Tetrapeptide-containing conjugates, preparation method therefor, and intermediate and use thereof

Abstract

The present invention relates to tetrapeptide-containing conjugates, a preparation method therefor, and an intermediate compound and use thereof.

Description

Tetrapeptide conjugates, their preparation methods, intermediates and uses

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to PCT International Patent Application No. PCT / CN2024 / 134552, filed on November 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, specifically to conjugates containing tetrapeptides, their preparation methods, intermediate compounds, and uses. Background Technology

[0004] Chemotherapy using cytotoxic agents was once the standard treatment for cancer, but highly lethal cytotoxic molecules can damage normal cells, causing severe toxic side effects. Targeted antitumor drugs, possessing both tumor-targeting and antitumor activity, have become a hot topic in current cancer research; however, the selectivity of targeted drugs often leads to significant toxic side effects, thus limiting their therapeutic efficacy. Biological macromolecular drugs, such as antibodies or antibody fragments, while highly targeted, have limited efficacy against solid tumors. Antibody-drug conjugates are novel targeted antitumor drugs, combining the properties of a targeted portion (e.g., antibody) that recognizes tumor cells and tissues with the toxic effects of small-molecule chemical drugs. Antibody-drug conjugates typically consist of three parts: 1) an antibody or antibody-like component that recognizes tumor cell-specific antigens; 2) a small molecule drug with potent cytotoxicity; and 3) a linker that connects the antibody to the small molecule drug. The linker can be either cleavable or non-cleavable. Cleavable linkers should possess sufficient plasma stability to ensure the integrity of the conjugate in the circulation system, preventing premature detachment of the small molecule toxin and causing toxic side effects to normal tissues in vivo. Simultaneously, after endocytosis by tumor cells, they should rapidly cleave to release the toxin molecules and kill the tumor cells. Non-cleavable linkers generally provide a stable linker after circulation and endocytosis; toxin release usually occurs after the antibody or antibody-like component has been completely broken down by lysosomes. Overall, cleavable linkers are more widely used in conjugate structures.

[0005] The linker portion of antibody-drug conjugates (ADCs) can largely determine the efficacy and toxicity. The linker may contain short peptide fragments that can be cleaved in cancer cells. Rapid cleavage of short peptides in cancer cells directly translates to increased efficacy, while stability in plasma or other tissue environments indicates low toxicity, thus allowing for increased dosage [Sassoon I, Blanc V. Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review. Antibody-Drug Conjugates 2013; 1–27].

[0006] Studies have found that cathepsin B is an important enzyme in organisms and plays a crucial role in tumor treatment. Substrate research targeting cathepsin B is of great significance. For example, introducing highly efficient cathepsin B hydrolysate fragments into the linkers of drug-adjuvant inhibitors (ADCs) will allow the ADC drug linkers to be rapidly cleaved within cancer cells, thereby improving drug efficacy. Furthermore, the linker remains stable in plasma or other tissue environments, reducing the toxic side effects caused by the shedding of small molecules. Substrates that can be efficiently and specifically hydrolyzed by cathepsin B, such as short peptides, also have other important uses and properties, such as being used to determine the activity of cathepsin B in samples.

[0007] GGFG (SEQ ID NO:30) is a tetrapeptide linker used for conjugating compounds, unlike previous cleavable linkers such as Val-Ala and Val-Cit. GGFG (SEQ ID NO:30) provides stable plasma stability and excellent intracellular lysis properties. Daiichi Sankyo DS 8201 ADC (Enhertu) utilizes the targeting effect of HER2 antibodies and contains the tetrapeptide linker GGFG sequence (SEQ ID NO:30). The bystander effect generated by the release of camptothecin toxoid further kills surrounding tumor cells, demonstrating good efficacy and safety.

[0008] Although GGFG (SEQ ID NO:30) exhibits good plasma stability and rapid intracellular lysosomal cleavage and release of camptothecin-like toxins in ADC conjugates, concerns remain regarding the safety of partial and premature release during in vivo circulation (Nguyen, TD; Bordeau, BM; Balthasar, JP. Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability, Cancer 2023, 15, 713.). Furthermore, the rapid release of lysosomal toxins is also considered one of the important properties of ADC activity in vivo. Enhancing the rate of rapid release of linkers while improving in vivo circulation stability will accelerate the killing process of tumor cells, thereby improving the efficacy and safety of ADC drugs (Zhang et al. Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues. Drug Metab Dispos 47:1146–1155, October 2019; Samantasinghar et al. A comprehensive review of key factors affecting the efficacy of antibody drug conjugate. Biomedicine & Pharmacotherapy 161(2023)114408).

[0009] Given the broad application prospects and unique properties of cathepsin B substrates, we conducted in-depth research, designing and studying a series of tetrapeptide sequences that can be specifically cleaved by cathepsin B and remain stable in human blood circulation. Enzymatic methods were used to validate enhanced release activity under simulated lysosomal protease degradation conditions, and stability assays were conducted to identify excellent plasma stability. The validation results provided feedback and model parameter correction for rapid iteration of the next round of molecular model development. Using GGFG (SEQ ID NO:30) as a reference, linkers with superior enzymatic activity and plasma stability were selected, resulting in short peptide linkers suitable for clinical application. These linkers were then used in a wide range of toxin linker-payload structures to create antibody-drug conjugates, producing more hydrophilic, effective, and stable ADC products, ultimately achieving remarkable in vivo antitumor efficacy.

[0010] Through extensive and in-depth research, the inventors have developed a series of antibody-drug conjugates containing specific tetrapeptide compounds, which can be efficiently and specifically hydrolyzed by cathepsin B and have high plasma stability, enhanced in vivo or in vitro efficacy and / or reduced toxicity, thus possessing extremely high application value. Summary of the Invention

[0011] On the one hand, this application provides drug conjugates of Formula I, or stereoisomers thereof, isotopic variants, pharmaceutically acceptable salts or solvates thereof.

[0012] Tg is the target region;

[0013] q represents DAR; and is an integer selected from 1 to 20.

[0014] Z stands for connector unit;

[0015] L1 is an extended unit or does not exist;

[0016] L2 is a self-destructing unit or does not exist;

[0017] L3 is a spacer subunit or does not exist;

[0018] D is a bioactive molecule; and

[0019] A1A2A3A4 is a tetrapeptide unit, in which

[0020] A1 is selected from R, G, K, W, N, P, F, I, M, L, T, E, Q, H, D or Y;

[0021] A2 is selected from L, G, Q, F, I, Y, W, S, A, T, N, K, D, M, H, or R;

[0022] A3 is selected from W, F, R, V, K, D, M, A, G, E, Y, P, L, T, I, Q, or S;

[0023] A4 is selected from Q, R, K, cit, W, G, N, D, E, H, L, V, S, T, F, Y, M, or A;

[0024] The premise is that A1A2A3A4 is not GGFG (SEQ ID NO:30).

[0025] In some implementations, A1A2A3A4 is not GGFG (SEQ ID NO:30), NYEE (SEQ ID NO:31), PNEE (SEQ ID NO:32), KIMR (SEQ ID NO:33), FKYK (SEQ ID NO:34), ILMK (SEQ ID NO:35), KFRL (SEQ ID NO:36), FGPD (SEQ ID NO:37), FDPV (SEQ ID NO:38), MMMK (SEQ ID NO:39), LDQF (SEQ ID NO:40), LDIQ (SEQ ID NO:41), FWRF (SEQ ID NO:42), HDGQ (SEQ ID NO:43), HGQT (SEQ ID NO:44), DKGT (SEQ ID NO:45), WIYF (SEQ ID NO:46), RDGT (SEQ ID NO:47), YRGT (SEQ ID NO:48), or LAVF (SEQ ID NO:39). NO:49), WGLS (SEQ ID NO:50), FDAY (SEQ ID NO:51) or HGIT (SEQ ID NO:52).

[0026] In some implementations, A1A2A3A4 is selected from the group consisting of:

[0027] (1) A1 is selected from R, T, E, F or G;

[0028] A2 is selected from D, A, H, K, G, L, or I;

[0029] A3 is selected from V, I, S, L, D, or W; and

[0030] A4 is selected from T, Q, G, L, or E;

[0031] (2) A1 is selected from R, E, F or G;

[0032] A2 is selected from D, H, K, G, I, or L;

[0033] A3 is selected from V, S, I, D, or W; and

[0034] A4 is selected from T, G, Q, L, or E;

[0035] (3) A1 is selected from R, E, F, G or T;

[0036] A2 is selected from D, H, K, G, I, or A;

[0037] A3 is selected from V, S, I, D, W, or L; and

[0038] A4 is selected from T, G, Q, L, or E;

[0039] (4) A1 is selected from R, E, F or G;

[0040] A2 is selected from D, H, K, G, or I;

[0041] A3 is selected from V, S, I, D, or W; and

[0042] A4 is selected from T, G, Q, L, or E;

[0043] (5) A1 is selected from R, E, or F;

[0044] A2 is selected from D, H, K, or G;

[0045] A3 is selected from V, S, I, or D; and

[0046] A4 is selected from T, G, Q, L, or E;

[0047] (6) A1 is selected from R, E, or F;

[0048] A2 is selected from D, H, or K;

[0049] A3 is selected from V, S, I, or D; and

[0050] A4 is selected from T, G, Q, L, or E;

[0051] (7) A1 is selected from R, E or F;

[0052] A2 is selected from D, H, or K;

[0053] A3 is selected from V, S, I, or D; and

[0054] A4 is selected from T, G, Q, or L;

[0055] (8) A1 is selected from R or E;

[0056] A2 is selected from D or H;

[0057] A3 is selected from V, S, I, or D; and

[0058] A4 is selected from T, G, Q, or L;

[0059] (9) A1 is R;

[0060] A2 is D;

[0061] A3 is selected from V, S, or I; and

[0062] A4 is selected from T, G, or Q;

[0063] (10) A1 is R;

[0064] A2 is D;

[0065] A3 is selected from V or S; and

[0066] A4 is selected from T or G;

[0067] (11) A1 is R;

[0068] A2 is D;

[0069] A3 is selected from V, S, or I; and

[0070] A4 is selected from T, G, Q, or E; and / or

[0071] (12) A1 is selected from R, T, E, F or G;

[0072] A2 is selected from D, A, H, K, G, L, or I;

[0073] A3 is selected from V, L, D, or W; and

[0074] A4 is selected from T, Q, G, L, or E.

[0075] In some implementations, A1 is selected from R, T, E, F, or G;

[0076] A2 is selected from D, A, H, K, G, L, or I;

[0077] A3 is selected from V, I, S, L, D, or W; and

[0078] A4 is selected from T, Q, G, L, or E.

[0079] In some implementations, A1 is selected from R, E, or F;

[0080] A2 is selected from D, H, or K;

[0081] A3 is selected from V, I, S, or D; and

[0082] A4 is selected from T, Q, G, L, or E.

[0083] In some implementations, A1 is selected from R;

[0084] A2 is selected from D;

[0085] A3 is selected from V, I, or S; and

[0086] A4 is selected from T, Q, G or E; preferably, A4 is selected from T, Q or G.

[0087] In some implementations, A1 is selected from R, T, E, F, or G;

[0088] A2 is selected from D, A, H, K, G, L, or I;

[0089] A3 is selected from V, L, D, or W; and

[0090] A4 is selected from T, Q, G, L, or E.

[0091] In some implementations, A1A2A3A4 is a tetrapeptide unit selected from:

[0092] RDVT(SEQ ID NO:53)、RLWQ(SEQ ID NO:54)、RDIQ(SEQ ID NO:55)、RDSG(SEQ ID NO:56)、TALQ(SEQ ID NO:57)、EHDL(SEQ ID NO:58)、FKVT(SEQ ID NO:59)、FGVQ(SEQ ID NO:60)、DKGT(SEQ ID NO:45)、HGIT(SEQ ID NO:52)、RDVA(SEQ ID NO:61)、QGAM(SEQ ID NO:62)、WIYF(SEQ ID NO:46)、ELVY(SEQ ID NO:63)、HDGQ(SEQ ID NO:43)、WGLS(SEQ ID NO:50)、FDAY(SEQ ID NO:51)、GGWG(SEQ ID NO:64)、GWRG(SEQ ID NO:65)、NYEE(SEQ ID NO:31)、NLAS(SEQ ID NO:66)、GGFA(SEQ ID NO:67)、GGFW(SEQ ID NO:68)、GFWG(SEQ ID NO:69)、PNEE(SEQ ID NO:32)、GIWG(SEQ ID NO:70)、GGRR(SEQ ID NO:71)、GGFR(SEQ ID NO:72)、GGFK(SEQ ID NO:73)、GGVR(SEQ ID NO:74)、GAAN(SEQ ID NO:75)、KIMR(SEQ ID NO:33)、FKYK(SEQ ID NO:34)、ILMK(SEQ ID NO:35)、KFRL(SEQ ID NO:36)、NLAS(SEQ ID NO:66)、WSME(SEQ ID NO:76)、GQKN(SEQ ID NO:77)、FGPD(SEQ ID NO:37)、GTGH(SEQ ID NO:78)、FDPV(SEQ ID NO:38)、MMMK(SEQ ID NO:39)、KYDD(SEQ ID NO:79)、GGVCit(SEQ ID NO:80),GFLG (SEQ ID NO:81), RDTL (SEQ ID NO:82), GFGS (SEQ ID NO:83), LDIL (SEQ ID NO:84), FDRQ (SEQ ID NO:85), LDQF (SEQ ID NO:40), WQAH (SEQ ID NO:86), LDIQ (SEQ ID NO:41), MNAL (SEQ ID NO:87), FWRF (SEQ ID NO:42), RDGT (SEQ ID NO:47), RDVE (SEQ ID NO:88), YRGT (SEQ ID NO:48), LAVF (SEQ ID NO:49), and HGQT (SEQ ID NO:44),

[0093] In some implementations, A1A2A3A4 is selected from: RDVT (SEQ ID NO:53), RDIQ (SEQ ID NO:55), RLWQ (SEQ ID NO:54), ELVY (SEQ ID NO:63), TALQ (SEQ ID NO:57), QGAM (SEQ ID NO:62), YDAM (SEQ ID NO:89), GGWG (SEQ ID NO:64), RDSG (SEQ ID NO:56), LDIL (SEQ ID NO:84), EHDL (SEQ ID NO:58), RDVE (SEQ ID NO:88), GGFK (SEQ ID NO:73), GWRG (SEQ ID NO:65), GFGS (SEQ ID NO:83), FKVT (SEQ ID NO:59), and FGVQ (SEQ ID NO:60).

[0094] In some implementations, A1A2A3A4 is selected from: RDVT (SEQ ID NO:53), RDSG (SEQ ID NO:56), RDIQ (SEQ ID NO:55), EHDL (SEQ ID NO:58), FKVT (SEQ ID NO:59), RDVE (SEQ ID NO:88), FGVQ (SEQ ID NO:60), GIWG (SEQ ID NO:70), RLWQ (SEQ ID NO:54), and TALQ (SEQ ID NO:57);

[0095] In some implementations, A1A2A3A4 is selected from: RDVT (SEQ ID NO:53), RDIQ (SEQ ID NO:55), RDSG (SEQ ID NO:56), TALQ (SEQ ID NO:57), EHDL (SEQ ID NO:58), FKVT (SEQ ID NO:59), FGVQ (SEQ ID NO:60), and RLWQ (SEQ ID NO:54).

[0096] In some implementations, A1A2A3A4 is RDVT (SEQ ID NO:53).

[0097] In some implementations, A1A2A3A4 is RDIQ (SEQ ID NO:55).

[0098] In some implementations, A1A2A3A4 is RDSG (SEQ ID NO:56).

[0099] In some implementations, A1A2A3A4 is TALQ (SEQ ID NO:57).

[0100] In some implementations, A1A2A3A4 is EHDL (SEQ ID NO:58).

[0101] In some implementations, A1A2A3A4 is FKVT (SEQ ID NO:59).

[0102] In some implementations, A1A2A3A4 is RDVE (SEQ ID NO:88).

[0103] In some implementations, A1A2A3A4 is GIWG (SEQ ID NO:70).

[0104] In some implementations, A1A2A3A4 is FGVQ (SEQ ID NO:60).

[0105] In some implementations, A1A2A3A4 is RLWQ (SEQ ID NO:54).

[0106] It should be understood that, unless otherwise specified, the left A1 (e.g., R in RLWQ (SEQ ID NO:54)) of the tetrapeptide A1A2A3A4 described herein is linked to L1, and when L1 is absent, it is linked to Z; correspondingly, the right A4 (e.g., Q in RLWQ (SEQ ID NO:54)) is linked to -L2-L3-D, for example, when L2 is present, it is linked to L2, and when L2 is absent but L3 is present, it is linked to L3.

[0107] As shown above, Z represents the connector unit. The "connector unit" is a structural unit that connects the target part (Tg) and the rest of the connector-payload (-L1-A1A2A3A4-L2-L3-D), which can be obtained by reacting the connector unit precursor (Z') in the connector-payload with the target part.

[0108] In some implementations, Z represents -Z1-Z2-, where Z1 is connected to the target portion (Tg).

[0109] In some embodiments, Z1 is selected from the following groups:

[0110] The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), for example, covalently connected to the sulfur atom of the target portion (Tg), indicated by a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0111] Each of X1 is independently selected from C 1-6 Alkyl, halogen, and nitro groups;

[0112] r is 0, 1, 2, or 3;

[0113] Alternatively, Z1 can be selected from the following groups:

[0114] The ends marked with an asterisk (*) are covalently connected to the target moiety (Tg), for example, to the primary or secondary amino group or hydroxyl group of the target moiety (Tg), and are marked with a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0115] Alternatively, Z1 can be selected from the following groups:

[0116] The ends marked with an asterisk (*) are covalently linked to the target region (Tg), such as to a sugar or a non-natural amino acid in the target region, and are indicated by a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0117] Each of X1 is independently selected from C 1-6 Alkyl, C 1-6 Alkoxy, halogen, and nitro;

[0118] r is 0, 1, 2, or 3;

[0119] Alternatively, Z1 can be selected from the following groups:

[0120] The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), such as the -C (=O)- on the target portion (Tg), and are marked with a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0121] in,

[0122] Z2 does not exist, or can be selected from: -C1-C 10 Alkylene -, -C2-C 10 alkenyl-, -C2-C 10 Ethyne-, -C1-C 10 Heteroalkyl-, -C3-C8 carbocycloalkyl-, -O-(C1-C8 alkylene)-, -C6-C 10 Aromatic-, -C1-C 10 Alkylene-C6-C 10 Aspartic-, -C6-C 10 Aspartic-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10Alkylene -C(=O)-, -C1-C 10 Alkylene-NH-, C1-C 10 Heteroalkyl-NH-, -C3-C8 carbocycloalkyl-NH-, -O-(C1-C8 alkylene)-NH-, -C6-C 10 aryl-NH-, -C1-C 10 Alkylene-C6-C 10 aryl-NH-,-C6-C 10 Aspartic-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkyl-S-, -C3-C8 carbocycloalkyl-S-, -O-(C1-C8 alkylene)-S-, -C6-C 10 Aromatic-S-,-C1-C 10 Alkylene-C6-C 10 Aromatic-S-,-C6-C 10 Aspartic-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S- or -(C3-C8 heterocyclic)-C1-C 10 Alkylene-S-;

[0123] The Z2 group is optionally replaced by one or more (e.g., 1, 2, 3, or 4) Bu groups, wherein the Bu groups are selected from: H, deuterium, halogen, nitro, -CN, -G1, -OR. a and -(CH2) x N(R a )2; where x is 0, 1, 2, 3, 4, 5, or 6; R a Independently H or -C optionally substituted with halogen 1-6 Alkyl groups, or two R atoms attached to the same N atom. aThe groups and the nitrogen atoms attached to them together form 4-7 membered heterocyclic groups, such as aza-heterocyclic butyl, pyrrolidinyl, or piperidinyl groups; wherein -G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers, preferably polyethylene glycol (PEG) units.

[0124] The Z2 group is optionally connected to -NH-(CH2CH2O) at its end (left and / or right). n1 - and / or -(CH2CH2O) n1 - or optionally, insert the group -NH-(CH2CH2O) at its intermediate position. n1 - and / or -(CH2CH2O) n1 -,and

[0125] n1 is an integer selected independently from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30, such as an integer selected independently from 1 to 10, such as 1 to 5;

[0126] It should be understood that the left side of the Z2 group is connected to Z1.

[0127] In some implementations, Z1 is as defined above, and Z1 is not...

[0128] In some embodiments, Z1 is selected from the following groups:

[0129] The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), for example, covalently connected to the sulfur atom of the target portion (Tg), indicated by a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0130] Each of X1 is independently selected from C 1-6 Alkyl, halogen, and nitro groups;

[0131] r is 0, 1, 2, or 3;

[0132] Alternatively, Z1 can be selected from the following groups:

[0133] The ends marked with an asterisk (*) are covalently connected to the target moiety (Tg), for example, to the primary or secondary amino group or hydroxyl group of the target moiety (Tg), and are marked with a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0134] Alternatively, Z1 can be selected from the following groups:

[0135] The ends marked with an asterisk (*) are covalently linked to the target region (Tg), such as to a sugar or a non-natural amino acid in the target region, and are indicated by a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0136] Each of X1 is independently selected from C 1-6 Alkyl, C 1-6 Alkoxy, halogen, and nitro;

[0137] r is 0, 1, 2, or 3;

[0138] Alternatively, Z1 can be selected from the following groups:

[0139] The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), such as the -C (=O)- on the target portion (Tg), and are marked with a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist);

[0140] in,

[0141] Z2 does not exist, or can be selected from: -C1-C 10 Alkylene -, -C2-C 10 alkenyl-, -C2-C 10 Ethyne-, -C1-C 10 Heteroalkyl-, -C3-C8 carbocycloalkyl-, -O-(C1-C8 alkylene)-, -C6-C 10 Aromatic-, -C1-C 10 Alkylene-C6-C 10 Aspartic-, -C6-C 10 Aspartic-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-NH-, C1-C 10 Heteroalkyl-NH-, -C3-C8 carbocycloalkyl-NH-, -O-(C1-C8 alkylene)-NH-, -C6-C 10 aryl-NH-, -C1-C 10 Alkylene-C6-C 10 aryl-NH-,-C6-C 10 Aspartic-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkyl-S-, -C3-C8 carbocycloalkyl-S-, -O-(C1-C8 alkylene)-S-, -C6-C 10 Aromatic-S-,-C1-C 10 Alkylene-C6-C 10 Aromatic-S-,-C6-C 10 Aspartic-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S- or -(C3-C8 heterocyclic)-C1-C 10 Alkylene-S-;

[0142] The Z2 group is optionally replaced by one or more (e.g., 1, 2, 3, or 4) Bu groups, wherein the Bu groups are selected from: H, deuterium, halogen, nitro, -CN, -G1, -OR. a and -(CH2) x N(R a )2; where x is 0, 1, 2, 3, 4, 5, or 6; R a Independently H or -C optionally substituted with halogen 1-6 Alkyl groups, or two R atoms attached to the same N atom. a The groups and the nitrogen atoms attached to them together form 4-7 membered heterocyclic groups, such as aza-heterocyclic butyl, pyrrolidinyl, or piperidinyl groups; wherein -G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers, preferably polyethylene glycol (PEG) units.

[0143] The Z2 group is optionally connected to -NH-(CH2CH2O) at its end (left and / or right). n1 - and / or -(CH2CH2O) n1 - or optionally, insert the group -NH-(CH2CH2O) at its intermediate position. n1 - and / or -(CH2CH2O) n1 -,and

[0144] n1 is an integer selected independently from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30, such as an integer selected independently from 1 to 10, such as 1 to 5;

[0145] It should be understood that the left side of the Z2 group is connected to Z1.

[0146] In some implementations, Z1 is selected from:

[0147] The symbols are defined as above.

[0148] In some implementations, Z1 is selected from:

[0149] The symbols are defined as above.

[0150] In some implementations, Z1 is

[0151] In some embodiments, Z2 is as defined above, and wherein the C1-C, either alone or as part of a group, 10 The heteroalkylene group has 1-3 (e.g., 1 or 2, preferably 1) cyclic heteroatoms selected from O, N, and S, and the heteroalkylene group is fully saturated or has 1 to 3 degrees of unsaturation, preferably fully saturated; and

[0152] The -C3-C8 heterocyclic group has 1-4 (e.g., 1-3, e.g., 1 or 2, e.g., 1) cyclic heteroatoms selected from O, N and S, preferably, the -C3-C8 heterocyclic group is a -C3-C8 heteroaryl group.

[0153] In some embodiments, the Z2 group is unsubstituted.

[0154] In some embodiments, the Z2 group is replaced by one or more (e.g., 1, 2, 3, or 4, e.g., 1) Bu groups, wherein the Bu groups are selected from: H, deuterium, halogen, nitro, -CN, -G1, and -OR. a ;R a Independently H or -C optionally substituted with halogen 1-6 Alkyl group; wherein -G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides and dendritic polymers, preferably polyethylene glycol (PEG) units.

[0155] In some embodiments, the Z2 group is connected to -NH-(CH2CH2O) at its end (left and / or right). n1 - and / or -(CH2CH2O) n1 - where n1 is as defined above;

[0156] In some embodiments, the Z2 group has a -NH-(CH2CH2O) group inserted at its intermediate position. n1 - and / or -(CH2CH2O) n1 - where n1 is as defined above.

[0157] In some implementations, Z2 is selected from: -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-C(=O)-.

[0158] In some implementations, Z2 is selected from: -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-C(=O)-,

[0159] The C1-C, either individually or as a part of a group, 10 The heteroalkylene group has 1-3 (e.g., 1 or 2, preferably 1) cyclic heteroatoms selected from O, N, and S, and the heteroalkylene group is fully saturated or has 1 to 3 degrees of unsaturation, preferably fully saturated; and

[0160] The -C3-C8 heterocyclic group has 1-4 (e.g., 1-3, e.g., 1 or 2, e.g., 1) cyclic heteroatoms selected from O, N and S, preferably, the -C3-C8 heterocyclic group is a -C3-C8 heteroaryl group.

[0161] In some implementations, Z2 is selected from: -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -3-10-membered heterocyclic-C(=O)-, -C1-C 10 Alkylene-(3-10-membered heterocyclic)-C(=O)-, -(3-10-membered heterocyclic)-C1-C 10 Alkylene-C(=O)-,

[0162] The C1-C 10 The heteroalkyl group (alone or as part of a group) has 1-3 (e.g., 1 or 2, preferably 1) cyclic heteroatoms selected from O, N, and S, and the heteroalkyl group is fully saturated or has 1 to 3 degrees of unsaturation, preferably fully saturated; and

[0163] The 3-10 membered heterocyclic group (alone or as part of a group) has 1-4 (e.g., 1-3, e.g., 1 or 2, e.g., 1) cyclic heteroatoms selected from O, N and S, and the remaining cyclic atoms are carbon atoms. Preferably, the 3-10 membered heterocyclic group is a 4-8 membered heterocyclic group, e.g., a 5-6 membered heterocyclic group, e.g., a 5-6 membered heteroaryl group.

[0164] In some embodiments, the Z2 group is optionally replaced by a Bu group selected from halogens and -(CH2). x N(R a )2; where x is 0, 1, 2, 3, 4, 5, or 6; R aIndependently H or -C optionally substituted with halogen 1-6 alkyl.

[0165] In some embodiments, the Bu group is located on the carbon atom of the Z2 group that is attached to the Z1 group.

[0166] In some implementations, Z2 is not present.

[0167] In some implementations, -Z1-Z2- has the following structure:

[0168] Each of n2 is an integer selected from 1 to 20, for example, from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 18 or 20, preferably 1, 2 or 5;

[0169] The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), and are marked with a wavy line. The end of it is covalently linked to L1 or a tetrapeptide unit (when L1 is not present).

[0170] In some implementations, -Z1-Z2- has the following structure:

[0171] The symbols are defined as above.

[0172] In some implementations, L1 is not present.

[0173] In some implementations, L1 is an extension unit. An "extension unit" is a group or chemical structure that connects the linker unit precursor (Z') or linker unit (Z) to the tetrapeptide unit.

[0174] In some embodiments, L1 is selected from the following groups:

[0175] R b Selected from -C 1-10 alkylene-, -C 6-10 Alpha- and -C 1-10 Heteroalkyl-, -C 3-8 heterocyclic group -, -C 1-10 Alkylene-C 6- 10 Alpha- and -C 6-10 Aspartic-C 1-10 alkylene-, -C 1-10 Alkylene-C 6-10 Aspartic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 subcarbocyclic -, -C 3-8subcarbocyclic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 subcarbocyclic-C 1-10 alkylene-, -C 1- 10 Alkylene-C 3-8 heterocyclic group -, -C 3-8 Heterocyclic-C 1-10 alkylene- and -C 1-10 Alkylene-C 3-8 Heterocyclic-C 1-10 alkylene-;

[0176] R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-;

[0177] n3 is independently selected from 0, 1, 2, 3 or 4; preferably 0 or 1, for example 1;

[0178] G2 is independently selected from: -O-, -S-, -NH-, -NH(CH2CH2O) n1 - and -(CH2CH2O) n1 - n1 are each independently an integer selected from 0 to 30, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30; preferably, G2 are each independently selected from: -NH(CH2CH2O) n1 - and -(CH2CH2O) n1 -, more preferably -(CH2CH2O) n1 - where n1 is as defined above;

[0179] An asterisk (*) indicates a connection to a precursor (Z') or a connector unit (Z), while a wavy line indicates a connection to a tetrapeptide unit.

[0180] G3 is selected independently from: (Preferred) ), Where R represents an amino acid side chain, and the amino acid is as defined herein, for example selected from alanine (Ala), aspartic acid (Asp), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), proline (Pro), and citrulline (Cit); where an asterisk (*) indicates a side chain of R. b Connection; G3 is preferably GGFG (SEQ ID NO:30);

[0181] G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers; for example, polyethylene glycol (PEG) units;

[0182] G4 is selected from non-existent -(CH2). n9 C(O)- and -NH-(CH2) n9 O(CH2) n9 C(O)-;

[0183] n8 and n9 are each independently selected from integers from 0 to 9, such as 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0184] G5 is selected from -S(O)-, -S(O)2-, Preferably selected from -S(O)- or -S(O)2-, for example -S(O)2-.

[0185] It should be understood that the left side of the G4 group shown is connected to G3.

[0186] In some embodiments, L1 is selected from the following groups:

[0187] R b Selected from -C 1-10 alkylene-, arylene- (e.g., -C) 6-10 -aryl, -C 1-10 Heteroalkyl-, -C 3-8 heterocyclic group -, -C 1-10 Alkylene-C 6-10 Alpha- and -C 6-10 Aspartic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 subcarbocyclic -, -C 3-8 subcarbocyclic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 Heterocyclic group- and -C 3-8 Heterocyclic-C 1-10 alkylene-;

[0188] R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-;

[0189] n3 is independently selected from 0, 1, 2, 3 or 4;

[0190] G2 is independently selected from: -O-, -S-, -NH-, -NH(CH2CH2O) n1-、-(CH2CH2O) n1 - n1 are each an integer selected from 0 to 30, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30;

[0191] An asterisk (*) indicates a connection to a precursor (Z') or a connector unit (Z), while a wavy line indicates a connection to a tetrapeptide unit.

[0192] G3 is selected independently from: (Preferred) ), Where R represents an amino acid side chain, and the amino acid is as defined herein, for example selected from alanine (Ala), aspartic acid (Asp), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), proline (Pro), and citrulline (Cit); where an asterisk (*) indicates a side chain of R. b Connection; G3 is preferably GGFG (SEQ ID NO:30);

[0193] G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers;

[0194] G4 is selected from non-existent -(CH2). n9 C(O)- and -NH-(CH2) n9 O(CH2) n9 C(O)-;

[0195] n8 and n9 are each independently selected from integers from 0 to 9, such as 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0196] G5 is selected from -S(O)-, -S(O)2-,

[0197] It should be understood that the left side of the G4 group shown is connected to G3.

[0198] In some embodiments, L1 is selected from the following groups:

[0199] The symbols are defined as above.

[0200] In some embodiments, L1 is selected from the following groups:

[0201] The symbols are defined as above.

[0202] In some embodiments, L1 is selected from the following groups:

[0203] The symbols are defined as above.

[0204] In some embodiments, L1 is absent or selected from the following groups: -C(=O)-C 3-8 Carbocyclic groups -C(=O)-, -C(=O)-C 3-8 Heterocyclic groups -C(=O)-, -C(=O)-C 1-10 Alkylene -C(=O)-, -C(=O)-C 3-8 Heterocyclic-C 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-(CH2CH2O) n1 -C 1-10 Alkylene -C(=O)-, -C 1-10 Alkylene -OC(=O)-, -C(=O)-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-S(O)2-, -N(R) c )-C 1-10 Alkylene-HP(O)4-, -N(R) c )-C 1-10 Alkylene-SC 1-10 Alkylene -C(=O)-, -SC 1-10 Alkylene-C(=O)-

[0205] Each of n is an integer selected from 0 to 9, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0206] n1 is an integer independently selected from 0 to 30, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28, or 30; and

[0207] R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-.

[0208] In some embodiments, L1 is absent or selected from the following groups: -C(=O)-C 3-8 Carbocyclic groups -C(=O)-, -C(=O)-C 3-8 Heterocyclic groups -C(=O)-, -C(=O)-C 1-10 Alkylene -C(=O)-, -C(=O)-C 3-8 Heterocyclic-C 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-(CH2CH2O) n1 -C 1-10 Alkylene -C(=O)-, -C 1-10 Alkylene -OC(=O)-, -C(=O)-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-S(O)2-, -N(R) c )-C 1-10 Alkylene-HP(O)4-, -N(R) c )-C 1-10 Alkylene-SC 1-10 Alkylene -C(=O)-, -SC 1-10 Alkylene-C(=O)-

[0209] Each of n is an integer selected from 0 to 9, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0210] n1 is an integer independently selected from 0 to 30, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28, or 30; and

[0211] R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-.

[0212] It should be understood that, unless otherwise specified and without contradiction with the context, the divalent groups shown in the text are connected to other groups in the direction shown in the text, for example, the left side of the L1 group is connected to Z, and the right side is connected to A1A2A3A4.

[0213] In some embodiments, L1 is as defined above, and wherein the C1-C, either alone or as part of a group, 10 The heteroalkylene group has 1-3 (e.g., 1 or 2, preferably 1) cyclic heteroatoms selected from O, N, and S, and the heteroalkylene group is fully saturated or has 1 to 3 degrees of unsaturation, preferably fully saturated; and

[0214] The -C3-C8 heterocyclic group has 1-4 (e.g., 1-3, e.g., 1 or 2, e.g., 1) cyclic heteroatoms selected from O, N and S, preferably, the -C3-C8 heterocyclic group is a -C3-C8 heteroaryl group.

[0215] In some embodiments, L1 is absent or selected from any of the following groups:

[0216] Where n10 and n11 are independently selected from 0, 1, 2 and 3, and n10 and n11 are not both 0;

[0217] The remaining symbols are as defined above.

[0218] In some embodiments, L1 is absent or selected from the following groups:

[0219] Where n10 and n11 are independently selected from 0, 1, 2 and 3, and n10 and n11 are not both 0;

[0220] The remaining symbols are as defined above.

[0221] In some implementations, L1 is selected from

[0222] The symbols such as n, n1, n8 and R are defined as above.

[0223] In some implementations, L1 is selected from

[0224] The symbols are defined as above.

[0225] In some implementations, L1 is selected from

[0226] The symbols are defined as above.

[0227] In some implementations, L1 is either absent or selected from...

[0228] The symbols are defined as above.

[0229] In some implementations, L1 is either absent or selected from...

[0230] In some implementations, L1 is not present.

[0231] In some implementations, L2 is a self-cleaving unit. A "self-cleaving unit" is a group or chemical structure that can facilitate the release of the active drug moiety from the antibody-drug conjugate.

[0232] In some implementations, L2 is either absent or selected from:

[0233] Where X is selected from -NH-, -O-, or -S-;

[0234] R1 is independently selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, cyano group, -PEG unit, -C 1-8 alkylene-PEG unit, -C 1-8 Alkylene-C(=O)NH-PEG unit, -C 1-8 Alkylene-NHC(=O)-PEG unit, -C 1-8 alkylene-C(=O)O-PEG units and -C 1-8 Alkylene-OC(=O)-PEG unit;

[0235] Su refers to the sugar portion;

[0236] r1 is 0, 1, 2, 3 or 4;

[0237] t can be 0, 1, 2, or 3.

[0238] In some implementations, R1 is independently selected from C. 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group.

[0239] In some implementations, R1 is independently selected from -C 1-8 alkylene-C(=O)NH-PEG units and -C 1-8 Alkylene-NHC(=O)-PEG unit.

[0240] In some implementations, R1 is independently selected from -C 1-8Alkylene-C(=O)NH-PEG unit.

[0241] In some implementations, R1 is independently selected from -C 1-8 Alkylene-NHC(=O)-PEG unit.

[0242] In some implementations, R1 is independently selected from -C 1-4 alkylene-C(=O)NH-PEG units and -C 1-4 Alkylene-NHC(=O)-PEG unit.

[0243] In some implementations, r1 is 0 or 1.

[0244] In some implementations, r1 is 0.

[0245] In some implementations, r1 is 1.

[0246] In some implementations, t is 0 or 1.

[0247] In some implementations, t is 0.

[0248] In some implementations, t is 1.

[0249] In some implementations, the PEG unit is -(CH2CH2O). b- R PEG R PEG a is the end-capping group of the PEG unit, preferably -CH3 or -CH2CH2COOH; b is independently selected from 2-72, for example 4-20, 2-10 or 4-10 integers, for example 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0250] In some embodiments, Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; in other embodiments, Su is independently selected from...

[0251] In some implementations, Su is independently:

[0252] Preferred More

[0253] In some implementations, L2 is either absent or selected from:

[0254] X, Su, and R1 are defined as above.

[0255] In some implementations, L2 is either absent or selected from:

[0256] X, Su, and R1 are defined as above.

[0257] In some implementations, preferably, L2 is absent or selected from... Where X and Su are as defined above; preferably, X is -NH- and Su is

[0258] In some implementations, L2 is either absent or selected from... Where X is defined as above.

[0259] In some implementations, L2 is Where X is defined as above.

[0260] In some implementations, L2 is

[0261] In some implementations, L2 is not present.

[0262] In some implementations, L2 is either absent or selected from:

[0263] t is 0 or 1, and the other symbols are as defined above.

[0264] It should be understood that, unless otherwise specified, the left side of the L2 group shown in the text is connected to A1A2A3A4, and the right side is connected to -L3-D.

[0265] In some implementations, L3 is not present.

[0266] In some implementations, L3 is a spacer unit. A "spacer unit" is a group or chemical structure that links L2 (when L2 is present) or a tetrapeptide unit (when L2 is absent) to D.

[0267] In some implementations, L3 is either absent or selected from...

[0268] -NR2-(CHR3)n 1a -L a -L b -L c -;

[0269] L a This represents -C(=O)-NR2- and -NR2-(CH2)n. 2a -, -O-, -S-, -CHR3-, -NH- or single bonds,

[0270] L b Represents -(CR2R3)n3a -, -O-, -NR2-, or a single bond, preferably represented as -(CHR3)n 3a -, -O-, -NR2- or a single bond,

[0271] L c It represents -CH2- or -C(=O)-;

[0272] R2 and R3 independently represent hydrogen atoms and -C atoms, respectively. 1-8 Alkyl groups, C groups substituted with groups selected from the following 1-8 Alkyl-:MeSO 2- , halogen, nitro and cyano, -(CH2)n 4a -COOH, -(CH2)n 5a -OH, -(CH2)n 6a -NH2;

[0273] n 1a n 2a n 3a n 4a n 5a and n 6a Each can independently represent 0, 1, 2, 3, 4, 5, or 6;

[0274] The left side of the L3 group shown is connected to -Z-L1-A1A2A3A4-L2-, and the right side is connected to -D;

[0275] Preferably, the right side of L3 is connected to the nitrogen atom of the amino group in the D group;

[0276] Preferably, the left side of L3 is connected to the carbonyl group in -Z-L1-A1A2A3A4-L2-.

