Disaccharide compound, antibody-drug conjugate containing disaccharide compound, and use thereof

Abstract

Provided are a disaccharide compound represented by formula I, an antibody-drug conjugate containing the disaccharide compound, and the use thereof as an anti-tumor drug, etc.

Description

Disaccharide compounds, antibody-drug conjugates containing disaccharide compounds, and uses thereof

[0001] Cross-reference to Related Applications

[0002] This disclosure claims priority to and the benefit of Chinese Patent Application No. 202410724880.0, filed on June 5, 2024, and Chinese Patent Application No. 202411267787.8, filed on September 11, 2024, which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure belongs to the field of biological medicine, and relates to a class of novel disaccharide compounds, antibody-drug conjugates containing the disaccharide compounds, and uses thereof as anti-tumor drugs and the like. BACKGROUND

[0004] Antibody drug conjugate (ADC) is a new type of drug treatment form in recent years. In terms of structure, ADC is composed of an antibody with targeting effect, a payload with pharmacological activity, and a linker connecting the antibody and the payload by chemical means. In terms of mechanism, ADC is through the specific recognition of the antibody to the antigen, enhances the enrichment of ADC in the lesion tissue with high expression of the antigen, and realizes the targeted release of the active payload in the lesion tissue through endocytosis, lysosomal degradation and other ways, and then produces a pharmacological effect. SUMMARY

[0005] The present disclosure provides a novel disaccharide compound, a glycoengineered antibody containing the disaccharide compound, and a ligand-drug conjugate containing the disaccharide compound.

[0006] In a first aspect, the present disclosure provides a disaccharide compound represented by Formula I or a pharmaceutically acceptable salt thereof:

[0007] wherein ring A is selected from C6-C 10 arylene, 5-10 membered heteroarylene, 4-14 membered heterocyclylene, or C3-C 10 cycloalkylene, the C6-C 10 arylene, 5-10 membered heteroarylene, 4-14 membered heterocyclylene, or C3-C 10 cycloalkylene is optionally substituted by one or more R a ;

[0008] each R aindependently selected from C1-C3alkyl, C3-C6cycloalkyl, 4-7 membered heterocyclyl, halogen, CN, OH, O(C1-C3alkyl), NH2, NH(C1-C3alkyl), N(C1-C3alkyl)2, C(=O)NH2, or COOH;

[0009] Y is selected from a bond, C1-C6alkylene, C=O, S(=O)2, C(=O)NR 10 alkylene, C=O, S(=O)2, C(=O)NR b , S(=O)2NR b , C3-C6cycloalkylene, 4-7 membered heterocyclylene, O, S, NR b , or any combination thereof, each R b is independently selected from H or C1-C6alkyl, each m1, m2 is independently selected from an integer between 0 and 20, the b terminus is independently covalently attached to Z;

[0010] each Z is independently selected from each R d is independently selected from H, C1-C6alkyl, C3-C6cycloalkyl, or 4-7 membered heterocyclyl;

[0011] m is an integer between 1 and 5;

[0012] n is an integer between 1 and 20.

[0013] In some embodiments, the ring A is selected from phenylene, 4-10 membered heterocyclylene, or C3-C 10 cycloalkylene, the phenylene, 4-10 membered heterocyclylene, or C3-C 10 cycloalkylene is optionally substituted with one or more R a , each R a is independently selected from C1-C3alkyl, C3-C6cycloalkyl, 4-7 membered heterocyclyl, halogen, CN, OH, O(C1-C3alkyl), NH2, NH(C1-C3alkyl), N(C1-C3alkyl)2, C(=O)NH2, or COOH.

[0014] In some embodiments, the ring A is selected from

[0015] In some embodiments, Y is selected from C1-C6alkylene, C=O, S(=O)2, C(=O)NR or any combination thereof, the b terminus is independently covalently attached to Z.

[0016] In some embodiments, each Z is independently selected from

[0017] In some embodiments, m is 1 or 2.

[0018] In some embodiments, the structural unit is selected from

[0019] In some embodiments, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0020] In some embodiments, the disaccharide compound of Formula I or a pharmaceutically acceptable salt thereof is selected from the following disaccharide compounds or a pharmaceutically acceptable salt thereof:

[0021] In a second aspect, the present disclosure provides a glycoengineered antibody of Formula V, wherein the sugar chain in the glycoengineered antibody is covalently linked to Asn at position 297 of the heavy chain of the antibody Ab:

[0022] wherein,

[0023] Ab is an antibody unit;

[0024] A, Y, Z, m, and n are defined as in the first aspect above;

[0025] R is selected from H or

[0026] y is selected from a real number from 1 to 2.

