Tumor microenvironment-activated drug conjugate and antibody-drug conjugate

Abstract

The present invention relates to a tumor microenvironment-activated drug conjugate structure and an antibody-drug conjugate. Specifically, the present invention provides a drug conjugate having a chemical structure represented by formula R1-R2-D or R2'-D, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein R1 is a chemical structure conjugated to a biomolecule or is absent, R2 and R2' are each independently an adjustment chemical structure for enhancing the structure-activity relationship, and D is selected from structures represented by X1-X5. The present invention also provides an antibody-drug conjugate comprising the drug conjugate.

Description

Tumor microenvironment-activated drug conjugates and antibody-drug conjugates Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a tumor microenvironment activated drug conjugate and an antibody drug conjugate. Background Art

[0002] To expand the therapeutic window of antibody or protein-drug conjugates, researchers have been working to improve their tumor specificity and release efficiency. In recent years, driven by advances in tumor molecular biology and other basic disciplines, the research and development of targeted tumor therapeutics has made significant progress in many areas.

[0003] The method of linking effector molecules to antibodies by utilizing the natural cysteine ​​residues in antibody sequences has been widely used, such as cytotoxins, immune agonists, and immune antagonists, further promoting the use of antibodies. Typically, antibody-drug conjugates must first enter tumor cells through endocytosis to produce their efficacy, where they are then degraded to release the cell-killing effector molecules, thereby achieving tumor inhibition. However, after years of development, only two types of linkers that can be activated by cathepsin B have been successfully used, and these must enter tumor cells through endocytosis, which has become a major factor limiting the drug development of various antibodies.

[0004] The tetrapeptide Gly-Gly-Phe-Gly (GGFG) is a cathepsin B-activatable linker, used in the ADC Enhertu. Daiichi Sankyo's Enhertu is a plasma-stable ADC with a DAR of 7.7. It undergoes proteolytic degradation in lysosomes, releasing the DX-8951f derivative, a potent topoisomerase I inhibitor derived from exatecan. Achieving such a high DAR is remarkable due to the unique properties of the GGFG linker, demonstrating that the structure-activity relationship between the linker and the toxin molecule is crucial for ADC development. However, the GGFG linker combined with the DX-8951f derivative has been associated with interstitial pneumonitis, a toxicity seen in clinical trials with Enhertu (T-Dxd), Datopotamab deruxtecan (Dato-DXd), Raludotatug deruxtecan (R-DXd), and Ifinatamab deruxtecan (I-DXd). Comprehensive analysis of 8 T-DXd monotherapy trials: 879 patients with different tumor types, 139 patients (15.8%) have been diagnosed with ILD. 108 people experienced grade 1 or 2 events, and 21 people experienced grade 5 events. This may be because cathepsin B is expressed in lung epithelial cells, and GGFG is the linker that activates cathepsin B, which will produce a certain release in the lungs. Comprehensive analysis of 23 PD-1 monotherapy trials: 3284 patients with different tumor types, 4% of patients developed ILD. In the future, if T-DXd is combined with PD-1 treatment, the incidence of ILD may be further increased.

[0005] We discovered that leucopenia is highly expressed in tumors and developed a new leucopenia-activated small molecule drug conjugate, leucopenia. Clinical trials have shown that hundreds of patients treated with novel chemotherapy have experienced no interstitial pneumonia, demonstrating that leucopenia activation is more tumor-specific than cathepsin B. DX-8951f derivatives and the immune agonist T785 are significantly more toxic than conventional chemotherapy drugs such as doxorubicin and paclitaxel, making their development into broad-spectrum small molecule drug conjugates more challenging. By leveraging the diverse linkages and enzymatic activation of leucopenia linkers and toxins, we identified promising AAN-toxin combinations. By screening linker and toxin combinations using R1 and R2, we identified small molecule drug conjugates with promising preclinical therapeutic indices. These small molecules can be developed as standalone drugs or conjugated to antibodies via EMC or other conjugation methods to form antibody-drug conjugates. Summary of the Invention

[0006] The present invention aims to provide drug conjugates and antibody drug conjugates with strong specificity and high stability, and their use in treating and / or preventing cancer and / or inflammation. The drug conjugates and antibody drug conjugates of the present invention are activated only in pathological microenvironments (e.g., tumor microenvironments or inflammatory sites), releasing active molecules, overcoming drug resistance and reducing toxicity.

[0007] Specifically, the present invention provides a drug conjugate having a chemical structure represented by formula (I) or (II), and stereoisomers thereof or pharmaceutically acceptable salts thereof, R1-R2-D (I) R2'-D (II)

[0008] Wherein, R1 is a chemical structure coupled with a biomolecule or does not exist; R2 and R2' are each independently a chemical structure adjusted to enhance the structure-activity relationship; D contains -AAN- and a cytotoxic structure, and D is selected from X1-X5.

[0009] The present invention also provides an antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is represented by formula (III): R3-(R1-R2-D) x Formula (III);

[0010] wherein R3 is an antibody; R1, R2, and D are as defined in any embodiment herein; and x is an integer selected from 1-8.

[0011] The present invention also provides a pharmaceutical composition comprising: (i) the drug conjugate and / or antibody drug conjugate according to any embodiment herein, or a stereoisomer or pharmaceutically acceptable salt thereof; and (iii) a pharmaceutically acceptable carrier.

[0012] The present invention also provides use of the drug conjugate and / or antibody drug conjugate described in any embodiment herein in the preparation of a medicament for treating and / or preventing tumors and / or inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1: Enzyme cleavage pattern of control compound Y.

[0014] Figure 2: Enzymatic cleavage pattern of compound S5.

[0015] Figure 3: Enzymatic cleavage pattern of compound S16.

[0016] Figure 4: Enzymatic cleavage pattern of Deruxtecan by Cathepsin B.

[0017] Figure 5: Enzymatic cleavage spectrum of S16 in lung tissue.

[0018] Figure 6: Enzymatic cleavage spectrum of Deruxtecan in lung tissue.

[0019] Figure 7: Hydrophobic interaction chromatography (HIC) of Trastuzumab-PEG6-AAN-AM-Dxd (DAR8).

[0020] Figure 8: MS spectrum of Atezolizumab Ab-C5-AAN-PABC-T785 (DAR8).

[0021] Figure 9: HIC spectrum of Atezolizumab Ab-AAN-T785 (DAR8).

[0022] Figure 10: MS spectrum of Atezolizumab Ab-C5-AAN-T785 (DAR8).

[0023] Figure 11: Efficacy of high-dose small molecule drug conjugates based on TLR7 / 8 agonists (S5) alone and in combination with legurubicin.

[0024] Figure 12: Efficacy of high-dose small molecule drug conjugates based on TLR7 / 8 agonists (S5) alone and in combination with CTLA-4 antibodies.

[0025] Figure 13: Inhibitory effect of anti-HER2 antibody coupled with Dxd drug molecule based on Affinity linker 2 on gastric cancer tumors.

[0026] Figure 14: Inhibitory effect of anti-LY6G6D antibody coupled with Dxd drug molecule based on Affinity linker2 on colon cancer tumor model.

[0027] Figure 15: Inhibitory effect of PDL1-Dxd in the MC38 tumor model.

[0028] Figure 16: Inhibitory effect of Affinity linker4-based anti-PD-L1 antibody coupled with T785 drug on large tumors.

[0029] FIG17 : Tumor growth inhibition effect of 7B6-DXD in a mouse subcutaneous HT-55 xenograft tumor model.

[0030] Figure 18: Comparison of the stability of S15 coupled to albumin and after coupling and ring opening.

[0031] Figure 19: Schematic diagram of the structure of bispecific antibody-drug conjugates.

[0032] FIG20 : Inhibitory effect of EGFR / Cmet-T785 in the NCI-H292 tumor model.

[0033] FIG. 21 : Inhibitory effect of EGFR / Cmet-Dxd in the NCI-H292 tumor model.

[0034] Figure 22: Tumor volume of human fibrosarcoma HT-1080 xenograft tumor model in nude mice after administration of S41.

[0035] Figure 23: Tumor volume of human fibrosarcoma HT-1080 xenograft tumor model in nude mice after administration of S41.

[0036] Figure 24: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41.

[0037] Figure 25: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41 and / or anti-PD1 antibodies.

[0038] Figure 26: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41. DETAILED DESCRIPTION

[0039] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0040] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of the terminology "solely," "only," and the like in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0041] The inventors previously developed the small molecule drugs legumain activated by the enzyme legumain, containing AANL-DOX, and legumataxel, containing AAN-PABC-taxel and paclitaxel. These drugs have been patented in China and are currently undergoing key Phase II / III and Phase I clinical trials, respectively. However, the toxicity of doxorubicin and paclitaxel cannot meet the more severe toxicity requirements used in ADCs, necessitating the innovation of conjugate structures with novel structure-activity relationships. Studies have found that legumain forms complexes with integrins, which can efficiently activate specific substrate structures on the surface of tumor cells. Therefore, the inventors used legumain as the activating enzyme and conducted extensive structural screening in combination with various toxin and immune agonist chemical structures, as well as auxiliary structures and linker structures. Through biological mechanisms of action and in vitro and in vivo efficacy, they obtained restrictive, specialized conjugate structures. These conjugate structures are highly stable in human plasma and can efficiently activate and release the compatible chemical molecules in the tumor microenvironment (outside of tumor cells). Therefore, compared with traditional ADCs, this new release method can better exert the bystander effect and activate adjacent immune cells when the combined payload drug is an immune agonist. It has a different drug-making mechanism and scalability from traditional ADCs.

[0042] Drug conjugates

[0043] Provided herein are compounds having a chemical structure represented by formula (I), and stereoisomers thereof or pharmaceutically acceptable salts thereof: R1-R2-D (I);

[0044] The compound can be used as a drug conjugate for preparing ADC.

[0045] In formula (I), R1 is a chemical structure coupled to a biomolecule or does not exist; R2 is a chemical structure adjusted to enhance the structure-activity relationship; and D is -AAN-linker-drug.

[0046] Herein, R1 is preferably selected from:

[0047] and;

[0048] The wavy line indicates the connection position between R1 and R2.

[0049] Preferably, R1 is selected from:

[0050] and

[0051] In some embodiments, R1 is absent.

[0052] In some embodiments, R2 is selected from:

[0053] -C 1-6 Alkylene-CO-*;

[0054] -C 1-6 Alkylene-O-*;

[0055] -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*;

[0056] -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*;

[0057] -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*;

[0058] -(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*;

[0059] -C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*;

[0060] -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*;

[0061] -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*;

[0062] -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*;

[0063] -NH-C1- 55 Alkylene-CO-*;

[0064] -NH-C 1-55 Alkylene-NH-*;

[0065] -NH-C 1-55 Alkylene-O-*;

[0066] -CO-C 1-55 Alkylene-CO-*;

[0067] -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*;

[0068] -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*;

[0069] -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*;

[0070] -CO-C 1-6 Alkylene-NH-*;

[0071] -CO-C 1-6 Alkylene-NHCO-SO3H substituted C 1-6 Alkylene-NH-*;

[0072] in:

[0073] * indicates the position connected to D;

[0074] R4 is a monovalent group or a divalent group, and when R4 is connected to other parts of the compound, it is a divalent group, selected from: C 1-6 Alkyl or C 1-6 Alkylene; C 1-6 Alkyl-C 3-8 Cycloalkylene- or C 1-6 Alkylene-C 3-8 Cycloalkylene-;-CO-C 1-6 Alkylene-; C 1-6 Alkyl-(O-CH2CH2-O) m -C 1-6 Alkylene- or C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-;-NH-C1-6 Alkylene; C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-; and -NH-C 1-6 Alkylene-C 3-8 cycloalkylene;

[0075] R5 is a monovalent group or a divalent group. When R5 is connected to other parts of the compound, it is a divalent group selected from: C 1-6 Alkyl-(O-CH2CH2-O) m -;C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -;-CO-C 1-6 Alkylene-(O-CH2CH2-O) n -;

[0076] One of R4 and R5 is a divalent group and the other is a monovalent group;

[0077] R6 does not exist or is -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylenecarbonyl or -NH-C 1-6 Alkylene-triazolyl-C 1- 6-alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkyleneoxy-C 1-6 alkylenecarbonyl;

[0078] L2 and L3 are each independently a bond or C 1-6 alkylene;

[0079] L4 is independently C 1-6 alkylene;

[0080] L5 is a key, C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl or C 1-6 alkylenecarbonyl;

[0081] L6 is C 1-6 alkylene;

[0082] L7 is C 1-6 Alkylene or -C 3-8 Cycloalkylene-C 1-6 alkylene;

[0083] Each of n and m is independently an integer from 1 to 50.