[0277] In some implementations, L3 is either absent or selected from...

[0278] R2 is independently selected from H and C. 1-8 Alkyl groups and C groups substituted with groups selected from the following 1-8 Alkyl-:MeSO 2- halogens, nitro groups, and cyano groups;

[0279] R3 is independently selected from H and C. 1-8 alkyl-; and

[0280] X4 is -CH2-, -NH-, -O-, or -S-;

[0281] n5 and n6 each independently represent 0, 1, 2, 3, 4, 5 or 6;

[0282] Or selected from

[0283] -NR 5 -(CH2)n 1 -L a -L b -L c -;

[0284] Where n 1 It represents 0, 1, 2, 3, 4, 5, or 6.

[0285] L a Represents -C(=O)-NR 1 -、-NR 1 -(CH2)n 2 -, -O-, or a single bond,

[0286] Where, n 2 It represents 1, 2, 3, 4, 5, or 6.

[0287] R 1 and R 5 Each independently represents a hydrogen atom and -C. 1-6 Alkyl group, -(CH2)n 3 -COOH or -(CH2)n 4 -OH, where n 3 Indicates 1, 2, 3, or 4, n 4 It represents 1, 2, 3, 4, 5, or 6.

[0288] L b Indicates -CR 2 (R 3 -, -O-, -NR 4 - or a single key,

[0289] R 2 and R 3 Each independently represents a hydrogen atom and -C. 1-6 Alkyl group, -(CH2)n a -NH2、-(CH2)n b -COOH, or -(CH2)n c -OH,R 4 Represents a hydrogen atom or C 1-6 Alkyl, n a n represents 0, 1, 2, 3, 4, 5, or 6. b Indicates 1, 2, 3, or 4, n c It represents 1, 2, 3, or 4, but n a When R is 0, 2 and R 3 They are not the same, and

[0290] L c It represents -CH2- or -C(=O)-.

[0291] In some implementations, L3 is either absent or selected from...

[0292] R2 is independently selected from H and C. 1-8 Alkyl groups and C groups substituted with groups selected from the following 1-8 Alkyl-:MeSO 2- halogens, nitro groups, and cyano groups;

[0293] R3 is independently selected from H and C. 1-8 alkyl-; and

[0294] X4 is -CH2-, -NH-, -O-, or -S-;

[0295] n5 and n6 each independently represent 0, 1, 2, 3, 4, 5, or 6.

[0296] In some implementations, L3 can also perform self-destruction.

[0297] In some implementations, L2 and L3 together or individually perform the self-destruction function.

[0298] In some implementations, L3 is absent while L2 is present.

[0299] In some implementations, L3 exists while L2 does not.

[0300] In some implementations, both L3 and L2 exist.

[0301] In some implementations, neither L3 nor L2 exists.

[0302] In some implementations, L3 is either absent or selected from...

[0303] R2 is independently selected from H and C. 1-8 Alkyl groups and C groups substituted with groups selected from the following 1-8 Alkyl groups: MeSO2-, halogens, nitro groups, and cyano groups;

[0304] R3 is independently selected from H and C. 1-8 alkyl-; and

[0305] X4 can be -CH2-, -NH-, -O-, or -S-.

[0306] In some embodiments, R2 is independently selected from H, methyl, and ethyl groups substituted with groups selected from MeSO2-, halogen, nitro, and cyano.

[0307] In some implementations, R3 is H.

[0308] In some implementations, X4 is -O- or -S-.

[0309] In some implementations, X4 is -O-.

[0310] In some implementations, L3 is selected from

[0311] The symbols are defined as above.

[0312] In some implementations, L3 is selected from The symbols are defined as above.

[0313] In some implementations, L3 is selected from:

[0314] It should be understood that, unless otherwise specified, the left side of the L3 group shown in the text is connected to Z-L1-A1A2A3A4-L2-, and the right side is connected to -D.

[0315] In embodiments of this disclosure, polydisperse PEG (a non-uniform mixture of size and molecular weight), monodisperse PEG (a single chain length and molecular weight) and discrete PEG can be used as PEG units in the ADC of this disclosure, with preferred PEGs being discrete PEGs, which are compounds synthesized stepwise rather than by a polymerization process, and are single molecules having a defined chain length.

[0316] The ADC disclosed herein may comprise one or more PEG chains consisting of at least two ethylene oxide (CH2CH2O) subunits. The PEG chains may be linked together, for example, in a linear, branched, or star configuration. Typically, at least one PEG chain is functionalized to enable covalent linkage with other components in the linker unit, including via, for example, amines, thiols, NHS esters, maleimides, alkynes, azides, carbonyl groups, or other functional groups. For example, one end of the PEG chain may be functionalized to covalently link to an appropriate site in the linker unit, while the other end (or ends) may be free and unbound, i.e., not linked to other components in the ADC, and may take the form of a methoxy, carboxylic acid, alcohol, or other suitable functional group; or the PEG chains may be covalently linked in tandem between two components of two linker units or inserted into the chain of a linker. The linkages between the PEG chains and the components within the linker unit are cleavable bonds, which may be bonds that are substantially insensitive to cleavage during circulation in plasma but sensitive to cleavage in the intracellular or tumor intracellular environment. Exemplary connections include, but are not limited to, amide bonds, ether bonds, ester bonds, hydrazone bonds, oxime bonds, disulfide bonds, peptide bonds, or triazole bonds.

[0317] Methods of attaching PEG units to the linker units of an ADC are well known to those skilled in the art. For example, PEG can be covalently bound to an amino acid residue via a reactive group. Reactive groups are those to which an activated PEG molecule can bind, such as free amino or carboxyl groups; thiol groups, for example, on cysteine ​​residues, can also be used as reactive groups for linking PEG. In some embodiments, methoxylated PEG (“mPEG”) with different reactive moieties can also be used to link PEG to an amino group. Non-limiting examples of such reactive moieties include succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imino ester, p-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuric chloride. Accordingly, non-limiting examples of mPEG include mPEG-succinimide succinate (mPEG-SS), mPEG-succinimide carbonate (mPEG-SC), mPEG-imide ester, mPEG-p-nitrophenyl carbonate (mPEG-NPC), mPEG-succinimide propionate (mPEG-SPA), mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG-cyanuric chloride, mPEG2-lysine alcohol-NPC, and mPEG2-Lys-NHS.

[0318] In some embodiments, the PEG unit comprises one or more linear PEG chains, each PEG chain having 2-72 (e.g., 2-60, 2-40, 2-24, 6-60, 8-40, 6-24, 2-30, 2-20, 2-12) ethylene oxide (CH2CH2O) subunits, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 ethylene oxide (CH2CH2O) subunits. In a preferred embodiment, the PEG unit comprises at least 2, at least 4, at least 6, at least 8, at least 10, or at least 12 ethylene oxide (CH2CH2O) subunits, for example, the PEG unit comprises 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 12, 6 to 10, 6 to 8, 8 to 12, or 8 to 10 subunits. For example, PEG... 4-20 This indicates a PEG unit having 4-20 CH2CH2O subunits.

[0319] Exemplary implementations of linear PEG units (i.e., one end connected to a connector unit and the other end a cap) include:

[0320] -NH-(CH2CH2O) b- CH2CH2COOH;

[0321] -NH-(CH2CH2O) b- CH2CH2C(=O)NH(CH2CH2O)-CH2CH2COOH;

[0322] -C(=O)-(CH2CH2O) b- CH3;

[0323] -(CH2CH2O) b CH3;

[0324] -NH-(CH2CH2O) b- CH2CH2NH-(CH2CH2O)-CH2CH2COOH;

[0325] Each b is independently selected from 2-72 integers, such as 2-60, 2-4, 2-24, 2-10, 4-10, 6-60, 8-40, 6-24, 2-30, 2-20, 2-12, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20, 24, 30, 35, 40.

[0326] In some implementations, -L2-L3- is selected from the following group:

[0327] (a) L2 is selected from non-existent, X, Su, and R1 are defined in the text. The left side of the L2 group is connected to A1A2A3A4, and the right side is connected to -L3-D; and L3 is absent.

[0328] (b) L2 does not exist; L3 is selected from... R2, R3, and X4 are each independently defined in the text, with the left side of the L3 group connected to -Z-L1-A1A2A3A4-L2- and the right side connected to -D; and / or

[0329] (c)L2 is selected from The symbols are as defined above; L3 is... The symbols are as defined above;

[0330] Preferably, -L2-L3- is selected from the group consisting of:

[0331] (a) L2 is either non-existent or selected from Where X and Su are as defined in the text, the left side of the L2 group is connected to A1A2A3A4, and the right side is connected to -L3-D; and L3 is absent; and

[0332] (b) L2 does not exist; and L3 is selected from... The left side of the L3 group shown is connected to -Z-L1-A1A2A3A4-L2-, and the right side is connected to -D.

[0333] In some implementations, -L2- and -L3- are not present.

[0334] In some implementations, -L2-L3- is selected from

[0335] The symbols, such as X, X4, R1, R2, and R3, are defined as above.

[0336] -L2-L3- indicates that it does not exist or is selected from [the specified range].

[0337] In some implementations, -L2-L3- is selected from:

[0338] Does not exist

[0339] In some implementations, -L2-L3- is

[0340] In some implementations, -L2-L3- is

[0341] In some implementations, -L2-L3- is

[0342] In some implementations, -L2-L3- is

[0343] In some implementations, -L2-L3- is In some implementations, -L2-L3- is

[0344] In some implementations, -L2-L3- is

[0345] In some implementations, -L2-L3- is

[0346] It should be understood that, unless otherwise specified, the left side (e.g., the end with an asterisk) of the -L2-L3- group shown in the text is connected to A1A2A3A4, and the right side (e.g., the end with a wavy line) is connected to -D.

[0347] In some implementations, -Z-L1-A1A2A3A4-L2-L3- is selected from:

[0348] The bonds marked with an asterisk (*) are connected to the target portion Tg, and the bonds marked with a wavy line are connected to D.

[0349] In some implementations, D is selected from cytotoxic agents, STING activators, glucocorticoid compounds, radionuclides, and siRNA.

[0350] In some implementations, D is selected from cytotoxic agents, STING activators, glucocorticoid compounds, protein degraders, and siRNA.

[0351] In some embodiments, D is selected from topoisomerase I inhibitors, topoisomerase II inhibitors, glycopeptide antibiotics, agents that interfere with DNA synthesis, microtubule inhibitors, anti-apoptotic agents, mitotic inhibitors, antitumor antibiotics, immunomodulators, alkylating agents, anti-angiogenic agents, antimetabolites, corticosteroids, photosensitizing agents, oligonucleotides, radionuclides, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, TLR7 / 8 agonists, PNU, Bcl-xl inhibitors, kinase inhibitors, transcription inhibitors (e.g., reverse transcriptase inhibitors), proteasome inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, protein degraders, glucocorticoid receptor modulators, and other small molecule compounds that inhibit tumor growth.

[0352] In some implementations, D is selected from camptothecin compounds, auristatin compounds, vinca alkaloids, taxane compounds, podophyllotoxin compounds, maytansine compounds, and benzo[a]benzodiazepines (PBD).

[0353] In some implementations, D is selected from: Dxd, Exatecan, SN38, MMAE, MMAF, DM1, DM4, eribulin, tubulysin, duocamycin, thailanstatin A, and amanitin.

[0354] In some implementations, D has the structure shown in equation (D-1a), equation (D-1b), or equation (D-1c):

[0355] Where R 1a Selected from H and C1-C6 alkyl-;

[0356] R 2a Selected from H, halogen, C1-C6 alkyl-, C1-C6 haloalkyl-, -OR 5a and -SR 5a ;

[0357] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, and -OR 5a ;and

[0358] R 4a and R 5a Independently selected from H and C1-C4 alkyl-;

[0359] or

[0360] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0361] R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0362] R 9b Selected from C 1-8 Alkyl group and COOH; preferably C 1-4 Alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0363] R 10b Selected from OH and H;

[0364] or

[0365] Where R 1c Selected from H, halogen, C1-C6 alkyl-, C1-C6 haloalkyl-, -OR 2c and -SR 2c ;and

[0366] R 2c Independently selected from H and C1-C4 alkyl-.

[0367] It should be understood that a key with a wavy line in the structure indicates that the key is connected to another structure or segment.

[0368] In some implementation schemes, R 1a For H; R 2a C1-C6 alkyl-; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl.

[0369] In some implementation schemes, R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl;

[0370] R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl-;

[0371] R 6b and R 7b Each was independently selected from C 1-2 alkoxy-; and

[0372] R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0373] In some implementation schemes, R 1c It is a C1-C6 alkyl-, preferably ethyl.

[0374] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b):

[0375] Where R 1a R 2a R 3a and R 4a As defined above; or

[0376] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined above;

[0377] Where R 1cAs defined above.

[0378] In some implementations, D has the structure shown in formula (D-3a) or (D-3b):

[0379] In some implementations, the unspecified chiral neutrals are each independently of the R or S configuration.

[0380] In some implementations, D is selected from the following structures:

[0381] In some embodiments, the drug conjugate of Formula I is selected from...

[0382] Tg and q are as defined in the implementation plan or definition section.

[0383] In some implementations, Tg is an antigen-binding molecule.

[0384] In some implementations, Tg is an antibody or its antigen-binding fragment.

[0385] In some implementations, Tg is a full-length antibody or its antigen-binding fragment.

[0386] In some implementations, Tg is a monoclonal antibody or its antigen-binding fragment.

[0387] In some implementations, Tg is a multispecific antibody, such as a bispecific antibody or its antigen-binding fragment.

[0388] In some implementations, Tg is an antibody or antigen-binding fragment thereof that targets tumor-specific antigens or tumor-associated antigens.

[0389] In some implementations, the target Tg is an antibody or antigen-binding fragment thereof that targets one or more of the following targets or antigens (e.g., 1, 2, 3, or 4):

[0390] Claudin18.2 (CLDN18.2), CDH17 (intestinal adhesion molecule 1), FOLR1, MSLN, B7H1, B7H3, B7H4, Her1, Her2, Her3, Her4, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET, c-Met, EGFRvIII, KIT, CTLA-4, GCPII, IL-13Ra, BCMA, GD2, 5T4,, VEGFR1, VEGFR2, VEGFR3, AOC3,, CA-125, FGFR1, FGFR2, FGFR3, FGFR4, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD16, CD18, CD19, CD20, CD21, CD22, CD25, CD30, CD32, CD33, CD37, CD38, CD40, CD44, CD45 (PTPRC), CD52, CD56, CD64, CD66e, CD70, CD72, CD74, CD79a, CD79b, CD80, CD123, CD138, CD142, CD174, CD223, CD276, CDH3, CCD79b, CLDN9 / CLDN6, IGF1R, IGF2R PDGFR-α, PDGFR-β, ENPP3, CA19-9, DPEP3, AGS-16, FcRH5, FRα, KAAG1, RPR1, CSF1R, EphA2, Mesothelin, ROR1, SLTRK6, TF, BMPR1B, E16, CLL-1, CA-IX, Somatostatin receptor, RANK, MUC1, TOP1, NCA, MDP, IL20R-α, Brevican, STEAP1, Sema 5b, PSCA hIg, ETBR, RNF124, TrpM4, C3DR, FcRH2, EphB2R, ASLG659, GEDA, BAFF-R, DLL4, EpCAM, FAP, CXCR5, HLA-DOB, P2X5, LY64, Mucin1. FcRH1, IRTA2, TENB2, FGF2, ALK, AXL, C242, PSMA, O772P, MUC16, Napi3b, SLAMF7, ITGB6, CEACAM5, CA9, EGFRvlll, IL2RA, CCL5, CXCL10, CXCL11, IFI6, TGF-βR, TNFRSF8, CLEC14A, GRP78, ASG-5, PRR4, GUCY2C, SLC39A6, Endothelin receptor, LIV-1, Integrin α5β6, Integrin α4β7, TPBG, CA242, FOLR1, GPNMB, HAVCR1, VTCN1, PTK7, TACSTD2, CA6, DLL3, DKK-1, Endoglin, VCAM1, GPC3, DR5, and ASCT2.

[0391] In some implementations, Tg is an antibody or its antigen-binding fragment that targets the following targets or antigens:

[0392] CLDN18.2, CDH17, FORR1, MSLN, B7H1, B7H3, B7H4, Her1, Her2, Her3, Her4, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET and / or c-Met.

[0393] In some implementations, Tg is an antibody or its antigen-binding fragment that targets the following targets or antigens:

[0394] CLDN18.2, CDH17, FORR1, MSLN, B7H3, Her2, Her3, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET and / or c-Met.

[0395] In some implementations, Tg is an anti-HER2 antibody or its antigen-binding fragment; the anti-HER2 antibody is, for example, trastuzumab or pertuzumab;

[0396] In some implementations, Tg is an anti-TROP2 antibody or its antigen-binding fragment; an anti-TROP2 antibody is, for example, Sacituzumab;

[0397] In some implementations, Tg is an anti-Nectin4 antibody or its antigen-binding fragment;

[0398] In some implementations, Tg is an anti-Nectin4 / anti-Trop2 bispecific antibody or its antigen-binding fragment;

[0399] In some implementations, Tg is an anti-EGFR and / or c-Met antibody or its antigen-binding fragment.

[0400] In some implementations, Tg is an antibody that specifically binds to EGFR and cMET (also referred to herein as an anti-EGFR / cMET antibody) or its antigen-binding fragment.

[0401] In some embodiments, the anti-EGFR / cMET antibody comprises a VHH domain that specifically binds to EGFR, a first VHH domain that specifically binds to cMET, and a second VHH domain that specifically binds to cMET.

[0402] In some implementations, the specific binding of the VHH domain (VHH) to EGFR EGFR The VHH comprises CDR1, CDR2, and CDR3, respectively, having amino acid sequences as shown in SEQ ID NOs:3, 4, and 5. In some embodiments, the VHH EGFR The domain comprises, or is composed of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence shown in SEQ ID NO:6, and preferably comprises the aforementioned CDR1, CDR2, and CDR3. In some embodiments, the VHH EGFR The domain contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0403] In some embodiments, the specific binding cMET first VHH domain (first VHH) cMET The VHH comprises CDR1, CDR2, and CDR3, respectively, having amino acid sequences as shown in SEQ ID NOs:7, 8, and 9. In some embodiments, the first VHH cMET The domain comprises, or is composed of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence shown in SEQ ID NO: 10, and preferably comprises the aforementioned CDR1, CDR2, and CDR3. In some embodiments, the first VHH cMET The domain contains, or is composed of, the amino acid sequence shown in SEQ ID NO:10.

[0404] In some embodiments, the second VHH domain (second VHH) that specifically binds to cMET cMET The VHH comprises CDR1, CDR2, and CDR3, respectively, having amino acid sequences as shown in SEQ ID NOs:11, 12, and 13. In some embodiments, the second VHHcMET The domain comprises, or is composed of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence shown in SEQ ID NO: 14, and preferably comprises the aforementioned CDR1, CDR2, and CDR3. In some embodiments, the second VHH cMET The domain contains, or is composed of, the amino acid sequence shown in SEQ ID NO:14.

[0405] In some preferred embodiments, the VHH domain that specifically binds to EGFR comprises CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs:3, 4, and 5, respectively; the first VHH domain that specifically binds to cMET comprises CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs:7, 8, and 9, respectively; and the second VHH domain that specifically binds to cMET comprises CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs:11, 12, and 13, respectively.

[0406] In some particularly preferred embodiments, the VHH domain that specifically binds to EGFR comprises or is composed of the amino acid sequence shown in SEQ ID NO:6, the first VHH domain that specifically binds to cMET comprises or is composed of the amino acid sequence shown in SEQ ID NO:10, and the second VHH domain that specifically binds to cMET comprises or is composed of the amino acid sequence shown in SEQ ID NO:14.

[0407] In some embodiments, the anti-EGFR / cMET antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: a VHH domain specifically binding to EGFR and a first immunoglobulin Fc region, and the second polypeptide chain comprises: a first VHH domain specifically binding to cMET, a peptide linker, a second VHH domain specifically binding to cMET, and a second immunoglobulin Fc region, and preferably the VHH domain specifically binding to EGFR, the first VHH domain specifically binding to cMET, and the second VHH domain specifically binding to cMET are as previously defined.

[0408] In some embodiments of the anti-EGFR / cMET antibody comprising the aforementioned first and second polypeptide chains, the first and second immunoglobulin Fc regions respectively contain Hole mutations and Knob mutations. In some embodiments, the first and second immunoglobulin Fc regions respectively contain LALA mutations. In some embodiments, the first and second immunoglobulin Fc regions are Fc regions of human IgG1, IgG2, IgG3, or IgG4 isotypes, or comprise, respectively, the amino acid sequences of SEQ ID NOs:15 and 16, or sequences having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, or are composed of, sequences thereof.

[0409] In some embodiments of the anti-EGFR / cMET antibody comprising the aforementioned first and second polypeptide chains, the peptide linker comprises (GGGGS)n (SEQ ID NO:17), where n is 1, 2, 3, 4, 5, 6, or 7, for example, GGGGSGGGGS (SEQ ID NO:18).

[0410] In some preferred embodiments of the anti-EGFR / cMET antibody comprising the aforementioned first and second polypeptide chains, the first polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and the second polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some particularly preferred embodiments, the first polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:1, and the second polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:2.

[0411] In some particularly preferred embodiments, Tg is an anti-EGFR / cMET antibody (referred to herein as V-F31) consisting of a first polypeptide chain with the amino acid sequence shown in SEQ ID NO:1 and a second polypeptide chain with the amino acid sequence shown in SEQ ID NO:2.

[0412] In some implementations, Tg is an antibody that specifically binds to Nectin4 and Trop2 (also referred to herein as an anti-Nectin4 / Trop2 antibody) or its antigen-binding fragment.

[0413] In some embodiments, the anti-Nectin4 / Trop2 antibody contains a VHH domain that specifically binds to Nectin4 (VHH). Nectin4 ) and the VHH domain that specifically binds to Trop2 (VHH Trop2 ).

[0414] In some implementations, the specific binding to the VHH domain of Nectin4 (VHH) Nectin4 The VHH comprises CDR1, CDR2, and CDR3, respectively, having amino acid sequences as shown in SEQ ID NOs:22, 23, and 24. In some embodiments, the VHH Nectin4 The domain comprises, or is composed of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence shown in SEQ ID NO:25, and preferably comprises the aforementioned CDR1, CDR2, and CDR3. In some embodiments, the VHH Nectin4 The domain contains, or is composed of, the amino acid sequence shown in SEQ ID NO:25.

[0415] In some implementations, the specific binding to the VHH domain of Trop2 (VHH) Trop2 The VHH comprises CDR1, CDR2, and CDR3, respectively, having amino acid sequences as shown in SEQ ID NOs:26, 27, and 28. In some embodiments, the VHH Trop2 The domain comprises, or is composed of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the amino acid sequence shown in SEQ ID NO:29, and preferably comprises the aforementioned CDR1, CDR2, and CDR3. In some embodiments, the VHH Trop2 The domain contains, or is composed of, the amino acid sequence shown in SEQ ID NO:29.

[0416] In some preferred embodiments, the VHH domain that specifically binds to Nectin4 comprises CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs:22, 23, and 24, respectively, and the VHH domain that specifically binds to Trop2 comprises CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs:26, 27, and 28, respectively.

[0417] In some particularly preferred embodiments, the VHH domain that specifically binds to Nectin4 comprises or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VHH domain that specifically binds to Trop2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:29.

[0418] In some embodiments, the anti-Nectin4 / Trop2 antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: a VHH domain specifically binding to Nectin4, a peptide linker, a VHH domain specifically binding to Trop2, and a second immunoglobulin Fc region; the second polypeptide chain comprises: a VHH domain specifically binding to Nectin4 and a first immunoglobulin Fc region, and preferably the VHH domain specifically binding to Nectin4 and the VHH domain specifically binding to Trop2 are as previously defined.

[0419] In some embodiments of the anti-Nectin4 / Trop2 antibody comprising the aforementioned first and second polypeptide chains, in some embodiments, the first and second immunoglobulin Fc regions respectively contain Knob mutations and Hole mutations. In some embodiments, the first and second immunoglobulin Fc regions respectively contain LALA mutations. In some embodiments, the first and second immunoglobulin Fc regions are Fc regions of human IgG1, IgG2, IgG3, or IgG4 isotypes, or comprise, respectively, the amino acid sequences of SEQ ID NOs:16 and 15, or sequences having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, or are composed of, sequences thereof.

[0420] In some embodiments of the anti-Nectin4 / Trop2 antibody comprising the aforementioned first and second polypeptide chains, the peptide linker comprises (GGGGS)n (SEQ ID NO:17), where n is 1, 2, 3, 4, 5, 6, or 7, for example, GGGGSGGGGS (SEQ ID NO:18).

[0421] In some preferred embodiments of the anti-Nectin4 / Trop2 antibody comprising the aforementioned first and second polypeptide chains, the first polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:20 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it; and the second polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:21 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. In some particularly preferred embodiments, the first polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:20, and the second polypeptide chain comprises, or is composed of, the amino acid sequence of SEQ ID NO:21.

[0422] In some particularly preferred embodiments, Tg is an anti-Nectin4 / Trop2 antibody (referred to herein as V-hu23) consisting of a first polypeptide chain with the amino acid sequence shown in SEQ ID NO:20 and a second polypeptide chain with the amino acid sequence shown in SEQ ID NO:21.

[0423] In some embodiments, the targeting moiety (Tg) is bonded to the linker unit via its heteroatoms. Heteratoms present on the targeting moiety for this bonding include sulfur (in one embodiment, a thiol group from the targeting ligand), oxygen (in one embodiment, a carboxyl or hydroxyl group from the targeting ligand), and optionally substituted nitrogen (in one embodiment, a primary or secondary amine functional group from the targeting ligand, or in another embodiment, an optionally substituted amide nitrogen). These heteroatoms may be present on the targeting moiety in a natural state, such as being naturally present in the targeting moiety, or may be introduced into the targeting moiety through chemical modification or bioengineering.

[0424] In some embodiments, the target moiety (Tg) has a thiol functional group, such that it is bonded to the linker unit via the sulfur atom of the thiol functional group. In other embodiments, the thiol group is generated by reducing the interchain disulfide of the target moiety. Therefore, in some embodiments, the linker unit is conjugated to a cysteine ​​residue from the reduced interchain disulfide in the target moiety. In other embodiments, the thiol group is chemically introduced into the target moiety, for example, by introducing a cysteine ​​residue. Accordingly, in some embodiments, the linker unit is conjugated to the target moiety via a cysteine ​​residue introduced into the target moiety.

[0425] In other embodiments, the targeting moiety (Tg) has one or more lysine residues that are capable of reacting with activated esters (including but not limited to N-hydroxysuccinimide, pentafluorophenyl and p-nitrophenyl esters) in the linker unit, thereby providing an amide bond consisting of a nitrogen atom of the targeting moiety and a C=O of the linker unit.

[0426] In other embodiments, the targeting moiety (Tg) has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In these embodiments, the targeting moiety is covalently attached to the linker unit via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysine in this manner include, but are not limited to, N-succinimide-S-acetylthioacetate (SATA) and 2-iminothiacyclopentane hydrochloride (Traut reagent).

[0427] In other embodiments, the targeting portion (Tg) has one or more carbohydrate groups that can be modified to provide one or more thiol functional groups. The chemically modified targeting portion in the ADC is bonded to the linker unit via the sulfur atom of the thiol functional group.

[0428] In other embodiments, the targeting portion (Tg) has one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) functional group. In these embodiments, the corresponding aldehyde interacts with reactive sites on the linker unit to form a chemical bond between the linker unit and the targeting portion unit.

[0429] In other implementations, artificial connection sites are introduced in the target portion (Tg) to achieve more targeted coupling.

[0430] In some embodiments, the antibody of this application may be a modified glycosylated antibody. In some embodiments, the antibody is an antibody obtained by in vitro enzymatic modification of the glycan chain (e.g., modification of the glycan chain by a glycosidase (e.g., glycosidase or glycosyltransferase)). In some embodiments, the modified glycosylated antibody refers to an antibody in which the glycan chain at the antibody glycosylation site is modified from a heterogeneous N-glycan chain to a single-structure N-glycan chain with a reactive group (e.g., any reactive group capable of reacting with the linker moiety, such as an azide, ketone, or alkynyl group). In a preferred embodiment, the N-glycosylation site of the antibody is a conserved N-glycosylation site on the antibody Fc domain, such as Asn297.

[0431] Methods suitable for the modification of antibody glycosylation in this application are, for example, PCT / NL2013 / 050744, PCT / EP2016 / 059194 or PCT / EP2017 / 052792, the entire contents of which are incorporated herein by reference.

[0432] In one embodiment, the targeting portion of the drug conjugate comprises q (where q is as defined in Formula I, preferably 1 or 2) structures as follows: Wherein GlcNAc is N-acetylglucosamine, and E is a sugar or modified sugar selected from galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), preferably Gal, GlcNAc, glucose and GalNAc, most preferably GalNAc, and b is 0 or 1;

[0433] An asterisk (*) indicates a connection to the rest of the target portion (Tg), and a wavy line indicates a connection to the target portion (Tg). This indicates a connection to Z.

[0434] If the modified antibody is an antibody fragment, such as a Fab or Fc fragment, the antibody may have only one It is optionally fucosylated. In one embodiment, the... The substituent is located in the Fc domain of the antibody, more preferably in the CH2 domain.

[0435] In a preferred embodiment It exists at the native N-glycosylation site (e.g., a naturally conserved N-glycosylation site) of the antibody, such as the glycosylation site of the Fc region (more preferably the CH2 domain). In another preferred embodiment, the antibody is an IgG antibody and the... It exists at the native N-glycosylation site (naturally conserved N-glycosylation site) of the IgG antibody. In another preferred embodiment, the native site is the Asn297-glycosylation site of the IgG antibody. The Asn297-glycosylation site is located in the Fc region of the heavy chain of the IgG antibody. In a preferred embodiment, the It exists at the Asn297-glycosylation site on both heavy chains of the antibody.

[0436] In one implementation, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

[0437] In some embodiments, the drug conjugate or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates have an average DAR of 1-10, 3-5, 5-7, 6-9, 6-10, 4-8, 7-9 or 2-4.

[0438] It should be understood that the average DAR can include both integers and decimals.

[0439] On the other hand, this application provides compounds of formula II or their stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates.

[0440] Z'-L1-A1A2A3A4-L2-L3-D

[0441] II

[0442] in

[0443] Z' is the connector unit precursor;

[0444] Other symbols, such as L1, A1A2A3A4, L2, L3, and D, are defined as described above (Equation I).

[0445] The connector unit precursor is a group or structure that can react with the target portion to form the connector unit Z.

[0446] In some embodiments, Z' is selected from groups capable of reacting with primary amines or cysteines (e.g., their thiol groups) on the target moiety (e.g., antibodies or their antigen-binding fragments) to link to the target moiety, and groups capable of click chemistry with the target moiety (including modified antibodies or their antigen-binding fragments) to link to the target moiety.

[0447] In some embodiments, the cysteine ​​is located in the hinge region of the antibody or its antigen-binding fragment.

[0448] In some embodiments, Z' has the following structure: Z1'-Z2-, where Z2 is as defined above (Formula I), and Z1' is selected from the following groups:

[0449] Each of X1 is independently selected from C 1-6 Alkyl, halogen, and nitro groups;

[0450] r is 0, 1, 2, or 3;

[0451] X2 is independently selected from Cl, Br, and I; and

[0452] X3 is independently selected from Cl, Br, I, and C. 1-6 Alkyl SO2- (preferably MeSO2-);

[0453] It contains wavy lines The end of is covalently connected to Z2 or L1 (when Z2 does not exist).

[0454] In some embodiments, Z' has the following structure: Z1'-Z2-, where Z2 is as defined above (Formula I), and Z1' is selected from the following groups:

[0455] The symbols (e.g., X1, r, X2, and X3) are defined as above.

[0456] In some implementations, Z1' is selected from:

[0457] X2 is independently selected from Cl, Br, and I; and

[0458] X3 is independently selected from Cl, Br, I and MeSO2-.

[0459] In some implementations, Z1' is selected from:

[0460] X2 is selected from Cl, Br and I.

[0461] In some implementations, Z1' is

[0462] It should be understood that the terminal bonds with wavy lines in the structure in the text represent covalent connections with other groups or structures through that bond.

[0463] In some embodiments, the compound of formula II is selected from...

[0464] On the other hand, this application provides a compound of formula III or a salt thereof:

[0465] P1-A 1b A 2b A 3b A 4b -P4

[0466] III

[0467] in

[0468] P1 is selected from H- and amino protecting groups;

[0469] P4 is selected from -OH, leaving group, and -OR. 3d -NR 4d R 5d ,

[0470] R 3d Selected from C 1-20Alkyl-, 6-10 aryl-, 3-10 heteroaryl- and 3-10 cycloalkyl-;

[0471] R 4d R 5d Independently selected from H and C 1-20 Alkyl-, 6-10 aryl-, 3-10 heteroaryl- and 3-10 cycloalkyl-;

[0472] Where X, R1, Su, r1, and t are defined as L2 above;

[0473] Where -A 1b A 2b A 3b A 4b -As defined above for the tetrapeptide unit -A1A2A3A4-, and the amino acids constituting the tetrapeptide unit (A 1b A 2b A 3b and / or A 4b )Optionally protected by the protective base;

[0474] P1 is located at the N-terminus of the peptide, and P4 is located at the C-terminus of the peptide.

[0475] In some embodiments, the amino protecting group is selected from: Boc, Fmoc, acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, formyl or benzoyl, more preferably Boc or Fmoc.

[0476] In some embodiments, the leaving group is a detectable leaving group, preferably a fluorescent leaving group.

[0477] In some embodiments, the fluorescent leaving group is selected from ACC- and AMC-.

[0478] ACC stands for 7-amino-4-carbamoylmethyl-coumarin;

[0479] AMC is 7-amino-4-methylcoumarin.

[0480] In some embodiments, the fluorescent leaving group is bonded via an amide bond.

[0481] In some implementations, P1 is selected from H-, Fmoc, and Boc-.

[0482] In some implementations, P1 is selected from H- and Ac-.

[0483] In some implementations, P4 is selected from -OH and In some implementations, P4 is selected from -OH and AMC-.

[0484] In some implementations, P1 is Ac and P4 is AMC-.

[0485] In some implementations, P1 is H and P4 is -OH.

[0486] In some implementations, the amino acids (A, B, C, D, E, F, G, M, C, M ... 1b A 2b A 3b and / or A 4b The protecting group of a peptide is a protecting group used in peptide synthesis, such as an amino protecting group, a guanidine protecting group, an amide protecting group, a carboxyl protecting group, and / or a hydroxy protecting group.

[0487] In some implementations, the protective base is selected from: Boc, Fmoc, Pbf, Trt, t-BuO, t-Bu, and Acm.

[0488] It should be understood that those skilled in the art are aware of the meaning of the abbreviations for the protection base.

[0489] In some embodiments, the compound of formula III is selected from:

[0490] P2 is independently selected from H and guanidinium protecting groups, for example, selected from H and Pbf;

[0491] P3 is independently selected from H and amide protecting groups, for example, selected from H and Tlt;

[0492] P5 groups are each independently selected from HO- and carboxyl protecting groups; for example, selected from HO- and C-. 1-8 Alkyl-O-, for example t-BuO;

[0493] P6 is independently selected from H and hydroxyl protecting groups, for example, selected from H and C. 1-8 Alkyl-O-, for example t-Bu; and

[0494] P1 and P4 are defined independently as described above.

[0495] In some embodiments, the compound of formula III is selected from:

[0496] P2 is independently selected from H and guanidinium protecting groups, for example, selected from H and Pbf;

[0497] P3 is independently selected from H and amide protecting groups, for example, selected from H and Tlt;

[0498] P5 groups are each independently selected from HO- and carboxyl protecting groups; for example, selected from HO- and C-. 1-8 Alkyl-O-, for example t-BuO;

[0499] P6 is independently selected from H and hydroxyl protecting groups, for example, selected from H and C. 1-8 Alkyl-O-, for example t-Bu; and

[0500] P1 and P4 are defined independently as described above.

[0501] In some implementations, P1 is independently selected from H, Boc, and Fmoc;

[0502] P2 is independently selected from H and Pbf;

[0503] P3 is independently selected from H and Trt;

[0504] P4 is independently selected from -OH and

[0505] P5 is independently selected from -OH and t-BuO;

[0506] P6 is independently selected from H and t-Bu.

[0507] In some embodiments, the compound of formula III is selected from...

[0508] On the other hand, this application provides the use of the compound of Formula III as described above or its salt for the preparation of linkers and / or drug conjugates.

[0509] In some embodiments, the linker is a linker used to prepare a drug conjugate or a linker in a drug conjugate.

[0510] On the other hand, this application provides a kit for analyzing the cathepsin B activity of a sample, comprising: a compound of formula III or a salt thereof as described above, wherein P4 is a detectable leaving group as defined above, and the remaining variables are as defined above; and a suitable container in which the compound of formula III or a salt thereof is placed.

[0511] In some embodiments, the compound of formula III or a salt thereof is provided in solution form, lyophilized, or bound to a solid support.

[0512] In another aspect, this application provides compositions, such as pharmaceutical compositions, comprising molecules of the present invention (including pharmaceutical conjugates of Formula I, compounds of Formula II, or stereoisomers, isotopic variants, pharmaceutically acceptable salts, or solvates thereof) formulated with pharmaceutically acceptable excipients. As used herein, “pharmaceuticalally acceptable excipients” include any physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the molecule of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the molecule of the present invention with one or more other therapeutic agents.

[0513] In another aspect, this application provides a pharmaceutical combination comprising the molecules of the present invention with one or more other therapeutic agents.

[0514] The therapeutic agents in the pharmaceutical compositions and pharmaceutical combinations applicable to the present invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; and (iv) drugs that have antitumor effects.

[0515] The pharmaceutical compositions of the present invention may contain a "therapeutic effective amount" or a "preventive effective amount" of the molecules of the present invention.

[0516] In another aspect, this application provides a kit comprising the molecules of the present invention. The kit may include one or more other elements, such as: instructions for use; other reagents, such as markers or agents for conjugation; pharmaceutically acceptable carriers or excipients; and a device or other material for administration to a subject.

[0517] On the other hand, this application provides a method for treating and / or preventing diseases or conditions, comprising administering an effective amount of the molecules of the present invention (including drug conjugates of Formula I, compounds of Formula II, or stereoisomers, isotope variants, pharmaceutically acceptable salts or solvates thereof) to a subject or patient in need.

[0518] In some implementations, the disease or condition is a proliferative disease, such as a tumor or cancer.

[0519] In some embodiments, the tumor is selected from: lung cancer (e.g., squamous cell carcinoma, adenocarcinoma, non-small cell lung cancer, or small cell lung cancer), breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, myeloma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, bone cancer, prostate cancer, esophageal cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma. Cellular carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

[0520] In some implementations, the tumor is selected from: lung cancer (e.g., squamous cell carcinoma, adenocarcinoma, non-small cell lung cancer, or small cell lung cancer), breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, and myeloma.

[0521] The targeting portion of this invention specifically binds to antigens on the surface of tumor cells, thereby enabling the drug conjugate to be targeted, with higher activity and reduced toxicity. In some embodiments, the tumor is an antigen-positive tumor (e.g., the antigens mentioned herein). In the application described, the molecules of this invention can be administered to the subject as the sole active agent or in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate the tumor by binding to and / or blocking these molecules; and drugs that activate the subject's immune system, prompting it to spontaneously eliminate the tumor.

[0522] In any of the above embodiments of the method of the present invention, the application of the molecules according to the present invention may include 1) a therapeutic measure that cures, alleviates, or reduces the symptoms of a diagnosed pathological condition or disease and / or stops the progression of the diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or alleviates the development of a pathological condition or disease. Therefore, in the method of the present invention, the subject may be an individual who already suffers from a disease, an individual susceptible to a disease, or an individual who wishes to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, this application relates to the treatment of a disease or condition; in other embodiments, this application relates to the prevention of a disease or condition.

