Disaccharide-acylhydrazone or disaccharide-hydrazide connexon, sugar engineering antibody based on disaccharide-acylhydrazone or disaccharide-hydrazide connexon, sugar chain fixed-point antibody-drug conjugate and application

Abstract

The invention relates to a disaccharide-acylhydrazone or disaccharide-hydrazide connexon, a sugar engineering antibody based on the disaccharide-acylhydrazone or disaccharide-hydrazide connexon, a sugar chain fixed-point antibody-drug conjugate and application. Specifically, the invention provides a disaccharide-acylhydrazone or disaccharide-acylhydrazone connexon as shown in the following formula I. The disaccharide-acylhydrazone or disaccharide-acylhydrazone connexon can effectively modify a functional group or a cytotoxin drug at a glycosylation site at the Asn297 site of an Fc structural domain of an antibody in a site-specific manner. The preparation method of the sugar chain fixed-point antibody-drug conjugate (ADC) provided by the invention is simple and efficient, can reduce the use amount of small molecular drugs, enhance the hydrophilicity of ADC molecules and enrich the structure of ADC compounds, and has a great application prospect in ADC preparation. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and biotechnology drugs, and in particular to a class of disaccharide-acylhydrazone linkers and disaccharide-hydrazide linkers, wherein the disaccharide-acylhydrazone linkers are used to prepare non-natural sugar engineering antibodies and antibody-drug conjugates with fixed-point and quantitative reaction based on acylhydrazone-oxime replacement reaction, as well as preparation methods and uses thereof. Background Art

[0002] Antibody-Drug Conjugates (ADCs) consist of three parts: antibodies, cytotoxins, and linkers. Cytotoxins are delivered to tumor tissues through antibodies to achieve targeted delivery of toxins, thereby exerting anti-tumor activity. Compared with traditional chemotherapy drugs, ADCs have lower body toxicity and better therapeutic index. In the early stage, ADC drugs mainly used random coupling to couple cytotoxins to lysine (Lys) or cysteine ​​(Cys) with high abundance in antibodies. The toxin coupling sites and quantities of ADCs formed in this way are uneven, with poor in vivo stability, efficacy and pharmacokinetic properties, and a narrow therapeutic window. Fixed-point ADCs can solve the above problems. The mainstream fixed-point coupling technologies currently used to prepare fixed-point ADCs include THIOMAB technology, non-natural amino acid insertion technology, enzyme catalysis technology, and sugar chain fixed-point coupling technology, each of which has its own characteristics.

[0003] Sugar chain-directed ADCs compounds are prepared by sugar chain-directed coupling technology, and their cytotoxins are site-directedly modified at the glycosylation site N297 of the antibody Fc domain. At present, the in vitro antibody glycosylation site modification methods mainly include glycosyltransferase technology and endoglycosidase technology.

[0004] Glycosyltransferase technology uses galactosyltransferase or sialyltransferase to transfer galactose or sialic acid with reactive functional groups to the glycosylation site of the antibody, and further couples cytotoxins to prepare sugar chain-directed ADC compounds. For example, Zhu et al. first used β1,4-galactosidase to hydrolyze the galactose at the terminal of the N-sugar chain at the glycosylation site of the antibody, and then used galactosyltransferase to transfer GalNAc containing a ketocarbonyl group to the terminal of the N-sugar chain, and further coupled toxins containing a hydroxylamine functional group to obtain sugar chain-directed ADC compounds. Qun Zhou et al. used galactosyltransferase and sialyltransferase to transfer galactose and sialic acid to the glycosylation site of the antibody in sequence, and then used sodium periodate to oxidize the terminal sialic acid, introducing an aldehyde group at the glycosylation site to provide a reaction site for toxin coupling. Floris L. van Delft et al. first used the endoglycosidase Endo-S to hydrolyze the heterogeneous N-glycans of antibodies, then used galactosyltransferase to transfer the azide-containing GalNAz to the antibody glycosylation sites, and finally used click chemistry to achieve the modification of the toxin at the glycosylation sites.

[0005] Endoglycosidase technology uses endoglycosidase and bioorthogonal reaction to prepare sugar chain-specific ADC. Professor Huang Wei's team and Davis' team used semi-synthetic modification to obtain azide-modified oligosaccharide oxazoline substrates, and used two endoglycosidases, Endo-S and its mutant enzyme Endo-SD233Q, to transfer the oligosaccharide modified with bioorthogonal groups to the antibody glycosylation site, and further used bioorthogonal reaction to obtain sugar chain-specific ADC compounds.

[0006] Existing glycosyltransferase technology and endoglycosidase technology can obtain ADC compounds that are more uniform than random coupling, but both have their own problems. In glycosyltransferase technology, it is necessary to synthesize sugar substrates with CMP or UDP activation forms, and glycosyltransferases often have weak catalytic activity, which makes the reaction time longer, and the preparation efficiency and cost are difficult to control. The oligosaccharide substrates used in endoglycosidase technology are also difficult to obtain. Semi-synthetic modification methods need to be extracted from egg yolks, and the purification steps are complicated, while the full synthesis method is more difficult. Whether it is glycosyltransferase or endoglycosidase, multiple enzymes and multi-step reactions are involved, and there are certain limitations in drug efficacy and production. At the same time, it is not conducive to the stability of antibodies. Both methods are highly dependent on bioorthogonal reactions to achieve the modification of toxins at antibody glycosylation sites, which limits the development of sugar chain-directed ADCs drugs.

[0007] Recently, scientists have developed a method for preparing sugar chain-directed antibody-drug conjugates based on disaccharide linkers of LacNAc, Manβ1,4GlcNAc and Glcβ1,4GlcNAc. 1) The disaccharide-toxin molecule is directly transferred to the antibody glycosylation site through the catalysis of endoglycosidase Endo-S2 to achieve the one-step preparation of sugar chain-directed antibody-drug conjugates. This method is simple to operate, but due to the limited Fc domain cavity and enzyme recognition ability, this method can often only achieve the preparation of ADC compounds with a DAR value of 2. The preparation of ADC compounds with a DAR value of 4 is highly dependent on It relies on the size of the structure and requires the consumption of a larger amount of disaccharide-toxin molecules, which is not conducive to industrial production and the diversity expansion research of sugar chain-directed ADC compounds; 2) The sugar structure with an azide group is transferred through the catalytic action of the endoglycosidase Endo-S2 to obtain the corresponding azide-modified glycoengineered antibody, which is further coupled with the drug-linker to obtain a sugar chain-directed ADC compound. Although this method can achieve the preparation of ADC compounds with high DAR values, the structures formed by DBCO, BCN, etc. after the click chemistry reaction are highly hydrophobic, which will enhance the hydrophobicity of the ADC molecules and affect the drugability of the ADC compounds.

[0008] Considering the above problems existing in the prior art, there is still a need to develop a class of disaccharide linkers that can introduce small molecule drugs into antibodies in a targeted and quantitative manner. Summary of the invention

[0009] The technical purpose of the present invention is to provide a class of disaccharide-acylhydrazone linkers and disaccharide-hydrazide linkers, both of which can achieve site-specific transfer to antibodies, wherein the disaccharide-acylhydrazone linker can achieve replacement with a small molecule drug with a hydroxylamine functional group, thereby achieving site-specific and quantitative introduction of the small molecule drug into the antibody.

[0010] Another technical purpose of the present invention is to provide a class of hydroxylamine small molecule drug conjugates.

[0011] Another technical purpose of the present invention is to provide a glycoengineered antibody prepared by the above disaccharide-acylhydrazone linker / disaccharide-hydrazide linker.

[0012] Another technical purpose of the present invention is to provide an antibody drug conjugate (ADC) prepared by coupling a small molecule drug with the disaccharide-acylhydrazone linker / disaccharide-hydrazide linker.

[0013] Another technical purpose of the present invention is to provide the use of the disaccharide-acylhydrazone linker / disaccharide-hydrazide linker in the preparation of ADC.

[0014] Another technical purpose of the present invention is to provide the use of ADC prepared by coupling the glycoengineered antibody with a small molecule drug containing hydroxylamine in the preparation of drugs or diagnostic reagents.

[0015] In one aspect, the present invention provides a disaccharide linker, which is a disaccharide-acylhydrazone linker or a disaccharide-hydrazide linker represented by the following formula I:

[0016]

[0017] In Formula I,

[0018] X can be selected from:

[0019] Structures containing positive and negative charges;

[0020] C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C10 cycloalkyl, C5-C10 aryl,

[0021] wherein the C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C10 cycloalkyl, C5-C10 aryl is optionally substituted by a substituent selected from aryl, hydroxyl, halogen, R'R"N-, quaternary ammonium ion, C1-C3 alkoxy, or a 3-10 membered saturated or unsaturated heterocyclic group containing 1-3 heteroatoms independently selected from N, O, and S, wherein R' and R" are each independently H or C1-C3 alkyl;

[0022] 3 to 10 membered alicyclic and heteroaryl groups containing 1 to 3 heteroatoms independently selected from N, O, and S; and

[0023] Sulfonic acid group, phosphoric acid group, PEG structure, C1-C6 alkoxycarbonyl group, amide group, hydroxyl group, amino group, azido group, aldehyde group,

[0024] in, represents a single bond or a double bond, where When there is a double bond, it is an acylhydrazone structure. As can be understood by those skilled in the art, the H on N does not exist at this time. When it is a single bond, it is a hydrazide structure.

[0025] In a specific embodiment, the structure containing positive and negative charges can be selected from quaternary ammonium ions and sulfonium ions.

[0026] In a specific embodiment, the heterocyclic group and heteroaryl group can be 2-thienyl, 5-pyrimidinyl, 2-tetrahydrofuranyl, tetrazinyl.

[0027] In a specific embodiment, X is selected from the following:

[0028]

[0029] R1-R 14 Each is independently selected from -OH, C1-C20 alkyl, H;

[0030] W1 is selected from -OH, -CF3;

[0031] W2-W8 are each independently selected from -[O-(CH2) q ]-、-(CH2) q -;

[0032] A1, A2, A5 are each independently selected from C, N, O, S;

[0033] A3 and A4 are selected from C or N; and

[0034] n and q are independently integers of 0-20, and r and t are independently integers of 1-20.

[0035] In a specific embodiment, X is a C1-C15 alkyl group, preferably a C1-C10 alkyl group, and more preferably a C1-C7 alkyl group. For example, X can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, or a decyl group.

