed in a constant temperature water bath at 37°C, and 30 μL samples were taken at 0, 2, 4, and 20 hours. 90 μL of protein precipitant was added and the samples were shaken uniformly. The samples were then centrifuged at 12000 rpm for 5 minutes, the supernatant was collected and transferred to a sample vial, and injected. The HPLC method was as described in 1.2 of Example 4.

[0218] 3. Experimental Results: The experimental results are shown in Figures 8 and 9.

[0219] 4. Conclusion: After placing MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD in monkey heart homogenate for 20 hours, the generated DXD (calculated according to the external standard method of the DXD standard curve) was calculated. The DXD generated by MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD accounted for 0% and 7.41% of the substrate respectively. It can be seen that MI-PEG6-GGFL-AM-DXD was not released into the heart homogenate, while EMC-GGFG-AM-DXD was partially released, indicating that MI-PEG6-GGFL-AM-DXD did not show cardiotoxicity.

[0220] Example 10 MI-PEG6-GGFL-AM-DXD shows high stability in monkey liver homogenate and is superior to EMC-GGFG-AM-DXD. 1. Preparation of experimental solutions: Detection buffer: 25 mM MES, pH 5.0.

[0221] Protein precipitant: DMSO / MEOH = 1:1 Preparation of sample solution: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL. Then take 10 uL and add it to 80 uL of DMSO to dilute it to 1 umol / mL. Further add purified water in a ratio of 1:4 to dilute the concentration of the sample solution to 0.2 umol / mL.

[0222] Preparation of liver homogenate: Collect an appropriate amount of liver rapidly frozen with liquid nitrogen, put it into a 4 mL EP tube, weigh it, add detection buffer with pH 5.0 at a mass-volume ratio of 1:5, use a tissue grinder to homogenize the tissue for 3 minutes, then centrifuge the liver homogenate at 12000 rpm for 5 minutes, collect the supernatant, and store it in a refrigerator at -40 °C for reserve.

[0223] 2. Stability of liver homogenate: Respectively collect the sample solution and liver homogenate, shake well at a volume ratio of 1:9, and also shake well another sample solution and buffer (pH 5.0) at a volume ratio of 1:9 for use as a blank control solution. Place them in a constant temperature water bath at 37 °C, collect 30 μL at the time points of 0 hour, 2 hours, 4 hours, and 20 hours respectively, add 90 μL of protein precipitant, shake well uniformly, centrifuge at 12,000 rpm for 5 minutes, collect the supernatant, transfer it to a sample vial, inject it, and the HPLC method is as described in 1.2 in Example 4.

[0224] 3. Experimental results: The experimental results are as shown in Figures 10 and 11.

[0225] 4. Conclusion: After MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD were placed in liver homogenate for 20 hours, the amount of DXD generated by EMC-GGFG-AM-DXD accounted for 27.6% of the substrate, and the amount of DXD generated by MI-PEG6-GGFL-AM-DXD accounted for 5.7% of the substrate, indicating that GGFL-AM-DXD is more stable than GGFG-AM-DXD in liver homogenate.

[0226] Example 11 GGFL-AM-DXD shows a high activation efficiency in human gastric cancer homogenate and is superior to GGFG-AM-DXD. 1. Preparation of experimental solutions: Detection buffer: 25 mM MES, pH 5.0.

[0227] Protein precipitant: DMSO / MEOH = 1:1 Preparation of sample solution: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 μmol / mL, then take 10 μL and add it to 80 μL of DMSO to dilute to 1 μmol / mL, and further add purified water at a ratio of 1:4 to dilute the concentration of the sample solution to 0.2 μmol / mL.

[0228] Preparation of tumor homogenate 1: A suitable amount of tumor tissue is rapidly frozen with liquid nitrogen, placed in a 4 mL EP tube, weighed, and detection buffer with pH 5.0 added in a mass-to-volume ratio of 1:5. The tissue is homogenized for 3 minutes using a tissue grinder, and then the tumor homogenate is centrifuged at 12,000 rpm for 5 minutes. The supernatant is collected and stored in a refrigerator at -40°C.

