Disaccharide-acylhydrazone or disaccharide-hydrazide linker, glyco-engineered antibody and sugar-chain site-directed antibody-drug conjugate based on same, and use thereof

Abstract

Provided are a disaccharide-acylhydrazone or disaccharide-hydrazide linker, a glyco-engineered antibody and a sugar-chain site-directed antibody-drug conjugate based on same, and the use thereof. Specifically, provided is a disaccharide-acylhydrazone or disaccharide-hydrazide linker as shown in the following formula I, which linker can effectively modify a functional group or a cytotoxic drug at an Asn297 glycosylation site in an antibody Fc domain in a site-directed manner. The provided sugar-chain site-directed antibody-drug conjugate (ADC) has a simple and efficient preparation method, which can reduce the amount of small-molecule drugs used, enhances the hydrophilicity of ADC molecules, enriches the structure of ADC compounds, and has great application prospects in ADC preparation.

Description

Disaccharide-acylhydrazone or disaccharide-acylhydrazide linker, glycoengineered antibody and sugar chain-directed antibody-drug conjugate based thereon and use thereof 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. Disaccharide-acylhydrazone linkers are used to prepare non-natural glycoengineered antibodies and antibody-drug conjugates with fixed-point and fixed quantity based on acylhydrazone-oxime displacement reactions, as well as their preparation methods and uses. Background Art

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

[0003] Glycan-directed ADCs are prepared using glycan-directed conjugation technology, where the cytotoxin is modified at the glycosylation site N297 in the antibody Fc domain. Currently, in vitro antibody glycosylation site modification methods primarily include glycosyltransferase technology and endoglycosidase technology.

[0004] Glycosyltransferase technology uses galactosyltransferase or sialyltransferase to transfer galactose or sialic acid with reactive functional groups to antibody glycosylation sites, which are then conjugated to cytotoxins to produce glycan-directed ADC compounds. For example, Zhu et al. first used β1,4-galactosidase to hydrolyze the galactose at the terminal end of the N-glycan at the antibody glycosylation site. They then used galactosyltransferase to transfer the ketocarbonyl-containing GalNAc to the N-glycan terminus, which was then conjugated to a toxin containing a hydroxylamine functional group to produce a glycan-directed ADC compound. Qun Zhou et al. used galactosyltransferase and sialyltransferase to sequentially transfer galactose and sialic acid to antibody glycosylation sites. They then used sodium periodate to oxidize the terminal sialic acid, introducing an aldehyde group at the glycosylation site, providing a reactive site for toxin conjugation. Floris L. van Delft et al. first used the endoglycosidase Endo-S to hydrolyze the heterogeneous N-glycan chains of antibodies, then used galactosyltransferase to transfer the azide-containing GalNAz to the antibody glycosylation site, and finally used click chemistry reaction to achieve the modification of the toxin at the glycosylation site.

[0005] Endoglycosidase technology utilizes endoglycosidases and bioorthogonal reactions to prepare glycan-directed ADCs. Professor Huang Wei's team and the Davis team used semisynthetic modification to obtain an azide-modified oligosaccharide oxazoline substrate. Using two endoglycosidases, Endo-S and its mutant Endo-S D233Q, they ultimately transferred the bioorthogonal group-modified oligosaccharide to the antibody glycosylation site, and further utilized bioorthogonal reactions to obtain a glycan-directed ADC compound.

[0006] Existing glycosyltransferase and endoglycosidase technologies can both produce more uniform ADC compounds than random conjugation, but each has its own problems. In glycosyltransferase technology, sugar substrates with CMP or UDP activation forms need to be synthesized, and glycosyltransferases often have weak catalytic activity, resulting in longer reaction times and difficult-to-control preparation efficiency and costs. The oligosaccharide substrates used in endoglycosidase technology are also difficult to obtain. Semi-synthetic modification methods require extraction from egg yolk, which involves complex purification steps, while full synthesis methods are even more difficult. Both glycosyltransferase and endoglycosidase methods involve multiple enzymes and multi-step reactions, and both have certain limitations in efficacy and production. They are also not conducive to antibody stability. Both methods rely heavily on bioorthogonal reactions to achieve toxin modification at antibody glycosylation sites, which limits the development of sugar chain-directed ADCs.

[0007] Recently, scientists have developed a method for preparing sugar chain-directed antibody-drug conjugates based on disaccharide linkers such as LacNAc, Manβ1,4GlcNAc and Glcβ1,4GlcNAc. 1) The disaccharide-toxin molecule is directly transferred to the antibody glycosylation site through the catalysis of the endoglycosidase Endo-S2, thereby achieving a 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 the recognition ability of the enzyme, 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 the This method 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 expansion of the diversity of sugar chain-directed ADC compounds. 2) The sugar structure with an azide group is transferred through the catalysis of the endoglycosidase Endo-S2 to obtain the corresponding azide-modified glycoengineered antibody, which is further coupled with a 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 molecule and affect the drugability of the ADC compound.

[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.

[0009] Summary of the Invention

[0010] 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. Among them, the disaccharide-acylhydrazone linker can be replaced with a small molecule drug with a hydroxylamine functional group, thereby achieving the site-specific and quantitative introduction of the small molecule drug into the antibody.

[0011] Another technical purpose of the present invention is to provide a class of hydroxylamine small molecule drug conjugates.

[0012] Another technical purpose of the present invention is to provide a glycoengineered antibody prepared by the above-mentioned disaccharide-acylhydrazone linker / disaccharide-hydrazide linker.

[0013] 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-acylhydrazine linker.

[0014] Another technical object of the present invention is to provide the use of the disaccharide-acylhydrazone linker / disaccharide-hydrazide linker in the preparation of ADC.

[0015] Another technical objective 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.

[0016] In one aspect, the present invention provides a disaccharide linker, which is a disaccharide-acylhydrazone linker or a disaccharide-acylhydrazine linker represented by the following formula I:

[0017] In Formula 1,

[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, wherein the C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C10 cycloalkyl, C5-C10 aryl is optionally substituted with a substituent selected from aryl, hydroxyl, halogen, R'R"N-, quaternary ammonium ion, C1-C3 alkoxy, or a 3- to 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;

[0021] 3- to 10-membered alicyclic and heteroaryl groups containing 1-3 heteroatoms independently selected from N, O, and S; and

[0022] Sulfonic acid group, phosphoric acid group, PEG structure, C1-C6 alkoxycarbonyl group, amide group, hydroxyl group, amino group, azide group, aldehyde group,

[0023] in, represents a single bond or a double bond, where When there is a double bond, it is an acylhydrazone structure. As those skilled in the art will understand, the H on N does not exist. When it is a single bond, it is a hydrazide structure.

[0024] In a specific embodiment, the structure containing positive and negative charges can be selected from quaternary ammonium ions and sulfonium ions.

[0025] In a specific embodiment, the heterocyclic group and heteroaryl group can be 2-thienyl, 5-pyrimidinyl, 2-tetrahydrofuranyl, tetrazinyl.

