Oligosaccharide linker, linker-payload containing oligosaccharide linker, and antibody-drug conjugate with remodeled sugar chains, method for producing the same and use

Abstract

The present invention relates to oligosaccharide groups, particularly linker-payload compounds containing disaccharide groups, wherein the oligosaccharide groups are linked to other parts of the compound via amide bonds. The present invention further relates to an antibody-drug conjugate (ADC) containing the linker-payload compound, and uses the oligosaccharide group in the linker-payload compound to remodel the sugar chain of the antibody. The present invention further relates to methods for producing and using the above-described substances.

Description

[Technical Field]

[0001] The present invention relates to oligosaccharide (particularly disaccharide) linkers. Furthermore, it relates to linker-payload compounds comprising an oligosaccharide group, particularly a disaccharide group, wherein the oligosaccharide group is bonded to other parts of the compound via an amide bond. The present invention further relates to antibody-drug conjugates (ADCs) comprising the linker-payload compound, wherein the oligosaccharide group in the linker-payload compound remodels the sugar chains in the antibody. The present invention further relates to methods for producing and using the above-mentioned substances. [Background technology]

[0002] Cancer is one of the leading causes of death in humans, accounting for approximately one-sixth of all deaths worldwide each year. In 2017, there were 24.5 million new cancer cases and 9.6 million cancer deaths worldwide. Cancer treatment primarily involves surgery, radiation therapy, and drug therapy. With the development and application of these therapies, the survival rates of cancer patients have improved dramatically. Cancer drug therapy has evolved through three generations: chemotherapy, targeted therapy, and immunotherapy. Chemotherapy plays a crucial role in cancer treatment, but it has relatively significant non-therapeutic toxic side effects, killing not only cancer cells but also a large number of normal cells. Targeted therapy mitigates some of the serious toxic side effects of conventional chemotherapy. This therapy primarily uses small molecule targeted drugs (currently mainly tyrosine kinase inhibitors) or monoclonal antibodies to target specific genes or proteins (i.e., target sites) involved in the growth and survival of tumor cells, thereby killing tumor cells and tissues. Antibody-drug conjugates (ADCs) are a novel type of targeted drug, formed by chemically linking a highly active small-molecule drug to a monoclonal antibody. ADCs combine the high activity of small-molecule drugs with the high specificity and targeting capabilities of antibody drugs. They can mitigate some of the non-therapeutic toxic side effects of small-molecule toxins on vital tissues and organs such as the liver, kidneys, nerves, and heart, and overcome the limitations of antibody therapy's therapeutic effect on solid tumors. As a result, ADCs are currently one of the hottest directions in the research and development of anti-tumor drugs.

[0003] The development of ADC drugs involves system engineering that carefully controls four elements: monoclonal antibodies, bioactive small molecules, linkers, and binding methods. Of these, the binding method significantly influences drug-related properties such as efficacy stability, metabolic consistency, and quality control (Non-Patent Literature 1). Currently, commercially available and clinically-stage ADC drugs mainly employ random binding via lysine or cysteine ​​residues, often resulting in random binding sites and highly heterogeneous mixtures with uneven drug / antibody ratios (DARs). This can easily lead to problems in terms of process stability, quality control, drug stability, metabolic consistency, and safety.

[0004] To address the aforementioned problems associated with random binding, since 2008, academia and industry have diligently researched the development and application of site-directed binding strategies, achieving several promising advances. These site-directed binding methods can be broadly categorized into three types: binding through genetic mutation to introduce specific amino acids, binding promoted by enzymes into which polypeptides are inserted, and site-directed binding techniques that promote glycan remodeling by enzymes. The exploration and application of these site-directed binding techniques in ADC drug development have successfully resolved many of the problems arising from random binding. However, the first two site-directed binding techniques typically require genetic engineering or modification of antibodies, lacking versatility in producing ADCs for different target antibodies. Each new antibody requires extensive and complex cell engineering, significantly reducing the efficiency of new drug development. On the other hand, site-directed binding through remodeling of antibody Fc glycans eliminates the need for antibody modification and cell engineering, greatly reducing the difficulty and workload of development and potentially becoming a versatile platform technology for antibody site-directed binding.

[0005] The asparagine at position 297 of the antibody Fc region has a highly preserved glycosylation (N-297Glycan), and site-specific binding of different molecules to the antibody can be achieved through glycosylation remodeling at this site (Non-Patent Literature 2). Site-specific binding techniques using glycosylation modification of the antibody Fc region can be broadly divided into the following two types.

[0006] 1) In the chemical method, core fucose (Non-Patent Literature 3) or o-diol of sialic acid at the sugar group terminus (Non-Patent Literature 4) is oxidized with sodium periodate to obtain the corresponding aldehyde, and the aldehyde carbonyl group can be used to conjugate with a low molecular weight toxin fragment to produce ADC. The limitation of this method is that the structure of the sugar group at position N-297 varies, and not all monoclonal antibodies have a reactable site in their sugar groups, resulting in relatively large substrate limitations.

[0007] 2) In the enzyme-catalyzed method, a catalytic relay of deglycosylation-transglycosylation by tool enzymes such as endoglycosidase and glycosyltransferase enables glycan remodeling and the introduction of bioorthogonal reactive groups, and subsequent chemical reactions enable the production of site-specific ADCs (Non-Patent Literature 2 and Non-Patent Literature 5).

[0008] In 2012, Wang Laixi et al. reported a site-specific binding technique for antibodies via glycosylation remodeling catalyzed by the endoglycosidase Endo S and its variants (Non-Patent Literature 6). This technique utilized the selective endo-deglycosylation activity of wild-type Endo S on the β-1,4-glycosidic bond between GlcNAc-GlcNAc in the N-297 oligosaccharide structure and the transglycosylation activity of the mutant enzyme Endo S D233Q to synthesize glycosylation remodeling antibodies with a single glycan structure modified with an azide group. Building on this work, Wang Laixi and Huang Wei et al. developed a broad category of ADC site-specific binding techniques based on a three-step method of deglycosylation-transglycosylation-click reaction catalyzed by tool enzymes such as endoglycosidase Endo S, Endo S2, and its variants (Non-Patent Literature 5). In 2021, Wang Laixi et al. reported site-specific binding of antibody glycan remodeling catalyzed by a one-pot Endo S2 reaction, introducing bioorthogonal azide functional groups into antibodies and then performing a click reaction to obtain ADC molecules (Non-Patent Literature 7). Currently, all of the above technologies are still in the basic research stage, and there have been no reports yet on the clinical development of ADC drugs based on the corresponding technologies.

[0009] Another commonly used tool enzyme in site-specific binding techniques for glycosylation remodeling is β-1,4-galactosyltransferase (β-1,4-Gal-T1) and its mutant (β-1,4-Gal-T1 Y289L). This enzyme uses uridine diphosphate galactose (Gal-UDP) as a donor to transfer the galactosyl group (Gal) to the non-reducing end of acetylglucosamine (Glc-NAc) in glycoproteins. In 2009, Qasba et al. (Non-Patent Literature 8) were the first to report site-specific antibody binding techniques using β-1,4-Gal-T1 and β-1,4-Gal-T1 Y289L as tool enzymes and C2-keto-Gal-UDP or N-azidoacetylgalactosamine-UDP (GalNAz-UDP) as donors, and produced antibodies modified with ketone carbonyl or azide groups. Based on this, in 2014 they reported the first method for synthesizing ADC molecules based on a three-step deglycosylation-transglycosylation-biorthogonal reaction using β-1,4-Gal-T1 and its variants as catalysts. It should be noted that the corresponding ADC molecules retained a considerable affinity for FcγRIIIa and FcγRI receptors, and their antibody-dependent cytotoxicity (ADCC) effect to some extent. The molecules showed potential killing power in the Her2-positive JIMT-1 breast cancer cell line (Non-Patent Literature 9). Building on the above work, Synaffix developed a three-step synthesis method for ADCs via deglycosylation-transglycosylation-biorthogonal reaction using a combination of the endoglycosidase Endo S and the tool enzyme β-1,4-Gal-T1 Y289L (Non-Patent Literature 10). Furthermore, the literature reports a four-step synthesis method for ADCs involving deglycosylation-transglycosylation-transglycosylation-biorthogonal reaction (Non-Patent Literature 11), which uses a total of three tool enzymes: β-1,4-Gal-T1, β-1,4-Gal-T1 Y289L, and sialic acid transferase.

[0010] While the above-described glycosylation remodeling reaction avoids the problem of needing to genetically modify antibodies in other site-specific binding technologies, it still has significant limitations. On the one hand, the binding process is very long and complicated, requiring at least two enzyme-catalyzed reactions plus one chemical reaction, i.e., at least three reactions and three complete purifications, before ADCs can be obtained. On the other hand, even trace amounts of tool enzymes remaining in the product can cause the ADC product to deglycosylate and degrade, releasing the toxin molecule and potentially causing a serious toxic reaction, posing a major challenge to drug research and development and production, and posing a significant risk to the safety of ADC drugs. More importantly, in the above-described glycosylation remodeling reaction, the methods for linking sugar groups and toxin molecules remain limited, and additional reactions may be needed to better suit the demand, and these additional reactions can also produce many undesirable byproducts. The objective of the present invention is to solve these problems. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Tsuchikama K,An Z.Antibody-drug conjugates:recent advances in conjugation and linker chemistries.Protein Cell 2018,9,33-46 [Non-Patent Document 2] Wang LX,Tong X,Li C.Glycoengineering of Antibcdies for Modulating Functions.Annu Rev Biochem2019,88,433-459 [Non-Patent Document 3] Zuberbuhler K,Casi G,Bernardes GJ,et al.Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format.Chem Commun 2012,48,7100-7102 [Non-Patent Document 4] Zhou Q,Stefano JE,Manning C,et al.Site-specific antibody-drug conjugation through glycoengineering.Bioconjugate Chem 2014,25,510-520 [Non-Patent Document 5] Zeng Y,Tang F,Shi W,et al.Recent advances in synthetic glycoengineering for biological applications.Current Opinion in Biotechnol.2022,74,247-255 [Non-Patent Document 6] Huang W, Giddens J, Fan SQ, et al.Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions.J.Am.Chem.Soc.2012,134,12308 [Non-Patent Document 7] Zhang X,Ou C,Liu H,et al.General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies:facile synthesis of homogeneous antibody-drug conjugates.ACS Chem.Biol.2021,16,11,2502-2514 [Non-Patent Document 8] Boeggeman E, Ramakrishnan B, Pasek M, et al. Site specific coniugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjugate Chem. 2009, 20, 6, 1228 - 1236

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

[0012] The present invention provides novel oligosaccharide (particularly disaccharide) linkers, as well as methods for producing and using the same. Furthermore, it provides linker-payload compounds containing oligosaccharide groups, where the oligosaccharide groups are linked to other parts of the compound via amide bonds. Specifically, the present invention provides a linker-payload compound having formula (I): [ka] During the ceremony, P is the payload; DC(O)-L- is a linker; DC(O)- is an oligosaccharide structure, consisting of -a first hexose group or its derivative moiety- (a second hexose group or its derivative moiety) f -β-DN-acetylglucosamine moiety, or -first hexose group or its derivative moiety- (second hexose group or its derivative moiety) f -β-D-glucose oxazoline moiety, the 6th carbon of the first hexose group or its derivative moiety exhibits the form -C(O)-, which is -C(O)- in DC(O)-, and the β-DN-acetylglucosamine moiety is [ka] The β-D-glucose oxazoline portion is [ka] And f is 0, 1, 2, 3, 4, 5, or 6, L is a linker end that can release P by severing its linkage with P, for example, by a chemical method (e.g., hydrolysis) or a biological method (e.g., enzyme-catalyzed method), and L is directly linked to the carbonyl group in DC(O)- via its -NH-, and if L is an unbranched linker end, L is linked to one P and t is 1, and if L is a branched linker end, each branch may be linked to one P and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0013] The present invention further relates to an antibody-drug conjugate (ADC) comprising the linker-payload compound, wherein the oligosaccharide group in the linker-payload compound is used to remodel the glycans of the antibody. Specifically, the present invention provides an antibody-drug conjugate having formula (II) through site-specific binding of the N-glycosylation site of the antibody Fc region: [ka] During the ceremony, P is the payload; R is either hydrogen or an α-L-fucosyl group; q is either 1 or 2; Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II) is derived from the asparagine at position 297 of the antibody Fc region), Of these, the carbon at position 6 of the first hexose group or its derivative portion exhibits the form -C(O)-; L is a linker end (which can be broken by chemical methods (e.g., hydrolysis) or biological methods (e.g., enzyme-catalyzed methods) to release P), and L is directly linked to the carbonyl group in the first hexose group or its derivative moiety via its -NH-, and if L is an unbranched linker end, L is linked to one P and t is 1, and if L is a branched linker end, each branch may be linked to one P and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10); f is 0, 1, 2, 3, 4, 5, or 6.

[0014] The present invention further relates to a method for producing and using the above-mentioned substance.

[0015] This invention expands the range of ADCs obtainable by glycosylation remodeling techniques by using a novel oligosaccharide structure that forms an amide bond. Under expected conditions, compounds having NH2 (e.g., L'-(P) t Compounds having the above structure (where P and t in the formula are as defined in the text, and L' is the same as L as defined in the text, except that the -NH- linked to DC(O)- in L is H2N- in L') all have a linker-payload containing an oligosaccharide linker formed by a simple amide reaction via the above oligosaccharide structure. [ka] It forms an antibody and an ADC, and [ka] The binding of the antibody to the target cell can be performed by a one-pot enzyme catalysis. Furthermore, the ADC having a novel oligosaccharide structure that forms an amide bond according to the present invention can be efficiently delivered to target cells and the payload can be efficiently released in the target cells. In the present invention, a unique oligosaccharide carboxylic acid substrate (for example, [ka] This method successfully achieves linkage with amine compounds in a single step using a mild and well-suited amide bond formation method, eliminating the need for additional reactions that often result in numerous undesirable byproducts, thereby simplifying and accelerating the synthesis of linker-payload compounds. [Brief explanation of the drawing]

[0016] [Figure 1] The SDS-PAGE measurement analysis of ADC-1 is shown. [Figure 2] The HIC-HPLC measurement and analysis of ADC-1 is shown. [Figure 3] The SEC-HPLC measurement and analysis of ADC-1 is shown. [Figure 4] This shows the measurement of ADC-1's affinity for cell surface ErbB2 / HER2 (EC50, nM). [Figure 5] ADC-1 and other different drugs exhibit inhibitory effects (IC50, nM) on the proliferation of tumor cells BT474. [Figure 6] ADC-1 and other different drugs exhibit inhibitory effects (IC50, nM) on the proliferation of tumor cells NCI-N87. [Figure 7] ADC-1 and other different drugs exhibit inhibitory effects (IC50, nM) on tumor cell HepG2 proliferation. [Figure 8] This demonstrates the inhibitory effect of ADC-1 on the NCI-N87 CDX mouse model. [Figure 9] The SDS-PAGE measurement analysis of ADC-2 is shown. [Figure 10] The HIC spectrum of ADC-2 is shown. [Figure 11] The SEC spectrum of ADC-2 is shown. [Figure 12] ADC-2 and different drugs exhibit inhibitory effects (IC50, nM) on tumor cell SK-BR-3 proliferation. [Figure 13] ADC-2 and different drugs exhibit inhibitory effects (IC50, nM) on the proliferation of tumor cells HCC1954. [Figure 14] ADC-2 and different drugs exhibit inhibitory effects (IC50, nM) on the proliferation of tumor cells MDA-MB-468. [Figure 15] The SEC-HPLC measurement analysis of ADC-3 is shown. [Figure 16] The high-resolution mass spectrometry deconvolution diagram of ADC-3 is shown. [Figure 17] ADC-3 and different drugs exhibit inhibitory effects (IC50, nM) on tumor cell BxPC-3 proliferation. [Figure 18]ADC-3 and different drugs exhibit inhibitory effects (IC50, nM) on tumor cell FaDu proliferation. [Figure 19] ADC-3 and different drugs exhibit inhibitory effects (IC50, nM) on tumor cell HepG2 proliferation. [Figure 20] This study demonstrates the tumor-suppressing effect of ADC-3 on NCI-N87 CDX mice. [Figure 21] This shows the effect of ADC-3 on body weight in NCI-N87 CDX mice. [Figure 22] The HIC-HPLC measurement analysis diagram for ADC-4 is shown. [Figure 23] The SEC-HPLC measurement analysis diagram for ADC-4 is shown. [Figure 24] The HIC-HPLC measurement analysis of ADC-5 is shown. [Figure 25] The SEC-HPLC measurement analysis of ADC-5 is shown. [Figure 26] ADC-4 and ADC-5 demonstrate in vitro inhibitory activity against BxPC-3. [Figure 27] ADC-4 and ADC-5 demonstrate in vitro inhibitory activity against FaDu. [Figure 28] The in vitro inhibitory activity of ADC-4 and ADC-5 against HepG2 was demonstrated. [Figure 29] The HIC-HPLC measurement analysis of ADC-6 is shown. [Figure 30] The SEC-HPLC measurement analysis of ADC-6 is shown. [Figure 31] This study demonstrates the in vitro inhibitory activity of ADC-6 against SK-BR-3. [Figure 32] This study demonstrates the in vitro inhibitory activity of ADC-6 against NCI-N87. [Modes for carrying out the invention]

[0017] general definition Unless otherwise defined later, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. "Technology" as used herein refers to techniques commonly understood in the art, including obvious modifications and equivalent substitutions. While the following terms are expected to be readily understood by those skilled in the art, their definitions are provided below for the purpose of better illustrating the present invention. Where a trade name is mentioned herein, it refers to the corresponding product or its active ingredient. All patents, disclosed patent applications, and publications cited herein are incorporated herein by reference.