[0229] 2. Activation status of tumor homogenate 1: Each sample solution and tumor homogenate 1 were collected and shaken thoroughly in a volume ratio of 1:9. Another sample solution and buffer (pH 5.0) were also shaken thoroughly in a volume ratio of 1:9 and used as a blank control solution. These were placed in a constant temperature water bath at 37°C, and 30 μL was collected at 0, 2, 4, and 20 hours. 90 μL of protein precipitant was added and shaken uniformly, and the mixture was centrifuged at 12000 rpm for 5 minutes. The supernatant was collected, transferred to a sample vial, and injected. The HPLC method was as described in 1.2 of Example 4.

[0230] 3. Experimental Results: The experimental results are shown in Figures 12 and 13.

[0231] 4. Conclusion: After GGFL-AM-DXD and GGFG-AM-DXD were placed in tumor homogenate 1 for a certain period of time, the amount of DXD produced by GGFG-AM-DXD accounted for 29.1% of the substrate after 48 hours, while the amount of DXD produced by GGFL-AM-DXD reached 94.2% after 20 hours. This indicates that GGFL-AM-DXD is released more readily than GGFG-AM-DXD in tumor homogenate 1.

[0232] Example 12 GGFL-AM-DXD shows high activation efficiency in CT26 tumor homogenates, outperforming GGFG-AM-DXD. 1. Preparation of experimental solutions: Detection buffer: 25 mM MES, pH 5.0.

[0233] Protein precipitant: DMSO / MEOH = 1:1 Preparation of sample solution: Weigh appropriate amounts of MI-PEG6-GGFL-AM-DXD and EMC-GGFG-AM-DXD, add a certain amount of DMSO solution to make the stock solution concentration 10 umol / mL, then take 10 uL and add it to 80 uL of DMSO to dilute to 1 umol / mL, and further dilute the sample solution to 0.2 umol / mL by adding purified water in a 1:4 ratio.

[0234] Preparation of tumor homogenate 2: A suitable amount of tumor tissue is rapidly frozen with liquid nitrogen, placed in a 4 mL EP tube, weighed, and detection buffer with pH 5.0 added in a mass-to-volume ratio of 1:5. The tissue is homogenized for 3 minutes using a tissue grinder, and then the tumor homogenate is centrifuged at 12,000 rpm for 5 minutes. The supernatant is collected and stored in a refrigerator at -40°C.

[0235] 2. Activation status of tumor homogenate 2: Each sample solution and tumor homogenate 2 were collected and thoroughly shaken in a volume ratio of 1:9. Another sample solution and buffer (pH 5.0) were also thoroughly shaken in a volume ratio of 1:9 and used as a blank control solution. These were placed in a constant temperature water bath at 37°C, and 30 μL samples were collected at 0, 2, 4, and 20 hours. 90 μL of protein precipitant was added and the samples were shaken uniformly. The samples were then centrifuged at 12000 rpm for 5 minutes, the supernatant was collected and transferred to a sample vial, and injected. The HPLC method was as described in 1.2.

[0236] 3. Experimental Results: The experimental results are shown in Figures 14 and 15.

[0237] 4. Conclusion: After GGFL-AM-DXD and GGFG-AM-DXD were placed in tumor homogenate 2 for a certain period of time, the amount of DXD produced by GGFG-AM-DXD accounted for 42.5% of the substrate after 20 hours, while the amount of DXD produced by GGFL-AM-DXD reached 98.7% after 20 hours. This indicates that GGFL-AM-DXD is released more readily than GGFG-AM-DXD in tumor homogenate 2.

[0238] Example 13. Antibody-drug conjugate 1. General preparation methods for antibody-drug conjugates Antibody target points include HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylyl cyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1. Selected from PDL1, HER2, NY-ESO-1, BCMA, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC1, and MAGE-A3.