[0036] In a specific embodiment, X is a C3-C8 cycloalkyl group, preferably a C3-C6 cycloalkyl group, for example, X can be a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group.

[0037] In a specific embodiment, the disaccharide-acylhydrazone linker or disaccharide-acylhydrazine linker is selected from the following compounds:

[0038]

[0039] Wherein, n is an integer from 0 to 20.

[0040] On the other hand, the present invention also provides a linker in the form of an oxazoline ring-opened linker at positions 1 and 2 of GlcNAc in the above disaccharide-acylhydrazone linker or disaccharide-hydrazide linker.

[0041] In another aspect, the present invention provides a method for preparing the above-mentioned disaccharide-acylhydrazone linker or disaccharide-acylhydrazone linker.

[0042] When the disaccharide linker is a disaccharide-acylhydrazone linker, it is prepared by the following method:

[0043]

[0044] As shown in the above reaction formula, the method comprises the following steps:

[0045] Step 1: Oxidation of galactose at position 6 to form an aldehyde disaccharide LacNAc (hereinafter referred to as CHO-LacNAc) and a hydrazide compound The reaction is carried out under alkaline conditions to obtain a disaccharide-acylhydrazone compound in which the 1 and 2 positions of GlcNAc are not cyclized;

[0046] Step 2: cyclizing the disaccharide-acylhydrazone compound under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to form oxazoline, thereby obtaining the disaccharide-acylhydrazone linker with cyclization at positions 1 and 2 of the GlcNAc;

[0047] When the disaccharide linker is a disaccharide-hydrazide linker, it is prepared by the following two routes:

[0048]

[0049] Among them, route one includes the following steps:

[0050] Step 1: directly reducing CHO-LacNAc and a hydrazide compound in the presence of a reducing agent to obtain a disaccharide-hydrazide compound;

[0051] Step 2: Cyclize the disaccharide-hydrazide compound to oxazoline under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to obtain a cyclized disaccharide-hydrazide linker at the 1,2 positions of GlcNAc.

[0052] Route 2 includes the following steps:

[0053] Step 1: reacting CHO-LacNAc with a hydrazide compound under alkaline conditions to obtain a disaccharide-acylhydrazone compound;

[0054] Step 2: adding a reducing agent to the reaction system of step 1 to obtain a disaccharide-hydrazide compound;

[0055] Step 3: Cyclize the disaccharide-hydrazide compound to oxazoline under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to obtain a disaccharide-hydrazide linker.

[0056] In a specific embodiment, the reducing agent used in the preparation of the disaccharide-hydrazide compound is sodium cyanoborohydride.

[0057] On the other hand, the present invention provides a drug-linker containing a hydroxylamine functional group, wherein the drug-linker can undergo a replacement reaction with the disaccharide-acylhydrazone linker described above through the hydroxylamine group at the linker site, thereby preparing a small molecule drug-disaccharide linker, wherein the drug-linker containing a hydroxylamine functional group is shown in the following formula II:

[0058]

[0059] In the above formula II, linker1 represents a linker having three or more connection sites derived from an amino acid or a short peptide, for example, it can be a linker derived from glutamic acid (Glu), β-glutamic acid, lysine (Lys), Lys-Glu dipeptide or Lysine-β-glutamic acid dipeptide,

[0060] Linker2 represents a short peptide linear linker with two or more connection sites that is connected or not connected to p-aminobenzyl formate, for example, it can be a tumor microenvironment cleavable or non-cleavable structural unit such as Val-Cit-PAB, Val-Ala-PAB, GGFG, etc.;

[0061] Wherein, linker1 and linker2 can further connect PEG chains at the ends of the groups they contain or between linker1 and linker2. For example, when linker1 is a linker derived from a Lys-Glu dipeptide, a PEG chain is further connected to the amino group of lysine.

[0062] Each R 15 Each independently is -NH2, NHR 16 、-NR 16 R 17 , -N3, fluorescent group, orthogonal group, where R 16 , R 17 Each is independently a C1-C20 alkyl group, a C1-C20 heteroaryl group, a C3-C20 cycloalkyl group, a C3-C20 heterocycloalkyl group, a C1-C20 acyl group, a C1-C20 acyloxy group, or a C1-C20 alkylamino group,

[0063] Or each R 15 are each independently a group derived from a toxin small molecule, a radioactive therapeutic, a nucleic acid, and a nuclide, and

[0064] n1 is 0 and n2 is 1; or

[0065] n1 is 1 and n2 is an integer of 1-4, for example, n1 is 1, and n2 is 1, 2, 3 or 4, preferably n2 is 2,

[0066] Among them, when n1 is 1, n2 linker2-R 15 They are covalently connected to linker1, and n2 = the number of connection sites of linker1 - 1,

[0067] Here, the "short peptide" refers to a peptide having 2-8 amino acids.

[0068] In a specific embodiment, the toxin small molecules include but are not limited to maytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, Dx8951, duokamycin, amanitine, PBD, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, olaparib, nocodazole, colchicine, estramustine, cematodin, and eloserol; preferably, the toxin small molecules are MMAE, MMAF, Dxd, and PBD.

[0069] In a specific embodiment, the nucleic acid includes but is not limited to antisense nucleic acid ASO, small interfering RNA (siRNA), micro RNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA).

[0070] In a specific embodiment, the nuclides include non-metallic element nuclides and metal nuclides. The non-metallic element nuclides are, for example, 131 I or 123 I.

[0071] In a specific embodiment, the hydroxylamine-small molecule drug conjugate is selected from any one of the following compounds:

[0072]

[0073]

[0074] In the above structures, the structure of the MMAE part is:

[0075]

[0076] In another aspect, the present invention provides a method for preparing a glycoengineered antibody, the method comprising the following method 1 and method 2:

[0077] Method 1:

[0078]

[0079] Wherein, in the above reaction formula, X is as defined above, m is 0 or 1,

[0080] Method 1:

[0081] The above-mentioned disaccharide linker, antibody and endoglycosidase, or a combination of endoglycosidase and fucosylation enzyme, are co-incubated, and the endoglycosidase has both hydrolysis activity on the sugar chain of the antibody Fc domain and transglycosylation activity on the disaccharide-acylhydrazone linker to prepare a glycoengineered antibody with the disaccharide linker shown in formula I connected to Asn297 of the antibody Fc domain. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0082] Method 2:

[0083] Method 2 includes the following two steps:

[0084] Step 1: The antibody is hydrolyzed by endoglycosidase or endoglycosidase combined with fucosylation enzyme to remove the non-uniform sugar chains at the conservative glycosylation sites of the natural antibody to obtain a deglycosylated antibody. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0085] Step 2:

[0086] The disaccharide linker described above, the deglycosylated antibody generated in the first step and an endoglycosidase are co-incubated, and the disaccharide linker is transferred to the glycosylation site of the deglycosylated antibody using the transglycosylation activity of the endoglycosidase on the disaccharide linker, thereby preparing a glycoengineered antibody in which the disaccharide linker shown in Formula I is connected to Asn297 of the Fc domain of the antibody. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0087] In another aspect, the present invention provides a glycoengineered antibody prepared by the above-mentioned glycoengineered antibody preparation method, which has a structure shown in the following formula III:

[0088]

[0089] Wherein, the definition of X is the same as that of X in Formula I above, and Ab represents an antibody.

[0090] In a specific embodiment, the antibody is an antibody having an N-glycosylation site,

[0091] In a specific embodiment, the antibody may be an IgG having a conserved N-glycosylation site at N297 in the Fc region.

[0092] In specific embodiments, the antibody may be IgG1, IgG2 or IgG4.

[0093] In a specific embodiment, the antibody can be a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species (mouse, rat, sheep, rabbit, human, etc.).

[0094] In a specific embodiment, the targets of the antibodies include but are not limited to

[0095] HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276(B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR 1. DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF , RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R.

[0096] In a specific embodiment, the antibody may include, but is not limited to, trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, nivolumab.

[0097] In a specific embodiment, the antibody may be selected from trastuzumab and rituximab.

[0098] In another aspect, the present invention provides a method for preparing a sugar chain-directed antibody-drug conjugate, as shown below:

[0099]

[0100] In the above reaction formula, the definition of X is the same as that of X in Formula I above, m is 0 or 1, and R represents the remaining part of the above hydroxylamine small molecule drug conjugate structure after removing the hydroxylamine group.

[0101] As shown in the above reaction formula, the method comprises: incubating the glycoengineered antibody III having a disaccharide linker as shown in formula I with a drug-linker (N2H-OR) having a hydroxylamine functional group under the following two conditions: 1) in the absence of a catalyst, the pH is 3-5, preferably 3-4, or 2) in the presence of a catalyst, the pH is 5-7.5, preferably 5.5-7, further preferably 6-6.5, and further preferably 6, to obtain a final antibody-drug conjugate,

[0102] Wherein, the catalyst has a structure shown in the following formula IV:

[0103]

[0104] In the above formula IV, Ra is independently selected from C1-C6 alkyl and carboxyl, and n is an integer of 0-3. Preferably, Ra is carboxyl.

[0105] In a specific embodiment, Ra is independently selected from C1-C3 alkyl and carboxyl, and n is 1 or 2.

[0106] In a specific embodiment, the catalyst is selected from the following compounds: aniline, m-aminobenzoic acid, p-aminobenzoic acid, 5-aminoisophthalic acid, 3-amino-5-methyl-benzoic acid.

[0107] In another aspect, the present invention provides a sugar chain-directed antibody-drug conjugate prepared by the above method, which is represented by the following formula V:

[0108]

[0109] In Formula V, R represents a linker-cytotoxic compound, n is 1 or 2, m is 0 or 1, and Ab represents an antibody.

[0110] In a specific embodiment, the antibody is an IgG having a conserved N-glycosylation site at position Asn297 in the Fc region of the antibody.

[0111] In specific embodiments, the antibody may be IgG1, IgG2 or IgG4.

[0112] In a specific embodiment, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody, a therapeutic antibody or a functional antibody from different species (mouse, rat, sheep, rabbit, human, etc.), or a fragment thereof.

[0113] In a specific embodiment, the targets of the antibodies include but are not limited to HER2, Trop-2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276 (B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, H LA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274 , IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factorIXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R.