[0026] In a specific embodiment, X is selected from the following:

[0027] R1-R 14 Each is independently selected from -OH, C1-C20 alkyl, H;

[0028] W1 is selected from -OH, -CF3;

[0029] W2-W8 are each independently selected from -[O-(CH2) q ]-、-(CH2) q -;

[0030] A1, A2, A5 are each independently selected from C, N, O, S;

[0031] A3 and A4 are selected from C or N; and

[0032] n and q are independently integers of 0-20, and r and t are independently integers of 1-20.

[0033] 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.

[0034] 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.

[0035] In a specific embodiment, the disaccharide-acylhydrazone linker or disaccharide-acylhydrazine linker is selected from the following compounds:

[0036] Wherein, n is an integer from 0 to 20.

[0037] On the other hand, the present invention also provides a linker in the form of an oxazoline ring-opened form at positions 1 and 2 of GlcNAc in the above-mentioned disaccharide-acylhydrazone linker or disaccharide-acylhydrazine linker.

[0038] On the other hand, the present invention provides a method for preparing the above-mentioned disaccharide-acylhydrazone linker or disaccharide-acylhydrazine linker.

[0039] When the disaccharide linker is a disaccharide-acylhydrazone linker, it is prepared by the following method:

[0040] As shown in the above reaction formula, the method comprises the following steps:

[0041] 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;

[0042] 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 an oxazoline, thereby obtaining the disaccharide-acylhydrazone linker with cyclized positions 1 and 2 of the GlcNAc;

[0043] When the disaccharide linker is a disaccharide-hydrazide linker, it is prepared by the following two routes:

[0044] Among them, route one includes the following steps:

[0045] Step 1: CHO-LacNAc and a hydrazide compound are directly reduced in the presence of a reducing agent to obtain a disaccharide-hydrazide compound;

[0046] Step 2: The disaccharide-hydrazide compound is cyclized 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 cyclized at the 1,2 positions of GlcNAc.

[0047] Route 2 includes the following steps:

[0048] Step 1: reacting CHO-LacNAc with a hydrazide compound under alkaline conditions to obtain a disaccharide-acylhydrazone compound;

[0049] Step 2: adding a reducing agent to the reaction system of step 1 to obtain a disaccharide-hydrazide compound;

[0050] Step 3: The disaccharide-hydrazide compound is cyclized 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.

[0051] In a specific embodiment, the reducing agent used in the preparation of the disaccharide-hydrazide compound is sodium cyanoborohydride.

[0052] In another aspect, the present invention provides a drug-linker containing a hydroxylamine functional group, wherein the drug-linker can undergo a displacement reaction with the disaccharide-acylhydrazone linker described above via the hydroxylamine group at the linker site, thereby preparing a small molecule drug-disaccharide linker. The drug-linker containing a hydroxylamine functional group is shown in the following formula II:

[0053] 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 group derived from glutamic acid (Glu), β-glutamic acid, lysine (Lys), Lys-Glu dipeptide or Lysine-β-glutamic acid dipeptide,

[0054] Linker2 represents a short peptide linear linker with two or more connection sites that is connected or not connected to p-aminobenzyl carboxylate, for example, it can be a tumor microenvironment cleavable or non-cleavable structural unit such as Val-Cit-PAB, Val-Ala-PAB, GGFG;

[0055] 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 Lys-Glu dipeptide, a PEG chain is further connected to the amino group of lysine.

[0056] Each R 15 Each independently represents -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,

[0057] Or each R 15 are each independently a group derived from a toxin, a small molecule, a radioactive therapeutic, a nucleic acid, and a nuclide, and

[0058] n1 is 0 and n2 is 1; or

[0059] n1 is 1 and n2 is an integer from 1 to 4, for example, n1 is 1, and n2 is 1, 2, 3 or 4, preferably n2 is 2,

[0060] Among them, when n1 is 1, n2 linker2-R 15 are covalently linked to linker1, and n2 = the number of linker1 connection sites - 1,

[0061] Here, the "short peptide" refers to a peptide having 2-8 amino acids.

[0062] 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, duocarmycin, amanitin, PBD, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, olaparib, nocodazole, colchicine, estramustine, cemadotin, and elaborinoside; preferably, the toxin small molecules are MMAE, MMAF, Dxd, and PBD.

[0063] In a specific embodiment, the nucleic acid includes but is not limited to antisense nucleic acid ASO, small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA).

[0064] In a specific embodiment, the nuclides include non-metallic element nuclides and metal nuclides, and the non-metallic element nuclides are, for example, 131 I or 123 I.

[0065] In a specific embodiment, the hydroxylamine-small molecule drug conjugate is selected from any one of the following compounds:

[0066] In the above structures, the structure of the MMAE part is:

[0067] In another aspect, the present invention provides a method for preparing a glycoengineered antibody, comprising the following method 1 and method 2:

[0068] Method 1:

[0069] Wherein, in the above reaction formula, X is as defined above, m is 0 or 1,

[0070] Method 1:

[0071] The disaccharide linker described above is co-incubated with an antibody and an endoglycosidase, or a combination of an endoglycosidase and a fucosylation enzyme. Utilizing the endoglycosidase's simultaneous hydrolysis activity on the antibody Fc domain sugar chain and transglycosylation activity on the disaccharide-acylhydrazone linker, a glycoengineered antibody is prepared in which the disaccharide linker represented by Formula I is linked to position 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 thereof, as well as fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0072] Method 2:

[0073] Method 2 includes the following two steps:

[0074] Step 1: The antibody is hydrolyzed using an endoglycosidase or a combination of an endoglycosidase and a fucosylation enzyme to remove the heterogeneous sugar chains at the conserved glycosylation sites of the native antibody, thereby obtaining 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, as well as fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0075] Step 2:

[0076] The disaccharide linker described above, the deglycosylated antibody produced in the first step, and an endoglycosidase are co-incubated. The transglycosylation activity of the endoglycosidase on the disaccharide linker is utilized to transfer the disaccharide linker to the glycosylation site of the deglycosylated antibody, thereby producing a glycoengineered antibody in which the disaccharide linker shown in Formula I is linked to position 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, as well as fusion proteins formed by fusing the functional regions of these different endoglycosidases.

[0077] In another aspect, the present invention provides a glycoengineered antibody prepared by the above-mentioned method for preparing a glycoengineered antibody, which has a structure shown in the following formula III:

[0078] Wherein, the definition of X is the same as that of X in Formula I above, and Ab represents an antibody.

[0079] In a specific embodiment, the antibody is an antibody having an N-glycosylation site,

[0080] In a specific embodiment, the antibody may be an IgG having a conserved N-glycosylation site at N297 in the Fc region.

[0081] In a specific embodiment, the antibody can be IgG1, IgG2 or IgG4.

[0082] 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 of different species (mouse, rat, sheep, rabbit, human, etc.).

[0083] In a specific embodiment, the targets of the antibody include but are not limited to

[0084] 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.