[0239] Add TCEP (tris(2-carbonylethyl)phosphine hydrochloride, 40 e.q.) to any of the above antibodies and reduce for 8 hours. Then, add one of the compounds S1 to S25 prepared in Examples 1 to 3 (40 e.q.) and react for 16 hours to form the corresponding antibody-drug conjugate.

[0240] 2. In vivo cancer and tumor treatment efficacy of antibody-drug conjugates According to the above general method, MI-PEG6-GGFL-AM-DXD and MI-PEG6-GGFG-AM-DXD are respectively coupled to the HER2 antibody, and then treated and compared in a gastric cancer tumor homogenate.

[0241] The in vivo tumor treatment experimental method is as follows.

[0242] Human gastric cancer cells NCI-N87 are subcutaneously transplanted into NCG mice to establish an in vivo model. The NCI-N87 cells are cultured according to the culture conditions, the cells are collected and counted during the exponential growth phase. 5×10 6 human gastric cancer cells NCI-N87 (suspended in 0.1 mL of basal medium) are inoculated into the right flank of each mouse. After inoculation, when the tumor grows to 70-150 mm 3 (8 days after tumor bearing), according to the body weight and tumor volume of the mice, they are randomly divided into groups, and the test is divided into a PBS group, a HER2-GGFG-DXD (5 mg / kg) group, and a HER2-GGFL-DXD (5 mg / kg) group.

[0243] Treatment starts on day 1. Treatment is performed twice a week until day 25, the body weight of the mice is weighed, and the tumor volume is measured. The formula for calculating the volume is as follows.

[0244] Tumor volume = 1 / 2 × length × width × width (mm 3 ).

[0245] Tumor growth inhibition rate TGI (%) = 100% - (Tt - T0) / (Ct - C0) × 100%, Tumor growth inhibition rate T / C (%) = (Tt / T0) / (Ct / C0) × 100%.

[0246] T0 is the tumor volume of the test antibody group during different administration periods, Tt is the tumor volume of the test antibody group during each measurement period, C0 is the tumor volume of the PBS group during the cage administration period, and CT is the average tumor volume of the PBS group during each measurement period.

[0247] The experimental results are shown in Figure 16. The anti-HER2 antibody coupled to the candidate Dxd drug molecule based on the GGFL linker showed superior curative and inhibitory effects against gastric cancer tumors compared to the GGFG linker, with 5 out of 6 mice cured, compared to only 2 mice cured with the GGFG linker.

[0248] Example 14 Efficacy studies of drug conjugates S7 (MI-PEG6-GGFL-AM-DXd), S8, S10, S18, S19, S23, and DXd at the same dose in human fibrosarcoma. 1. Animals: Nude mice, 6-8 weeks old, all female (Shanghai SLAC ANIMAL Co., Ltd.).

[0249] 2. Generation of a tumor model 1) The corresponding cells were purchased from the American Type Culture Collection (ATCC), and identified according to the instructions provided by the ATCC. The cells were cultured at 37°C under 5% carbon dioxide conditions using Dulbecco's Modified Eagle Medium (abbreviated as DMEM culture medium) containing 10% fetal bovine serum. Cells were passaged every 3 days, and cells up to 15 passages were used.

[0250] 2) A tumor is formed, 5 × 10 6 Individual corresponding cells were subcutaneously injected into the back of nude mice, and the tumor grew to approximately 100 mm. 3 Once the animals reach a certain age, they are randomly divided into groups, and treatment is initiated. The day treatment begins is designated as day 1 of treatment.

[0251] 3) Treatment process Use 1 / 10th of the MTD dose (5 μmol / Kg) and administer once a week for a total of 3 weeks.

[0252] 4) The grouping and measurement of results are as shown in Table 4.