[0114] In a specific embodiment, the antibody includes but is not limited to trastuzumab, inituzumab, zenidatuzumab, pertuzumab, sacituzumab, datopotamab, zotuzumab, cetuximab, panitumumab, nimotuzumab, nexituzumab, ervantumab, telisotuzumab, enfortumab, vobramitamab, mirzotamab, deparetumab, muromonab, catumaxomab, belintoumab, blinatumomab, mirvetuximab, belantuzumab, atuximab, rituximab, lovatuzumab, talatumomab, clivatuzumab, sontuzumab, mab, Gatipotuzumab, Vofatamab, Atezolizumab, Avelumab, Durvalumab, Durvalumab, Atezolizumab, Zilovertamab, Tisotumab, Denintuzumab, Inotuzumab, Moxetumomab, Brentuximab, Vorsetuzumab, Polatuzumab, Iladatuzumab, Burosumab, Bematuzumab, Efruxifermin, Anetumab, Amatuximab, MEDI9447, Adalimumab, Infliximab, Golimumab, Zi ralimumab, ATG-031, daratumumab, daratumumab, letaplimab, leszolimab, lefalimab, tusamitamab, labetuzumab, IMAB027, tremelimumab, zeflalimab, botensilimab, nullimab, ipilimumab, ipilimumab, tigatuzumab, lexatumumab, vofatamab, talquetamab, bersanlimab, enlimomab, inolimomab, ozriftamab, ladiratuzumab, gemt uzumab, Camidanlumab, Nirsevimab, Felvizumab, Bevacizumab, Ramucirumab, Ranibizumab, Denosumab, Denizumab, Tremelimumab, Zevolizumab, Zevolizumab, Alemtuzumab, Palivizumab, Nivolumab, Pembrolizumab, Nivolumab, Pembrolizumab, Teplizumab, Sintilimab, Carrelizumab, Lesabelimab, Omalizumab, Tocilizumab, Ustekinumab, Basiliximab, Eculizumab, Brolimumab, Exetiluzumab, Secukinumab, Daclizumab, Elotuzumab, Emicizumab, Iomecilizumab, Elotumumab_, Emicizumab, Belimumab,Alirocumab, Guselkumab, Vedolizumab, Dupilumab, Lanadelumab, Brodalumab, Satralizumab.

[0115] In a specific embodiment, the antibody may be selected from trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, nivolumab.

[0116] In a specific embodiment, the antibody may be trastuzumab or rituximab.

[0117] In a specific embodiment, the DAR value of the glycan site-specific antibody-drug conjugate is 0 < DAR ≤ 4, preferably 0.5 ≤ DAR ≤ 4, more preferably, the DAR is 2 - 4, still more preferably, the DAR is 2 or 4.

[0118] In a specific embodiment, the glycan site-specific antibody-drug conjugate may be selected from the following:

[0119]

[0120]

[0121] As described above, the disaccharide linker of the present invention can specifically link the target molecule to the glycosylation site at Asn297 of the antibody Fc domain, wherein the target molecule includes but is not limited to small molecule toxins, radiotherapy agents, fluorescent molecules, nucleic acids, and radionuclides.

[0122] Therefore, in another aspect, the present invention provides the use of the above-mentioned disaccharide linker or the above-mentioned drug-linker containing a hydroxylamine functional group in the preparation of antibody-drug conjugates.

[0123] In another aspect, the present invention provides the use of the above-mentioned disaccharide linker in introducing small molecule toxins, functional molecules, radionuclides, nucleic acids, or orthogonal groups into antibodies, and the functional molecules include but are not limited to small molecule toxins, fluorescent molecules, radiotherapy agents.

[0124] In another aspect, the present invention provides the use of the above-mentioned disaccharide linker, the above-mentioned drug-linker containing a hydroxylamine functional group, the above-mentioned glycoengineered antibody, and the above-mentioned glycan site-specific antibody-drug conjugate in the preparation of drugs or diagnostic agents.

[0125] In a specific embodiment, the drug is an anti-tumor drug, and the tumors include but are not limited to: breast cancer, colon cancer, non-small cell carcinoma, brain astrocytoma, chronic myeloid leukemia, pancreatic cancer, acute monocytic leukemia, liver cancer, gastric cancer, non-small cell lung cancer, malignant glioblastoma, and prostate adenocarcinoma.

[0126] Beneficial effects

[0127] The present invention develops a novel disaccharide linker containing an acylhydrazone / acylhydrazide structure, which can effectively site-modify the target molecule (including all functional molecules, cytotoxic molecules, etc. in the prior art) at the glycosylation site Asn297 of the antibody Fc domain. The preparation method of the sugar chain site-specific ADC compound provided by the present invention is simple and efficient, can reduce the usage of small molecule drugs, enhance the hydrophilicity of ADC molecules, and enrich the structure of ADC compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1 The results of screening of some disaccharide-acylhydrazone oxazolines of the present application are shown. The ability of different disaccharide-acylhydrazone oxazolines substrates to transfer to antibody glycosylation sites is screened.

[0129] Figure 2 The catalytic effect of the catalyst used for the replacement reaction of the acylhydrazone-oxime structure involved in the present application is shown.

[0130] Figure 3 The in vitro activity experimental data results of some ADCs obtained by acylhydrazone-hydroxylamine substitution reaction of the present application are shown.

[0131] A: The inhibition rate of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on SK-Br-3 cells; B: The inhibition rate of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on NCI-N87 cells; C: The effect of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on the survival rate of MDA-MB-231 cells.

[0132] Figure 4 The in vivo activity experimental data results of some ADCs prepared by the acylhydrazone-hydroxylamine replacement method of the present application are shown, respectively showing the inhibition of tumor volume (A) in vivo and the effect on nude mouse body weight (B) of ADC-1, ADC-2, ADC-4 and ADC-5.

[0133] Figure 5 The structural characterization of the non-natural glycoengineered antibody of rituximab and the sugar chain-directed antibody-drug conjugate ADC-6 prepared therefrom is shown, wherein, left figure: Rituximab; middle figure: Rituximab-C5; right figure: ADC-6 conjugated with 4 Dxd toxins: Rituximab-LacNAc-C=NOE-(GGFG-Dxd)2.

[0134] Figure 6The DAR values ​​of ADCs prepared by Acylhydrazone-LacNAc-ox and Az-LacNAc-ox of the present application are compared.

[0135] From the experimental results, it can be seen that under the conditions of antibody concentration of 15 mg / mL and oxazoline of 5 equivalents, the antibody glycosylation effect using acylhydrazone disaccharide oxazoline substrate is better than the conversion effect of monoazide substrate, indicating that fewer equivalents are required to achieve high DAR conversion using acylhydrazone disaccharide substrate.

[0136] Figure 7 The in vivo antitumor activity test results of antibody-drug conjugates obtained by acylhydrazone displacement reaction and click chemistry reaction are shown. DETAILED DESCRIPTION

[0137] Terminology: "Multivalent linker" in this application refers to a linker with a valency greater than two.

[0138] The small molecule cytotoxic drugs PBD, Dxd and MMAE used in the present invention were purchased from Shanghai Ruishan Pharmaceutical Technology Co., Ltd.; N-acetyl-D-lactosamine and acetonitrile were purchased from Shanghai Jizhi Biochemical Co., Ltd.; galactose oxidase, catalase, and horseradish peroxidase were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; amino acid compounds were purchased from Shanghai Jier Biochemical Co., Ltd.; various hydrazide compounds were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. Other compounds and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. unless further explained.

[0139] The instruments and chromatographic columns used in the present invention include: Waters Xevo G2-XS QTOF, analytical high performance liquid chromatograph (Thermo ultimate 3000), analytical high performance liquid chromatograph (Beijing Chuangxin Tongheng LC3000), preparative high performance liquid chromatograph (Beijing Chuangxin Tongheng LC3000); ThermoC18 (AcclaimTM 120, 5μm, 4.6x250mm), Agilent SB-C18 (5μm, 4.6x 150mm), Waters C18 column (ACQUITY UPLC BEHC18, 1.7μm, 2.1x50mm).

[0140] Antibody molecular weight determination instrument: Liquid chromatography-mass spectrometry (LC-MS), Waters Xevo G2-XS QTOF, equipped with Waters C4 (ACQUITY UPLC Protein BEH C4, 1.7 μm, 2.1 mm x 50 mm).

[0141] The following reaction schematics generally illustrate the preparation routes of the disaccharide-acylhydrazone linker, disaccharide-hydrazide linker, glycoengineered antibody, and sugar chain-directed antibody-drug conjugate (ADC) based on acylhydrazone-oxime replacement chemistry in the present application.

[0142] General Preparation Example

[0143] The synthetic route of disaccharide-acylhydrazone linker is as follows:

[0144]

[0145] in,

[0146] Note: a. Goase, catalase, horseradish peroxidase, HRP, O2, pH 7.0, 30°C; b. pH 7.4, 30°C; d. 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate.

[0147] The synthetic route of disaccharide-hydrazide linker is as follows:

[0148]

[0149] in,

[0150] Note: a. Goase, catalase, horseradish peroxidase HRP, O2, pH 7.0, 30℃; b. pH 7.4, 30℃; c. NaBH3CN, pH 6.0; d. 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate.

[0151] The preparation route of non-natural glycoengineered antibodies is as follows:

[0152]

[0153] Wherein, in the above reaction formula, the definitions of X are as described above, and m is selected from 0 or 1.

[0154] Preparation method 1:

[0155] Glycoengineered antibody: The above-mentioned disaccharide linker, antibody and endoglycosidase (or combined endoglycosidase and fucosylation enzyme) are co-incubated, and the endoglycosidase has both hydrolysis activity on the antibody Fc domain sugar chain and transglycosylation activity on the disaccharide linker to prepare a glycoengineered antibody containing a disaccharide linker. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing these different endoglycosidase functional regions.

[0156] Preparation method 2:

[0157] Step 1: The antibody is hydrolyzed by endoglycosidase or endoglycosidase and fucosylation enzyme to remove the non-uniform sugar chains at the conservative glycosylation sites of the natural antibody to obtain a deglycosylated antibody. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing these different endoglycosidase functional regions.