[0523] The molecules according to the invention, and optionally other therapeutic agents used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0524] For the prevention or treatment of disease, the appropriate dosage of the molecule according to the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0525] On the other hand, this application provides molecules of the present invention (including drug conjugates of Formula I, compounds of Formula II, or stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof) for use as medicines, for example for treatment and / or prevention.

[0526] In some embodiments, the molecules of the present invention are used to treat and / or prevent diseases or conditions, preferably as defined above.

[0527] On the other hand, this application provides a pharmaceutical composition comprising the molecules of the present invention (including pharmaceutical conjugates of Formula I, compounds of Formula II, or stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof), said pharmaceutical composition being used as a drug.

[0528] In some embodiments, the pharmaceutical composition is used to treat and / or prevent diseases or conditions, preferably as defined above.

[0529] On the other hand, this application provides the use of the molecules of the present invention (including drug conjugates of Formula I, compounds of Formula II, or stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof) in the preparation of medicaments for treating and / or preventing diseases or conditions.

[0530] Preferably, the disease or symptom is as defined above.

[0531] The beneficial effects of this invention are:

[0532] Molecules containing the tetrapeptide structure of the present invention can be efficiently and specifically hydrolyzed by cathepsin B and / or exhibit good plasma stability, thus possessing extremely high application value. For example, they can be used as linkers for ADCs or as linking fragments between target proteins and other functional molecules. Furthermore, antibody-drug conjugates containing the tetrapeptide structure of the present invention exhibit lower hydrophobicity and / or better sidekilling activity, and / or improved stability, such as improved shelf life, and enhanced drug development potential, thereby resulting in improved efficacy and / or reduced toxicity.

[0533] definition:

[0534] The term "targeting moiety" refers to a portion of a cell surface that can specifically bind to a target (such as an antigen) or a portion of a target. Through the interaction between the targeting moiety and the target, conjugates can be delivered to specific cell populations. Targeting moieties include antigen-binding molecules, such as antibodies or antigen-binding fragments thereof.

[0535] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or peptide, or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions constructed based on antibodies or antigen-binding fragments, such as VHH-Fc antibodies, multi / bispecific antibodies, and chimeric antigen receptors (CARs). As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR".

[0536] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy chain antibodies, chimeric or humanized antibodies, intact antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0537] In this article, "intact antibody" or "full-length antibody" may be used interchangeably, referring to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this article) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL in this article) and a light chain constant region.

[0538] In this document, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to a molecule distinct from the intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; camelid antibodies (heavy chain antibodies) or fragments thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise stated herein or explicitly contradicted by the context, the term "antibody" as used herein is equivalent to "antibody or an antibody fragment thereof." In some embodiments of the invention, the antibody fragment includes cysteine ​​residue portions for forming interchain disulfide bonds between heavy chains, such as cysteine ​​residues in the antibody hinge region, to provide amino acid residue sites usable for thiol coupling chemistry. In other embodiments of the invention, the antibody fragment includes cysteine ​​residues introduced into the Fc region to provide amino acid residue sites usable for thiol coupling chemistry.

[0539] In this paper, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region in an antibody molecule that actually binds to the antigen.

[0540] In this document, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) comprising at least two antigen-binding sites, each of which binds to a different antigen or a different epitope, for example, to different epitopes on different antigens or different epitopes on the same antigen. Correspondingly, "single-specific" refers to the ability to bind to only one epitope. "Dual-specific" refers to the ability to bind to two different antigens or epitopes.

[0541] In this article, the terms "target" or "antigen" may be selected from Claudin18.2 (CLDN18.2), CDH17 (gut adhesion molecule 1), FOLR1, MSLN, B7H1, B7H3, B7H4, Her1, Her2, Her3, Her4, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET, c-Met, EGFRvIII, KIT, CTLA-4, GCPII, IL- 13Ra, BCMA, GD2, 5T4,, VEGFR1, VEGFR2, VEGFR3, AOC3,, CA-125, FGFR1, FGFR2, FGFR3, FGFR4, CCL11, CCR 5. CD2, CD3, CD4, CD5, CD15, CA15-3, CD16, CD18, CD19, CD20, CD21, CD22, CD25, CD30, CD32, CD33, CD37, C D38, CD40, CD44, CD45(PTPRC), CD52, CD56, CD64, CD66e, CD70, CD72, CD74, CD79a, CD79b, CD80, CD123, CD138, CD142, CD174, CD223, CD276, CDH3, CCD79b, CLDN9 / CLDN6, IGF1R, IGF2RPDGFR-α, PDGFR-β, ENPP3 , CA19-9, DPEP3, AGS-16, FcRH5, FRα, KAAG1, RPR1, CSF1R, EphA2, Mesothelin, ROR1, SLTRK6, TF, BMP R1B, E16, CLL-1, CA-IX, Somatostatin receptor, RANK, MUC1, TOP1, NCA, MDP, IL20R-α, Brevican, STEAP1, Sema 5b, PSCA hIg, ETBR, RNF124,, TrpM4,, C3DR, FcRH2, EphB2R, ASLG659, GEDA, BAFF-R, DLL4, EpCAM, FAP, CXCR5, HLA-DOB, P2X5, LY64, Mucin1. FcRH1, IRTA2, TENB2, FGF2, ALK, AXL, C242, PSMA, O772P, MUC16, Napi3b, SLAMF7, ITGB6, CEACAM5, CA9, EGFRvlll, IL2RA, CCL5, CXCL10, CXCL11, IFI6, TGF-βR, TNFRSF8, CLEC14A, GRP78, ASG-5, PRR4, GUCY2C, SLC39A6, Endothelin receptor, LIV-1, Integrin α5β6, Integrin α4β7, TPBG, CA242, FOLR1, GPNMB, HAVCR1, VTCN1, PTK7, TACSTD2, CA6, DLL3, DKK-1, Endoglin, VCAM1, GPC3, DR5, and ASCT2.

[0542] As used herein, a "drug conjugate" refers to a compound in which a target moiety and a biologically active moiety are linked together by a linker. In this invention, when the target moiety in the conjugate is an antibody or its antigen-binding fragment, the conjugate may be referred to as an "antibody-drug conjugate" or an ADC.

[0543] "Linker-payload" is a term well known to those skilled in the art, referring to a compound formed by linking a linker to a payload (e.g., a bioactive molecule such as a drug).

[0544] The term "linker" refers to a structural segment that connects a drug to a target portion. It should be understood that a linker has functional groups that can form bonds with functional groups of the target portion before being attached to it. Non-limiting examples of linkers include those involved in embodiments and examples of the present invention.

[0545] A "connector unit" is a structural unit that connects the target portion (Tg) and the remainder of the linker-payload (-L1-A1A2A3A4-L2-L3-D), and can be obtained by reacting a connector unit precursor (Z') in the linker-payload with the target portion. That is, the connector unit has functional groups that can form bonds with functional groups of the target ligand before being connected to the target portion (i.e., the connector unit precursor Z'). In some embodiments, the connector unit precursor (Z') has an electrophilic group capable of interacting with reactive nucleophilic groups present on the target portion (e.g., an antibody) to form a covalent bond between the target portion and the connector unit. Nucleophilic groups on the target portion with this capability include, but are not limited to, thiol, hydroxyl, and amino functional groups. The heteroatoms of the nucleophilic group of the target portion are reactive with the electrophilic group on the connector unit precursor and form a covalent bond between the target portion and the connector unit of the linker-payload portion. Electrophilic groups available for this purpose include, but are not limited to, maleimide compounds, haloamides (e.g., iodine, bromine, or chlorinated); haloesters (e.g., iodine, bromine, or chlorinated); halomethyl ketones (e.g., iodine, bromine, or chlorinated); benzyl halides (e.g., iodine, bromine, or chlorinated); vinyl sulfones, pyridyl sulfides, and methanesulfonyl pyrimidines. The electrophilic group provides a convenient site for linking the target moiety to form a conjugate or target moiety-linker intermediate. In some embodiments, the linker unit precursor has a reactive site with a nucleophilic group that reacts with the electrophilic group present on the target moiety (e.g., an antibody). Electrophilic groups available for this purpose on the target moiety include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group of the linker unit precursor can react with the electrophilic group on the target moiety and form a covalent bond with the target moiety. Nucleophilic groups available for this purpose on the linker unit precursor include, but are not limited to, acylhydrazides, hydroxylamines, amino groups, hydrazides, thioureas, carboxylic acid hydrazides, and aryl acylhydrazides. Electrophilic groups on the targeting moiety provide convenient sites for the targeting moiety to connect to form drug conjugates or targeting moiety-linker intermediates. In some embodiments, the sulfur atom of the targeting moiety is bonded to the linker unit by a succinimide ring system formed by the thiol functional group of the targeting ligand and the maleimide moiety of the corresponding linker unit precursor via a Michael addition reaction. In some embodiments, the thiol functional group of the targeting moiety is provided with a sulfur-bonded linker unit by a nucleophilic substitution reaction with the α-haloacetamide moiety to provide a linker unit by nucleophilic substitution of its halogen substituent. In some embodiments, the azide or alkynyl group on the modified targeting moiety undergoes a click chemistry reaction with the alkynyl or azide group of the linker unit precursor (Z') to provide a linker unit comprising a triazole structure. More specific “linker unit” and “linker unit precursor Z'” include, but are not limited to, the embodiments shown for Z herein.

[0546] An "extension unit" is a group or chemical structure that links a linker unit precursor (Z') or linker unit (Z) to a tetrapeptide unit. In some embodiments, an extension unit is included between the linker unit precursor (Z') or linker unit (Z) and the tetrapeptide unit to add additional distance between them, if necessary. In some embodiments, this additional distance may facilitate the activity of an enzyme such as cathepsin B on the tetrapeptide unit. Those skilled in the art will understand that the extension unit can be any group used to provide the linker unit precursor (Z') or linker unit (Z) to the tetrapeptide unit. More specific "extension units" include, but are not limited to, the embodiments shown in L1 herein.

[0547] A “self-cleaving unit” is a group or chemical structure capable of facilitating the release of a bioactive molecule from a drug conjugate. In some embodiments, a “self-cleaving unit” is a self-eliminating group capable of releasing -D without a separate hydrolysis step or subsequent self-elimination event. In some embodiments, a “self-cleaving unit” can provide additional structural components to further facilitate the release of the bioactive molecule from the remainder of the ADC. In some embodiments, a “self-cleaving unit” is a p-aminobenzyloxycarbonyl (PAB) group linked to the carbonyl group of a tetrapeptide unit and directly linked to the PAB moiety of L3 or -D via a carbonate group. More specific “self-cleaving units” include, but are not limited to, the embodiments shown for L2 herein.

[0548] A “spacer unit” is a group or chemical structure that links L2 (when L2 is present) or a tetrapeptide unit (when L2 is absent) to D. In some embodiments, a spacer unit (L3) is present between L2 (when L2 is present) or the tetrapeptide unit (when L2 is absent) and D. The spacer unit may be a functional group that facilitates the connection of L2 or the tetrapeptide unit to D, or it may provide additional structural components to further facilitate the release of D from the remainder of the conjugate (e.g., a methyl carbamate unit), or increase the additional distance between L2 (when L2 is present) or the tetrapeptide unit (when L2 is absent) and D. In some embodiments, L3 is also capable of self-cleavage. In some embodiments, L2 and L3 together or individually function as self-cleavage units. More specific “spacer units” include, but are not limited to, the embodiments shown herein with respect to L3.

[0549] In this document, the term "polysaccharide" refers to a polysaccharide composed of two or more monosaccharide groups linked by glycosidic bonds, which can be α-, β-, or a mixture of α and β. Structurally, it can be linear, branched, or cyclic. The monosaccharides constituting polysaccharides are selected from, but are not limited to, pentose and hexose sugars, such as glucose, galactose, mannose, arabinose, or xylose. Examples of polysaccharides include, but are not limited to, hyaluronic acid, hydroxyethyl starch, xylan, water-soluble starch, water-soluble cellulose, and carboxymethyl cellulose.

[0550] The term "dendritic polymer," also known as a dendritic polymer, refers to linear polymers in which each repeating unit has a dendron.

[0551] The term "cyclodextrin" is a collective term for a series of cyclic oligosaccharides formed from linear starch by cyclodextrin glucosyltransferase produced by Bacillus, typically containing 6 to 12 D-glucan units. Preferably, molecules containing 6, 7, or 8 glucose units are referred to as alpha-, beta-, and gama-cyclodextrins, respectively. The "cyclodextrin" portion attached to a drug conjugate is called a "cyclodextrin unit."

[0552] As used in this article, "stereoisomer" refers to compounds that have the same chemical composition but differ in the spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, etc.

[0553] The terms “enantiomer” and “enantiomer” used in this article are used interchangeably and refer to two stereoisomers of a compound that are non-overlapping mirror images of each other.

[0554] As used herein, the terms "diastereomer" and "diastereomer" are used interchangeably and refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, or biological activity. Mixtures of diastereomers can be separated using high-resolution analytical methods such as electrophoresis and chromatography such as HPLC.

[0555] In some embodiments, this application provides compounds containing multiple stereoisomer purities, i.e., enantiomeric or diastereomeric purities expressed in different "ee" or "de" values. In some embodiments, the compounds described herein have an enantiomeric purity of at least 60% ee (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% ee, or any value between these listed values). In some embodiments, the compounds described herein have an enantiomeric purity greater than 99.9% ee. In some embodiments, the compounds described herein have a diastereomeric purity of at least 60% de (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100% de, or any value between these listed values). In some embodiments, the compounds described herein have a diastereomeric purity greater than 99.9% de.

[0556] The determination of diastereomer excess and enantiomeric excess can be performed using a variety of analytical techniques (including nuclear magnetic resonance spectroscopy, chiral column chromatography and / or optical rotation determination) and according to conventional procedures familiar to those skilled in the art.

[0557] Racemic mixtures can be used in their original form or can be resolved into their individual isomers. Resolution yields stereochemically pure compounds or mixtures enriched with one or more isomers.

[0558] The term "salt" refers to a salt formed by the molecules of the present invention (e.g., pharmaceutical conjugates) with an organic or inorganic acid, or a salt formed with an organic or inorganic base, including pharmaceutically acceptable salts.

[0559] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the molecules of the present invention (e.g., drug conjugates (ADCs)) and is not biologically or otherwise undesirable. The ADCs of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0560] The term "solvent" refers to an association formed by one or more solvent molecules with the drug conjugate of this invention. Solvents that form solvates include solvents commonly used in the pharmaceutical industry, such as water, ethanol, diethyl ether, isopropanol, ethyl acetate, and dimethyl sulfoxide.

[0561] The term "DAR" refers to the ratio of the biologically active portion (D) to the target portion (Tg) in the drug conjugate molecule (e.g., the compound of formula I) described herein. It should be understood that, in this document, DAR refers to antibody-drug conjugate molecules and is an integer; for example, the DAR of the drug conjugate molecule of the present invention can be 1 to 20, such as an integer from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The prepared drug conjugates typically contain one or more drug conjugate molecules with different DARs, which are usually characterized by the average DAR, i.e., the overall ratio of the bioactive portion (D) conjugated to the Tg portion described herein to the Tg portion in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis and / or HPLC). This is referred to as the average DAR in this document, and the average DAR may be a decimal. In some embodiments, the average DAR value of the drug conjugate of the present invention is 1 to 16, for example 2-16, 4-12, 3-5, 5-7, 7-9, 3-8, 2-6, 4-6, for example 3.0-8.0, 3.5-4.5, 5.5-6.5 or 7.5-8.5, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. As described above, it should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules (which may also be referred to as a mixture of drug conjugates) containing ADC molecules having the same and / or different DAR values.

[0562] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0563] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0564] In this article, "pharmaceutically acceptable" means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious and intolerable side effects.

[0565] The term "prodrug" refers to a compound with low or no biological activity that is converted in vivo into a compound with higher biological activity via a chemical or biological process (i.e., a chemical reaction or enzymatic biotransformation). Typically, the biological activity of a biologically active compound is reduced (i.e., converted to a prodrug) by chemically modifying the compound with a prodrug portion. In some aspects, the prodrug is a type II prodrug, which is bioactivated extracellularly (e.g., in digestive fluids) or in the circulatory system of the human body (e.g., in the blood). In this document, the term "isotope variant" refers to a compound in which one or more atoms constituting the compound are replaced by atoms having an atomic mass or mass number different from those normally found in nature. Examples of isotopes that may be incorporated into one or more atoms of the compounds disclosed herein include, for example... 2 H, 3 H, 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S and 18 F, thereby forming isotopic variations of the compounds disclosed herein, whether or not they are radioactive, are intended to be covered within the scope of this disclosure. In some embodiments, the compounds of this disclosure are provided in an unlabeled form, and in other embodiments, the compounds of this disclosure are provided in an isotopically labeled form, such as in the form of hydrogen isotope D (deuterium) labeling, for example, one or more (e.g., 1, 2, 3, 4, 5, or 6) hydrogens substituted with deuterium. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) hydrogens of the bioactive molecular portion are substituted with deuterium.

[0566] In this disclosure, "bioactive molecule" refers to any molecule known in the art, including those used for diagnosing and / or treating diseases or for detecting and / or labeling targets, lesions, or diseases, including but not limited to drugs such as antitumor drugs, small molecule drugs, and small molecule antitumor drugs. In drug conjugates, after the linker is cleaved / degraded / enzymatically digested between tumor tissues or within tumor cells, a portion (fragment or group) of a biologically active compound, such as a drug (e.g., a small molecule cytotoxic drug, said drug including a group after losing an atom or group of atoms) or its derivative (e.g., its precursor), can be formed. To avoid ambiguity, "drug" does not only refer to "medicines" approved by pharmaceutical regulatory authorities, but also includes any molecule with potential therapeutic biological activity in clinical practice, or in research and development and academic studies. It should be understood that the bioactive molecule in a drug conjugate refers to a bioactive molecular fragment linked to the linker. Exemplary bioactive molecules for drug conjugates described herein include: cytotoxic agents, STING activators, glucocorticoid compounds, siRNA, topoisomerase I inhibitors, topoisomerase II inhibitors, glycopeptide antibiotics, DNA synthesis interfering agents, microtubule inhibitors, anti-apoptotic agents, mitotic inhibitors, antitumor antibiotics, immunomodulators, alkylating agents, anti-angiogenic agents, antimetabolites, corticosteroids, photosensitizing agents, oligonucleotides, radionuclides, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, TLR7 / 8 agonists, PNU, Bcl-xl inhibitors, kinase inhibitors, transcription inhibitors (e.g., reverse transcriptase inhibitors), proteasome inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, protein degraders, glucocorticoid receptor modulators, and other small molecule compounds that inhibit tumor growth. In some embodiments, the bioactive molecule is selected from camptothecin compounds, auristatin compounds, vinca alkaloids, taxane compounds, podophyllotoxin compounds, maytansine compounds, and benzobenzodiazepines (PBDs). In some embodiments, the bioactive molecules include Dxd, Exatecan, SN38, MMAE, MMAF, DM1, DM4, eribulins, tubulysin, duocamycin, thailanstatin A, and amanitin.

[0567] In this application, the term "small molecule" refers to molecules with a molecular weight of less than 2000 Daltons (Da), such as less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, or less than 500 Da.

[0568] For drug conjugates linked to a sugar in the targeting moiety (e.g., an antibody including a sugar-modified antibody) by a linker, the term "sugar" refers to a monosaccharide, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc), and includes modified monosaccharides, i.e., monosaccharides containing substituents and / or functional groups. Examples of modified sugars include amino sugars and sugar acids, such as glucosamine (GlcN), galactosamine (GalN), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). The sugar may be linked to the targeting moiety (e.g., other monosaccharides or polysaccharides on the targeting moiety). For example, it may be linked to a core-N-acetylglucosamine substituent on an antibody.

[0569] The core-N-acetylglucosamine substituent (core-GlcNAc substituent) is defined herein as GlcNAc bonded to the antibody via a C1 bond, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of the antibody's asparagine amino acid. The core-GlcNAc substituent may be present at the antibody's native glycosylation site, but it may also be introduced at different sites on the antibody. In this context, the core-N-acetylglucosamine substituent is a monosaccharide substituent, or (if the core-GlcNAc substituent is fucosylated) a disaccharide core-(Fucα1-6)GlcNAc substituent—also referred to as GlcNAc(Fuc).

[0570] "Glycosylation modification" refers to the process of altering the glycan chains of an antibody through glycosylation engineering. Antibody glycosylation can be further modified for various purposes to obtain antibodies with novel glycosylations. For example, glycosylation can be removed to eliminate FcγR affinity and complement binding / effective function; fucose and sialic acid groups can be reduced and dimeric N-acetylglucosamine, galactose, and mannose can be added to enhance Fc-mediated ADCC and CDC effects. Glycosylation modification methods are known in the art, such as increasing or decreasing the glycan chains on the antibody surface by altering the glycosylation sites, modifying the glycan chains in vitro via chemical or enzymatic methods, catalyzing antibody glycosylation by altering the glycosylation pathway of the expression system (e.g., composed of enzymes such as glycosidases and glycosyltransferases), and altering antibody glycosylation by influencing cell culture conditions. In some embodiments, the "glycosylation modification" of this invention is performed by in vitro enzymatic modification of the glycan chains. Preferably, the glycosylation modification of the present invention is carried out by modifying the glycan chain with glycosidases (e.g., glycosidases or glycosyltransferases).

[0571] The modified glycosylated antibody of the present invention refers to an antibody whose glycosylation pattern has been modified compared to an antibody with a natural glycosylation pattern. Preferably, the modified glycosylated antibody refers to an antibody obtained by in vitro enzymatic modification of the glycan chain (e.g., by modification of the glycan chain by a glycosidase (e.g., an endonuclease or glycosyltransferase)) after expression in an expression system (e.g., mammalian cells). More preferably, the modified glycosylated antibody of the present invention refers to an antibody comprising a core -GlcNAc and a sugar derivative E(A)x linked thereto, wherein GlcNAc is bonded to the antibody via a C1 bond, preferably via an N-glycosidic bond to the amide nitrogen atom on the side chain of the asparagine amino acid of the antibody. If the -GlcNAc substituent in the GlcNAc-E(A)x substituent is fucose-substituted, typically fucose is linked to the C6 of the -GlcNAc substituent via α-1,6. The fucosylated -GlcNAc substituent refers to the core -GlcNAc(Fuc), and the fucosylated GlcNAc-E(A)x substituent refers to GlcNAc(Fuc)-E(A)x, where E is a sugar as defined above, and A is a group capable of undergoing a click chemical reaction with the linker unit precursor (Z'), such as an azide group.

[0572] The term "site-specific conjugation" as used in this article refers to the conjugation that specifically links a drug / active substance to a specific site on an antibody via a linker.

[0573] "STING activator" refers to a compound that can activate STING (interferon gene stimulating factor), thereby activating antiviral and antitumor immune responses.

[0574] "Glucocorticoids" also known as "steroids" refer to steroid hormones secreted by the adrenal cortex or synthesized artificially. They are used for anti-inflammatory, immunosuppressive, anti-shock, and anti-tumor treatments. Representative drugs include dexamethasone, prednisone, and hydrocortisone.

[0575] "Radioactive nuclides," also known as "radionuclides," refer to targeted therapeutic agents or targeted diagnostic agents that destroy diseased tissues by using particles emitted by radioactive nuclides (such as β, α, and γ).

[0576] "VHH," also known as a single-domain antibody (sdAb), refers to a genetically engineered antibody composed solely of the variable region of the heavy chain antibody, containing only the three HCDRs of the heavy chain variable region. VHH possesses antigen specificity and high affinity with just these three HCDRs, while ordinary antibodies require six HCDRs. Crystal structure analysis shows that VHH consists of a scaffold composed of two β-sheets, similar to the folding of traditional antibody VH immunoglobulins.

[0577] The term "amino acid" as used herein has the meaning conventionally understood in the art, including both D- and L-amino acids. The notation of amino acids follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. For ease of understanding, the names of amino acids and their corresponding abbreviations are listed in the table below:

[0578] It should be understood that, unless otherwise specified, the amino acids in this invention preferably refer to L-amino acids.

[0579] In this article, "amino acid residue" refers to the amino acid unit that makes up a peptide. Compared to an amino acid, an amino acid residue lacks a hydrogen atom at its N-terminus and a hydroxyl group at its C-terminus. It should be understood that the amino acid mentioned in the structure (e.g., A1A2A3A4) refers to the amino acid residue.

[0580] "Amino protecting group" is a suitable group known in the art for amino protection, see, for example, the literature ("Protective Groups in Organic Synthesis", 5). Th The amino protecting group in Ed.TW Greene & P. ​​GMWuts, preferably, is a (C 1-10 Alkyl or aryl acyl groups, such as formyl, acetyl (Ac), benzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; or (C 1-6 Alkoxy or C 6-10Aryloxycarbonyl, such as tert-butoxycarbonyl (Boc) or Cbz; can also be substituted or unsubstituted alkyl, such as triphenylmethyl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn); also includes 9-fluorenylmethoxycarbonyl (Fmoc), methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, etc.

[0581] "Acylamino protecting group" refers to the protecting group of the acylamino group on the side chain of an amino acid used in peptide synthesis, such as solid-phase peptide synthesis, for example, Trt.

[0582] "Guidinium protecting group" refers to the guanidinium group protecting the amino acid side chain used in peptide synthesis, such as solid-phase peptide synthesis, for example, Pbf.

[0583] "Carboxyl protecting group" is a suitable group known in the art for carboxyl protection, see, for example, the literature ("Protective Groups in Organic Synthesis", 5). Th The carboxyl protecting group in Ed.TW Greene & P.GMWuts, preferably, is a C14 carboxyl protecting group. 1-8 Alkyl-O-, C 6-10 Aryl-O- or C 6-10 Aryl-C 1-8 Alkylene-O-.

[0584] "Hydroxy protecting group" is a suitable group known in the art for the protection of hydroxyl groups, for example, see reference ("Protective Groups in Organic Synthesis", 5). Th The hydroxyl protecting group in Ed.TW Greene & P.GMWuts. Preferably, the hydroxyl protecting group can be C. 1-8 Alkyl, such as tert-butyl (t-Bu).

[0585] In this paper, the “leaving group” in Formula III refers to a group that can be easily removed under physiological or chemical conditions, causing the carboxyl terminus of the peptide to become a carboxyl group.

[0586] Unless otherwise specified, the term "alkyl" on its own or as part of another term refers to a substituted or unsubstituted straight-chain or branched, saturated or unsaturated hydrocarbon having a specified number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C...). 10"alkyl refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms. When the number of carbon atoms is not specified, the alkyl group has 1 to 8 carbon atoms. Alkyl in this document is, for example, but not limited to, alkyl groups containing 1-6, 1-5, 1-3, 1-2, 2-5, 2-4, 2-3, or 1 carbon atom. Representative straight-chain '-C1-C8 alkyl' groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; while branched -C3-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl." Unsaturated -C2-C8 alkyl groups include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexyl, 2-hexyl, -3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl. Sometimes the alkyl group is unsubstituted. The alkyl group may be substituted by one or more groups. In other respects, the alkyl group will be saturated.

[0587] Unless otherwise specified, "alkylene" itself, or as part of another term, refers to a substituted or unsubstituted saturated, branched, straight-chain, or cyclic hydrocarbon group having the stated number of carbon atoms, typically 1-10, and having two monovalent groups at the center obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Alkylenes include divalent groups formed by losing one hydrogen atom from an alkyl group as defined above. Typical alkylene groups include, but are not limited to, methylene (CH2), 1,2-ethylene (CH2CH2), 1,3-propylene (CH2CH2CH2), 1,4-butylene (CH2CH2CH2CH2), etc. In a preferred aspect, the alkylene is a branched or straight-chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).

[0588] Unless otherwise specified, "aryl" itself, or as part of another term, refers to a monovalent carbon cyclic aromatic hydrocarbon group having the referred number of carbon atoms, typically 6-20 (e.g., 6-10), obtained by removing a hydrogen atom from a single carbon atom of the parent aromatic ring system, whether substituted or unsubstituted. Some aryl groups are represented by "Ar" in exemplary structures. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc. An exemplary aryl group is a phenyl group.

[0589] Unless otherwise indicated, “arylene” on its own or as part of another term refers to an aryl group as defined above that has two covalent bonds (i.e., it is divalent) and can be oriented in the ortho, meta, or para positions, including, for example, p-phenylene, ortho-phenylene, and meta-phenylene.

[0590] Unless otherwise specified, "C3-C8 heterocyclic group" itself or as part of another term refers to a monocyclic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 (e.g., 1-3, 3-6, 3-5, 5-6, e.g., 1, 2, 3, or 4) carbon atoms (also called ring members) and one to four (1, 2, 3, or 4) heteroatom ring members independently selected from N, O, P, or S, obtained by removing a hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in the heterocycle may be oxidized. The ring containing the heteroatom can be aromatic or non-aromatic. A heterocycle in which all ring atoms are involved in the structure of an aromatic compound is called a heteroaryl; otherwise, it is called a heterocarbon ring.

[0591] Unless otherwise specified, heterocycles are attached to their side groups at any heteroatom or carbon atom that will produce a stable structure. Thus, heteroaryl groups can be bonded to the aromatic carbons of their aromatic ring system, referred to as C-linked heteroaryl groups, or to non-double-bonded nitrogen atoms (i.e., not =N-) in their aromatic ring system, referred to as N-linked heteroaryl groups. Therefore, nitrogen-containing heterocycles can be C-linked or N-linked and include pyrrole moieties such as pyrrole-1-yl (N-linked) and pyrrole-3-yl (C-linked), and imidazole moieties such as imidazole-1-yl and imidazole-3-yl (both N-linked) and imidazole-2-yl, imidazole-4-yl, and imidazole-5-yl moieties (all C-linked).

[0592] Unless otherwise specified, “C3-C8 heteroaryl” refers to an aromatic C3-C8 heterocycle, where the subscript indicates the total number of carbons in the cyclic system of the heterocycle or the total number of aromatic carbons in the aromatic ring system of the heteroaryl group, and does not imply the size of the ring system or the presence or absence of ring fusion. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azacyclic butyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolidinyl, thiophene, furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, isothiazolyl, and isoxazolyl.

[0593] When explicitly given, the size of a heterocyclic or heteroaryl ring system is described by the total number of atoms in the rings. For example, specifying a 5- or 6-membered heteroaryl indicates the total number of aromatic atoms (i.e., 5 or 6) in the heteroaromatic ring system of the heteroaryl, but does not imply the number of aromatic heteroatoms or aromatic carbons in the ring system. Fused heteroaryls are explicitly referred to or so implied by the context, and are generally described by the number of aromatic atoms in each aromatic ring fused together to form a fused heteroaromatic ring system. For example, a 5,6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring, wherein one or both rings have one or more aromatic heteroatoms or wherein heteroatoms are shared between the two rings. Heterocyclic groups include, but are not limited to, 3-10-membered, 3-8-membered, 3-6-membered, and 5-6-membered heterocyclic groups, i.e., heterocyclic groups containing 3-10, 3-8, 3-6, or 5-6 ring members (including carbons and heteroatoms).

[0594] A heterocycle that is fused with an aryl or heteroaryl group through connection to a non-aromatic portion of a fused ring system, thereby maintaining the heterocycle's non-aromatic nature and being part of a larger structure, is an example of an optionally substituted heterocycle, wherein the heterocycle is replaced by ring fusion with the aryl or heteroaryl group. Similarly, an aryl or heteroaryl group fused with a heterocycle or carbocyclic ring that is part of a larger structure through connection to an aromatic portion of a fused ring system is an example of an optionally substituted aryl or heterocycle, wherein the aryl or heterocycle is replaced by ring fusion with the heterocycle or carbocyclic ring.

[0595] Unless otherwise stated, "C3-C8 heterocyclic group" itself or as part of another term refers to the C3-C8 heterocyclic group as defined above, in which one of the hydrogen atoms of the heterocycle is replaced by a bond (i.e., it is divalent). Unless otherwise stated, "C3-C8 heteroaryl group" itself or as part of another term refers to the C3-C8 heteroaryl group as defined above, in which one of the hydrogen atoms of the heteroaryl group is replaced by a bond (i.e., it is divalent).

[0596] Unless otherwise specified, "C3-C8 carbocyclic" itself, or as part of another term, refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered, monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring obtained by removing a hydrogen atom from a ring atom of the parent ring system. Representative -C3-C8 carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3,5-cycloheptadienyl, cyclooctyl, and cyclooctadienyl. -C3-C8 carbocyclic groups include -C3-C8 cycloalkyl groups, such as -C3-C6 cycloalkyl or -C3-C5 cycloalkyl groups.

[0597] "Cycloalkyl" refers to the carbocyclic group as defined above, but it is saturated.

[0598] Unless otherwise indicated, “C3-C8 subcarbocyclic group” either by itself or as part of another term refers to another C3-C8 carbocyclic group as defined above, in which the hydrogen atom of the carbocyclic group is replaced by a bond (i.e., it is divalent).

[0599] In this document, the term "heteroalkyl" refers to a stable straight-chain or branched hydrocarbon that is fully saturated or contains 1 to 3 degrees of unsaturation, consisting of the indicated number of carbon atoms and one to five, preferably one to three, heteroatoms selected from O, N, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, Si, and S may be located at any internal position of the heteroalkyl group or at the position where the heteroalkyl group is attached to the rest of the molecule. Representative examples of heteroalkyl groups include –CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and –CH=CH-N(CH3)-CH3. At most two heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and –CH2-O-Si(CH3)3. In some embodiments, the heteroalkyl group has 1-10 (e.g., 1-8, 1-6, 1-5, 1-4, or 1-3) carbon atoms and 1-5 (e.g., 1, 2, 3, or 4) heteroatoms. Typically, C1 to C4 heteroalkyl or heteroalkylene compounds have 1 to 4 carbon atoms and 1 or 2 heteroatoms, while C1 to C3 heteroalkyl compounds have 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some respects, heteroalkyl and heteroalkylene compounds are saturated.

[0600] Unless otherwise specified, the term "heteroalkylene" on its own or in combination with another term refers to a divalent group derived from a heteroalkylene group as defined above, such as –CH2-CH2-S-CH2-CH2- and –CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms may also occupy any one or both of the chain ends.

[0601] In this document, the term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, containing at least one double bond. Specifically, an alkenyl group has 2 to 10, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, etc. The carbon atoms in the alkenyl group that are attached to the rest of the molecule can be saturated or alkene-bonded carbon atoms.

[0602] In this document, the term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, comprising at least one triple bond. Specifically, the alkynyl group has 2 to 10, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. For example, as used herein, the term "C 2-6 "Alynyl" refers to a straight-chain or branched alkynyl group with 2 to 6 carbon atoms, such as ethynyl, propynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl-1-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, etc. The carbon atom in the alkynyl group that is attached to the rest of the molecule can be saturated or an alkyne bond.

[0603] In this document, the term "alkenyl" refers to a divalent group obtained by removing one hydrogen atom from an alkenyl group as defined above. Specifically, examples of alkenyl groups include, but are not limited to, vinylene, propenylene, allylene, butenylene, pentenylene, and hexenylene.

[0604] In this document, the term "ynynyl" refers to a divalent group obtained by removing one hydrogen atom from an ynyl group as defined above. Specifically, examples of ynynyl groups include, but are not limited to, etynylene, propynylene, propynylene, butynylene, penynylene, and hexynylene.

[0605] Unless otherwise specified, the term "substituted" as used in defining various groups herein means that the corresponding group can be substituted for, for example, but not limited to, the following groups as defined herein or conventional in the art: alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic, halogen, cyano, nitro, azide, carboxyl, hydroxyl, mercapto, amino, mono- or dialkylamino, mono- or dicycloalkylamino. Examples of substituents include, but are not limited to, one or more groups independently selected from: halogen, -OH, -SH, -CN, -NH2, -NO2, -N3, -C(O)CH3, -COOH, -C(O)-amino, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino, methanesulfonylamino, SO, SO2, phenyl, piperidinyl, piperazine, and pyrimidinyl.

[0606] In this document, the term “substitution” or “substituted” means that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom are replaced by a specified group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and forms a stable compound. Combinations of substituents and variables are only permitted if such combinations form a stable compound.

[0607] As used herein, the term "optionally substituted" means, unless otherwise indicated, that a group may be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4, or 5 or more, or any range thereof) of the listed substituents, wherein said substituents may be the same or different. In one embodiment, the optionally substituted group has 1 substituent. In another embodiment, the optionally substituted group has 2 identical or different substituents. In another embodiment, the optionally substituted group has 3 identical or different substituents. In another embodiment, the optionally substituted group has 4 identical or different substituents. In yet another embodiment, the optionally substituted group has 5 identical or different substituents.

[0608] The term "halogen" refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0609] In this document, the term "PEG unit" refers to an organic moiety comprising repeating ethylene-oxygen subunits (PEG or PEG subunits), which can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxygen subunits). Polydisperse PEG is a non-homogeneous mixture of size and molecular weight, while monodisperse PEG is typically purified from a non-homogeneous mixture and therefore has a single chain length and molecular weight. Preferred PEG units comprise discrete PEG, which is a compound synthesized stepwise rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0610] The PEG unit described herein comprises one or more polyethylene glycol chains, each consisting of one or more enylethoxy subunits covalently linked together. The polyethylene glycol chains may be linked together, for example, in a linear, branched, or star configuration. Typically, prior to incorporation into the conjugate, at least one polyethylene glycol chain is derivatized at one end with an alkyl portion substituted with an electrophilic group to covalently link to the urethane nitrogen of the methylene carbamate unit. Typically, the terminal ethoxy groups in each polyethylene glycol chain that do not participate in the covalent linking to the remainder of the linker unit are modified with PEG end-capping units, typically optionally substituted alkyl groups such as -CH3, CH2CH3, or CH2CH2CO2H. In some implementations, the PEG unit has 2-72, such as 2-70, 2-60, 2-50, 2-24, 2-20, 2-18, 2-16, 2-10, 2-8, 4-50, 4-20, 4-18, 4-16, 4-10, or 4-8 -CH2CH2O- subunits. PEG 4-20 This refers to a PEG unit having 4-20 -CH2CH2O- subunits, which may be linear or branched. Preferably, the PEG unit is linear. Exemplary embodiments of a linear PEG unit (i.e., one end connected to a linker unit and the other end a cap) include:

[0611] -NH-(CH2CH2O) b- CH2CH2COOH;

[0612] -NH-(CH2CH2O) b- CH2CH2C(=O)NH(CH2CH2O)-CH2CH2COOH;

[0613] -C(=O)-(CH2CH2O) b- CH3;

[0614] -(CH2CH2O) b CH3;

[0615] -NH-(CH2CH2O) b-CH2CH2NH-(CH2CH2O)-CH2CH2COOH;

[0616] Each b is independently selected from 2-72 integers, such as 2-60, 2-4, 2-24, 2-10, 4-10, 6-60, 8-40, 6-24, 2-30, 2-20, 2-12, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20, 24, 30, 35, 40.

[0617] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0618] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective dose can vary depending on various factors such as disease state, individual age, sex, and weight. Therapeutic effective dose is the amount at which any toxic or harmful effects are less than the beneficial therapeutic effect. Relative to untreated subjects, "therapeutic effective dose" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the antibody of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors.

[0619] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0620] The term "pharmaceuticalally acceptable excipient" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are administered together with the active substance. It may also be called "pharmaceutical excipient."

[0621] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0622] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term “non-fixed combination” means that the active ingredients (e.g., (i) the molecules of the present invention (e.g., ADCs) comprising their pharmaceutically acceptable salts, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the molecules of the present invention (e.g., ADCs) used in the drug combination comprise their pharmaceutically acceptable salts and other therapeutic agents administered at levels not exceeding those obtained when used alone. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0623] The terms "combination therapy" or "treatment in combination" refer to the administration of two or more therapeutic agents or modalities of treatment (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0624] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0625] When used herein, the terms “molecule of the invention” or “compound of the invention” include, for example, drug conjugates (compounds of formula I), compounds of formula II, compounds of formula III or their stereoisomers, isotopic variants, prodrugs, pharmaceutically acceptable salts or solvates, etc., as defined herein (especially in the embodiments and examples sections). Attached Figure Description

[0626] Figure 1. Enzyme activity curve of GQKN (SEQ ID NO:77) short peptide.