[0018] When a quantity, concentration, or other numerical value or parameter is described in terms of a range, preferred range, preferred upper limit, or preferred lower limit, any range formed by combining any upper limit or preferred value with any lower limit or preferred value should be understood as equivalent to a specifically disclosed range, regardless of whether such range is explicitly stated or not. Unless otherwise stated, numerical ranges mentioned herein include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the expression "i is an integer between 1 and 20" means that i is any integer between 1 and 20, for example, i could be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions, such as j, g, k, etc., should be understood in a similar manner.

[0019] Unless otherwise explicitly stated in the preamble or concluding clause, singular forms such as "one type (item)" and "the said type (item)" also include plural forms. Expressions such as "one type (item) or more types (items)" or "at least one type (item)" refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0020] When the terms "approximately" and "about" are used with numerical variables, it generally means that the value of the variable, and all values ​​of the variable, are within the range of experimental error (e.g., within the 95% confidence interval of the mean), or within a wide range of ±10% or more of the specified value.

[0021] The term "optional / may" means that such circumstances may occur, but do not necessarily occur, and includes both cases where such circumstances or situations occur and cases where they do not.

[0022] Expressions such as “include,” “contain,” “contain,” and “have” are open language and do not exclude other elements, processes, or components not mentioned. The expression “composed of…” does not include elements, processes, or components not specified. The expression “basically composed of…” means that its scope is limited to the specified elements, processes, or components, and any elements, processes, or components that may be present but do not substantially affect the basis and novel features of the subject for protection. The expression “includes” should be understood to include expressions such as “basically composed of…” and “composed of…”.

[0023] The term "targeting molecule" refers to a molecule that has affinity for a specific target (e.g., receptors, cell surface proteins, cytokines, tumor-specific antigens, etc.). Targeting molecules can deliver payloads to specific sites in vivo through targeted delivery. Targeting molecules can identify one or more targets. A specific target site is defined by the target identified by the targeting molecule. For example, a targeting molecule that targets receptors can deliver cytotoxins to sites containing many receptors. Examples of targeting molecules include, but are not limited to, antibodies, antigen-binding proteins, antibody mimetic compounds, scaffold proteins with affinity for specific targets, and ligands. The targets identified by the targeting molecules are CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, and EGFRv This includes, but is not limited to, III, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin6, FGFR2, FGFR3, CA6, CanAg, IntegrinαV, TDGF1, EphrinA4, TROP2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, TF (Tissue Factor), and ROR1 / 2.

[0024] HER2 refers to the human epidermal growth factor receptor-2, and belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0025] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. TROP2 is a type of intracellular calcium signaling sensor and is overexpressed in multiple types of tumors.

[0026] CLDN18.2 (Claudin-18 type 2) is a member of the human claudin family. CLDN18.2 is a pan-cancer target expressed in primary and metastatic lesions of multiple human cancer types.

[0027] As used in this text, the term “antibody” is used in a broad sense, and its definition includes general antibodies, recombinant antibodies / genetically modified antibodies, and in particular, complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they possess the required biological activity. Antibodies may be any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass, and may originate from any appropriate species. In some embodiments, antibodies are derived from humans or mice. Antibodies may be fully human antibodies, humanized antibodies, or chimeric antibodies produced by recombinant methods.

[0028] The monoclonal antibodies used in this text are antibodies obtained from a fundamentally homogeneous antibody population; that is, with the exception of a few possible spontaneous mutations, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific to a single antigen site. The term "monoclonal" means that the characteristics of the antibody originate from a fundamentally homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced by some specific method.

[0029] A complete or full-length antibody essentially consists of an antigen-binding variable region, a light chain constant region (CL), and a heavy chain constant region (CH), which may include CH1, CH2, CH3, and CH4 depending on the antibody subtype. The antigen-binding variable region (also called a fragment variable region or Fv fragment) typically contains a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region with a native sequence (e.g., a constant region with a human native sequence) or a variant of the amino acid sequence. The variable region identifies and interacts with the target antigen. The constant region can be identified and interacted with by the immune system.

[0030] Antibody fragments may include a portion of a complete antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments composed of VH and CH1 domains, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting full-length immunoglobulin with papain, or fragments with the same structure produced, for example, by recombinant expression. Fab fragments include a light chain (containing VL and CL) and another chain, the other chain containing a variable region (VH) of the heavy chain and one constant region (CH1) of the heavy chain. F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulin with pepsin at pH 4.0-4.5, or fragments with the same structure produced, for example, by recombinant expression. An F(ab')2 fragment essentially consists of two Fab fragments, of which each heavy chain portion contains several extra amino acids and a cysteine ​​that forms a disulfide bond linking the two fragments. A Fab' fragment is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain). The antibody fragment may also contain multiple chains linked by, for example, disulfide bonds and / or peptide linker ends. Examples of antibody fragments further include single-chain Fv(scFv), Fv, dsFv, bispecific antibodies, Fd and Fd' fragments, and other fragments including modified fragments. Antibody fragments typically contain at least 50 or about 50 amino acids, and typically at least 200 or about 200 amino acids. Antigen-binding fragments may include fragments of any antibody from which an immunospecific binding antigen is obtained when inserted into the antibody framework (for example, by substitution of a corresponding region).

[0031] In particular, the antibody-drug conjugate of this invention performs site-specific binding based on any site containing a natural N-glycosylation modification in the antibody FC region. Any molecule containing an antibody FC region containing a sugar chain (including, but not limited to, antibodies, bispecific antibodies, FC fusion proteins, single-chain antibodies, nanoantibodies, etc.) is manufactured in a one-step method using the linker-payload containing the oligosaccharide of this invention. Therefore, the antibody of this invention is not particularly limited; it may be a natural antibody as long as its FC region contains a sugar chain.

[0032] Furthermore, the antibodies of the present invention may also be manufactured using technologies well known in the industry, such as recombinant / genetic engineering, phage display, synthesis, or other technologies known in the industry, or combinations thereof. For example, recombinant antibodies produced by genetic engineering can be expressed in a suitable culture system (e.g., Escherichia coli (E. coli) or mammalian cells). The genetic engineering may involve, for example, introducing a ligase-specific identification sequence to its terminal.

[0033] As used in this text, the term "targeting molecule-drug conjugate" will be referred to simply as "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0034] Low molecular weight compounds are molecules whose size is comparable to that of organic molecules commonly used in drugs. This term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.), but it does include low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of low molecular weight compounds may be, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0035] Cytotoxins are substances that inhibit or prevent the expression activity or function of cells, and / or cause cell destruction. Currently, cytotoxins commonly used in ADCs are more toxic than chemotherapeutic drugs. Examples of cytotoxins include drugs that target, but are not limited to, the microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and apoptosis. Drugs that target the microtubule cytoskeleton may be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastine, colchicine, and apricyatoxins. Drugs that target DNA may be, for example, drugs that directly disrupt DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylating agents, and DNA intercalators. DNA double-strand breakers may include, for example, enediyne antibiotics, including, but are not limited to, dynemycin, esperamycin, neocarutinostatin, and uncialamycin. DNA alkylating agents may include, for example, DNA bis-alkylators (i.e., DNA cross-linkers) or DNA mono-alkylators. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, indoline benzodiazepine (IGN) dimers, and duocarmycin-like compounds.Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1), and DXd-(2), whose structures are shown below), camptothecin derivatives, and anthracycline derivatives. Drugs targeting RNA may include, for example, drugs that inhibit cleavage, and examples include, but are not limited to, pladienolides. Drugs targeting kinesin-mediated protein transport may include, for example, mitotic kinesin inhibitors, and include, but are not limited to, kinesin spindle protein (KSP) inhibitors.

[0036] A "spacer" is a structure located between different structural modules, spatially separating them. The definition of a spacer does not limit whether it has a certain function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, and non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" is an amino acid sequence that acts as a spacer, and examples include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, a sequence containing two amino acids such as GA, or sequences such as GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGGS. A self-destructing spacer (e.g., self-destructing spacer Sp1) is a covalent component that causes two chemical bonds to break in succession after a protective region in the precursor is activated: specifically, the protective region (e.g., a cleavable sequence) is removed after activation, triggering a cascade of degradation reactions that sequentially release relatively small molecules. Examples of self-destructing spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl group), acetals, heteroacetals, and combinations thereof.

[0037] As used in this text, the term "amino acid" includes both "natural amino acids" and "unnatural amino acids."

[0038] The term "natural amino acids" refers to amino acids that are constituent amino acids of proteins, including the 20 commonly found amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less commonly found selenocysteine ​​and pyrrolicin.

[0039] As used in this text, the term "non-natural amino acid" refers to an amino acid that is not a protein-forming amino acid. Specifically, the term means an amino acid that is not a natural amino acid as defined above.

[0040] The term "alkyl group" refers to a straight-chain or branched saturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms, linked to other parts of the molecule via single bonds. Alkyl groups may have 1 to 20 carbon atoms, and are defined as "C1-C20". 20"Alkyl group" means, for example, C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, C3-C6 alkyl groups. Non-exclusive examples of alkyl groups include, but are not limited to, methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, isopropyl group, isobutyl group, s-butyl group, t-butyl group, isopentyl group, 2-methylbutyl group, 1-methylbutyl group, 1-ethylpropyl group, 1,2-dimethylpropyl group, neopentyl group, 1,1-dimethylpropyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 2-ethylbutyl group, 1-ethylbutyl group, 3,3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 2,3-dimethylbutyl group, 1,3-dimethylbutyl group, or 1,2-dimethylbutyl group, or their isomers. A divalent free radical is a group obtained by removing one hydrogen atom from a carbon atom that has free valence electrons in its corresponding monovalent free radical. A divalent free radical has two linkage sites that connect to other parts of the molecule. For example, an "alkylene group" or "alkylidene group" is a saturated, straight-chain or branched-chain divalent hydrocarbon group. Examples of alkylene groups include, for example, the methylene group (-CH2-), the ethylene group (-C2H4-), the propylene group (-C3H6-), the butylene group (-C4H8-), and the pentylene group (-C5H 10 -), hexylene group (-C6H 12 This includes, but is not limited to, a 1-methylethylene group (-CH(CH3)CH2-), a 2-methylethylene group (-CH2CH(CH3)-), a methylpropylene group, or an ethylpropylene group.

[0041] The term "cycloalkyl group" refers to a cyclic saturated aliphatic group composed of carbon atoms and hydrogen atoms, linked to other parts of a molecule via single bonds. A cycloalkyl group may have 3 to 10 carbon atoms, i.e., "C3-C 10It is a "cycloalkyl group", for example, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, or a cyclodecyl group. The "cycloalkylene group" is a divalent cycloalkyl group.

[0042] The term "heterocyclyl group" refers to a group in which one or more carbon atoms in the above cycloalkyl group are replaced by heteroatoms selected from nitrogen, oxygen, and sulfur. For example, an aza, oxa, or thiocyclopropyl group, an aza, oxa, or thiocyclobutyl group, a pyrrolidinyl group, a pyrazolidinyl group, an imidazolidinyl group, a tetrahydrofuranyl group, a tetrahydrothiophenyl group, a piperidyl group, a piperazinyl group, a tetrahydropyranyl group, or a tetrahydrothiopyranyl group. The "heterocyclylene group" is a divalent cycloalkyl group.

[0043] When "substituted" is mentioned in the present text, unless otherwise specified, the relevant substituents are selected from an alkyl group, a halogen, an amino group, a monoalkylamino group, a dialkylamino group, a nitro group, a cyano group, a formyl group, an alkylcarbonyl group, a carboxyl group, an alkyloxycarbonyl group, an alkylcarbonyloxy group, an aminocarbonyl group, a monoalkylaminocarbonyl group, a dialkylaminocarbonyl group, a formylamino group, an alkylcarbonylamino group, a formyl(monoalkyl)amino group, or an alkylcarbonyl(monoalkyl)amino group.

[0044] As used in the present text, when combining a group with another group, on the premise of forming a chemically stable structure, the connection between the groups may be linear or branched. The structure formed by such a combination may be connected to other parts of the molecule via any appropriate atom in the structure, and preferably, it is connected via a predetermined chemical bond. For example, -CR 1 R <, C 2 1~10 alkylene group, C 4~10 cycloalkylene group, C 4~10A heterocyclylene group and two or more divalent groups selected from -(CO)- combine to form a combination, and the two or more divalent groups are in a linear combination, for example, -CR 1 R 2 -C 1~10 Alkylene-(CO)-,-CR 1 R 2 -C 4~10 Cycloalkylene-(CO)-,-CR 1 R 2 -C 4~10 Cycloalkylene-C 1~10 Alkylene-(CO)-,-CR 1 R 2 -CR 1 R 2’ -(CO)-, -CR 1 R 2 -CR 1’ R 2’ -CR 1” R 2” It may form -(CO)- or similar structures. The resulting divalent structure may further be linked to other parts of the molecule.

[0045] When a single chemical structural formula contains multiple identical letters representing chemical groups, they are selected independently and are not necessarily the same. For example, the multiple M's in formula I-2 are each independently LKa-L 2 -L 1 - Selected from BP; and multiple L 2 These groups are independent of each other and are not necessarily the same.

[0046] As used in the text, the expressions "antibody-drug complex" and "antibody-drug conjugate" have the same meaning.

[0047] Linker-payload compound In the first aspect, the present invention provides a linker-payload compound having formula (I): [ka] During the ceremony, P is the payload; DC(O)-L- is a linker; DC(O)- is an oligosaccharide structure, consisting of -a first hexose group or its derivative moiety- (a second hexose group or its derivative moiety) f -β-DN-acetylglucosamine moiety, or -first hexose group or its derivative moiety- (second hexose group or its derivative moiety) f -β-D-glucose oxazoline moiety, the 6th carbon of the first hexose group or its derivative moiety exhibits the form -C(O)-, which is -C(O)- in DC(O)-, and the β-DN-acetylglucosamine moiety is [ka] The β-D-glucose oxazoline portion is [ka] And f is 0, 1, 2, 3, 4, 5, or 6; L is a linker end (for example, it can be broken from its linkage to P by a chemical method (e.g., a hydrolysis method) or a biological method (e.g., an enzyme-catalyzed method), releasing P), and L is directly linked to the carbonyl group in DC(O)- via its -NH-, and if L is an unbranched linker end, L is linked to one P and t is 1, and if L is a branched linker end, each branch may be linked to one P and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0048] In one embodiment, -L-(P) t is, -L 2 -L 1 -BP, that is, equation (I) is, [ka] And: During the ceremony, B is either not present, or is either 1) or 2) below, or a combination of 1) and 2): 1) a self-destructing spacer Sp1; 2) one divalent group, or a combination of two or more divalent groups, wherein the divalent group is -CR 1 R 2 , C 1~10 Alkylene group, C 4~10 Cycloalkylene group, C 4~10 Selected from heterocyclylene groups and -(CO)-; L 1 It is either independently absent; or an incleavable sequence, e.g., a thioether linkage coupling the payload and the antibody; or a cleavable sequence containing an enzymatically cleavable amino acid sequence, the enzymatically cleavable amino acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; L 2 Independently, it does not exist; or it is 1) below; or it is 2) below; or it is a combination of 1) and 2) below: 1)-NH-C 2~20 It is an alkylene group, and one or more -CH2- structures in the alkylene group are -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -, [ka] C 4~10 Cycloalkylene group, C 4~10 They may be substituted with heterocyclylene or phenylene groups: the cycloalkylene group, heterocyclylene group, and phenylene group can each be independently unsubstituted or with halogens, -C 1~10 Alkyl alkyl group, -C 1~10 Haloalkyl group, -C 1~10 Alkilen-NH-R 8 , and -C 1~10 Alkilen-OR 9 Substituted with at least one substituent selected from; 2) Amino acid residue sequence, i.e., -*(AA) n **- where n is an integer from 1 to 100, AA is an amino acid residue each time it appears independently, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) between the amino group and α-carbon of one amino acid. m -(CH2) p - may be present in the formula, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, p is 0, 1, 2, or 3, and the * end forms an amide bond with the carbonyl group in the oligosaccharide structure; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 Each of these is independently hydrogen, halogen, substituted or unsubstituted -C 1~10 Alkyl alkyl group, C 4~10 Selected from cycloalkylene groups: or R 1 and R 2 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkylene group, and / or, R 3 and R 4 These, together with the carbon atoms linked to them, form a 3-6 membered cycloalkylene group; P is part B, or L 1 Part, or L 2 This is a payload that connects to a part.