[0253] (TIFF2026510850000111.tif1261655) Results and Discussion: Compared to DXd, S7 (MI-PEG6-GGFL-AM-DXd), S8, S10, S18, S19, and S23 showed significantly improved treatment efficacy and nearly achieved tumor cure.

[0254] Example 15. Coupling of the glycosylation site of an antibody with a drug. Antibody target points include HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylyl cyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1. Selected from PDL1, HER2, NY-ESO-1, BCMA, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC1, and MAGE-A3.

[0255] Add β1,4-galactosidase to any of the above antibodies and incubate for 24 hours. Add the purified Protein A antibody to β-1,4-galactosyltransferase and UDP-C2 keto-Gal and incubate for 48 hours. Change to 50 mM NaOAc (pH 4.5), add Aminoo-azide (10 e.q.) and incubate for 8 hours. After filtration, add the above alkynyl group-containing linker-payload (10 eq) and react for 4 hours.

[0256] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. A conjugate or its stereoisomer or a pharmaceutically acceptable salt thereof, The aforementioned conjugate is as shown in equation (II), Formula (II): R1-LR2 Here, L is the linker (-GGFL-) shown in equation (I), R1 is a group having one or more functions selected from the group consisting of activating the tumor microenvironment, improving the water solubility of tumor-targeted drugs, and providing a coupling site with the antibody element. The conjugate is characterized in that R2 is a group derived from a cytotoxic drug or a cytotoxic-carrying drug.

2. The aforementioned conjugate is selected from Table B1 below, Alternatively, the conjugate is characterized by being selected from Table B2 below. The conjugate according to claim 1.

3. (i) Providing a coupling site with the antibody means that R1 contains a reactive group that can be coupled with the antibody, and that the reactive group that can be coupled with the antibody is This refers to being selected from a group consisting of and / or (ii) The activating groups in the tumor microenvironment are Characterized by being selected from the group consisting of The conjugate according to claim 1.

4. R1 is selected from the following tables C1-A and C1-B. In C1-B, o is 0 or an integer between 1 and 20, and m and n are independently 0 or an integer between 1 and 20. Or, R1 is selected from the following tables C2-A and C2-B. In C2-B, m and n are each independently 0 or an integer from 1 to 20. The conjugate according to claim 1.

5. The aforementioned conjugate is characterized by being selected from Table A1, Table A2, or Table A3. The conjugate described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

6. An antibody conjugate or its stereoisomer or a pharmaceutically acceptable salt thereof, The antibody conjugate is as shown in formula (III), Formula (III): R3 - (R1' - L - R2) ｘ Here, R3 represents the antibody portion. R1'-L-R2 represents a group formed when the conjugate shown in formula (II) is coupled to the antibody element via the R1 group, where R1, L, and R2 are as defined in claim 1, and R １ ' is R １ It is a group formed by coupling to the antibody element, and The antibody-drug conjugate is characterized in that x is 1 to 8.

7. The antibodies mentioned above are HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, glycoprotein NMB, guanylate cyclase C, CEA, AXL, GCC, CD79b, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, and FOLR. １ , DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, B CMA, pCadherin, Ephrin-A, LAMP1, MUC1, PDL1, HER2, NY-ESO-1, BCMA, WT1, MUC1, CD20, CD23, ROR １ , CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFR, EGFRvIII, VEGFR ２ Characterized by being selected from the group consisting of NY-ESO-1, MUC-1 and MAGE-A3, or compositions thereof. The antibody-drug conjugate according to claim 6.

8. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising (i) a conjugate or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or an antibody conjugate or stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 7, and (ii) a pharmaceutically acceptable carrier thereof.

9. Uses of the conjugate according to claim 1 or the antibody conjugate according to claim 6 in the preparation of a drug for treating or preventing tumors or inflammation.

10. The tumor is characterized by being selected from the group consisting of bladder cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and hematological cancer, or a combination thereof. The use described in claim 9.

11. It is a linker, The linker is characterized by being as shown in formula (I).

Images