[0084] Herein, alkylene has a well-known meaning in the art and refers to a divalent alkyl group, such as methylene (-CH2-), ethylene (-CH2CH2-), etc. The number of carbon atoms in the alkylene group can be, for example, 1-55, 1-20, 1-10, 1-6 or 1-4.

[0085] As used herein, cycloalkyl has the meaning generally known herein and refers to a saturated carbocyclic ring. Cycloalkyl groups generally have 3 to 8, such as 3 to 6, ring carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. As used herein, cycloalkylene groups are divalent cycloalkyl groups, i.e., groups having two ring carbon atoms for attachment to the rest of the compound.

[0086] In some embodiments, R2 is:

[0087] in:

[0088] R4 is a monovalent group or a divalent group, and when R4 is connected to other parts of the compound, it is a divalent group, selected from: C 1-6 Alkyl or C 1-6 Alkylene; and C 1-6 Alkyl-C 3-8 Cycloalkylene- or C 1-6 Alkylene-C 3-8 Cycloalkylene-;

[0089] R5 is a monovalent group or a divalent group. When R5 is connected to other parts of the compound, it is a divalent group selected from: C 1-6 Alkyl-(O-CH2CH2-O) m -; or C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -;

[0090] One of R4 and R5 is a divalent group and the other is a monovalent group;

[0091] R6 does not exist;

[0092] L2 and L3 are each independently a bond or C 1-6 alkylene;

[0093] L4 is independently C 1-6 alkylene;

[0094] L5 is a key, C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl or C 1-6 alkylenecarbonyl;

[0095] L6 is C 1-6alkylene;

[0096] L7 is C 1-6 Alkylene or -C 3-8 Cycloalkylene-C 1-6 alkylene;

[0097] Each of n and m is independently an integer from 1 to 50.

[0098] In some embodiments, R2 is:

[0099] wherein L4, L5, L6, L7 and n are as described in any of the above embodiments.

[0100] In formula (I), preferably, R2 is selected from:

[0101] wherein o is independently an integer of 1-20, and m and n are each independently an integer of 1-50.

[0102] Preferably, in R2, o is independently an integer of 1 to 10. Preferably, in R2, m and n are each independently an integer of 1 to 40, or each independently an integer of 1 to 30, 2 to 40, 5 to 35, 10 to 25, 15 to 20, or 2 to 10.

[0103] Preferably, R2 is selected from:

[0104] Here, o is independently an integer of 1-10, and n and m are each independently an integer of 2-20.

[0105] In formula (I), the drug can be selected from: camptothecin, camptothecin derivatives (such as 10-hydroxycamptothecin, irinotecan and SN38 compounds), topotecan, floxuridine, doxifluridine, cytarabine, etoposide, fludarabine, capecitabine, vincristine, epothilone B, daunorubicin, epirubicin, methotrexate, gemcitabine, melphalan, nimustine, mitoxantrone, paclitaxel, docetaxel, mitomycin, exitecan, exitecan derivative DXd, Sting agonists, resiquimod and Toll-like receptor agonists, etc.

[0106] In some embodiments, the drug is a TLR7 / 8 agonist, such as T785, resiquimod, and STING agonists, or a topoisomerase I inhibitor, such as exitecan and exitecan derivatives (DXd), etc.

[0107] In some embodiments, the drug is selected from:

[0108] In formula (I), the drug can be directly linked to -AAN- or linked through a suitable linker. Typically, the drug is linked to the -AAN- linker via a suitable chemical bond, which can be a peptide bond (-NH-CO-) or -COO-. In some embodiments, the linker is a PABC (-NH-phenyl-CH2-O-CO-) group or an AM (-NH-CH2-) group.

[0109] In some embodiments, D of formula (I) is selected from the following X1 to X5:

[0110] The wavy line indicates the position where D and R2 are connected.

[0111] In some embodiments, the drug conjugate represented by formula (I) is selected from:

[0112] In each formula, R1 is as described in any embodiment herein.

[0113] In some embodiments, the compound of formula (I) is selected from the following compounds S1 to S59:

[0114] In some embodiments, the drug conjugates described herein have a chemical structure represented by R2'-D (Formula II), wherein R2' is a modified chemical structure to enhance the structure-activity relationship, and the structure of D is represented by any one of X1-X5.

[0115] Preferably, said R2' is selected from: C 1-6 Alkyl-CO-*; C 1-6 Alkyl-O-*; C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O)n -C 1-6 Alkylene-CO-*; C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; NH2-C 1-55 Alkylene-CO-*; NH2-C 1-55 Alkylene-NH-*; NH2-C 1-55 Alkylene-O-*; HOOC-C 1-55 Alkylene-CO-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; HOOC-C 1-6 Alkylene-NH-*; HOOC-C 1-6Alkylene-NHCO-SO3H substituted C 1-6 Alkylene-NH-*; N3-C 1-6 Alkylene-CO-*; N3-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkylene-OC 1-6 Alkylene-CO-*; HOOC-C 1-6 Alkylene-OC 1-6 Alkylene-CO-*;

[0116] in:

[0117] * indicates the position connected to D;

[0118] R4 is selected from: C 1-6 Alkyl; C 1-6 Alkyl-C 3-8 Cycloalkylene-; COOH-C 1-6 Alkylene-; C 1-6 Alkyl-(O-CH2CH2-O)mC 1-6 Alkylene-; NH2-C 1-6 Alkylene; C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-; and NH2-C 1-6 Alkylene-C 3-8 cycloalkylene;

[0119] R5 is selected from: C 1-6 Alkyl-(O-CH2CH2-O) m -;C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -;COOH-C 1-6 Alkylene-(O-CH2CH2-O) n -;

[0120] R6 does not exist or is -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylenecarbonyl, or -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkyleneoxy-C1-6 alkylenecarbonyl;

[0121] L2 and L3 are each independently a bond or C 1-6 alkylene;

[0122] L4 is independently C 1-6 alkylene;

[0123] L5 is a key, C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl or C 1-6 alkylenecarbonyl;

[0124] L6 is C 1-6 alkylene;

[0125] L7 is C 1-6 Alkylene or -C 3-8 Cycloalkylene-C 1-6 alkylene;

[0126] Each n and m is independently an integer of 1-50, p is 0 or 1; when p is 0, the -NH-[CO-R4] p The group is -NH2.

[0127] Preferably, R2' is selected from: wherein o is independently an integer of 1-20, and m and n are each independently an integer of 1-50.

[0128] More preferably, R2' is selected from:

[0129] Preferably, n and m are each independently an integer of 2-20.

[0130] In some embodiments, the drug conjugate represented by formula (II) is selected from:

[0131] Antibody Drug Conjugates

[0132] Provided herein are antibody drug conjugates represented by the following formula (III), stereoisomers thereof, or pharmaceutically acceptable salts thereof: R3-(R1-R2-D)x Formula (III);

[0133] In the formula: R3 is an antibody; R1, R2 and D are as described in any of the above embodiments; x is an integer selected from 1-8.

[0134] In formula III, when R1 is:

[0135] hour,

[0136] The connection method of R3-R1 is:

[0137] Wherein, S is the S atom of the cysteine ​​residue in R3;

[0138] When R1 is:

[0139] hour,

[0140] The connection method of R3-R1 is:

[0141] Among them, R3 is coupled to the alkynyl group in the R1 group.

[0142] An azide modification can be introduced into the sugar chain of the antibody or the reconstructed sugar chain, which undergoes a click reaction with DBCO (dibenzocyclooctyne) or BCN (bicyclo[6,1,0]nonyne) to form a triazole ring, thereby achieving the connection between R1 and R3.

[0143] The drug conjugate or antibody drug conjugate of the present invention may exist in the form of any one of geometric isomers or a mixture thereof.

[0144] Antibody

[0145] In the present disclosure, "antibody" is used in the broadest sense and covers biological molecules such as monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they can exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170: 4854-4861).

[0146] Antibodies specifically bind to antigens and are composed of at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), which comprises three constant domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL), which comprises one constant domain. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Generally speaking, from N-terminus to C-terminus, both light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids are typically assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al., (1987) J Mol. Biol. 196: 901-917 or Chothia et al., (1989) Nature 341: 878-883.

[0147] The carboxyl-terminal portion of the heavy chain defines the constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda chains. Human heavy chains are typically classified as μ, δ, γ, α, or ε. Antibodies are typically categorized as IgM, IgD, IgG, IgA, and IgE, respectively. IgG subclasses are well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG3, and IgG4.

[0148] Herein, the antibody may be a naturally occurring antibody or a non-naturally occurring antibody; may be a monoclonal antibody or a polyclonal antibody; may be a mouse antibody or a humanized antibody; or may be a chimeric antibody.

[0149] A chimeric antibody is an antibody in which a portion of its heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species (such as human) or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (such as mouse) or belonging to another antibody class or subclass. A humanized antibody is an antibody form that contains sequences from non-human (such as murine) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins.

[0150] As used herein, an antibody fragment or antigen-binding fragment refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability of a full-length antibody to specifically bind to an antigen, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibodies (scFv); nanobodies; and multispecific antibodies formed from antibody fragments.

[0151] The antibody used in the present invention can be an antibody known in the art or a functional fragment thereof. For example, the antibody or its functional fragment can be selected from: anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, or anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR6 antibody, anti-CD38 antibody, anti-CD5 2 antibody, anti-complement C5 antibody, anti-RSV F protein antibody, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib / Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 antibody, anti-α4 integrin antibody, anti-IgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody, anti-SSTR2 antibody, anti-LY6G6D antibody, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody.

[0152] In some embodiments, the antibody used herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies refer to artificial antibodies containing two or more specific antigen binding sites, which can build a bridge between target cells and functional molecules (cells) to stimulate a directional immune response. An antibody molecule comprising at least two antigen binding domains can be referred to as a bispecific antibody molecule, wherein each antigen binding domain is capable of binding to a different target. The VH and VL regions comprise framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDR. The structure of an exemplary bispecific antibody is shown in Figure 19. As shown in Figure 19 (left), from N-terminus to C-terminus, the VH region contains the following structure: {N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]}-linker-{[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus}, and the VL region contains the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus. As shown in Figure 19 (right), from N-terminus to C-terminus, the VH region contains the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region contains the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0153] In a preferred embodiment, the antibodies used in the present invention are selected from the group consisting of: Utomilumab, Urelumab, ADG106, Poteligeo TM (Mogamulizumab), Poteligeo TM (Mogamulizumab), Bexxar TM (tositumomab), Zevalin TM (ibritumomab tiuxetan), Rituxan TM (rituximab), Arzerra TM (Ofatumumab), Gazyva TM (Obinutuzumab), Besponsa TM<h2 style=";text-align:left;direction:ltr">(Inotuzumab ozogamicin),Zenapax<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (daclizumab),Varlilumab,Theralizumab,Adcetris<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Brentuximab vedotin),Myelotarg<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (gemtuzumab),Darzalex<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Daratumumab),CDX-1140,SEA-CD40,RO7009789,JNJ-64457107,APX-005M,Chi Lob 7 / 4,Campath<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (alemtuzumab),Raptiva<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (efalizumab),Soliris<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (eculizumab),Yervoy<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (ipilimumab),tremelimumab,Erbitux<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (cetuximab),Vectibix<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (panitumumab),Portrazza<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Necitumumab),TheraCIM<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Nimotuzumab),Synagis<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (palivizumab) and Unituxin<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Dinutuximab),TRX-518,MK-4166,MK-1248,GWN-323,INCAGN0186,BMS-986156,AMG-228,ReoPro<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (abiciximab),Herceptin<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (trastuzumab),Perjeta<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Pertuzumab),Kadcyla<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (Ado-trastuzumab emtansine),GSK-3359609,JTX-2011,Simulect<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (basiliximab),Tysabri<h2 style=";text-align:left;direction:ltr"> TM <h2 style=";text-align:left;direction:ltr"> (natalizumab), BMS-986016, REGN3767, LAG525, Xolair<h2 style=";text-align:left;direction:ltr"> TM(omalizumab), Tavolimab, PF-04518600, BMS-986178, MOXR-0916, GSK-3174998, INCAGN01949, IBI-101, Keytruda TM (Pembrolizumab), Opdivo TM (Nivolumab), Lartruvo TM (Olaratumab), Tencentriq TM (Atezolizumab), BMS-936559, Bavencio TM (Avelumab), Imfinzi TM (Duralumab), Prolia TM (Denosumab), Empliciti TM (Elotuzumab), MTIG7192A, TSR-022, MBG-453, Remicade TM (infliximab), Humira TM (adalimumab), Avastin TM (bevacizumab), Lucentis TM (ranibizumab), Cyramza TM (Ramucirumab), and JNJ-61610588.