[0085] In a specific embodiment, the antibody may include, but is not limited to, trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, and nivolumab.

[0086] In a specific embodiment, the antibody may be selected from trastuzumab and rituximab.

[0087] In another aspect, the present invention provides a method for preparing a sugar chain-directed antibody-drug conjugate, as shown below:

[0088] 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 portion of the above hydroxylamine small molecule drug conjugate structure after removing the hydroxylamine group.

[0089] As shown in the above reaction formula, the method comprises: incubating a 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 at a pH of 3-5, preferably 3-4, or 2) in the presence of a catalyst at a pH of 5-7.5, preferably 5.5-7, further preferably 6-6.5, and even more preferably 6, to obtain a final antibody-drug conjugate.

[0090] Wherein, the catalyst has the structure shown in the following formula IV:

[0091] In the above formula IV, Ra is independently selected from C1-C6 alkyl and carboxyl, and n is an integer from 0 to 3. Preferably, Ra is a carboxyl.

[0092] In a specific embodiment, Ra is independently selected from C1-C3 alkyl and carboxyl, and n is 1 or 2.

[0093] 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.

[0094] 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:

[0095] In Formula V, R represents a linker-cytotoxin compound, n is 1 or 2, m is 0 or 1, and Ab represents an antibody.

[0096] 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.

[0097] In a specific embodiment, the antibody can be IgG1, IgG2 or IgG4.

[0098] 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 of different species (mouse, rat, sheep, rabbit, human, etc.), or a fragment thereof.

[0099] In a specific embodiment, the targets of the antibody 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, 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.

[0100] In a specific embodiment, the antibodies include but are not limited to trastuzumab, initutuzumab, zenidatuzumab, pertuzumab, sacituzumab, datopotamab, zotocilizumab, cetuximab, panitumumab, nimotuzumab, nexitozumab, ervantuzumab, telisotuzumab, enfortumab, vobramitamab, mirzotamab, deparetumab, muromonab, catumaxomab, belintozumab, blinatumomab, mirvetuximab, belantuzumab, atuximab, rituximab, lovacumab, 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, Lezolimab, Lefarimab, Tusamitamab, Labetuzumab, IMAB027, Tremelimumab, Zevolimab, Botensilimab, Nurulimab, Ipilimumab, Ipilimumab, Tigatuzumab, Lexatumumab, Vofatamab, Talquetamab, Bersanlimab, Enlimomab, Inolimomab, Ozuriftamab, Ladiratuzumab, Gemt Uzumab, Camidanlumab, Nirsevimab, Felvizumab, Bevacizumab, Ramucirumab, Ranibizumab, Denosumab, Denizumab, Tremelimumab, Zevolizumab, Zevolizumab, Alemtuzumab, Palivizumab, Nivolumab, Pembrolizumab, Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Camrelizumab, Lesabelimab, Omalizumab, Tocilizumab, Ustekinumab, Basiliximab, Eculizumab, Brolimumab, Ixetine, Secukinumab, Daclizumab, Elotuzumab, Emicizumab, Iomeclizumab, Elotuzumab, Emicizumab, Belimumab,Alirocumab, Guselkumab, Vedolizumab, Dupilumab, Lanadelumab, Brodalumab, Satralizumab.

[0101] In a specific embodiment, the antibody can be selected from trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, nivolumab.

[0102] In a specific embodiment, the antibody can be trastuzumab or rituximab.

[0103] 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, and still more preferably, the DAR is 2 or 4.

[0104] In a specific embodiment, the glycan-site-specific antibody-drug conjugate can be selected from the following:

[0105] 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 toxin molecules, radiotherapy agents, fluorescent molecules, nucleic acids, and radionuclides.

[0106] 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 an antibody-drug conjugate.

[0107] In another aspect, the present invention provides the use of the above-mentioned disaccharide linker in introducing a small toxin molecule, a functional molecule, a radionuclide, a nucleic acid, or an orthogonal group into an antibody, and the functional molecule includes but is not limited to a small toxin molecule, a fluorescent molecule, and a radiotherapy agent.

[0108] 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 a drug or a diagnostic agent.

[0109] 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 myelogenous leukemia, pancreatic cancer, acute monocytic leukemia, liver cancer, gastric cancer, non-small cell lung cancer, malignant glioblastoma, and prostate adenocarcinoma. Beneficial effects

[0110] The present invention has developed a novel disaccharide linker containing an acylhydrazone / acylhydrazide structure. This disaccharide linker can effectively site-specifically modify target molecules (including all functional molecules and cytotoxic molecules known in the art) at the glycosylation site Asn297 in the Fc domain of an antibody. The preparation method of the sugar chain-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

[0111] Figure 1 shows the screening results of some disaccharide-acylhydrazone oxazolines of the present application. Different disaccharide-acylhydrazone oxazoline substrates were screened for their ability to transfer to antibody glycosylation sites.

[0112] FIG2 shows the catalytic effect of the catalyst used in the replacement reaction of the acylhydrazone-oxime structure involved in this application.

[0113] FIG3 shows the in vitro activity experimental data results of some ADCs obtained by acylhydrazone-hydroxylamine displacement reaction of the present application.

[0114] A: Inhibitory rate of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on SK-Br-3 cells; B: Inhibitory rate of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on NCI-N87 cells; C: Effect of some ADCs (ADC-1, ADC-2, ADC-4, ADC-5) on the survival rate of MDA-MB-231 cells.

[0115] Figure 4 shows the in vivo activity data of some ADCs prepared by the acylhydrazone-hydroxylamine replacement method of this application. The data show the inhibition of tumor volume (A) and the effect of ADC-1, ADC-2, ADC-4, and ADC-5 on nude mouse body weight (B).

[0116] Figure 5 shows the structural representation of the non-natural glycoengineered antibody of rituximab and the sugar chain-directed antibody-drug conjugate ADC-6 prepared therefrom, wherein, left: Rituximab; middle: Rituximab-C5; right: ADC-6 conjugated with four Dxd toxins: Rituximab-LacNAc-C=NOE-(GGFG-Dxd)2.

[0117] FIG6 shows a comparison of the DAR values ​​of ADCs prepared by Acylhydrazone-LacNAc-ox and Az-LacNAc-ox of the present application.

[0118] The experimental results show that under the conditions of an antibody concentration of 15 mg / mL and 5 equivalents of oxazoline, the antibody glycosylation effect using the acylhydrazone disaccharide oxazoline substrate is better than the conversion effect using the monoazide substrate, indicating that fewer equivalents are required to achieve high DAR conversion using the acylhydrazone disaccharide substrate.

[0119] FIG7 shows the in vivo anti-tumor activity test results of the antibody-drug conjugates obtained by acylhydrazone displacement reaction and click chemistry reaction. DETAILED DESCRIPTION

[0120] Terminology: "Multivalent linker" in this application refers to a linker with a valency greater than two.