[0049] In another embodiment, -L-(P) t teeth, [ka] Therefore, equation (I) is, [ka] And: During the ceremony, Ld2 and each Ld1 are each independently a bond; or, -NH-C 1~20 alkylene-(CO)-, -NH-(PEG) i -(CO)-; or, each independently on the side chain is unsubstituted or -CO-(PEG) j -R 11 and is a natural amino acid substituted with, or an oligo natural amino acid having a degree of polymerization of 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10); -(PEG) i - and -(PEG) j - are each a PEG fragment, containing a predetermined number of consecutive -(O-C2H4)- structural units, or consecutive -(C2H4-O)- structural units, and may have a C 1~10 alkylene group added to one end; M is hydrogen, or LKa-L 2 -L 1 -B-P; Q is NH2, or L 2 -L 1 -B-P; provided that M is not hydrogen and Q is not NH2 at the same time;

[0050] Each LKa is each independently

Chemical formula

Chemical formula

[0051] In one embodiment, B, L 1 and L 2 At least one of them is not "non-existent".

[0052] In one embodiment, L 2 is -NH-(CH2) a -(CH2)2(CO)-(a is an integer of 0, 1, 2, 3, 4, or 5); [ka] (b is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In one embodiment, the above L 2 The carbonyl group in the structure is L 1 It is connected to the other. In one embodiment, a is 0, 1, 2, or 3, preferably 3.

[0053] In one embodiment, L 2 This is an amino acid residue sequence, i.e., -*(AA) n **- where n is an integer from 1 to 100, AA is an amino acid residue each time it appears independently, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) between the amino group and α-carbon of one amino acid. m -(CH2) p- may be present in the formula, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 0, 1, 2, or 3, and the * end forms an amide bond with the carbonyl group in the oligosaccharide structure. In one embodiment, AA is independently one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof. In one embodiment, n is an integer from 1 to 50, preferably an integer from 1 to 30, preferably an integer from 1 to 20, preferably an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0054] In one embodiment, L 1 The enzyme-cleavable amino acid sequence comprises an enzymatically cleavable amino acid sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the enzymatically cleavable amino acid sequence is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof; preferably, the enzymatically cleavable amino acid sequence is -Gly-Gly-Phe-Gly-. In one embodiment, L 1 is one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn, or any combination thereof, preferably -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof. In one embodiment, L 1 This represents -Val-Cit-.

[0055] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl group), ethyl acetal, heteroacetal, and combinations thereof; preferably, Sp1 is an acetal, heteroacetal, or PABC; more preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is linked to an enzymatically cleavable amino acid sequence, U is absent or CH2, O, S, or NH, preferably O or S.

[0056] In one embodiment, B is absent, or is -NH-CH2-U-, or -NH-CH2-U-(CH2) g The formula is -(CO)-, where g is 1, 2, 3, 4, 5, or 6, and U is absent or CH2, O, S, or NH, preferably O or S. In one embodiment, B is absent. In one embodiment, B is 1), 2), or a combination of 1) and 2): 1) a self-destructing spacer Sp1; 2) one divalent group, or a combination of two or more divalent groups, wherein the divalent group is -CR 1 R 2 -, C 1~10 Selected from alkylene groups and -(CO)-. In one embodiment, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g It is -(CO)-, and U is absent or is CH2, O, S, or NH, preferably O or S. In one embodiment, B is linked to the payload via an amide bond, an ester bond, or an ether bond. In one embodiment, B is [ka] (-PABC-), -NH-CH2-U-, or -NH-CH2-U-(CH2) g-(CO)- (where g is 1, 2, 3, 4, 5, or 6) is selected; U is absent or is CH2, O, S, or NH, preferably O or S.

[0057] In one embodiment, -L 1 -B- represents -Val-Cit-PABC- or -Gly-Gly-Phe-Gly-.

[0058] In one embodiment, -L 2 -L 1 -B- is -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C2H4-O) m -(CH2) p -Gly-Gly-Phe-Gly- represents

[0059] In one embodiment, Ld2 and each Ld1 are independently combined, or [ka] Selected from; Each of i, j, and k is independently selected from integers between 1 and 100.

[0060] In one embodiment, each i, j, and k is independently selected from integers between 1 and 20. In another embodiment, each i, j, and k is independently selected from integers between 1 and 12.

[0061] In one embodiment, each i is independently selected from integers between 2 and 8; in particular, 4.

[0062] In one embodiment, each j is independently selected from integers 8 to 12; in particular, 8 or 12.

[0063] In one embodiment, each k is independently selected from integers 1 to 7; in particular, it is 1, 3, or 5.

[0064] In one embodiment, Ld2 and each Ld1 are independently bonded; or C having an amino group and a carbonyl group at each of its ends. 1~20 A PEG fragment of a certain length having an alkylene group, or an amino group and a carbonyl group at each end (-(PEG) i (represented by -), or selected from one or more natural amino acids, each of which is independently unsubstituted or a PEG fragment of a certain length (-CO-(PEG)) on the side chain. j It is replaced by (represented by -).

[0065] In one embodiment, -(PEG) i - is -(O-C2H4) i -, or -(C2H4-O) i - contains and has C at one end 1~10 An alkylene group may be added; -(PEG) j - is -(O-C2H4) j -, or -(C2H4-O) j - contains and has C at one end 1~10 An alkylene group may be added. In a very specific embodiment, -(PEG) i - is -C2H4-(O-C2H4) i -, or -(C2H4-O) i Contains -C2H4-.

[0066] In one embodiment, the payload may be selected from the group consisting of small molecule compounds (e.g., small molecule drugs of various mechanisms of action, including each conventional small molecule drug, photoacoustic therapy drug, photothermotherapy drug, etc., e.g., conventional cytotoxic drugs such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, e.g., conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine; small molecule targeted drugs such as imatinib mesylate, gefitinib, and anlotinib; immune agonists such as STING agonists and TLR agonists), nucleic acids and nucleic acid analogs, probe molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes, and radiolabeling), oligopeptides, polypeptides, peptidomimetic compounds, and proteins. In one embodiment, the payload is selected from the group consisting of small molecule compounds and nucleic acid molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from the group consisting of cytotoxins and fragments thereof.

[0067] In one embodiment, the payload is a cytotoxin or a fragment thereof, comprising the L portion of formula (I), or the B portion of the compound of formula (I-1) or formula (I-2), L 2 or L 1 It has arbitrary derivation for connecting to parts.

[0068] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is a taxane, a tansinoid, auristatin, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamide, vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 10) and its analogues, halichondrin B, indole-3-oxoacetamide derivatives, podophyllotoxin derivatives, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discormolide, and laulimalide are selected from the group. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, e.g., camptothecin derivatives and their derivatives, mitoxantrone, and mitogwazone. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustard derivatives, e.g., chlorambucil, chlornafadin, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrovin hydrochloride, melphalan, nobembitin, fenamet, fenesterine, prednimustine, trophosphamide, and uramustine. In another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosourea derivatives, such as carmustine, flubenzuron, formoterol, lomustine, nimustine, and ranimustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridine derivatives.In one preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carbocone, metsuredepa, and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In one preferred embodiment, the cytotoxin is selected from the group consisting of engine antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarutinostatin, and acrasinomycin. In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycin derivatives, actinomycin C, carabicin, carminomycin, sarcomycin, carminomycin, actinomycin D, daunorubicin, detrubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin derivatives, nogaramycin, olibomycin, peplomycin, porphyromycin, promycin, keramycin, rhodorubicin, streptonigrin, streptozocin, dinostatin, and sorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenese derivatives. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, loridine A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In one preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azacerin, and 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In one preferred embodiment, the cytotoxin is selected from the group consisting of denopterin, methotrexate, pteropterin, trimethrexate, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In one preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine. In yet another embodiment, the cytotoxin is selected from the group consisting of pyrimidine analogs.In one preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In one preferred embodiment, the cytotoxin is selected from the group consisting of carsterone, dromostanolone propionate, epithiostanol, mepithiostan, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenergics. In one preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In one preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In yet another embodiment, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, maytansinoid, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine, and exatecan. In one embodiment, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, and maytansinoid.

[0069] In one embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f.

[0070] In one embodiment, the cytotoxin is a maytansinoid, such as DM1 and the like. It should be noted that when using a cytotoxin containing a thiol group moiety, the thiol group moiety can react with a maleimide moiety to form succinimide. For example, a cytotoxin such as a maytansinoid such as DM1 can be directly linked via succinimide. In such cases, in some embodiments, since the payload and the thiol group moiety together constitute the cytotoxin, in such cases, the payload can be understood to represent the other part of the cytotoxin molecule excluding the thiol group moiety.

[0071] In one embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), and the like. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0072] The payload contains an active group that can react with the active group in the compound of formula (I), thereby covalently bonding the payload and the compound of formula (I). A compound that does not contain an active group cannot obtain a payload unless appropriately derivatized.

[0073] In one embodiment, the cytotoxin is a compound of the following formula (i).

Chemical formula

[0074] In one embodiment, the cytotoxin is selected from the following Compounds 1 to 16, and the wavy bond represents the linking site linked to the compound of formula (I).

[0075]

Chemical formula

[0076] In some embodiments, the payload is selected from DX8951f (Compound 9), DXd-(1) (Compound 10), and DXd-(2) (Compound 14), Compound 15, preferably DX8951f, or DXd-(1), more preferably DXd-(1).

[0077] In one embodiment, the first hexosyl group or its derivative moiety is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a glucosyl group, an idosyl group, or derivatives thereof, and its carbon 6 is in the form of -C(O)-.

[0078] In one embodiment, each monosaccharide moiety in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond.

[0079] In one embodiment, each monosaccharide moiety in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond.

[0080] In one embodiment, the derivatives are independently selected from derivatives in which the uronic acid or the hydroxyl group of the monosaccharide is replaced by an acylamino group (for example, an alkanoylamino group, for example, a formylamino group, an acetylamino group, a propionylamino group, etc., particularly an acetylamino group).

[0081] In one embodiment, the first hexosyl group or its derivative moiety is

Chemical Structure

Chemical Structure

[0082] In one embodiment, f is 0.

[0083] In one embodiment, D-C(O)- is a disaccharide structure

Chemical formula

[0084] In one embodiment, D-C(O)- is a disaccharide structure

Chemical formula

[0085] In one embodiment, the linker-payload compound having formula (I) is:

Chemical formula

Chemical formula

[0086] In another aspect, the present invention provides a linker-payload compound obtained by a method comprising the following steps.

[0087] (i) Oxidizing the primary alcohol at the 6-position of the terminal first hexosyl unit in an oligosaccharide containing a terminal first hexosyl unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein there may be 1, 2, 3, 4, 5, or 6 second hexosyl units or derivative moieties between the terminal first hexosyl unit and the terminal N-acetylglucosamine (GlcNAc) unit; (ii) Reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group of a linker-terminal-payload compound (b) having a reactive group at the terminal to obtain the linker-payload compound.

[0088] In one embodiment of this other aspect, The first hexose group unit is selected from a glucosyl group, a mannosyl group, a galactosyl group, or a fructosyl group; and / or, Each time the second hexose unit appears, it is independently selected from a glucosyl group, a mannosyl group, a galactosyl group, or a fructosyl group, and / or Each monosaccharide portion in the oligosaccharide is linked via a β-(1→4) glycosidic bond; and / or, The aforementioned derivatives are independently selected from derivatives in which the hydroxyl group of a monosaccharide is replaced with an acylamino group (e.g., an alkanoylamino group, e.g., a formylamino group, an acetylamino group, a propionylamino group, etc., in particular an acetylamino group).

[0089] In one embodiment of this other aspect, The first hexose unit is a mannosyl group or a glucosyl group; and / or, The second hexose group unit or its derivative portion is absent.

[0090] In one embodiment of the other aspect, the oligosaccharide in step (i) has the following structure: [ka] It has.

[0091] In one embodiment of the other aspect, the oligosaccharide in step (i) has the following structure: [ka] It has.

[0092] In one embodiment of the other aspect, the reactive group of the linker-terminated payload compound (b) having a reactive group at its terminus in step (ii) is an amino group.

[0093] In one embodiment of the other aspect, the method may further include the step of converting an intermediate compound (a) having a carboxyl group into an acid halide, and further reacting it with a linker-terminated payload compound (b) having a reactive group at its terminus to obtain the linker-payload compound.

[0094] In one embodiment of this other aspect, the linker-payload compound is the linker-payload compound defined in the first aspect or each embodiment thereof.

[0095] Method for producing linker-payload compounds In the second aspect, the present invention provides a method for producing a linker-payload compound having formula (I), where, unless otherwise described, each variable is as defined in the first aspect or in each embodiment thereof.

[0096] In one embodiment, the method involves DC(O)-OH and L'-(P) t By subjecting them to an amide formation reaction, [ka] The process includes the step of forming a such that L' is the same as L as defined in the first aspect or each embodiment thereof, except that the -NH- linked to DC(O)- in L is H2N- in L'.

[0097] In one embodiment, DC(O)- in the linker-payload compound of formula (I) that is produced, [ka] If that is the case, the method further, [ka] Furthermore, [ka] The process may include a step of condensation. In one embodiment, the condensation is carried out in the presence of water, a base, and 2-chloro-1,3-dimethylimidazoline chloride (DMC, cas: 37091-73-9). In one embodiment, the base is an inorganic base or an organic base. In one embodiment, the inorganic base is potassium carbonate, potassium phosphate, etc. In one embodiment, the organic base is an amine, for example, a tertiary amine, for example, triethylamine (Et3N).

[0098] In one embodiment, the amide formation reaction is carried out in the presence of an organic solvent, an organic base, and a condensation reagent. In one embodiment, the organic solvent is selected from N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA). In one embodiment, the organic base is selected from diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM). In one embodiment, the condensation reagent is selected from HATU, HBTU, TBTU, and PyBOP.

[0099] In one embodiment, DC(O)-OH is [ka] That is the case.

[0100] In one embodiment, [ka] teeth, [ka] It is manufactured from. In one embodiment, the manufacturing is carried out under conditions that debenzylate the substrate. In one embodiment, the deprotection is carried out in the presence of hydrogen gas and a palladium catalyst. In one embodiment, the palladium catalyst is selected from palladium carbon and palladium carbon hydroxide.

[0101] In one embodiment, [ka] teeth, [ka] It is manufactured from. In one embodiment, the manufacture is carried out in the presence of an oxidizing agent and an optional oxidation catalyst. In one embodiment, the oxidizing agent is iodobenzene diacetate. In one embodiment, the oxidation catalyst is 2,2,6,6-tetramethyl-1-piperidine oxide.

[0102] In one embodiment, [ka] teeth, [ka] It is manufactured from. In one embodiment, the manufacture is carried out in the presence of an acid. In one embodiment, the acid is p-toluenesulfonic acid.

[0103] In one embodiment, [ka] teeth, [ka] It is manufactured from. In one embodiment, the manufacture is carried out in the presence of thioacetic acid (AcSH) and an organic solvent. In one embodiment, the organic solvent is selected from chloroform and pyridine, or a mixture thereof.

[0104] In one embodiment, [ka] teeth, [ka] It is produced by forming a β-(1→4) glycosidic bond. In one embodiment, the production is carried out under glycosidic bond formation conditions. In one embodiment, the glycosidic bond formation conditions include using trifluoromethanesulfonic acid anhydride in an anhydrous state.

[0105] The present invention further relates to the following compounds: [ka] To provide.

[0106] Antibody-drug conjugates In a third aspect, the present invention provides an antibody-drug conjugate having formula (II) through site-specific binding of the N-glycosylation site of the antibody Fc region: [ka] During the ceremony, P is the payload; R is either hydrogen or an α-L-fucosyl group; q is either 1 or 2; Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II) is derived from the asparagine at position 297 of the antibody Fc region); The carbon at position 6 of the first hexose group or its derivative moiety exhibits the form -C(O)-; L is a linker end (which can be broken by chemical means (e.g., hydrolysis) or biological means (e.g., enzyme-catalyzed methods) to release P), and L is directly linked to the carbonyl group in the first hexose group or its derivative moiety via its -NH-, and if L is an unbranched linker end, L is linked to one P and t is 1, and if L is a branched linker end, each branch may be linked to one P and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10); f is 0, 1, 2, 3, 4, 5, or 6.

[0107] In one embodiment, the first hexose group or its derivative portion is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a grosyl group, an idosyl group, or a derivative thereof, and its 6th carbon exhibits the form -C(O)-.

[0108] In one embodiment, the second hexose group or its derivative moiety is independently selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, or a derivative thereof each time it appears.

[0109] In one embodiment, each monosaccharide portion is linked via a β-(1→4) glycosidic bond.

[0110] In one embodiment, the derivative is independently selected from derivatives in which the hydroxyl group of a uronic acid or monosaccharide is replaced with an acylamino group (e.g., an alkanoylamino group, e.g., a formylamino group, an acetylamino group, a propionylamino group, etc., in particular an acetylamino group).

[0111] In one embodiment, the first hexose group or its derivative portion is [ka] Selected from.

[0112] In one embodiment, f is 0.

[0113] In one embodiment, the -NHC(O)CH2- in formula (II) is derived from the asparagine at position 297 of the antibody Fc region.

[0114] In one embodiment, targets identified by Ab include CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, and Nectin. n)-4, EGFRvIII, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin6, FGFR2, FGFR3, CA6, CanAg, IntegrinαV, TDGF1, EphrinA4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, TF (Tissue Factor), and ROR1 / 2, but not limited to these.

[0115] In some embodiments, Ab is a monoclonal antibody. In some embodiments, Ab is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include, but are not limited to, pertuzumab and trastuzumab. In one embodiment, Ab is trastuzumab.