[0627] Figure 2. Enzyme kinetics curve of GQKN (SEQ ID NO:77) short peptide.

[0628] Figure 3: Killing activity of V-F31 antibody-based ADCs in EBC-1 cells in vitro.

[0629] Figure 4: Killing activity of V-F31 antibody-based ADCs in NCI-H1975 cells in vitro.

[0630] Figure 5: In vitro killing activity of ADCs based on V-hu23 antibody.

[0631] Figure 6: Tumor growth inhibition results of ADCs in mouse MDA-MB-468 subcutaneous transplantation model.

[0632] Figure 7: Tumor growth inhibition results of ADCs in mouse MDA-MB-453 model.

[0633] Figure 8: Tumor growth inhibition results and body weight curve of ADCs in mouse NCI-N87 model.

[0634] Figure 9: Tumor growth inhibition results and body weight curves of ADCs in the mouse Aspc-1 model.

[0635] Figure 10: Tumor growth inhibition results and body weight curves of ADCs in the mouse SNU620 model.

[0636] Figure 11: FACS combination experiment results of ADCs.

[0637] Figure 12: In vitro killing activity of ADCs against T47D(+) cells.

[0638] Figure 13: In vitro killing activity of ADCs against SNU620(+ / -) cells.

[0639] Figure 14: Results of in vitro side-by-side killing experiments of ADCs. Detailed Implementation

[0640] Example

[0641] The present disclosure is further illustrated below through a description of specific embodiments, but this is not intended to limit the scope of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this disclosure without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0642] The abbreviations used in this disclosure have the following meanings:

[0643] Example 1: Short Peptide Design and Research

[0644] The inventors used protein-short peptide modeling and molecular docking algorithms to model and screen different short peptides, designed the optimized tetrapeptide linker sequence of the present invention, and tested the effect through the following experiments.

[0645] Example 2: Synthesis of a tetrapeptide compound

[0646] Steps for solid-phase synthesis of peptide linkers:

[0647] Fmoc is prepared using a tetrapeptide solid-phase synthesis process, utilizing commercially available protected amino acids as raw materials. Examples of these raw materials include: Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-S... er(t-Bu)-OH,Fmoc-Thr(t-Bu)-OH,Fmoc-Tyr(t-Bu)-OH,Fmoc-Asp(t-Bu)-OH,Fmoc-Glu(t-Bu)-OH,Fmoc-A la-OH,Fmoc-Gly-OH,Fmoc-Leu-OH,Fmoc-Pro-OH,Fmoc-Met-OH,Fmoc-Val-OH,Fmoc-Phe-OH,Fmoc-Ile-OH.

[0648] 1. Peptide sequence:

[0649] A general synthesis method using Ac-RLWQ-AMC (Ac-Arg-Leu-Trp-Gln-AMC) as an example

[0650] 2. Synthesis of resin peptides:

[0651] Ac-Arg(Pbf)-Leu-Trp(Boc)-Gln(Trt)-2-Cl resin

[0652] 2.1 Synthesis steps of resin peptides

[0653] Using the Fmoc- process, 2-Cl resin (full loading substitution degree = 1.06 mmol / g) was used to condense amino acids sequentially from the C-terminus to the N-terminus (from right to left) according to the above peptide sequence until peptide chain condensation was completed.

[0654] Add 1g of 2-Cl resin to a clean reactor, add an appropriate amount of DCM, stir and swell for 20 minutes, then stop and filter to remove the liquid.

[0655] 2.2 Resin modification, end cap

[0656] Add the material at a rate of 3 times the molar amount of resin substitution. Weigh an appropriate amount of Fmoc-Gln(Trt)-OH and pour it into the reactor. Add an appropriate amount of DMF to dissolve it. After complete dissolution, add DIC dropwise and react for 3.0 h. Add 4 ml of methanol and react for 15 min. Filter to remove the liquid and wash with DMF 5 times.

[0657] 2.3 Removal of FMOC

[0658] Add 20% hexahydropyridine / DMF solution, stir and react for 10 minutes, remove the liquid, add 20% hexahydropyridine / DMF solution, stir and react for 5 minutes, remove the liquid, wash 6 times with DMF; take a sample for ninhydrin colorimetric detection, and record the detection color.

[0659] 2.4 Condensation Reaction

[0660] Add the following materials in an amount three times the molar amount of resin substitution: weigh Fmoc-Trp(Boc)-OH and HoBt, dissolve them in an appropriate amount of DMF, activate with DIC for 10 minutes, and then add to the reactor. React for 1.5 hours (no color development is detected by the ninhydrin colorimetric method), then remove the residue. Wash five times with DMF.

[0661] 2.5 Cyclic Reaction

[0662] Repeat steps 2.3 to 2.4 until the condensation of the last amino acid Fmoc-Arg(Pbf)-OH is complete. After condensation, remove Fmoc by repeating step 2.3, and wash 6 times with DMF; take a sample for ninhydrin colorimetric detection and record the detection color.

[0663] 2.6N-end Ac end cap

[0664] Add the resin at a rate of 5 times the molar amount of the degree of resin substitution. Measure acetic anhydride and add it to the reactor. Use DCM as a solvent and add an appropriate amount of DIEA. React for 30 minutes, filter to remove the liquid, wash with DCM 5 times, and then dry.

[0665] 2.7 Fully Protected Cleavage of Peptides

[0666] Prepare a fully protective cutting fluid (DCM, trifluoroethanol), add an appropriate amount of the dried resin to the cutting fluid, cut for three hours, then rotary evaporate and freeze dry.

[0667] Synthesis of 2.8Ac-Arg(Pbf)-Leu-Trp(Boc)-Gln(Trt)-AMC

[0668] Weigh out the lyophilized peptide, an equimolar amount of HATU, and twice the amount of DIEA, and dissolve them in an appropriate amount of DMF. Add AMC (i.e., 1 molar volume) at three times the molar volume. After reacting overnight, the mixture was settled with ether.

[0669] 3. Remove side chain protectants

[0670] The precipitated peptides were lysed with an appropriate amount of lysis reagent (TFA and H2O) and stirred at 20–25 °C for 1 hour. The precipitate was then precipitated with 10 times the volume of the filtrate in ice-cold diethyl ether, centrifuged and washed four times, and the precipitate was dried under reduced pressure at room temperature to obtain the crude product.

[0671] 4. Purification

[0672] The preparative liquid chromatography system used 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. Separation was performed using a reversed-phase C18 packed column (50×250 mm, 10 μm particles), with a UV detector set to 220 nm and a flow rate of 40 mL / min. The column was equilibrated with 10% acetonitrile aqueous solution for 10 minutes before injection. 100 mg of crude peptide was ground and dissolved in 10% acetonitrile aqueous solution, microwaved until completely dissolved, filtered through a 0.45 μm filter membrane, and then injected.

[0673] Elution was performed at room temperature using a 28%–38% (0.01 min–40 min) acetonitrile concentration gradient. Note the changes in absorbance, remove impurity peaks, collect the target product with >98% purity, and freeze-dry the combined products to obtain the target product.

[0674] Characterization data of fluorescently labeled 4-peptides

[0675] Example 3. Determination of enzymatic cleavage activity and stability of tetrapeptide compounds

[0676] Fluorescence and liquid chromatography were used to characterize the experimental results. The main reagents and consumables were cathepsin B (Beijing Yiqiao Shenzhou Technology Co., Ltd.), microplate reader (MD ID3), high-performance liquid chromatography (Shimadzu LC-2010, Agilent), and chromatographic column (AQ C18). Other routine reagents were commercially available analytical grade chemicals.

[0677] Example 3.1 Detection of enzyme digestion activity using fluorescence method

[0678] Detection principle

[0679] The substrate short peptide has the structure Ac-tetrapeptide-AMC. The N-terminus is protected by an acetyl group, which protects the free amino group. The tetrapeptide is recognized and bound by cathepsin. The AMC molecule does not fluoresce when bound to the short peptide, but it fluoresces when cleaved (excitation light 380 nm, emission light 440 nm). Utilizing this property of the AMC molecule, the fluorescence intensity after enzyme cleavage can represent the efficiency of cathepsin B cleavage of the short peptide [Richard JA, Meyer Y, Jolivel V, et al. Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing. Bioconjug Chem 2008; 19:1707–1718].

[0680] Enzyme activity detection methods

[0681] Enzyme activity (U) is defined as 1 U, representing the breakdown of 1 μmol of short peptides by cathepsin B per minute. The reaction was performed in a 96-well microplate. The 1x reaction solution contained 100 mM acetate / sodium acetate buffer (pH 5.5), 1 mM DTT, 1 mM EDTA, cathepsin B (activated), and short peptide substrate. The 1x reaction buffer was prepared first, followed by the substrate in each well, then the cathepsin B, and the plate was placed in the microplate reader to begin the reaction. The cathepsin B was activated by incubating it in pH 5.5 buffer for 15 min [Mach L, Mort JS, ...]. J. Maturation of human procathepsin B. Proenzyme activation and proteolytic processing of the precursor to the mature proteinase, in vitro, are primarily unimolecular processes. J Biol Chem 1994; 269:13030–13035. Fluorescence detection was performed with excitation light at 380 nm and absorption light at 440 nm, for 1 hour, with readings every 10 minutes. Each reaction was performed in triplicate.

[0682] Enzyme activity kinetic parameters were determined. Substrate concentrations of 200, 400, 600, and 800 μM were used to detect enzyme activity, and the results were plotted as kinetic curves. If the Km value was low, the concentrations were reduced to 50, 75, 100, 150, and 200 μM for enzyme activity detection, and kinetic curves were plotted as well. Kinetic parameters Kcat, Km, and Kcat / Km were fitted based on the kinetic curves.

[0683] For example, the enzyme activity curve (Figure 1) and enzyme kinetic curve (Figure 2) of Ac-GQKN-AMC (SEQ ID NO:77).

[0684] Fluorescence detection results

[0685] The results of the fluorescence assay are summarized in Table 1 below. Dozens of short peptide sequences (Ac-tetrapeptide-AMC, which are represented as tetrapeptide sequences in the following results for simplicity) were detected by fluorescence. Several short peptide sequences with enzyme digestion efficiencies far exceeding (more than 10 times the activity) of GGFG (SEQ ID NO:30) were successfully obtained, such as RLWQ (SEQ ID NO:54), RDVT (SEQ ID NO:53), and RDIQ (SEQ ID NO:55), which are completely different from those reported in the literature [Eastman P, Swails J, Chodera JD, et al. OpenMM 7: Rapid development of high performance algorithms for molecular dynamics. PLOS Computational Biology 2017; 13:e1005659; Richard JA, Meyer Y, Jolivel V, et al. Bioconjug Chem 2008; 19:1707–1718; Mach L, Mort JS, J., J Biol Chem 1994;269:13030–13035; Choe Y, Leonetti F, Greenbaum DC, et al.Substrate profiling of cysteine ​​proteases using a combinatorial peptide library identifies functionally unique specificities. Y,et al.Multiple proteolytic action of rat liver cathepsin B:specificities and pH-dependences of the endo-and exopeptidase activities.J Biochem 1991;110:179–188;Anami Y,Yamazaki CM,Xiong W,et al.Glutamic acid–valine–citrulline linkers ensure stability and efficacy of antibody–drug conjugates in mice.Nat Commun 2018; 9:2512; Chowdhury MA, Moya IA, Bhilocha S, et al. Prodrug-inspired probes selective to cathepsin B over other cysteine ​​cathepsins. J Med Chem 2014; 57:6092–6104] substrate sequences.

[0686] Table 1. Results of enzyme activity detection by fluorescence method.

[0687] Example 3.2 Enzyme digestion activity detection by HPLC and LC-MS

[0688] HPLC was reused to detect enzyme activity. HPLC can more accurately identify the concentration of substrate and product, but the experimental throughput is relatively low. Enzyme activity is defined as enzyme activity, where 1 U represents the breakdown of 1 μmol of short peptides by cathepsin B per minute.

[0689] The reaction was carried out in 1.5 mL EP tubes; the 1x reaction solution contained 100 mM acetate / sodium acetate buffer (pH 5.5), 1 mM DTT, 1 mM EDTA, cathepsin B (activated), and short peptide substrate. The 1x reaction buffer was prepared in the following order: first, the substrate was added to each well, then cathepsin B was added, and the tubes were placed in a shaker to start the reaction. The cathepsin B was activated by incubating it in pH 5.5 buffer for 15 min [Musil D, Zucic D, Turk D, et al. The refined 2.15A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. The EMBO Journal 1991; 10:2321–2330]. The liquid chromatography method used an Agilent 1260 system with a C18 AQ column, water (A) and acetonitrile (B) as the mobile phase, as shown in the table below (gradually increasing from 5% to 90% within a 13-minute time interval), a flow rate of 0.4 mL / min, and a 224 nm detector. The elution time and peak shape could be adjusted by varying the mobile phase ratio for different short peptides.

[0690] Mass spectrometry identification: The enzyme concentration and reaction time were adjusted to achieve a conversion rate between 20% and 80% before sample detection by mass spectrometry. The mass spectrometry method used was a triple quadrupole mass spectrometer (4500 MD), with a gradient method employed in the liquid phase. The mass spectrometry portion detected molecular weights from 0 to 1000 Da.

[0691] Table 2. Injection conditions for HPLC detection.

[0692] HPLC and LC-MS detection results

[0693] The HPLC results were consistent with those obtained by fluorescence assay. RLWQ (SEQ ID NO:54), GGVCit (SEQ ID NO:80), GGFG (SEQ ID NO:30), GQKN (SEQ ID NO:77), and NYEE (SEQ ID NO:31) were selected for mass spectrometry identification, confirming the cleavage site as the amide bond linked to AMC. The final mass spectrometry results indicate that the cleavage mode is consistent with the theoretical assumption. The enzyme activity detection results by HPLC are shown in Table 3 below.

[0694] Table 3. Results of enzyme activity detection by HPLC method.

[0695] Example 3.3 Plasma stability test

[0696] 3.3.1. Human plasma stability test

[0697] The stability of the tetrapeptide in plasma was determined using a fluorescence method. 50 μM of the short peptide was incubated at 37 degrees Celsius, and the values ​​were determined at 2-hour intervals.

[0698] 10% plasma provides suitable sensitivity for detection and demonstrates clear differences in plasma stability between different sequences. All plasma stability assays were performed by incubation in 10% and 1% plasma.

[0699] The specific experimental conditions are as follows:

[0700] 1. Experimental reagents

[0701] Human plasma, 10X PBS, dH2O, AMC

[0702] 2. Experimental apparatus

[0703] Microplate reader, 96-well black fluorescent microplate

[0704] 3. Experimental Procedure

[0705] 3.1 Creating the AMC Standard Curve

[0706] ELISA reader detection standard curve

[0707] 1) Prepare 200 μL solutions of AMC at concentrations of 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM in 1x PBS using 1.5 mL EP tubes.

[0708] The absorbed light intensity was measured under the set conditions of the ELISA reader, and a standard curve was plotted with this intensity as the ordinate and the AMC concentration as the abscissa.

[0709] Liquid chromatography standard curve

[0710] 1) Repeat the steps above for preparing the standard sample.

[0711] 2) Inject 100 μL of sample and detect the elution time and peak area using HPLC.

[0712] 3) Plot a standard curve with peak area on the ordinate and AMC concentration on the abscissa.

[0713] 3.2 Dilution of short peptides

[0714] Prepare a 5mM short peptide stock solution using 1xPBS.

[0715] 3.3 Plasma Treatment

[0716] Centrifuge the plasma at 14000 rpm for 5 minutes.

[0717] 3.4 Prepare the reaction system (200 μL) according to the table below --- short peptide reaction concentration 50 μM

[0718] Plasma, 10x PBS and dH2O were added to 96-well microplates, and short peptide substrate was added last. The reaction was started and the microplate was measured at 37°C, with excitation light at 380nm and absorption light at 440nm for 24 hours. Each group had four replicates.

[0719] Data is read every 10 minutes for the first 2 hours, then exported. Parameters are then changed to read data every 2 hours, for a total of 24 hours.

[0720] 3.5 Liquid Chromatography Detection

[0721] At 2 hours, one set of liquid phase measurements was taken, and the remaining three sets were used to observe errors. At 24 hours, another set of liquid phase measurements was taken.

[0722] Note: " / " indicates no degradation.

[0723] 3.3.2. Mouse plasma stability test

[0724] ●Experimental Objective

[0725] The degradation of 20 sequences was determined in 10% mouse plasma.

[0726] ●Experimental Methods

[0727] Similar to the method for detecting the stability of human plasma, the degradation of short peptides is determined by either enzyme-linked immunosorbent assay (ELISA) or liquid chromatography.

[0728] a. Microplate reader detection: Three parallel groups, excitation light 380nm, absorption light 440nm, 37℃ for 24h, detection once every 10min for the first 2h, and once every 2h thereafter.

[0729] b. Liquid phase detection was performed according to Example 3.2, and the results are shown in the table below:

[0730] "-" indicates no degradation.

[0731] Z-RR is a standard for measuring enzyme digestion efficiency, and its structure is shown below:

[0732] Example 4: Antibody used to synthesize the ADC of Example 6

[0733] Each polypeptide chain of V-hu23 and V-F31 used to construct the ADC was constructed into a pcDNA 3.4 expression vector (containing one, two, or three vectors for each polypeptide chain, depending on the number of polypeptide chains) and transfected into HEK293F cells. Cells were cultured for 3 days, and the culture supernatant was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetate-sodium acetate solution (pH 3.5) and immediately neutralized with 2M Tris. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0734] The sequences of V-hu23 and V-F31 are shown below:

[0735] In this application, unless otherwise specified, Trastuzumab is derived from Biointron, B7432; Enhertu (also known as Trastuzumab deruxtecan or DS8201, a linker with GGFG (SEQ ID NO:30) as its main structure and an Exatecan toxin) is derived from Biointron, B23603201.

[0736] In this application, the toxin Exatecan may be abbreviated as Exd.

[0737] Example 5: Synthesis of Linker-Payload

[0738] Example 5.1 Synthesis of MC-RLWQ-Dxd

[0739] 5.1.1 Preparation of HM-1276A_6

[0740] Procedure: Compound HM-1276A_1 (1 g, 2.52 mmol) and TCFH (0.85 g, 3.03 mmol) were dissolved in DMF (10 mL), followed by the addition of NMI (0.62 g, 7.57 mmol). After stirring for 5 min, HM-384_8 (0.36 g, 2.77 mmol) was added. The reaction solution was reacted at 25 °C for 2 h. The reaction was monitored by LC-MS, and the starting material disappeared. After the reaction was complete, 1 M HCl was added to the reaction solution to adjust the pH to 5-6. The reaction solution was then directly purified by reverse column chromatography in an H2O (0.1% TFA) / ACN system. After lyophilization, 410 mg of white solid was obtained, yield: 32%. LC-MS (ES-API) m / z: 510.2 [M+H] +

[0741] 5.1.2 Preparation of HM-1276A_6

[0742] Procedure: Compound HM-1276A_3 (1.35 g, 2.65 mmol) and TCFH (0.89 g, 3.18 mmol) were dissolved in DMF (15 mL), followed by the addition of NMI (0.65 g, 7.95 mmol). After stirring for 5 min, HM-1276A_5 (0.60 g, 2.9 mmol) was added. The reaction solution was reacted at 25 °C for 2 h. The reaction was monitored by LC-MS, and the starting material disappeared. After the reaction was complete, 1 M HCl was added to the reaction solution to adjust the pH to 5-6. The reaction solution was then directly purified by reverse column chromatography in an H2O (0.1% TFA) / ACN system. After lyophilization, 770 mg of white solid was obtained, yield: 40%. LC-MS (ES-API) m / z: 696.4 [M+H] + .

[0743] 5.1.3 Preparation of HM-1276A_7

[0744] Procedure: Compound HM-1276A_6 (770 mg, 1.11 mmol) was dissolved in DMF (10 mL), followed by the addition of piperidine (471.2 mg, 5.53 mmol). The reaction solution was reacted at 25 °C for 2 hours. The reaction was monitored by LC-MS, and the starting material disappeared. After the reaction was complete, the reaction solution was directly purified by reverse-phase column chromatography in an H2O (0.1% TFA) / ACN system. After lyophilization, 250 mg of white solid was obtained, yield: 47%. LC-MS (ES-API) m / z: 474.3 [M+H] + .

[0745] 5.1.4 Preparation of HM-1276A_8

[0746] Procedure: Compounds HM-1276A_7 (80 mg, 0.169 mmol) and HM-297Q_6 (62.5 mg, 0.203 mmol) were dissolved in DMF (2 mL), followed by the addition of DIEA (65.5 mg, 0.507 mmol). The reaction solution was reacted at 25 °C for 1 hour. The reaction was monitored by LC-MS, and the starting material disappeared. After the reaction was complete, 1 M CH3COOH solution was added to the reaction solution to adjust the pH to 5-6, and the solution was directly purified by RP-HPLC in an H2O (0.1% TFA) / ACN system. After lyophilization, 65 mg of yellow solid was obtained, yield: 50%. LC-MS (ES-API) m / z: 667.3 [M+H] + .

[0747] 5.1.5 Preparation of HM-1276A_11

[0748] Procedure: Compound HM-1276A_9 (15 g, 40.72 mmol) and HATU (18.58 g, 48.86 mmol) were dissolved in DMF (150 mL), followed by the addition of Py (9.67 g, 122.2 mmol). After stirring for 5 min, HM-1276A_10 (5.88 g, 44.79 mmol) was added. The reaction solution was reacted at 25 °C for 2 h. The reaction was monitored by LC-MS, and the starting material disappeared. After the reaction was completed, the reaction solution was directly purified by reverse-phase column chromatography in an H2O (0.1% TFA) / ACN system. After lyophilization, 19 g of white solid was obtained, yield: 80%. LC-MS (ES-API) m / z: 482.2 [M+H] + .

[0749] 5.1.6 Preparation of HM-1276A_12

[0750] Procedure: Compound HM-1276A_11 (18g) was dissolved in DCM:TFA (4:1, 10mL), and the reaction solution was reacted at 25℃ for 18 hours. The reaction was monitored by LCMS, and the starting material disappeared. After the reaction was complete, the mixture was directly evaporated to dryness three times with DCM, slurried with 300mL of methyl ether, stirred at 25℃ for 1 hour, filtered, and the filter cake was collected to obtain 13g of yellow solid, yield: 80%. LC-MS (ES-API) m / z: 426.2 [M+H] + .

[0751] 5.1.7 Preparation of HM-1276A_13

[0752] Procedure: Compound HM-1276A_12 (10 g, 23.51 mmol) was dissolved in DMF (100 mL), and AcOH (2.8 g, 47.1 mmol) was added. This solution was then slowly added dropwise to a DMF (100 mL) solution of Pb(OAc)4 (15.6 g, 35.26 mmol). The reaction mixture was incubated at 25 °C for 2 hours. The reaction was monitored by LCMS, and the starting material disappeared. After the reaction was complete, water (400 mL) was added to the reaction mixture, followed by extraction with EA (3 × 200 mL). The organic phases were combined, washed with water (3 × 100 mL), washed with saturated sodium chloride (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. 100 mL of MTBE was added to the crude product, and the mixture was stirred at 25 °C for 1 hour, then filtered. The filter cake was collected to obtain 7.5 g of white solid, yield: 70%. LC-MS(ES-API)m / z:380.2[Μ-HOAc+H] + .

[0753] 5.1.8 Preparation of HM-1276A_14

[0754] Procedure: Compound HM-1276A_13 (6.5 g, 14.8 mmol) was dissolved in THF (70 mL), followed by the addition of HM-297Q_11 (7.4 g, 44.4 mmol). The mixture was cooled to 0 °C under N2 protection, and then 2.2 M t-BuoLi / THF (10.1 mL, 22.2 mmol) was added. The reaction was stirred at 0 °C for 0.5 h. The reaction was stopped by LCMS. After the reaction was complete, water (100 mL) was added to the reaction solution, followed by extraction with EA (3 × 50 mL). The organic phases were combined, washed with saturated sodium chloride (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was then added to 80 mL MTBE, stirred at 25 °C for 1 h, filtered, and the filter cake was collected to obtain 6.5 g of light brown solid. The yield of both batches was 70%. LC-MS(ES-API)m / z:380.2[Μ-166+H] + .

[0755] 5.1.9 Preparation of HM-1276A_15

[0756] Procedure: 3 g of compound HM-1276A_14 was added to 30 mL of EA, followed by 0.8 g of 10% Pd / C. The reaction mixture was incubated at 25 °C for 12 hours. LC-MS monitoring showed that 15% of the starting material remained. After the reaction, the reaction mixture was filtered through a diatomaceous earth pad and washed with copious amounts of methanol until the product was completely rinsed away. The filtrate was concentrated to obtain the crude product. 20 mL of MTBE was added to the crude product, and the mixture was stirred at 25 °C for 1 hour before filtration. The filter cake was collected to obtain 1.1 g of white solid; the yield was negligible. LC-MS (ES-API) m / z: 380.1 [M-76+H] + .

[0757] 5.1.10 Preparation of HM-1276A_16

[0758] Procedure: Compounds HM-1276A_15 (500 mg, 1.10 mmol) and HM-582_10 (583.53 mg, 1.10 mmol) were added, followed by DMF (5 mL), then HATU (500.86 mg, 1.32 mmol). After stirring for 5 min, DIEA (425.64 mg, 3.29 mmol) was added, and the reaction was carried out at 25 °C with stirring for 1 h. LCMS analysis showed that the starting material disappeared and the main peak was the product, at which point the reaction was stopped. After the reaction was complete, water (20 mL) was added to the reaction solution, followed by extraction with EA (3 × 10 mL). The organic phases were combined, washed with water (3 × 10 mL), washed with saturated sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. 20 mL of MTBE was added to the crude product, and after stirring at 30 °C for 1 h, the mixture was filtered, and the filter cake was collected to obtain 630 mg of pale yellow solid, yield: 60%. LC-MS (ES-API) m / z: 874.3 [M+H] + .

[0759] 5.1.11 Preparation of HM-1276A_17

[0760] Procedure: Compound HM-1276A_16 (580 mg, 0.665 mmol) was added to DMF (6 mL), followed by piperidine (282.9 mg, 3.32 mmol). The reaction was stirred at 25 °C for 1 h. LC-MS analysis showed that the starting material disappeared and the main peak was the product; the reaction was then stopped. The crude product was directly purified by reverse-phase column chromatography to obtain 300 mg of a pale yellow oil, yield: 65%. LC-MS (ES-API) m / z: 651.2 [M+H] + .

[0761] 5.1.12 Preparation of HM-1276A_18

[0762] Procedure: Take compounds HM-1276A_17 (48.8 mg, 0.075 mmol) and HM-1276A_8 (50 mg, 0.075 mmol), add DMF (0.25 mL), then add DIEA (29.1 mg, 0.225 mmol) dissolved in 0.3 mL, stir for 5 minutes, and finally add HATU (34.2 mg, 0.090 mmol). Stir the reaction at 25 °C for 10 minutes.

[0763] LC-MS analysis showed that the starting material disappeared, and the main peak represented the product; the reaction was then stopped. The pH of the reaction solution was adjusted to 5-6 with 1M HCl aqueous solution; the reaction solution was then directly used for preparation, yielding 35 mg of a yellowish-brown solid, yield: 35%. LC-MS (ES-API) m / z: 650.4 [M+2H] 2+ .

[0764] 1 HNMR(400MHz,DMSO)δ10.76(s,1H),8.64(t,J=8.0Hz,1H),8.55(t,J=8.0H z,1H),8.28–8.05(m,2H),8.02–7.89(m,2H),7.80(d,J=12.0Hz,1H),7.62– 7.50(m,1H),7.48–7.38(m,1H),7.35–7.23(m,4H),7.14–7.07(m,1H),7.0 5–6.97(m,4H),6.97–6.88(m,1H),6.80(s,1H),5.66–5.56(m,1H),5.49–5. 39(m,2H),5.22(s,2H),4.71–4.58(m,2H),4.55–4.46(m,1H),4.35–4.14( m,4H),4.04(s,2H),3.23–3.15(m,2H),3.14–3.05(m,3H),2.99–2.87(m,1H ),2.40(s,3H),2.21–2.05(m,6H),1.92–1.75(m,4H),1.54–1.36(m,11H),1 .27–1.10(m,4H),0.93–0.82(m,6H),0.82–0.76(m,2H),0.74–0.65(m,2H).

[0765] Example 5.2 Synthesis of MC-RDIQ-PAB-Exd (HM-2100C_14)

[0766] 5.2.1 Preparation of HM-2100C_3

[0767] Weigh 10 g (15 mmol, ~1.5 mmol / g) of HM-2100C_2 into a 500 mL peptide tube, add 200 mL of DMF solution containing 12.2 g (20 mmol) of HM-2100C_1, and then add DIEA (4.3 g, 33.35 mmol). Shake on a shaker for 16 hours. Add 30 mL of MeOH to the peptide tube, shake for 1 hour, and then dry. Wash the resin sequentially with 3 x 200 mL of DMF, 2 x 2 x 2 x 3 x 2 ...

[0768] LC-MS (ESI) m / z: 611.2 [M+H] + .

[0769] 5.2.2 Preparation of HM-2100C_5

[0770] (1) Take 45% of 5.2.1 loaded resin HM-2100C_3 (6.67 mmol) into a polypeptide tube, add 20% piperidine / DMF (150 mL), shake on a shaker for 20 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 20 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0771] (2) Add a DMF (150 mL) solution of HM-2100C_4 (7.07 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100C_5. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the attachment of HM-2100C_4.

[0772] LC-MS (ESI) m / z: 724.3 [M+H] + .

[0773] 5.2.3 Preparation of HM-2100C_6

[0774] (1) Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100C_5 (6.67 mmol), shake on a shaker for 20 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 20 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0775] (2) Add a DMF (150 mL) solution of HM-588_3A (8.3 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100C_6. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the attachment of HM-588_3A.

[0776] LC-MS (ESI) m / z: 895.4 [M+H] + .

[0777] 5.2.4 Preparation of HM-2100C_8

[0778] (1) Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100C_6 (6.67 mmol), shake on a shaker for 20 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 20 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0779] (2) Add a DMF (150 mL) solution of HM-2100C_7 (10.5 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100C_8. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the incorporation of HM-2100C_7.

[0780] LC-MS (ESI) m / z: 1181.6 [M+H] + .

[0781] 5.2.5 Preparation of HM-2100C_9

[0782] The resin HM-2100C_8 (6.67 mmol) in the peptide tube was transferred to a single-necked flask, and HFIP / DCM (1 / 4) (100 mL) was added. The mixture was stirred at room temperature (15 °C) for 2 hours. The reaction solution was filtered, washed with DCM (200 mL x 3), concentrated, and the residual HFIP was repeatedly removed with DCM to obtain the target product HM-2100C_9 (6.67 mmol, light white solid). LC-MS (ESI) m / z: 1181.4 [M+H] + .

[0783] 5.2.6 Preparation of HM-2100C_10

[0784] Under ice bath conditions (5°C), COMU (5.62 g, 13.13 mmol) and 2,6-Lutidine (4.21 g, 39.39 mmol) were added to a DMF (150 mL) solution of HM-2100C_9 (15.5 g, 13.13 mmol) and HM-297D_10 (1.62 g, 13.13 mmol). The mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was poured into water (1 L), the precipitated solid was filtered, collected, dissolved in ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to 100 mL. The remaining 100 mL suspension was added to MTBE (1 L), the precipitated solid was filtered, collected, and dried to obtain the target product HM-2100C_10 (15 g, yield: 88%, pale yellow solid). LC-MS (ESI) m / z: 1286.5 [M+H] + .

[0785] 5.2.7 Preparation of HM-2100C_11

[0786] DIEA (2.24 g, 17.26 mmol) was added to a DMF (300 mL) solution of HM-2100C_10 (14.8 g, 11.51 mmol) and HM-297D_13 (7.03 g, 23.02 mmol) under ice bath conditions (5 °C), and the mixture was stirred at room temperature (15 °C) for 16 hours. The reaction solution was poured into water (1 L), extracted with ethyl acetate, concentrated, and the residue was added to MTBE (1 L). The precipitated solid was filtered, collected, and dried to obtain the target product HM-2100C_11 (10 g, yield: 60%, pale yellow solid). LC-MS (ESI) m / z: 1451.6 [M+H] + .

[0787] 5.2.8 Preparation of HM-2100C_12

[0788] At room temperature (15°C), DIEA (263 mg, 2.04 mmol) was added to a mixture of HM-582_10 (450 mg, 0.847 mmol), HM-2100C_11 (1.476 g, 1.02 mmol), Py (4.5 mL), and HOAt (114.3 mg, 0.847 mmol) in 18 mL of DMF. The mixture was stirred at room temperature (15°C) for 16 hours. The reaction mixture was poured into water (200 mL), the precipitated solid was filtered, collected, redissolved in DCM, dried, and directly subjected to silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the target product HM-2100C_12 (1.4 g, yield: 94%, light green solid).

[0789] LC-MS (ESI) m / z: 1747.6 [M+H] + .

[0790] 5.2.9 Preparation of HM-2100C_13

[0791] At room temperature (15°C), TFA (12 mL) was added to a mixture of HM-2100C_12 (600 mg, 0.3435 mmol) and DCM (12 mL), and the mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was concentrated, the residue was dissolved in acetonitrile, purified by reverse-phase column chromatography (ACN / H2O with 0.1% TFA), and lyophilized to obtain the target product HM-2100C_13 (350 mg, yield: 84%, yellow solid, TFA salt). LC-MS (ESI) m / z: 1097.5 [M+H] + .

[0792] 5.2.10 Preparation of HM-2100C_14

[0793] At room temperature (15°C), 2,6-Lutidine (53.1 mg, 0.493 mmol) was added to a DMF (3 mL) solution of HM-2100C_13 (150 mg, 0.124 mmol) and HM-297Q_6 (57 mg, 0.186 mmol), and the mixture was stirred at room temperature (15°C) for 16 hours. The reaction solution was purified by reverse-phase reaction (ACN / H2O with 0.1% TFA) and lyophilized to obtain the target product HM-2100C_14 (45 mg, yield: 28%, yellow solid). LC-MS (ESI) m / z: 1290.5 [M+H] + .

[0794] 1 H NMR (400MHz, DMSO) δ12.43(s,1H),9.94(s,1H),8.31(d,J=7.8Hz,1H),8.14(d,J=7.1Hz,1H),8.11-8.00(m,2H),7.80(d,J=10 .9Hz,1H),7.66-7.60(m,3H),7.43-7.37(m,3H),7.34-7.31(m,2H),7.02(s,2H),6.82(s,1H),6.54(s,1H),5.47(s,2H),5.31 (s,3H),5.10(s,2H),4.59(d,J=6.2Hz,1H),4.35-4.27(m,2H),4.24–4.17(m,1H),3.09(s,3H),2.81–2.66(m,2H),2.56(s,1H ),2.40(s,3H),2.25–2.12(m,6H),1.98–1.85(m,4H),1.77-1.65(m,2H),1.49(s,8H),1.25–1.06(m,4H),0.93-0.77(m,13H).

[0795] MS(ES-API): 1290.5(M+H) +

[0796] Example 5.3 Synthesis of MC-RDVT-PAB-Exd

[0797] 5.3.1 Preparation of HM-2100D_2

[0798] Weigh 10 g (15 mmol, ~1.5 mmol / g) of HM-2100C_2 into a 500 mL peptide tube, add 200 mL of DMF solution containing 7.94 g (20 mmol) of HM-2100D_1, and then add DIEA (4.3 g, 33.35 mmol). Shake on a shaker for 16 hours. Add 30 mL of MeOH to the peptide tube, shake for 1 hour, and then dry under vacuum. Wash the resin sequentially with 200 mL x 3 DMF, 200 mL x 2 MeOH, and 200 mL x 3 DMF to obtain approximately 15 mmol of HM-2100D_2. Take a small sample, treat with HFIP / DCM (1 / 4), remove the resin, and send to LCMS to confirm resin incorporation.

[0799] LC-MS (ESI) m / z: 342.1 [M-56+H] + .

[0800] 5.3.2 Preparation of HM-2100D_3

[0801] (1) Take 45% 5.3.1 loaded resin HM-2100D_2 (6.67 mmol) into a polypeptide tube, add 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0802] (2) Add a DMF (150 mL) solution of HM-297D_1 (6.78 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_3. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the incorporation of HM-297D_1.

[0803] LC-MS (ESI) m / z: 497.3 [M+H] + .

[0804] 5.3.3 Preparation of HM-2100D_4

[0805] (1) Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_3 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0806] (2) Add a DMF (150 mL) solution of HM-588_3A (8.22 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_4. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the attachment of HM-588_3A.

[0807] LC-MS (ESI) m / z: 668.3 [M+H] + .

[0808] 5.3.4 Preparation of HM-2100D_5

[0809] (1) Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_4 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence.

[0810] (2) Add a DMF (150 mL) solution of HM-2100C_7 (10.52 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_5. Take a small sample and treat it with HFIP / DCM (1 / 4), cut off the resin, and send it to LCMS to confirm the incorporation of HM-2100C_7.

[0811] LC-MS (ESI) m / z: 954.5 [M+H] + .

[0812] 5.3.5 Preparation of HM-2100D_6

[0813] HFIP / DCM (1 / 4) (100 mL) was added to a peptide tube containing HM-2100D_5 (6.67 mmol). The mixture was shaken on a shaker for 2 hours, filtered, washed with DCM (200 mL x 4), concentrated, and the residual HFIP was removed with DCM (400 mL x 4) to obtain the target product HM-2100D_6 (6.67 mmol, light pink solid). LC-MS (ESI) m / z: 954.5 [M+H] + .

[0814] 5.3.6 Preparation of HM-2100D_7

[0815] Under ice bath conditions (5°C), COMU (7.41 g, 17.3 mmol) and 2,6-Lutidine (5.05 g, 47.19 mmol) were added to a DMF (150 mL) solution of HM-2100D_6 (15 g, 15.73 mmol) and HM-297D_10 (2.13 g, 17.3 mmol). The mixture was stirred at room temperature (15°C) for 1 hour. The reaction solution was poured into water (1 L), the precipitated solid was filtered, collected, dissolved in ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the target product HM-2100D_7 (8.8 g, yield: 53%, light white solid). LC-MS (ESI) m / z: 1059.5 [M+H] + .

[0816] 5.3.7 Preparation of HM-2100D_8

[0817] Under ice bath conditions (5°C), DIEA (1.6 g, 12.18 mmol) was added to an 86 mL DMF solution of HM-2100D_7 (8.6 g, 8.12 mmol) and NPC (5 g, 16.24 mmol), and the mixture was stirred at room temperature (15°C) for 16 hours. The reaction mixture was poured into water (1 L), extracted with ethyl acetate, concentrated, and the residue was dissolved in a small amount of ethyl acetate and added to MTBE (1 L). The precipitated solid was filtered, collected, and dried to obtain the target product HM-2100D_8 (8.5 g, yield: 85%, light white solid). LC-MS (ESI) m / z: 1224.5 [M+H]+ .

[0818] 5.3.8 Preparation of HM-2100D_9

[0819] At room temperature (15°C), DIEA (263 mg, 2.034 mmol) was added to a mixture of HM-582_10 (450 mg, 0.8475 mmol), HM-2100D_8 (1.25 g, 1.017 mmol), Py (4.5 mL), and HOAt (115 mg, 0.8475 mmol) in DMF (18 mL). The mixture was stirred at room temperature (15°C) for 16 hours. The reaction solution was poured into water (200 mL), the precipitated solid was filtered, collected, redissolved in DCM, dried, and directly subjected to silica gel column chromatography (DCM / MeOH = 20 / 1) to obtain the target product HM-2100D_9 (1.1 g, yield: 85%, light green solid).

[0820] LC-MS (ESI) m / z: 1520.6 [M+H] + .

[0821] 5.3.9 Preparation of HM-2100D_10

[0822] At room temperature (15°C), 10 mL of TFA was added to a mixture of HM-2100D_9 (500 mg, 0.329 mmol) and DCM (10 mL), and the mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was concentrated, and the residue was purified by reverse-phase column chromatography (ACN / H2O with 0.1% TFA) and lyophilized to obtain the target product HM-2100D_10 (360 mg, yield: 93%, yellow solid, TFA salt). LC-MS (ESI) m / z: 1056.6 [M+H] + .