[0121] The small molecule cytotoxic drugs PBD, Dxd, and MMAE used in this 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 Shengong Bioengineering (Shanghai) Co., Ltd.; amino acid compounds were purchased from Shanghai Jier Biochemical Co., Ltd.; and various hydrazide compounds were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. All other compounds and reagents, unless otherwise specified, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0122] 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 Innovation Tongheng LC3000), preparative high performance liquid chromatograph (Beijing Innovation Tongheng LC3000); ThermoC18 (Acclaim™ 120, 5 μm, 4.6 x 250 mm), Agilent SB-C18 (5 μm, 4.6 x 150 mm), Waters C18 column (ACQUITY UPLC BEHC18, 1.7 μm, 2.1 x 50 mm).

[0123] 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).

[0124] The following reaction schematics generally illustrate the preparation routes of the disaccharide-acylhydrazone linker, disaccharide-hydrazide linker, glycoengineered antibodies, and sugar chain-directed antibody-drug conjugates (ADCs) based on acylhydrazone-oxime replacement chemistry in this application.

[0125] General Preparation Example

[0126] The synthetic route of the disaccharide-acylhydrazone linker is as follows:

[0127] in,

[0128] 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-benzimidazol-3-yl chloride, potassium phosphate.

[0129] The synthetic route of disaccharide-hydrazide linker is as follows:

[0130] in,

[0131] Note: a. Goase, catalase, horseradish peroxidase (HRP), O2, pH 7.0, 30°C; b. pH 7.4, 30°C; c. NaBH3CN, pH 6.0; d. 2-chloro-1,3-dimethyl-1H-benzimidazol-3-yl chloride, potassium phosphate.

[0132] The preparation route of non-natural glycoengineered antibodies is as follows:

[0133] Wherein, in the above reaction formula, X is defined as above, and m is selected from 0 or 1.

[0134] Preparation method 1:

[0135] Glycoengineered antibodies: Glycoengineered antibodies containing the disaccharide linker are prepared by incubating the above-described disaccharide linker, an antibody, and an endoglycosidase (or a combination of an endoglycosidase and a fucosylation enzyme). The endoglycosidase possesses both hydrolysis activity on the antibody Fc domain sugar chains and transglycosylation activity on the disaccharide linker. Specifically, the endoglycosidase includes, but is not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and mutants of these enzymes, as well as fusion proteins formed by fusing the functional domains of these different endoglycosidases.

[0136] Preparation method 2:

[0137] Step 1: The antibody is hydrolyzed with an endoglycosidase or a combination of an endoglycosidase and a fucosylation enzyme to remove the heterogeneous sugar chains at the conserved glycosylation sites of the native antibody, thereby obtaining 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, as well as fusion proteins formed by fusing the functional domains of these different endoglycosidases.

[0138] Step 2:

[0139] Glycoengineered antibodies: The disaccharide linker described above is co-incubated with a deglycosylated antibody and an endoglycosidase. The endoglycosidase's transglycosylation activity on the disaccharide linker is utilized to transfer the disaccharide linker to the glycosylation site of the deglycosylated antibody, thereby preparing a glycoengineered antibody containing the disaccharide linker. Specifically, the endoglycosidase includes, but is not limited to, Endo-S, Endo-S2, Endo-F3, Endo-M, and mutants of these enzymes, as well as fusion proteins formed by fusing the functional regions of different endoglycosidases.

[0140] The preparation route of sugar chain-directed antibody-drug conjugates is as follows:

[0141] Wherein, X is defined as described above, m is selected from 0 or 1, and R represents the remaining portion of the hydroxylamine small molecule drug conjugate structure after removing the hydroxylamine group.

[0142] Preparation method:

[0143] The glycoengineered antibody with a disaccharide linker is incubated with a drug-linker with a hydroxylamine functional group in a neutral buffer, and a catalyst is added to promote the displacement reaction to obtain the final antibody-drug conjugate. The catalyst has the structure shown in Formula IV described above.

[0144] General operation 1:

[0145] A method for preparing a non-natural glycoengineered antibody based on disaccharide-acylhydrazone or disaccharide-acylhydrazine comprises preparing the prepared disaccharide-acylhydrazone oxazoline (i.e., compounds G1 to G27) or disaccharide-acylhydrazine oxazoline (i.e., compound G2'), a wild-type antibody, and a wild-type endoglycosidase Endo-S2, adjusting the concentrations of each component to 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 1–2 hours. After LC-MS confirmation of conversion to product, small molecule compounds are removed by ultrafiltration to obtain the desired non-natural glycoengineered antibodies Ab-1 to Ab-7, as shown in Examples 34-40 below.

[0146] General operation two:

[0147] Method 1 for preparing site-directed sugar chain antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry: Non-natural glycoengineered antibodies (i.e., Ab-1, Ab-4) prepared with disaccharide-acylhydrazone linkers, m-aminobenzoic acid, and drug-linkers containing hydroxylamine structures (i.e., compounds D1 and D2) are added at concentrations of 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 4–16 hours. After LC-MS confirmation of conversion to an ADC product with a specific DAR value, low-molecule compounds are removed by ultrafiltration to obtain the desired site-directed sugar chain antibody-drug conjugates ADC-1 to ADC-2, as shown in Examples 41 to 42 below.

[0148] General operation three:

[0149] Method 2 for preparing sugar chain-directed antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry: Non-natural glycoengineered antibodies (i.e., Ab-1, Ab-4) prepared with disaccharide-acylhydrazone linkers, 5-aminoisophthalic acid, and drug-linkers containing a hydroxylamine structure (i.e., compounds D3-D5) were prepared at concentrations of 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 4–16 h. After LC-MS confirmation of conversion to an ADC product with a specific DAR value, low-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.

[0150] General operation four:

[0151] Method 3 for preparing sugar chain-directed antibody-drug conjugates based on acylhydrazone-oxime replacement chemistry (catalyst-free): Non-natural glycoengineered antibodies (i.e., Ab-1, Ab-4) prepared with disaccharide-acylhydrazone linkers and drug-linkers containing hydroxylamine structures (i.e., compounds D1-D2) were prepared at concentrations of 5 mg / mL and 0.34 mM, respectively. The pH of the reaction system was adjusted to 3-5, and the mixture was incubated at 30°C for 2-16 hours. After LC-MS confirmation of conversion to an ADC product with a specific DAR value, low-molecule compounds were removed by ultrafiltration to obtain the desired sugar chain-directed antibody-drug conjugates ADC-1 to ADC-2, as shown in Examples 47-48 below.

[0152] The following describes the specific process of preparing each disaccharide linker, glycoengineered antibody, and sugar chain-directed antibody-drug conjugate using the above general preparation method through specific examples.

[0153] I: Preparation of disaccharide linkers

[0154] Example 1: Synthesis of Compound G1

[0155] The structure and synthesis method of compound G1 are as follows:

[0156] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer (pH 7.4). Compound 3 (15.24 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. Compound 2 (22.6 mg, 90% yield) was obtained after separation and purification using a semi-preparative C18 column. HRMS, calculated value C 14 H 33 N3O 11 [M+H] + 480.2193, measured value 480.2424.