[0158] Step 2:

[0159] Glycoengineered antibody: The above-mentioned disaccharide linker, deglycosylated antibody and endoglycosidase are co-incubated, and the disaccharide linker is transferred to the glycosylation site of the deglycosylated antibody by utilizing the transglycosylation activity of the endoglycosidase on the disaccharide linker to prepare a glycoengineered antibody containing a disaccharide linker. Specifically, the endoglycosidase includes but is not limited to Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and includes fusion proteins formed by fusing these different endoglycosidase functional regions.

[0160] The preparation route of sugar chain-directed antibody-drug conjugates is as follows:

[0161]

[0162] Wherein, X is defined as described above, m is selected from 0 or 1, and R represents the remaining part of the above-mentioned hydroxylamine small molecule drug conjugate structure after removing the hydroxylamine group.

[0163] Preparation method:

[0164] The glycoengineered antibody with a disaccharide linker is incubated with a drug-linker with a hydroxylamine functional group in a neutral buffer, and a certain catalyst is added to promote the displacement reaction to obtain the final antibody-drug conjugate. The catalyst has the structure shown in Formula IV above.

[0165] General operation 1:

[0166] A method for preparing a non-natural glycoengineered antibody based on disaccharide-acylhydrazone or disaccharide-hydrazide: the prepared disaccharide-acylhydrazone oxazoline (i.e., compound G1 to G27) or disaccharide-hydrazide oxazoline (i.e., compound G2'), wild-type antibody, and wild-type endoglycosidase Endo-S2 are adjusted to a concentration of 1 mM, 15 mg / mL, and 0.1 mg / mL, respectively, the pH of the reaction system is adjusted to 7.0, and the mixture is incubated at 30°C for 1h-2h. After LC-MS confirms the conversion to the product, the small molecule compound is removed by ultrafiltration to obtain the desired non-natural glycoengineered antibodies Ab-1 to Ab-7, as shown in Examples 34-40 below.

[0167] General operation two:

[0168] Method 1 for preparing sugar chain-directed antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry: non-natural sugar engineering antibodies (i.e., Ab-1, Ab-4), m-aminobenzoic acid, and drug-linkers containing hydroxylamine structures (i.e., compounds D1 and D2) prepared by disaccharide-acylhydrazone linkers are added, and the concentrations of each are adjusted to 5 mg / mL, 136 mM, and 0.34 mM, respectively. The pH of the reaction system is adjusted to 6.0, and the mixture is incubated at 30°C for 4h-16h. After LC-MS confirms the conversion into an ADC product with a specific DAR value, small molecule compounds are removed by ultrafiltration to obtain the desired sugar chain-directed antibody-drug conjugates ADC-1 to ADC-2, as shown in Examples 41 to 42 below.

[0169] General operation three:

[0170] Method 2 for preparing sugar chain-directed antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry: non-natural sugar engineering antibodies (i.e., Ab-1, Ab-4), 5-aminoisophthalic acid, and drug-linkers containing hydroxylamine structures (i.e., compounds D3-D5) prepared by disaccharide-acylhydrazone linkers, with concentrations of 5 mg / mL, 136 mM, and 0.34 mM, respectively, and the pH of the reaction system was adjusted to 6.0, incubated at 30°C for 4h-16h, and after LC-MS confirmed the conversion into an ADC product with a specific DAR value for coupling, small molecule compounds were removed by ultrafiltration to obtain the desired sugar chain-directed antibody-drug conjugates ADC-3 to ADC-5, as shown in Examples 43 to 45 below.

[0171] General operation four:

[0172] Preparation method 3 of sugar chain-directed antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry (without catalyst): non-natural sugar engineering antibodies (i.e., Ab-1, Ab-4) prepared by disaccharide-acylhydrazone linkers and drug-linkers containing hydroxylamine structures (i.e., compounds D1-D2) are prepared with concentrations of 5 mg / mL and 0.34 mM, respectively, and the pH of the reaction system is adjusted to 3-5. The mixture is incubated at 30°C for 2h-16h. After LC-MS confirms the conversion into an ADC product with a specific DAR value, the small molecule compounds are removed by ultrafiltration to obtain the desired sugar chain-directed antibody-drug conjugates ADC-1 to ADC-2, as shown in Examples 47 to 48 below.

[0173] The specific process of preparing each disaccharide linker, glycoengineered antibody and sugar chain-directed antibody-drug conjugate by the above general preparation method is described below through specific examples.

[0174] I: Preparation of disaccharide linkers

[0175] Example 1: Synthesis of Compound G1

[0176] The structure and synthesis method of compound G1 are as follows:

[0177]

[0178] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution pH 7.4 buffer. Add compound 3 (15.24 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete. After separation and purification using a semi-preparative C18 column, compound 2 (22.6 mg, yield 90%) was obtained. HRMS, calculated value C 14 H 33 N3O 11 [M+H] + 480.2193, measured value 480.2424.

[0179] Step 2: Weigh compound 2 (20 mg, 41.8 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (136 mg, 0.627 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (265 mg, 1.25 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitation was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G1. After separation and purification using a semi-preparative C18 column, compound G1 (12 mg, yield 63%) was obtained. HRMS, calculated value C 19 H 31 N3O 10[M+H] + 462.2088, measured value 462.1957.

[0180] Example 2: Synthesis of Compound G2

[0181] The structure and synthesis method of compound G2 are as follows:

[0182]

[0183]

[0184] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 5 (47.47 mg, 183.75 μmoL) to the above system and react at 30°C overnight to obtain compound 4. LC-MS monitored the reaction to be complete. HRMS, calculated value C 24 H 39 N5O 12 S[M+H] + 622.2394, measured value 622.2370.

[0185] Step 2: Weigh compound 4 (20 mg, 32.2 μmoL) and dissolve it in 1.6 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (105 mg, 0.483 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (205 mg, 0.966 mmoL), add ddH2O to a total volume of 3.2 mL, and react at 0°C for 2 h. A large amount of precipitation was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G2. Compound G2 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 24 H 37 N 11 S[M+H] + 604.2289, measured value 604.2928.

[0186] Example 3: Synthesis of Compound G3

[0187] The structure and synthesis method of compound G3 are as follows:

[0188]

[0189] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 7 (17.85 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 6 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 29 N3O 11 [M+H] + 500.1880, measured value 500.1891.

[0190] Step 2: Weigh compound 6 (20 mg, 40.07 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (131 mg, 0.603 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (255 mg, 1.20 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G3. Compound G3 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 27 N3O 10 [M+H] + 482.1775, measured value 482.1739.

[0191] Example 4: Synthesis of Compound G4

[0192] The structure and synthesis method of compound G4 are as follows:

[0193]

[0194] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 9 (18.65 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 8 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 27 N3O 11 S[M+H] + 506.1445, measured value 506.2019.

[0195] Step 2: Weigh compound 8 (20 mg, 39.59 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (129 mg, 0.594 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (253 mg, 1.19 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G4. Compound G4 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 25 N3O 10 S[M+H] + 488.1339, measured value 488.1459.

[0196] Example 5: Synthesis of Compound G5

[0197] The structure and synthesis method of compound G5 are as follows:

[0198]

[0199] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 11 (13.14 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 10 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 11 [M+H] + 464.1880, measured value 464.1795.

[0200] Step 2: Weigh compound 10 (20 mg, 43.18 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (141 mg, 0.648 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (276 mg, 1.30 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G5. Compound G5 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 27 N3O 10 [M+H] + 446.1775, measured value 446.1291.

[0201] Example 6: Synthesis of Compound G6

[0202] The structure and synthesis method of compound G6 are as follows:

[0203]

[0204] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 13 (15.24 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 12 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 33 N3O 11 [M+H] + 480.2193, measured value 480.1732.

[0205] Step 2: Weigh compound 12 (20 mg, 41.74 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (135 mg, 0.626 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (265 mg, 1.25 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G6. Compound G6 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 31 N3O 10 [M+H] + 462.2088, measured value 462.2106.

[0206] Example 7: Synthesis of Compound G7

[0207] The structure and synthesis method of compound G7 are as follows:

[0208]

[0209] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 15 (21.55 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 14 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 23 H 33 N3O 11 [M+H] +528.2193, measured value 528.2027.

[0210] Step 2: Weigh compound 14 (20 mg, 7.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G7. Compound G7 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 23 H 31 N3O 10 [M+H] + 510.2088, measured value 510.2107.

[0211] Example 8: Synthesis of Compound G8

[0212] The structure and synthesis method of compound G8 are as follows:

[0213]

[0214] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 17 (15.37 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 16 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 32 N4O 11 [M+H] + 481.2146, measured value 481.2217.

[0215] Step 2: Weigh compound 16 (20 mg, 41.65 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add DMC (211 mg, 1.25 mmoL) to the above reaction system. After the system is mixed evenly, add triethylamine (210 mg, 2.08 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h to obtain a reaction system containing compound G8. HRMS, calculated value C 18 H 30 N4O 10 [M+H] + 463.2030, measured value 463.2040.

[0216] Example 9: Synthesis of Compound G9

[0217] The structure and synthesis method of compound G9 are as follows:

[0218]

[0219] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 19 (17.35 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 18 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 35 N4O 11 + [M] + 496.2374, measured value 496.2320.

[0220] Step 2: Weigh compound 18 (20 mg, 40.39 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add DMC (205 mg, 1.21 mmoL) to the above reaction system. After the system is mixed evenly, add triethylamine (204 mg, 2.02 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h to obtain a reaction system containing compound G9. HRMS, calculated value C 19 H 33 N4O 10 + [M]+478.2269, measured value 478.2196.

[0221] Example 10: Synthesis of Compound G10

[0222] The structure and synthesis method of compound G10 are as follows:

[0223]

[0224] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 21 (9.72 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 20 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 27 N3O 11 [M+H] + 438.1724, measured value 438.1739.

[0225] Step 2: Weigh compound 20 (17 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G10. Compound G10 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 25 N3O 10 [M+H] + 420.1618, measured value 420.1629.

[0226] Example 11: Synthesis of Compound G11

[0227] The structure and synthesis method of compound G11 are as follows:

[0228]

[0229] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 23 (11.56 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 22 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 29 N3O 11 [M+H] + 452.1880, measured value 452.1896.

[0230] Step 2: Weigh compound 22 (17 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G11. Compound G11 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 10 [M+H] + 434.1775, measured value 434.1698.