[0823] 5.3.10 Preparation of HM-2100D_11

[0824] At room temperature (15°C), DIEA (50 mg, 0.384 mmol) was added to a 3 mL solution of DMF containing HM-2100D_10 (150 mg, 0.128 mmol) and HM-297Q_6 (60 mg, 0.193 mmol), and the mixture was stirred at room temperature (15°C) for 2 hours. The reaction solution was neutralized with HOAc, purified by reverse-phase reaction (ACN / H2O with 0.1% TFA), and lyophilized to obtain the target product HM-2100D_11 (25 mg, yield: 15.6%, yellow solid). LC-MS (ESI) m / z: 1249.4 [M+H] + .

[0825] 1 H NMR (400MHz, DMSO) δ12.41 (s, 1H), 9.83 (s, 1H), 8.33 (d, J = 7.4Hz, 1H), 8.12-7 .99(m,2H),7.85(d,J=8.0Hz,1H),7.80(d,J=10.9Hz,1H),7.73(d,J=8.6Hz,1H ),7.62(d,J=8.5Hz,2H),7.45(t,J=5.6Hz,1H),7.39(d,J=8.4Hz,2H),7.33(s, 1H),7.26-6.72(m,5H),6.55(s,1H),5.47(s,2H),5.30(d,J=4.0Hz,3H),5.17- 4.91(m,3H),4.66-4.56(m,1H),4.36–4.24(m,3H),4.12–4.04(m,1H),3.33–3. 21(m,2H),3.17–3.05(m,3H),2.79-2.69(m,1H),2.57(d,J=8.0Hz,1H),2.40(s ,3H),2.26–2.10(m,4H),2.09-1.99(m,1H),1.97-1.83(m,2H),1.66(s,1H),1. 54–1.44(m,7H),1.24-1.16(m,2H),1.10(d,J=6.3Hz,3H),0.95–0.78(m,10H).

[0826] MS(ES-API): 1249.4(M+H) +

[0827] Example 5.4 Synthesis of Mal-PEG4-RDSG-PAB-Exd

[0828] 5.4.1 Preparation of HM-2368_2

[0829] Under ice-water bath conditions, HATU (438 mg, 1.15 mmol) was added to 8 mL of DMF solution of HM-2368_1 (940 mg, 1.05 mmol, prepared from appropriate raw materials according to the methods of Examples 5.2.1-5.2.5). The mixture was stirred at this temperature for 15 min. Then, HM-297D_10 (155 mg, 1.26 mmol) and DIEA (270 mg, 2.1 mmol) were added to the above reaction solution, and the reaction was allowed to return to room temperature for 1 h. The reaction solution was purified by reverse-phase column chromatography (H2O: 0.1% TFA: ACN = 63%: 37%). After lyophilization, 893 mg of white solid was obtained, yield: 85%. LC-MS (ES-API) m / z: 1003.4 [M+H] + .

[0830] 5.4.2 Preparation of HM-2368_4

[0831] Under a nitrogen atmosphere, DIEA (541 mg, 4.19 mmol) was added to 15 mL of a DMF solution containing HM-2368_2 (840 mg, 0.84 mmol) and PNP (510 mg, 1.68 mmol). After stirring at room temperature for 1 h, HM-582_10 (890 mg, 1.68 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 2 h. The reaction solution was poured into 150 mL of water and extracted with EA (80 mL × 3). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. After evaporation, 2 g of a yellow-green oily substance was obtained. The crude product was not considered in yield. LC-MS (ES-API) m / z: 1466.4 [M+H] + .

[0832] 5.4.3 Preparation of HM-2368_5

[0833] At 0°C, 3 mL of TFA (26 mg, 0.2 mmol) was slowly added to 3 mL of DCM solution containing HM-2368 (300 mg, 0.21 mmol), and the reaction was maintained at 0°C for 10 h. The reaction solution was then suspended and evaporated to dryness. After dissolving in DMF, the solution was passed through a reverse-phase column with H2O (0.1% TFA):ACN = 72%:28%. The solution was lyophilized to obtain 38 mg of yellow powder, yield: 18%. LC-MS (ES-API) m / z: 1000.3 [M+H] + .

[0834] 5.4.4 Preparation of HM-2368_6

[0835] DIEA (10 mg, 0.08 mmol) was added to 1 mL of DMF solution containing HM-2368_5 (38 mg, 0.04 mmol) and HM-1559_3 (21 mg, 0.04 mmol), and the mixture was reacted at room temperature for 1 h. The reaction solution was then purified by reverse-phase chromatography.

[0836] H₂O (0.1% TFA):ACN system, lyophilized to give 11 mg of pale yellow solid. Yield: 53%. LC-MS (ES-API) m / z: 1398.4 [M+H] + .

[0837] 1 H NMR (400MHz, DMSO) δ9.67 (s, 1H), 8.24 (d, J = 7.4Hz, 1H), 8.19 (t, J = 5.7Hz, 1H), 8.0 5(d,J=7.7Hz,1H),8.00(d,J=8.9Hz,1H),7.94(t,J=5.5Hz,1H),7.81(d,J=7.1Hz, 1H),7.71(d,J=10.9Hz,1H),7.53(d,J=8.3Hz,2H),7.32(t,J=8.4Hz,3H),7.25(s, 1H),7.18–6.63(m,6H),6.47(s,1H),5.38(s,2H),5.22(s,3H),5.01(s,3H),4.55( d,J=5.6Hz,1H),4.26–4.13(m,3H),3.82(d,J=5.8Hz,2H),3.54–3.50(m,6H),3.42 (d,J=7.2Hz,12H),3.08(dd,J=11.6,5.8Hz,3H),3.01(s,2H),2.72(dd,J=16.7,5. 3Hz,1H),2.28(dt,J=11.5,6.6Hz,8H),2.11(d,J=11.7Hz,1H),1.86–1.76(m,2H), 1.62(dd,J=12.5,7.2Hz,2H), 1.43(dd,J=13.1,6.5Hz,3H), 0.79(d,J=2.0Hz,3H).

[0838] Example 5.5 Synthesis of Mal-PEG4-TALQ-PAB-Exd

[0839] 5.5.1 Preparation of HM-2368_8

[0840] EEDQ (774 mg, 3.13 mmol) was added to a DCM / MeOH (20 mL) solution of HM-2368_7 (920 mg, 1.57 mmol, prepared from suitable starting materials according to the methods of Examples 5.2.1-5.2.5; unless otherwise specified, similar tetrapeptide compounds in the following examples can be prepared similarly from suitable starting materials), HM-297D_10 (231 mg, 1.88 mmol), and the mixture was stirred at room temperature for 16 h. The reaction solution was evaporated to dryness and purified by reverse-phase column chromatography (H₂O (0.1% TFA):ACN = 73%:27%). After lyophilization, 680 mg of white solid was obtained, yield: 62%. LC-MS (ES-API) m / z: 693.4 [M+H] + .

[0841] 5.5.2 Preparation of HM-2368_10

[0842] Under N2 atmosphere, DIEA (587 mg, 4.55 mmol) was added to 10 mL of DMF solution containing HM-2368_8 (630 mg, 0.91 mmol) and PNP (554 mg, 1.82 mmol). After stirring at room temperature for 1 h, HM-582_10 (967 mg, 1.82 mmol) was added to the above reaction solution, and the mixture was stirred at room temperature for 2 h. A solid precipitated when the reaction solution was poured into 200 mL of methyl ether; filtration yielded 1.52 g of a grayish-black solid. The crude product was not considered in yield. LC-MS (ES-API) m / z: 1154.5 [M+H] + .

[0843] 5.5.3 Preparation of HM-2368_11

[0844] At 0°C, 2 mL of TFA was slowly added to 10 mL of DCM solution containing HM-2368_10 (1 g, 0.87 mmol), and the reaction was maintained at this temperature for 16 h. After removing the solvent by evaporation at low temperature, the DMF was dissolved and passed through a reverse-phase column.

[0845] H₂O (0.1% TFA):ACN = 78%:22%, lyophilized to give 256 mg of pale yellow solid, yield: 29%. LC-MS (ES-API) m / z: 998.3 [M+H] + .

[0846] 5.5.4 Preparation of HM-2368_12

[0847] DIEA (26 mg, 0.2 mmol) was slowly added to 1 mL of DMF solution containing HM-2368_11 (100 mg, 0.1 mmol) and HM-1559_3 (57 mg, 0.11 mmol), and the mixture was allowed to react at room temperature for 1 h after the addition was complete. The reaction solution was then analyzed by reversed-phase HPLC.

[0848] H2O (0.1% TFA): ACN system, lyophilized to obtain 33 mg of pale yellow solid with a purity of 91%. Further purification and lyophilization yielded 13 mg of pale yellow solid with a purity of 90%. The decrease in purity may be due to structural factors of the compound itself causing deterioration during lyophilization. Yield: 9%.

[0849] LC-MS (ES-API) m / z: 1396.8 [M+H] + .

[0850] 1 H NMR (400MHz, DMSO) δ9.87(s,1H),8.10–7.92(m,4H),7.85(d,J=8.1Hz,2H),7.77(d,J=11.0Hz,1H),7.61(d,J=8.4Hz,2H),7.37(d,J=8 .4Hz,2H),7.30(d,J=7.1Hz,2H),6.99(s,2H),6.79(s,1H),6.51(s,1H),5.44(s,2H),5.28(s,3H),5.08(s,2H),4.31–4.22(m,4H),4. 01–3.96(m,1H),3.59(d,J=5.4Hz,4H),3.48(d,J=3.3Hz,12H),3.17–3.10(m,3H),2.37(s,3H),2.32(dd,J=9.7,4.7Hz,2H),2.24–2.1 0(m,4H),1.89–1.80(m,4H),1.73–1.67(m,3H),1.58(dd,J=9.8,5.3Hz,3H),1.24(s,3H),0.94(d,J=6.6Hz,6H),0.83(d,J=2.2Hz,9H).

[0851] Example 5.6 Synthesis of Mal-PEG4-FKVT-PAB-Exd

[0852] 5.6.1 Preparation of HM-2368_21

[0853] EEDQ (527 mg, 2.14 mmol) was added to 10 mL of a DCM:MeOH solution containing HM-2368_20 (930 mg, 1.07 mmol) and HM-297D_10 (328 mg, 2.67 mmol) at a ratio of 5:1. The reaction mixture was allowed to react at room temperature for 16 h. The reaction solution was evaporated to dryness, dissolved in DMF, and passed through a reverse-phase column. The resulting H₂O (0.1% TFA):ACN system was 67%:33%, and lyophilized to give 296 mg of a pale yellow solid. Yield: 28%. LC-MS (ES-API) m / z: 977.4 [M+H] + .

[0854] 5.6.2 Preparation of HM-2368_24

[0855] DIEA (63 mg, 0.49 mmol) was added to 10 mL of DMF solution containing HM-2368_21 (240 mg, 0.25 mmol) and PNP (150 mg, 0.49 mmol). After addition, the reaction was allowed to proceed at room temperature for 1 h. Then, HM-582_10 (196 mg, 0.37 mmol) was added to the reaction solution, and the reaction was allowed to proceed at room temperature for 2 h. LCMS detected the target product and some of the product undergoing Fmoc removal. Triethylenediamine (83 mg, 0.74 mmol) was then added to the reaction solution, and the mixture was stirred at room temperature for 2 h. The reaction solution was then passed through a reverse-phase column.

[0856] H₂O (0.1% TFA):ACN = 53%:47%, lyophilized to give 70 mg of gray solid. Yield: 23%. LC-MS (ES-API) m / z: 1216.8 [M+H] + .

[0857] 5.6.3 Preparation of HM-2368_25

[0858] Add 2,6-dimethylpyridine (18 mg, 0.17 mmol) to 1 mL of DMF solution containing HM-2368_24 (70 mg, 0.06 mmol) and HM-1559_3 (35 mg, 0.07 mmol), and react at room temperature for 1 h. Pass the reaction solution through a reverse-phase column.

[0859] H₂O (0.1% TFA):ACN = 45%:55%, lyophilized to give 45 mg of a light brown solid. Yield: 48%. LC-MS (ES-API) m / z: 1615.7 [M+H] + .

[0860] 5.6.4 Preparation of HM-2368_26

[0861] At 0°C, 0.2 mL of TFA was added to 1 mL of DCM solution containing HM-2368_25 (42 mg, 0.03 mmol), and the reaction was maintained at 0°C for 5 h. The reaction solution was then evaporated to dryness, and the DMF was dissolved and purified by reverse-phase chromatography.

[0862] H₂O (0.1% TFA): ACN system, lyophilized to give 16 mg of near-white solid. Yield: 42%. LC-MS (ES-API) m / z: 1458.5 [M+H] + .

[0863] 1 H NMR(400MHz, DMSO)δ9.80(s,1H),8.25(d,J=7.5Hz,1H),8.08–7.98(m,3H),7.91(d,J= 8.6Hz,1H),7.83(d,J=7.9Hz,1H),7.78(d,J=10.8Hz,1H),7.60(d,J=8.1Hz,5H),7.36 (d,J=8.5Hz,2H),7.31(s,1H),7.24(d,J=4.3Hz,4H),7.18(d,J=4.4Hz,1H),6.99(s,2 H),6.53(s,1H),5.44(s,2H),5.28(s,3H),5.08(s,2H),5.01(d,J=4.9Hz,1H),4.54(s, 1H),4.36–4.26(m,3H),4.07(s,1H),3.58(t,J=7.2Hz,2H),3.47(d,J=7.3Hz,12H),3. 43(d,J=3.9Hz,3H),3.17–3.08(m,3H),3.00(d,J=10.0Hz,1H),2.73(d,J=13.4Hz,3H) ,2.67(s,1H),2.38(s,3H),2.32(t,J=7.3Hz,5H),2.19(s,2H),2.04(d,J=6.5Hz,1H), 1.94–1.78(m,4H),1.70(d,J=7.3Hz,4H),1.07(d,J=6.1Hz,3H),0.81(t,J=3.2Hz,9H).

[0864] Example 5.7 Synthesis of Mal-PEG4-EHDL-PAB-Exd

[0865] 5.7.1 Preparation of HM-2368_14

[0866] At 0-5°C, 2,6-Lutidine (265 mg, 2.48 mmol) and COMU (354 mg, 0.827 mmol) were added to a DMF (10 mL) solution of compounds HM-2368_13 (900 mg, 0.827 mmol) and HM-297D_10 (112 mg, 0.909 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours.

[0867] The reaction was monitored by LCMS until it ended. The reaction solution was dropped into ice-cold HCl (0.5 mmol / L, 100 mL), and a solid precipitated out. After filtration and drying, the solid was obtained by column chromatography (50% EA in DCM) to give 790 mg of white solid. Yield: 80%.

[0868] LC-MS (ES-API) m / z: 1194.4 [M+H] + .

[0869] 5.7.2 Preparation of HM-2368_15

[0870] At 0-5℃, DIEA (130 mg, 1.005 mmol) was added to an 8 mL DMF solution of compound HM-2368_14 (400 mg, 0.335 mmol) and PNP (204 mg, 0.671 mmol), and the reaction was carried out at room temperature for 4 h after the addition was complete.

[0871] The reaction was monitored by LC-MS until completion. The reaction solution was added dropwise to ice-cold HCl (0.5 mol / L, 80 mL), resulting in the precipitation of a white solid. After filtration and drying, the solid was subjected to column chromatography (25%-50% EA in PE) to obtain 240 mg of white solid, yield: 52%. LC-MS (ES-API) m / z: 1359.5 [M+H] + .

[0872] 5.7.3 Preparation of HM-2368_16

[0873] DIEA (44 mg, 0.338 mmol) was added to a DMF (4 mL) solution of compounds HM-2368_15 (230 mg, 0.169 mmol), HM-582_10 (90 mg, 0.169 mmol), and HOBt (35 mg, 0.254 mmol). The reaction was carried out at room temperature (35 °C) for 3 h after the addition was complete.

[0874] The reaction was monitored by LCMS until it ended. The reaction solution was added dropwise to an ice-cold HCl solution (0.5 mol / L, 40 mL). A yellow-green solid precipitated out. After filtration and drying, 220 mg of yellow-green solid was obtained. Yield: 78%.

[0875] LC-MS (ES-API) m / z: 1655.6 [M+H] + .

[0876] 5.7.4 Preparation of HM-2368_17

[0877] At 0-5°C, 0.2 mL of piperidine was added to a 1.8 mL DCM solution of compound HM-2368_16 (200 mg, 0.121 mmol), and the mixture was stirred at this temperature for 4 hours after the addition was complete.

[0878] The reaction was monitored by LCMS until it ended. When the reaction solution was dropped into PE (20 ml), a solid precipitated out. After centrifugation, 130 mg of pale yellow solid was obtained, yield: 73%.

[0879] LC-MS (ES-API) m / z: 1434.5 [M+H] + .

[0880] 5.7.5 Preparation of HM-2368_18

[0881] DIEA (23 mg, 0.176 mmol) was added to a DMF (2 ml) solution of HM-1559_3 (68 mg, 0.132 mmol) containing compound HM-2368_17 (130 mg, 0.088 mmol) and the mixture was reacted at room temperature (30 °C) for 1 h after the addition was complete.

[0882] The reaction was monitored by LCMS until it ended. The pH of the reaction solution was adjusted to 5-6 with acetic acid and then purified by reverse phase (0.05% FA aqueous solution of 50%-55% acetonitrile) to obtain 80 mg of white solid, yield: 49%.

[0883] LC-MS (ESI) m / z: 1855.8 [M+Na] + 916.6[Μ+2H] 2+ .

[0884] 5.7.6 Preparation of HM-2368_19

[0885] 2 ml of a solution (TFA / DCM = 1 / 1) was added to compound HM-2368_18 (40 mg, 0.022 mmol), and the reaction was allowed to proceed at room temperature for 0.5 h. The reaction was monitored by LCMS until completion. The reaction solution was then added dropwise to 10 ml of diethyl ether, resulting in the precipitation of a yellow solid. After centrifugation, the solid was lyophilized to obtain 16 mg of a white solid. The remaining 30 mg was added using the same method and sent together with the solid for further preparation.

[0886] (4) A total of 23 mg of white solid was obtained, yield: 25%.

[0887] 1 H NMR(400MHz, DMSO)δ9.76(s,1H),8.64(brs,1H),8.34(d,J=7.0Hz,1H),8.24–7.85(m,5H),7.78-7.57(m,4H),7.36(d,J=8.2Hz ,2H),7.31(s,1H),6.98(brs,2H),6.90(s,1H),6.52(s,1H),5.44(s,2H),5.29(s,3H),5.07(s,2H),4.55(d,J=7.1Hz,1H),4.48 -4.36(m,2H),4.28-4.19(m,1H),3.60–3.56(m,8H),3.50-3.46(m,J=4.0Hz,14H),3.16–3.11(m,4H),2.95-2.85(m,1H),2.71(d ,J=10.3Hz,1H),2.37(s,3H),2.32(t,J=7.1Hz,3H),2.25-2.15(m,3H),1.95-1.80(m,3H),1.80-1.50(m,4H),0.88–0.83(m,9H)

[0888] Example 5.8 Synthesis of Mal-PEG4-FGVQ-PAB-Exd

[0889] 5.8.1 Preparation of HM-2368_28

[0890] At 0-5°C, 2,6-Lutidine (497 mg, 4.64 mmol) and COMU (663 mg, 1.55 mmol) were added to a DMF (9 mL) solution of compounds HM-2368_27 (850 mg, 1.55 mmol) and HM-297D_10 (248 mg, 2.01 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour.

[0891] The reaction was monitored by LCMS until it ended. The reaction solution was directly purified by reverse phase (30%-35% acetonitrile in 0.1% TFA aqueous solution) to obtain the product, which was lyophilized to give 750 mg of white solid. Yield: 74%.

[0892] LC-MS (ES-API): 655.3 (M+H) +

[0893] 5.8.2 Preparation of HM-2368_29

[0894] DIEA (237 mg, 1.83 mmol) was added to a DMF (8 mL) solution of compound HM-2368_28 (400 mg, 0.612 mmol) and PNP (372 mg, 1.22 mmol), and the reaction was carried out at room temperature for 4 h after the addition was complete.

[0895] The reaction was monitored by LCMS until it ended. The reaction solution was added dropwise to an ice-cold HCl solution (0.5 mol / L, 100 ml). A white solid precipitated out. After filtration and drying, 365 mg of white solid was obtained, yield: 73%.

[0896] LC-MS (ES-API): 820.2 (M+H)+

[0897] 5.8.3 Preparation of HM-2368_30

[0898] DIEA (109 mg, 0.84 mmol) was added to a DMF (6 mL) solution of compounds HM-2368_29 (345 mg, 0.42 mmol), HM-582_10 (224 mg, 0.42 mmol), and HOBt (85 mg, 0.63 mmol). The reaction was carried out at room temperature (35 °C) for 3 h after the addition was complete.

[0899] The reaction was monitored by LCMS until it ended. The reaction solution was then added dropwise to an ice-cold HCl solution (1 mol / L, 60 mL). A yellow-green solid precipitated out. After filtration and drying, 410 mg of the yellow-green solid was obtained, with a yield of 87%.

[0900] LC-MS (ES-API): 1116.4 (M+H)+

[0901] 5.8.4 Preparation of HM-2368_31

[0902] Add 2 ml of a solution (TFA / DCM = 1 / 5) to compound HM-2368_30 (100 mg, 0.0897 mmol), and allow the mixture to react at room temperature (35 °C) for 1 h after the addition is complete.

[0903] The reaction was monitored by LC-MS until completion. The reaction solution was directly concentrated to obtain 110 mg of a yellow solid crude product; the yield was negligible. LC-MS (ES-API): 1016.3 (M+H)+

[0904] 5.8.5 Preparation of HM-2368_32

[0905] DIEA (38 mg, 0.196 mmol) was added to a DMF (2 ml) solution of HM-1559_3 (56 mg, 0.108 mmol) containing compound HM-2368_31 (100 mg, 0.099 mmol) and the mixture was reacted at room temperature (35 °C) for 1 h after the addition was complete.

[0906] The reaction was monitored by LCMS until it ended. The pH of the reaction solution was adjusted to 5-6 with acetic acid and then purified by reverse phase (45% acetonitrile in 0.1% TFA water) to obtain the product. After lyophilization, 40 mg of yellow solid with a purity of 93% was obtained and sent directly to the preparation.

[0907] (3) After freeze-drying, 10 mg of yellow solid was obtained, yield: 7%.

[0908] LC-MS (ES-API): 1414.5 (M+H)+

[0909] 1 H NMR(400MHz, DMSO)δ9.93(s,1H),8.26(dd,J=12.1,6.5Hz,2H),8.11(d,J=8.1Hz,1H),8 .07–7.98(m,2H),7.78(dd,J=9.5,5.2Hz,2H),7.60(d,J=8.4Hz,2H),7.36(d,J=8.5Hz,2 H),7.31(s,2H),7.25–7.21(m,4H),7.19–7.15(m,1H),6.99(s,2H),6.78(s,1H),6.52( d,J=2.5Hz,1H),5.44(s,2H),5.29(s,3H),5.07(s,2H),4.48(d,J=12.2Hz,1H),4.32(m, J=5.7Hz,1H),4.26–4.20(t,1H),3.78(m,2H),3.57(t,J=7.4Hz,3H),3.47(s,12H),3.1 4(dd,J=5.7Hz,4H),3.04(d,J=4.2Hz,1H),3.00(d,J=4.1Hz,1H),2.74(dd,J=13.8,10.1 Hz,2H),2.38(s,3H),2.33(m,J=1.8Hz,2H),2.29(d,J=4.8Hz,2H),2.15(m,J=14.9,8.3 Hz,4H),2.02–1.95(m,2H),1.94–1.83(m,4H),0.89–0.85(m,6H),0.83(d,J=6.8Hz,3H).

[0910] MS(ES-API): 1414.5(M+H)+

[0911] Example 5.9 Synthesis of Mal-PEG8-RLWQ-Exd(HM-1738B_6)

[0912] 5.9.1 Preparation of HM-1738B_3

[0913] (1) Cl resin (5g, 6.85mmol) was packed into a solid-phase polypeptide tube, and anhydrous DMF (100ml), Fmoc-L glutamine (13g, 34.25mmol), and DIEA (5g, 34.25mmol) were added. The mixture was shaken in a shaker for 17h, 100ml of methanol was added and the mixture was shaken for another 1h. The solvent was then removed by vacuuming. The resin in the column was washed 5 times with DMF (50ml*2), methanol (100ml*1), and DMF (100ml*2) in sequence to obtain 5g of HM-1738B_10.

[0914] (2) HM-1738B_10 (5g, 6.85mmol) was placed in a solid-phase peptide tube, and anhydrous DMF (50ml) was added. The reaction was carried out under nitrogen bubbling for 30 minutes to allow the amino resin to swell completely. The solvent in the peptide tube was then removed, and DBLK (20% piperidine DMF solution) (50ml) was added to remove the Fmoc protecting group twice, 20 minutes each time. The removal was qualitatively detected by ninhydrin. After the removal was detected, the DBLK solution in the peptide tube was directly filtered off, and the resin was washed five times successively with DMF (50ml*2), methanol (50ml*1), and DMF (50ml*2). After drying, DMF (20 ml), HM-1738B_11A (15 g, 34.25 mmol), and HOBT (4.6 g, 34.25 mmol) were added sequentially to the peptide tube, followed by DIC (5 ml, 34.25 mmol). The reaction was carried out under nitrogen bubbling for 3 hours. Qualitative analysis using ninhydrin showed no residual raw material. The solvent was then removed, and the resin in the reaction column was washed five times with DMF (50 ml * 2), methanol (50 ml * 1), and DMF (50 ml * 2). After drying, 5 g of HM-1738B_11 was obtained as crude product.

[0915] (3) HM-1738B_11 (5g, 6.85mmol) was placed in a solid-phase peptide tube, and anhydrous DMF (50ml) was added. The reaction was carried out under nitrogen bubbling for 30 minutes to allow the amino resin to swell completely. The solvent in the peptide tube was then removed, and DBLK (20% piperidine DMF solution) (50ml) was added to remove the Fmoc protecting group twice, 20 minutes each time. The removal was qualitatively detected by ninhydrin. After the removal was detected, the DBLK solution in the peptide tube was directly filtered off, and the resin was washed five times successively with DMF (50ml*2), methanol (50ml*1), and DMF (50ml*2). After drying, DMF (20 ml), HM-1738B_12A (10 g, 34.25 mmol), and HOBT (4.6 g, 34.25 mmol) were added sequentially to the peptide tube, followed by DIC (5 ml, 34.25 mmol). The reaction was carried out under nitrogen bubbling for 3 hours. Qualitative analysis using ninhydrin showed no residual raw material. The solvent was then dried, and the resin in the reaction column was washed five times with DMF (50 ml * 2), methanol (50 ml * 1), and DMF (50 ml * 2). After drying, 5 g of HM-1738B_12 was obtained as crude product.

[0916] (4) HM-1738B_12 (5g, 6.85mmol) was placed in a solid-phase peptide tube, and anhydrous DMF (50ml) was added. The mixture was bubbled under nitrogen for 30 minutes to allow the amino resin to fully swell. The solvent in the peptide tube was then removed, and DBLK (20% piperidine DMF solution) (50ml) was added twice to remove the Fmoc protecting group, each time for 20 minutes. The removal was qualitatively detected by ninhydrin. After the DBLK solution in the peptide tube was removed by direct filtration, the resin was washed five times sequentially with DMF (50 ml * 2), methanol (50 ml * 1), and DMF (50 ml * 2). After drying, DMF (20 ml), HM-1738B_13A (15 g, 34.25 mmol), and HOBT (4.6 g, 34.25 mmol) were added sequentially to the peptide tube, and finally DIC (5 ml, 34.25 mmol) was added. The reaction was carried out under nitrogen bubbling for 3 hours. Qualitative analysis by ninhydrin showed no residue of the raw material. The solvent was dried, and the resin in the reaction column was washed five times with DMF (50 ml * 2), methanol (50 ml * 1), and DMF (50 ml * 2). After drying, 5 g of HM-1738B_13, crude product, was obtained.

[0917] (5) HM-1738B_13 (5g crude) was dissolved in 50ml (DCM / HFIP=4 / 1), stirred at room temperature for 1h and then filtered. The mother liquor was concentrated and then mixed with ether and freeze-dried to obtain 2g of white powder, with a yield of 32%.

[0918] MS(ES-API): 824.4(M+H) +

[0919] 5.9.2 Preparation of HM-1738B_4

[0920] (1) Dissolve HM-582_10 (531mg, 1mmol), HM-1738B_3 (823mg, 1mmol), and HATU (380mg, 1mmol) in 10ml of DMF, add DIEA (390mg, 3mmol) and react at room temperature for 0.5h.

[0921] (2) The reaction was monitored by LCMS until it ended, and the product was obtained by reverse phase column purification (acetonitrile:water = 55%:45%).

[0922] (3) 400 mg of yellow solid was obtained by freeze-drying, yield: 41%.

[0923] MS(ES-API): 1242.4(M+H) +

[0924] 5.9.3 Preparation of HM-1738B_5

[0925] (1) Dissolve HM-1738B_4 (400mg, 0.32mmol) in 3ml of DMF, add DBU (97mg, 0.64mmol) and react at room temperature for 1h.

[0926] (2) The reaction was monitored by LCMS until it ended, and the product was obtained by reverse phase column purification (acetonitrile:water = 65%:35%).

[0927] (3) 100 mg of yellow solid was obtained by freeze-drying, yield: 33%.

[0928] MS(ES-API): 1019.4(M+H) +

[0929] 5.9.4 Preparation of HM-1738B_6

[0930] (1) Dissolve HM-1738B_5 (100mg, 0.10mmol) in DMF, add DIEA (38mg, 0.3mmol), and then add DMF solution of HM-1738A_4 (68mg, 0.10mmol). React at room temperature for 0.5h.

[0931] (2) The reaction was monitored by LCMS until it ended, and then sent to the preparation site.

[0932] (3) The preparation solution was freeze-dried to obtain 21 mg of yellow solid, yield: 14%.

[0933] 1 H NMR (400MHz, DMSO) δ10.78(s,1H),8.35(d,J=8.4Hz,1H),8.10–7.99(m,3H),7.90(d,J=8.0Hz,1H),7.80(t,J =8.7Hz,2H),7.43(d,J=7.1Hz,2H),7.34–6.80(m,12H),6.77(s,1H),6.51(s,1H),5.52–5.26(m,4H),5.19-5 .05(m,1H),4.50-4.40(m,1H),4.27–4.04(m,3H),3.70-3.53(m,4H),3.53–3.47(m,26H),3.46–3.44(m,3H), 3.20-3.00(m,7H),2.93(s,1H),2.42–2.30(m,7H),2.20-1.70(m,9H),1.67–1.21(m,8H),0.86–0.75(m,9H).

[0934] MS(ES-API): 1593.8(M+H) +

[0935] HPLC: 99.2555%.

[0936] Example 5.10 Synthesis of Mal-PEG4-GGFG-PAB-Exatecan (HM-5164_69)

[0937] 5.10.1 Preparation of HM-5164_66

[0938] HM-5164_65 (1.9 g, 3.40 mmol, 1.0 eq.) and HM-297D_10 (503 mg, 4.08 mmol, 1.2 eq.) were dissolved in 40 mL of DMF. DIEA (1.32 g, 10.20 mmol, 3.0 eq.) and COMU (2.19 g, 5.10 mmol, 1.5 eq.) were added under ice bath conditions. The mixture was removed from the ice bath and stirred at room temperature for 1 h. The reaction solution was diluted with saturated brine and extracted three times with ethyl acetate. The organic phases were combined. The solution was washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, purified by reversed-phase chromatography (TFA / ACN), and lyophilized to give 1.85 g of a pale yellow solid (81% yield).

[0939] LCMS m / z: 664.3 (M+H).

[0940] 5.10.2 Preparation of HM-5164_67

[0941] HM-5164_66 (1.8 g, 2.71 mmol, 1.0 eq.) was dissolved in DMF (18 mL), and di(p-nitrobenzene) carbonate (1.24 g, 4.07 mmol, 1.5 eq.) and DIEA (876 mg, 6.78 mmol, 2.5 eq.) were added under ice bath conditions. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with saturated brine, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, purified by reversed-phase chromatography (H2O / ACN), and lyophilized to give 1.6 g of a yellow solid, yield 71%.

[0942] LCMS m / z: 829.3 (M+H).

[0943] 5.10.3 Preparation of HM-5164_68

[0944] HM-5164_67 (1.6 g, 1.93 mmol, 1.0 eq.) and HM-582_10 (1.03 g, 1.93 mmol, 1.0 eq.) were dissolved in DMF (16 mL), and DIEA (1.5 g, 11.58 mmol, 6 eq.) was slowly added under ice bath conditions. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was diluted with saturated brine, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, purified by reversed-phase chromatography (ACN / H2O), and lyophilized to give 1.2 g of a yellow solid. Yield: 90%.

[0945] LCMS m / z: 1125.4 (M+H)

[0946] 5.10.4 Preparation of HM-1725E_5

[0947] HM-5164_68 (600 mg, 0.0089 mmol, 1.0 eq.) was dissolved in DMF (6 mL), and ethylenediamine (0.6 mL) was added under ice bath conditions. The mixture was stirred at room temperature for 2 h. The reaction solution was directly purified by reversed-phase chromatography (H2O / ACN) and lyophilized to give 400 mg of a white solid, yield: 83%.

[0948] LCMS m / z: 903.3(M+H)

[0949] 5.10.5 Preparation of HM-5164_69

[0950] HM-1725E_5 (380 mg, 0.421 mmol, 1.0 eq.) and HM-1559_3 (259.3 mg, 0.505 mmol, 1.2 eq.) were dissolved in DMF (4 mL). DIEA (163.1 mg, 1.26 mmol, 3 eq.) and COMU (216.3 mg, 0.505 mmol, 1.2 eq.) were added under ice bath conditions. After addition, the mixture was stirred at room temperature for 1 h. Acetic acid was added to the reaction solution under ice bath conditions to adjust the pH to 6-7. The solution was then sent for preparative purification (NH4OAc / ACN system) and lyophilized to obtain 79.2 mg of a white solid, yield 14%.

[0951] LCMS m / z: 1301.4 (M+H)

[0952] 1 H NMR (400MHz, DMSO) δ9.90(s,1H),8.42(s,1H),8.18(d,J=4.9Hz,2H),8.06(s,3H),7.80(d,J=10.9Hz,1H),7.64(d,J=8.5Hz,2 H),7.40(d,J=8.4Hz,2H),7.33(s,1H),7.28(d,J=4.2Hz,4H),7.21(s,1H),7.02(s,2H),6.55(s,1H),5.47(s,2H),5.31(s,3H ),5.10(s,2H),4.53(s,1H),3.90(s,2H),3.78(s,2H),3.71(d,J=4.9Hz,2H),3.60(t,J=7.0Hz,5H),3.49(d,J=7.3Hz,14H),3 .16(d,J=5.6Hz,3H),3.08(s,1H),2.86(d,J=10.3Hz,1H),2.43–2.34(m,7H),2.21(s,2H),1.89(s,2H),0.89(t,J=7.2Hz,3H).

[0953] Example 5.11 Synthesis of Mal-PEG4-RDVT-PAB-Gluc-Exatecan-o (also known as VPL70)

[0954] 5.11.1 Preparation of HM-2100D_2

[0955] Add 10 g (6.67 mmol, ~1.5 mmol / g) of HM-2100C_2-chlorotriphenylmethyl chloride resin to a 250 mL peptide tube, then add 200 mL of DMF solution containing 7.94 g (20 mmol) of HM-2100D_1, followed by DIEA (4.3 g, 33.35 mmol). Shake on a shaker for 16 hours. Add 30 mL of MeOH to the peptide tube, shake for 1 hour, and then dry under vacuum. Wash the resin sequentially with 3 x 200 mL of DMF, 2 x 2 x 2 x 3 x 2 ...

[0956] LC-MS (ESI) m / z: 342.1 [M-56+H] + .

[0957] 5.11.2 Preparation of HM-2100D_3

[0958] Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_2 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Add another 20% piperidine / DMF (150 mL), shake on a shaker for 30 minutes and then dry. Wash the resin sequentially with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3).

[0959] Add a 150 mL solution of DMF containing HM-297D_1 (6.78 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to a polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Add a small sample to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin sequentially with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) to obtain approximately 6.67 mmol of HM-2100D_3; yield is not considered. Treat a small sample with HFIP / DCM (1 / 4), remove the resin, and send to LCMS to confirm HM-297D_1 incorporation.

[0960] LC-MS (ESI) m / z: 497.3 [M+H] + .

[0961] 5.11.3 Preparation of HM-2100D_12

[0962] (1) Add 20% piperidine / DMF (150 mL) to the polypeptide tube containing HM-2100D_3 (6.67 mmol), shake on a shaker for 30 minutes and then dry. Repeat this process once. Wash the resin with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) in sequence and then dry.

[0963] (2) Add a DMF (150 mL) solution of HM-588_3A (8.22 g, 20 mmol) and HOBt (4.5 g, 33.35 mmol) to the polypeptide tube, then add DIC (4.2 g, 33.35 mmol) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C, and when no blue color appears, dry under vacuum. Wash the resin successively with DMF (200 mL x 3), MeOH (200 mL x 2), and DMF (200 mL x 3) and dry under vacuum to obtain approximately 6.67 mmol of HM-2100D_4. The yield is not considered.

[0964] (3) Add HFIP / DCM (1 / 1) (100mL) to the polypeptide tube containing HM-2100D_4 (6.67mmol), shake on a shaker for 2 hours, filter, wash with DCM (200mLx4), concentrate the filtrate, and remove the residual HFIP with DCM (400mLx4) to obtain the target product HM-2100D_12 (8.5g, yield not considered, light pink solid).

[0965] LC-MS (ESI) m / z: 668.3 [M+H] + .

[0966] 5.11.4 Preparation of HM-2933_2

[0967] HM-707C_1 (20 g, 50.36 mmol) was dissolved in ACN (250 ml), followed by HM-2933_1 (8.42 g, 50.36 mmol, 1 eq) and Ag₂O (11.67 g, 50.36 mmol, 1 eq). The reaction mixture was reacted at room temperature for 18 h in the dark. LC-MS showed that the reaction was complete. The reaction mixture was directly filtered to remove Ag₂O. The filtrate was concentrated to obtain the crude product, which was purified by normal silica gel (PE:EtOAc = 2:1) to give 20 g of the product, 82.17% yield, as a white solid. LC-MS (ESI) m / z: 506 [M + Na] + .

[0968] 5.11.5 Preparation of HM-2933_15

[0969] HM-707C_1 (20 g, 50.36 mmol) was dissolved in ACN (250 ml), and HM-2933_14 (8.42 g, 50.36 mmol, 1 eq) and Ag₂O (11.67 g, 50.36 mmol, 1 eq) were added. The reaction solution was reacted at room temperature for 18 h in the dark. LC-MS showed that the reaction was complete. The reaction solution was directly filtered to remove Ag₂O. The filtrate was concentrated to obtain the crude product, which was purified by normal silica gel (PE:EtOAc = 2:1) to give 21 g of product, 86.27% yield, as a white solid. LC-MS (ESI) m / z: 506 [M + Na] + .

[0970] 5.11.6 Preparation of HM-2933_3

[0971] Add NaBH4 (0.391 g, 10.34 mmol, 0.5 eq, MeOH 5 ml) to HM-2933_2 (10 g, 20.69 mmol, 100 ml DCM). React at room temperature for 0.5 h. LC-MS showed the reaction was complete. The reaction solution was quenched with saturated NH4Cl, extracted with DCM, dried, and concentrated to obtain a crude product. The crude product was purified with normal silica gel (PE:EtOAc = 2:1) to give 6.9 g of white solid, 68.71% yield. LC-MS (ESI) m / z: 508 [M+Na] + .