[0157] Step 2: Weigh compound 2 (20 mg, 41.8 μmoL) and dissolve it in 2 mL of 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) and add ddH2O to a total volume of 4 mL. React at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G1. Compound G1 (12 mg, 63% yield) 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 462.1957.

[0158] Example 2: Synthesis of Compound G2

[0159] The structure and synthesis method of compound G2 are as follows:

[0160] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 5 (47.47 mg, 183.75 μmol) was added to the above system and reacted at 30°C overnight to obtain compound 4. The reaction was monitored by LC-MS. HRMS, calculated value C 24 H 39 N5O 12 S[M+H] + 622.2394, measured value 622.2370.

[0161] Step 2: Compound 4 (20 mg, 32.2 μmoL) was weighed and dissolved in 1.6 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (105 mg, 0.483 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (205 mg, 0.966 mmol) was added. ddH2O was added to a total volume of 3.2 mL and the reaction was carried out at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G2. Compound G2 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 24 H 37 N 11 S[M+H] + 604.2289, measured value 604.2928.

[0162] Example 3: Synthesis of Compound G3

[0163] The structure and synthesis method of compound G3 are as follows:

[0164] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 7 (17.85 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 6. HRMS, calculated value C 21 H 29 N3O 11 [M+H] + 500.1880, measured value 500.1891.

[0165] Step 2: Compound 6 (20 mg, 40.07 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (131 mg, 0.603 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (255 mg, 1.20 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G3. Compound G3 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 21 H 27 N3O 10 [M+H] + 482.1775, measured value 482.1739.

[0166] Example 4: Synthesis of Compound G4

[0167] The structure and synthesis method of compound G4 are as follows:

[0168] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 9 (18.65 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 8 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 27 N3O 11 S[M+H] + 506.1445, measured value 506.2019.

[0169] Step 2: Compound 8 (20 mg, 39.59 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (129 mg, 0.594 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (253 mg, 1.19 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G4. Compound G4 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 25 N3O 10 S[M+H] + 488.1339, measured value 488.1459.

[0170] Example 5: Synthesis of Compound G5

[0171] The structure and synthesis method of compound G5 are as follows:

[0172] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 11 (13.14 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 10. HRMS, calculated value C 18 H 29 N3O 11 [M+H] + 464.1880, measured value 464.1795.

[0173] Step 2: Compound 10 (20 mg, 43.18 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (141 mg, 0.648 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (276 mg, 1.30 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G5. Compound G5 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 18 H 27 N3O 10 [M+H] + 446.1775, measured value 446.1291.

[0174] Example 6: Synthesis of Compound G6

[0175] The structure and synthesis method of compound G6 are as follows:

[0176] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 13 (15.24 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 12. HRMS, calculated value C 19 H 33 N3O 11 [M+H] + 480.2193, measured value 480.1732.

[0177] Step 2: Compound 12 (20 mg, 41.74 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (135 mg, 0.626 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (265 mg, 1.25 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G6. Compound G6 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 31 N3O 10 [M+H] + 462.2088, measured value 462.2106.

[0178] Example 7: Synthesis of Compound G7

[0179] The structure and synthesis method of compound G7 are as follows:

[0180] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 15 (21.55 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 14 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 23 H 33 N3O 11 [M+H] + 528.2193, measured value 528.2027.

[0181] Step 2: Compound 14 (20 mg, 7.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added, and ddH2O was added to a total volume of 4 mL. The reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G7. Compound G7 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 23 H 31 N3O 10 [M+H] + 510.2088, measured value 510.2107.

[0182] Example 8: Synthesis of Compound G8

[0183] The structure and synthesis method of compound G8 are as follows:

[0184] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 17 (15.37 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 16. HRMS, calculated value C 18 H 32 N4O 11 [M+H] + 481.2146, measured value 481.2217.

[0185] Step 2: Compound 16 (20 mg, 41.65 μmol) was weighed and dissolved in 2 mL of 50 mM phosphate buffer, pH 8.0. DMC (211 mg, 1.25 mmol) was added to the above reaction system. After the system was mixed evenly, triethylamine (210 mg, 2.08 mmol) was added. ddH2O was added to the total volume of 4 mL. The reaction was carried out 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.

[0186] Example 9: Synthesis of Compound G9

[0187] The structure and synthesis method of compound G9 are as follows:

[0188] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 19 (17.35 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 18 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 35 N4O 11 + [M] + 496.2374, measured value 496.2320.

[0189] Step 2: Compound 18 (20 mg, 40.39 μmol) was weighed and dissolved in 2 mL of 50 mM phosphate buffer, pH 8.0. DMC (205 mg, 1.21 mmol) was added to the above reaction system. After the system was mixed evenly, triethylamine (204 mg, 2.02 mmol) was added. ddH2O was added to the total volume of 4 mL. The reaction was carried out 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.

[0190] Example 10: Synthesis of Compound G10

[0191] The structure and synthesis method of compound G10 are as follows:

[0192] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 21 (9.72 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 20. HRMS, calculated value C 16 H 27 N3O 11 [M+H] + 438.1724, measured value 438.1739.

[0193] Step 2: Compound 20 (17 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G10. Compound G10 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 16 H 25 N3O 10 [M+H] + 420.1618, measured value 420.1629.

[0194] Example 11: Synthesis of Compound G11

[0195] The structure and synthesis method of compound G11 are as follows:

[0196] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 23 (11.56 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 22. HRMS, calculated value C 17 H 29 N3O 11 [M+H] + 452.1880, measured value 452.1896.

[0197] Step 2: Compound 22 (17 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G11. Compound G11 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 10 [M+H] + 434.1775, measured value 434.1698.

[0198] Example 12: Synthesis of Compound G12

[0199] The structure and synthesis method of compound G12 are as follows:

[0200] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 25 (13.40 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 24. HRMS, calculated value C 18 H 31 N3O 11 [M+H] + 466.2037, measured value 466.2039.

[0201] Step 2: Compound 24 (18 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G12. Compound G12 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 10 [M+H] + 448.1931, measured value 448.1990.

[0202] Example 13: Synthesis of Compound G13

[0203] The structure and synthesis method of compound G13 are as follows:

[0204] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 27 (17.06 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 26. HRMS, calculated value C 20 H 35 N3O 11 [M+H] + 494.2350, measured value 494.2409.

[0205] Step 2: Compound 26 (19 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G13. Compound G13 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 20 H 33 N3O 10 [M+H] + 476.2244, measured value 476.2278.

[0206] Example 14: Synthesis of Compound G14

[0207] The structure and synthesis method of compound G14 are as follows:

[0208] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 29 (18.92 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 28. HRMS, calculated value C 21 H 37 N3O 11 [M+H] + 508.2506, measured value 508.2590.

[0209] Step 2: Compound 28 (19.2 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G14. Compound G14 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 21 H 35 N3O 10 [M+H] + 490.2401, measured value 490.2399.