[0154] In some embodiments, the bioactive macromolecular portion (A) used in the ADC described herein is a fusion protein containing an antibody antigen binding domain and an optional cytokine. The antigen binding domain in the fusion protein can be selected from the antigen binding domains for the following antigens: 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, 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, MUC-1, SSTR2, LY6G6D, MAGE-A3, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or an antigen-binding domain (such as an antigen-binding fragment) of any one of the antibodies mentioned herein.

[0155] In some embodiments, the fusion protein can be a bispecific antibody comprising an antigen binding domain that specifically binds to an antigen selected from the group consisting of HER2, CD19, EGFR, CD22, CD3, TROP2, Glycoprotein NMB, Guanylylcyclase 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, CD19, PDL1, NY-ESO-1, WT1, MUC1, CD20, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, EGFR, FAP, EphA2, GD2, GPC3, IL-13Ra2, LewisY, Mesothelin, SS1, CD171, EGFR, EGFRvIII, VEGFR2, NY-ESO-1, MUC-1, MAGE-A3, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or an antigen-binding domain selected from any of the antibodies mentioned in the present invention. Preferably, the bispecific antibody is a single-chain bispecific antibody comprising two scFvs from the same or different antibodies.

[0156] Pharmaceutical composition

[0157] The pharmaceutical composition of the present invention comprises: (i) a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and / or an antibody drug conjugate, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier. The carrier can be any pharmaceutically acceptable carrier or excipient, which can vary depending on the dosage form and mode of administration. Pharmaceutically acceptable carriers are generally safe and non-toxic and may contain any known substance used in the pharmaceutical industry to formulate pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, and disintegrants. Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate; starches, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; silicon dioxide, talc, stearic acid or a salt thereof, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the primary consideration is the mode of administration of the pharmaceutical dosage form. This is well known in the art.

[0158] The pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and / or an antibody drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. An "effective amount" means that the amount of a component is sufficient to produce the desired response. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as weight, age and sex, the duration of treatment, co-administered treatments (if any), and the specific formulation used. Typically, an "effective amount" as described herein is a conventional amount of a biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the conjugate contained in the pharmaceutical composition of the present invention may be lower than the conventional amount of the biomolecule but may produce a better therapeutic or prophylactic effect because the biomolecule is protected by a protecting group before reaching the pathological microenvironment to bind to its ligand or receptor.

[0159] The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the intended purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally, or topically. The dosage of the drug can depend on the patient's age, health status and weight, concurrent treatments, and the frequency of treatment. The pharmaceutical composition of the present invention can be administered to any subject in need thereof, for example, a mammal, particularly a human.

[0160] The conjugate of the present invention can release the toxin (D) either through the pathway of biomolecule endocytosis into lysosomes or through the pathway of hydrolysis by proteolytic enzymes in the acidic microenvironment of the tumor, especially legumain or granzymes. For example, the biomolecule in the antibody-drug conjugate of the present invention is trastuzumab, anti-Trop-2 antibody or anti-PD-L1 antibody, etc. In terms of toxin release, the proteolytic enzyme hydrolysis pathway in the acidic microenvironment of the tumor can release the toxin more efficiently, thereby exerting better drug efficacy. Therefore, the conjugate of the present invention can effectively overcome the problem of bioconjugate drug release efficiency, and the toxin is efficiently released in the pathological microenvironment.

[0161] use

[0162] Each drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof, and each antibody drug conjugate, stereoisomer thereof, or pharmaceutically acceptable salt thereof disclosed in the present invention can be used to treat and / or prevent tumors or inflammation, or can be used as an active ingredient in the preparation of a drug for treating tumors or inflammation.

[0163] The diseases that can be treated by the drug conjugates, stereoisomers, or pharmaceutically acceptable salts thereof, and antibody drug conjugates, stereoisomers, or pharmaceutically acceptable salts thereof disclosed herein are related to the active ingredients contained in the conjugates. The indications of these active ingredients are well known in the art.

[0164] In some embodiments, the tumors described herein may include hematological tumors and solid tumors, including but not limited to sarcomas (such as fibrosarcomas), bladder cancer, brain cancer, 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 blood cancer, etc.

[0165] The present invention also includes a method for treating or preventing tumors or inflammation, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a drug conjugate as described herein, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and an antibody drug conjugate, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. This method can be used in combination with any known radiotherapy or immunotherapy.

[0166] The present invention also provides the use of the drug conjugates or stereoisomers thereof or pharmaceutically acceptable salts thereof, and / or antibody drug conjugates, or stereoisomers thereof or pharmaceutically acceptable salts thereof, as described herein, in the preparation of anti-tumor drugs. Preferably, the tumor is selected from blood tumors and solid tumors. Preferably, the tumor includes but is not limited to sarcomas (such as fibrosarcoma), bladder cancer, brain cancer, 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 blood cancer.

[0167] Advantages of the present invention include:

[0168] Through continuous experimental screening, the inventors have prepared trastuzumab-drug conjugates, atezolizumab-drug conjugates, anti-SSTR2 antibody-drug conjugates, and anti-LY6G6D antibody-drug conjugates, which are tumor-targeted and efficiently activated macromolecular conjugates with innovative multifunctional targeting activation properties: (1) The tumor microenvironment-targeted activated antibody-drug conjugates of the present invention have aggregation, retention, and activation effects at the tumor site, and have the characteristics of targeting the tumor microenvironment and efficiently releasing the load. (2) The tumor microenvironment-targeted drug conjugates of the present invention can be specifically activated or broken by tumor tissue to locally generate effective drugs. (3) Ixitecan, resiquimod, or their derivatives containing the AAN sequence in the linker are directly converted into macromolecules by conjugation with trastuzumab or certolizumab, changing the various properties of the drug, improving the efficacy, and changing the indication restrictions of the drug. (4) When tumor cells metastasize, they usually secrete a large amount of protease hydrolases to degrade the extracellular matrix, so the protease-activated conjugates have a special therapeutic effect on the treatment of tumor metastasis. The antibody-drug conjugates of the present invention have high activation efficiency.

[0169] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0170] It should be understood that the terms "comprising" and "including" or similar expressions used in the present invention also mean "consisting of..." etc. The sum of all weight percentages or volume percentages should equal 100%. Unless otherwise stated, the various reagents and products used in the examples are commercial products. Unless otherwise stated, the methods mentioned in the examples are carried out according to conventional techniques. The following examples are not intended to limit the scope of the present invention.

[0171] Example 1: Preparation of drug conjugates S1-S21, S26-S59

[0172] A typical preparation method of the compound is as follows:

[0173] Synthesis route general formula (I)

[0174] 1) Take the compound R1-R2-Ala-Ala-Asn-PABOH (1.0 eq) and dissolve it in an appropriate amount of DMF solvent. Then add the compound Bis-PNP (2.0 eq). Add DIPEA (2.0 eq) to the reaction solution under an ice bath, then slowly return to room temperature and continue stirring for 10-15 hours. The resulting reaction solution is removed from most of the solvent under high vacuum, and then dissolved and diluted with an appropriate amount of dichloromethane. The organic phase is washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and then column chromatography with dichloromethane / methanol as the eluent. The mixture is spin-dried to obtain R1-R2-Ala-Ala-Asn-PABC-PNP.

[0175] 2) Compound R1-R2-Ala-Ala-Asn-PABC-PNP (1.0 eq) was dissolved in an appropriate amount of DMF, followed by the addition of compound D-NH2 (1.0 eq). DIPEA (2.0 eq) was added to the reaction mixture under an ice bath, and the mixture was slowly returned to room temperature and stirred for 10-15 hours. The resulting reaction mixture was mostly stripped of solvent under high vacuum, then diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to yield the desired product, R1-R2-Ala-Ala-Asn-PABC-(C=O)-NHD.

[0176] The compounds shown in Table 1 can be obtained by using the corresponding raw materials according to the above method. The properties of each compound in Table 1 are shown in Table 2.

[0177] Table 1

[0178] Table 2

[0179] Synthesis route general formula (II)

[0180] Compound R1-R2-Ala-Ala-Asn-OH (1.0 eq) was dissolved in an appropriate amount of DMF, followed by the addition of compound D-NH2 and the condensing agent HBTU. DIPEA was added to the reaction mixture while it was ice-cooled, then slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly stripped of solvent under high vacuum, diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to obtain the desired product.

[0181] The corresponding raw materials were used to obtain the compounds shown in Table 3 according to the above method. The properties of each compound in Table 3 are shown in Table 4.

[0182] Table 3

[0183] Table 4

[0184] Synthesis route general formula (III)

[0185] 1) Synthesis of the compound Fmoc-Ala-Ala-Asn-AM-OAc

[0186] Dissolve Fmoc-Ala-Ala-Asn(Trt)-Gly-OH (1.0 eq) in dichloromethane / tetrahydrofuran (15 / 5 by volume) and cool to 0°C. Add AcOH (1.0 eq), lead acetate (1.2 eq), and copper acetate (0.2 eq) under nitrogen. Stir at 40-50°C under nitrogen for 2-3 hours. Monitor the reaction for completion by TLC. Dilute the reaction solution with dichloromethane, wash with water, separate the layers, extract the aqueous phase with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, spin dry, and analyze by column chromatography to obtain Fmoc-Ala-Ala-Asn-AM-OAc.

[0187] 2) Synthesis of the compound Fmoc-Ala-Ala-Asn(Trt)-AM-Ac-OBn

[0188] To a three-necked flask, add Fmoc-Ala-Ala-Asn-AM-OAc (1.0 eq) and benzyl glycolate (2.0 eq). Under nitrogen and in an ice bath, add trifluoroacetic acid / dichloromethane (TFA / DCM, volume ratio = 1 / 5) and stir at room temperature for 2-3 hours. After completion of the reaction, the reaction mixture is concentrated and separated by high-pressure preparative chromatography to afford Fmoc-Ala-Ala-Asn-AM-Ac-OBn as a white solid.

[0189] 3) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-Ac-OH

[0190] The compound Fmoc-Ala-Ala-Asn-AM-Ac-OBn (1.0 eq) was dissolved in an appropriate amount of MeOH, and Pd / C (10 wt%, 0.1 eq) was slowly added. The mixture was stirred under hydrogen for 2-3 hours. The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH was obtained as a light yellow product under high pressure.

[0191] 4) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-DXd

[0192] The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (1.0 eq) and isoproterenol methanesulfonate (1.0 eq) were dissolved in DMF and cooled to 0°C. Under nitrogen, the condensing agent DEPBT (1.2 eq) and the organic base DIPEA (2.0 eq) were added sequentially, and the mixture was stirred at room temperature for 2-3 hours. TLC confirmed the reaction was complete. The reaction mixture was concentrated in vacuo, redissolved in DMF, filtered, and purified by high-pressure liquid chromatography to obtain the compound Fmoc-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0193] 5) Synthesis of compound H-Ala-Ala-Asn-AM-DXd

[0194] The compound Fmoc-Ala-Ala-Asn-AM-DXd (1.0 eq) was dissolved in DMF, and piperidine (2.0 eq) was added. The mixture was stirred at room temperature under nitrogen for 2-3 hours. TLC confirmed the reaction was complete. The reaction solution was redissolved, filtered, and then purified by high-pressure liquid chromatography to obtain the compound H-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0195] 6) Synthesis of compound R1-R2-Ala-Ala-Asn-AM-DXd

[0196] Compound R1-R2-OH (1.0 eq) was dissolved in an appropriate amount of DMF, and H-Ala-Ala-Asn-AM-DXd (1.0 eq) was added. HBTU (1.2 eq) and DIPEA (2.0 eq) were then added under nitrogen, and the mixture was stirred at room temperature for 1-2 hours. TLC confirmed the reaction was complete. The crude reaction mixture was filtered, and then purified by high-pressure liquid chromatography to yield compound R1-R2-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0197] The compounds shown in Table 5 can be obtained by using the corresponding raw materials according to the above method. The properties of each compound in Table 5 are shown in Table 6.