[0972] 5.11.6 Preparation of HM-2933_16

[0973] Add NaBH4 (0.704 g, 18.62 mmol, 0.5 eq, MeOH 10 ml) to HM-2933_15 (18 g, 37.24 mmol, DCM 200 ml). React at room temperature for 0.5 h. LC-MS showed the reaction was complete. The reaction solution was quenched with saturated NH4Cl, extracted with DCM, dried, and concentrated to obtain a crude product. The crude product was purified with normal silica gel (PE:EtOAc = 2:1) to give 11 g of white solid, 60.86% yield. LC-MS (ESI) m / z: 508 [M+Na] + .

[0974] 5.11.7 Preparation of HM-2933_4

[0975] HM-2933_3 (6.9 g, 14.22 mmol) was dissolved in MeOH (70 ml), and Pd / C (1 g) was added. The reaction solution was reacted at room temperature under H2 for 1 h. LC-MS showed that the reaction was complete. The reaction solution was directly filtered to remove Pd / C, and the filtrate was concentrated to obtain the crude product. The crude product was purified by positive silica gel (PE:EtOAc = 1:1) to give the product: 3.5 g white solid, 54.06% yield. LC-MS (ESI) m / z: 478 [M+Na] + .

[0976] 5.11.8 Preparation of HM-2933_17

[0977] HM-2933_16 (10 g, 20.6 mmol) was dissolved in MeOH (10 ml), and Pd / C (1 g) was added. The reaction solution was reacted at room temperature under H2 for 1 h. LC-MS showed that the reaction was complete. The reaction solution was directly filtered to remove Pd / C, and the filtrate was concentrated to obtain the crude product. The crude product was purified by positive silica gel (PE:EtOAc = 1:1) to give the product: 4 g white solid, 42.63% yield. LC-MS (ESI) m / z: 456 [M+H] + .

[0978] 5.11.9 Preparation of HM-2933_5

[0979] HM-2933_4 (2.05 g, 4.49 mmol, 1.3 eq) was dissolved in DMF (30 mL), and HM-2100D_12 (2 g, 2.99 mmol, 1 eq), COMU (2.5 g, 5.98 mmol, 2 eq), and 2,6-Lutidine (1.44 g, 7.49 mmol, 2.5 eq) were added. The reaction mixture was reacted at 0 °C to room temperature for 2 h. LC-MS showed that the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified with normal silica gel (DCM:EtOAc = 3:1) to give the product: 2 g white solid, 60.42% yield. LC-MS (ESI) m / z: 1087 [M-17] + .

[0980] 5.11.10 Preparation of HM-2933_18

[0981] HM-2933_17 (3.19 g, 7.01 mmol, 1.3 eq) was dissolved in DMF (50 mL), and HM-2100D_12 (3.6 g, 5.39 mmol, 1 eq), COMU (4.51 g, 10.78 mmol, 2 eq), and 2,6-Lutidine (1.44 g, 13.48 mmol, 2.5 eq) were added. The reaction mixture was reacted at 0 °C to room temperature for 2 h. LC-MS showed that the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified by normal silica gel (DCM:EtOAc = 3:1) to give the product: 2.7 g white solid, 45.52% yield. LC-MS (ESI) m / z: 1105.4 [M+H] + .

[0982] 5.11.11 Preparation of HM-2933_7

[0983] HM-2933_5 (2.0 g, 1.81 mmol) was dissolved in DMF (30 mL), and NPC (0.605 g, 1.99 mmol, 1.1 eq) and DIPEA (0.467 g, 3.62 mmol, 2 eq) were added. The reaction mixture was reacted at room temperature for 4 h, and LCMS showed that the reaction was complete. The reaction mixture was used directly in the next step without purification. HM-528_10 (1.15 g, 2.17 mmol, 1.2 eq) and DIPEA (0.468 g, 3.62 mmol, 2 eq) were added to the reaction mixture, and the reaction mixture was reacted at room temperature for 8 h. LCMS showed that the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated to obtain a crude product. The crude product was purified with normal silica gel (DCM:EtOAc = 2:1) to give 1.7 g of gray solid, 59.93% yield. LC-MS (ESI) m / z: 1566.6 [M+H] + .

[0984] 5.11.12 Preparation of HM-2933_8

[0985] Diethylamine (5 ml) was added to a DCM (15 ml) solution of HM-2933_7 (1.7 g, 1.09 mmol), and the reaction mixture was reacted at room temperature for 4 h. LC-MS showed that the reaction was complete. The reaction mixture was then added dropwise to petroleum ether (100 ml) until the product precipitated. The mixture was stirred for 1 h, and filtered to give the product: 1 g of gray solid, 69.27% ​​yield. LC-MS (ESI) m / z: 1345.4 [M+H] + .

[0986] 5.11.13 Preparation of HM-2933_10

[0987] HM-2933_8 (1 g, 0.743 mmol) and HM-2933_9 (0.245 g, 0.892 mmol, 1.2 eq) were dissolved in DMF (12 mL), and HATU (0.425 g, 1.12 mmol, 1.5 eq) and 2,6-Lutidine (0.159 g, 1.49 mmol, 2 eq) were added. The reaction mixture was reacted at 0 °C to room temperature for 1 h. LC-MS showed that the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated to obtain a crude product. The crude product was purified with normal silica gel (DCM:MeOH = 20:1) to give the product: 0.95 g gray solid, 79.79% yield. LC-MS (ESI) m / z: 1600.6 [M+H] + .

[0988] 5.11.14 Preparation of HM-2933_11

[0989] HM-2933_10 (0.9 g, 0.562 mmol) was dissolved in THF / i-PrOH (10 / 2 ml), and H2O2 (35.4 mg, 5.62 mmol, 10 eq) was added. The reaction solution was cooled to 0 °C, and LiOH aqueous solution (80.79 mg, 3.37 mmol, 6 eq, 3 ml water) was added dropwise. The reaction solution was reacted at 0 °C to room temperature for 4 h. LC-MS showed that the reaction was complete. TFA / MeOH (30%) was added dropwise to bring the pH of the reaction solution to 6-7. The reaction solution was directly purified by reversed-phase HPLC (0.1% TFA H2O / ACN) to give the product: 0.5 g yellow solid, 60.89% yield. LC-MS (ESI) m / z: 1460.6 [M+H] + .

[0990] 5.11.15 Preparation of HM-2933_12

[0991] HM-2933_11 (0.5 g, 0.342 mmol) was added to TFA / DCM (70%, 5 mL). The reaction solution was reacted at 0 °C for 2 h. LC-MS showed that the reaction was complete. Triethylamine was added dropwise to the reaction solution, and the pH of the reaction solution was adjusted to 6-7. The reaction solution was concentrated to remove DCM, and purified by reversed-phase HPLC (0.1% TFA in H2O / ACN) to give 50 mg of the product as a yellow solid, 11.7% yield. LC-MS (ESI) m / z: 1248.4 [M+H] + .

[0992] 5.11.16 Preparation of HM-2933_13

[0993] HM-2933_12 (50 mg, 40.0 μmol) was dissolved in DMF (2 mL), and HM-1559_3 (61.7 mg, 0.12 mmol, 3 eq) and 2,6-Lutidine (21.4 mg, 0.2 mmol, 5 eq) were added. The reaction mixture was reacted at room temperature for 18 h. LCMS showed that the reaction was complete. The reaction mixture was directly purified by RP-HPLC (0.1% TFA in H2O / ACN) to give the product: 45 mg of yellow solid, 68.22% yield.

[0994] LC-MS (ESI) m / z: 1646.6 [M+H] + .

[0995] HPLC: 95.42%

[0996] 1 H NMR (400MHz, DMSO) δ12.58 (s, 1H), 9.19 (s, 1H), 8.33 (d, J = 7.2Hz, 1H), 8.07 -7.95(m,3H),7.77(d,J=10.8Hz,1H),7.65(d,J=8.8Hz,1H),7.42(d,J=8.8Hz,,1H),7.40-7.22(m,6H),7.01-6.75(m,4H),6.51(s,1H),5.67 -5.46(m,3H),,5.40-5.28(m,5H),5.06(m,1H),4.95(d,J=7.0Hz,1H),4.60 -4.57(m,2H),4.43 -4.41(m,1H),4.38–4.28(m,2H),4.09-4.05(m,1H),3.97(d,J=9.2Hz,1H),3.63-3.59(m,4 H),3.56-3.49(m,13H),3.37-3.35(m,4H),3.30-3.23(m,2H),3.20-3.14(m,3H),3.12-3.07 (m,2H),2.82–2.69(m,1H),2.62-2.54(m,1H),2.45-2.38(m,8H),2.26–2.05(m,3H),1.95-1 .81(m,2H),1.71-1.65(m,1H),1.52-1.41(m,3H),1.09(d,J=6.2Hz,3H),0.85-0.81(m,9H).

[0997] 5.12.1 Preparation of HM-2933_20

[0998] HM-2933_18 (2.5 g, 2.26 mmol) was dissolved in DMF (30 mL), and NPC (0.757 g, 2.49 mmol, 1.1 eq) and DIPEA (0.643 g, 4.98 mmol, 2.2 eq) were added. The reaction mixture was reacted at room temperature for 4 h, and LCMS showed that the reaction was complete. The reaction mixture was used directly in the next step without purification. HM-528_10 (1.29 g, 2.42 mmol, 1.1 eq) and DIPEA (0.284 g, 2.2 mmol, 1 eq) were added to the reaction mixture, and the reaction mixture was reacted at room temperature for 8 h. LCMS showed that the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified with normal silica gel (DCM:EtOAc = 2:1) to give 2 g of gray solid, 57.92% yield. LC-MS (ESI) m / z: 1566.4 [M+H] + .

[0999] 5.12.2 Preparation of HM-2933_21

[1000] Diethylamine (8 ml) was added to a 20 ml solution of HM-2933_20 (2 g, 1.28 mmol) in DCM. The reaction mixture was reacted at room temperature for 4 h. LC-MS showed that the reaction was complete. Petroleum ether (100 ml) was added dropwise until the product precipitated. The mixture was stirred for 1 h and filtered to give 1.6 g of a gray solid, 93.22% yield. LC-MS (ESI) m / z: 1344.4 [M+H] +

[1001] 5.12.3 Preparation of HM-2933_22

[1002] HM-2933_21 (1.5 g, 1.12 mmol) and HM-2933_9 (0.367 g, 1.34 mmol, 1.2 eq) were dissolved in DMF (20 mL). HATU (0.634 g, 1.67 mmol, 1.5 eq) and 2,6-Lutidine (0.239 g, 2.23 mmol, 2 eq) were added, and the reaction mixture was reacted at 0 °C to room temperature for 1 h. LC-MS showed the reaction was complete. Deionized water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried, and concentrated to obtain the crude product. The crude product was purified using a normal silica gel chromatography (DCM:MeOH = 20:1) to give the product: 1.2 g gray solid, 67.19% yield. LC-MS (ESI) m / z: 1600.6 [M+H]+

[1003] 5.12.4 Preparation of HM-2933_23

[1004] HM-2933_22 (1.2 g, 0.749 mmol) was dissolved in THF / isopropanol (10:2 v / v, 12 ml), and H2O2 (85 mg, 9.5 mmol, 10 eq) was added. The reaction solution was cooled to 0 °C, and LiOH aqueous solution (107 mg, 4.5 mmol, 6 eq, 3 ml) was added dropwise. The reaction solution was allowed to react at 0 °C to room temperature for 4 h. LC-MS showed that the reaction was complete. TFA / MeOH (30%) was added dropwise to bring the pH of the reaction solution to 6-7. The reaction solution was then purified directly by reversed-phase HPLC (0.1% TFA in H2O / ACN) to give the product: 0.6 g yellow solid, 54.8% yield. LC-MS (ESI) m / z: 1460.6 [M+H]+

[1005] 5.12.5 Preparation of HM-2933_24

[1006] HM-2933_23 (0.6 g, 0.41 mmol) was added to TFA / DCM (70%, 6 ml), and the reaction solution was reacted at 0 °C for 2 h. LC-MS showed that the reaction was complete. Triethylamine was added dropwise to bring the pH of the reaction solution to 6-7. The reaction solution was concentrated to remove DCM, and purified by reversed-phase HPLC (0.1% TFA in H2O / ACN) to give 200 mg of the product as a yellow solid, 39.0% yield. LC-MS (ESI) m / z: 1248.4 [M+H]+

[1007] 5.12.6 Preparation of HM-2933_25

[1008] HM-2933_24 (50 mg, 40.0 μmol) was dissolved in DMF (2 mL), and HM-1559_3 (61.7 mg, 0.12 mmol, 3 eq) and 2,6-Lutidine (21.4 mg, 0.2 mmol, 5 eq) were added. The reaction mixture was reacted at room temperature for 18 h. LCMS showed that the reaction was complete. The reaction mixture was directly purified by RP-HPLC (0.1% TFA in H2O / ACN) to give the product: 30 mg of yellow solid, 45.48% yield.

[1009] LC-MS(ESI)m / z:1647.6[M+H]+, HPLC:99.89%

[1010] 1 H NMR (400MHz, DMSO) δ12.58(d,J=175.2Hz,2H),9.19(s,1H),8.33(d,J=7.2Hz,1H),8.17(d,J=8.2Hz,1H), 8.07(t,J=8.4Hz,2H),8.05-7.95(m,2H),7.77(d,J=10.8Hz,1H),7.65(d,J=8.8Hz,1H),7.42(s,1H),7.31 (s,1H),7.17(s,1H),7.11(d,J=8.2Hz,1H),7.01-6.75(m,4H),6.51(s,1H),5.67(s,1H),5.51-5.46(m,2 H),5.40–5.32(m,1H),5.32-5.28(m,3H),5.17(s,1H),5.06(q,J=12.4Hz,2H),4.95(d,J=7.0Hz,1H),4.60 -4.57(m,1H),4.43 -4.41(m,1H),4.38–4.28(m,2H),4.09-4.05(m,1H),3.97(d,J=9.2Hz,1H),3.63-3.59(m,4H),3. 56-3.49(m,13H),3.37-3.35(m,4H),3.30-3.23(m,2H),3.20-3.14(m,3H),3.12-3.07(m,2H),2.8 2–2.69(m,1H),2.62-2.54(m,1H),2.45-2.38(m,8H),2.26–2.05(m,3H),1.95-1.81(m,2H),1.71 -1.65(m,1H),1.52-1.41(m,3H),1.09(d,J=6.2Hz,3H),0.93–0.85(m,6H),0.81(d,J=6.8Hz,3H).

[1011] 5.13.1 Synthesis and Preparation of HM-1276H_2

[1012] HM-1276F_1 (1.55 g, 5.7 mmol), HM-1276H_1 (1.0 eq, 1.8 g, 5.7 mmol), and DIEA (1.1 eq, 805 mg, 6.26 mmol) were added sequentially to DMF (15 mL), and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by LCMS until completion. The product was purified by reverse-phase preparative column chromatography (0.1% TFA in H2O / ACN) and lyophilized to give a white solid in 87% yield.

[1013] LC-MS (ESI) m / z: 475.2 [M+H] + .

[1014] 5.13.2 Preparation of HM-5164_85

[1015] HM-5164_84 (24 g, 80.8 mmol), HM-297D_10 (1.0 eq, 10 g, 80.8 mmol), HATU (1.2 eq, 36 g, 97.0 mmol), and 2,6-dimethylpyridine (3.0 eq, 10.4 g, 242.4 mmol) were added sequentially to DMF (200 mL), and the reaction was carried out at 25 °C for 16 h s. The reaction was monitored by LC-MS until completion. The reaction solution was poured into 1 mM dilute hydrochloric acid, and a solid precipitated. The solid was filtered and lyophilized overnight to obtain a white-yellow solid, with a yield of 95%. LC-MS (ESI) m / z: 403.2 [M+H] + .

[1016] 5.13.3 Preparation of HM-5164_86

[1017] HM-5164_85 (10 g, 24.8 mmol) and DBU (0.5 eq, 1.9 g, 12.4 mmol) were added sequentially to DMF (50 mL), and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by LCMS until completion. The reaction solution was poured into MTBE and stirred for 10 minutes. The product adhered to the flask wall. After evaporation, a yellow-brown oily substance was obtained, with a yield of 90%.

[1018] LC-MS (ESI) m / z: 181.0 [M+H] + .

[1019] 5.13.4 Preparation of HM-5164_87

[1020] HM-5164_86 (2.0 eq, 1.74 g, 9.6 mmol), HM-1276H_2 (1.0 eq, 2.3 g, 4.8 mmol), TSTU (1.2 eq, 1.75 g, 5.8 mmol), and DIEA (2.0 eq, 1.25 g, 9.6 mmol) were sequentially added to DMF (20 mL) and reacted at 0 °C for 2 hours. The reaction was monitored by LC-MS until completion. The product was then purified directly using a reverse-phase preparative column (0.1% TFA in H₂O / ACN) and lyophilized to obtain a white solid in 68.6% yield. LC-MS (ESI) m / z: 637.3 [M+H] + .

[1021] 5.13.5 Preparation of HM-5164_88

[1022] HM-5164_87 (1.0 eq, 2.2 g, 3.5 mmol), NPC (1.5 eq, 1.6 g, 5.2 mmol), and DIEA (1.5 eq, 670 mg, 5.2 mmol) were added sequentially to DMF (20 mL), and the reaction was carried out at room temperature for 16 hours. The reaction was monitored by LCMS to indicate completion. The reaction solution was poured into deionized H2O (100 mL), extracted twice with EA (200 mL), and the organic phase was washed twice with deionized H2O (100 mL). The organic phases were combined and separated by normal phase chromatography (EA / PE = 40%-55%, Me / DCM = 10%) to obtain a yellow solid in 83% yield.

[1023] LC-MS (ESI) m / z: 802.3 [M+H] + .

[1024] 5.13.6 Preparation of HM-5164_89

[1025] HM-5164_88 (1.0 eq, 1.25 g, 1.6 mmol), HM-582_10 (1.0 eq, 830 mg, 1.6 mmol), and DIEA (2.0 eq, 400 mg, 3.2 mmol) were added sequentially to DMF (10 mL). The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by LC-MS to indicate completion. The product was then purified directly using a reverse-phase preparative column (0.1% TFA in H2O / ACN), and lyophilized to obtain a yellow solid with a yield of 95.9%. LC-MS (ESI) m / z: 1098.4 [M+H] + .

[1026] 5.13.7 Preparation of HM-5164_90

[1027] HM-5164_89 (1.0 eq, 0.9 g, 0.82 mmol) was added to DCM (12 mL), and TFA (6 mL) was added in an ice bath. The reaction was carried out at 0 °C for 2 hours, and the reaction was monitored by LC-MS until completion. The solution was poured directly into MTBE and filtered to obtain a brown solid with a yield of 85%. LC-MS (ESI) m / z: 998.4 [M+H] + .

[1028] 5.13.8 Preparation of HM-5164_91

[1029] HM-5164_90 (1.0 eq, 400 mg, 0.4 mmol), HM-1559_3 (1.0 eq, 187 mg, 0.4 mmol), and DIEA (2.0 eq, 94 mg, 0.8 mmol) were added sequentially to DMF (4 mL). The reaction was carried out at 25 °C for 2 hours, and the reaction was monitored by LC-MS until completion. After adjusting the pH to neutral with acetic acid, 25 mg of white solid was obtained by HPLC purification, with a yield of 4%.

[1030] LC-MS (ESI) m / z: 1396.5 [M+H] + .

[1031] 1H NMR (400MHz, DMSO) δ10.79(s,1H),9.79(s,1H),8.26(s,1H),8.17(d,J=6.7Hz,1H ),8.12(s,1H),8.06(d,J=8.5Hz,1H),8.02-7.98(m,1H),7.83–7.75(m,2H),7.63 (d,J=8.4Hz,2H),7.57(d,J=7.7Hz,1H),7.37(d,J=8.3Hz,2H),7.31(t,J=4.0Hz, 2H),7.16(s,1H),7.05(d,J=6.8Hz,1H),7.02–6.92(m,3H),6.51(s,1H),5.44(s,2 H),5.29(s,3H),5.08(s,2H),4.61-4.44(m,1H),4.17(d,J=7.6Hz,1H),3.9-3.8( m,2H),3.73(d,J=5.4Hz,2H),3.61-.356(m,6.9Hz,4H),3.47(d,J=3.9Hz,14H),3. 18–3.10(m,4H),2.38(s,6H),2.33(d,J=7.5Hz,2H),2.18(s,2H),1.87(s,3H),1. 67(s,1H),1.28(s,1H),0.99(s,1H),0.87(t,J=7.1Hz,3H),0.73(t,J=5.8Hz,6H).

[1032] Example 5.14 Synthesis of MC-RDVT-Diamine-Exd (HY-X02503)

[1033] 5.14.1 Preparation of HM-64-37

[1034] HM-582_10 (1 eq, 2 g, 3.77 mmol) and DIEA (2.0 eq, 1 g, 7.5 mmol) were added to DMF (20 mL) to replace N2. A solution of CDI (1.2 eq, 732 mg, 4.5 mmol) in DMF (2 mL) was added dropwise at 0 °C, and the reaction was carried out at this temperature for 30 mins. LC-MS was used to monitor the reaction until complete. HY-1544_7 (2 eq, 1.2 g, 7.5 mmol) in DMF (2 mL) was added dropwise at 0 °C, and the reaction was carried out at 0 °C for 30 mins. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into 0.5 mM dilute hydrochloric acid, filtered, and evaporated to dryness to obtain a yellow solid with a purity of 85% and a yield of 68%. LC-MS (ESI) m / z: 622.2 [M+H] + .

[1035] 5.14.2 Preparation of HM-5164_38

[1036] HM-5164_37 (1.0 eq, 1.5 g, 2.4 mmol) was added to DCM (12 mL), followed by TFA (6 mL). The reaction was carried out at 0 °C for 2 hours, and the reaction was monitored by LC-MS until completion. After rotary evaporation, the product was purified directly by reverse-phase column chromatography (0.1% TFA in H2O / ACN). Lyophilization yielded a brown solid, 40% yield. LC-MS (ESI) m / z: 522.2 [M+H] + .

[1037] 5.14.3 Preparation of HM-5164_47

[1038] HM-5164_3 (1.0 eq, 668 mg, 0.65 mmol), HM-5164_38 (1.0 eq, 340 mg, 0.65 mmol), HATU (1.1 eq, 0.72 mmol), and DIEA (1.5 eq, 126 mg, 0.98 mmol) were added sequentially to DMF (10 mL), and the reaction was carried out at 25 °C for 2 hours. The reaction was monitored by LCMS until completion.

[1039] Piperidine (2 mL) was added to the reaction solution from the previous step, and the reaction was carried out at 25 °C for 1 hour. The reaction was monitored by LC-MS until completion. The solution was then purified directly using a reverse-phase column (0.1% TFA in H₂O / ACN), and lyophilized to obtain 535 mg. LC-MS (ESI) m / z: 1305.7 [M+H] + .

[1040] 5.14.4 Preparation of CF55460-043

[1041] HM-5164_47 (1.0 eq, 520 mg, 0.41 mmol) was added to FA (5 mL) and reacted at 25 °C for 48 hrs. The reaction was monitored by LC-MS until completion. The mixture was poured into MTBE and filtered to obtain a brown solid with a yield of 70%. After purification, 110 mg was obtained. LC-MS (ESI) m / z: 993.4 [M+H] + .

[1042] 5.14.5 Preparation of HM-5164_49

[1043] CF55460-043 (100 mg, 0.1 mmol), HM-297Q_6 (34 mg, 0.11 mmol, 1.1 eq), and 2,6-dimethylpyridine (30 mg, 0.3 mmol, 3 eq) were added sequentially to DMF (1 mL). The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by LCMS to indicate completion. The product was lyophilized to give a yellow solid in 18% yield.

[1044] LC-MS (ESI) m / z: 1186.5 [M+H] + .

[1045] 1 H NMR (400MHz, DMSO) δ12.52–12.24(m,1H),8.31(d,1H),8.03(d,J=7.6Hz,1H),7.9-7.8(m,2H),7.75(d,J=8.8Hz,1H),7.68(d,J =7.7Hz,1H),7.50–6.59(m,9H),6.55(s,1H),6.08(s,1H),5.45(s,2H),5.41-5.35(m,2H),5.29-5.21(m,1H),4.84(s,1H),4.58 (s,1H),4.29(s,1H),4.20(s,1H),4.15(s,1H),3.98(s,1H),3.21-3.05(m,7H),2.71(s,1H),2.42(s,3H),2.14(s,4H),2.03(s, 1H),1.88(s,2H),1.65(s,1H),1.49(s,8H),1.31-1.15(m,4H),1.02(d,J=5.9Hz,3H),0.88(d,J=7.3Hz,3H),0.86–0.81(m,6H).

[1046] Example 5.15 Synthesis of Mal-SO2-RDVT-Diamine-Exd (HY-X02504)

[1047] 5.15.1 Preparation of HM-5164_35

[1048] HM-297N_6 (4.8 g, 18 mmol), glycine tert-butyl ester (2.36 g, 18 mmol, 1.0 eq), and 2,6-dimethylpyridine (3.86 g, 36 mmol, 2 eq) were sequentially added to DMF (50 mL), and the reaction was carried out at 25 °C for 16 hrs. The reaction was monitored by LC-MS to indicate completion. The product was directly purified by reverse-phase column chromatography (0.1% TFA in H2O / ACN) and lyophilized to give 5.8 g of a brown oily substance, with a yield of 88%. LC-MS (ESI) m / z: 227.0 [M-56+H] + .

[1049] HM-5164_35A (5.8 g, 20 mmol) was added to TFA (25 mL) and DCM (50 mL), and reacted at 25 °C for 2 hours. The reaction was monitored by LCMS until completion. The solution was evaporated to dryness using a water pump, dissolved in DCM (20 mL), and evaporated to dryness twice. The solution was then lyophilized to give 5 g of a brown oily substance, with a yield of 82%.

[1050] LC-MS (ESI) m / z: 227.0 [M+H] + .

[1051] 5.15.2 Preparation of HM-5164_41

[1052] HM-5164_39 (5 ​​g, 28.2 mmol), HM-5164_40 (6.5 g, 31.1 mmol, 1.1 eq), and K2CO3 (7.8 g, 56.5 mmol, 2 eq) were added sequentially to DMF (50 mL), and the reaction was carried out at 25 °C for 16 h s. The reaction was monitored by LC-MS to indicate completion. The reaction solution was poured into deionized water (200 mL), and extracted three times with EA (200 mL). The organic phases were combined and washed twice with saturated brine (200 mL). The organic phase was concentrated and separated by normal phase chromatography (EA / PE = 10%) to obtain a yellow oily substance in 66.9% yield. LC-MS (ESI) m / z: 206.0 [M-100+H] + .

[1053] 5.15.3 Preparation of HM-5164_42

[1054] HM-5164_41 (9 g, 29.5 mmol, 1 eq) was added to DCM (100 mL) to replace N2. m-CPBA (10 g, 59 mmol, 2 eq) was added at 0 °C, and the reaction was carried out at 25 °C for 2 hours. The reaction mixture was monitored by TLC to ensure complete reaction. The reaction solution was quenched with saturated sodium thiosulfate solution (200 mL), and extracted three times with DCM (100 mL). The organic phases were combined and purified by normal phase chromatography (EA / PE = 40%) to obtain a yellow oily substance in 45% yield.

[1055] 5.15.4 Preparation of HM-5164_43

[1056] HM-5164_42 (2.7 g, 8 mmol, 1.0 eq) was added to DCM (20 mL), followed by TFA (10 mL). The reaction was carried out at 25 °C for 2 hours, and the reaction was monitored by LC-MS until completion. The product was evaporated to dryness to obtain a brown oil. LC-MS (ESI) m / z: 182.0 [M+H] + .

[1057] 5.15.5 Preparation of HM-5164_44

[1058] HM-5164_35 (2.1 g, 14.9 mmol, 1.0 eq), TSTU (3.8 g, 16.4 mmol, 1.1 eq), and DIEA (3 g, 29.8 mmol, 2.0 eq) were added sequentially to DMF (20 mL), and the reaction was carried out at 0 °C for 2 hours. The reaction was monitored by LC-MS to indicate completion. HM-5164_43 (2.7 g, 14.9 mmol, 1.0 eq) in DMF (3 mL) was added dropwise, and the reaction was carried out at 0 °C for 2 hours. The reaction was monitored by LC-MS to indicate completion. The pH was adjusted to weakly acidic to prepare a white solid with a yield of 11%. LC-MS (ESI) m / z: 390.0 [M+H] +

[1059] 5.15.6 Preparation of HM-5164_47

[1060] HM-5164_3 (927 mg, 0.9 mol, 1.1 eq), HM-5164_38 (430 mg, 0.82 mmol, 1.0 eq), HATU (375 mg, 0.99 mmol, 1.2 eq), and DIEA (150 mg, 1.23 mmol, 1.5 eq) were added sequentially to DMF (10 mL) and reacted at 25 °C for 2 hours. The reaction was monitored by LCMS to indicate completion.

[1061] Piperidine (2 mL) was added to the reaction solution from the previous step, and the reaction was carried out at 25 °C for 1 hour. The reaction was monitored by LC-MS to indicate completion. The reaction solution was poured into deionized water, filtered, and lyophilized. The product was slurried with MTBE (10 mL), filtered, and yielded 680 mg (P: 70%). LC-MS (ESI) m / z: 1305.6 [M+H] +

[1062] 5.15.7 Preparation of HM-5164_50

[1063] HM-5164_44 (388 mg, 0.99 mmol, 2.0 eq), TSTU (240 mg, 0.79 mmol, 1.6 eq), and DIEA (128 mg, 0.99 mmol, 2.0 eq) were added sequentially to DMF (10 mL) and reacted at 0 °C for 2 hours. The reaction was monitored by LCMS to indicate completion. HM-5164_47 (650 mg, 0.5 mol, 1.0 eq) was added to the reaction solution and reacted at 25 °C for 2 hours. The reaction was monitored by LCMS to indicate completion. The crude product was directly purified by reversed-phase HPLC (0.1% TFA in H2O / ACN), and lyophilized to obtain 450 mg of product, with a yield of 54%. LC-MS (ESI) m / z: 1677.6 [M+H] +

[1064] 5.15.8 Preparation of HM-5164_51

[1065] HM-5164_50 (200 mg, 0.12 mmol, 1.0 eq) was added to FA (3 mL), and the reaction was carried out at 25 °C for 6 hours. The reaction was monitored by LC-MS to indicate completion. After preparation and lyophilization, 25.8 mg of product was obtained, with a yield of 16%. LC-MS (ESI) m / z: 1364.4 [M+H] +

[1066] 1H NMR(400MHz,DMSO)δ12.33(s,1H),8.34(s,2H),8.26(s,1H),8.07(s,1H),7.83 (s,1H),7.8-7.76(m,1H),7.74(d,J=8.9Hz,1H),7.69-7.63(m,1H),7.39(s,1H ),7.32(s,1H),7.30-7.66(m,6H),6.53(s,1H),6.07(s,1H),5.43(s,2H),5.4- 5.32(m,2H),5.28-5.18(m,1H),4.83(s,1H),4.58(s,1H),4.30(s,1H),4.19(s ,1H),4.13(s,1H),3.96(s,1H),3.69–3.56(m,4H),3.48–3.42(m,3H),3.25-3. 21(m,2H),3.17(s,4H),3.07(s,4H),2.75(s,1H),2.70(s,1H),2.63(s,2H),2. 39(s,4H),2.33(s,1H),2.16(s,2H),2.01(s,1H),1.86(s,2H),1.65(s,1H),1. 48(s,4H),1.00(d,J=5.9Hz,3H),0.87(t,J=7.2Hz,3H),0.81(t,J=7.4Hz,6H).

[1067] Example 5.16 Synthesis of MC-RDVT-PAB-MMAE (HY-X02502)

[1068] 5.16.1 Preparation of HM-5164_6

[1069] HM-5164_5 (1.8 g, 1.6 mmol, 1.0 eq), NPC (720 mg, 2.4 mmol, 1.5 eq), and DIEA (410 mg, 3.2 mmol, 2 eq) were added sequentially to DMF (20 mL). The reaction mixture was reacted at room temperature for 16 hours, and the reaction was monitored by LC-MS to indicate completion. The reaction solution was transferred to EA (200 mL) and extracted twice with deionized H2O (100 mL). The organic phases were combined. After concentration, the solution was separated by normal phase chromatography (EA / PE = 40%-55%) to obtain a yellow solid in 49% yield. LC-MS (ESI) m / z: 1294.5 [M+H] +

[1070] 5.16.2 Preparation of HM-5164_32

[1071] HM-5164_6 (560 mg, 0.43 mmol, 1.0 eq), HM-297A_2 (310 mg, 0.43 mmol, 1.0 eq), HOAT (65 mg, 0.47 mmol, 1.1 eq), DIEA (56 mg, 0.43 mmol, 1.0 eq), and 2,6-dimethylpyridine (231 mg, 2.2 mmol, 5.0 eq) were added sequentially to DMF (5 mL), and the reaction was carried out at 25 °C for 2 hours. The reaction was monitored by LCMS to indicate completion.

[1072] Piperidine (1 mL) was added to the reaction solution from the previous step, and the reaction was carried out at 25 °C for 1 hour. The reaction was monitored by LC-MS to indicate completion. The crude product was directly purified by reverse-phase column chromatography (0.1% TFA / ACN = 40%-50%), and after lyophilization, 700 mg (P: 40%) was obtained. LC-MS (ESI) m / z: 1650.9 [M+H] +

[1073] 5.16.3 Preparation of HM-5164_33

[1074] HM-5164_32 (700 mg, 0.42 mmol), HM-297Q_6 (131 mg, 0.42 mmol, 1.0 eq), and DIEA (60 mg, 0.46 mmol, 1.1 eq) were added sequentially to DMF (5 mL). The reaction mixture was allowed to react at room temperature for 2 hours, and the reaction was monitored by LC-MS until completion. The pH was adjusted to weakly acidic by adding AcOH (60 mg). The reaction mixture was poured into deionized water (20 mL), extracted three times with EA (20 mL), and the organic phases were combined. The mixture was washed once with water (30 mL) and once with saturated brine (30 mL). After concentration under reduced pressure, the mixture was purified by normal-phase column chromatography (Me / DCM = 10%). The solid was evaporated to dryness to give a yellow solid (partial loss of Boc), with a yield of 14%. LC-MS (ESI) m / z: 1844.0 [M+H] +

[1075] 5.16.4 Preparation of HM-5164_43

[1076] HM-5164_33 (100 mg, 0.054 mmol) was added to a mixed solution of TFA (1 mL) and DCM (3 mL) at -15 °C, and the reaction was carried out at -15 °C for 7 days. The reaction was monitored by LCMS to indicate completion. The pH was adjusted to neutral (pH = 6) by adding a DIEA / DCM (1:1) mixed solution dropwise at -15 °C. The reaction solution was evaporated to dryness at 0 °C, and the solid was directly sent to a reverse-phase column for preparation (ammonium acetate / H2O / ACN). After lyophilization, 7 mg of white solid was obtained, with a yield of 8.3%.

[1077] LC-MS (ESI) m / z: 1531.7 [M+H] +

[1078] 1 H NMR(400MHz,DMSO)δ9.73(s,1H),8.61(s,1H),8.20(s,1H),8.15-7.95(m,2H),7.94-7.85(m,2H),7.75-7.62(m,2H),7.35–7.26 (m,7H),7.17(s,4H),7.01(s,2H),5.5-5.40(m,1H),5.06(s,1H),4.64(s,1H),4.55-4.35(m,2H),4.30-4.10(m,4H),4.10-3.90( m,3H),3.60(s,1H),3.26–3.19(m,7H),3.13(s,2H),3.10-2.95(m,5H),2.92-2.93(m,3H),2.41(s,2H),2.25(s,2H),2.11(s,4H ),2.01(s,2H),1.85-1.65(m,4H),1.49(s,8H),1.28-1.15(m,4H),1.11(d,J=5.7Hz,3H),1.07–0.99(m,6H),0.93-0.72(m,30H).

[1079] Example 5.17 Synthesis of Mal-PEG-RDVE-PAB-Exd(HM-5164_83)

[1080] 5.17.1 Preparation of HM-5164_77

[1081] The polypeptide sequence was prepared using a standard solid-phase synthesis method.

[1082] 1) Add DMF (30 mL) to a container containing CTC-resin (7 g, 7 mmol, degree of substitution: 1.0 mmol / g).

[1083] Allow the resin to swell for 30 min, then add Fmoc-L-Glu(OtBu)-OH (2 eq) and DIEA (3 eq), and react on a shaker overnight at room temperature. After drying, add methanol, DMF and DIEA (5 eq), and react on a shaker for 30 min.

[1084] 2) Drain and then rinse three times with DMF.

[1085] 3) Add 20% piperidine / DMF, react for 15 minutes, dry under vacuum, and repeat step 3 once.

[1086] 4) Rinse five times alternately with DMF and methanol, each time with nitrogen agitation for 30 seconds.

[1087] 5) Add Fmoc-protected amino acids (Fmoc-L-Valine (2.0 eq)) to another reaction flask, add condensing agent, stir and react for 30 min, then add to the resin and react on a shaker for 4-18 hours.

[1088] 6) Repeat steps 2 to 5 to condense amino acids Fmoc-Asp(OtBu)-OH(2eq) and FMOC-Arg(Boc)2-OH(2eq) in the order shown in the table.

[1089] Peptide cleavage and purification:

[1090] 1) Add cleavage buffer (20% DCM solution of hexafluoroisopropanol) to a flask containing a peptide with side chain protection, and stir at room temperature for 1.5 hours.

[1091] 2) Filter, evaporate the filtrate to dryness, and purify with a reverse-phase column to obtain 700 mg of light yellow solid, yield: 4.75%. Proceed directly to the next step.

[1092] LCMS(ESI)[M+H] + =1052.5

[1093] 5.17.2 Preparation of HM-5164_78

[1094] HM-5164_77 (650 mg, 0.618 mmol, 1.0 eq), HM-297D_10 (84 mg, 0.68 mmol, 1.1 eq), and HATU (282 mg, 0.742 mmol, 1.2 eq) were dissolved in 15 mL of DMF. DIEA (240 mg, 1.854 mmol, 3.0 eq) was added, and the reaction was carried out at room temperature for 1 hour. LC MS showed that the reaction was complete. The solution was purified by reversed-phase column chromatography (TFA system) to give 600 mg of white solid, yield: 84%. LCMS (ESI) [M+H] + =1157.6

[1095] 5.17.3 Preparation of HM-5164_79

[1096] HM-5164_78 (550 mg, 0.475 mmol, 1.0 eq) and NPC (433 mg, 1.425 mmol, 3.0 eq) were dissolved in 10 mL of DMF. DIEA (306 mg, 2.375 mmol, 5.0 eq) was added, and the reaction was carried out at room temperature for 16 h. LC-MS showed that the reaction was complete. Passing the solution through a reversed-phase column (formic acid system) gave 250 mg of a white solid. Yield: 39.8%. LC-MS (ES-API): 1322.5 (M+H) +

[1097] 5.17.3 Preparation of HM-5164_80

[1098] HM-5164_79 (230 mg, 0.174 mmol, 1.0 eq), HM-582_10 (76 mg, 0.174 mmol, 1.0 eq), and HOBT (23.5 mg, 0.174 mmol, 1.0 eq) were dissolved in 5 mL of DMF. DIEA (67 mg, 0.522 mmol, 3.0 eq) was added, and the mixture was stirred at room temperature for 4 hours. LC-MS showed that the reaction was complete. Reversed-phase preparative chromatography (formic acid system) purified the solid to 160 mg, yield 57%. LC-MS (ES-API): 1118.6 (M+H) +

[1099] 5.17.4 Preparation of HM-5164_81

[1100] HM-5164_80 (150 mg, 0.927 mmol, 1.0 eq) was dissolved in 4 mL of DMF, and DEA (34 mg, 0.464 mmol, 5.0 eq) was added. The mixture was stirred at room temperature for 4 hours, and LC-MS showed that the reaction was complete. Purification by reversed-phase preparative chromatography (formic acid system) yielded 90 mg of a white solid, 69.6% yield. LC-MS (ES-API): 1396.6 (M+H) +

[1101] 5.17.5 Preparation of HM-5164_82

[1102] HM-5164_81 (80 mg, 0.057 mmol, 1.0 eq) and HM-1559_3 (29 mg, 0.057 mmol, 1.0 eq) were dissolved in 2 mL of DMF, and DIEA (22 mg, 0.171 mmol, 3.0 eq) was added. The mixture was stirred at room temperature for 2 hours. LC-MS showed that the reaction was complete. HPLC purification (TFA system) yielded 65 mg of colorless gel, with a yield of 63.5%. LC-MS (ES-API): 961.5 (1 / 2 M + H) +

[1103] 5.17.6 Preparation of HM-5164_83

[1104] HM-5164_82 (60 mg, 0.033 mmol, 1.0 eq) was dissolved in 30 mL of DCM, cooled to -20 °C, and TFA was slowly added dropwise. The reaction was carried out at -10 °C for 48 h. LC-MS showed that the reaction was complete. The product was purified by reversed-phase preparative HPLC (ammonia formate system) to give 20 mg of white solid, with a yield of 40.8%.