[0116] In one embodiment, formula (II) is formula (II-1): [ka]

[0117] In one embodiment, formula (II) is formula (II-2): [ka]

[0118] In one embodiment, formula (II) is formula (II-3): [ka]

[0119] In one embodiment, formula (II) is formula (II-4): [ka]

[0120] In one embodiment, formula (II) is formula (II-5): [ka] During the ceremony, R is either hydrogen or an α-L-fucosyl group; q is either 1 or 2; Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II-1) is derived from the asparagine at position 297 of the antibody Fc region); The other variables are as defined in the first phase or each of its embodiments.

[0121] In one embodiment, formula (II) is selected from formulas (II-1), (II-2), (II-3), (II-4), and (II-5): [ka] During the ceremony, R is either hydrogen or an α-L-fucosyl group; q is either 1 or 2, for example, q is 2; Ab is an anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-Nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-Nectin4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-Mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-Cadherin6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-IntegrinαV antibody, anti-TDGF1 antibody, anti-EphrinA4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TF (Tissue Factor) antibody, anti-ROR1 / 2 antibody; preferably, an anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-Nectin-4 antibody, anti-FGFR3 antibody, anti-Trop2 antibody; more preferably, an anti-ErbB2 / HER2 antibody, anti-Trop2 antibody; particularly preferably, an anti-ErbB2 / HER2 antibody (e.g., trastuzumab);

[0122] (P) t -L- is

Chemical formula

[0123] In one embodiment, R in formula (II) is an α-L-fucosyl group; q is 2; Ab is trastuzumab;

[0124] (P) t -L- is [ka] for example, [ka] and; In formula (II), -NHC(O)CH2- is derived from the asparagine at position 297 of the antibody Fc region.

[0125] Method for producing antibody-drug conjugates In a fourth aspect, the present invention provides a method for producing an antibody-drug conjugate having formula (II), comprising conjugating a linker-payload compound having formula (I) with an antibody Ab, wherein, unless otherwise described, each variable is as defined in the third aspect or each embodiment thereof.

[0126] In one embodiment, the method includes the following steps.

[0127] a) Catalytic action of glycosidase or a variant thereof is used to excise the N-glycan portion of antibody Ab, thereby obtaining an antibody in which the N-glycosylation site of its Fc region is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine.

[0128] b) The modified antibody obtained in step a) is conjugated to the linker-payload compound by catalytic action of glycosidase or a variant thereof.

[0129] In the formula, the glycosidase or its variant used in steps a) and b) may be the same or different.

[0130] In one embodiment, the glycosidase or its variant used in steps a) and b) is fucose hydrolase, N-acetylglucosamine endohlase, or a variant thereof. In one embodiment, the N-acetylglucosamine endohydrolase comprises at least one selected from Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); preferably, the endoglycosidase is Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2. In one embodiment, the enzyme is Endo S2.

[0131] In one embodiment, steps a) and b) are carried out by a one-pot enzyme catalytic reaction.

[0132] In one embodiment, steps a) and b) are carried out by a one-pot enzyme catalysis, and the enzyme is Endo S2.

[0133] Pharmaceutical compositions and pharmaceutical preparations Another object of the present invention is to provide a pharmaceutical composition comprising a preventive or therapeutically effective dose of the complex of the present invention and at least one pharmaceutically acceptable carrier.

[0134] The pharmaceutical composition of the present invention may be administered by any method that has the effect of preventing, alleviating, preventing or treating symptoms in humans or animals. For example, it may be manufactured in various appropriate dosage forms depending on the route of administration, in particular injectable preparations, such as lyophilized powder injections, or sterile injections / powder preparations.

[0135] The term "pharmaceutically acceptable" means that, when in contact with patient tissue within the bounds of normal medical judgment, it does not cause unintended toxicity, irritation, or allergic reactions, has a reasonable advantage-in-disadvantage ratio, and is effective for its intended use.

[0136] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and performance of the complex. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers also include carrier substances that bring the composition closer to physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and substances such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate.

[0137] In one embodiment, the drug-to-antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer between 1 and 20, for example, about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2. In a particular embodiment, the DAR of the complex of the present invention is about 2, about 4, about 6, or about 8.

[0138] Treatment methods and use The antibody-drug conjugates of the present invention can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to treatment with antibody-drug conjugates include tumors characterized by specific tumor-associated antigens or cell surface receptors, and these tumor cells can be identified by the targeting molecule in the antibody-drug conjugate and further killed by the payload / cytotoxin in the antibody-drug conjugate.

[0139] Therefore, in another aspect, the present invention further provides the use of the antibody-drug conjugate or the pharmaceutical composition of the present invention for the production of therapeutic drugs for diseases, illnesses or conditions, wherein the disease, illness or condition is selected from tumors or autoimmune diseases.

[0140] In another aspect, the present invention provides antibody-drug conjugates or pharmaceutical compositions of the present invention for treating tumors or autoimmune diseases.

[0141] In a further aspect, the present invention provides a method for treating a tumor or autoimmune disease, comprising administering an effective amount of the antibody-drug conjugate or pharmaceutical composition of the present invention to an individual in need thereof.

[0142] In one embodiment, an antibody-drug conjugate formed by linking an anti-human HER2 or Trop2 antibody according to the present invention with a low molecular weight cytotoxin specifically binds to HER2 or Trop2 on the surface of tumor cells, selectively killing tumor cells that express HER2. In another embodiment, the present invention provides the use of the antibody-drug conjugate or pharmaceutical composition of the present invention for the production of a therapeutic agent for a disease, illness or condition, the disease, illness or condition being selected from HER2 or Trop2-positive tumors. In a more preferred embodiment, the disease, illness or condition being selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma, among others.

[0143] The dose of antibody-drug conjugate administered to subjects can be adjusted over a wide range. The dose can be modified depending on the specific route of administration and the subject's needs, and can also be determined by a medical or healthcare professional.

[0144] Examples Production of monosaccharide linkers

[0145] Example 1 Preparation of disaccharide substrate compound 1 Compound 1 was prepared using the following procedure, and its structure is as follows: [ka]

[0146] (1) Preparation of compound 1c [ka] Under vacuum evacuation conditions using an oil pump, a 100 mL Schlenk reaction flask was baked with a heat gun for 5 minutes. After cooling, activated molecular sieves were added to the system and baked for another 5 minutes. Vacuum evacuation and nitrogen gas purging were performed three times. Compound 1a (4.41 g, 7.96 mmol) was added to the system under nitrogen protection and stirred for 3 minutes, then anhydrous dichloromethane (30 mL) was added and stirred for 0.5 hours. Separately, following a similar procedure, compound 1b (1.89 g, 3.98 mmol, dissolved in 20 mL of anhydrous dichloromethane) was added to another 50 mL Schlenk reaction flask containing activated molecular sieves and pre-dried by stirring for 1 hour to remove any remaining moisture in the system.

[0147] Under nitrogen protection, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-t-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added to the above-mentioned dried compound 1a solution at room temperature, and the mixture was stirred for a further 20 minutes. The reaction flask was placed in a dry ice / ethyl acetate bath and cooled to -65°C, and trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was added to the system. After 2 minutes, the pre-dried dichloromethane solution of compound 1b was added to the system, and the resulting reaction mixture was stirred at -65°C until the reaction was complete (approximately 3 hours) under TLC (eluent: siRNA / PE = 1 / 8). The reaction was stopped by adding saturated sodium bicarbonate solution to the system, and the mixture was extracted with dichloromethane and separated (150 mL x 3). The combined organic phases were sequentially washed with water and saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the sample was fed by a wet method. Separation was performed by column chromatography (eluent: dimethyl / PE = 1 / 12-1 / 10) to obtain compound 1c (2.72 g, yield 75.4%, colorless viscous oily liquid). 1 H NMR (400 MHz, chloroform-d) δ 7.55 (dd, J = 7.6, 2.1 Hz, 2H), 7.51 - 7.28 (m, 28H), 5.60 (s, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 11.8 Hz, 1H), 4.90 - 4.80 (m, 2H), 4.76 (d, J = 12.0 Hz, 1H), 4.74 - 4.63 (m, 3H), 4.57 (s, 1H, H 1’ ), 4.48 (d, J = 12.1 Hz, 1H), 4.37 (d, J = 8.1 Hz, 1H,H 1), 4.22 - 4.10 (m, 2H), 4.05 (t, J = 9.3 Hz, 1H), 3.80 (d, J = 3.1 Hz, 1H), 3.72 (dd, J = 11.2, 2.2 Hz, 1H), 3.67 - 3.52 (m, 3H), 3.49 (dd, J = 9.8, 3.1 Hz, 1H), 3.41 (t, J = 9.3 Hz, 1H), 3.35 (dt, J = 9.8, 2.9 Hz, 1H), 3.16 (td, J = 9.7, 4.8 Hz, 1H). C 54 H 56 N3O 10 + [M+H] + In MS(ESI)m / z, the calculated value was 906.4, while the measured value was 906.7.

[0148] (2) Preparation of compound 1d [ka] At room temperature, compound 1c (228 mg, 0.252 mmol), chloroform (1.2 mL), pyridine (1 mL), and AcSH (1.2 mL) were sequentially added to a 25 mL Schlenk reaction flask and dissolved. The stopcock was closed, and the system was stirred at 60°C for approximately 18 hours under HPLC monitoring until the reaction was almost complete. After concentration under reduced pressure and removal of most of the solvent, ethyl acetate (50 mL) was added to the system, and the mixture was sequentially washed with saturated sodium bicarbonate solution (30 mL), 1 M hydrochloric acid (10 mL x 4), and saturated sodium bicarbonate solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: dimethyl / PE = 1 / 10 - 1 / 1) to obtain compound 1d (200 mg, yield 86%, white solid). 1H NMR (400 MHz, chloroform-d) δ 7.57 - 7.49 (m, 2H), 7.48 - 7.39 (m, 5H), 7.39 - 7.24 (m, 23H), 5.80 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 5.00 (d, J = 6.7 Hz, 1H), 4.97 - 4.87 (m, 3H), 4.87 - 4.75 (m, 2H), 4.69 - 4.59 (m, 4H), 4.57 (s, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.19 - 4.05 (m, 3H), 3.96 (t, J = 7.3 Hz, 1H), 3.85 - 3.76 (m, 2H), 3.73 - 3.59 (m, 4H), 3.49 (dd, J = 9.8, 3.1 Hz, 1H), 3.18 (td, J = 9.7, 4.8 Hz, 1H), 1.77 (d, J = 1.2Hz, 3H).C 56 H 60 NO 11 + [M+H] + In MS(ESI)m / z, the calculated value was 922.4, and the measured value was also 922.4.

[0149] (3) Preparation of compound 1e [ka] At room temperature, compound 1d (168 mg, 0.168 mmol), p-toluenesulfonic acid monohydrate (35 mg, 0.168 mmol), methanol (5 mL), and tetrahydrofuran (5 mL) were sequentially added to a 100 mL single-necked flask. The flask was purged three times with nitrogen gas, and the reaction system was stirred overnight at room temperature until the reaction was complete under HPLC monitoring. The reaction system was terminated with saturated sodium bicarbonate solution (20 mL), extracted with dichloromethane (20 mL x 3), and separated. The combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: MeOH / DCM = 1 / 20) to obtain compound 1e (136 mg, yield 89%, white solid). 1H NMR (400 MHz, chloroform-d) δ 7.43 - 7.24 (m, 29H), 5.76 (d, J = 7.9 Hz, 1H), 5.01 (d, J = 6.9 Hz, 1H), 4.94 (dd, J = 11.8, 2.3 Hz, 2H), 4.85 (d, J = 11.7 Hz, 1H), 4.77 (d, J = 11.8 Hz, 1H), 4.70 - 4.59 (m, 3H), 4.58 - 4.47 (m, 3H), 4.36 (d, J = 11.7 Hz, 1H), 4.20 (t, J = 7.9 Hz, 1H), 3.94 (t, J = 7.5 3.91 - 3.80 (m, 3H), 3.80 - 3.71 (m, 3H), 3.63 - 3.50 (m, 2H), 3.22 - 3.14 (m, 2H), 1.78 (s, 3).C 49 H 56 NO 11 + [M+H] + In MS(ESI)m / z, the calculated value was 834.4, while the measured value was 834.5.

[0150] (4) Preparation of compound 1f [ka] At room temperature, compound 1e (130 mg, 0.156 mmol), dichloromethane, t-butyl alcohol, and water were sequentially added to a 100 mL single-necked flask. Iodobenzene diacetate (0.5-10 equivalents) and 2,2,6,6-tetramethyl-1-piperidine oxide (0.01-1 equivalent) were added to the system, and the reaction system was stirred overnight at room temperature until the reaction was complete under TLC monitoring. The mixture was extracted with dichloromethane and separated, and the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 1f (100 mg, yield 75.1%, white solid). 1H NMR (400 MHz, chloroform-d) δ 7.42 - 7.31 (m, 18H), 7.30 - 7.27 (m, 2H), 7.25 - 7.20 (m, 3H), 7.15 (dd, J = 6.7, 2.9 Hz, 2H), 6.06 (d, J = 8.3 Hz, 1H), 4.96 (d, J = 6.6 Hz, 1H), 4.93 (d, J = 11.9 Hz, 1H), 4.86 (d, J = 11.1 Hz, 1H), 4.81 (d, J = 12.1 Hz, 1H), 4.73 (d, J = 11.1 Hz, 1H), 4.71 - 4.65 (m, 3H), 4.65 - 4.57 (m, 3H), 4.49 (s, 1H), 4.46 (d, J = 12.0 Hz, 1H), 4.23 (t, J = 7.1 Hz, 1H), 4.15 (t, J = 9.5 Hz, 1H), 3.89 (t, J = 6.5 Hz, 1H), 3.86 - 3.65 (m, 6H), 3.54 (d, J = 9.7 Hz, 1H), 3.30 (dd, J = 9.3, 2.8 Hz, 1H), 1.65 (s, 3H).C 49 H 52 NO 12 - [MH + ] - In MS(ESI)m / z, the calculated value was 846.3, and the measured value was also 846.3.

[0151] (5) Preparation of compound 1 [ka] At room temperature, compound 1f (160 mg, 0.189 mmol), tetrahydrofuran, methanol, and palladium-carbon catalyst were sequentially added to a 50 mL single-necked flask, and the reaction system was stirred under a hydrogen atmosphere until all the starting materials were consumed, under TLC measurement. The mixture was filtered, concentrated under reduced pressure, and extracted and dried using an oil pump to obtain compound 1 (75 mg, 100% yield, white solid). 14 H 22 NO 12 -[MH + ] - In MS(ESI)m / z, the calculated value was 396.1, and the measured value was also 396.1.

[0152] Example 2 Preparation of disaccharide substrate compound 2 Compound 2 was prepared using the following procedure, and its structure is as follows: [ka]

[0153] (1) Synthesis of compound 2-1 [ka]

[0154] Step A: Synthesis of compound 2-1b In a single-necked flask, compound 2-1a (1.0 equivalent, commercially available, CAS: 959153-39-0), MeOH, and sodium methoxide (0.1 equivalent, 5 mol / L in MeOH) were added and stirred at room temperature under a nitrogen atmosphere. The reaction was monitored by TLC, and once the reaction was complete, 1 M hydrochloric acid was added to stop the reaction. The reaction system was then neutralized, the solvent was removed under reduced pressure, a small amount of toluene was added, and water was removed by azeotropic effect under reduced-pressure rotational evaporation conditions to obtain the crude compound 2-1b, a pale yellow, viscous oily liquid. Further purification was not necessary, and it was used directly in the next reaction.

[0155] Step B: Synthesis of Compound 2-1c Crude compound 2-1b (1 equivalent), acetonitrile, camphor sulfonic acid (0.1 equivalent), and benzaldehyde dimethyl acetal (4 equivalents) were sequentially added to a single-necked flask, and the reaction system was stirred overnight at room temperature. The progress of the reaction was monitored by TLC, and when the reaction was complete (approximately 24 hours), saturated sodium bicarbonate solution was added to the system to stop the reaction, and the mixture was extracted with ethyl acetate and separated. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography using a wet sample preparation (eluent: PE / siRNA = 5:1) to obtain compound 2-1c (white solid, 83% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51-7.49 (m, 2H, Ar-H), 7.45 (d, J= 8.0 Hz, 2H, Ar-H), 7.42-7.37 (m, 5H, Ar-H), 7.36-7.31 (m, 3H, Ar-H), 7.15 (d, J = 8.0 Hz, 2H, Ar-H), 5.56 (s, 1H, PhCH), 4.97 (d, J = 11.6 Hz, 1H, PhCH2), 4.81 (d, J = 11.6 Hz, 1H, PhCH2), 4.58 (d, J = 9.6 Hz, 1H), 4.40 (dd, J = 10.2, 4.8 Hz, 1H), 3.80 (dd, J = 10.0, 10.4 Hz, 1H), 3.70 (dd, J = 9.2, 9.2 Hz, 1H), 3.65 (dd, J = 9.2, 9.2 Hz, 1H), 3.53-3.48 (m, 2H), 2.64(br s, 1H, -OH), 2.36 (s, 3H).C 27 H 29 O5S + [M+H] + The MS(ESI) m / z values ​​were calculated at 465.2 and measured at 465.3. The NMR data was consistent with the literature report (see compound 2b1 described in Nature 2007, 446, 896).