[0198] Table 5

[0199] Table 6

[0200] Example 2: Synthesis of Compound S22

[0201] 1) Synthesis of compound Boc-Glu(PEG8)-OtBu

[0202] Boc-Glu(OH)-OtBu (300 mg, 0.989 mmol) and PEG8-NH2 (379 mg, 0.988 mmol) were dissolved in ultra-dry DMF (5 ml). HBTU (450 mg, 1.19 mmol) was added as a condensing agent. DIPEA (255 mg, 1.98 mmol) was added to the reaction mixture in an ice bath and stirred at room temperature for 2-3 hours. The reaction mixture was evaporated to dryness under reduced pressure, and the crude product was added to silica gel and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1 to 1 / 1) to afford Boc-Glu(PEG8)-OtBu (600 mg, 91% yield) as an oil.

[0203] 2) Synthesis of compound H-Glu(PEG8)-OH

[0204] Boc-Glu(PEG8)-OtBu (600 mg, 0.897 mmol) was dissolved in a 1 / 1 DCM / TFA mixture (10 mL) and stirred for 1 hour. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was dissolved in DCM and repeatedly concentrated to remove residual TFA. This afforded H-Glu(PEG8)-OH (450 mg, 98% yield) as an oil.

[0205] 3) Synthesis of Compound S22-1

[0206] H-Glu(PEG8)-OH (300 mg, 0.585 mmol) and BCN-PNP (185 mg, 0.587 mmol) were dissolved in ultra-dry DMF (5 ml). Diisopropylethylamine (151 mg, 1.17 mmol) was added dropwise and stirred at room temperature for 12-16 hours. The reaction mixture was evaporated to dryness under reduced pressure and then slurried with MTBE to obtain compound S22-1 (350 mg, 87% yield) as an oily liquid.

[0207] 4) Synthesis of Compound S22

[0208] Compound S22-1 (100 mg, 0.145 mmol) and X3 (82 mg, 0.144 mmol) were dissolved in ultra-dry DMF (5 ml). HBTU (66 mg, 0.174 mmol) was added dropwise under an ice bath. Diisopropylethylamine (38 mg, 0.29 mmol) was then added dropwise. The mixture was stirred at room temperature for 2-3 hours. LC-MS confirmed the reaction was complete. The reaction mixture was filtered and the filtrate was purified by high-pressure reverse-phase preparative chromatography to afford S22 (69 mg, 38% yield) as a white solid.

[0209] Example 3: Synthesis of Compound S23

[0210] 1) Synthesis of compound Cbz-Glu(PEG8)-OtBu

[0211] Cbz-Glu(OH)-OtBu (334 mg, 0.991 mmol) and PEG8-NH2 (379 mg, 0.988 mmol) were dissolved in ultra-dry DMF (5 ml). HBTU (450 mg, 1.19 mmol) was added as a condensing agent. DIPEA (255 mg, 1.98 mmol) was added to the reaction mixture in an ice bath and stirred at room temperature for 2-3 hours. The reaction mixture was evaporated to dryness under reduced pressure, and the crude product was added to silica gel and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1 to 1 / 1) to afford Cbz-Glu(PEG8)-OtBu (610 mg, 88% yield) as an oil.

[0212] 2) Synthesis of compound H-Glu(PEG8)-OtBu

[0213] The compound Cbz-Glu(PEG8)-OtBu (600 mg, 0.854 mmol) was dissolved in a 1 / 1 MeOH / THF mixture (10 mL) and stirred for 1 hour. Pd / C (60 mg, 10 wt%) was then added and the reaction mixture was stirred for 2-3 hours. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure to obtain the oily liquid compound H-Glu(PEG8)-OtBu (480 mg, 99% yield).

[0214] 3) Synthesis of Compound S23-1

[0215] Compounds H-Glu(PEG8)-OtBu (400 mg, 0.703 mmol) and MI-C2-COOH (119 mg, 0.704 mmol) were dissolved in ultra-dry DMF (8 ml). The condensing agent HBTU (320 mg, 0.844 mmol) was added. DIPEA (181 mg, 1.40 mmol) was added to the reaction mixture in an ice bath, and the mixture was stirred at room temperature for 2-3 hours. The reaction mixture was evaporated to dryness under reduced pressure, and the crude product was added to silica gel and purified by column chromatography (dichloromethane:methanol = 100 / 1 to 20 / 1) to afford compound S23-1 (400 mg, yield: 79%) as an oil.

[0216] 4) Synthesis of Compound S23-2

[0217] Compound S23-1 (400 mg, 0.556 mmol) was dissolved in a 1 / 1 DCM / TFA mixture (10 mL) and stirred for 2-3 h. The reaction solution was evaporated to dryness under reduced pressure, and the residue was dissolved in DCM and repeatedly concentrated to remove residual TFA, yielding compound S23-2 (360 mg, 97.5% yield) as an oil.

[0218] 5) Synthesis of Compound S23

[0219] Compound S23-2 (100 mg, 0.150 mmol) and X3 (80 mg, 0.141 mmol) were dissolved in ultra-dry DMF (5 ml). HBTU (65 mg, 0.172 mmol) was added dropwise under an ice bath. Diisopropylethylamine (36 mg, 0.28 mmol) was then added dropwise, and the mixture was stirred at room temperature for 2-3 hours. LC-MS confirmed the reaction was complete. The reaction mixture was filtered, and the filtrate was purified by high-pressure reverse-phase preparative chromatography to afford S23 (70 mg, yield: 41%) as a white solid. LCMS: [M+H] = 1213.64.

[0220] Example 4: Synthesis of Compound S24

[0221] Compound S23-2 (100 mg, 0.151 mmol) was dissolved in an appropriate amount of DMF, followed by the addition of compound X2 (100 mg, 0.140 mmol) and condensing agent HBTU (63 mg, 0.166 mmol). DIPEA (40 mg, 0.31 mmol) was added to the reaction mixture under an ice bath, and the mixture was slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly stripped of solvent under high vacuum, diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to afford S24 (50 mg, 26% yield), a white powder. LCMS: [M+H] = 1362.69.

[0222] Example 5: Preparation of Compound S25

[0223] 1) Compound S25-1 (2 g, 4.9 mmol), HBTU (2.24 g, 5.9 mmol), DIPEA (3.2 g, 24.5 mmol) and compound S25-2 (2.24 g, 5.9 mmol) were dissolved in DMF (30 mL). The mixture was stirred at room temperature for 3 h. The reaction was complete as determined by TLC. Water (20 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried to obtain the target compound S25-3 (3.5 g, yield 89.7%).

[0224] 2) Compound S25-3 (3.5 g, 4.4 mmol) was dissolved in 1% DBU in DCM (40 mL), and the mixture was stirred at room temperature for 1.5 h. The reaction was confirmed to be complete by TLC. The organic phase was dried and mixed with silica gel. The target product S25-4 (2.35 g, 92.1% yield) was obtained by normal phase column separation.

[0225] 3) Compound S25-4 (2.35 g, 4.06 mmol), DEPBT (1.7 g, 5.7 mmol), DIEA (1.5 g, 12 mmol) and compound S25-5 (1.77 g, 5.7 mmol) were dissolved in DMF (30 mL), and the mixture was stirred at room temperature for 3 h. The reaction was confirmed to be complete by TLC. Water (20 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water, then with saturated brine, and dried over anhydrous sodium sulfate. The organic phase was spin-dried and separated on a silica gel column to give the target product S25-6 (2.5 g, yield 71.4%).

[0226] 4) Compound S25-6 (1.5 g, 1.72 mmol) was added to 15 mL of a solution of hydrogen chloride in dioxane, and stirred at room temperature for 2 hours. The reaction solution was concentrated and dried to give the product compound S25-7 (1.2 g, yield 85.7%).

[0227] 5) To a solution of compound S25-7 (1.2 g, 1.47 mmol) in 100 mL of DMF were added DIPEA (379 mg, 2.94 mmol), HBTU (1.1 g, 2.94 mmol), and compound S25-8 (516 mg, 1.76 mmol, TFA Salt). The reaction was stirred at 25°C for 0.5 h. The reaction solution was concentrated and purified by silica gel column chromatography (DCM / MeOH = 20:1) to afford compound S25-9 (800 mg, 54% yield).

[0228] 6) Compound S25-9 (800 mg, 0.806 mmol) and 0.1 eq of tetrakistriphenylphosphine palladium (73 mg) were placed in a dry three-necked flask. Anhydrous dichloromethane was added under nitrogen in an ice bath. The mixture was allowed to return to room temperature and allowed to react for 1 h. The mixture was filtered, concentrated, and purified by silica gel column chromatography (DCM / MeOH = 20:1) to obtain compound S25-10 (500 mg, 65% yield).

[0229] 7) Compound S25-10 (120 mg, 0.126 mmol) and compound X5 (76 mg, 0.098 mmol) were dissolved in DMF (3 mL). HBTU (57 mg, 0.150 mmol) and DIPEA (49 mg, 0.38 mmol) were added and reacted for 2 hours. The reverse reaction afforded compound S25 (57 mg, 26.3% yield) as a pale yellow solid. LCMS: [M+H] + =1713.81.

[0230] Example 6: Enzyme cleavage assay of S1-S59 drug conjugates

[0231] The stability and enzymatic cleavage efficiency testing methods for drug conjugate compounds are as follows, and the test results are shown in Table 7.

[0232] Accurately weigh each drug conjugate compound and add a sufficient amount of DMSO solution to a stock solution concentration of 10 μmol / mL. Then, a 10 μL aliquot was diluted to 1 μmol / mL by adding 80 μL of DMSO. This was then diluted to a sample solution concentration of 0.2 μmol / mL by adding purified water at a 1:4 ratio. After the sample clarified, it was placed in a 25°C / 37°C water bath. Samples were taken at 0 and 24 hours, and analyzed by HPLC (Agilent 1260, Eclipse Plus C18 column, 4.6 x 250 mm, 5 μm, mobile phase A: 0.1% TFA + H2O, mobile phase B: CH3CN, flow rate: 1.0 ml / min, column temperature: 30°C, detection wavelength: 214 nm). The drug conjugate content relative to the 0-hour concentration was used to obtain solution stability data for each compound.

[0233] An appropriate amount of sample was weighed and prepared into a 1 mM solution with DMSO. Human plasma was added at a volume ratio of 1:9 and the solution was incubated in a 37°C thermostat. Samples were taken after 0, 2, 4, 6, and 24 h. DMSO / MeOH (1:1) was added at a volume ratio of 1:3 to remove protein. The solution was centrifuged at 12,000 rpm for 5 min and the supernatant was collected. The HPLC method was as described above (Agilent 1260, chromatographic column: Eclipse Plus C18, 4.6*250 mm, 5 μm, mobile phase A: 0.1% TFA+H2O, mobile phase B: CH3CN, flow rate: 1.0 ml / min, column temperature: 30°C, detection wavelength: 214 nm).

[0234] Weigh a certain amount of drug conjugate, dissolve it, and dilute it tenfold to a concentration of 0.1mM / ml. Add the drug conjugate at a concentration of 1mg / ml to 100μg of acidified CT-26 tumor tissue homogenate (pH 5.0) at 37°C. The enzyme in the tumor tissue homogenate can be released and detected by HPLC to compare the activation efficiency of the linker by the tumor tissue.

[0235] Table 7

[0236] Control compound C

[0237] As can be seen from Table 7, compounds S1-S59 of the present invention have high stability, with 24-hour aqueous solution stability exceeding 85%, 24-hour plasma stability exceeding 75%, and 2-hour enzymatic cleavage efficiency exceeding 90%. The control compound C (compound S1 disclosed in CN104262455B) has lower 24-hour aqueous solution stability and plasma stability than compounds S1-S59 of the present invention, and its 2-hour enzymatic cleavage efficiency is lower than that of compounds S1-S59 of the present invention. This is because the amide bond and urethane bond between the linker and the payload in the present invention are much more stable than the carbonate bond in the control compound C, while the ester bond is more susceptible to hydrolysis.