[0231] Example 12: Synthesis of Compound G12

[0232] The structure and synthesis method of compound G12 are as follows:

[0233]

[0234] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 25 (13.40 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 24 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 31 N3O 11 [M+H] + 466.2037, measured value 466.2039.

[0235] Step 2: Weigh compound 24 (18 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G12. Compound G12 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 10 [M+H] + 448.1931, measured value 448.1990.

[0236] Example 13: Synthesis of Compound G13

[0237] The structure and synthesis method of compound G13 are as follows:

[0238]

[0239] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 27 (17.06 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 26 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 20 H 35 N3O11 [M+H] + 494.2350, measured value 494.2409.

[0240] Step 2: Weigh compound 26 (19 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G13. Compound G13 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 20 H 33 N3O 10 [M+H] + 476.2244, measured value 476.2278.

[0241] Example 14: Synthesis of Compound G14

[0242] The structure and synthesis method of compound G14 are as follows:

[0243]

[0244] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 29 (18.92 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 28 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 37 N3O 11 [M+H] + 508.2506, measured value 508.2590.

[0245] Step 2: Weigh compound 28 (19.2 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G14. Compound G14 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 35 N3O10 [M+H] + 490.2401, measured value 490.2399.

[0246] Example 15: Synthesis of Compound G15

[0247] The structure and synthesis method of compound G15 are as follows:

[0248]

[0249]

[0250] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 31 (20.76 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 30 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 22 H 39 N3O 11 [M+H] + 522.2663, measured value 522.2679.

[0251] Step 2: Weigh compound 30 (20 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G15. Compound G15 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 22 H 37 N3O 10 [M+H] + 504.2557, measured value 504.2564.

[0252] Example 16: Synthesis of Compound G16

[0253] The structure and synthesis method of compound G16 are as follows:

[0254]

[0255] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 33 (13.66 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 32 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 29 N3O 12 [M+H] + 468.1829, measured value 468.1796.

[0256] Step 2: Weigh compound 32 (18.5 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G16. Compound G16 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 10 [M+H] + 450.1724, measured value 450.1793.

[0257] Example 17: Synthesis of Compound G17

[0258] The structure and synthesis method of compound G17 are as follows:

[0259]

[0260] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 35 (11.82 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 34 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 27 N3O 12 [M+H] + 454.1673, measured value 454.1791.

[0261] Step 2: Weigh compound 34 (17.2 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G17. Compound G17 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 25 N3O 11 [M+H] + 436.1567, measured value 436.1575.

[0262] Example 18: Synthesis of Compound G18

[0263] The structure and synthesis method of compound G18 are as follows:

[0264]

[0265] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 37 (13.52 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 36 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 26 N4O 12 [M+H] + 467.1625, measured value 467.1644.

[0266] Step 2: Weigh compound 36 (17.7 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G18. Compound G18 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 16 H 24 N4O 11 [M+H] + 449.1520, measured value 449.1518.

[0267] Example 19: Synthesis of Compound G19

[0268] The structure and synthesis method of compound G19 are as follows:

[0269]

[0270] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 39 (15.5 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 38 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 13 [M+H] + 482.1622, measured value 482.1634.

[0271] Step 2: Weigh compound 38 (18.3 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G19. Compound G19 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 25 N3O 12 [M+H] + 464.1516, measured value 464.1598.

[0272] Example 20: Synthesis of Compound G20

[0273] The structure and synthesis method of compound G20 are as follows:

[0274]

[0275] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 41 (15.6 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 40 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 30 N4O 12 [M+H]+ 483.1938, measured value 483.2004.

[0276] Step 2: Weigh compound 40 (18.3 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G20. Compound G20 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 17 H 28 N4O 11 [M+H] + 465.1833, measured value 465.1918.

[0277] Example 21: Synthesis of Compound G21

[0278] The structure and synthesis method of compound G21 are as follows:

[0279]

[0280] Step 1: Compound 1 (20 mg, 52.5 μmoL) was weighed and dissolved in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer, and compound 43 (15.0 mg, 131.3 μmoL) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 42 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 31 N3O 11 [M+H] + 478.2037, measured value 478.2047.

[0281] Step 2: Weigh compound 42 (18.1 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G21. Compound G21 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 29 N3O 10 [M+H]+ 460.1931 Measured value 460.1979.

[0282] Example 22: Synthesis of Compound G22

[0283] The structure and synthesis method of compound G22 are as follows:

[0284]

[0285] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 45 (15.5 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 44 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 31 N3O 12 [M+H] + 482.1986, measured value 482.2001.

[0286] Step 2: Weigh compound 44 (18.3 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G22. Compound G22 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 11 [M+H] + 464.1880, measured value 464.1624.

[0287] Example 23: Synthesis of Compound G23

[0288] The structure and synthesis method of compound G23 are as follows:

[0289]

[0290] Step 1: Compound 1 (20 mg, 52.5 μmoL) was weighed and dissolved in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer, and compound 47 (17.08 mg, 131.3 μmoL) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 46 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 31 N3O 12 [M+H] + 494.1986, measured value 494.1998.

[0291] Step 2: Weigh compound 46 (18.7 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G23. Compound G23 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 29 N3O 11 [M+H] + 476.1880, measured value 476.1869.

[0292] Example 24: Synthesis of Compound G24

[0293] The structure and synthesis method of compound G24 are as follows:

[0294]

[0295] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 49 (18.66 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 48 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 35 N3O 11 [M+H] + 506.2350, measured value 506.2379.

[0296] Step 2: Weigh compound 48 (19.2 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G24. Compound G24 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 21 H 33 N3O 10 [M+H] + 487.2244, measured value 487.2196.

[0297] Example 25: Synthesis of Compound G25

[0298] The structure and synthesis method of compound G25 are as follows:

[0299]

[0300]

[0301] Step 1: Compound 1 (20 mg, 52.5 μmoL) was weighed and dissolved in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer, and compound 51 (18.13 mg, 131.3 μmoL) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 50 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 27 N5O 11 [M+H] + 502.1785, measured value 502.1820.

[0302] Step 2: Weigh compound 50 (19 mg, 37.94 μmoL) and dissolve it in 2 mL 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G25. Compound G25 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 19 H 25 N5O 10 [M+H] +484.1680, measured value 484.1705

[0303] Example 26: Synthesis of Compound G26

[0304] The structure and synthesis method of compound G26 are as follows:

[0305]

[0306] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 53 (17.3 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 52 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 13 [M+H] + 496.1779, measured value 496.1727.

[0307] Step 2: Weigh compound 52 (18.79 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G26. Compound G26 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 18 H 27 N3O 12 [M+H] + 478.1673, measured value 478.1653.

[0308] Example 27: Synthesis of Compound G27

[0309] The structure and synthesis method of compound G27 are as follows:

[0310]

[0311] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL 50 mM phosphate buffer solution, pH 7.4 buffer. Add compound 55 (19.7 mg, 131.3 μmoL) to the above system and react at 30°C for 4 h. LC-MS monitored the reaction to be complete, and compound 54 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C22 H 31 N3O 11 [M+H] + 514.2037, measured value 514.2119.

[0312] Step 2: Weigh compound 54 (19.47 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G27. Compound G27 was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 22 H 29 N3O 10 [M+H] + 496.1931, measured value 496.1990.

[0313] Example 28: Synthesis of Compound G2'

[0314] The structure and synthesis method of compound G2' are as follows:

[0315]

[0316] Step 1: Weigh compound 1 (20 mg, 52.5 μmoL) and dissolve it in 1 mL of 50 mM phosphate buffer solution, pH 7.4 buffer, add compound 5 (47.47 mg, 183.75 μmoL) to the above system, react at 30°C overnight, and monitor the reaction completion by LC-MS; freeze-dry the reaction system, dissolve the system in 50 mM phosphate buffer solution, pH 6.0, add sodium cyanoborohydride to the system, react at 0°C overnight, monitor the reaction completion by LC-MS, and separate and purify using a semi-preparative C18 column to obtain compound 56. HRMS, calculated value C 24 H 42 N5O 12 S[M+H] + 624.2551, measured value 624.2479.

[0317] Step 2: Weigh compound 56 (23.64 mg, 37.94 μmoL) and dissolve it in 2 mL of 50 mM phosphate buffer solution, pH 8.0. Add CDMBI (124 mg, 0.569 mmoL) to the above reaction system. After the system is mixed evenly, add potassium phosphate (242 mg, 1.14 mmoL), add ddH2O to a total volume of 4 mL, and react at 0°C for 2 h. A large amount of precipitate was observed, and the precipitate was removed by centrifugation to obtain a reaction system containing compound G2'. Compound G2' was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 24 H 40 N5O 11 S[M+H] + 606.2445, measured value 606.2576.

[0318] II. Preparation of small molecule drug-linkers

[0319] Example 29: Synthesis of Compound D1

[0320] The structure and synthesis method of compound D1 are as follows:

[0321]

[0322]

[0323] Step 1: Weigh compound 56 (100 mg, 0.13 mmoL) and dissolve it in 1 mL DMF. Add MMAE (112.4 mg, 0.157 mmoL), HOBt (3.53 mg, 26 μmoL) and pyridine (314 μL, 3.9 mmoL) to the above system in turn, react at 30°C overnight, add 500 μL piperidine to the above system, mix well and react at room temperature for 10 min. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound 57 (95 mg, yield 65%). HRMS, calculated value C 58 H 94 N 10 O 12 [M+H] + 1123.7131, [M+2H] 2+ 562.3605, measured value 1123.7145, 562.3623.

[0324] Step 2: Weigh compound 57 (20 mg, 17.8 μmoL) and dissolve it in 200 μL DMF. Add HATU (13.5 mg, 35.6 μmoL), compound 58 (6.7 mg, 21.4 μmoL), and N,N-diisopropylethylamine DIPEA (93 μL, 53.4 μmoL) to the above system in sequence, mix well and react at 30°C for 3 hours, add 124 μL piperidine to the above system, mix well and react at room temperature for 10 minutes. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound D1 (16 mg, yield 75%). HRMS, calculated value C 60 H 97 N 11 O 14 [M+H] + 1196.7295, [M+2H] 2+ 598.8686, measured values ​​1196.7263, 598.8622.