[1105] LC-MS (ES-API): 1482.3 (M+H) +

[1106] 1 H NMR (400MHz, DMSO) δ9.95(s,1H),8.42-8.27(m,2H),8.25-8.15(m,2H),8.11–8.04(m,2H),7.95-7.85(m,1H),7.79(d,J=10 .7Hz,1H),7.67(d,J=8.5Hz,2H),7.41–7.31(m,4H),7.30-7.20(m,3H),7.01(s,2H),6.55(s,1H),5.47(s,2H),5.30(s,3H) ,5.09(s,2H),4.60-4.50(m,1H),4.32-4.20(m,3H),4.15-4.05(m,1H),3.63–3.58(m,7H),3.55-3.45(m,10H),3.18–3.05( m,6H),2.47–2.32(m,9H),2.30-2.05(m,5H),2.02-1.81(m,4H),1.75-1.65(m,1H),1.60-1.45(m,3H),0.92–0.83(m,11H).

[1107] Example 5.18 Synthesis of MC-RDVT-PAB(PEG8)-Exatecan(HM-5164_64)

[1108] 5.18.1 Preparation of HM-5164_53

[1109] Add NaOH (100 mL, 2N aqueous solution) to a solution of H2O (100 mL) containing 24 g, 125.0 mmol, 1.0 eq, and allow the mixture to react at room temperature for 30 minutes after the addition is complete.

[1110] After the reaction was completed, the pH of the reaction solution was adjusted to 6-7 with hydrochloric acid. The reaction solution was then freeze-dried to obtain 33g of white solid. The yield of the crude product was not considered.

[1111] LC-MS(ESI)m / z:211.1[Μ+H]+.

[1112] 5.18.2 Preparation of HM-5164_55

[1113] At 0°C, borane dimethyl sulfide (13 mL, 10 M in DMS) was slowly added dropwise to a solution of HM-564_53 (14 g, 66.67 mmol, 1.0 eq) in 100 mL of THF. After the addition was complete, the temperature was raised to 70°C and the reaction was carried out for 5 hours. The reaction solution was then cooled to 0°C, and HCl (23 mL, 4 M MeOH solution) was added. After the addition was complete, the temperature was raised to 65°C and the mixture was stirred for 16 hours.

[1114] The reaction solution was purified by reversed-phase column chromatography (C18, 0.1% TFA·H2O / ACN·gradient 5%-15%) and lyophilized to give 2.3 g of brown solid, with a three-step yield of 24%.

[1115] LC-MS(ESI) m / z: 183.1 [M+H]+.

[1116] 5.18.3 Preparation of HM-5164_57

[1117] Under ice-water bath conditions, 2,6-dimethylpyridine (1.714 g, 16.02 mmol, 3.0 eq) was added to a 22 mL DMF solution containing HM-5164_56 (2.2 g, 5.34 mmol, 1.0 eq) and HATU (2.232 g, 5.87 mmol, 1.1 eq). The mixture was stirred for 15 min while maintaining the temperature. Then, HM-5164_55 (1.069 g, 5.87 mmol, 1.1 eq) was added to the above reaction solution. After the addition was complete, the mixture was allowed to return to room temperature and reacted for 2 hours.

[1118] Post-processing: The reaction solution was purified by reversed-phase column chromatography (C18, 0.01% TFA, H2O / CAN, gradient 15%-30%), and lyophilized to obtain 2.2 g of micro-yellow oily substance, yield 71%.

[1119] LC-MS(ESI) m / z: 577.3 [M+H]+.

[1120] 5.18.4 Preparation of HM-5164_58

[1121] Under H2 protection, Pd / C (5 wt.%) was added to a DCM / MeOH (22 ml, 10 v / 1 v) solution of HM-5164_57 (2.2 g, 3.82 mmol, 1.0 eq), and H2 was replaced after the addition was complete. The reaction was carried out at room temperature for 3 hours.

[1122] The reaction solution was filtered to remove Pd / C, and the mother liquor was evaporated to dryness to obtain 2.2 g of grayish-brown oily substance. The yield of the crude product was not considered.

[1123] LC-MS(ESI) m / z: 547.3 [M+H]+.

[1124] 5.18.5 Preparation of HM-5164_59

[1125] Under ice-water bath conditions, COMU (2.022 g, 4.73 mmol, 1.2 eq) was added to a 22 mL solution of DMF containing HM-5164_58 (2.15 g, 3.94 mmol, 1.0 eq.), HM-5164_3 (4.028 g, 3.94 mmol, 1.0 eq.), and 2,6-dimethylpyridine (1.264 g, 11.81 mmol, 3.0 eq.). After the addition was complete, the mixture was allowed to return to room temperature and reacted for 2 hours.

[1126] Post-processing: The reaction solution was purified by reversed-phase column chromatography (C18, 0.1% FA, H2O / CAN, gradient 40%-55%) and lyophilized to give 2.3 g of pink solid, yield 39%.

[1127] LC-MS(ESI) m / z: 1552.8 [M+H]+.

[1128] 5.18.6 Preparation of HM-5164_61

[1129] At 0°C, p-nitrophenyl chloroformate (0.259 g, 1.29 mmol, 2.0 eq), TEA (0.13 g, 1.29 mmol, 2.0 eq), and DMAP (0.008 g, 0.06 mmol, 0.1 eq.) were added to a 10 mL solution of HM-5164_59 (1.0 g, 0.64 mmol, 1.0 eq). After the addition was complete, the mixture was allowed to return to room temperature and reacted for 4 h. Then, HM-582_10E (0.685 g, 1.29 mmol, 2.0 eq) and DIEA (0.25 g, 1.93 mmol, 3.0 eq) were added to the above reaction solution, and the mixture was stirred at room temperature for 16 h.

[1130] The reaction solution was evaporated to dryness to obtain 1.3 g of brown solid. The crude product was not considered in terms of yield and was used directly in the next step.

[1131] LC-MS(ESI)m / z:1008.1[1 / 2M+H]+

[1132] 5.18.7 Preparation of HM-5164_62

[1133] At 0 °C, triethylenediamine (0.217 g, 1.94 mmol, 3.0 eq) was added to a DMF (13 mL) solution of HM-5164_61 (1.3 g, 0.65 mmol, 1.0 eq). After addition, the mixture was allowed to return to room temperature and reacted for 16 h.

[1134] The reaction solution was purified by reversed-phase column chromatography (C18, 0.1% TFA, H2O / CAN, gradient 55%-75%) and lyophilized to give 380 mg of brown solid, yield 34%.

[1135] LC-MS(ESI) m / z: 1792.7 [M+H]+.

[1136] 5.18.8 Preparation of HM-5164_64

[1137] DIEA (0.052 g, 0.4 mmol, 2.0 eq) was added to a DMF (4 mL) solution of HM-5164_62 (0.36 g, 0.2 mmol, 1.0 eq) and HM-297Q_6 (0.062 g, 0.2 mmol, 1.0 eq), and the reaction was carried out at room temperature for 2 h. The reaction solution was then cooled to 0 °C, and TFA (4 mL) was added to the reaction solution. The mixture was then brought back to room temperature and stirred for another 2 h.

[1138] The reaction solution was subjected to reversed-phase HPLC (Shimadzu LH-40 Liquid Hander, Shimadzu LC-20AP Pump, Shimadzu SPD-20AP UV Detector, Phenxyl-Hexyl, 50x250 mm, 10 μm, 0.1% TFAH2O / ACN, gradient 30-60%) and lyophilized to obtain 22 mg of a near-white solid with a purity of 95.01%, a two-step yield of 6%.

[1139] LC-MS(ESI) m / z: 1672.6 [M+H]+.

[1140] 1H NMR (400MHz, DMSO) δ12.31(s,1H),9.81(s,1H),8.3–8.24(m,1H),7.99(d,J=8.0Hz,1H),7.82–7.76(m,1H),7.67–7.57(m,1H ),7.46(s,1H),7.36–7.26(m,2H),7.25–6.81(m,10H),6.51(s,1H),5.44(s,1H),5.29(s,2H),5.15(s,1H),4.95(s,1H),4.62 –4.53(m,1H),4.33–4.20(m,4H),4.04(s,1H),3.67–3.53(m,2H),3.52–3.32(m,37H),3.23(s,4H),3.18–3.03(m,3H),2.77– 2.64(m,1H),2.38(s,5H),2.26–1.78(m,7H),1.74–1.38(m,8H),1.36–1.12(m,3H),1.07(d,J=6.0Hz,3H),0.88–0.81(m,6H).

[1141] Example 5.19 Synthesis of MC-RDVT-PAB-SN38 (HM-5164_30)

[1142] 5.19.1 Preparation of HN-082_6

[1143] Add imidazole (0.52 g, 7.65 mmol, 3.0 eq) to a DMF (10 ml) solution of HN-082_5 (1.0 g, 2.55 mmol, 1.0 eq) and TBSCl (0.497 g, 3.32 mmol, 1.3 eq). Allow the mixture to react at room temperature for 16 hours after the addition.

[1144] The reaction solution was poured into 100 mL of deionized water, filtered to collect the solid, and the solid was freeze-dried to remove water, yielding 1.2 g of nearly white solid. The yield of the crude product was not considered.

[1145] LC-MS(ESI) m / z: 507.2[M+1]+.

[1146] 5.19.2 Preparation of HM-5164_24

[1147] Under ice-water bath conditions, 2,6-dimethylpyridine (0.705 g, 6.59 mmol, 3.0 eq) and COMU (1.035 g, 2.42 mmol, 1.1 eq) were added to 10 mL of DMF solutions of HM-5164_22 (1.0 g, 2.20 mmol, 1.0 eq) and HM-297D_10 (0.297 g, 2.42 mmol, 1.1 eq). After the addition was complete, the mixture was allowed to return to room temperature and reacted for 2 hours.

[1148] Add DEA (3 mL) to the above reaction solution and stir at room temperature for 1 hour after the addition is complete.

[1149] After the reaction was completed, the product was purified by reversed-phase column chromatography (C18, 0.1% TFA H2O / ACN, gradient 30%-45%) and lyophilized to give 640 mg of a near-white solid, yield 86%.

[1150] LC-MS(ESI) m / z: 339.2 [M+H]+.

[1151] 5.19.3 Preparation of HM-2493_16

[1152] The peptides were prepared using a standard solid-phase synthesis method: 1) DCM (125 mL) was added to a container containing Merrifield-resin (5.00 g, substitution degree: 1.6) and the resin was allowed to swell for 30 min. After swelling, DMF was added to wash three times, 100 mL each time. After filtering off the DMF, HM-297D_1 (2 eq) and DIEA (2 eq) were added, and 125 mL of DMF was added to dissolve them. The mixture was shaken and reacted overnight. The mixture was dried and quenched with methanol. The mixture was then shaken and reacted for 1 h.

[1153] 2) Dry the container and rinse it with MeOH, DCM and DMF three times each in sequence.

[1154] 3) Add 20% piperidine / DMF, react for 2 hours, dry under vacuum, and repeat the above steps once.

[1155] 4) Rinse with MeOH, DCM and DMF in sequence, three times each, with nitrogen agitation for 30 seconds each time.

[1156] 5) Add the Fmoc-protected amino acid and condensing agent to another reaction flask, stir for 30 minutes, then add it to the resin and react on a shaker for 2 hours.

[1157] 6) Repeat steps 2 to 5 to condense the next amino acid.

[1158] Table 1 Feeding sequence

[1159] Peptide cleavage and purification:

[1160] 1) Add cleavage buffer (20% DCM solution of hexafluoroisopropanol) to a flask containing a peptide with side chain protection, and stir at room temperature for 1.5 hours.

[1161] 2) Filter, and evaporate the filtrate to dryness to obtain 4.20 g of white solid. Purity: 95%; Yield: 68%.

[1162] LC-MS (ESI) m / z: 819.3 [M+H] + .

[1163] 5.19.4 Preparation of HM-2493_16-A

[1164] HM-2493_16 (3.70 g, 4.5 mmol, 1.0 eq) was added to DEA (40 mL) / DCM (40 mL) and stirred at room temperature for 2 h. LCMS showed that the reaction was complete. The reaction solution was purified by reverse phase. 2.30 g of white solid was obtained, purity: 98%; yield: 85%.

[1165] LC-MS (ESI) m / z: 597.3 [M+H] + .

[1166] 5.19.5 Preparation of HM-2493_16-B

[1167] HM-2493_16-A (2.50 g, 8.75 mmol, 1.0 eq) was dissolved in DMF (25 mL), cooled to 0 °C, and then DIEA (525 mg, 8.75 mmol, 1.0 eq) and Alloc-OSu (825 mg, 8.75 mmol, 1.0 eq) were added. The mixture was stirred at room temperature for 1 h. LC-MS showed that the reaction was complete. The crude product was purified by reverse-phase chromatography to give 2.65 g of white solid, purity: 93%; yield: 91%.

[1168] LC-MS (ESI) m / z: 681.3 [M+H] + .

[1169] 5.19.6 Preparation of HM-5164_25-A

[1170] HM-2493_16-B (1.20 g, 1.76 mmol, 1.0 eq) and HM-5164_24 (600 mg, 1.76 mmol, 1.0 eq) were added to DMF (12 mL), and the mixture was cooled to 0 °C. Then, DIEA (720 mg, 5.29 mmol, 3.0 eq) and HATU (480 mg, 1.18 mmol, 1.0 eq) were added. The mixture was stirred at room temperature for 1 h, and LC-MS showed that the reaction was complete. After quenching with water, the mixture was extracted with EA (30 mL × 3), washed with brine, dried over Na2SO4, and purified with DCM:MeOH (0–8%) to give 1.58 g of white solid with a purity of 83% and a yield of 74%.

[1171] (LC-MS(ESI) m / z: 1001.5 [M+H]) + .)

[1172] 5.19.7 Preparation of HM-5164_26-A

[1173] Triphosgene (250 mg, 0.83 mmol, 1.2 eq) was dissolved in DCM (2 mL) and cooled to 0 °C. DMAP (735 mg, 6.01 mmol, 8.6 eq) was added at low temperature and stirred for 5 min. HM-5164_25-A (700 mg, 0.69 mmol, 1.0 eq) was added at low temperature and reacted for 5 min. HN-082_6 (355 mg, 0.69 mmol, 1.0 eq) was added and stirred at room temperature for 1 h. LC-MS showed the reaction was complete. The reaction was quenched with saturated ammonium chloride solution, extracted with DCM, and concentrated to obtain the crude product. The crude product was purified by reverse-phase chromatography and lyophilized to give 500 mg of a yellow solid with a purity of 93% and a yield of 46%.

[1174] LC-MS (ESI) m / z: 1534.6 [M+H] +

[1175] 5.19.8 Preparation of HM-5164_27-B

[1176] HM-5164_26-A (200 mg, 0.13 mmol, 1.0 eq) was dissolved in DCM (4 mL), followed by the addition of a DCM mixture of piperidine (66 mg, 0.78 mmol, 6.0 eq) and AcOH (40 mg, 0.78 mmol, 6.0 eq) (4 mL). Then, Pd(PPh3)4 (20 mg, 0.017 mmol, 0.13 eq) was added and the mixture was reacted at room temperature for 2 h. LC-MS showed complete reaction. The crude product was purified by reverse-phase chromatography and lyophilized to give 155 mg of a yellow solid with a purity of 85% and a yield of 83%.

[1177] LC-MS (ESI) m / z: 1127.5 [M+H] +

[1178] 5.19.9 Preparation of HM-5164_29

[1179] HM-5164_27-B (390 mg, 0.34 mmol, 1.0 eq) was dissolved in DCM (4 mL), followed by the addition of pyridine hydrogen fluoride (103 mg, 1.04 mmol, 3.0 eq). The reaction was carried out at room temperature for 2 h, and LC-MS showed that the reaction was complete. The crude product was purified by reverse-phase chromatography and lyophilized to give 160 mg of a yellow solid with a purity of 92% and a yield of 45%.

[1180] LC-MS (ESI) m / z: 1013.4 [M+H] +

[1181] 5.19.10 Preparation of HM-5164_30

[1182] HM-5164_29 (160 mg, 0.158 mmol, 1.0 eq) and HM-297Q_6 (63 mg, 0.205 mmol, 1.3 eq) were dissolved in DMF (2 mL), and then DIEA (25 mg, 0.19 mmol, 1.2 eq) was added at low temperature (0 °C). LC-MS showed that the reaction was complete. The solution was prepared and purified (NH4AcO system) and lyophilized to give 42 mg of a yellow solid with a purity of 98% and a yield of 22%.

[1183] LC-MS (ESI) m / z: 1207.5 [M+H] +

[1184] 1 H NMR (400MHz, DMSO-d6)

[1185] δ10.35(s,1H),9.83(s,1H),8.31(d,J=7.1Hz,1H),8.05(d,J=8.8Hz,1H),7.99(d,J=8.0Hz,1H),7.82(d,J=7.7Hz,1H),7.70( d,J=8.1Hz,1H),7.59(d,J=8.4Hz,2H),7.45–7.28(m,6H),7.02–6.94(m,4H),5.50(s,2H),5.31(s,2H),5.15–4.95(m,3H),4.5 9(d,J=5.9Hz,1H),4.33–4.23(m,3H),4.05(s,1H),3.10(d,J=8.0Hz,5H),2.78–2.65(m,2H),2.13(d,J=5.8Hz,4H),2.03(d,J= 6.2Hz,1H),1.64(s,1H),1.47(s,8H),1.30(t,J=7.4Hz,3H),1.19(d,J=7.0Hz,3H),1.07(d,J=6.0Hz,3H),0.91–0.80(m,10H).

[1186] Example 5.20 Synthesis of MC-RDVT-DXD (also known as MC-RDVT-Aminobutoxyl-Exatecan) (HM-2493_21)

[1187] 5.20.1 Preparation of HM-2493_3

[1188] DIEA (7.95 g, 61.566 mmol, 1 eq) and HATU (12.26 g, 32.249 mmol, 1.1 eq) were added to a DMF (200 mL) solution of HM-2493_1 (10 g, 29.317 mmol, 1 eq) and HM-2493_2 (5.9 g, 29.317 mmol, 1 eq) in an ice bath (5 °C), and the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into deionized water (1 L), the precipitated solid was filtered, collected, slurried with ethyl acetate, and dried to obtain 13 g of the target product HM-2493_3, a white solid, with a yield of 90.8%. LC-MS (ESI) m / z: 489.2 [M+H] + .

[1189] 5.20.2 Preparation of HM-2493_4

[1190] TBSCl (5.974 g, 39.635 mmol, 1.5 eq) was added to a DMF (130 mL) solution of HM-2493_3 (12.9 g, 26.424 mmol, 1 eq) and Imidazole (3.6 g, 52.848 mmol, 2 eq) under ice bath conditions (5 °C), and the mixture was stirred at room temperature for 16 hours. The reaction solution was poured into deionized water (1 L), and the precipitated viscous substance was filtered, dissolved in ethyl acetate, dried, concentrated, and subjected to silica gel column chromatography (PE / EtOAc = 5 / 1) to obtain the target product HM-2493_4 (14.6 g, yield 91.7%, white foamy viscous substance). LC-MS (ESI) m / z: 603.4 [M+H] + .

[1191] 5.20.3 Preparation of HM-2493_5

[1192] At room temperature, 5% Pd / C (1.44 g, 10% wt) was added to a MeOH (360 mL) solution of HM-2493_4 (14.4 g, 23.908 mmol, 1 eq). The mixture was purged three times with hydrogen gas, and the reaction was stirred at room temperature for 2 hours under a hydrogen balloon atmosphere. The reaction solution was filtered, washed with methanol, and concentrated to obtain the target product HM-2493_5 (12 g, 98% yield, white solid). LC-MS (ESI) m / z: 513.2 [M+H] + .

[1193] 5.20.4 Preparation of HM-2493_6

[1194] Pb(OAc)4 (14.4 g, 90% wt, 29.286 mmol, 1.5 eq) was added to a THF (550 mL) solution of HM-2493_5 (10 g, 19.524 mmol, 1 eq) and Cu(OAc)2 (355 mg, 1.9524 mmol, 0.1 eq) under a nitrogen atmosphere and stirred at room temperature for 2 hours. The reaction solution was neutralized with saturated sodium bicarbonate aqueous solution, filtered, washed with EtOAc, and the filtrate was concentrated to remove most of the THF. The solution was then extracted with EtOAc. The combined organic phases were dried, concentrated, and subjected to silica gel column chromatography (PE / EtOAc = 5 / 1) to obtain the target product HM-2493_6 (6.8 g, yield: 66%, white solid). LC-MS (ESI) m / z: 549.2 [M+Na] + .

[1195] 5.20.5 Preparation of HM-2493_7

[1196] TsOH (233 mg, 1.34 mmol, 0.1 eq) was added to a THF (140 mL) solution of HM-2493_6 (7.05 g, 13.4 mmol, 1 eq) and HM-297Q_61 (11.14 g, 67 mmol, 5 eq) under an ice bath at 5 °C, and the reaction was stirred at room temperature for 16 hours. The reaction solution was neutralized with saturated sodium bicarbonate aqueous solution, most of the THF was concentrated, and the solution was extracted with EtOAc. The combined organic phases were dried and concentrated, and purified by reverse-phase column chromatography (ACN / H2O with 0.1% FA). The eluent containing the product was neutralized with saturated sodium bicarbonate aqueous solution, concentrated to remove most of the acetonitrile, and extracted with EtOAc. The combined organic phases were dried and concentrated to obtain the target product HM-2493_7 (6.6 g, yield: 93.5%, white oil). LC-MS (ESI) m / z: 655.2 [M+Na] + .

[1197] 5.20.6 Preparation of HM-2493_8

[1198] DABCO (6.48 g, 57.726 mmol, 5 eq) was added to a DMF (70 mL) solution of HM-2493_7 (7.3 g, 11.545 mmol, 1 eq) at room temperature, and the mixture was stirred for 2 hours at room temperature. The reaction solution was neutralized with HOAc, purified by reverse-phase column chromatography (C18) (ACN / H2O with 0.1% FA), and lyophilized to obtain the target product HM-2493_8 (4.5 g, yield: 85%, white oil, formate). LC-MS (ESI) m / z: 411.2 [M+H] + .

[1199] 5.20.7 Preparation of HM-2493_9

[1200] At room temperature, 5% Pd / C (450 mg, 10% wt) was added to a MeOH (180 mL) solution of HM-2493_8 (4.5 g, 9.864 mmol, 1 eq). The gas was purged three times with a hydrogen balloon, and the mixture was stirred at room temperature for 2 hours in a hydrogen balloon atmosphere. The reaction solution was filtered, washed with methanol, and the filtrate was concentrated to obtain the target product HM-2493_9 (3.29 g, 91% yield, white solid, formate).

[1201] 5.20.8 Preparation of HM-2493_11

[1202] Weigh 5 g (7.5 mmol, 1.5 mmol / g, 1 eq) of HM-2493_10 into a 500 mL peptide tube, add 3.8 g (11.25 mmol, 1.5 eq) of HM-2493_11A in 100 mL of DMF solution, then add 2.42 g (18.75 mmol, 2.5 eq) and shake on a shaker for 16 hours. Add 15 mL of MeOH to the peptide tube, shake for 1 hour, and then dry under vacuum. Wash the resin sequentially with 80 mL x 3 DMF, 20 mL of MeOH, and 30 mL of DMF to obtain approximately 7.5 mmol of HM-2493_11. The yield is not considered. Take a small sample, treat with HFIP / DCM (1 / 4), remove the resin, and send to LCMS to confirm resin incorporation.

[1203] LC-MS (ESI) m / z: 340.2 [M+H] + .

[1204] 5.20.9 Preparation of HM-2493_13

[1205] Add 20% piperidine / DMF (100 mL) to the polypeptide tube containing HM-2493_11 (7.5 mmol, 1 eq), shake on a shaker for 30 minutes, then dry. Repeat the above steps once. Then wash the resin sequentially with DMF (80 mL x 3), MeOH (80 mL x 2), and DMF (80 mL x 3).

[1206] Add 80 mL of DMF solution containing HM-2493_12 (5.93 g, 15 mmol, 2 eq) and HOBt (3 g, 22.5 mmol, 3 eq) to the polypeptide tube, then add DIC (2.84 g, 22.5 mmol, 3 eq) and shake on a shaker for 16 hours. Add a small sample to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin sequentially with DMF (80 mL x 3), MeOH (80 mL x 2), and DMF (80 mL x 3) to obtain approximately 7.5 mmol of HM-2493_13; yield is not considered. Treat a small sample with HFIP / DCM (1 / 4), remove the resin, and send to LCMS to confirm HM-2493_12 incorporation.

[1207] LC-MS (ESI) m / z: 495.2 [M+H] + .

[1208] 5.20.10 Preparation of HM-2493_15

[1209] Add 20% piperidine / DMF (100 mL) to the polypeptide tube containing HM-2493_13 (7.5 mmol, 1 eq), shake on a shaker for 30 minutes and then dry. Repeat the above steps once, and then wash the resin with DMF (80 mL x 3), MeOH (80 mL x 2), and DMF (80 mL x 3) in sequence.

[1210] Add 80 mL of DMF solution containing HM-2493_14 (5 g, 9 mmol, 1.2 eq) and HOBt (2 g, 15 mmol, 2 eq) to the polypeptide tube, then add DIC (1.89 g, 15 mmol, 2 eq) and shake on a shaker for 16 hours. Take a small sample and add it to ninhydrin hydrate, heat to 110 °C until no blue color appears, then dry under vacuum. Wash the resin sequentially with DMF (80 mL x 3), MeOH (80 mL x 2), DMF (80 mL x 3), and DCM (80 mL x 3) to obtain approximately 7.5 mmol of HM-2493_15; yield is not considered. Take a small sample and treat with HFIP / DCM (1 / 4), remove the resin, and send to LCMS to confirm HM-2493_14 incorporation.

[1211] LC-MS (ESI) m / z: 819.4 [M+H] + .

[1212] 5.20.11 Preparation of HM-2493_16

[1213] HM-2493_15 (7.5 mmol, 1 eq) was transferred from the peptide tube to a flask, and HFIP / DCM (1 / 4) (100 mL) was added. The mixture was stirred at room temperature for 2 hours, filtered, washed with DCM, concentrated, and the residual HFIP was removed with DCM to obtain the target product HM-2493_16 (7 g, yield not considered, light yellow solid).

[1214] LC-MS (ESI) m / z: 819.4 [M+H] + .

[1215] 5.20.12 Preparation of HM-2493_17

[1216] HATU (3.07 g, 8.066 mmol, 1.1 eq) was added to a DMF (60 mL) solution of HM-2493_16 (6 g, 7.332 mmol, 1 eq) and 2,6-dimethylpyridine (3.15 g, 29.328 mmol, 4 eq) for 1 hour under ice bath conditions (5 °C), and the mixture was stirred. Then, a DMF (30 mL) solution of HM-2493_9 (3 g, 8.066 mmol, 1.1 eq) was added to the above reaction mixture at 5 °C, and the mixture was stirred at room temperature for 1 hour. The reaction solution was purified by reverse-phase column chromatography (C18) (ACN / H2O with 0.1% FA) and lyophilized to obtain the target product HM-2493_17 (2.39 g, yield 29%, white solid). LC-MS (ESI) m / z: 1121.5 [M+H] + .

[1217] 5.20.13 Preparation of HM-2493_18

[1218] HATU (774 mg, 2.022 mmol, 1.1 eq) and DIEA (475 mg, 3.676 mmol, 2 eq) were added to a DMF mixture (50 mL) of HM-2493_17 (2.06 g, 1.838 mmol, 1 eq) and HM-582_10 (980 mg, 1.838 mmol, 1 eq) under ice bath conditions (5 °C), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into deionized water (500 mL), and the precipitated solid was filtered and collected. The solid was dissolved in DCM, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (DCM / MeOH = 30 / 1) to obtain the target product HM-2493_18 (2.7 g, 95% yield, pale yellow solid).

[1219] LC-MS (ESI) m / z: 1538.5 [M+H] + .

[1220] 5.20.14 Preparation of HM-2493_19

[1221] Dimedone (410 mg, 2.926 mmol, 3 eq) and Pd(PPh3)4 (113 mg, 0.0976 mmol, 0.1 eq) were added to a THF (30 mL) solution of HM-2493_18 (1.5 g, 0.9755 mmol, 1 eq) at room temperature and stirred at room temperature for 2 hours under nitrogen protection. Water (5 mL) and HOAc (1 mL) were added to the reaction solution to clarify the solution, which was then purified by reverse-phase column chromatography (C18) (ACN / H2O with 0.1% FA) and lyophilized to obtain the target product HM-2493_19 (1.07 g, yield 82%, yellow solid).

[1222] LC-MS (ESI) m / z: 1330.4 [M+H] + .

[1223] 5.20.15 Preparation of HM-2493_19A

[1224] DABCO (445 mg, 3.95 mmol, 5 eq) was added to a DMF (15 mL) solution of HM-2493_19 (1.05 g, 0.79 mmol, 1 eq), and the mixture was stirred at 25 °C for 2 hours. The reaction solution was neutralized with HOAc, purified by reverse-phase column chromatography (C18) (ACN / H2O with 0.1% FA), and lyophilized to obtain the target product HM-2493_19A (600 mg, yield 66%, yellow solid, FA salt). LC-MS (ESI) m / z: 1108.4 [M+H] + .

[1225] 5.20.16 Preparation of HM-2493_20

[1226] 3HF·Et3N (2.74 mL) was added to a THF / H2O (11.4 mL / 2.28 mL) solution of HM-2493_19A (570 mg, 0.494 mmol, 1 eq) under an ice bath at 5 °C, and the reaction was stirred at room temperature for 5 hours. The reaction solution was purified by reverse-phase column chromatography (C18) (ACN / H2O with 0.1% FA) and lyophilized to obtain the target product HM-2493_20 (460 mg, yield 89%, yellow solid, FA salt). LC-MS (ESI) m / z: 994.2 [M+H] + .

[1227] 5.20.17 Preparation of HM-2493_21

[1228] 2,6-Dimethylpyridine (124 mg, 1.155 mmol, 5 eq) was added to a 4 mL DMF solution of HM-2493_20 (240 mg, 0.231 mmol, 1 eq) and HM-297Q_6 (107 mg, 0.346 mmol, 1.5 eq) at room temperature, and the mixture was stirred for 2 hours at room temperature. The reaction solution was purified by reverse-phase reaction (ACN / H2O with 0.1% FA) and lyophilized to obtain the target product HM-2493_21 (36 mg, purity 97.7%, yield: 13%, white solid). LC-MS (ESI) m / z: 1187.4 [M+H] + .

[1229] 1 H NMR (400MHz, DMSO) δ8.83(t,J=6.3Hz,1H),8.66(d,J=8.7Hz,1H),8.24(d,J=8.5Hz,1H),8.10(s,1H),8.02(d,J=7.4Hz,1H),7.93(d,J=6.8H z,1H),7.85(d,J=8.0Hz,1H),7.78(d,J=11.0Hz,1H),7.33(s,1H),7.18(s,4H),7.01(s,2H),6.53(s,1H),5.64-5.56(m,1H),5.44(s,2H),5. 23(d,J=6.8Hz,2H),4.64–4.47(m,3H),4.22-4.14(m,1H),4.05–3.95 (m,4H),3.90–3.85(m,1H),3.21–3.03(m,4H),2.41(s,3H),2.39–2.03 (m,8H),1.94–1.83(m,2H),1.66-1.37(m,9H),1.23-1.14(m,2H),0.98(d,J=6.3Hz,3H),0.89(t,J=7.3Hz,3H),0.80(dd,J=10.3,6.9Hz,6H).

[1230] Example 5.21 Synthesis of Mal-PEG4-RDVT-PAB-Exatecan (HM-5164_71)

[1231] 5.21.1 Preparation of HM-5164_3

[1232] The polypeptide was prepared using a standard solid-phase synthesis method. 1) DCM (500 mL) was added to a container containing Merrifield-resin (40 g, substitution degree: 1.6) and the resin was allowed to swell for 30 min. After swelling, DMF was added to wash three times, 500 mL each time. After filtering off the DMF, HM-2100D_1 (2 eq) and DIEA (5 eq) were added, and 500 mL of DMF was added to dissolve them. The mixture was reacted on a shaker overnight, dried under vacuum, quenched with methanol, and reacted on a shaker for 1 h.

[1233] 2) Dry the container and rinse it with MeOH, DCM and DMF three times each in sequence.

[1234] 3) Add 20% piperidine / DMF, react for 2 hours, dry under vacuum, add another 20% piperidine / DMF, and react for another 2 hours.

[1235] 4) Drain the solution, then rinse it with MeOH, DCM and DMF in sequence, three times each, with nitrogen agitation for 30 seconds each time.

[1236] 5) Add the Fmoc-protected amino acid and condensing agent to another reaction flask, stir for 30 minutes, then add it to the resin and react on a shaker for 2 hours.

[1237] 6) Repeat steps 2 through 5 to condense the next amino acid.

[1238] Table 1 Feeding sequence

[1239] Peptide cleavage and purification:

[1240] 1) Add cleavage buffer (20% DCM solution of hexafluoroisopropanol) to a flask containing a peptide with protected side chains, and place at room temperature.

[1241] Stir at room temperature for 1.5 hours.

[1242] 2) Filter, evaporate the filtrate to dryness, and obtain 43g of yellow solid, which is directly added to the next step. Yield: 66%

[1243] LC-MS (ESI) m / z: 1024.6 [M+H] + .

[1244] 5.21.2 Preparation of HM-5164_5

[1245] HM-5164_3 (6 g, 5.86 mmol, 1.0 eq.) and HM-297D_10 (1.08 g, 8.79 mmol, 1.5 eq.) were dissolved in 60 mL of DCM. EEDQ (2.17 g, 8.79 mmol, 1.5 eq.) was added under ice bath conditions, and the reaction was carried out for 2 h under ice bath conditions. LC-MS showed that the reaction was complete. The reaction solution was added dropwise to 600 mL of petroleum ether to precipitate a solid product. The supernatant was removed by centrifugation to give 5.1 g of a yellow solid. The yield of the crude product was negligible. LC-MS (ESI) m / z: 1129.6 [M+H] + .

[1246] 5.21.3 Preparation of HM-5164_6

[1247] HM-5164_5 (5.1 g, 4.52 mmol, 1.0 eq) and bis(4-nitrophenyl) carbonate (2.75 g, 9.03 mmol, 2.0 eq) were dissolved in 51 mL of DMF. DIEA (1.17 g, 9.03 mmol, 2.0 eq) was added under ice bath conditions. The mixture was then removed from the ice bath and reacted at room temperature for 2 h. LC-MS showed the reaction was complete. The mixture was extracted three times with EA, washed with saturated brine, and the organic phases were combined and evaporated to dryness to give 5 g of a yellow solid. The yield of the crude product was negligible. LC-MS (ESI) m / z: 1294.6 [M+H] + .

[1248] 5.21.4 Preparation of HM-5164_7

[1249] HM-5164_5 (5 g, 3.96 mmol, 1.0 eq) and HM-582_10 (4.52 g, 8.50 mmol, 1.5 eq) were dissolved in 50 mL of DMF. DIEA (3.99 g, 30.90 mmol, 8.0 eq) was added under ice bath conditions. The mixture was then removed from the ice bath and reacted at room temperature for 6 h. LC-MS showed complete reaction. The mixture was extracted three times with EA and washed with saturated brine. The organic phases were combined, evaporated to dryness, and purified by normal-phase chromatography (PE / EA = 1:1) to give 2.5 g of a yellow solid (LC-MS (ESI) m / z: 1590.6 [M+H]). + HM-5164_7 (yield: 27%) and 1.2 g of yellow solid (LC-MS (ESI) m / z: 1368.6 [M+H)). + HM-5164_8 (yield: 15%).

[1250] 5.21.5 Preparation of HM-5164_8

[1251] HM-5164_7 (1.3 g, 0.628 mmol, 1.0 eq) was dissolved in 10 mL of DCM. 5 mL of ethylenediamine was added under ice bath conditions. The ice bath was removed, and the reaction was allowed to proceed at room temperature for 2 h. LC-MS showed the reaction was complete. The reaction solution was added dropwise to 200 mL of petroleum ether, causing crystals to precipitate. The supernatant was removed by centrifugation to give 850 mg of a brownish-yellow solid. The yield of the crude product was negligible. LC-MS (ESI) m / z: 1368.6 [M+H] +

[1252] 5.21.6 Preparation of HM-5164_70

[1253] HM-5164_8 (400 mg, 0.292 mmol, 1.0 eq) and HM-1559_3 (150 mg, 0.292 mmol, 1.0 eq) were dissolved in 4 mL of DMF. 2,6-Dimethylpyridine (313 mg, 2.920 mmol, 10.0 eq) was added under ice bath conditions. The mixture was then removed from the ice bath and reacted at room temperature for 2 h. LC-MS showed that the reaction was complete. The reaction solution was purified by reversed-phase chromatography (FA / H2O / ACN) and lyophilized to give 130 mg of a yellow solid. Two-step yield: 25%. LC-MS (ESI) m / z: 1766.8 [M+H] +

[1254] 5.21.7 Preparation of HM-5164_71

[1255] HM-5164_70 (130 mg, 0.107 mmol, 1.0 eq) was placed in a 100 mL single-necked flask. TFA (6 mL) and DCM (18 mL) were added at -15 °C, and the reaction was carried out at -15 °C for 48 h. LC-MS showed that the reaction was complete. The reaction solution was evaporated to dryness under an ice-water bath. Five additional 30 mL DCM solutions were added and evaporated again. After evaporation to dryness, the solution was sent to a preparation (0.01% TFA, H₂O / ACN) and lyophilized to obtain 46 mg of a white solid with a purity of 96.27% and a yield of 18.6%. LC-MS (ESI) m / z: 1454.6 [M+H] +

[1256] 1H NMR (400MHz, DMSO) δ12.68–12.05(m,1H),9.84(s,1H),8.36(d,J=7.0Hz,1H),8.3-8.0(m,3H),7.88–7.70(m,3H),7.62(d,J =8.5Hz,2H),7.42–7.32(m,4H),7.02(s,2H),6.56(s,1H),5.47(s,2H),5.31(s,3H),5.10(s,2H),5.02(s,1H),4.62(s,1H), 4.34(s,2H),4.26(s,1H),4.08(s,1H),3.62(d,J=7.2Hz,5H),3.55–3.42(m,18H),3.21–3.04(m,5H),2.79–2.69(m,1H),2. 45–2.31(m,7H),2.21(s,2H),2.04(s,1H),1.89(s,2H),1.67(s,1H),1.49(s,3H),1.10(d,J=6.3Hz,3H),0.91–0.84(m,9H).

[1257] Example 6. Synthesis of ADC

[1258] General Method A

[1259] 2.86 mg / ml of antibody (e.g., the bispecific antibody of this application) in 50 mM NaOAc-HAc (pH 5.5) was placed in a Biofil tube. 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM) were added to the antibody buffer (TCEP:antibody = 6:1). The Biofil tube containing the reaction mixture was placed on a shaker (x500 rpm) and reacted at 37°C for 2 hours. An additional 6 molar equivalents of TCEP (5 mM) were added to the mixture, and the reaction mixture was shaken on a shaker (500 rpm) at 37°C for another 2 hours. Then, the mixture was ultrafiltered (MWCO 30 kDa) 6 times to remove TECP, replenishing to the initial volume with 10 mM His-Hac (pH 6.2) buffer after each ultrafiltration. 12 molar equivalents of linker-payload (5 mg / ml in DMA) were added dropwise to the fully reduced antibody while maintaining the DMA concentration below 20% (v / v). The reaction was incubated at room temperature on a shaker for 1 hour (linker-payload:antibody = 6:1). The conversion was assessed using HIC-HLPC, and purification was performed immediately after complete conversion.