[0210] Example 15: Synthesis of Compound G15

[0211] The structure and synthesis method of compound G15 are as follows:

[0212] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 31 (20.76 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 30. HRMS, calculated value C 22 H 39 N3O 11 [M+H] + 522.2663, measured value 522.2679.

[0213] Step 2: Compound 30 (20 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G15. Compound G15 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 22 H 37 N3O 10 [M+H] + 504.2557, measured value 504.2564.

[0214] Example 16: Synthesis of Compound G16

[0215] The structure and synthesis method of compound G16 are as follows:

[0216] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 33 (13.66 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 32. HRMS, calculated value C 17 H 29 N3O 12 [M+H] + 468.1829, measured value 468.1796.

[0217] Step 2: Compound 32 (18.5 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G16. Compound G16 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 10 [M+H] + 450.1724, measured value 450.1793.

[0218] Example 17: Synthesis of Compound G17

[0219] The structure and synthesis method of compound G17 are as follows:

[0220] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 35 (11.82 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 34. HRMS, calculated value C 16 H 27 N3O 12 [M+H] + 454.1673, measured value 454.1791.

[0221] Step 2: Compound 34 (17.2 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G17. Compound G17 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 16 H 25 N3O 11 [M+H] + 436.1567, measured value 436.1575.

[0222] Example 18: Synthesis of Compound G18

[0223] The structure and synthesis method of compound G18 are as follows:

[0224] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 37 (13.52 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 36 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 16 H 26 N4O 12 [M+H] + 467.1625, measured value 467.1644.

[0225] Step 2: Compound 36 (17.7 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G18. Compound G18 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 16 H 24 N4O 11 [M+H] + 449.1520, measured value 449.1518.

[0226] Example 19: Synthesis of Compound G19

[0227] The structure and synthesis method of compound G19 are as follows:

[0228] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 39 (15.5 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 38 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 17 H 27 N3O 13 [M+H] + 482.1622, measured value 482.1634.

[0229] Step 2: Compound 38 (18.3 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G19. Compound G19 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 17 H 25 N3O 12 [M+H] + 464.1516, measured value 464.1598.

[0230] Example 20: Synthesis of Compound G20

[0231] The structure and synthesis method of compound G20 are as follows:

[0232] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 41 (15.6 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 40. HRMS, calculated value C 17 H 30 N4O 12 [M+H] + 483.1938, measured value 483.2004.

[0233] Step 2: Compound 40 (18.3 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G20. Compound G20 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 17 H 28 N4O 11 [M+H] + 465.1833, measured value 465.1918.

[0234] Example 21: Synthesis of Compound G21

[0235] The structure and synthesis method of compound G21 are as follows:

[0236] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 43 (15.0 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 42. HRMS, calculated value C 19 H 31 N3O 11 [M+H] + 478.2037, measured value 478.2047.

[0237] Step 2: Compound 42 (18.1 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G21. Compound G21 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 29 N3O 10 [M+H] + 460.1931 Measured value 460.1979.

[0238] Example 22: Synthesis of Compound G22

[0239] The structure and synthesis method of compound G22 are as follows:

[0240] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 45 (15.5 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 44. HRMS, calculated value C 18 H 31 N3O 12 [M+H] + 482.1986, measured value 482.2001.

[0241] Step 2: Compound 44 (18.3 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G22. Compound G22 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 11 [M+H] + 464.1880, measured value 464.1624.

[0242] Example 23: Synthesis of Compound G23

[0243] The structure and synthesis method of compound G23 are as follows:

[0244] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 47 (17.08 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 46. HRMS, calculated value C 19 H 31 N3O 12 [M+H] + 494.1986, measured value 494.1998.

[0245] Step 2: Compound 46 (18.7 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G23. Compound G23 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 29 N3O 11 [M+H] + 476.1880, measured value 476.1869.

[0246] Example 24: Synthesis of Compound G24

[0247] The structure and synthesis method of compound G24 are as follows:

[0248] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 49 (18.66 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 48. HRMS, calculated value C 21 H 35 N3O 11 [M+H] + 506.2350, measured value 506.2379.

[0249] Step 2: Compound 48 (19.2 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G24. Compound G24 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 21 H 33 N3O 10 [M+H] + 487.2244, measured value 487.2196.

[0250] Example 25: Synthesis of Compound G25

[0251] The structure and synthesis method of compound G25 are as follows:

[0252] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 51 (18.13 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 50. HRMS, calculated value C 19 H 27 N5O 11 [M+H] + 502.1785, measured value 502.1820.

[0253] Step 2: Compound 50 (19 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G25. Compound G25 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 19 H 25 N5O 10 [M+H] + 484.1680, measured value 484.1705

[0254] Example 26: Synthesis of Compound G26

[0255] The structure and synthesis method of compound G26 are as follows:

[0256] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 53 (17.3 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. LC-MS monitored the reaction completion. Compound 52 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 18 H 29 N3O 13 [M+H] + 496.1779, measured value 496.1727.

[0257] Step 2: Compound 52 (18.79 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer solution, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to the total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G26. Compound G26 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 18 H 27 N3O 12 [M+H] + 478.1673, measured value 478.1653.

[0258] Example 27: Synthesis of Compound G27

[0259] The structure and synthesis method of compound G27 are as follows:

[0260] Step 1: Compound 1 (20 mg, 52.5 μmol) was weighed and dissolved in 1 mL of 50 mM phosphate buffer, pH 7.4. Compound 55 (19.7 mg, 131.3 μmol) was added to the above system and reacted at 30°C for 4 h. The reaction was monitored by LC-MS and purified using a semi-preparative C18 column to obtain compound 54. HRMS, calculated value C 22 H 31 N3O 11 [M+H] + 514.2037, measured value 514.2119.

[0261] Step 2: Compound 54 (19.47 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G27. Compound G27 was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 22 H 29 N3O 10 [M+H] + 496.1931, measured value 496.1990.

[0262] Example 28: Synthesis of Compound G2'

[0263] The structure and synthesis method of compound G2' are as follows:

[0264] 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 and react at 30°C overnight. LC-MS monitors the reaction to be complete. After freeze-drying 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, LC-MS monitors the reaction to be complete, 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.

[0265] Step 2: Compound 56 (23.64 mg, 37.94 μmoL) was weighed and dissolved in 2 mL of 50 mM phosphate buffer, pH 8.0. CDMBI (124 mg, 0.569 mmol) was added to the above reaction system. After the system was mixed evenly, potassium phosphate (242 mg, 1.14 mmol) was added. ddH2O was added to a total volume of 4 mL and the reaction was incubated at 0°C for 2 h. A large amount of precipitate was observed. The precipitate was removed by centrifugation to obtain a reaction system containing compound G2'. Compound G2' was isolated and purified using a semi-preparative C18 column. HRMS, calculated value C 24 H 40 N5O 11 S[M+H] + 606.2445, measured value 606.2576.