[0325] Example 30: Synthesis of Compound D2

[0326] The structure and synthesis method of compound D2 are as follows:

[0327]

[0328]

[0329] Step 1: Weigh compound 58 (6.8 mg, 21.7 μmoL) and dissolve it in 68 μL DMF. Add compound 60 (3.75 mg, 32.5 μmoL), compound 61 (6.71 mg, 32.5 μmoL), compound 59 (10 mg, 10.85 mmoL), N, N-diisopropylethylamine DIPEA (11.3 μL, 65.1 μmoL) to the above system in turn, and react at 37°C overnight. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound D2 (6.47 mg, yield 60%). HRMS, calculated value C 51 H 62 N8O 13 [M+H] + 995.4515, measured value 995.4523.

[0330] Example 31: Synthesis of Compound D3

[0331] The structure and synthesis method of compound D3 are as follows:

[0332]

[0333] Step 1: Weigh compound 62 (30 mg, 81.2 μmoL) and dissolve it in 300 μL DMF. Add HATU (92.8 mg, 0.244 mmoL), compound 57 (200 mg, 0.179 mmoL), and N,N-diisopropylethylamine DIPEA (141 μL, 0.812 mmoL) to the above system in sequence, mix well and react at 30°C for 3 hours, add 592 μL piperidine to the above system, mix well and react at room temperature for 10 minutes. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound 63 (348 mg, yield 80%). HRMS, calculated value C 121 H 192 N 21 O 26 [M+H] + 2357.4504, [M+2H] 2+ 1179.2291, [M+3H] 3+ 786.4886, measured values ​​2357.4529, 1179.2371, 786.4456.

[0334] Step 2: Weigh compound 63 (42 mg, 17.8 μmoL) and dissolve it in 200 μL DMF. Add HATU (13.5 mg, 35.6 μmoL), compound 58 (6.7 mg, 21.4 μmoL), and N,N-diisopropylethylamine DIPEA (93 μL, 53.4 μmoL) to the above system in sequence, mix well and react at 30°C for 3 hours, add 124 μL piperidine to the above system, mix well and react at room temperature for 10 minutes. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound D3 (32.4 mg, yield 75%). HRMS, calculated value C 123 H 196 N 22 O 28 [M+H] + 2430.4668, [M+2H] 2+ 1215.7373, [M+3H] 3+ 810.8274, measured values ​​2430.4798, 1215.7412, 810.8620.

[0335] Example 32: Synthesis of Compound D4

[0336] The structure and synthesis method of compound D4 are as follows:

[0337]

[0338] Step 1: Weigh compound 63 (191 mg, 81.2 μmoL) and dissolve it in 1 mL DMF. Add HATU (92.8 mg, 0.244 mmoL), compound 64 (97.7 mg, 97.44 μmoL), and N,N-diisopropylethylamine DIPEA (42.5 μL, 0.244 mmoL) to the above system in sequence, mix well and react at 30°C for 3 h, add 736 μL of piperidine to the above system, mix well and react at room temperature for 10 min. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound 65 (348 mg, yield 82%). HRMS, calculated value C 155 H 259 N 23 O 41 [M+H] + 3099.8967, [M+2H] 2+ 1550.4522, [M+3H] 3+ 1033.9708, measured values ​​3099.9242, 1550.4542, 1033.9698.

[0339] Step 2: Weigh compound 65 (55.1 mg, 17.8 μmoL) and dissolve it in 200 μL DMF. Add HATU (13.5 mg, 35.6 μmoL), compound 58 (6.7 mg, 21.4 μmoL), and N,N-diisopropylethylamine DIPEA (93 μL, 53.4 μmoL) to the above system in sequence, mix well and react at 30°C for 3 hours, add 124 μL piperidine to the above system, mix well and react at room temperature for 10 minutes. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound D4 (42.9 mg, yield 76%). HRMS, calculated value C 123 H 196 N 22 O 28 [M+H] + 3172.9131, [M+2H] 2+ 1586.9609, [M+3H] 3+ 1058.3101, measured values ​​3172.9235, 1586.9719, 1058.3296.

[0340] Example 33: Synthesis of Compound D5

[0341] The structure and synthesis method of compound D5 are as follows:

[0342]

[0343] Step 1: Weigh compound 66 (4.2 mg, 11.4 μmoL) and dissolve it in 42 μL DMF. Add HATU (13 mg, 34.2 μmoL), compound 67 (21 mg, 25 μmoL), and N,N-diisopropylethylamine DIPEA (11.88 μL, 68.2 μmoL) to the above system in sequence. The total volume of the system is 394 μL. Mix well and react at 30°C for 3 hours. Add 394 μL triethylamine to the above system and mix well and react at 30°C for 2 hours. LC-MS monitoring of the reaction system showed that the reaction was complete. Semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound 68 (24 mg, yield 53%). HRMS, calculated value C 89 H 96 F2N 17 O 22 [M+H] + 1792.6884, [M+2H] 2+ 896.8481, measured values ​​1792.3795, 896.4799.

[0344] Step 2: Weigh compound 58 (2.94 mg, 9.39 μmoL) and dissolve it in 30 μL DMF. Add HATU (10.71 mg, 28.2 μmoL), compound 68 (14 mg, 7.82 μmoL), and N,N-diisopropylethylamine DIPEA (4.08 μL, 28.2 μmoL) to the above system in sequence. Mix well and react at 30°C for 3 hours. The total volume of the system is 292 μL. Mix well and react at 30°C for 3 hours. Add 292 μL triethylamine to the above system and mix well and react at 30°C for 2 hours. LC-MS monitoring of the reaction system showed that the reaction was complete. A semi-preparative C18 column was used for separation and purification, and then freeze-dried to obtain compound D5 (10 mg, yield 69%). HRMS, calculated value C 91 H 98 F2N 18 O 24 [M+H] + 1865.7048, [M+2H] 2+ 933.3563, measured values ​​1865.8045, 933.4976.

[0345] III. Preparation of Glycoengineered Antibodies Synthesis of Ab-1 to Ab-7

[0346] Example 34: Synthesis of glycoengineered antibody Ab-1

[0347] The non-natural glycoengineered antibody Ab-1 was obtained by general operation 1 from compound G1 and wild-type antibody Herceptin.

[0348] Example 35: Synthesis of glycoengineered antibody Ab-2

[0349] The non-natural glycoengineered antibody Ab-2 was obtained by general operation 1 using compound G2 and wild-type antibody Herceptin.

[0350] Example 36: Synthesis of glycoengineered antibody Ab-3

[0351] The non-natural glycoengineered antibody Ab-3 was obtained by general operation 1 from compound G3 and wild-type antibody Herceptin.

[0352] Example 37: Synthesis of Glycoengineered Antibody Ab-4

[0353] The non-natural glycoengineered antibody Ab-4 was obtained by general operation 1 from compound G4 and wild-type antibody Herceptin.

[0354] Example 38: Synthesis of glycoengineered antibody Ab-5

[0355] The non-natural glycoengineered antibody Ab-5 was obtained by general operation 1 from compound G5 and wild-type antibody Herceptin.

[0356] Example 39: Synthesis of glycoengineered antibody Ab-6

[0357] The non-natural glycoengineered antibody Ab-6 was obtained by general operation 1 from compound G6 and wild-type antibody Herceptin.

[0358] Example 40: Synthesis of glycoengineered antibody Ab-7

[0359] The non-natural glycoengineered antibody Ab-7 was obtained by general operation 1 from compound G7 and wild-type antibody Herceptin.

[0360] IV. Synthesis of sugar chain-directed antibody-drug conjugates ADC-1 to ADC-5

[0361] Example 41: Synthesis of ADC-1

[0362] Method 1:

[0363] The antibody-drug conjugate ADC-1 is obtained by combining compound D1 and non-natural glycoengineered antibody Ab-1 through operation 2.

[0364] Method 2:

[0365] The antibody-drug conjugate ADC-1 can also be obtained from compound D1 and non-natural glycoengineered antibody Ab-4 through operation 2.

[0366]

[0367] Example 42: Synthesis of ADC-2

[0368] The antibody-drug conjugate ADC-2 is obtained by combining compound D2 and non-natural glycoengineered antibody Ab-1 through operation 2.

[0369]

[0370] Example 43: Synthesis of ADC-3

[0371] The antibody-drug conjugate ADC-3 is obtained by combining compound D3 and non-natural glycoengineered antibody Ab-1 through operation three.

[0372]

[0373] Example 44: Synthesis of ADC-4

[0374] The antibody-drug conjugate ADC-4 is obtained by combining compound D4 and non-natural glycoengineered antibody Ab-1 through operation three.

[0375]

[0376] Example 45: Synthesis of ADC-5

[0377] Method 1:

[0378] The antibody-drug conjugate ADC-5 is obtained by combining compound D5 and non-natural glycoengineered antibody Ab-1 through operation three.

[0379] Method 2:

[0380] The antibody-drug conjugate ADC-5 can also be obtained from compound D5 and non-natural glycoengineered antibody Ab-4 through operation 2.

[0381]

[0382] Example 46: Synthesis of ADC-6

[0383] first step:

[0384] Valerylhydrazone-disaccharide oxazoline (G1), rituximab, and wild-type endoglycosidase Endo-S2 were added to the reaction system in sequence to make the concentrations of each item 1 mM, 15 mg / mL, and 0.1 mg / mL, respectively. The pH of the reaction system was adjusted to 7.0 and incubated at 30°C for 1 h–2 h. After LC-MS confirmed the conversion to the product, small molecule compounds were removed by ultrafiltration to obtain a non-natural glycoengineered antibody based on rituximab.

[0385] Step 2:

[0386] The non-natural glycoengineered antibody based on rituximab prepared above, m-aminobenzoic acid, and compound D5 were added in sequence to make the concentrations of each item 5 mg / mL, 136 mM, and 0.34 mM, respectively. The pH of the reaction system was adjusted to 6.0, and the mixture was incubated at 30°C for 4 h. After LC-MS confirmed the conversion into an ADC product with a specific DAR value, the reaction system was purified by Protein A to obtain the desired sugar chain-directed antibody-drug conjugate. The structural characterization is shown in FIG. Figure 5 shown.

[0387] Example 47: Synthesis of ADC-1 without catalyst

[0388] To a 50 mM phosphate buffer solution, pH 6.0 buffer, non-natural sugar engineering antibody Ab-1 and compound D1 were added in sequence to make the final concentrations of each item 5 mg / mL and 0.34 mM, respectively. The final pH of the reaction system was adjusted to 3-4. After incubation at 30°C for 2 h, mass spectrometry detection results showed that ADC-1 with a DAR value of more than 1.7 was prepared.