[1260] Ultrafiltration purification method: Transfer the reaction mixture to an ultrafiltration tube (MWCO 30kd), and add 20% DMA / 10mM His-HAc (pH 6.2) to the maximum volume of the ultrafiltration tube. Centrifuge the sample at 3000 rpm for 10 minutes. When approximately half the maximum volume of the ultrafiltration tube remains, replenish the solution with 20% DMA / 10mM His-HAc to the initial volume. Discard the flow-through. Repeat the washing step 10 times. Then, replace the solution with 10mM His-HAc (pH 6.2) 10 times until the organic solvent DMA is completely replaced. Transfer the remaining solution and add an appropriate amount of 10mM His-HAc (pH 6.2). Rinse the filter membrane twice, and combine the solutions to obtain the final coupling product. Determine the concentration using the BCA method. If the final concentration is lower than the desired concentration, concentrate it to the expected concentration using an ultrafiltration membrane (MWCO 30kd).

[1261] General Method B

[1262] Place 6.31 mg / ml of antibody (e.g., the Trastuzumab antibody of this application) in an Eppendorf tube in PBS buffer. Add 8 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 10 mM concentration) to the antibody buffer (TCEP:antibody molar ratio = 8:1). Place the Eppendorf tube containing the reaction mixture on a shaker (x500 rpm) at 37°C for 4 hours, then ultrafilter (MWCO 30 kDa, Millipore filter) 6 times, each time to half the solution volume. Replenish the reduced antibody solution to the initial volume with 10 mM His-hac buffer to remove TECP. Add 10 molar equivalents of linker-payload (i.e., linker-payload, 5 mg / ml DMA solution) dropwise to the completely reduced antibody until the linker-payload to antibody molar ratio reaches 10:1. During the addition, keep the DMA concentration below 10% (v / v), otherwise adjust with buffer. The reaction was maintained at room temperature (500 RPM) for 1 hour using a shaker. Conversion was assessed using HIC-HLPC. Once conversion was complete, 10 molar equivalents of cysteine ​​(10 mM) were added. The reaction was then maintained at room temperature (cysteine:antibody = 10:1) on a shaker for 30 minutes to deplete excess free linker-load. After completion, the reaction mixture was transferred to an ultrafiltration tube (MWCO 30 kDa), and the sample was centrifuged at 10,000 rpm for 5 minutes to half the solution volume. The volume was then replenished with 10 mM His-HAc (10% DMA). The flow-through was discarded. The washing step was repeated 10 times. The solution was then replaced by washing 10 times with 10 mM His-HAc at pH 6.0. The remaining solution was transferred, and an appropriate amount of 10 mM His-HAc (pH 6.2) was added. The filter membrane was rinsed twice, and the solutions were combined to obtain the final conjugated product.

[1263] Using the methods described in General Method A or General Method B above, an ADC as shown below is prepared with appropriate antibodies and appropriate adapter-payload.

[1264] The distribution of free antibody and DAR was detected using the HIC-HPLC method. The HIC-HPLC conditions and parameters are shown below.

[1265] Perform elution according to the table below.

[1266] The purity of the ADC was determined using the SEC-HPLC method. The SEC-HPLC conditions and parameters are shown below.

[1267] Reversed-phase HPLC (RP HPLC) method (for the detection of free drugs)

[1268] Perform elution according to the table below.

[1269] Information and detection data related to the prepared ADC.

[1270] The Isotype-RDVT-Exd information is as follows:

[1271] The Isotype antibody is named Anti-HEL IgG1 LALA-Kappa and is sourced from Baiying Biotechnology (CAT: B09802).

[1272] Example 7. In vitro experiments of ADC

[1273] Example 7.1 In vitro killing experiment

[1274] Target cells EBC-1 at 3x10 3 Placing samples in 96-well plates (100 μL / well) and incubating overnight at 37°C with 5% CO2. Diluting the sample to be tested to 200 nM with complete culture medium, performing 4-fold serial dilutions, and adding 100 μL / well to each plate, then incubating for 5 days at 37°C with 5% CO2. Adding 20 μL / well of CCK-8 and incubating for 3 hours at 37°C with 5% CO2. Reading OD values. 450。

[1275] As shown in Figure 3, V-F31-RDVT-Exd exhibits strong killing activity.

[1276] Target cells NCI-H1975 at 3x10 3 Placing samples at 100 μL / well in a 96-well plate and incubating overnight at 37°C with 5% CO2. Diluting the sample to be tested to 200 nM with complete culture medium, performing a 4-fold serial dilution, and adding 100 μL / well to each plate, incubating for 5 days at 37°C with 5% CO2. Adding 20 μL / well of CCK-8 and incubating for 3 hours at 37°C with 5% CO2. Reading OD450.

[1277] As shown in Figure 4, V-F31-RDVT-Exd exhibits strong killing activity.

[1278] Example 7.2 In vitro killing experiment

[1279] 1) Target cells MDA-MB-468 (TCHu136, Chinese Academy of Sciences Cell Bank) or T-47D (TCHu 87, Chinese Academy of Sciences Cell Bank) at 1-3 x 10⁻⁶ 5 1) Spread 100 μL of the sample to a plate in 96 wells and incubate overnight at 37°C with 5% CO2. Dilute the sample to be tested to 400 nM wit...

Claims

Drug conjugates of Formula I, or their stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof. Tg is the target region; q represents DAR; and is an integer selected from 1 to 20. Z stands for connector unit; L1 is an extended unit or does not exist; L2 is a self-destructing unit or does not exist; L3 is a spacer subunit or does not exist; D is a bioactive molecule; and A1A2A3A4 is a tetrapeptide unit, in which: A1 is selected from R, G, K, W, N, P, F, I, M, L, T, E, Q, H, D or Y; A2 is selected from L, G, Q, F, I, Y, W, S, A, T, N, K, D, M, H, or R; A3 is selected from W, F, R, V, K, D, M, A, G, E, Y, P, L, T, I, Q, or S; A4 is selected from Q, R, K, cit, W, G, N, D, E, H, L, V, S, T, F, Y, M, or A; The premise is that A1A2A3A4 is not GGFG (SEQ ID NO:30). The drug conjugate of claim 1, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, The A1A2A3A4 is not GGFG (SEQ ID NO:30), NYEE (SEQ ID NO:31), PNEE (SEQ ID NO:32), KIMR (SEQ ID NO:33), FKYK (SEQ ID NO:34), ILMK (SEQ ID NO:35), KFRL (SEQ ID NO:36), FGPD (SEQ ID NO:37), FDPV (SEQ ID NO:37) NO:38), MMMK (SEQ ID NO:39), LDQF (SEQ ID NO:40), LDIQ (SEQ ID NO:41), FWRF (SEQ ID NO:42), HDGQ (SEQ ID NO:43), HGQT (SEQ ID NO:44), DKGT (SEQ ID NO:45), WIYF (SEQ ID NO:46), RDGT (SEQ ID NO:46) NO:47), YRGT (SEQ ID NO:48), LAVF (SEQ ID NO:49), WGLS (SEQ ID NO:50), FDAY (SEQ ID NO:51) or HGIT (SEQ ID NO:52). The drug conjugate, or stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate, as described in any one of claims 1-2, wherein A1A2A3A4 is selected from the group consisting of: (1) A1 is selected from R, T, E, F or G; A2 is selected from D, A, H, K, G, L, or I; A3 is selected from V, I, S, L, D, or W; and A4 is selected from T, Q, G, L, or E; (2) A1 is selected from R, E, F or G; A2 is selected from D, H, K, G, I, or L; A3 is selected from V, S, I, D, or W; and A4 is selected from T, G, Q, L, or E; (3) A1 is selected from R, E, F, G or T; A2 is selected from D, H, K, G, I, or A; A3 is selected from V, S, I, D, W, or L; and A4 is selected from T, G, Q, L, or E; (4) A1 is selected from R, E, F or G; A2 is selected from D, H, K, G, or I; A3 is selected from V, S, I, D, or W; and A4 is selected from T, G, Q, L, or E; (5) A1 is selected from R, E, or F; A2 is selected from D, H, K, or G; A3 is selected from V, S, I, or D; and A4 is selected from T, G, Q, L, or E; (6) A1 is selected from R, E, or F; A2 is selected from D, H, or K; A3 is selected from V, S, I, or D; and A4 is selected from T, G, Q, L, or E; (7) A1 is selected from R, E or F; A2 is selected from D, H, or K; A3 is selected from V, S, I, or D; and A4 is selected from T, G, Q, or L; (8) A1 is selected from R or E; A2 is selected from D or H; A3 is selected from V, S, I, or D; and A4 is selected from T, G, Q, or L; (9) A1 is R; A2 is D; A3 is selected from V, S, or I; and A4 is selected from T, G, or Q; (10) A1 is R; A2 is D; A3 is selected from V or S; and A4 is selected from T or G; (11) A1 is R; A2 is D; A3 is selected from V, S, or I; and A4 is selected from T, G, Q, or E; and / or (12) A1 is selected from R, T, E, F or G; A2 is selected from D, A, H, K, G, L, or I; A3 is selected from V, L, D, or W; and A4 is selected from T, Q, G, L, or E. The drug conjugate, or stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to any one of claims 1-3, wherein A1 is selected from R, E, or F; A2 is selected from D, H, or K; A3 is selected from V, I, S, or D; and A4 is selected from T, Q, G, L, or E. The drug conjugate, or stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to any one of claims 1-4, wherein A1 is selected from R; A2 is selected from D; A3 is selected from V, I, or S; and A4 is selected from T, Q, G or E; preferably, A4 is selected from T, Q or G. The drug conjugate, or stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to any one of claims 1-5, wherein A1 is selected from R, T, E, F, or G; A2 is selected from D, A, H, K, G, L, or I; A3 is selected from V, L, D, or W; and A4 is selected from T, Q, G, L, or E. The drug conjugate of claim 1, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, wherein A1A2A3A4 is selected from: RDVT(SEQ ID NO:53)、RLWQ(SEQ ID NO:54)、RDIQ(SEQ ID NO:55)、RDSG(SEQ ID NO:56)、TALQ(SEQ ID NO:57)、EHDL(SEQ ID NO:58)、FKVT(SEQ ID NO:59)、FGVQ(SEQ ID NO:60)、DKGT(SEQ ID NO:45)、HGIT(SEQ ID NO:52)、RDVA(SEQ ID NO:61)、QGAM(SEQ ID NO:62)、WIYF(SEQ ID NO:46)、ELVY(SEQ ID NO:63)、HDGQ(SEQ ID NO:43)、WGLS(SEQ ID NO:50)、FDAY(SEQ ID NO:51)、GGWG(SEQ ID NO:64)、GWRG(SEQ ID NO:65)、NYEE(SEQ ID NO:31)、GGFA(SEQ ID NO:67)、GGFW(SEQ ID NO:68)、GFWG(SEQ ID NO:69)、PNEE(SEQ ID NO:32)、GIWG(SEQ ID NO:70)、GGRR(SEQ ID NO:71)、GGFR(SEQ ID NO:72)、GGFK(SEQ ID NO:73)、GGVR(SEQ ID NO:74)、GAAN(SEQ ID NO:75)、KIMR(SEQ ID NO:33)、FKYK(SEQ ID NO:34)、ILMK(SEQ ID NO:35)、KFRL(SEQ ID NO:36)、NLAS(SEQ ID NO:66)、WSME(SEQ ID NO:76)、GQKN(SEQ ID NO:77)、FGPD(SEQ ID NO:37)、GTGH(SEQ ID NO:78)、FDPV(SEQ ID NO:38)、MMMK(SEQ ID NO:39)、KYDD(SEQ ID NO:79)、GGVcit(SEQ ID NO:80),GFLG (SEQ ID NO:81), RDTL (SEQ ID NO:82), GFGS (SEQ ID NO:83), LDIL (SEQ ID NO:84), FDRQ (SEQ ID NO:85), LDQF (SEQ ID NO:40), WQAH (SEQ ID NO:86), LDIQ (SEQ ID NO:41), MNAL (SEQ ID NO:87), FWRF (SEQ ID NO:42), RDGT (SEQ ID NO:47), RDVE (SEQ ID NO:88), YRGT (SEQ ID NO:48), LAVF (SEQ ID NO:49), WIYF (SEQ ID NO:46) and HGQT (SEQ ID NO:44);, Preferably, the A1A2A3A4 is selected from RDVT (SEQ ID NO:53), RLWQ (SEQ ID NO:54), RDIQ (SEQ ID NO:55), RDSG (SEQ ID NO:56), TALQ (SEQ ID NO:57), EHDL (SEQ ID NO:58), FKVT (SEQ ID NO:59), FGVQ (SEQ ID NO:59) NO:60), RDVA (SEQ ID NO:61), QGAM (SEQ ID NO:62), ELVY (SEQ ID NO:63), GGWG (SEQ ID NO:64), GWRG (SEQ ID NO:65), GGFA (SEQ ID NO:67), GGFW (SEQ ID NO:68), GFWG (SEQ ID NO:69), GIWG (SEQ ID NO:70), GGRR (SEQ ID NO:71), GGFR (SEQ ID NO:72), GGFK (SEQ ID NO:73), GGVR (SEQ ID NO:74), GAAN (SEQ ID NO:75), NLAS (SEQ ID NO:66), WSME (SEQ ID NO:76), GQKN (SEQ ID NO:77), GTGH (SEQ ID NO:78), KYDD (SEQ ID NO:79), GGVcit (SEQ ID NO:80), GFLG (SEQ ID NO:81), RDTL (SEQ ID NO:82), GFGS (SEQ ID NO:83), LDIL (SEQ ID NO:84), FDRQ (SEQ ID NO:85), WQAH (SEQ ID NO:86), MNAL (SEQ ID NO:87), RDVE (SEQ ID NO:88), LAVF (SEQ ID NO:49) and HGQT (SEQ ID NO:44); Preferably, A1A2A3A4 is selected from RDVT (SEQ ID NO:53), RDSG (SEQ ID NO:56), RDIQ (SEQ ID NO:55), EHDL (SEQ ID NO:58), FKVT (SEQ ID NO:59), RDVE (SEQ ID NO:88), FGVQ (SEQ ID NO:60), GIWG (SEQ ID NO:70), RLWQ (SEQ ID NO:54), and TALQ (SEQ ID NO:57). The drug conjugate, stereoisomer, isotope variant, pharmaceutically acceptable salt, or solvate of any one of claims 1-7 Where L2 is either non-existent or selected from: Where X is selected from -NH-, -O-, or -S-; R1 is independently selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, cyano group, -PEG unit, -C 1-8 alkylene-PEG unit, -C 1-8 Alkylene-C(=O)NH-PEG unit, -C 1-8 Alkylene-NHC(=O)-PEG unit, -C 1-8 alkylene-C(=O)O-PEG units and -C 1-8 Alkylene-OC(=O)-PEG unit; Su refers to the sugar portion; r1 is 0, 1, 2, 3 or 4; t is 0, 1, 2, or 3; Preferably, R1 is independently selected from C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; more preferably, each R1 is independently selected from -C 1-8 alkylene-C(=O)NH-PEG units and -C 1-8 Alkylene-NHC(=O)-PEG unit; Preferably, r1 is 0 or 1; more preferably, r1 is 0. Preferably, the PEG unit is -(CH2CH2O). b- R PEG R PEG b is the end-capping group of the PEG unit, preferably -CH3 or -CH2CH2COOH; b is independently selected from 2-72, for example 4-20, 2-10 or 4-10 integers; Preferably, each of Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; for example, each is independently selected from... For example, Su is independently: More The drug conjugate of claim 8, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, Where L2 is either non-existent or selected from: Wherein X, Su, and R1 are as defined in claim 8. The L2 group is connected to A1A2A3A4 on the left and to -L3-D on the right. Preferably, L2 is either non-existent or selected from: Wherein X, Su and R1 are as defined in claim 8; Preferably, L2 is absent or selected from Wherein X and Su are as defined in claim 8; preferably, where X is -NH- and Su is Preferably, L2 is absent or selected from Wherein X is defined as in claim 8; Preferably, L2 is or Preferably, L2 is non-existent. The drug conjugate, or its stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate, as described in any one of claims 1-9 L3 is either non-existent or selected from [the specified value]. -NR2-(CHR3)n 1a -L a -L b -L c -; L a This represents -C(=O)-NR2- and -NR2-(CH2)n. 2a -, -O-, -S-, -CHR3-, -NH- or single bonds, L b Represents -(CR2R3)n 3a -, -O-, -NR2-, or a single bond, preferably represented as -(CHR3)n 3a -, -O-, -NR2- or a single bond, L c It represents -CH2- or -C(=O)-; R2 and R3 independently represent hydrogen atoms and -C atoms, respectively. 1-8 Alkyl groups, C groups substituted with groups selected from the following 1-8 Alkyl-:MeSO 2- , halogen, nitro and cyano, -(CH2)n 4a -COOH, -(CH2)n 5a -OH, -(CH2)n 6a -NH2 n 1a n 2a n 3a n 4a n 5a and n 6a Each can independently represent 0, 1, 2, 3, 4, 5, or 6; L3 is preferred from R2 is independently selected from H and C. 1-8 Alkyl groups and C groups substituted with groups selected from the following 1-8 Alkyl-:MeSO 2- halogens, nitro groups, and cyano groups; R3 is independently selected from H and C. 1-8 alkyl-; X4 is -CH2-, -NH-, -O-, or -S-; and n5 and n6 each independently represent 0, 1, 2, 3, 4, 5 or 6; Preferably, L3 is absent or selected from... R2 is independently selected from H and C. 1-8 Alkyl groups and C groups substituted with groups selected from the following 1-8 Alkyl groups: MeSO2-, halogens, nitro groups, and cyano groups; R3 is independently selected from H and C. 1-8 alkyl-; and X4 is -CH2-, -NH-, -O-, or -S-; Preferably, R2 is independently selected from H, methyl and ethyl groups substituted with groups selected from: MeSO2-, halogen, nitro and cyano; Preferably, R3 is H; Preferably, X4 is -O- or -S-; Preferably, X4 is -O-; Preferably, L3 is selected from: The drug conjugate of claim 10, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, Among them, -L2-L3- are selected from the following groups: (a) L2 is selected from non-existent, Wherein X, Su and R1 are as defined in claim 8, the left side of the L2 group is connected to A1A2A3A4, and the right side is connected to -L3-D; and L3 is absent; (b) L2 does not exist; L3 is selected from... Wherein R2, R3, and X4 are each independently defined as in claim 10, wherein the left side of the L3 group is connected to -Z-L1-A1A2A3A4-L2- and the right side is connected to -D; and / or (c)L2 is selected from The symbols are defined as above; L3 is... The symbols are defined as above; Preferably, -L2-L3- is selected from the group consisting of: (a) L2 is either non-existent or selected from Wherein X and Su are defined as in claim 8, the left side of the L2 group is connected to A1A2A3A4, and the right side is connected to -L3-D; and L3 is absent; (b) L2 does not exist; and L3 is selected from... The L3 group shown is connected to -Z-L1-A1A2A3A4-L2- on the left and to -D on the right; and / or (c)-L2-L3-Selected from Wherein X, X4, R1, R2 and R3 are as defined in any one of claims 8-12; Preferably, -L2-L3- is absent or selected from The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate, as described in any one of claims 1-11 in, The connector unit Z is represented by the formula -Z1-Z2-, where Z1 is connected to the target portion (Tg); Z1 is selected from the following groups: The ends marked with an asterisk are covalently connected to the target portion (Tg), for example, to the sulfur atom of the target portion (Tg); the ends marked with a wavy line are covalently connected to Z2, or to L1 when Z2 is absent. Each of X1 is independently selected from C 1-6 Alkyl, halogen, and nitro groups; r is 0, 1, 2, or 3; Alternatively, Z1 can be selected from the following groups: The ends marked with an asterisk (*) are covalently connected to the target moiety (Tg), for example, to the primary or secondary amino group or hydroxyl group of the target moiety (Tg), and are marked with a wavy line. The end of is covalently connected to Z2, or covalently connected to L1 when Z2 does not exist; Alternatively, Z1 can be selected from the following groups: The ends marked with an asterisk (*) are covalently linked to the target region (Tg), such as to a sugar or a non-natural amino acid in the target region, and are indicated by a wavy line. The end of is covalently connected to Z2 or L1 (when Z2 does not exist); Each of X1 is independently selected from C 1-6 Alkyl, C 1-6 Alkoxy, halogen, and nitro; r is 0, 1, 2, or 3; Alternatively, Z1 can be selected from the following groups: The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), such as the -C (=O)- on the target portion (Tg), and are marked with a wavy line. The end of is covalently connected to Z2, or covalently connected to L1 when Z2 does not exist; in, Z2 does not exist, or can be selected from: -C1-C 10 Alkylene -, -C2-C 10 alkenyl-, -C2-C 10 Ethyne-, -C1-C 10 Heteroalkyl-, -C3-C8 carbocycloalkyl-, -O-(C1-C8 alkylene)-, -C6-C 10 Aromatic-, -C1-C 10 Alkylene-C6-C 10 Aspartic-, -C6-C 10 Aspartic-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-, -(C3-C8 carbocyclic)-C1-C 10 alkylene-, -C3-C8 heterocyclic-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-, -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-NH-, C1-C 10 Heteroalkyl-NH-, -C3-C8 carbocycloalkyl-NH-, -O-(C1-C8 alkylene)-NH-, -C6-C 10 aryl-NH-, -C1-C 10 Alkylene-C6-C 10 aryl-NH-,-C6-C 10 Aspartic-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-NH-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclic-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-NH-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene -NH-, -C1-C 10 Alkylene -S-, -C1-C 10 Heteroalkyl-S-, -C3-C8 carbocycloalkyl-S-, -O-(C1-C8 alkylene)-S-, -C6-C 10 Aromatic-S-,-C1-C 10 Alkylene-C6-C 10 Aromatic-S-,-C6-C 10 Aspartic-C1-C 10 Alkylene -S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-S-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclic-S-, -C1-C 10 alkylene-(C3-C8 heterocyclic)-S- or -(C3-C8 heterocyclic)-C1-C 10 Alkylene-S-; The Z2 group is optionally replaced by one or more Bu groups, wherein the Bu groups are selected from: H, deuterium, halogen, nitro, -CN, -G1, -OR. a and -(CH2) x N(R a )2; where x is 0, 1, 2, 3, 4, 5, or 6; R a Independently H or -C optionally substituted with halogen 1-6 Alkyl groups, or two R atoms attached to the same N atom. a The groups and the nitrogen atoms they are attached to form 4-7 membered heterocyclic groups, such as aza-heterocyclic butyl, pyrrolidinyl, or piperidinyl groups; wherein -G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers; The Z2 group is optionally connected to -NH-(CH2CH2O) at its left and / or right ends. n1 - and / or -(CH2CH2O) n1 - or optionally, insert the group -NH-(CH2CH2O) at its intermediate position. n1 - and / or -(CH2CH2O) n1 -,and n1 is an integer selected independently from 0 to 30, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30; The left side of the Z2 group is connected to the Z1 group. The drug conjugate of claim 12, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, Z1 is selected from: The asterisk and wavy line have the meanings described in claim 6; Preferably, Z1 is selected from: Preferably, Z1 is The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate, as described in any one of claims 12-13 Z2 is selected from: -C1-C 10 Alkylene C(=O)-, -C2-C 10 alkenyl C(=O)-, -C2-C 10 Ethyne C(=O)-, -C1-C 10 Heteroalkyl-C(=O)-, -C3-C8 carbocycloalkyl-C(=O)-, -O-(C1-C8 alkylene)-C(=O)-, -C6-C 10 aryl-C(=O)-, -C1-C 10 Alkylene-C6-C 10 aryl-C(=O)-, -C6-C 10 Aspartic-C1-C 10 Alkylene -C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic)-C(=O)-, -(C3-C8 carbocyclic)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclic-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic)-C(=O)-, -(C3-C8 heterocyclic)-C1-C 10 Alkylene-C(=O)-; Preferably, the Z2 group is optionally replaced by one Bu group, wherein the Bu group is selected from halogens and -(CH2). x N(R a )2; where x is 0, 1, 2, 3, 4, 5, or 6; R a Independently H or -C optionally substituted with halogen 1-6 alkyl; Preferably, the Bu group is located on the carbon atom of the Z2 group that is connected to the Z1 group; Alternatively, Z2 does not exist. The drug conjugate, or stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to any one of claims 12-14, Where -Z1-Z2- has the following structure: Each of n2 is an integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2 or 5; The ends marked with an asterisk (*) are covalently connected to the target portion (Tg), and a wavy line is expected. The end of it is covalently linked to L1, or covalently linked to a tetrapeptide unit when L1 is absent. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate, as described in any one of claims 1-15 in, L1 is absent or selected from the following groups: Where R b Selected from -C 1-10 alkylene-, -C 6-10 Alpha- and -C 1-10 Heteroalkyl-, -C 3-8 heterocyclic group -, -C 1-10 Alkylene-C 6-10 Alpha- and -C 6-10 Aspartic-C 1-10 alkylene-, -C 1-10 Alkylene-C 6-10 Aspartic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 subcarbocyclic -, -C 3-8 subcarbocyclic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 subcarbocyclic-C 1-10 alkylene-, -C 1-10 Alkylene-C 3-8 heterocyclic group -, -C 3-8 Heterocyclic-C 1-10 alkylene- and -C 1-10 Alkylene-C 3-8 Heterocyclic-C 1-10 alkylene-; R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-; n3 is independently selected from 0, 1, 2, 3 or 4; G2 is independently selected from: -O-, -S-, -NH-, -NH(CH2CH2O) n1 -、-(CH2CH2O) n1 - n1 are each an integer selected from 0 to 30, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28 or 30; An asterisk (*) indicates a connection to a precursor (Z') or a connector unit (Z), while a wavy line indicates a connection to a tetrapeptide unit. G3 is selected independently from: Where R represents an amino acid side chain, for example, the amino acid is selected from alanine (Ala), aspartic acid (Asp), glycine (Gly), glutamic acid (Glu), glutamine (Gln), phenylalanine (Phe), proline (Pro), and citrulline (Cit); where the asterisk (*) indicates that it is related to R. b Connection; G3 is preferably GGFG (SEQ ID NO:30); G1 is selected from polyethylene glycol (PEG) units, hydrophilic peptides, cyclodextrin units, polyamides, polysaccharides, and dendritic polymers; G4 is selected from non-existent -(CH2). n9 C(O)- and -NH-(CH2) n9 O(CH2) n9 C(O)-; and n8 and n9 are each independently selected from integers from 0 to 9, such as 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; G5 is selected from -S(O)-, -S(O)2-, The left side of the G4 group is connected to G3; Preferably, L1 is absent or selected from the following groups: -C(=O)-C 3-8 Carbocyclic groups -C(=O)-, -C(=O)-C 3-8 Heterocyclic groups -C(=O)-, -C(=O)-C 1-10 Alkylene -C(=O)-, -C(=O)-C 3-8 Heterocyclic-C 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-(CH2CH2O) n1 -C 1- 10 Alkylene -C(=O)-, -C 1-10 Alkylene -OC(=O)-, -C(=O)-C 1-10 Alkylene-OC 1-10 Alkylene -C(=O)-, -N(R) c )-C 1-10 Alkylene-S(O)2-, -N(R) c )-C 1-10 Alkylene-HP(O)4-, -N(R) c )-C 1-10 Alkylene-SC 1-10 Alkylene -C(=O)-, -SC 1-10 Alkylene-C(=O)-, Each of n is an integer selected from 0 to 9, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n1 is an integer independently selected from 0 to 30, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 28, or 30; and R c Each is independently selected from H and C. 1-6 Alkyl group, preferably H or C 1-3 alkyl-; Preferably, L1 is selected from any of the following groups: Where n10 and n11 are independently selected from 0, 1, 2 and 3, and n10 and n11 are not both 0; The remaining symbols are as defined above; Preferably, L1 is selected from any one of the following groups: The symbols, such as n, n1, n8, and R, are defined as above; Preferably, L1 is selected from any one of the following groups: Where n, n1, n8 and R are defined as above; Preferably, L1 is absent or selected from Where n and n1 are defined as above; Preferably, L1 is absent or selected from Preferably, L1 does not exist. The drug conjugate of claim 1, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, Where -Z-L1-A1A2A3A4-L2-L3- is selected from: The bonds marked with an asterisk (*) are connected to the target portion Tg, and the bonds marked with a wavy line are connected to D. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate, as described in any one of claims 1-17 in, D is selected from cytotoxic agents, STING activators, glucocorticoid compounds, radioactive nuclides, and siRNA; Optionally, D is selected from topoisomerase I inhibitors, topoisomerase II inhibitors, glycopeptide antibiotics, DNA synthesis interfering agents, tubulin inhibitors, anti-apoptotic agents, mitotic inhibitors, antitumor antibiotics, immunomodulators, alkylating agents, anti-angiogenic agents, antimetabolites, corticosteroids, photosensitizing agents, oligonucleotides, radionuclides, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, TLR7 / 8 agonists, PNU, Bcl-xl inhibitors, kinase inhibitors, transcription inhibitors (e.g., reverse transcriptase inhibitors), proteasome inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, protein degraders, glucocorticoid receptor modulators, and other small molecule compounds that inhibit tumor growth. Preferably, D is selected from camptothecin compounds, auristatin compounds, vinca alkaloids, taxane compounds, podophyllotoxin compounds, maytansine compounds, and benzo[a]benzodiazepines (PBDs). Preferably, D is selected from: Dxd, Exatecan, SN38, MMAE, MMAF, DM1, DM4, eribulin, tubulysin, duocamycin, thailanstatin A, and amanitin. The drug conjugate of claim 18, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, Where D has the structure shown in equation (D-1a), equation (D-1b), or equation (D-1c): Where R 1a Selected from H and C1-C6 alkyl-; R 2a Selected from H, halogen, C1-C6 alkyl-, C1-C6 haloalkyl-, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl-; or Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl group and COOH; preferably C 1-4 Alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H; or Where R 1c Selected from H, halogen, C1-C6 alkyl-, C1-C6 haloalkyl-, -OR 2c and -SR 2c ;and R 2c Independently selected from H and C1-C4 alkyl-. The drug conjugate of claim 19, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, Where R 1a For H; R 2a C1-C6 alkyl-; R 3a It is a halogen, preferably -F; R 4a It is C1-C4 alkyl-, preferably ethyl; R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl-; R 6b and R 7b Each was independently selected from C 1-2 alkoxy-; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H; and R 1c It is a C1-C6 alkyl-, preferably ethyl. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to claim 19 or 20, Where D has the structure shown in equation (D-2a) or equation (D-2b): Where R 1a R 2a R 3a and R 4a As defined in claim 13 or 14; or Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in claim 19 or 20; and Where R 1c As defined in claim 19 or 20. The drug conjugate of claim 18, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, in, D is selected from the structure shown below: The drug conjugate of claim 1, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, wherein the drug conjugate is selected from... Wherein Tg and q are as defined in claim 1. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate according to any one of claims 1-23, in, Tg is an antigen-binding molecule; Preferably, Tg is an antibody or its antigen-binding fragment; Preferably, Tg is a full-length antibody or its antigen-binding fragment; Preferably, Tg is a monoclonal antibody or its antigen-binding fragment; Preferably, Tg is a multispecific antibody, such as a bispecific antibody or its antigen-binding fragment; Preferably, Tg is an antibody or antigen-binding fragment thereof that targets tumor-specific antigens or tumor-associated antigens; Preferably, the target Tg is an antibody or its antigen-binding fragment that targets one or more targets or antigens selected from the following: Claudin18.2 (CLDN18.2), CDH17 (intestinal adhesion molecule 1), FOLR1, MSLN, B7H1, B7H3, B7H4, Her1, Her2, Her3, Her4, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET, c-Met, EGFRvIII, KIT, CTLA-4, GCPII, IL-13Ra, BCMA, GD2, 5T4, VEGFR1, VEGFR2, VEGFR3, AOC3, CA-125, FGFR1, FGFR2, FGFR3, FGFR4, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD16, CD18, CD19, CD20, CD21, CD22, CD25, CD30, CD32, CD33, CD37, CD38, CD40, CD44, CD45 (PTPRC), CD52, CD56, CD64, CD66e, CD70, CD72, CD74, CD79a, CD79b, CD80, CD123, CD138, CD142, CD174, CD223, CD276, CDH3, CCD79b, CLDN9 / CLDN6, IGF1R, IGF2R PDGFR-α, PDGFR-β, ENPP3, CA19-9, DPEP3, AGS-16, FcRH5, FRα, KAAG1, RPR1, CSF1R, EphA2, Mesothelin, ROR1, SLTRK6, TF, BMPR1B, E16, CLL-1, CA-IX, Somatostatin receptor, RANK, MUC1, TOP1, NCA, MDP, IL20R-α, Brevican, STEAP1, Sema 5b, PSCA hIg, ETBR, RNF124, TrpM4, C3DR, FcRH2, EphB2R, ASLG659, GEDA, BAFF-R, DLL4, EpCAM, FAP, CXCR5, HLA-DOB, P2X5, LY64, Mucin1. FcRH1, IRTA2, TENB2, FGF2, ALK, AXL, C242, PSMA, O772P, MUC16, Napi3b, SLAMF7, ITGB6, CEACAM5, CA9, EGFRvlll, IL2RA, CCL5, CXCL10, CXCL11, IFI6, TGF-βR, TNFRSF8, CLEC14A, GRP78, ASG-5, PRR4, GUCY2C, SLC39A6, endothelin receptor, LIV-1, integrin α5β6, integrin α4β7, TPBG, CA242, FOLR1, GPNMB, HAVCR1, VTCN1, PTK7, TACSTD2, CA6, DLL3, DKK-1, Endoglin, VCAM1, GPC3, DR5, and ASCT2. Preferably, Tg is an antibody or its antigen-binding fragment that targets the following targets or antigens: CLDN18.2, CDH17, FORR1, MSLN, B7H1, B7H3, B7H4, Her1, Her2, Her3, Her4, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET and / or c-Met; Preferably, Tg is an antibody or its antigen-binding fragment that targets the following targets or antigens: CLDN18.2, CDH17, FORR1, MSLN, B7H3, Her2, Her3, TROP2, Nectin4, EGFR, PD-L1, PD-1, MET and / or c-Met; Preferably, Tg is an anti-HER2 antibody or its antigen-binding fragment; the anti-HER2 antibody is, for example, trastuzumab or pertuzumab; Preferably, Tg is an anti-TROP2 antibody or its antigen-binding fragment; the anti-TROP2 antibody is, for example, Sacituzumab; Preferably, Tg is an anti-Nectin4 antibody or its antigen-binding fragment; Preferably, Tg is a bispecific antibody against Nectin4 and / or against Trop2 or an antigen-binding fragment thereof; Preferably, Tg is an anti-EGFR and / or c-Met antibody or its antigen-binding fragment; Preferably, Tg is an antibody against CLDN18.2 and / or CDH17 or an antigen-binding fragment thereof; Preferably, Tg is an anti-FOLR1 and / or MSLN antibody or its antigen-binding fragment. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate, according to any one of claims 1-24 Where q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The drug conjugate, or its stereoisomer, isotopic variant, pharmaceutically acceptable salt, or solvate, as described in any one of claims 1-25 The drug conjugate or its stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate has an average DAR of 1-10, for example 2-5, 6-10. Compounds of Formula II or their stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates Z'-L1-A1A2A3A4-L2-L3-D II in Z' is the precursor of the connector unit; and L1, A1A2A3A4, L2, L3, and D are as defined in any one of claims 1-23; Preferably, Z' has the structure Z1'-Z2-, wherein Z2 is as defined in any one of claims 1-23, and preferably, Z1' is selected from the following groups: Each of X1 is independently selected from C 1-6 Alkyl, halogen, and nitro groups; r is 0, 1, 2, or 3; X2 is independently selected from Cl, Br, and I; and X3 is independently selected from Cl, Br, I, and C. 1-6 Alkyl SO2-, preferably MeSO2-; It contains wavy lines The end of it is covalently connected to Z2, and when Z2 does not exist, it is covalently connected to L1; Preferably, Z1' is selected from: X2 is independently selected from Cl, Br, and I; and X3 is independently selected from Cl, Br, I and MeSO2-; Preferably, Z1' is selected from: X2 is selected from Cl, Br, and I; Preferably, Z1' is The compound of formula II as claimed in claim 27, or its stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates thereof, The compound of formula II is selected from Compound of formula III or its salt: P1-A 1b A 2b A 3b A 4b -P4 III in P1 is selected from H- and amino protecting groups; P4 is selected from -OH, leaving group, and -OR. 3d -NR 4d R 5d , R 3d Selected from C 1-20 Alkyl-, 6-10 aryl-, 3-10 heteroaryl- and 3-10 cycloalkyl-; R 4d R 5d Independently selected from H and C 1-20 Alkyl-, 6-10 aryl-, 3-10 heteroaryl- and 3-10 cycloalkyl-; Where X, R1, Su, r1, and t are as defined in the text for L2; Where -A 1b A 2b A 3b A 4b -As defined above for the tetrapeptide unit -A1A2A3A4-, and the amino acids constituting the tetrapeptide unit (A 1b A 2b A 3b and / or A 4b )Optionally protected by the protective base; P1 is located at the N-terminus of the peptide, and P4 is located at the C-terminus of the peptide. Preferably, the amino protecting group is selected from: Boc, Fmoc, acetyl, methoxyacetyl, trifluoroacetyl, trichloroacetyl, neopentanoyl, formyl or benzoyl, more preferably acetyl; Preferably, the leaving group is a detectable leaving group, and more preferably a fluorescent leaving group; more preferably, the fluorescent leaving group is selected from 7-amino-4-carbamoylmethyl-coumarin and 7-amino-4-methylcoumarin. The compound of formula III or a salt thereof according to claim 29, wherein the compound of formula III is selected from: in, P2 is independently selected from H and guanidinium protecting groups, for example, selected from H and Pbf; P3 is independently selected from H and amide protecting groups, for example, selected from H and Tlt; P5 groups are each independently selected from HO- and carboxyl protecting groups; for example, selected from HO- and C-. 1-8 Alkyl-O-, for example t-BuO; P6 is independently selected from H and hydroxyl protecting groups, for example, selected from H and C. 1-8 Alkyl-O-, for example t-Bu; and P1 and P4 are each independently defined as in claim 29. The compound of formula III of claim 29 or a salt thereof, wherein the compound of formula III is selected from... A pharmaceutical composition comprising the pharmaceutical conjugate of claims 1-26, or a stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate thereof, or the compound of formula II of claims 27-28, or a stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate thereof, and optionally a pharmaceutically acceptable excipient. A pharmaceutical combination comprising the pharmaceutical conjugate of claims 1-26, or a stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate thereof, or the compound of formula II of claims 27-28, or a stereoisomer, isotope variant, pharmaceutically acceptable salt or solvate thereof, and other therapeutic agents. Use of the drug conjugates of claims 1-26, or their stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, or the compounds of formula II of claims 27-28, or their stereoisomers, isotopic variants, pharmaceutically acceptable salts or solvates, or the pharmaceutical compositions of claim 32 in the preparation of medicaments for treating and / or preventing diseases or conditions; Preferably, the disease or symptom is a proliferative disease, such as a tumor; Preferably, the tumor is selected from: lung cancer (e.g., squamous cell carcinoma, adenocarcinoma, non-small cell lung cancer, or small cell lung cancer), breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, myeloma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, bone cancer, prostate cancer, esophageal cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, and renal cell carcinoma. Liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, bladder cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma; lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma; Preferably, the tumor is selected from: lung cancer (e.g., squamous cell carcinoma, adenocarcinoma, non-small cell lung cancer, or small cell lung cancer), breast cancer, gastric cancer, ovarian cancer, pancreatic cancer, and myeloma.

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