[0156] Step C: Synthesis of compound 2-1d Compound 2-1c (1 equivalent), dichloromethane, and triethylamine (5 equivalents) were sequentially added to a single-necked flask. The system was cooled to 0°C, and anhydrous acetic acid (2 equivalents) was added. After stirring for 10 minutes, the reaction system was raised to room temperature and stirred. The progress of the reaction was monitored by TLC. Once the reaction was complete (approximately 6 hours), saturated sodium bicarbonate solution was added to the system to stop the reaction. The mixture was extracted with dichloromethane and separated. The combined organic phase was washed with water, then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography using a wet sample preparation method (eluent: PE / siRNA = 5:1) to obtain compound 2-1d (white solid, 89% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51-7.48 (m, 2H, Ar-H), 7.43-7.36 (m, 5H, Ar-H), 7.34-7.25 (m, 5H, Ar-H), 7.13 (d, J = 8.0 Hz, 2H, Ar-H), 5.58 (s, 1H, PhCH), 5.00 (dd, J = 8.0, 8.0 Hz, 1H, H-2), 4. 87 (d, J = 12.0 Hz, 1H, PhCH2), 4.67 (d, J = 12.0 Hz, 1H, PhCH2), 4.64 (d, J = 10.4 Hz, 1H, H-1), 4.39 (dd, J = 10.8 Hz, J = 5.2 Hz, 1H), 3.81 (t, J = 10.4 Hz, 1H), 3.78-3.70 (m, 2H), 3.48 (ddd, J = 9.6, 5.4, 10.0 Hz, 1H), 2.35 (s, 3H, Ar-CH3), 2.05 (s, 3H, OAc). C 29 H 31 O6S + [M+H] +The MS(ESI) m / z values ​​were calculated at 507.2 and measured at 507.4. The NMR data was consistent with the literature report (see compound 3b1 described in Nature 2007, 446, 896).

[0157] Step D: Synthesis of Compound 2-1e Under a nitrogen atmosphere, compound 2-1d (1 equivalent), dichloromethane, and boranetetrahydrofuran (10 equivalents, 1M in THF) were sequentially added to a 100 mL two-necked flask. The system was cooled to 0°C, and dibutylboron trifluoromethanesulfonate (1.4 equivalents, 1.0 M in DCM) was added dropwise. The system was kept at 0°C until the TLC indicated the completion of the reaction (approximately 5 hours). Then, triethylamine solution was added to the system at 0°C to halt the reaction of dibutylboron trifluoromethanesulfonate in the system, and methanol was slowly added. The reaction of boranetetrahydrofuran is stopped by adding it dropwise, and after the system no longer produces a large amount of bubbles, water is added to ensure complete cessation of the reaction. Then, the mixture is extracted with ethyl acetate and separated. The combined organic phase is washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the sample is fed by a wet method. It is purified by flash column chromatography (eluent:eluent:PE / siRNA = 4:1) to obtain 2-1e crude product (white solid). The structure of the product is confirmed to be correct by LCMS. 29 H 33 O6S + [M+H] + In MS(ESI) m / z measurements, the calculated value was 509.2 and the measured value was 509.3. Therefore, there was no need to precisely purify the crude product, and it was used directly in the next reaction.

[0158] Step E / F: Synthesis of compound 2-1f Step E: Compound 2-1e (1 equivalent), iodobenzene acetate (3 equivalents), TEMPO (0.5 equivalents), and t-butyl alcohol / dichloromethane / water (volume ratio 4:4:1) were sequentially added to a single-necked flask. The resulting mixture was stirred at room temperature, and the progress of the reaction was monitored by TLC (eluent: PE / siRNA = 2 / 1, containing 1 v / v% acetic acid). Once the reaction was complete, saturated sodium thiosulfate was added to the system to stop the reaction, and the mixture was extracted with dichloromethane and separated. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting carboxylic acid intermediate did not require further purification and was used directly in the next reaction.

[0159] Step F: The carboxylic acid intermediate obtained in the previous step was dissolved in DMF, methyl iodide (3 equivalents) and potassium carbonate (5 equivalents) were added, and the mixture was stirred at room temperature. The progress of the reaction was monitored by TLC (eluent: PE / Ã=5 / 1). After the reaction was complete (approximately 2 hours), water was added to the system, and the mixture was extracted with ethyl acetate and separated. The organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / Ã=7:1) to obtain compound 2-1f (white solid, total yield of 3 steps 59%). 1 H NMR (400 MHz, chloroform-d) δ, 7.42-7.26 (m, 12H, Ar-H), 7.16 (d, J= 7.6 Hz, 2H, Ar-H), 5.02 (dd, J = 8.4, 8.0 Hz, 1H), 4.83 (d, J = 11.6 Hz, 1H, PhCH2), 4.79 (d, J = 11.2 Hz, 1H, PhCH2), 4.70 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.62 (d, J = 10.0 Hz, 1H), 3.97 (d, J = 10.0 Hz, 1H), 3.90 (dd, J = 8.4, 8.0 Hz, 1H), 3.79 (s, 3H, OMe), 3.72 (dd, J = 8.8, 8.8 Hz, 1H), 2.38 (s, 3H, Ar-CH3), 2.04 (s, 3H, OAc). C 30 H 33 O7S + [M+H] + In MS(ESI)m / z, the calculated value was 537.2, while the measured value was 537.4.

[0160] Step G: Synthesis of 2-1g of compound Compound 2-1f (1.0 equivalent) and acetone were sequentially added to a 50 mL single-necked flask, the system was cooled to 0°C, N-bromosuccinimide (1.4 equivalents) was added, and the mixture was stirred at 0°C for approximately 1 hour until TLC (eluent: PE / Ã=4 / 1) indicated completion of the reaction. Saturated sodium thiosulfate solution was added to the system to halt the reaction, and the acetone was removed by rotary evaporation under reduced pressure. Water and ethyl acetate were added, and the mixture was extracted by liquid-liquid extraction. The organic phase was combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / Ã=6:1) to obtain compound 2-1 g (white solid, yield 90%). 23 H 27 O8 + [M+H] + In MS(ESI)m / z, the calculated value was 431.2, while the measured value was 431.5.

[0161] Step H: Synthesis of Compound 2-1 Under a nitrogen atmosphere, 2-1 g (1 equivalent) of compound, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 0.1 equivalent), and dichloromethane were sequentially added to a 50 mL single-necked flask. The system was cooled to 0°C and mixed uniformly, then trichloroacetonitrile (4 equivalents) was added. The ice bath was removed, and the mixture was allowed to rise naturally to room temperature while mixing. The reaction was monitored by TLC (eluent: PE / siRNA=2 / 1), and after the reaction was complete (approximately 2 hours), the solvent was removed by rotary evaporation under reduced pressure. The sample was fed by wet chromatography and purified by column chromatography (eluent: PE / siRNA=4:1) to obtain compound 2-1 (pale yellow viscous oily liquid, yield 87%). 23 H 25 O7 + [M-Cl3CC(NH)O - ] + In MS(ESI)m / z, the calculated value was 413.2, and the measured value was also 413.2.

[0162] (2) Synthesis of compound 2-2 [ka]

[0163] Step A: Synthesis of compound 2-2b Compound 2-2a (CAS: 1235137-45-7) is commercially available and was prepared by referring to the synthesis method of Compound 2 described in the literature Carbohydr Res 2016, 426, 33.

[0164] At room temperature, compound 2-2a (1 equivalent), methanol, and sodium methoxide (0.1 equivalent, 5M in MeOH) were added to a single-necked flask and stirred at room temperature. The reaction was monitored by TLC (eluent: PE / siRNA = 2 / 1). After the reaction was complete (approximately 1 hour), dilute hydrochloric acid (1M) was added to neutralize the solution to pH = 7. The reaction mixture was concentrated, then toluene was added, and the system was evaporated under reduced pressure by rotation to remove residual water by azeotropic effect, yielding a light brown oily crude product 2-2b, which was used directly in the next reaction without purification.

[0165] Step B: Synthesis of Compound 2-2c At room temperature, p-toluenesulfonic acid monohydrate (0.2 equivalents) and anhydrous acetonitrile were sequentially added to the crude reaction product 2-2b (1 equivalent) from the previous step, and the mixture was thoroughly stirred until homogeneous. The system was then replaced with a nitrogen atmosphere, and benzaldehyde dimethyl acetal (5 equivalents) was added. The resulting reaction liquid was stirred overnight at room temperature until TLC (eluent: PE / siRNA=5 / 1) indicated completion of the reaction. Then, saturated sodium carbonate solution was added to the reaction system to halt the reaction, and the mixture was extracted with dichloromethane and separated. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the sample was fed by wet chromatography. Purification by silica gel column chromatography (eluent: PE / siRNA=5 / 1) was obtained to obtain compound 2-2c (white solid, total yield of 85% from the two steps). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.54 (m, 2H), 7.52 - 7.39 (m, 5H), 7.25 - 7.14 (m, 2H), 5.60 (s, 1H), 5.53 (d, J = 5.4 Hz, 1H), 4.52 - 4.39 (m, 1H), 4.28 (dd, J = 10.4, 4.9 Hz, 1H), 4.07 (t, J = 9.5 Hz, 1H), 3.92 (dd, J = 10.0, 5.6 Hz, 1H), 3.79 (t, J = 10.3 Hz, 1H), 3.60 (t, J = 9.3 Hz, 1H), 3.08 (br s, 1H), 2.40 (s, 3H).C 20 H 22 N3O4S + [M+H] + The MS(ESI) m / z values ​​were calculated at 400.1 and measured at 399.9. The NMR data was consistent with the literature report (see compound 47 in Angew.Chem.Int.Ed.2021,60,12413).

[0166] Step C: Synthesis of compound 2-2d Under a nitrogen atmosphere, compound 2-2c (1 equivalent) and anhydrous tetrahydrofuran (reaction concentration 0.2 M) were sequentially added to a dry two-necked flask, and the mixture was cooled to 0°C in an ice bath. Sodium hydride (1.2 equivalents, 60% content, dispersed in mineral oil) was added, and after the addition was complete, the ice bath was removed and the mixture was heated to room temperature and stirred. After 1 hour, tetrabutylammonium iodide (0.1 equivalent) and benzyl bromide (1.5 equivalents) were added, and the resulting reaction system was stirred at room temperature until TLC (eluent: PE / siRNA=8 / 1) indicated the completion of the reaction (approximately 6 hours). After the reaction was complete, water was added dropwise to stop the reaction, and the mixture was extracted with ethyl acetate and separated. The combined organic phases were sequentially washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE / siRNA=5 / 1) to obtain compound 2-2d (white solid, yield 97%). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.55 (m, 2H), 7.53 - 7.33 (m, 10H), 7.20 (d, J = 8.4 Hz, 2H), 5.67 (s, 1H), 5.56 (d, J = 4.6 Hz, 1H), 5.05 (d, J = 10.9 Hz, 1H), 4.90 (d, J = 10.9 Hz, 1H), 4.59 - 4.47 (m, 1H), 4.31 (dd, J = 10.4, 4.9 Hz, 1H), 4.10 - 3.97 (m, 2H), 3.90 - 3.77 (m, 2H), 2.41 (s, 3H). C 27 H 28 N3O4S + [M+H] + The MS(ESI) m / z values ​​were calculated at 490.2 and measured at 490.5. The NMR data was consistent with the literature report (see compound 20 listed in Bioorg.Med.Chem.2011,19,30).

[0167] Step D: Synthesis of compound 2-2e In a single-necked flask, compound 2-2d (1 equivalent), tetrahydrofuran / methanol (v / v=1:1, reaction concentration 0.5M), and p-toluenesulfonic acid monohydrate (0.2 equivalents) were added and stirred overnight at room temperature. The reaction was monitored by TLC (eluent: PE / siRNA=8 / 1), and once the reaction was complete (approximately 12 hours), saturated sodium bicarbonate solution was added to the system to halt the reaction. The mixture was extracted with ethyl acetate and separated. The combined organic phase was washed with water, then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the sample was fed by wet chromatography. The mixture was purified by silica gel column chromatography (eluent: siRNA / PE=1 / 1) to obtain compound 2-2e. 1 H NMR (400 MHz, chloroform-d) δ 7.41 - 7.34 (m, 7H), 7.14 (d, J = 8.0 Hz, 2H), 5.49 (d, J = 5.2 Hz, 1H), 5.01 (d, J = 11.2 Hz, 1H), 4.77 (d, J = C 20 H 27 N4O4S + [M+NH4] + In MS(ESI)m / z, the calculated value was 419.2, and the measured value was also 419.2.

[0168] Step E: Synthesis of Compound 2-2 Compound 2-2e (1 equivalent), dichloromethane (reaction concentration 0.5 M), imidazole (2 equivalents), and t-butyldiphenylchlorosilane (1.5 equivalents) were sequentially added to a single-necked flask. The mixture was stirred at room temperature, and the reaction was monitored using TLC (eluent: siRNA / PE = 1 / 12). Once the reaction was complete (approximately 6 hours), saturated ammonium chloride solution was added to the system to halt the reaction. The mixture was then extracted with dichloromethane and separated. The combined organic phase was washed with water, then with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the sample was fed by a wet process. Purification by silica gel column chromatography (eluent: PE / siRNA / DCM = 15 / 1 / 1) yielded compound 2-2 (yellow viscous oily liquid, yield 66%). 1 H NMR (400 MHz, chloroform-d) δ 7.73 -7.68 (m, 4H), 7.48-7.35 (m, 13H), 7.06 (d, J = 7.6 Hz, 1H), 5.49 (d, J = 5.2 Hz, 1H), 4.94 (ABq, J = 10.8 Hz, 2H), 4.32-4.27 (m, 1H), 3.96-3.90 (m, 2H), 3.87 (dd, J = 4.8, 5.2 Hz, 1H), 3.82 (dt, J = 2.8, 8.8 Hz, 1H), 3.74-3.69 (m, 1H), 2.70 (d, J = 2.8 Hz, OH), 2.33 (s, 3H), 1.09 (s, 9H).C 36 H 42 N3O4SSi + [M+H] + In MS(ESI)m / z, the calculated value was 640.3, while the measured value was 640.2.

[0169] (3) Synthesis of Compound 2 [ka]

[0170] Step A: Synthesis of Compound 2a A pre-activated molecular sieve was added to a dry two-necked flask, baked with a heat gun under vacuum conditions using an oil pump, allowed to cool, and then replaced with nitrogen gas. The vacuum-evacuation-nitrogen gas replacement procedure described above was repeated three times. Then, under nitrogen protection, compound 2-1 (2 equivalents), compound 2-2 (1 equivalent), and dry toluene were added to the system, and the resulting system was stirred at room temperature for 0.5 hours to thoroughly remove any remaining moisture. Subsequently, the reaction system was cooled to -40°C and stirred, a fixed amount of TMSOTf was added, and the system was stirred while maintaining the temperature until TLC measurements indicated completion of the reaction (approximately 3 hours). Triethylamine was added to the system to stop the reaction, filtered, concentrated, and the sample was fed by wet chromatography. Compound 2a (white solid, 60% yield) was obtained by separation by silica gel column chromatography. 1 H NMR (400 MHz, chloroform-d) δ 7.72 (d, J = 6.4 Hz, 2H), 7.64 (d, J = 6.4 Hz, 2H), 7.49 - 7.47 (m, 2H), 7.44 - 7.23 (m, 21H), 7.04 (d, J = 7.8 Hz, 2H), 5.57 (d, J = 7.2 Hz, 1H), 5.17 (d, J = 7.2 Hz, 1H), 5.10 (dd, J = 9.7, 8.1 Hz, 1H), 4.93 (d, J = 8.0 Hz, 1H), 4.82 (d, J = 11.4 Hz, 1H), 4.74 (d, J = 10.9 Hz, 1H), 4.66 - 4.60 (m, 3H), 4.20 (dd, J = 9.2, 9.2 Hz, 1H), 4.05 - 4.02 (m, 2H), 3.97 (dd, J = 9.3, 9.2 Hz, 1H), 3.92 - 3.76 (m, C 59 H 66 N3O 11 SSi + [M+H] +In MS(ESI)m / z, the calculated value was 1052.4, while the measured value was 1052.7.

[0171] Step B: Synthesis of Compound 2b In a reaction flask, compound 2a (1 equivalent) and thioglycolic acid (CAS: 68-11-1) / pyridine / trichloromethane (v / v / v = 1:1:1) were added, the system was heated to 60°C and stirred, and the progress of the reaction was monitored by TLC. Once the reaction was complete (approximately 12 hours), the mixture was concentrated under reduced pressure to remove most of the solvent, an appropriate amount of ethyl acetate was added, and the mixture was washed with saturated sodium bicarbonate solution. After extraction and liquid-liquid separation, the organic phase was washed with 1 M hydrochloric acid solution, liquid-liquid separation was performed, and the mixture was washed again with saturated sodium bicarbonate solution. The resulting organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 2b (white solid, 71% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.70 (d, J = 6.8 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.44 - 7.28 (m, 20H), 7.26 - 7.21 (m, 3H), 7.00 (d, J = 7.9 Hz, 2H), 5.68 (d, J = 4.8 Hz, 1H), 5.36 - 5.30 (m, 1H), 5.09 (dd, J = 9.5, 8.1 Hz, 1H), 4.92 (d, J = 12.4 Hz, 1H), 4.85 (d, J = 11.5 Hz, 1H), 4.75 - 4.66 (m, 3H), 4.63- 4.57 (m, 2H), 4.29 (ddd, J = 9.3, 7.7, 4.8 Hz, 1H), 4.10 (t, J = 7.8 Hz, 1H), 4.01 (dd, J = 11.5, 3.2 Hz, 1H), 3.94 - 3.89 (m, 2H), 3.86 - 3.84 (m, 1H), 3.80 (dd, J = 11.5, 2.5 Hz, 1H), 3.67 (s, 3H), 3.60 - 3.51 (m, 2H), 2.28 (s, 3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H). C61 H 70 NO 12 SSi + [M+H] + In MS(ESI)m / z, the calculated value was 1068.4, and the measured value was also 1068.4.