[0238] These results demonstrate that compounds S1-S59 provided herein exhibit enhanced stability in aqueous solution and plasma, are less susceptible to degradation or deterioration into other compounds upon entry into the body, and exhibit a stable targeted therapeutic effect upon entry into tumor cells or tumor tissue. Furthermore, compounds S1-S59 provided herein exhibit enhanced cell-killing efficacy by releasing toxic drugs after entry into tumor cells and cleavage by specific enzymes within 2 hours.

[0239] Example 7: Comparison of activation and release of Dxd by S5, S16, and control compound Y

[0240] A certain amount of drug conjugates (S5, S16, Deruxtecan, and control compound Y, where control compound Y is compound S1 in CN103011521B) were weighed, dissolved, and diluted tenfold to a concentration of 0.1 mmol / ml. The drug conjugates were added to 100 μg of acidified CT-26 tumor tissue homogenate (pH 5.0) at 37°C at a concentration of 1 mg / ml. The enzyme in the tumor tissue homogenate was released and detected by HPLC to compare the activation efficiency of the conjugates by the tumor tissue.

[0241] The results are shown in Figures 1 to 4. Figure 1 shows that the control compound Y begins to release Leudox at 0.5 hours under the action of the enzyme (no Leudox release was detected at 5 minutes), and the release is complete in about 2 hours. Figure 2 shows that compound S5 begins to release T785 at 5 minutes. Figure 3 shows that compound S16 begins to release Dxd at 5 minutes. Figure 4 shows that Deruxtecan begins to release Dxd at 6 hours under the action of Cathepsin B enzyme.

[0242] Therefore, the activation efficiency of compounds S5 and S16 was significantly better than that of the control compounds Y and Deruxtecan.

[0243] Example 8: Comparison of lung tissue stability between S16 and Deruxtecan

[0244] method:

[0245] Preparation of experimental solution:

[0246] Assay buffer: 25 mM MES, pH 5.0.

[0247] Protein precipitant: DMSO / MEOH = 1:1.

[0248] Preparation of sample solution: Weigh appropriate amounts of 6PEG-lys(MI-6PEG)-AAN-AM-Dxd and EMC-GGFG-AM-Dxd, add a certain amount of DMSO solution to make the stock solution concentration 10umol / mL, then take 10uL and add 80uLDMSO to dilute to 1umol / mL, then add pure water at a ratio of 1:4 to dilute to a sample solution concentration of 0.2umol / mL.

[0249] Preparation of lung tissue homogenate:

[0250] Take an appropriate amount of liquid nitrogen-frozen lungs and place them in a 4 mL EP tube. Weigh them and add pH 5.0 detection buffer at a mass-to-volume ratio of 1:5. Use a tissue grinder to homogenize the tissue for 3 minutes. Then centrifuge the lung tissue homogenate at 12,000 rpm for 5 minutes, take the supernatant, and place it in a -40°C refrigerator for later use.

[0251] Stability of lung tissue homogenate:

[0252] Take the sample solution and lung tissue homogenate in a volume ratio of 1:9 and shake them thoroughly. Take another sample solution and buffer solution (pH 5.0) in a volume ratio of 1:9 and shake them thoroughly as a blank control solution. Place it in a 37°C constant temperature water bath. Take 30uL at 0h, 2h, 4h, and 20h, add 90uL protein precipitant and shake well. Centrifuge at 12000rpm for 5min, take the supernatant into the injection bottle, and inject it into HPLC.

[0253] The percentage of DXD released after the drug enters the lung tissue homogenate is calculated using the following formula:

[0254] Drug loading% = (Compound molar amount - newly generated Dxd molar amount) / Compound molar amount * 100%

[0255] The results are shown in Figures 5 and 6 and Table 8 below.

[0256] Table 8

[0257] The results showed that S16 produced less Dxd in lung tissue than Deruxtecan and had less impact on normal lung tissue.

[0258] Example 9: Preparation of conjugate intermediates L1-L51

[0259] Some compounds were prepared using the following reaction scheme:

[0260] 1) Compound Fmoc-R2'-OH (1.0 eq) was dissolved in an appropriate amount of DMF, followed by the addition of compound D-NH2 and the condensing agent HBTU. DIPEA was added to the reaction mixture while it was ice-cooled, then slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly stripped of solvent under high vacuum, diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to obtain the desired product.

[0261] 2) Dissolve compound Fmoc-R2'-D (1.0 eq) in DMF and stir for 30 min. Then add piperidine (3.0 eq) and continue stirring at room temperature for 2-3 h. Evaporate the reaction mixture to dryness under reduced pressure, and slurry the residue with a mixture of MTBE to obtain the desired product, NH2-R2'-D.

[0262] The compounds shown in Table 9 can be obtained using the corresponding raw materials according to the above method. The properties of each compound in Table 9 are shown in Table 10.

[0263] Table 9

[0264] Table 10

[0265] Some compounds were prepared using the following reaction scheme:

[0266] 1) Compound Cbz-R2'-OH (1.0 eq) was dissolved in an appropriate amount of DMF, followed by the addition of compound D-NH2 and the condensing agent HBTU. DIPEA was added to the reaction mixture while it was ice-cooled, then slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly stripped of solvent 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 desired product.

[0267] 2) Compound Cbz-R2'-D (1.0 eq) was dissolved in methanol and stirred for 30 min. Pd / C (0.1 eq) was then added and the reaction mixture was stirred at room temperature for 2-3 h. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was slurried with a mixture of MTBE and ethyl acetate to obtain the desired product, NH2-R2'-D.

[0268] The compounds shown in Table 11 can be obtained using the corresponding raw materials according to the above method. The properties of each compound in Table 11 are shown in Table 12.

[0269] Table 11

[0270] Table 12

[0271] Compounds L14 and L23 were prepared using the following method:

[0272] Compound L14-1 (100 mg, 0.299 mmol) was dissolved in an appropriate amount of DMF, followed by the addition of compound X4 (250 mg, 0.297 mmol) and condensing agent HBTU (136 mg, 0.359 mmol). DIPEA (116 mg, 0.899 mmol) was added to the reaction mixture under an ice bath, which was then slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly stripped of solvent under high vacuum, diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to afford L14 (100 mg, 29% yield), a light yellow powder. LCMS: [M+H] = 1157.46.

[0273] By replacing X4 with X5, compound L23 was prepared under the same reaction conditions.

[0274] Compounds L13 and L22 were prepared using the following method:

[0275] Compound diglycolic anhydride (14 mg, 0.12 mmol) was dissolved in an appropriate amount of DMF solvent, followed by the addition of compound X4 (100 mg, 0.119 mmol). DIPEA (31 mg, 0.24 mmol) was added to the reaction mixture under an ice bath, and the mixture was slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly freed of solvent under high vacuum, then diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to obtain a light yellow powder L14 (80 mg, 70% yield). LCMS: [M+H] = 957.34

[0276] By replacing X4 with X5, compound L22 can be prepared under the same reaction conditions.

[0277] Compounds L28 and L29 were prepared using the following method:

[0278] Compound succinic anhydride (3 mg, 0.03 mmol) was dissolved in an appropriate amount of DMF solvent, followed by the addition of compound L26 (20 mg, 0.015 mmol). DIPEA (31 mg, 0.24 mmol) was added to the reaction mixture under an ice bath, and the mixture was slowly returned to room temperature and stirred for 3-5 hours. The resulting reaction mixture was mostly freed of solvent under high vacuum, then diluted with an appropriate amount of methanol, purified by reverse-phase high-pressure preparative chromatography, and finally lyophilized to afford a light yellow powder L28 (20 mg, 95% yield). LCMS: [M+H] = 1431.65

[0279] L26 was replaced by L27 and compound L29 was prepared under the same reaction conditions.

[0280] Example 10: Preparation of Compounds X1-X5

[0281] Synthesis of compound X1:

[0282] 1) Synthesis of Compound X1-1

[0283] The compound Fmoc-Ala-Ala-Asn-OH (500 mg, 1.01 mmol) and resiquimod (314 mg, 1.00 mmol) were dissolved in ultra-dry DMF (5 ml). The condensing agent HBTU (455 mg, 1.20 mmol) and diisopropylethylamine (387 mg, 3.00 mmol) were added, and the mixture was stirred at room temperature for 16 h. The reaction solution was evaporated to dryness under reduced pressure, slurried with a mixed solvent of ethanol and MTBE, and filtered to obtain compound X1-1 (500 mg, yield: 63%) as a white solid.

[0284] 2) Synthesis of Compound X1

[0285] Compound X1-1 (500 mg, 0.631 mmol) was dissolved in DMF (10 mL) and stirred for 30 min. Piperidine (160 mg, 1.88 mmol) was then added and the reaction mixture was stirred at room temperature for 2-3 h. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was slurried with a mixture of MTBE to obtain Compound X1 (300 mg, 83% yield) as a white solid.

[0286] Synthesis of compound X2:

[0287] 1) Synthesis of the compound Fmoc-Ala-Ala-Asn-PABC-PNP

[0288] The compounds Fmoc-Ala-Ala-Asn-PABC (800 mg, 1.33 mmol) and Bis-PNP (808 mg, 2.66 mmol) were dissolved in ultra-dry DMF (5 ml), and diisopropylethylamine (343 mg, 2.66 mmol) was added and stirred at room temperature for 16 h. The reaction solution was evaporated to dryness under reduced pressure, and then slurried and filtered with a mixed solvent of ethanol and MTBE to obtain a white solid compound Fmoc-Ala-Ala-Asn-PABC-PNP (820 mg, yield: 80%).

[0289] 2) Synthesis of Compound X2-1

[0290] The compound Fmoc-Ala-Ala-Asn-PABC-PNP (200 mg, 0.261 mmol) was dissolved in DMF solvent (20 mL) and stirred for 1 h. Then, the compound T785 (80 mg, 0.257 mmol) and diisopropylethylamine (60 mg, 0.465 mmol) were added. The reaction solution was evaporated to dryness under reduced pressure, and the residue was slurried with a mixed solvent of MTBE to give a white solid compound X2-1 (200 mg, yield 83%).

[0291] 3) Synthesis of Compound X2

[0292] Compound X2-1 (200 mg, 0.213 mmol) was dissolved in DMF solvent (5 mL) and stirred for 1 h. Then piperidine (55 mg, 0.647 mmol) was added and the reaction solution was evaporated to dryness under reduced pressure. The residue was slurried with a mixed solvent of MTBE to obtain a white solid compound X2 (110 mg, yield 72%).

[0293] Synthesis of compound X3:

[0294] 1) Synthesis of Compound X3-1

[0295] Compounds Fmoc-Ala-Ala-Asn-OH (450 mg, 0.907 mmol) and T785 (188 mg, 0.605 mmol) were dissolved in ultra-dry DMF (5 ml), and condensing agent DEPBT (217 mg, 0.726 mmol) and diisopropylethylamine (234 mg, 1.82 mmol) were added and stirred at room temperature for 16 h. The reaction solution was evaporated to dryness under reduced pressure and then slurried and filtered with a mixed solvent of ethanol and MTBE to obtain a white solid compound X3-1 (300 mg, yield: 63%).

[0296] 2) Synthesis of Compound X3

[0297] Compound X3-1 (300 mg, 0.380 mmol) was dissolved in DMF solvent (20 mL) and stirred for 1 h. Then piperidine (100 mg, 1.18 mmol) was added and the reaction solution was evaporated to dryness under reduced pressure. The residue was slurried with a mixed solvent of MTBE to obtain a white solid compound X3 (200 mg, yield 93%).

[0298] Synthesis of compound X4:

[0299] 1) Synthesis of Compound X4-1

[0300] Compound Fmoc-Ala-Ala-Asn-PABC-PNP (500 mg, 0.652 mmol) and isotecan mesylate (346 mg, 0.652 mmol) were dissolved in ultra-dry DMF (8 ml). Diisopropylethylamine (168 mg, 1.30 mmol) was added and stirred at room temperature for 16 h. The reaction solution was evaporated to dryness under reduced pressure and then slurried and filtered with a mixture of ethyl acetate and MTBE to afford compound X4-1 (600 mg, 86.6% yield) as a yellow solid.