[0389] Example 48: Synthesis of ADC-2 without catalyst

[0390] The antibody-drug conjugate ADC-2 is obtained by combining compound D2 and non-natural glycoengineered antibody Ab-1 through operation four.

[0391] V. Screening of Disaccharide-Acylhydrazone Compounds for Preparation of Glycoengineered Antibodies

[0392] Disaccharide-acylhydrazone oxazoline substrates (G1-G27), wild-type Herceptin and endoglycosidase Endo-S2 were added to 50mM phosphate buffer solution, pH 7.0 buffer, and the final concentrations were 0.5mM, 5mg / mL and 0.4mg / mL, respectively. After incubation at 30℃ for 2h, mass spectrometry was performed. A control group without endoglycosidase was set up to deduct the effect of non-enzymatic reaction. The results showed that under the catalysis of endoglycosidase Endo-S2, the DAR values ​​of disaccharide-acylhydrazone compounds G1 and G4 transferred to the antibody were as high as 1.7 or more (such as Figure 1 As shown in the figure); G1-G7 DAR values ​​are above 0.5, and all of them can achieve sugar conversion; other disaccharide-acylhydrazone oxazolines (G2, G3, G5~G27) can also achieve good antibody conversion effects.

[0393] In addition, the conversion conditions of G1 and G4 were further optimized, and it was found that when the concentrations of G1 / G4, wild-type antibody, and wild-type endoglycosidase Endo-S2 were 1 mM, 15 mg / mL, and 0.1 mg / mL, respectively, a high DAR value conversion antibody effect could still be achieved. Therefore, these two disaccharide-acylhydrazone compounds were used to prepare glycoengineered antibodies Ab-1 and Ab-4, and the two glycoengineered antibodies Ab-1 and Ab-4 were used as the non-natural glycosylated antibody part of the sugar chain-directed antibody-drug conjugate.

[0394] VI. Selection and Optimization of Metathesis Reaction Catalysts

[0395]

[0396] Glycoengineering antibodies (Ab-1 / Ab-4), benzylhydroxylamine and various catalysts (69a-69g) were added to 50mM His-HCl, pH 6.0 buffer in sequence, with the final concentrations of each being 0.34mM, 5mg / mL and 136mM, respectively. After incubation at 30°C for 2h, mass spectrometry was performed, and a control group without catalyst was set up to eliminate the reaction effect of non-catalyst. The results showed that under the action of catalysts 69a, 69e-69g, the acylhydrazone-oxime structure achieved complete quantitative exchange; while other catalysts (69b-69d) did not exchange the acylhydrazone-oxime structure under the same conditions, as shown in the following figure. Figure 2 shown.

[0397] It should be noted that, although the above only demonstrates the situation where Ab-1 and Ab-4 act under a catalyst, those skilled in the art will understand that the glycoengineered antibodies prepared using other disaccharide linkers of the present application also have this effect.

[0398] VII. Comparative Experiments of Displacement Reaction and Click Reaction

[0399] Experimental process: According to the general operation 4, under the conditions shown in Table 1 below, ADC was prepared using Acylhydrazone-LacNAc-ox (G1) of the present application and Az-LacNAc-ox (G3 disclosed in CN114949236A), and the DAR value of the transglycosylated antibody was determined. The results are as follows: Figure 6 shown.

[0400] Table 1:

[0401]

[0402] From the results in the figure, it can be seen that at low sugar substrate equivalents, the acylhydrazone sugar substrate can be transferred to the antibody more.

[0403] Pharmacological Example 1

[0404] Experimental process and result analysis of in vitro activity detection

[0405] SK-Br-3 cells and NCI-N87 cells are Her2-positive cells, and MDA-MB-231 cells are Her2-negative cells. The above cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The MTT method was used to test the cell activity and toxicity of the ADC molecules (ADC-1 to ADC-5) prepared above.

[0406] The specific operation is as follows: 100 μL PBS is added to the outermost circle of the 96-well plate, three other wells are added with only culture medium, and about 6,000 corresponding cells are added to each of the remaining wells, and incubated at 37°C in a CO2 incubator overnight. 10 μL of each ADC molecule, including ADC-1, ADC-2, ADC-4, ADC-5, and Herceptin (various ADC molecules are diluted 5-fold from the highest concentration of 100 nM, with a total of 9 concentrations, and 3 replicates for each concentration), and 10 μL of culture medium is added to the remaining 3 wells with cells and the other 3 wells with only culture medium in each 96-well plate as the control group and blank group, and the 96-well plate is placed in a CO2 incubator and incubated at 37°C for 72 hours. After adding 10 μL 5mg / mL MTT to each well, incubate at 37°C for 4 hours, then add 90 μL SDS lysis solution to each well, and incubate at 37°C for 7 hours to fully lyse the cells. Finally, the OD value of each well was measured at 570 nm, and data were processed using GraphPad Prism 6. The results are shown in Figure 3 .from Figure 3 It can be seen from the results in that the ADC prepared by the two-step replacement strategy has good cell activity against Her2-positive cells and is non-toxic in Her2-negative cells.

[0407] Pharmacological Example 2:

[0408] In vivo antitumor activity experimental process and result analysis

[0409] We used gastric cancer cell line NCI-N87 to construct a BALB / c nude mouse xenograft tumor model and adopted the principle of ear hole marking and large, medium and small grouping, with six mice in each group.

[0410] Four ADC compounds ADC-1, ADC-2, ADC-4 and ADC-5 obtained by acylhydrazone-hydroxylamine substitution were selected for animal level activity evaluation, one-step site-coupled ADC compound (DAR≈2) was used as positive control, and PBS was used as negative control. All samples were diluted to 0.2 mg / mL with 1x PBS before administration and sterilized with a 0.22 mm filter membrane for later use.

[0411] All samples were intraperitoneally administered at a concentration of 1 mg / kg, once a week for a total of three times. Starting from the first administration, the tumor size and mouse weight were measured every three days using a vernier caliper. The experimental procedures complied with animal ethics requirements. The measured data were analyzed using GraphPad Prism 6 software. Figure 4 As shown, the sugar chain-directed ADC compound prepared using the patented technology has good in vivo activity.

[0412] Pharmacological Example 3:

[0413] Results and analysis of in vivo antitumor activity of antibody-drug conjugates obtained by acylhydrazone displacement reaction and click chemistry reaction

[0414] Experimental process: ADC-1 obtained by acylhydrazone replacement reaction; ADC-7 obtained by click reaction, the structures are as follows Figure 7 shown.

[0415] A BALB / c nude mouse xenograft tumor model was constructed using gastric cancer cell line NCI-N87, and ear hole marking and large, medium and small grouping principles were used, with six mice in each group.

[0416] ADC-1 obtained by acylhydrazone replacement reaction and ADC-7 obtained by click reaction were selected for animal level activity evaluation. All samples were diluted to 0.2 mg / mL with 1x PBS before administration and sterilized with 0.22 mm filter membrane before use.

[0417] All samples were intraperitoneally administered at a concentration of 1 mg / kg, twice a week for a total of two weeks. Tumor size and mouse weight were measured using a vernier caliper every three days starting from the first administration. The experimental process complied with animal ethics requirements. The measured data were analyzed using GraphPad Prism 6 software.

[0418] Experimental results: Figure 7 As shown, the sugar chain-directed ADC compound ADC-1 prepared by the present application has better in vivo tumor inhibition activity than ADC-7 prepared by click chemistry reaction. The tumor volume of ADC-7 prepared by click chemistry reaction reached the maximum on the 31st day. In contrast, the tumor volume of ADC-1 prepared by replacement reaction was well controlled.

[0419] Experimental conclusion: ADC-1 obtained by acylhydrazone replacement reaction is more hydrophilic than ADC-7 obtained by click reaction and has better tumor inhibition effect in mice.

Claims

1. A disaccharide linker, which is a disaccharide-acylhydrazone linker or a disaccharide-hydrazide linker represented by the following formula I: In Formula I, X is selected from: Structures containing positive and negative charges; C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C10 cycloalkyl, C5-C10 aryl, in, The C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C10 cycloalkyl, C5-C10 aryl is optionally substituted by a substituent selected from aryl, hydroxyl, halogen, R'R"N-, quaternary ammonium ion, C1-C3 alkoxy, 3 to 10 membered saturated or unsaturated heterocyclic group containing 1 to 3 heteroatoms independently selected from N, O, S, wherein R' and R" are each independently H or C1-C3 alkyl; 3 to 10 membered alicyclic and heteroaryl groups containing 1 to 3 heteroatoms independently selected from N, O, and S; and Sulfonic acid group, phosphoric acid group, PEG structure, C1-C6 alkoxycarbonyl group, amide group, hydroxyl group, amino group, azido group, aldehyde group, in, represents a single bond or a double bond, where When there is a double bond, the disaccharide linker is an acylhydrazone structure. The H on the connected N does not exist. When it is a single bond, the disaccharide linker is a hydrazide structure.

2. The disaccharide linker according to claim 1, wherein The structure containing positive and negative charges is selected from quaternary ammonium ions, sulfonium ions, and The alicyclic group and the heteroaryl group are selected from 2-thienyl, 5-pyrimidinyl, 2-tetrahydrofuranyl and tetrazinyl.

3. The disaccharide linker according to claim 1, wherein X is selected from the following: R1-R 14 Each is independently selected from -OH, C1-C20 alkyl, H; W1 is selected from -OH, -CF3; W2-W8 are each independently selected from -[O-(CH2) q ]-、-(CH2) q -; A1, A2, A5 are each independently selected from C, N, O, S; A3 and A4 are each independently selected from C or N; and n and q are independently integers of 0-20, and r and t are independently integers of 1-20.

4. The disaccharide linker according to claim 1, wherein X is a C1-C15 alkyl group, preferably a C1-C10 alkyl group, more preferably a C1-C7 alkyl group, for example, X is a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, or a decyl group; or X is a C3-C8 cycloalkyl group, preferably a C3-C6 cycloalkyl group, for example, X is a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, or a cyclohexyl group.