[0172] Step C: Synthesis of Compound 2c After adding compound 2b (1 equivalent) and tetrahydrofuran to the reaction flask, add TBAF (2 equivalents, 1M in THF) to the system, stir at room temperature, monitor the progress of the reaction by TLC, and when the reaction is complete (approximately 10 hours), add saturated ammonium chloride solution to the system to stop the reaction, extract with ethyl acetate and separate, wash the combined organic phase with water, wash with saturated brine, dry with anhydrous sodium sulfate, filter, concentrate, feed the sample by wet method, and pre-purify by silica gel column chromatography to obtain compound 2c (white solid, 56% crude yield). 45 H 52 NO 12 S + [M+H] + In MS(ESI)m / z, the calculated value was 830.3, while the measured value was 830.6.

[0173] Step D: Synthesis of compound 2d Compound 2c was dissolved in THF / MeOH (v / v=3:1), and a 1M aqueous sodium hydroxide solution was slowly added to adjust the system to pH=12. The resulting reaction mixture was stirred at room temperature, and the progress of the reaction was monitored by HPLC. Once the reaction was complete (approximately 12 hours), 1M hydrochloric acid was added to the system to adjust the pH to 7-8. The mixture was concentrated under reduced pressure to remove most of the organic solvent, and the resulting crude product was purified by HPLC to obtain compound 2d (white solid, 82% yield). 1H NMR (400 MHz, chloroform-d) δ 7.43 - 7.27 (m, 17H), 7.07 (d, J = 7.9 Hz, 2H), 5.54 (d, J = 5.0 Hz, 1H), 5.33 (d, J = 8.0 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.86 (d, J = 11.3 Hz, 1H), 4.82 - 4.77 (m, 3H), 4.74 (d, J = 6.8 Hz, 1H), 4.69 (d, J = 12.2 Hz, 1H), 4.36 (ddd, J = 10.6, 7.9, 5.0 Hz, 1H), 4.25 - 4.22 (m, 1H), 4.13 - 4.02 (m, 3H), 3.88 (t, J = 8.6 Hz, 1H), 3.81 (dd, J = 12.7, 2.2 Hz, 1H), 3.74 (dd, J = 10.7, 8.5 Hz, 1H), 3.68 - 3.59 (m, 2H), 3.40 (br s, 1H), 2.30 (s, 3H), 1.77 (s, 3H). 13 ¹³C NMR (100 MHz, chloroform-d) δ 170.73, 169.33, 138.20, 138.06, 137.81, 137.55, 132.37, 129.99, 129.44, 128.84, 128.76, 128.58, 128.42, 128.30, 127.90, 103.24, 88.40, 83.43, 78.89, 77.85, 77.35, 77.24, 77.03, 76.92, 76.72, 75.17, 74.93, 74.57, 74.02, 73.93, 72.95, 60.66, 52.95, 23.08, 21.06 (In the aromatic region, three carbon signals overlapped and no peak was observed). C 42 H 46 NO 11 S - [MH] - In MS(ESI)m / z, the calculated value was 772.3, while the measured value was 772.2.

[0174] Step E: Synthesis of Compound 2e Compound 2d was weighed and dissolved in acetone, then stirred in an ice bath for 5 minutes and allowed to cool. NBS was weighed and added to the reaction mixture, and the mixture was reacted in an ice bath for approximately 1 hour. A sample was taken and subjected to HPLC measurement to confirm the completion of the reaction. After stopping the reaction with a saturated sodium thiosulfate solution, the mixture was concentrated to remove the acetone, prepared by prep-HPLC, and freeze-dried to obtain compound 2e (white solid, 45% yield). 35 H 40 NO 12 - [MH] - In MS(ESI)m / z, the calculated value was 666.3, while the measured value was 666.4.

[0175] Step F: Synthesis of Compound 2 At room temperature, compound 2e, tetrahydrofuran, methanol, and palladium-carbon catalyst were sequentially added to a 50 mL single-necked flask, and the reaction system was stirred under a hydrogen atmosphere until all the starting materials were consumed, under TLC measurement. The mixture was filtered, concentrated under reduced pressure, and extracted and dried with an oil pump to obtain compound 2 (white solid, 95% yield). 14 H 22 NO 12 - [MH + ] - In MS(ESI)m / z, the calculated value was 396.1, and the measured value was also 396.1.

[0176] Example 3 Preparation of disaccharide substrate compound 3 Compound 3 was prepared using the following procedure, and its structure is as follows: [ka]

[0177] The manufacturing process for compound 3 is based on a similar method to the synthesis of compound 2, and the specific route is as follows: [ka]

[0178] The final product, compound 3, was verified by mass spectrometry, and C 14 H 22 NO 12 - [MH + ] - In MS(ESI)m / z, the calculated value was 396.1, while the measured value was 396.0.

[0179] Example 4 Preparation of disaccharide substrate compound 4 Compound 4 was prepared using the following procedure, and its structure is as follows: [ka]

[0180] The manufacturing process for compound 4 is based on a similar method to the synthesis of compound 2, and the specific route is as follows: [ka]

[0181] The final product, compound 4, was verified by mass spectrometry, and C 14 H 22 NO 12 - [MH + ] - In MS(ESI)m / z, the calculated value was 396.1, and the measured value was also 396.1.

[0182] Example 5 Preparation of disaccharide substrate compound 5 Compound 5 was prepared using the following procedure, and its structure is as follows: [ka]

[0183] The manufacturing process for compound 5 is based on a similar method to the synthesis of compound 2, and the specific route is as follows: [ka]

[0184] The final product, compound 5, was verified by mass spectrometry, and C 14 H 22 NO 12 - [MH + ] - In MS(ESI)m / z, the calculated value was 396.1, while the measured value was 396.0.

[0185] 2. Manufacturing of the linker-payload (hereinafter abbreviated as LP)

[0186] Example 6 Manufacturing of LP-1 The structure of the linker-payload 1 (LP-1) is as follows: [ka]

[0187] (1) Production of compound LP-1b [ka] At room temperature, compound 1 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS: 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were sequentially added to a 10 mL single-necked flask. The resulting reaction mixture was stirred at room temperature under HPLC monitoring until the reaction was complete. The reaction mixture was purified by semi-preparative HPLC to obtain compound LP-1b (white solid, yield 81.2%). 78 H 126 N 14 O 26 2+ [M+2H] 2+ In MS(ESI)m / z, the calculated value was 837.4, while the measured value was 837.9.

[0188] (2) Production of compound LP-1 [ka] At room temperature, compound LP-1b (21.7 mg, 0.013 mmol, 1 equivalent), H2O, Et3N (1 to 100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1 to 100 equivalents) were sequentially added to a 10 mL single-necked flask. The reaction was monitored by HPLC, and the resulting reaction mixture was stirred at room temperature until the reaction was complete. The reaction mixture was purified by semi-preparative HPLC to obtain compound LP-1 (15.3 mg, yield 71.3%, white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ In MS(ESI)m / z, the calculated value was 828.4, while the measured value was 828.6.

[0189] Example 7 Manufacturing of LP-2 The structure of the linker-payload 2 (LP-2) is as follows: [ka]

[0190] The manufacturing process is as follows: [ka]

[0191] 20.1 Step A: Preparation of Compound LP-2a At room temperature, compound 2 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS: 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were sequentially added to a 10 mL single-necked flask. The resulting reaction mixture was stirred at room temperature under HPLC monitoring until the reaction was complete. The reaction mixture was purified by semi-preparative HPLC to obtain compound LP-2a (white solid, yield 84%). 78 H126 N 14 O 26 2+ [M+2H] 2+ In MS(ESI)m / z, the calculated value was 837.4, while the measured value was 837.8.

[0192] 20.2 Step B: Preparation of Compound LP-2 At room temperature, compound LP-2a (1 equivalent), H2O, Et3N (1-100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1-100 equivalents) were sequentially added to a 10 mL single-necked flask. The resulting reaction mixture was stirred at 0°C, and the reaction was monitored by HPLC until complete. The reaction mixture was purified by semi-preparative HPLC to obtain compound LP-2 (yield 87%, white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ In MS(ESI)m / z, the calculated value was 828.4, while the measured value was 828.7.

[0193] Example 8 Manufacturing of LP-3, LP-4, and LP-5 Linker-payloads LP-3, LP-4, and LP-5 were manufactured using a procedure similar to that used for LP-2, with the following structures. [ka]

[0194] Example 9 Manufacturing of LP-6 [ka]

[0195] (1) Synthesis of LP-6-1 [ka]

[0196] Step A: Synthesis of intermediate LP-6-1b Compound LP-6-1a (1.0 equivalent) and DMF (solubility 1 g / mL) were added to a reaction flask and stirred under a nitrogen atmosphere to dissolve. After cooling to 0-5°C, DIEA (3 equivalents) was added dropwise, and the resulting system was stirred at 5°C for 10 minutes. Then, benzyl bromide (1.3 equivalents) was added dropwise. After the addition was complete, the reaction system was allowed to rise naturally to room temperature and stirred for 16 hours. The reaction mixture was slowly poured into ice water, methyl-t-butyl ether was added and stirred, and then the mixture was allowed to stand and separated. The aqueous phase was extracted four times with methyl-t-butyl ether, and the organic phase was combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude yellow oily product. The sample was fed by wet method and purified by silica gel column chromatography (eluent:PE / EA=6:1) to obtain product LP-6-1b (pale yellow oily product, quantitative yield).

[0197] Step B: Synthesis of intermediate LP-6-1d Under nitrogen protection, intermediate LP-6-1b (2.0 equivalents), compound LP-6-1c (1.0 equivalent), and THF (solubility 10 g / mL) were added to the reaction flask and stirred to dissolve. TsOH (0.1 equivalent) was then added to the reaction, and the system was reacted at room temperature for 4 hours. The reaction mixture was slowly poured into ice water and extracted three times with ethyl acetate. The combined organic phases were sequentially washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. This was purified by silica gel column chromatography (eluent:PE / EA = 1:1) to obtain product LP-6-1d (white solid, yield 40%).

[0198] Step C: Synthesis of intermediate LP-6-1e Under nitrogen protection, compound LP-6-1d and N,N-dimethylacetamide (DMAc, solubility 10 g / mL) were added to the reaction flask and stirred to dissolve. The system was then cooled to 14-18°C, and DBU (0.5 equivalents) was added dropwise. The reaction was carried out while maintaining the temperature until TLC indicated completion of the reaction (approximately 1.5 hours) to obtain the intermediate LP-6-1e. Purification was not necessary, and it was used directly in the next reaction.

[0199] Step D: Synthesis of intermediate LP-6-1g The reaction mixture from the previous step was cooled to 0-5°C, and pyridinium 4-methylbenzenesulfonate (PPTS, 0.5 equivalents), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI, 1.0 equivalent), 1-hydroxybenzotriazole (HOBT, 1.0 equivalent), and LP-6-1f (0.85 equivalents) were added sequentially. The reaction system was allowed to react at 0-10°C until LCMS indicated completion of the reaction (approximately 4 hours). The reaction mixture was added to ice water, extracted once with 2-methyltetrahydrofuran, and the aqueous phase was further extracted twice with 2-methyltetrahydrofuran. The organic phase was combined and sequentially washed with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: DCM / MeOH) to obtain product LP-6-1g (white solid, yield 78%).

[0200] Step E: Synthesis of intermediate LP-6-1h Under nitrogen protection, 1 g of LP-6 and DMAc (solubility concentration 10 g / mL) were added to the reaction flask and stirred to dissolve. The temperature was lowered to 14-18°C, and 0.5 equivalents of DBU were added dropwise. The mixture was stirred at this temperature for 1.5 hours to allow the reaction to proceed. The progress of the reaction was monitored by TLC, and once the reaction was complete, the intermediate LP-6-1h was obtained. Purification was not necessary, and it was used directly in the next reaction.

[0201] Step F: Synthesis of intermediate LP-6-1j The reaction solution of LP-6-1h from the previous step was cooled to 0-5°C, PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent), and compound 3i (0.85 equivalent) were added, and the reaction was carried out at 0-10°C for 3-4 hours. The progress of the reaction was monitored by LC-MS, and once the reaction was complete, the reaction solution was added to ice water, 2-methyltetrahydrofuran was added and extracted once, the aqueous phase was further extracted twice with 2-methyltetrahydrofuran, the organic phase was combined, and sequentially washed with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, stirred by dry method, and purified by column chromatography (eluent: DCM / MeOH) to obtain LP-6-1j (white solid, yield 50%).

[0202] Step G: Synthesis of compound LP-6-1 Under nitrogen protection, the intermediate LP-6-1j was dissolved in DCM (concentration 15 g / mL), and DBU (0.5 equivalents) was added dropwise at 20°C. The reaction was stirred while maintaining the temperature until HPLC indicated completion of the reaction. Subsequently, DCM was added to the system to dilute the reaction mixture, and the sample was directly fed by a wet method and purified by column chromatography (eluent: DCM / MeOH) to obtain compound LP-6-1 (white solid, yield 82%). 34 H 47 N6O 10 + [M+H] + In MS(ESI)m / z, the calculated value was 699.4, while the measured value was 699.6.

[0203] (2) Synthesis of LP-6-2 [ka] LP-6-2 was synthesized using a peptide solid-phase synthesis method, and the procedure is as shown below.

[0204] Step 1: Preparation of NH2-Asp(OtBu)-Rink amide resin 400 g of Rink amide resin was weighed and placed in a reaction vessel, then immersed in 2400 mL of DCM for 0.5 hours to allow the resin to swell sufficiently, followed by extraction and drying. 2400 mL of decapping reagent was added, the resin was washed, and then extracted and dried. 2400 mL of decapping reagent was added, and the mixture was stirred at 25±1°C for 0.5 hours with nitrogen gas flowing through, followed by extraction and drying. The mixture was then sequentially washed twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, each time followed by extraction and drying. Kaiser measurement showed a blue color.

[0205] 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The mixture was stirred at room temperature for 2 hours with nitrogen gas flowing through it, and then extracted and dried. The mixture was washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and then extracted and dried. The Kaiser assay showed a pale blue color. 2400 mL of DCM was added, followed by 60 mL of capping reagent. The mixture was stirred at 25±1°C for 1 hour with nitrogen gas flowing through it, and then extracted and dried. The mixture was washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and then extracted and dried. The Kaiser measurement showed it was colorless.

[0206] The sample was washed with 2400 mL of decapping reagent and extracted and dried. The sample was reacted by stirring for 0.5 hours at 25±1°C with nitrogen gas passing through, and then extracted and dried. The sample was then washed twice each with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, extracted and dried, and the Kaiser assay showed a blue color.

[0207] Step 2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80.0 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The reaction was continued at room temperature with nitrogen gas flowing through for 2-4 hours, followed by extraction and drying. The solution was then washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed the solution was colorless.

[0208] 2400 mL of decapping reagent was added, the mixture was washed, and extracted and dried. Another 2400 mL of decapping reagent was added, and the mixture was stirred at 25±1°C for 0.5 hours with nitrogen gas flowing through it, followed by extraction and drying. The mixture was then washed twice each with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed a blue color.

[0209] Step 3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The reaction was continued at room temperature with nitrogen gas flowing through for 2-4 hours with stirring, followed by extraction and drying. The solution was then washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed the solution was colorless.

[0210] 2400 mL of decapping reagent was added, the mixture was washed, and extracted and dried. Another 2400 mL of decapping reagent was added, and the mixture was stirred at 25±1°C for 0.5 hours with nitrogen gas flowing through it, followed by extraction and drying. The mixture was then washed twice each with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed a blue color.

[0211] Step 4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80.0 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The reaction was continued at room temperature with nitrogen gas flowing through for 2-4 hours, followed by extraction and drying. The solution was then washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed the solution was colorless.

[0212] 2400±100 mL of decapping reagent was added, the mixture was washed, and extracted and dried. Another 2400 mL of decapping reagent was added, and the mixture was stirred at 25±1°C for 0.5 hours with nitrogen gas flowing through it, followed by extraction and drying. The mixture was then washed twice each with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, extracted and dried, and the Kaiser assay showed a blue color.

[0213] Step 5: Manufacturing of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80.0 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The reaction was continued at room temperature with nitrogen gas flowing through for 2-4 hours, followed by extraction and drying. The solution was then washed twice sequentially with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, and extracted and dried. The Kaiser assay showed the solution was colorless.

[0214] Step 6: Production of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 2400 mL of dde removal reagent was added, and the mixture was stirred at 25±1°C for 10 minutes with nitrogen gas flowing through it to allow the reaction to proceed. The mixture was then extracted and dried, and this procedure was repeated three times. After that, the mixture was washed twice each with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, respectively, and then extracted and dried. The Kaiser assay showed a blue color.