[0301] 2) Synthesis of Compound X4

[0302] Compound X4-1 (600 mg, 0.564 mmol) was dissolved in DMF (10 mL) and stirred for 1 h. Piperidine (144 mg, 1.69 mmol) was then added. The reaction mixture was allowed to continue for 2 h, monitored by TLC, and terminated upon completion. The product was evaporated to dryness under reduced pressure, and the residue was slurried with a mixture of MTBE and ethyl acetate to afford Compound X4 (450 mg, 95% yield) as a pale yellow solid.

[0303] Synthesis of compound X5:

[0304] 1) Synthesis of Compound X5-1

[0305] 1) Compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (500 mg, 0.857 mmol) and isotecan methanesulfonate (455 mg, 0.857 mmol) were dissolved in DMF and cooled to 0°C. Under nitrogen, the condensing agent DEPBT (307 mg, 1.03 mmol) and the organic base DIPEA (332 mg, 2.57 mmol) were added sequentially, and the mixture was stirred at room temperature for 2-3 hours. TLC indicated that the reaction was complete. The reaction solution was concentrated in vacuo, redissolved in DMF, filtered, and purified by high-pressure liquid chromatography to obtain compound X5-1 as a pale yellow product (510 mg, 60% yield).

[0306] 2) Synthesis of Compound X5

[0307] Compound X5-1 (500 mg, 0.50 mmol) was dissolved in DMF, and piperidine (128 mg, 1.51 mmol) was added. The mixture was stirred at room temperature under nitrogen for 2-3 hours. TLC indicated that the reaction was complete. The reaction solution was redissolved, filtered, and then purified by high-pressure liquid chromatography to obtain compound X5 as a pale yellow product (300 mg, 77% yield).

[0308] Example 11: Preparation of Antibody Drug Conjugates

[0309] The antibody-drug conjugate is prepared by adding tris(2-carboxyethyl)phosphine (TCEP) (1-40 eq) to an antibody buffer for reduction (0.5-16 hours), then adding the aforementioned drug conjugate (2-40 eq) for reaction (0.5-16 hours) to form the antibody-drug conjugate. The pure antibody-drug conjugate is then purified by column chromatography and filtration.

[0310] The following ADCs were prepared using the above method: trastuzumab-S15 (HER2-S15), anti-LY6G6D antibody-S15 (LY6G6D-PEG6-AAN-AM-DXD), atezolizumab-S5 (PD-L1-TLR7 / 8), atezolizumab-S16 (PD-L1-PEG-K(MI-PEG)-AAN-AM-DXd), and anti-GCC antibody-S16. The DAR value of each ADC was approximately 8. Note: The names in parentheses correspond to those in the figure.

[0311] In the above-mentioned ADCs, "HER2" refers to trastuzumab (Caihui Ruizhi Biotechnology (Shanghai) Co., Ltd.); "PD-L1" refers to atezolizumab (the amino acid sequences of its light chain variable region and heavy chain variable region are shown in SEQ ID NOs: 1 and 2, respectively; the light chain and heavy chain are shown in SEQ ID NOs: 3 and 4, respectively); "Anti-SSTR2" refers to an anti-SSTR2 antibody; "LY6G6D" refers to an anti-LY6G6D antibody (the amino acid sequences of its light chain variable region and heavy chain variable region are shown in SEQ ID NOs: 5 and 6, respectively); the amino acid sequences of the light chain variable region and heavy chain variable region of the anti-GCC antibody are shown in SEQ ID NOs: 7 and 8, respectively.

[0312] In the above ADC, the drug conjugates used are as follows:

[0313] Affinity linker2=EMC-PEG6-AAN-AM-Dxd(S15);

[0314] Affinity linker4=EMC-C5-AAN-T785(S5).

[0315] Affinity linker 5 = PEG-K(MI-PEG)-AAN-AM-DXd (S16) Figure 7 shows the hydrophobic interaction chromatography (HIC) of trastuzumab-S15; Figure 8 shows the MS spectrum of atezolizumab-S5; Figure 9 shows the HIC spectrum of atezolizumab-S5; and Figure 10 shows the MS spectrum of atezolizumab-S5.

[0316] Example 12

[0317] 1. Animals: 8-week-old female BALB / c mice (Shanghai Lingchang Biotechnology Co., Ltd.).

[0318] 2. Cells: Cells were obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every 3 days and used within 15 generations.

[0319] 3. Tumor production: 0.3×10 6 CT26 cells were injected subcutaneously into the back of BALB / c mice and the tumors were grown to 160 mm. 3 The patients were randomly divided into groups and treatment was started.

[0320] 4. Treatment process: Administer the drug through the tail vein once a week for three weeks.

[0321] The groups and outcome measures are shown in Table 13 and Figure 11.

[0322] Table 13

[0323] In the present invention, TGI% is the tumor growth inhibition rate of the drug. TMEA-TLR7 / 8 is compound S5.

[0324] Example 13

[0325] 1. Animals: 8-week-old female BALB / c mice (Shanghai Lingchang Biotechnology Co., Ltd.).

[0326] 2. Cells: Cells were obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every 3 days and used within 15 generations.

[0327] 3. Tumor production: 0.3×10 6 CT26 cells were injected subcutaneously into the back of BALB / c mice and the tumors were grown to 160 mm. 3 The patients were randomly divided into groups and treatment was started.

[0328] 4. Treatment process: Administer through tail vein, CTLA-4 twice a week, TMEA-TRL7 / 8 once a week, for a total of three weeks.

[0329] The groups and outcome measures are shown in Table 14 and Figure 12 .

[0330] Table 14

[0331] Example 14

[0332] 1. Animals: NCG mice, 8-week-old female (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

[0333] 2. Cells: Cells were obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every 7 days and used within the 15th generation.

[0334] 3. Tumor production: 5×10 6 NCI-N87 cells were injected subcutaneously into the back of NCG mice and the tumors were grown to 100 mm. 3 The patients were randomly divided into groups and treatment was started.

[0335] 4. Treatment process: Administer the drug through the tail vein, once.

[0336] The groups and outcome measures are shown in Table 15 and Figure 13 .

[0337] Table 15

[0338] Example 15

[0339] 1. Animals: 8-week-old female BALB / c mice (Shanghai Lingchang Biotechnology Co., Ltd.).

[0340] 2. Cells: Wuhan Huamei Biotechnology Co., Ltd. Cells were cultured in RPMI Medium 1640 (1x) containing 10% fetal bovine serum and 10 μg / ml Puromecin (10 mg / ml). The cells were cultured at 37°C in 5% CO2 and passaged every 3 days. Cells were used within 15 generations.

[0341] 3. Tumor production: 0.3×10 6 CT26 cells were subcutaneously injected into the back of BALB / c mice and the tumors were grown to 100 mm. 3 The patients were randomly divided into groups and treatment was started.

[0342] 4. Treatment course: Intraperitoneal administration, twice a week for 3 weeks.

[0343] The groups and outcome measures are shown in Table 16 and Figure 14.

[0344] Table 16

[0345] Example 16

[0346] 6-8 week old female C57BL / 6J-Pdedleml (hPDCD1) / Smoc mice were purchased from Shanghai Model Organism Center. After the mice arrived and were unpacked, they were housed in an SPF animal breeding center at a density of 5 per cage. The general condition of the animals (mental state, activity, hair color) was observed, and the weight of the first week was recorded. MC38 (mPD-L1 knockout; hPD-L1 knockin) cells were harvested during the logarithmic growth phase and placed in sterile saline before inoculation of the animals. 1×10 6 The cells were subcutaneously inoculated in the upper groin of the lateral hind limb of mice. The administration method was: Gr1-control PBS (twice a week, for a total of 2 times); Gr2-PDL1-PEG-K(MI-PEG)-AAN-AM-DXd (10 mg / kg, twice a week, for a total of 2 times); Gr3-Atezolizumab (10 mg / kg, twice a week, for a total of 2 times). Tumor volume and mouse body weight were measured twice a week, where tumor volume was calculated using the formula (0.5×L×W 2 ), where L and W refer to the length and width of the tumor, respectively. 3At the end of the experiment, the mice were killed by cervical dislocation.

[0347] The results are shown in FIG15 and Table 17 below.

[0348] Table 17

[0349] As shown in Figure 15 and Table 17, the TGI of the drug PDL1-PEG-K(MI-PEG)-AAN-AM-DXd in the MC38 tumor model was 98.6% on day 18, and PDL1-PEG-K(MI-PEG)-AAN-AM-DXd completely inhibited tumor growth between days 7 and 18. Therefore, the drug PDL1-Dxd exhibited a good tumor inhibitory effect in the MC38 tumor model.

[0350] Example 17

[0351] 1. Animals: NCG mice, 8-week-old female (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.).

[0352] 2. Cells: Cells were obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in DMEM (1x) containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were passaged every 3 days and were used within 15 generations.

[0353] 3. Tumor production: 5×10 6 MDA-MB-231 cells were subcutaneously injected into the back of NCG mice and the tumors were grown to 1000 mm. 3 The patients were randomly divided into groups and treatment was started.

[0354] 4. Treatment process: Administer the drug through the tail vein, once.

[0355] The groups and outcome measures are shown in Table 18 and Figure 16 .

[0356] Table 18

[0357] Example 18: In vivo characterization of GCC TMEAbody in a mouse tumor model

[0358] Human colon cancer cell line HT-55 was subcutaneously transplanted into NDG mice to establish an in vivo model. HT-55 cells were cultured according to the culture conditions, and cells were collected and counted during the exponential growth phase. Each mouse was inoculated with 1×10 6 Human colon cancer cells HT-55 (suspended in 0.1 ml of basal medium). After inoculation, wait for the tumor to grow to 56-86 mm. 3At 7 days after tumor loading, mice were randomly divided into PBS group and 7B6-S16 (5 mg / kg) group according to their body weight and tumor volume.

[0359] Treatment started on the day of grouping. The mice were weighed and the tumor volume was measured twice a week. The volume was calculated using the formula: tumor volume = 1 / 2 × length × width × width (mm 3 Tumor inhibition rate (TGI) (%) = [1-(Tt-T0) / (Ct-C0)] × 100%; tumor inhibition rate (T / C) (%) = (Tt / T0) / (Ct / C0) × 100%. T0 is the tumor volume of the experimental group at the time of grouping; Tt is the tumor volume of the experimental group during each measurement; C0 is the average tumor volume of the PBS group at the time of grouping; Ct is the average tumor volume of the PBS group during each measurement.

[0360] The results are shown in Figure 17. 7B6-S16 demonstrated significant tumor growth inhibition in a subcutaneous HT-55 xenograft mouse model. After 35 days of treatment, the 7B6-S16 (5 mg / kg) group achieved a TGI inhibition rate of 106% and a T / C ratio of 2%. These results demonstrate that 7B6-S16 can activate and stimulate anti-tumor immune responses within the tumor microenvironment.

[0361] Example 19

[0362] 1. Animals: Nude mice, 6-8 weeks old, all female (Shanghai Lingchang Biotechnology Co., Ltd.).

[0363] 2. Cells: Cells were purchased from the American Type Culture Collection (ATCC) and authenticated according to ATCC instructions. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every 3 days and used within 15 passages.

[0364] 3. Tumor generation, 1.5×10 6 HT1080 cells were subcutaneously injected into the back of nude mice. When the tumor grew to about 100 mm3, the mice were randomly divided into groups and treatment began. The day of treatment was regarded as the first day.

[0365] 4. Treatment Process

[0366] The dose was 5 μmol / Kg and administered once a week for 3 weeks.

[0367] The groups and outcome measures are shown in Table 19.

[0368] Table 19: Effects of corresponding compounds and control groups on tumor inhibition

[0369] 5. Results and Discussion: The compounds S26, S41, S43, S49, S57 and S58 of the present invention have greatly improved therapeutic effects compared to the exotecan derivatives and can almost achieve the effect of curing tumors.

[0370] Example 20

[0371] 1. Experimental purpose: To understand the acute toxicity of the drug of the present invention in vivo by determining the MTD experiment of intravenous administration in mice.

[0372] 2. Test drugs: Exitecan derivatives, compounds S26, S41, S43, S49, S57 and S58 were dissolved in DMSO and diluted with physiological saline to the corresponding dose during the test.

[0373] 3. Animals: BALB / C mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), weighing 19-21 g, all female.