[0266] II. Preparation of Small Molecule Drug-Linkers

[0267] Example 29: Synthesis of Compound D1

[0268] The structure and synthesis method of compound D1 are as follows:

[0269] Step 1: Weigh compound 56 (100 mg, 0.13 mmol) and dissolve it in 1 mL of 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 sequence. React at 30°C overnight. Add 500 μL of 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. Use a semi-preparative C18 column for separation and purification and lyophilization 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.

[0270] 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. Compound D1 (16 mg, yield 75%) was obtained after separation and purification using a semi-preparative C18 column and lyophilization. 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.

[0271] Example 30: Synthesis of Compound D2

[0272] The structure and synthesis method of compound D2 are as follows:

[0273] 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 sequence, and react at 37°C overnight. LC-MS monitoring of the reaction system showed that the reaction was complete. The compound D2 (6.47 mg, 60% yield) was obtained after separation and purification using a semi-preparative C18 column and lyophilization. HRMS, calculated value C 51 H 62 N8O 13 [M+H] + 995.4515, measured value 995.4523.

[0274] Example 31: Synthesis of Compound D3

[0275] The structure and synthesis method of compound D3 are as follows:

[0276] 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. The semi-preparative C18 column was used for separation and purification and then lyophilized 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.

[0277] 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. The compound D3 (32.4 mg, yield 75%) was separated and purified using a semi-preparative C18 column and then lyophilized to obtain. 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.

[0278] Example 32: Synthesis of Compound D4

[0279] The structure and synthesis method of compound D4 are as follows:

[0280] Step 1: Weigh compound 63 (191 mg, 81.2 μmoL) and dissolve it in 1 mL of 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 hours. Add 736 μL of 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. The product was separated and purified using a semi-preparative C18 column and then lyophilized 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.

[0281] 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. The compound D4 (42.9 mg, yield 76%) was separated and purified using a semi-preparative C18 column and then lyophilized to obtain. 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.

[0282] Example 33: Synthesis of Compound D5

[0283] The structure and synthesis method of compound D5 are as follows:

[0284] 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, 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 lyophilization was performed 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.

[0285] 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 lyophilization was performed 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.

[0286] III. Preparation of Glycoengineered Antibodies Synthesis of Ab-1 to Ab-7

[0287] Example 34: Synthesis of glycoengineered antibody Ab-1

[0288] The non-natural glycoengineered antibody Ab-1 was obtained by reacting compound G1 and wild-type antibody Herceptin through general operation 1.

[0289] Example 35: Synthesis of glycoengineered antibody Ab-2

[0290] The non-natural glycoengineered antibody Ab-2 was obtained by general operation 1 using compound G2 and wild-type antibody Herceptin.

[0291] Example 36: Synthesis of glycoengineered antibody Ab-3

[0292] The non-natural glycoengineered antibody Ab-3 was obtained by general operation 1 using compound G3 and wild-type antibody Herceptin.

[0293] Example 37: Synthesis of glycoengineered antibody Ab-4

[0294] The non-natural glycoengineered antibody Ab-4 was obtained by general operation 1 using compound G4 and wild-type antibody Herceptin.

[0295] Example 38: Synthesis of glycoengineered antibody Ab-5

[0296] The non-natural glycoengineered antibody Ab-5 was obtained by general operation 1 using compound G5 and wild-type antibody Herceptin.

[0297] Example 39: Synthesis of glycoengineered antibody Ab-6

[0298] The non-natural glycoengineered antibody Ab-6 was obtained by general operation 1 by reacting compound G6 and wild-type antibody Herceptin.

[0299] Example 40: Synthesis of glycoengineered antibody Ab-7

[0300] The non-natural glycoengineered antibody Ab-7 was obtained by general operation 1 by reacting compound G7 and wild-type antibody Herceptin.

[0301] IV. Synthesis of Glycan-Directed Antibody-Drug Conjugates ADC-1 to ADC-5

[0302] Example 41: Synthesis of ADC-1

[0303] Method 1:

[0304] The antibody-drug conjugate ADC-1 is obtained by combining compound D1 and non-natural glycoengineered antibody Ab-1 through operation 2.

[0305] Method 2:

[0306] Antibody-drug conjugate ADC-1 can also be obtained from compound D1 and non-natural glycoengineered antibody Ab-4 through operation 2.

[0307] Example 42: Synthesis of ADC-2

[0308] The antibody-drug conjugate ADC-2 is obtained by combining compound D2 and the non-natural glycoengineered antibody Ab-1 through operation 2.

[0309] Example 43: Synthesis of ADC-3

[0310] The antibody-drug conjugate ADC-3 is obtained by combining compound D3 and the non-natural glycoengineered antibody Ab-1 through operation three.

[0311] Example 44: Synthesis of ADC-4

[0312] The antibody-drug conjugate ADC-4 is obtained by combining compound D4 and the non-natural glycoengineered antibody Ab-1 through operation three.

[0313] Example 45: Synthesis of ADC-5

[0314] Method 1:

[0315] The antibody-drug conjugate ADC-5 is obtained by combining compound D5 and the non-natural glycoengineered antibody Ab-1 through operation three.

[0316] Method 2:

[0317] Antibody-drug conjugate ADC-5 can also be obtained from compound D5 and non-natural glycoengineered antibody Ab-4 through operation 2.

[0318] Example 46: Synthesis of ADC-6

[0319] first step:

[0320] Pentylhydrazone-disaccharide oxazoline (G1), rituximab, and wild-type endoglycosidase Endo-S2 were added to the reaction system in sequence to adjust the concentrations to 1 mM, 15 mg / mL, and 0.1 mg / mL, respectively. The pH of the reaction system was adjusted to 7.0, and the mixture was incubated at 30°C for 1–2 h. After LC-MS confirmed the conversion to the product, the small molecule compounds were removed by ultrafiltration to obtain a non-natural glycoengineered antibody based on rituximab.

[0321] Step 2:

[0322] The non-natural glycoengineered antibody based on rituximab prepared above, m-aminobenzoic acid, and compound D5 were added in sequence to adjust the concentrations to 5 mg / mL, 136 mM, and 0.34 mM, respectively. The pH of the reaction system was adjusted to 6.0 and incubated at 30°C for 4 h. After LC-MS confirmed the conversion to 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 Figure 5.

[0323] Example 47: Synthesis of ADC-1 without catalyst

[0324] To a 50 mM phosphate buffer solution, pH 6.0, the non-natural glycoengineered antibody Ab-1 and compound D1 were added sequentially to final concentrations of 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 analysis revealed that ADC-1 with a DAR value of greater than 1.7 was prepared.

[0325] Example 48: Synthesis of ADC-2 without Catalyst

[0326] The antibody-drug conjugate ADC-2 is obtained by combining compound D2 and the non-natural glycoengineered antibody Ab-1 through operation four.