[0215] Step 7: Manufacturing of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF solution and 80.0 mL of DIC. After being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction vessel and reacted. The reaction was carried out at room temperature with nitrogen gas flowing through for 2-4 hours with stirring, and then extracted and dried. The mixture was sequentially washed twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, extracted and dried, and was colorless by Kaiser measurement. The resin peptide was washed three times with 2400 mL of anhydrous ethanol, extracted and dried, and prepared for cutting.

[0216] Step 8: Manufacturing LP-6-2 10,000 mL of cutting reagent (TFA:TIS:H2O = 95:2.5:2.5) was added to a 10 L reaction vessel, cooled to -10 ± 2 °C, and the dried and weighed resin was added. The temperature was then raised, and the mixture was stirred for 2 hours at 20 ± 5 °C with nitrogen gas flowing through it. The mixture was filtered, the resin was washed once with 100 mL of TFA, and the filtrate and washing solution were combined.

[0217] 40 L of pre-cooled (-10°C or below) cold ethyl ether was added, stirred for 10 minutes, and then centrifuged to precipitate. After centrifuging, the supernatant was discarded, the precipitate was mixed with cold ethyl ether and shaken uniformly, and then centrifuged again (this process was repeated three times, with 10 L, 10 L, and 10 L used each time. The centrifugal speed was set to 3600 rpm, the centrifugal time to 5 minutes, and the centrifuge chamber temperature to -5°C each time).

[0218] The precipitate was collected, which was the crude LP-6-2 product. After purification by Prep-HPLC and lyophilization, LP-6-2 was obtained. 74 H 121 N9O 31 2+ [M+2H] 2+ In MS(ESI)m / z, the calculated value was 815.9, while the measured value was 816.3.

[0219] (3) Synthesis of LP-6-3 [ka]

[0220] Step A: Synthesis of intermediate LP-6-3a Compounds LP-6-1 (2.2 equivalents) and LP-6-2 (1.0 equivalent) were added to a reaction flask and dissolved in DMF. Then, DIPEA (5.0 equivalents) was added and the mixture was mixed uniformly. After adding HATU (2.5 equivalents) to the system, the reaction was allowed to proceed at room temperature and monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was prepared directly by prep-HPLC. The prepared solution was freeze-dried to obtain compound LP-6-3a (white solid, yield 52%). 143 H 212 O 48 N 21 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 997.2, while the measured value was 875.9 (some functional groups may be lost after ionization).

[0221] Step B: Synthesis of intermediate LP-6-3b Compound LP-6-3a was dissolved in purified water, a certain amount of palladium hydroxide (10 wt% Pd(OH)2 supported on carbon) was added, the system was purged three times with hydrogen gas, and the mixture was stirred at room temperature for 1.5 hours. The progress of the reaction was monitored during the reaction, and the reaction was stopped immediately after the starting materials were used up to prevent an increase in the amount of Fmoc-free product. The reaction mixture was filtered and prepared by prep-HPLC to obtain compound LP-6-3b (white solid, yield 76%). 128 H 200 O48 N 21 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 937.1, while the measured value was 875.9 (some functional groups may be lost after ionization).

[0222] Step C: Synthesis of intermediate LP-6-3d Compounds LP-6-3b (1.0 equivalent) and LP-6-3c (2.2 equivalents) were weighed and dissolved in DMF. DIPEA (5.0 equivalents) was added and the mixture was mixed uniformly. Then HATU (2.5 equivalents) was added, and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until complete (approximately 16 hours). The reaction system was prepared directly by prep-HPLC. After lyophilization of the prepared solution, compound LP-6-3d (yellow solid, yield 66%) was obtained. 174 H 232 O 55 Cl2F2N 27 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1229.2, while the measured value was 1229.3.

[0223] Step D: Synthesis of compound LP-6-3 Compound LP-6-3d is dissolved in DMF, diethylamine is added, and the reaction is carried out at room temperature. The reaction is monitored by HPLC until it is complete (approximately 0.5 hours). After the reaction is complete, the pH is adjusted to neutral, and the compound is prepared by prep-HPLC. After lyophilization, compound LP-6-3 (yellow solid, yield 73%) is obtained. 159 H 222 O 53 Cl2F2N 27 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1155.2, while the measured value was 1155.3.

[0224] For details on the synthesis of compound LP-6-3c, please refer to Chinese patent CN202211428194.6.

[0225] (4) Synthesis of LP-6 [ka]

[0226] Synthesis of LP-6a Compound 1 (0.5-5.0 equivalents) and compound LP-6-3 (1.0 equivalent) were taken and dissolved in DMF, DIPEA (1-10 equivalents) was added and the mixture was uniformly stirred at 0°C, HATU (0.5-10 equivalents) was added, and the reaction system was stirred at 0°C and monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was prepared directly by prep-HPLC, and after lyophilization of the prepared solution, compound LP-6a (yellow solid, yield 63%) was obtained. 173 H 243 O 64 Cl2F2N 28 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1281.5, while the measured value was 1281.8.

[0227] LP-6 synthesis Compound LP-6a is weighed and dissolved in purified water, then stirred in an ice bath and cooled. Et3N (1-100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1-100 equivalents) are added, the reaction is monitored by HPLC, and after the reaction is complete, it is purified by prep-HPLC. The prepared solution is freeze-dried to obtain compound LP-6 (pale yellow solid, yield 74%). 173 H 241 O 63 Cl2F2N 28 3+ [M+3H] 3+ The MS(ESI)m / z values ​​were calculated to be 1275.5 and measured to be 1275.9.

[0228] Example 9 Manufacturing of the LP-7 The structure of the LP-7 is as follows: [ka]

[0229] The synthesis pathway is as follows: [ka]

[0230] Step A: Synthesis of compound LP-7b Compound LP-7a was synthesized by solid-phase peptide synthesis, following a similar method to that used for LP-6-2.

[0231] Compound LP-7a (1.0 equivalent) and compound LP-6-1 (3.6 equivalents) were added to a reaction flask and dissolved in DMF. Then DIPEA (6.0 equivalents) was added and mixed uniformly. HATU (3.6 equivalents) was added to the system and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until complete (approximately 2 hours). The reaction system was prepared directly by prep-HPLC. After lyophilization of the prepared solution, compound LP-7b (white solid, yield 56%) was obtained. 191 H 280 O 66 N 29 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1345.3, while the measured value was 1345.8.

[0232] Step B: Synthesis of intermediate LP-7c Compound LP-7b is dissolved in purified water, a certain amount of palladium hydroxide (10 wt% Pd(OH)2 supported on activated carbon) is added, the system is purged with hydrogen gas three times, and the mixture is stirred at room temperature for 1.5 hours. The reaction is monitored during the reaction, the reaction solution is filtered, and compound LP-7c (white solid, yield 80%) is obtained by direct prep-HPLC. 170 H 262 O 66 N 29 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1255.3, while the measured value was 1255.9.

[0233] Step C: Synthesis of intermediate LP-7d Weigh compound LP-7c (1.0 equivalent) and compound LP-6-3 (1-10 equivalents) and dissolve them in DMF. Add DIPEA (1-20 equivalents) and stir uniformly. Then add HATU (1-10 equivalents) and react at room temperature. Monitor by HPLC until the reaction is complete (approximately 2 hours). Use the reaction product directly in the next reaction. 239 H 313 O 75 Cl3F3N 38 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1692.4, while the measured value was 1692.9.

[0234] Step D: Synthesis of compound LP-7e Compound LP-7d is dissolved in DMF, diethylamine is added, and the reaction is carried out at room temperature. The reaction is monitored by HPLC until it is complete (approximately 0.5 hours). After the reaction is complete, the pH is adjusted to neutral, and the compound is prepared by prep-HPLC. After lyophilization, compound LP-7e (yellow solid, yield 73%) is obtained. 224 H 303 O 73 Cl3F3N 38 3+ [M+3H] 3+ In MS(ESI)m / z, the calculated value was 1618.3, while the measured value was 1618.5.

[0235] Step E: Synthesis of compound LP-7f Compound 1 (0.5-5.0 equivalents) and compound LP-7e (1.0 equivalent) were dissolved in DMF, DIPEA (1-10 equivalents) was added, and the mixture was uniformly stirred at 0°C. HATU (0.5-10 equivalents) was added, and the reaction system was stirred at 0°C. The reaction was monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was prepared directly by prep-HPLC. After lyophilization of the prepared solution, compound LP-7f (yellow solid, 65% yield) was obtained. 238 H 325 O 84 Cl3F3N 39 4+ [M+4H] 4+In MS(ESI)m / z, the calculated value was 1308.8, while the measured value was 1309.0.

[0236] 15.6 Step F: Synthesis of compound LP-7 Compound LP-7f was weighed and dissolved in purified water. After stirring in an ice bath and cooling, Et3N (1-100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1-100 equivalents) were added. The progress of the reaction was monitored by HPLC. After the reaction was complete, the mixture was purified by prep-HPLC, and the prepared solution was freeze-dried to obtain compound LP-7 (pale yellow solid, yield 50%).

[0237] 3. Selection or manufacture of antibodies Antibody selection The antibody-drug conjugate produced by the substrate and compounding method of the present invention may have a polymeric portion that includes any antibody FC region sugar-containing chain, and may include, but is not limited to, antibodies, bispecific antibodies, FC fusion proteins, single-chain antibodies, etc.

[0238] In the specific examples below, commercially available trastuzumab may be selected.

[0239] Furthermore, genetically modified antibodies may be selected. An example of their manufacturing is shown below:

[0240] Example 10 Production of genetically modified anti-HER2 and Trop2 antibodies The production, purification, and identification of anti-human ErbB2 / HER2 antibody mAb-1 and anti-human TROP2 antibody mAb-2 were carried out with reference to Example 1 of Chinese Patent CN106856656B, and the entire contents of CN106856656B are incorporated herein by reference.

[0241] IV. Manufacturing, characterization, and activity testing of ADCs

[0242] Example 11 Manufacturing and characterization of ADC-1 The following steps involved promoting the remodeling of the sugar chain at the Fc end of antibody mAb-1 using endoglycosidase, specifically binding the linker-payload to the antibody, and forming the corresponding ADC drug.

[0243] 1) Processing of antibody mAb-1: The antibody was processed by ultrafiltration, dialysis, or desalting column chromatography, and its storage buffer was changed to 50 mM Tris-HCl (pH between 5 and 8) and 150 mM NaCl.

[0244] 2) Production of ADC-1: ADC-1 was produced by accelerating the compound reaction of antibody mAb-1 and LP-1 using endoglycosidase Endo S2 as a catalyst. In 1× endonuclease buffer, antibody mAb-1 and LP-1 were thoroughly mixed in an appropriate molar ratio (1:1 to 1:100), and endoglycosidase Endo S2 was added and mixed homogeneously. The compound reaction in the homogeneously mixed state was carried out at 4 to 40°C for 0.5 to 20 hours. After the reaction was complete, purification was performed. The purified ADC-1 was stored in 1× PBS at pH 7.4 at 4°C or -80°C.

[0245] The characterization data for the antibody-drug conjugate ADC-1 is as follows:

[0246] SDS-PAGE measurement and analysis of ADC-1 After the compounding reaction was complete, the purity and compounding efficiency of ADC-1 were measured by SDS-PAGE. As shown in Figure 1, the SDS-PAGE results for ADC-1 indicated that the compounding reaction occurred at a predetermined position on the antibody heavy chain, and that the heavy chain bound to the cytotoxin showed a clear molecular weight shift compared to the heavy chain not bound to the cytotoxin. The purity of the compound product was as expected.

[0247] HIC-HPLC measurement and analysis of ADC-1 HIC-HPLC: A Proteomix HIC Butyl-NP5 4.6×100 mm 5 μm non-porous chromatography column was used; the column temperature was set to 30°C; mobile phase A consisted of 1.5 M ammonium sulfate + 20 mM phosphate buffer at pH 7.0; mobile phase B consisted of 20 mM phosphate buffer:isopropanol = 7:3 (v / v); the flow rate was set to 0.8 mL / min; gradient method: phase B was increased from 10% to 100% within 8 mins; the measurement wavelength was set to 280 nm, and the DAR distribution of ADC-1 was measured.

[0248] The measurement results are shown in Figure 2. <1% of the antibody was not bound to LP-1; the complex product was mainly DAR 2, and the mean DAR value of the ADC drug was 1.93.

[0249] High precision molecular weight mass spectrometry (ESI-MS) analysis of ADC-1 High-precision molecular weight mass spectrometry analysis of ADC-1 revealed an apparent molecular weight of 150539.27, and a theoretical molecular weight of 150538.78, which was as expected. This also proved that one cytotoxin molecule is bound to the Fc end of each heavy chain.

[0250] SEC-HPLC measurement and analysis of ADC-1 The high molecular weight aggregation degree of ADC-1 was analyzed and measured using an SEC chromatography column. As shown in Figure 3, the measurement results showed that 6.7 min of ADC-1 was <5% high molecular weight polymer, indicating that the ADC sample mainly existed as monomers, and the damage to the antibody from the complex reaction was almost negligible.

[0251] Measurement of ADC-1 affinity for cell surface ErbB2 / HER2 1) HER2 ECD was taken, prepared to 1 μg / mL with CBS coating solution (0.1 M carbonate buffer, pH 9.6), and coated at 100 μL / well at 25°C for 60 min. 2) After coating was complete, the plate was washed three times with PBST, blocked with blocking solution (5% skim milk powder) (300 μL / well), and incubated at 25°C and 200 rpm for 60 min. 3) After blocking was complete, the plate was washed three times with PBST, antibody mAb-1 and ADC-1 (100 μL / well) were added, and incubated at 25°C and 200 rpm for 60 min. 4) After incubation with the sample was complete, the plate was washed three times with PBST, anti-human IgG Fc-HRP (100 μL / well) was added, and incubated at 25°C and 200 rpm for 60 min. 5) After the 60-minute incubation was complete, the reaction was further controlled with TMB (100 μL / well) for 5 minutes, then stopped with stop solution, and the reading was taken using OD450.

[0252] Referring to Figure 4, the Elisa measurement results show no significant difference in antigen affinity between mAb-1-naked monoclonal antibody and ADC-1 against HER2 ECD.

[0253] Example 12 In vitro activity testing of ADC-1 (BT-474, NCI-N87, HepG2) The effect of ADC-1 on tumor cell proliferation at different levels of ErbB2 / HER2 expression was measured according to the following method.

[0254] 1) ErbB2 / HER2-positive human breast cancer cells BT-474, ErbB2 / HER2-positive human gastric cancer cells NCI-N87, and ErbB2 / HER2-negative human liver cancer cells HepG2 were inoculated into 96-well cell plates at 100 μL (containing 1,000 to 10,000 cells) per well and cultured overnight in a cell incubator at 37°C, 5% CO2, 95% air, and 100% humidity.

[0255] 2) Each of the ErbB2 / HER2-positive cells cultured overnight was treated with different concentrations (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, 0.00051 nM) of ADC-1 or antibody mAb-1, or different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of MMAE (monomethyl auristatin E); cultured overnight. Each group of ErbB2 / HER2-negative cells was treated with either ADC-1 or antibody mAb-1 at different concentrations (100, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) or MMAE at different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM); the control group was treated with puromycin at a final concentration of 5 μM. The cells were incubated at 37°C for 72–120 hours.

[0256] 3) The cell plates were removed from the 37°C cell incubator and allowed to equilibrate to room temperature for approximately 30 minutes. 100 μL of CellTiter Glo reagent was added to each well, and the plates were shaken for 2 minutes using a shaker. After standing at room temperature in the dark for 10 minutes, the luminescence values ​​(RLU) were measured using a Cytation 3 microplate reader.

[0257] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 1, 2 and Figures 5-7. Both ADC-1 and MMAE small molecular weight toxin had a clear inhibitory effect on the proliferation of ErbB2 / HER2-positive cells, the mAb-1 monoclonal antibody had a slight inhibitory effect on the proliferation of ErbB2 / HER2-positive cells, and ADC-1 was clearly superior to both the mAb-1 monoclonal antibody and the MMAE small molecular weight toxin. On the other hand, ADC-1 and mAb-1 monoclonal antibody had no inhibitory effect on ErbB2 / HER2-negative cells.

[0258] [Table 1]

[0259] [Table 2]

[0260] Example 13 In vivo activity testing of ADC-1 (NCI-N87 CDX mouse model) The effect of ADC-1 on tumor growth in the ErbB2 / HER2 NCI-N87 CDX mouse model was measured according to the following method.