[0374] 4. Methods and Results: Forty-eight female BALB / C mice, weighing 19-21 g, were randomly divided into seven groups of six mice each. A single intravenous injection of an ixetidecan derivative, compounds S26, S41, S43, S49, S57, and S58 was administered at the doses shown in Table 20. A saline control group was also administered, with each mouse receiving a 0.2 ml dose. The animals were observed daily for 17 days for signs of erect piloerection, matted luster, lethargy, hunched back, or hyperreactivity. Body weight and mortality were recorded. Blood samples were collected for complete blood counts on days 3, 5, and 14. On day 14, the animals were dissected and the heart, liver, kidneys, lungs, spleen, and pancreas were analyzed for hematoxylin and eosin staining.

[0375] Table 20: Comparison of mortality rates of mice receiving different doses of compound injections and saline

[0376] 5. Results and Discussion: Compared with the ixetine derivatives, when the animals were injected with S26, S41, S43, S49, S57 and S58 of the present invention, there was no evidence of piloerection, matted luster, lethargy, hunchback, overreaction or death, indicating that the toxicity of the small molecule conjugated drug was significantly lower than that of the unconjugated drug.

[0377] Example 21: Ring opening method after S15 coupling to albumin and antibody

[0378] Coupling process: 1.8 mL of albumin solution (1.43 μmol / mL) was measured and placed into a 5 mL PEEK tube, and then 2 mL of S15 (2 μmol / mL) was added and mixed thoroughly. The mixture was placed at room temperature. After 2 hours, the mixture was dialyzed overnight using a 10K dialysis card with 10 mM NaH2PO4 buffer (pH 5.0) to obtain a compound coupled to albumin.

[0379] Ring-opening process: After coupling the compound to albumin, incubate at 25-50°C for 24-60 hours in 30-80 mM sodium bicarbonate buffer at a pH of 5.0-10.0. After incubation, ring-opening was detected using 3K ultrafiltration tubes and LC-MS high-resolution mass spectrometry. The molecular weight after coupling to albumin was 67702.78, and the molecular weight after ring-opening of the albumin-coupled compound was 67721.57.

[0380] Experimental steps for plasma stability of compounds after coupling and ring opening process:

[0381] 1. Measure 20 μL of HSA-Compound and HSA-Compound-opened ring sample (hereinafter referred to as HSA-Compound-OR) (0.5 μmol / mL) and add them to 380 μL of human plasma, mix thoroughly, and place in a 37°C water bath;

[0382] 2. Take 50uL of the mixed solution at 0h, 2h, 4h, 24h, 48h and 96h, add 150uL of protein precipitant (CH3OH / DMSO=1:1), centrifuge at 12000rpm for 5min, and take the supernatant for HPLC injection.

[0383] The percentage of DXD released after the drug enters the lung tissue homogenate is calculated using the following formula:

[0384] The results are shown in Figure 18.

[0385] Example 22: Preparation of bispecific antibody-drug conjugates

[0386] The bispecific conjugate drug can target two antigens simultaneously, and its targeting mode is the two forms shown in FIG19 , wherein Anti-Antigen1 is selected from any one of EGFR, Trop2, and Cmet; Anti-Antigen2 is selected from any one of EGFR, Trop2, and Cmet.

[0387] In the prepared bispecific antibody drug conjugate, the heavy chain amino acid sequence of the bispecific antibody in EGFR / Cmet-T785 is shown in SEQ ID NO: 9, and the light chain amino acid sequence is shown in SEQ ID NO: 11; the heavy chain amino acid sequence of the bispecific antibody in EGFR / Cmet-Dxd is shown in SEQ ID NO: 10, and the light chain amino acid sequence is shown in SEQ ID NO: 12; the light chain amino acid sequence of the bispecific antibody in EGFR / Trop2 is shown in SEQ ID NO: 13, and the heavy chain amino acid sequence is shown in SEQ ID NO: 14.

[0388] 1. EGFR / Cmet-S5:

[0389] 2.0×10 6 NCI-H292 cells expressing human HGF were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in a total volume of 100 μL into the right flank of SCID Beige mice. Treatment was initiated when tumors reached an average size of 139 mm³. The dose of test article and the number of animals in each study group are shown in Table 21 below. The date of tumor cell inoculation was designated as Day 0.

[0390] Table 21

[0391] Before starting treatment, all animals were weighed and tumor volume was measured. Because tumor volume can influence the effectiveness of any given treatment, mice were assigned to groups using a randomized block design based on tumor volume. This ensured that all groups were comparable at baseline. A randomized block design was used to assign experimental animals to groups. First, the experimental animals were divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals were randomized to a specific treatment. Using a randomized block design to assign experimental animals ensures that each animal has an equal probability of being assigned to a given treatment, thereby reducing systematic error.

[0392] During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimation of food and water consumption, weight gain / loss (weight was measured twice weekly), eye / hair matting, and any other abnormal effects.

[0393] The endpoint of the study was whether the tumor growth could be delayed or whether the tumor-bearing mice could be cured. Tumor size was measured twice a week in two dimensions using calipers, and volume was expressed in mm. 3 The formula is: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively.

[0394] The results are shown in Figure 20. The results show that on day 18, the TGI of EGFR / Cmet-T785 in the NCI-H292 tumor model was 76.9%, indicating that EGFR / Cmet-S5 can significantly slow tumor growth and effectively prolong the survival of mice. Therefore, the drug EGFR / Cmet-S5 demonstrated a good tumor-suppressing effect in the NCI-H292 tumor model.

[0395] 2. EGFR / Cmet-S16:

[0396] 2.0×10 6 NCI-H292 cells expressing human HGF were mixed with Matrigel in a 1:1 ratio in PBS and inoculated subcutaneously in the right flank of SCID Beige mice in a total volume of 100 μL. Treatment was initiated when tumors reached an average size of 139 mm³. The dose of test article and the number of animals in each study group are shown in Table 22 below. The date of tumor cell inoculation was designated as Day 0.

[0397] Table 22

[0398] Before starting treatment, all animals were weighed and tumor volume was measured. Because tumor volume can influence the effectiveness of any given treatment, mice were assigned to groups using a randomized block design based on tumor volume. This ensured that all groups were comparable at baseline. A randomized block design was used to assign experimental animals to groups. First, the experimental animals were divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals were randomized to a specific treatment. Using a randomized block design to assign experimental animals ensures that each animal has an equal probability of being assigned to a given treatment, thereby reducing systematic error.

[0399] During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimation of food and water consumption, weight gain / loss (weight was measured twice weekly), eye / hair matting, and any other abnormal effects.

[0400] The endpoint of the study was whether the tumor growth could be delayed or whether the tumor-bearing mice could be cured. Tumor size was measured twice a week in two dimensions using calipers, and volume was expressed in mm. 3 The formula is: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively.

[0401] The results are shown in Figure 21. The results show that on day 18, the TGI of the drug EGFR / Cmet-S16 in the NCI-H292 tumor model was 87.2%, and EGFR / Cmet-S16 completely inhibited tumor growth between days 7 and 18. Therefore, the drug EGFR / Cmet-S16 exhibited a good tumor inhibitory effect in the NCI-H292 tumor model.

[0402] 3. EGFR / Trop2-S5:

[0403] 2.0×10 6 NCI-H292 (TROP2 high EGFR high ) cells were mixed with Matrigel in a 1:1 ratio in PBS, and a total volume of 100 μL was subcutaneously inoculated into the right flank of SCID Beige mice. Treatment was initiated when tumors reached an average size of approximately 100 mm³. The dosage of the test article and the number of animals in each study group are shown in Table 23 below. The date of tumor cell inoculation was designated as Day 0.

[0404] Table 23

[0405] Before starting treatment, all animals were weighed and tumor volume was measured. Because tumor volume can influence the effectiveness of any given treatment, mice were assigned to groups using a randomized block design based on tumor volume. This ensured that all groups were comparable at baseline. A randomized block design was used to assign experimental animals to groups. First, the experimental animals were divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals were randomized to a specific treatment. Using a randomized block design to assign experimental animals ensures that each animal has an equal probability of being assigned to a given treatment, thereby reducing systematic error.

[0406] During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimation of food and water consumption, weight gain / loss (weight was measured twice weekly), eye / hair matting, and any other abnormal effects.

[0407] The endpoint of the study was whether the tumor growth could be delayed or whether the tumor-bearing mice could be cured. Tumor size was measured twice a week in two dimensions using calipers, and volume was expressed in mm. 3 The formula is: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively.

[0408] The results are shown in Table 24 below.

[0409] Table 24

[0410] 4. EGFR / Trop2-S16:

[0411] 2.0×10 6 NCI-H292 (TROP2 high EGFR high ) cells were mixed with Matrigel in PBS at a ratio of 1:1 and a total volume of 100 μL was subcutaneously inoculated into the right flank of SCID Beige mice. 3 Treatment was started around 2:00 p.m. The dosage of the test article and the number of animals in each study group are shown in Table 25 below. The date of tumor cell inoculation was designated as Day 0.

[0412] Table 25

[0413] Before starting treatment, all animals were weighed and tumor volume was measured. Because tumor volume can influence the effectiveness of any given treatment, mice were assigned to groups using a randomized block design based on tumor volume. This ensured that all groups were comparable at baseline. A randomized block design was used to assign experimental animals to groups. First, the experimental animals were divided into uniform blocks based on their initial tumor volume. Second, within each block, the experimental animals were randomized to a specific treatment. Using a randomized block design to assign experimental animals ensures that each animal has an equal probability of being assigned to a given treatment, thereby reducing systematic error.

[0414] During routine monitoring, animals were examined for tumor growth and any effects of treatment on normal behavior, such as activity, visual estimation of food and water consumption, weight gain / loss (weight was measured twice weekly), eye / hair matting, and any other abnormal effects.

[0415] The endpoint of the study was whether the tumor growth could be delayed or whether the tumor-bearing mice could be cured. Tumor size was measured twice a week in two dimensions using calipers, and volume was expressed in mm. 3 The formula is: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively.

[0416] The results are shown in Table 26 below.

[0417] Table 26

[0418] Through the above experiments, it was found that small molecule drugs or protein-coupled drugs whose linkers contain the AAN sequence can release the load more efficiently, thereby improving the therapeutic effect.

[0419] Example 23: Pharmacodynamics and safety evaluation of S41 (QHL-1618) on human fibrosarcoma cells HT-1080 in a BALB / c nude mouse subcutaneous tumor model

[0420] In this study, 32 female Balb / c nude mice were subcutaneously inoculated with human fibrosarcoma cells HT-1080 to construct a transplanted tumor model. 3 The animals were randomly divided into four groups, each with eight mice, and given vehicle (Vehicle), 2.47 mg / kg or 5 μmol / kg CPT-780 (positive control, S41 payload Dxd), 7.12 mg / kg or 5 μmol / kg S41, and 14.25 mg / kg or 10 μmol / kg S41, respectively. The drugs were injected into the tail vein at a volume of 10 ml / kg once a week for three weeks.

[0421] The results are shown in Figure 22. S41 showed a significant inhibitory effect on the growth of HT-1080 tumor cells. On day 38 of the experiment, 10 μmol / kg of S41 was an effective dose, and no significant drug toxicity was observed in mice.

[0422] Example 24: In vivo pharmacodynamic study of the test compound on human fibrosarcoma HT-1080 in a BALB / c nude mouse subcutaneous transplant tumor model

[0423] This study used 30 female Balb / c mice and subcutaneously inoculated human fibrosarcoma HT-1080 cells to construct a transplanted tumor model. The average volume of the transplanted tumor reached 161 mm. 3 The animals were randomly divided into five groups, with six mice in each group, and given lysozyme (Vehicle), 1.63 mg / kg or 3.3 μmol / kg CPT-780 (positive control, S41 payload Dxd), 1.57 mg / kg or 1.1 μmol / kg S41, 4.70 mg / kg or 3.3 μmol / kg S41, and 14.25 mg / kg or 10 μmol / kg S41, respectively. The drugs were injected into the tail vein at a volume of 10 μl / g once a week for 17 days.

[0424] The results are shown in Figure 23. The results showed that S41 exhibited a significant inhibitory effect on the growth of HT-1080 xenograft tumor cells. On day 17 of the experiment, tumor regression was observed in all mice (N=6) treated with S41 at 4.70 mg / kg and 14.25 mg / kg, with 80% of the mice in the S41 4.70 mg / kg and 14.25 mg / kg treatment groups, respectively, experiencing complete tumor regression to zero.