5. The disaccharide linker according to claim 1, wherein The disaccharide linker is selected from the following compounds: Wherein, n is an integer of 0-20, preferably, n is an integer of 1-15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. A method for preparing a disaccharide linker according to any one of claims 1 to 5, When the disaccharide linker is a disaccharide-acylhydrazone linker, it is prepared by the following method: As shown in the above reaction formula, the method comprises the following steps: Step 1: Oxidation of galactose at position 6 to form aldehyde-containing disaccharide LacNAc (CHO-LacNAc) and hydrazide compound The reaction is carried out under alkaline conditions to obtain a disaccharide-acylhydrazone compound in which the 1 and 2 positions of GlcNAc are not cyclized; Step 2: cyclizing the disaccharide-acylhydrazone compound under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to form oxazoline, thereby obtaining the disaccharide-acylhydrazone linker with cyclization at positions 1 and 2 of the GlcNAc; When the disaccharide linker is a disaccharide-hydrazide linker, it is prepared by the following two routes: As shown above, route one includes the following steps: Step 1: directly reducing CHO-LacNAc and a hydrazide compound in the presence of a reducing agent to obtain a disaccharide-hydrazide compound; Step 2: Cyclize the disaccharide-hydrazide compound to oxazoline under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to obtain a cyclized disaccharide-hydrazide linker at positions 1 and 2 of GlcNAc; Route 2 includes the following steps: Step 1: reacting CHO-LacNAc with a hydrazide compound under alkaline conditions to obtain a disaccharide-acylhydrazone compound; Step 2: adding a reducing agent to the reaction system of step 1 to obtain a disaccharide-hydrazide compound; Step 3: Cyclize the disaccharide-hydrazide compound to oxazoline under the conditions of 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride, potassium phosphate or 2-chloro-1,3-dimethylimidazoline chloride and triethylamine to obtain a disaccharide-hydrazide linker.

7. A drug-linker containing a hydroxylamine functional group, wherein the drug-linker can undergo a replacement reaction with the disaccharide linker according to any one of claims 1 to 5 through the hydroxylamine group at the linker site to prepare a small molecule drug-disaccharide linker, which has a structure shown in the following formula II: In the above formula II, linker1 represents a linker having three or more connection sites derived from an amino acid or a short peptide, for example, it is a linker derived from glutamic acid (Glu), β-glutamic acid, lysine (Lys), Lys-Glu dipeptide or Lysine-β-glutamic acid dipeptide, Linker2 represents a short peptide linear linker with two or more connection sites connected or not connected to p-aminobenzyl carboxylate, for example, it is a tumor microenvironment cleavable or non-cleavable structural unit such as Val-Cit-PAB, Val-Ala-PAB, GGFG, etc.; in, Linker1 and linker2 can further connect PEG chains at the ends of the groups they contain or between linker1 and linker2. For example, when linker1 is a linker derived from a Lys-Glu dipeptide, a PEG chain is further connected to the amino group of lysine. Each R 15 Each independently is -NH2, NHR 16 、-NR 16 R 17 , -N3, fluorescent group, orthogonal group, where R 16 , R 17 Each is independently C1-C20 alkyl, C1-C20 heteroaryl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl, C1-C20 acyl, C1-C20 acyloxy, C1-C20 alkylamino, or each R 15 are each independently a group derived from a toxin small molecule, a radioactive therapeutic, a nucleic acid, and a nuclide, and n1 is 0 and n2 is 1; or n1 is 1 and n2 is an integer of 1-4, for example, n1 is 1, and n2 is 1, 2, 3 or 4, preferably n2 is 2, Among them, when n1 is 1, n2 linker2-R 15 They are covalently connected to linker1, and n2 = the number of connection sites of linker1 - 1, The "short peptide" is a peptide having 2-8 amino acids.

8. The drug-linker containing a hydroxylamine functional group according to claim 7, wherein: The toxin small molecules include maytansine, DM-1, DM-4, ​​MMAE, MMAF, SN-38, Dxd, Dx8951, duocarmycin, amanitin, PBD, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, olaparib, nocodazole, colchicine, estramustine, cemadotin, eloserol; preferably, the toxin small molecules are MMAE, MMAF, Dxd, PBD; The nucleic acid includes antisense nucleic acid ASO, small interfering RNA (siRNA), micro RNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA); and The nuclides include non-metallic element nuclides and metal nuclides. For example, the non-metallic element nuclides are 131 I or 123 I.

9. The drug-linker containing a hydroxylamine functional group according to claim 7, wherein: The hydroxylamine-small molecule drug conjugate is selected from any one of the following compounds: In the above structures, the structure of the MMAE part is:

10. A method for preparing a glycoengineered antibody, the method comprising the following method 1 and method 2: Method 1: in, In the above reaction formula, group X is as defined in claim 1, m is 0 or 1, Method 1: The disaccharide linker, antibody and endoglycosidase according to any one of claims 1 to 5, or a combined endoglycosidase and fucosylation enzyme are co-incubated, and the glycoengineered antibody having the disaccharide linker of formula I connected to Asn297 of the antibody Fc domain is prepared by using the endoglycosidase having both hydrolysis activity on the sugar chain of the antibody Fc domain and transglycosylation activity on the disaccharide-acylhydrazone linker, specifically, the endoglycosidase is selected from Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and fusion proteins formed by fusing the functional regions of these different endoglycosidases; Method 2: Method 2 includes the following two steps: Step 1: The antibody is hydrolyzed by endoglycosidase or endoglycosidase and fucosylation enzyme to remove the heterogeneous sugar chains at the conservative glycosylation sites of the natural antibody to obtain a deglycosylated antibody. Specifically, the endoglycosidase is selected from Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and fusion proteins formed by fusing the functional regions of these different endoglycosidases; Step 2: The disaccharide linker according to any one of claims 1 to 5, the deglycosylated antibody produced in the first step and an endoglycosidase are co-incubated, and the disaccharide linker is transferred to the glycosylation site of the deglycosylated antibody by utilizing the transglycosylation activity of the endoglycosidase on the disaccharide linker, so as to prepare a glycoengineered antibody in which the disaccharide linker shown in formula I is connected to position Asn297 of the Fc domain of the antibody, specifically, the endoglycosidase is selected from Endo-S, Endo-S2, Endo-F3, Endo-M and mutants of these enzymes, and fusion proteins formed by fusing the functional regions of these different endoglycosidases.

11. A glycoengineered antibody prepared by the method of claim 10, which has a structure shown in the following formula III: in, The definition of X is the same as that of X in claim 1, Ab represents an antibody, m is 0 or 1, n is 1 or 2, Preferably, the antibody is an antibody having an N-glycosylation site, More preferably, the antibody is an IgG having a conserved N-glycosylation site at N297 in the Fc region, Specifically, the antibody is IgG1, IgG2 or IgG4, Specifically, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody of different species (mouse, rat, sheep, rabbit, human, etc.), Specifically, the targets of the antibodies include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276 (B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR 1. DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG(5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF , RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R, Preferably, the antibody is selected from trastuzumab and rituximab.

12. A method for preparing a sugar chain-directed antibody-drug conjugate, as follows: In the above reaction formula, X is defined as in claim 1, m is 0 or 1, and R represents the remainder of the drug-linker structure containing a hydroxylamine functional group as described in claim 7 after removing the hydroxylamine group. As shown in the above reaction formula, the method comprises: The glycoengineered antibody of formula III as claimed in claim 11 is incubated with the drug-linker H2N-OR containing a hydroxylamine functional group of formula IV as claimed in claim 7 under the following two conditions: 1) in the absence of a catalyst, the pH is 3-5, preferably 3-4, or 2) in the presence of a catalyst, the pH is 5-7.5, preferably 5.5-7, further preferably 6-6.5, and further preferably 6, to obtain a final antibody-drug conjugate, Wherein, when a catalyst is added, the catalyst has a structure shown in the following formula IV: In the above formula IV, Ra is independently selected from C1-C6 alkyl, carboxyl, n is an integer of 0-3, Preferably, Ra is independently selected from C1-C3 alkyl and carboxyl, and n is 1 or 2, Specifically, the catalyst is selected from the following compounds: aniline, m-aminobenzoic acid, p-aminobenzoic acid, 5-aminoisophthalic acid, 3-amino-5-methyl-benzoic acid.

13. A sugar chain-directed antibody-drug conjugate prepared by the method of claim 12, which is represented by the following formula V: In Formula V, R represents the remaining part of the drug-linker structure containing a hydroxylamine functional group as described in claim 7 after removing the hydroxylamine group, n is 1 or 2, m is 0 or 1, Ab represents an antibody, Preferably, the antibody is an antibody having an N-glycosylation site, More preferably, the antibody is an IgG having a conserved N-glycosylation site at N297 in the Fc region, Specifically, the antibody is IgG1, IgG2 or IgG4, Specifically, the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, trispecific antibody, nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species (such as mouse, rat, sheep, rabbit, human, etc.), Specifically, the targets of the antibody include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276 (B7H3), HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG (5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R, Preferably, the antibody is selected from trastuzumab and rituximab, Preferably, the drug-to-antibody ratio (DAR) of the glycan-site-specific antibody-drug conjugate is 0 < DAR ≤ 4, preferably 0.5 ≤ DAR ≤ 4. More preferably, the DAR is 2 - 4. Even more preferably, the DAR is 2 or 4, Preferably, the glycan-site-specific antibody-drug conjugate is selected from the following:

14. Use of the disaccharide linker as claimed in claim 1 and / or the drug-linker containing a hydroxylamine functional group as claimed in claim 7 in the preparation of an antibody-drug conjugate.

15. Use of the disaccharide linker as claimed in claim 1 in introducing a small toxin molecule, a functional molecule, a radionuclide, a nucleic acid or an orthogonal group into an antibody. Preferably, the functional molecule includes a fluorescent molecule and a radiotherapeutic agent.

16. Use of the disaccharide linker according to claim 1, the drug-linker containing a hydroxylamine functional group according to claim 7, the glycoengineered antibody according to claim 11, and the sugar chain-directed antibody-drug conjugate according to claim 13 in the preparation of a drug or a diagnostic agent, Preferably, the drug is an anti-tumor drug, and preferably, the tumor comprises: Breast cancer, colon cancer, non-small cell carcinoma, brain astrocytoma, chronic myeloid leukemia, pancreatic cancer, acute monocytic leukemia, liver cancer, gastric cancer, non-small cell lung cancer, malignant glioblastoma and prostate adenocarcinoma.

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