[0261] 1) Cell Culture: Logarithmic growth phase NCI-N87 human gastric cancer tumor cells (ATCC, Manassas, VA, cat # CRL-5822) were collected, and cell density was adjusted with Matrigel buffer (PBS:Matrigel = 1:1). 0.2 mL of the prepared NCI-N87 cell suspension was subcutaneously injected into the right scapula region of 7-9 week old SPF-grade female BALB / c nude mice, with a cell inoculation of 10 × 10⁴ 6 / animal

[0262] 2) Measure the diameter of the tumor with calipers, and use the formula V = 0.5a × b 2 The tumor volume was calculated using the formula (where a is the longest diameter of the tumor and b is the shortest diameter). Five days after cell inoculation, the average tumor volume ranged from 100 to 300 mm. 3 At that time, animals were randomly divided into a solvent control group and an ADC-1 3 mg / kg group, with 5 animals per group. The animals were administered by tail vein injection, and the control group was given an equal volume of solvent (vehicle). The day of group division and administration was defined as Day 0. From administration to day 35, the tumor volume of the animals in each group was measured twice a week, and the tumor volumes of the animals on day 35 were compared between the groups. The T / C value and TGI value were calculated using the tumor volume. The formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :Treatment group RTV, C RTV(Solvent control group RTV). Relative tumor volume (RTV) is calculated from the tumor measurement results, and the formula is RTV = Vt / VO, where VO is the average tumor volume measured at the time of group division, Vt is the average tumor volume at a certain measurement, and T RTV and C RTV The data used was from the same day. Calculation of TGI(%): TGI(%) = [1 - (average tumor volume at the end of treatment for a certain treatment group - average tumor volume at the start of administration for that treatment group) / (average tumor volume at the end of treatment for the solvent control group - average tumor volume at the start of treatment for the solvent control group)] × 100%.

[0263] 3) Data were statistically analyzed between groups using independent sample t-tests, and all analyses were performed using SPSS 17.0. A difference is considered statistically significant if P < 0.05.

[0264] 4) Table 3 and Figure 8 show that ADC-1 significantly suppresses tumor growth in the NCI-N87 CDX mouse model compared to the solvent control group.

[0265] [Table 3]

[0266] Example 14 Manufacturing and characterization of ADC-2 Except for the use of LP-6 as the linker-payload, the manufacturing and characterization were carried out by following the method described in Example 11. The characterization data for the antibody-drug conjugate ADC-2 are as follows.

[0267] SDS-PAGE Analysis of Antibody-Drug Conjugate ADC-2: After the completion of the conjugation reaction, the purity and conjugation efficiency of ADC-2 were measured by SDS-PAGE. As shown in Figure 9, the SDS-PAGE results for ADC-2 indicated that the conjugation reaction occurred at a predetermined position on the antibody heavy chain, and that the ADC-2 heavy chain bound to the linker-payload showed a clear molecular weight shift compared to the monoclonal antibody heavy chain from which the sugar chains had been removed, demonstrating that the linker-payload was able to bind to the monoclonal antibody heavy chain molecule at the predetermined position. The conjugation product contained almost no antibodies unbound to the linker-payload, the conjugation efficiency was high at over 95%, and the purity of the conjugation product was as expected.

[0268] The HIC-HPLC analysis of ADC-2 yielded the results shown in Figure 10. As illustrated, the results showed that <5% of the antibody was not bound to the cytotoxin, the complex product was mainly DAR4, and the overall DAR value of the ADC-2 drug was approximately 3.8.

[0269] SEC-HPLC analysis of ADC-2 revealed that, as shown in Figure 11, high molecular weight polymers were <5% in the ADC drug, and the ADC sample was mainly present as monomers at 8.1 min.

[0270] Measurement of ADC-2 plasma stability Test design: Mixed plasma from healthy humans (equal volumes from 5 men and 5 women) was taken, ADC-2 was added to achieve a specific final concentration, and the sample was divided into four sets of 450 μL each. These sets were incubated in a 37°C incubator, with sample collection times at 0h, 24h, 48h, and 96h. After collection, the samples were stored in a refrigerator at -60 to -90°C in preparation for measurement of free load loss rate and DAR change rate.

[0271] LC-MS measurement of low molecular weight toxins (the low molecular weight toxin of LP-6 in this example is represented by the payload). For each sample set (except the double blank), 40 μL was taken, a fixed amount of internal standard precipitant (1 ng / mL DXd) was added, and the mixture was shaken for at least 10 minutes; for the double blank, 40 μL of the blank matrix was taken, 120 μL of precipitant without the internal standard was added, and the mixture was shaken for at least 10 minutes; 3600 g was centrifuged at 4°C for 15 minutes, 60 μL of the supernatant was aspirated into an injector, a fixed amount of 0.1% FA ultrapure water was added, and the mixture was shaken for at least 3 minutes before HPLC measurement.

[0272] DAR measurement using hybrid LC-MS method 1 g of CNBr-activated agarose (Sigma, Cat# C9142) was weighed into a 50 mL centrifuge tube, 50 mL of pre-cooled 1 mM HCl was added, and the mixture was incubated in a rotary mixer for at least 30 minutes at 4°C and 10 RPM. The mixture was centrifuged for a set time to remove the 1 mM HCl. The sol was washed once with 5 to 10 times its volume of deionized water, and then washed three times with 0.1 M NaHCO3. An appropriate amount of HER2 ECD was taken, and the buffer was replaced with 0.1 M NaHCO3 using a 30 kD ultrafiltration tube. The replaced HER2 ECD was then mixed with the packing material and incubated in a rotary mixer at 25°C and 10 RPM for 2 hours ± 10 minutes to allow binding. Next, 5 mL of 0.1 M NaHCO3 buffer was added, and unbound proteins were removed by centrifugation. The mixture was then centrifuged for a set time and washed three times. A fixed amount of glycine (pH 8.0) was taken and added to the packing material, and it was allowed to stand at 2-8°C for 16 hours to block the unreacted chemical groups on the packing material. First, the packing material was washed with 5 mL of 0.1 M NaHCO3, centrifuged for a set time, then washed with a fixed amount of acetate buffer, centrifuged for a set time, and this washing cycle was repeated several times. 0.1 mL of each sample was placed in a 1.5 mL EP tube, 0.1 mL of packing material bound to HER2 ECD was added, and the mixture was incubated at 25°C and 10 RPM for 2 hours. The mixture was then washed three times with 1 mL PBST and centrifuged for a set time. The supernatant was collected after elution and centrifugation, the concentration was measured, and LC-MS analysis was performed.

[0273] Result analysis: When ADC-2 was incubated in healthy human mixed plasma for 0, 24, 48, and 96 hours, the DAR change rates were 100.00%, 106.7%, 104.2%, and 106.5%, respectively; the low molecular weight toxin shedding rates were only 0.006%, 0.179%, 0.373%, and 1.07%, respectively. As can be seen from the results described above, after incubation of ADC-2 at 37°C for 96 hours in healthy human mixed plasma, the amount of toxin shedding was extremely low, the DAR remained stable, and the low toxin shedding suggests a significant reduction in toxic side effects due to free toxins in clinical settings. The high DAR stability indicates that the antibody molecule can deliver more toxin molecules to the target site, thereby improving drug efficacy. The synergistic effect of both can significantly expand the therapeutic range of ADC-2.

[0274] Example 15 In vitro activity testing of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468) The effect of ADC-2 on tumor cell proliferation at different levels of ErbB2 / HER2 expression was measured according to the following method.

[0275] The inhibitory effect of the antibody-drug conjugate ADC-2 on the proliferation of cancer cells with various different ErbB2 / HER2 expression levels was measured, based on the method described in Example 12. For example, ErbB2 / HER2-positive human tumor cells such as SK-BR-3 and HCC1954, and MDA-MB-468 ErbB2 / HER2-negative human tumor cells were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 4, 5 and Figures 12-14. Both ADC-2 and the small molecular weight toxin had a clear inhibitory effect on the proliferation of ErbB2 / HER2-positive cells, the mAb-1 monoclonal antibody had some inhibitory effect on the proliferation of ErbB2 / HER2-positive cells, and ADC-2 was clearly superior to mAb-1. On the other hand, ADC-2 and the mAb-1 monoclonal antibody did not have an inhibitory effect on ErbB2 / HER2-negative cells, showing good targeting ability.

[0276] [Table 4]

[0277] [Table 5]

[0278] Example 16 Manufacturing and characterization of ADC-3 Antibody-drug conjugate ADC-3 was manufactured and characterized using the method described in Example 11, except that the antibody used was an anti-TROP2 antibody, i.e., mAb-2, and the linker-payload used was LP-6. The characterization data for antibody-drug conjugate ADC-3 is as follows.

[0279] In the SEC-HPLC analysis of ADC-3, the high molecular weight aggregation degree of ADC-3 was analyzed using an SEC chromatography column. As shown in Figure 15, the measurement results showed that the high molecular weight polymer was <5%, and the ADC sample mainly existed as monomers, with the damage to the antibody from the complex reaction being almost negligible.

[0280] High-resolution mass spectrometry (ESI-MS) analysis of the DAR value of antibody-drug conjugate ADC-3. The molecular weight of ADC-3 was analyzed using a high-resolution mass spectrometer, and the mass spectrum after deconvolution is shown in Figure 16. By comparing the measured molecular weight information with the theoretical molecular weight, each major molecular weight variant could be assigned. Using the abundance of each major molecular weight variant determined by mass spectrometry, DAR value analysis was performed, and the calculated average DAR value was 3.93.

[0281] Example 17 In vitro activity testing of ADC-3 (BxPC-3, FaDu, HepG2) By following a similar procedure to that described in Example 12, the inhibitory effect of the antibody-drug conjugate ADC-3 on the proliferation of cancer cells with various different TROP2 expression levels was measured at different concentration gradients. For example, human tumor cells such as BxPC-3, FaDu, and HepG2 were selected. The results of the tumor cell proliferation inhibitory effects of the different drugs are shown in Table 6 and Figures 17-19. Both ADC-3 and the small molecular weight toxin had a clear inhibitory effect on TROP2-positive cells, while the TROP2 antibody, i.e., mAb-2, had no clear inhibitory effect on TROP2-positive cells. ADC-3 was clearly superior to the monoclonal antibody and the small molecular weight toxin. Both ADC-3 and the mAb-2 monoclonal antibody showed good targeting ability against TROP2-negative cells, with no inhibitory effect against them.

[0282] [Table 6]

[0283] Example 18 In vivo activity testing of ADC-3 (NCI-N87 CDX mouse model) Referring to a similar method described in Example 13, the tumor growth inhibitory effect of ADC-3 in the NCI-N87 CDX mouse model was evaluated. The tumor growth curves and weight change curves after administration are shown in Figures 20-21. ADC-3 demonstrated good tumor growth inhibitory effect and good safety, and no toxicity related to weight loss was observed in the experimental mice.

[0284] Example 19 Manufacturing and characterization of ADC-4 In the manufacturing and characterization of ADC-4, the method described for ADC-1 was followed, except that the antibody used was an anti-TROP2 antibody, i.e., mAb-2. The characterization data for the antibody-drug conjugate ADC-4 is as follows.

[0285] HIC-HPLC analysis of ADC-4 showed that, as shown in Figure 22, <5% of the antibody was not bound to the cytotoxin; the complex product was mainly DAR2, and the overall DAR value of the ADC-4 drug was 1.89. SEC-HPLC analysis of ADC-4 showed that, as shown in Figure 23, <5% of the ADC drug was high molecular weight polymer, and the ADC sample mainly existed as monomers.

[0286] Example 20 Manufacturing and characterization of ADC-5 In the manufacturing and characterization of ADC-5, the antibody used was the anti-TROP2 antibody mAb-2, and the linker-payload used was LP-2. The manufacturing and characterization were carried out using the same method as described for ADC-1, and the characterization data for the antibody-drug conjugate ADC-5 is as follows.

[0287] HPLC analysis of ADC-5 showed that, as shown in Figure 24, <5% of the antibody was not bound to the cytotoxin; the complex product was mainly DAR2, and the overall DAR value of the ADC-5 drug was 1.87. SEC-HPLC analysis of ADC-5 showed that, as shown in Figure 25, <5% of the ADC drug was high molecular weight polymer, and the ADC sample mainly existed as monomers.

[0288] Example 21 In vitro activity comparison of ADC-4 and ADC-5 (BxPC-3, FaDu, HepG2) Following the procedure described in Example 12, the effects of antibody-drug conjugates ADC-4 and ADC-5 on the proliferation of cancer cells with various different TROP2 expression levels were measured. For example, TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells) and FaDu (human pharyngeal cancer cells), and TROP2-negative tumor cells such as HepG2 (human liver cancer cells) were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 7-8 and Figures 26-28. In particular, ADC-4, ADC-5, and MMAE small molecule toxin all had a clear inhibitory effect on the proliferation of positive cells, and there was no clear difference in the activity of ADC-4 and ADC-5. Monoclonal antibodies did not have a clear inhibitory effect on the proliferation of TROP2-positive cells. ADC-4, ADC-5, and monoclonal antibodies did not have an inhibitory effect on antigen-negative cells, showing good targeting ability.

[0289] [Table 7]

[0290] [Table 8]

[0291] Example 22 Manufacturing and characterization of ADC-6 In the manufacturing and characterization of ADC-6, the antibody used was mAb-3 (trastuzumab), and the linker-payload used was LP-6. The manufacturing and characterization were carried out using the same method as described for ADC-1, and the characterization data for the antibody-drug conjugate ADC-6 are as follows: HIC-HPLC analysis of ADC-6 showed that, as shown in Figure 29, <5% of the antibody was not bound to the cytotoxin; the conjugate product was mainly DAR4, and the overall DAR value of the ADC-6 drug was 3.93. SEC-HPLC analysis of ADC-6 showed that, as shown in Figure 30, <5% of the ADC drug was high molecular weight polymer, and the ADC sample mainly existed as monomers.

[0292] Example 23 In vitro activity testing of ADC-6 (SK-BR-3, NCI-N87) Following the procedure described in Example 12, the effects of the antibody-drug conjugate ADC-6 on the proliferation of cancer cells with different HER2 expression levels were measured. For example, two groups of HER2 cell lines, SK-BR-3 and NCI-N87, were selected, and the difference in activity between ADC-2 (containing Srt A tag) and ADC-6 (not containing Srt A tag) was compared. The results showed that, regardless of the presence or absence of the Srt A enzyme-specific recognition oligopeptide sequence, there was no difference in the cell-killing activity of the target ADC in the two cell lines (see Figures 31-32).

[0293] The arrangement of the present application is as follows: SEQ ID No.1: mAb-1 light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No.2: mAb-1 heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.3: mAb-2 light chain: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No.4: mAb-2 Heavy Chain QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.5: mAb-3 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.6: mAb-3 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A linker-payload compound having formula (I). 【Chemistry 1】 During the ceremony, P is the payload; D-C(O)- is a disaccharide structure. 【Chemistry 2】 And; and / or, D-C(O)- is a disaccharide structure. 【Transformation 3】 And; L is a linker end, and L is directly linked to the carbonyl group in D-C(O)- via its -NH-, and if L is an unbranched linker end, L is linked to one P and t is 1, and if L is a branched linker end, each branch may be linked to one P and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).

2. -L-(P) t teeth, 【Chemistry 4】 【Transformation 5】 A linker-payload compound according to claim 1, selected from the following.

3. D-C(O)-OH and L'-(P) t By subjecting them to an amide formation reaction, 【Transformation 6】 A method for producing the linker-payload compound according to claim 1 or 2, comprising the step of forming a linker. During the ceremony, In L, the -NH- connected to D-C(O)- is H in L'. 2 Except for being N-, L' is the same as L as defined in claim 1 or 2.

4. An antibody-drug conjugate having a structure selected from formulas (II-1) and (II-2), characterized by site-specific binding to the N-glycosylation site of the antibody Fc region. 【Transformation 7】 During the ceremony, R is hydrogen or an α-L-fucosyl group; q is either 1 or 2; Ab is an antibody or antigen-binding fragment; -L-(P) t This is as defined in claim 1 or 2.

5. (P) t -L- is, 【Transformation 8】 An antibody-drug conjugate according to claim 4, selected from the above.

6. A method for producing an antibody-drug conjugate according to claim 4 or 5, a) A step of obtaining an antibody in which antibody Ab excises its N-glycan portion under the catalytic action of glycosidase or a variant thereof, and the N-glycosylation site of its Fc region is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine; b) a step of conjugating the modified antibody obtained in step a) with the linker-payload compound according to claim 1 or 2 under the catalytic action of a glycosidase or a variant thereof; Includes, A method wherein the glycosidase or its variant used in steps a) and b) may be the same or different.

7. The method according to claim 6, wherein in steps a) and b), the glycosidase or its variant is fucose hydrolase, N-acetylglucosamine endohlase, or a variant thereof.

8. The N-acetylglucosamine endohylase comprises at least one selected from Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); preferably, the endoglycosidase comprises Endo H, Endo D, Endo F2, Endo F3, Endo The method according to claim 7, wherein M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

9. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 4 or 5.

10. An antibody-drug conjugate according to claim 4 or 5, for use in the treatment or prevention of tumors or autoimmune diseases.

11. The structure is 【Chemistry 9】 It is a compound.

12. The following steps: (i) A step of oxidizing the primary alcohol at the 6-position of the terminal first hexose group unit in an oligosaccharide containing a terminal first hexose group unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group; (ii) A step of reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group in the linker-terminated payload compound (b) having a reactive group at its terminus to obtain the linker-payload compound; A method for producing a linker-payload compound, comprising: The oligosaccharide in step (i) above has the following structure: 【Chemistry 10】 It has, A method wherein the reactive group of the linker-terminated payload compound (b) having a reactive group at its terminus in step (ii) is an amino group.

13. The method according to claim 12, further comprising the step of converting an intermediate compound (a) having a carboxyl group into an acid halide, and further reacting it with a linker-terminated payload compound (b) having a reactive group at its terminus to obtain the linker-payload compound.

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