[0425] Example 25: Pharmacodynamics and safety evaluation of S41 against mouse colon cancer cell line CT-26 in a BALB / c mouse subcutaneous tumor model

[0426] This study used 30 female Balb / c mice and subcutaneously inoculated them with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 127 mm. 3 The animals were randomly divided into five groups, with six mice in each group, and given lysozyme (Vehicle), 3 mg / kg or 6 μmol / kg CPT-780 (positive control, S41 payload Dxd), 3 mg / kg or 6 μmol / kg S41, and 9 mg / kg or 1 μmol / kg S41, respectively. The drugs were injected into the tail vein at a volume of 10 ml / kg, once a week for four weeks.

[0427] The results are shown in Figure 24. The results show that S41 exhibited a significant inhibitory effect on the growth of CT-26 tumor cells. On day 27 of the experiment, S41 (18 μmol / kg) was an effective dose. At the same time, no significant drug toxicity was observed in mice.

[0428] Example 26: Pharmacodynamics and safety evaluation of combined treatment with S41 and anti-mPD-1 on mouse colon cancer cell CT-26 in a BALB / c mouse subcutaneous tumor model

[0429] This study used 24 female Balb / c mice and subcutaneously inoculated them with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 127 mm. 3 The mice were randomly divided into four groups, each with six mice. Each group was given lysozyme (Vehicle), 9 mg / kg or 18 μmol / kg S41, 5 mg / kg anti-mPD-1 antibody, or 9 mg / kg S41 combined with 5 mg / kg anti-mPD-1 antibody, all via tail vein injection at a volume of 10 ml / kg. S41 was administered once a week, and anti-mPD-1 antibody was administered twice a week for four weeks.

[0430] The results are shown in Figure 25. The results showed that S41 and anti-mPD-1 exhibited a synergistic inhibitory effect on the growth of CT-26 tumor cell transplants.

[0431] Example 27: Pharmacodynamics and safety evaluation of S41 against mouse colon cancer cells CT-26 in a BALB / e mouse subcutaneous tumor model

[0432] This study used 24 female Balb / c mice subcutaneously inoculated with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 150-200 mm. 3The mice were randomly divided into 4 groups, with 6 mice in each group, and given lysozyme (Vehicle), 10 mg / kg T-DXD analog (positive control), 16 mg / kg S41, and 24 mg / kg S41 (HSNTD dose) respectively. The drugs were injected into the tail vein with a volume of 10 ml / kg, once a week for 2 weeks.

[0433] The results are shown in Figure 26. The results show that S41 significantly inhibited the growth of CT-26 xenograft tumor cells. Based on rat toxicity studies, 16 mg / kg S41 and 10 mg / kg T-DXD showed similar toxicity. At higher doses, S41 was significantly more effective than T-DXD.

[0434] Example 28: Toxicity study of SD rats after intravenous administration of S41 for four weeks and repeated administration for four weeks during the recovery period

[0435] This study selected 160 rats and randomly divided them into 4 groups, each with 40 rats, half male and half female, including 30 main test groups and 10 TK groups. They were given normal saline, 3, 6, and 12 mg / kg of S41, respectively, once a week for 4 consecutive weeks. The drug was then stopped and recovered for 4 weeks. The following examinations were performed during the experiment, including general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 12 mg / kg.

[0436] Example 29: Toxicity study of Beagle dogs after intravenous administration of S41 for four weeks with a recovery period of four weeks

[0437] This study selected 40 beagle dogs and randomly divided them into 4 groups, with 10 dogs in each group, half male and half female. They were given normal saline, 1, 2.5, and 5 mg / kg of S41, respectively, once a week for 4 consecutive weeks. The drugs were then discontinued for 4 weeks. The following examinations were performed during the experiment: general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, and coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 2.5 mg / kg.

[0438] In summary, the antibody-drug conjugate provided by the present invention can target and aggregate around tumor cells, and can only activate the drug on the surface of tumor cells, effectively killing tumors, and the toxicity of the drug is also reduced to a certain extent, which has very good application prospects.

Claims

1. A drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that: The drug conjugate has a chemical structure shown in formula (I) or (II), R1-R2-D (I) R2'-D (II) in: R1 is a chemical structure coupled to a biomolecule, or R1 does not exist; R2 and R2' are each independently an adjusted chemical structure to enhance the structure-activity relationship; D is selected from the following structures represented by X1-X5: The wavy line indicates the position where D is connected to R2 or R2'.

2. The drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R1 is absent or selected from: Preferably, R1 is selected from: The wavy line indicates the position where R1 and R2 are connected.

3. The drug conjugate according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The R2 is selected from: -C 1-6 Alkylene-CO-*; -C 1-6 Alkylene-O-*; -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-* -C 1-6 Alkylene-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-* -(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; -C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; -NH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; -NH-C 1-55 Alkylene-CO-*; -NH-C 1-55 Alkylene-NH-*; -NH-C 1-55 Alkylene-O-*; -CO-C 1-55 Alkylene-CO-*; -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; -CO-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; -CO-C 1-6 Alkylene-NH-*; -CO-C 1-6 Alkylene-NHCO-SO3H substituted C 1-6 Alkylene-NH-*; in: * indicates the position connected to D; R4 is a monovalent group or a divalent group. When R4 is connected to other parts of the compound, it is a divalent group, selected from: C 1-6 Alkyl or C 1-6 Alkylene; C 1-6 Alkyl-C 3-8 Cycloalkylene- or C 1-6 Alkylene-C 3-8 Cycloalkylene-; -CO-C 1-6 Alkylene-; C 1-6 Alkyl-(O-CH2CH2-O) m -C 1-6 Alkylene- or C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-; -NH-C 1-6 Alkylene; C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-; and -NH-C 1-6 Alkylene-C 3-8 Cycloalkylene; R5 is a monovalent group or a divalent group. When R5 is connected to other parts of the compound, it is a divalent group, selected from: C 1-6 Alkyl-(O-CH2CH2-O) m -;C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -;-CO-C 1-6 Alkylene-(O-CH2CH2-O) n -; One of R4 and R5 is a divalent group, and the other is a monovalent group; R6 does not exist or is -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylenecarbonyl or -NH-C 1-6 Alkylene-triazolyl-C 1- 6-Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl; L2 and L3 are each independently a bond or C 1-6 Alkylene; L4 is independent of C 1-6 Alkylene; L5 is a key, C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl or C 1-6 Alkylenecarbonyl; L6 is C 1-6 Alkylene; L7 is C 1-6 Alkylene or -C 3-8 Cycloalkylene-C 1-6 Alkylene; Each n and m is independently an integer from 1 to 50; Preferably, R2 is selected from: Wherein, o is independently an integer of 1-20, and m and n are each independently an integer of 1-50; More preferably, R2 is selected from: Preferably, n and m are each independently an integer of 2-20.

4. The drug conjugate according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The drug conjugate represented by formula (I) is selected from: In each formula, R1 is as described in claim 1 or 2.

5. The drug conjugate according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The drug conjugate of formula (I) is selected from:

6. The drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R2' is selected from: C 1-6 Alkyl-CO-*; C 1-6 Alkyl-O-*; C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-* C 1-6 Alkyl-OCONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; NH2-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; NH2-C 1-55 Alkylene-CO-*; NH2-C 1-55 Alkylene-NH-*; NH2-C 1-55 Alkylene-O-*; HOOC-C 1-55 Alkylene-CO-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-CO-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NH-*; HOOC-C 1-6 Alkylene-CONH-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-O-*; HOOC-C 1-6 Alkylene-NH-*; HOOC-C 1-6 Alkylene-NHCO-SO3H substituted C 1-6 Alkylene-NH-*; N3-C 1-6 Alkylene-CO-*; N3-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkylene-OC 1-6 Alkylene-CO-*; HOOC-C 1-6 Alkylene-OC 1-6 Alkylene-CO-*; in: * indicates the position connected to D; R4 is selected from: C 1-6 Alkyl; C 1-6 Alkyl-C 3-8 Cycloalkylene-; COOH-C 1-6 Alkylene-; C 1-6 Alkyl-(O-CH2CH2-O) m -C 1-6 Alkylene-; NH2-C 1-6 Alkylene; C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -C 1-6 Alkylene-; and NH2-C 1-6 Alkylene-C 3-8 Cycloalkylene; R5 is selected from: C 1-6 Alkyl-(O-CH2CH2-O) m -;C 1-6 Alkyl-CONH-C 1-6 Alkylene-(O-CH2CH2-O) m -;COOH-C 1-6 Alkylene-(O-CH2CH2-O) n -; R6 does not exist or is -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylenecarbonyl, or -NH-C 1-6 Alkylene-triazolyl-C 1-6 Alkylene-(O-CH2CH2-O) n -C 1-6 Alkylene-NHCO-C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl; L2 and L3 are each independently a bond or C 1-6 Alkylene; L4 is independent of C 1-6 Alkylene; L5 is a key, C 1-6 Alkyleneoxy-C 1-6 Alkylenecarbonyl or C 1-6 Alkylenecarbonyl; L6 is C 1-6 Alkylene; L7 is C 1-6 Alkylene or -C 3-8 Cycloalkylene-C 1-6 Alkylene; Each n and m is independently an integer of 1-50; p is 0 or 1; when p is 0, the -NH-[CO-R4] p The group is -NH2; Preferably, R2' is selected from: Wherein, o is independently an integer of 1-20, and m and n are each independently an integer of 1-50; More preferably, R2' is selected from: Preferably, n and m are each independently an integer of 2-20.

7. The drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug conjugate represented by formula (II) is selected from:

8. An antibody-drug conjugate, a stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The antibody-drug conjugate is as shown in formula (III), R3-(R1-R2-D) x Formula (III); Wherein, R3 is an antibody; R1, R2, D as described in any one of claims 1-4; x is an integer selected from 1-8.

9. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt thereof according to claim 8, characterized in that: R1 is: The connection method of R3-R1 is: wherein S is the S atom of a cysteine ​​residue in R3; or R1 is: The connection method of R3-R1 is: Among them, R3 containing an azide group is coupled with the alkynyl group in the R1 group.

10. The antibody-drug conjugate, its stereoisomer or pharmaceutically acceptable salt according to claim 8, characterized in that: The antibody is a monoclonal antibody or a multispecific antibody; Preferably, the antibody is selected from the group consisting of anti-Her2 antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD33 antibody, anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, anti-TNFα antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, anti-CD27 antibody, anti-b-CD40 antibody, anti-ICOS antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD3 antibody, anti-CD22 antibody, anti-CCR6 antibody, anti-CD38 antibody, anti-CD52 antibody, anti-complement C5 antibody, anti-RSV F protein antibody, anti-GD2 antibody, anti-GITR antibody, anti-glycoprotein receptor lib / Illa antibody, anti-ICOS antibody, anti-IL2R antibody, anti-LAG3 anti-body, anti-α4 integrin antibody, anti-lgE antibody, anti-PDGFRa antibody, anti-RANKL antibody, anti-SLAMF7 antibody, anti-LTIGIT antibody, anti-TIM-3 antibody, anti-VEGFR2 antibody, anti-VISTA antibody, anti-SSTR2 antibody and anti-LY6G6D antibody, anti-GCC antibody, anti-Trop2 antibody, anti-Cmet antibody, or a multispecific antibody formed by any two or more of these antibodies.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) the drug conjugate according to any one of claims 1 to 7, or a stereoisomer or a pharmaceutically acceptable salt thereof and / or the antibody drug conjugate according to any one of claims 8 to 10, or a stereoisomer or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

12. A drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that: The drug conjugate is selected from:

13. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the drug conjugate according to claim 12, its stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to any one of claims 11 to 13, characterized in that The pharmaceutical composition further contains one or more other anticancer drugs; preferably, the other anticancer drug is legubicin.

15. Use of the drug conjugate or its stereoisomer or pharmaceutically acceptable salt according to any one of claims 1 to 7 or the antibody drug conjugate or its stereoisomer or pharmaceutically acceptable salt according to any one of claims 8 to 11 in the preparation of a medicament for treating and / or preventing tumors and / or inflammation; preferably, the tumor is a blood tumor or a solid tumor; more preferably, the tumor is selected from the group consisting of sarcoma (such as fibrosarcoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.

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