[0327] V. Screening of Disaccharide-Acylhydrazone Compounds for Preparation of Glycoengineered Antibodies

[0328] Disaccharide-acylhydrazone oxazoline substrates (G1-G27), wild-type Herceptin, and endoglycosidase Endo-S2 were sequentially added to 50mM phosphate buffer, pH 7.0, to final concentrations of 0.5mM, 5mg / mL, and 0.4mg / mL, respectively. After incubation at 30°C for 2h, mass spectrometry was performed. A control group without endoglycosidase was also set up to eliminate the effects of non-enzymatic reactions. 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 above (as shown in Figure 1); G1-G7 had DAR values ​​of over 0.5, indicating that transglycosylation was achieved. Other disaccharide-acylhydrazone oxazolines (G2, G3, G5-G27) also achieved good transglycosylation.

[0329] In addition, the conversion conditions of G1 and G4 were further optimized. 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, high DAR-valued antibody conversion effects could still be achieved. Therefore, these two disaccharide-acylhydrazone compounds were used to prepare glycoengineered antibodies Ab-1 and Ab-4, and these two glycoengineered antibodies Ab-1 and Ab-4 were used as the non-natural glycosylated antibody portion of the sugar chain-directed antibody-drug conjugates.

[0330] VI. Selection and Optimization of Metathesis Reaction Catalysts

[0331] Glycoengineered antibodies (Ab-1 / Ab-4), benzylhydroxylamine, and various catalysts (69a-69g) were sequentially added to 50 mM His-HCl, pH 6.0 buffer to final concentrations of 0.34 mM, 5 mg / mL, and 136 mM, respectively. The reaction was incubated at 30°C for 2 hours before mass spectrometry analysis. A control group without catalyst was also established to eliminate the effects of the reaction. The results showed that catalysts 69a and 69e-69g achieved complete and quantitative exchange of the acylhydrazone-oxime structure. However, the other catalysts (69b-69d) did not exchange the acylhydrazone-oxime structure under the same conditions, as shown in Figure 2.

[0332] It should be noted that although the above only demonstrates the effects of Ab-1 and Ab-4 under a catalyst, those skilled in the art will appreciate that glycoengineered antibodies prepared using other disaccharide linkers of the present application also have this effect.

[0333] VII. Comparative Experiments of Displacement Reaction and Click Reaction

[0334] Experimental procedure: According to general procedure 4 under the conditions shown in Table 1 below, ADCs were prepared using Acylhydrazone-LacNAc-ox (G1) of the present application and Az-LacNAc-ox (G3 disclosed in CN114949236A), and the DAR values ​​of the transglycosylated antibodies were measured. The results are shown in Figure 6.

[0335] Table 1:

[0336] From the results in the figure, it can be seen that at low sugar substrate equivalents, more acylhydrazone sugar substrates can be transferred to the antibody.

[0337] Pharmacological Example 1

[0338] Experimental process and result analysis of in vitro activity detection

[0339] 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 cell activity and cytotoxicity of the ADC molecules (ADC-1 to ADC-5) prepared above were tested by MTT assay.

[0340] 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 approximately 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, is added (various ADC molecules are diluted 5-fold from the highest concentration of 100 nM, for a total of 9 concentrations, with 3 replicates for each concentration). 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 control and blank groups. The 96-well plate is placed in a CO2 incubator and incubated at 37°C for 72 hours. 10 μL 5 mg / mL MTT is added to each well and incubated at 37°C for 4 hours. Subsequently, 90 μL SDS lysis buffer is added to each well and incubated 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. As can be seen from the results in Figure 3, the ADC prepared using the two-step displacement strategy has good cell activity against Her2-positive cells and is non-toxic in Her2-negative cells.

[0341] Pharmacological Example 2:

[0342] In vivo antitumor activity experimental process and result analysis

[0343] We used gastric cancer cell line NCI-N87 to establish a BALB / c nude mouse xenograft tumor model and used ear hole marking and large, medium and small grouping principles, with six mice in each group.

[0344] Four ADC compounds, ADC-1, ADC-2, ADC-4, and ADC-5, derived from acylhydrazone-hydroxylamine displacement, were evaluated for activity in animals. A one-step site-coupled ADC compound (DAR ≈ 2) served as a positive control, and PBS served as a negative control. All samples were diluted to 0.2 mg / mL in 1x PBS prior to administration and sterilized using a 0.22 mm filter.

[0345] All samples were administered intraperitoneally at a concentration of 1 mg / kg, once weekly for three doses. Tumor size and mouse body weight were measured every three days starting after the first dose. The experimental procedures adhered to animal ethics standards. The measured data were analyzed and plotted using GraphPad Prism 6 software. As shown in Figure 4, the sugar chain-directed ADC compound prepared using this patented technology exhibited excellent in vivo activity.

[0346] Pharmacological Example 3:

[0347] Results and analysis of in vivo antitumor activity of antibody-drug conjugates obtained via acylhydrazone displacement reaction and click chemistry

[0348] Experimental process: ADC-1 obtained by acylhydrazone displacement reaction; ADC-7 obtained by click reaction, the structures of which are shown in Figure 7 respectively.

[0349] A BALB / c nude mouse xenograft tumor model was established using gastric cancer cell line NCI-N87. Ear hole marking and large, medium and small grouping principles were used, with six mice in each group.

[0350] ADC-1 obtained by acylhydrazone displacement 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.

[0351] All samples were administered intraperitoneally at a concentration of 1 mg / kg twice weekly for two weeks. Tumor size and mouse body weight were measured every three days starting after the first dose. Experimental procedures adhered to animal ethics guidelines. Data were analyzed using GraphPad Prism 6 software.

[0352] Experimental results: As shown in Figure 7, the sugar chain-directed ADC compound ADC-1 prepared using the present application has better in vivo tumor inhibitory activity than ADC-7 prepared by click chemistry reaction. The tumor volume of ADC-7 prepared by click chemistry reaction reached its maximum on day 31. In contrast, the tumor volume of ADC-1 prepared by displacement reaction was well controlled.

[0353] 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 and the drug-linker H2N-OR containing a hydroxylamine functional group of formula IV as claimed in claim 7 are in the following two situations: 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, more preferably 6-6.5, and even more 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 portion of the drug-linker structure containing a hydroxylamine functional group according to 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, bifunctional antibody, trifunctional antibody, nanobody fused with an Fc domain, therapeutic antibody or 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 sugar chain-directed 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 molecule toxin, functional molecule, radionuclide, nucleic acid or orthogonal group into an antibody. Preferably, the functional molecule includes a fluorescent molecule and a radiotherapeutic agent.

16. Use of the disaccharide linker as claimed in claim 1, the drug-linker containing a hydroxylamine functional group as claimed in claim 7, the glycoengineered antibody as claimed in claim 11, and the glycan site-specific antibody-drug conjugate as claimed in claim 13 in the preparation of a drug or 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 myelogenous leukemia, pancreatic cancer, acute monocytic leukemia, liver cancer, gastric cancer, non-small cell lung cancer, malignant glioblastoma, and prostate adenocarcinoma.

Images