[0027] In some embodiments, the antibody unit Ab can specifically bind to one or more target antigens selected from the group consisting of: HER2 (ErbB2), p95HER2, HER3 (ErbB3), CD3, CD16, DLL3, CDH6, CDH11, CDH17, CD5, CD20, BCMA, EGFR, VEGF, Trop-2, Claudin6, Claudin18.2, c-MET, CD30, MUC1, Nectin-4, CD22, CD74, CD19, CD79b, MSLN, TOPO2, EpCAM, CEACAM-5, Mesothelin, PD-L1, PSMA, ROR1, ROR2, CD25, CD33, CD70, CD123, FLT3, CD174, CD166, CD326, CD71, LIV-1, MUC16, MUC17, ENPP3, TDGF1, ETBR, TIM1, TIM3, LRRC15, CanAg / AFP, FAP, SLITRK6, KIT / CD117, STEAP1, SLAMF7 / CS1, NaPi2B / SLC34A2, GPNMB, AXL, ALPP / ALPPL2, ADAM9, B7-H3 (CD276), B7H4, PTK7 / CCK4, PRLR, EFNA4, 5T4, LRG5, NOTCH3, CD142, CA6, GPR20, GPC-1, EphA2, LYPD3, ITGB6, FGFR2, FGFR3, FRa, CEACAMs, GUCY2C, SEZ6, Integrin Av, CAIX, P-cadherin, GD3, cadherin6, LAMP1, KLK2, PD-L1, CD56, CD37, CD47, CD138, CD228, or CD352.

[0028] In some embodiments, the antibody unit Ab is an antibody or an antigen binding fragment that can specifically bind to a target antigen.

[0029] In some embodiments, the antibody unit Ab has a heavy chain variable region VH, a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and / or the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3.

[0030] In some embodiments, the HCDR1-3 are respectively HCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 1, or HCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 1.

[0031] In some embodiments, the HCDR1-3 are, respectively, HCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 11, or HCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 11.

[0032] In some embodiments, the HCDR1-3 are, respectively, HCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 21, or HCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 21.

[0033] In some embodiments, the HCDR1-3 are, respectively, the amino acid sequences set forth in SEQ ID NOs: 5-7, or a sequence having at least 70% identity or at most 3 mutations compared thereto.

[0034] In some embodiments, the HCDR1-3 are, respectively, the amino acid sequences set forth in SEQ ID NOs: 15-17, or a sequence having at least 70% identity or at most 3 mutations compared thereto.

[0035] In some embodiments, the HCDR1-3 are, respectively, the amino acid sequences set forth in SEQ ID NOs: 25-27, or a sequence having at least 70% identity or at most 3 mutations compared thereto.

[0036] In some embodiments, the LCDR1-3 are, respectively, LCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 2, or LCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 2.

[0037] In some embodiments, the LCDR1-3 are, respectively, LCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 12, or LCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 12.

[0038] In some embodiments, the LCDR1-3 are, respectively, LCDR1-3 of the amino acid sequence set forth in SEQ ID NO: 22, or LCDR1-3 of a sequence having at least 70% identity or at most 3 mutations compared to SEQ ID NO: 22.

[0039] In some embodiments, the LCDR1-3 are, respectively, the amino acid sequences set forth in SEQ ID NOs: 8-10, or a sequence having at least 70% identity or at most 3 mutations compared thereto.

[0040] In some embodiments, the LCDR1-3 are the amino acid sequences set forth as SEQ ID NOs: 18-20, respectively, or sequences having at least 70% identity or at most 3 mutations thereto.

[0041] In some embodiments, the LCDR1-3 are the amino acid sequences set forth as SEQ ID NOs: 28-30, respectively, or sequences having at least 70% identity or at most 3 mutations thereto.

[0042] In some embodiments, the heavy chain variable region VH has the amino acid sequence set forth as SEQ ID NO: 1, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0043] In some embodiments, the heavy chain variable region VH has the amino acid sequence set forth as SEQ ID NO: 11, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0044] In some embodiments, the heavy chain variable region VH has the amino acid sequence set forth as SEQ ID NO: 21, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0045] In some embodiments, the light chain variable region VL has the amino acid sequence set forth as SEQ ID NO: 2, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0046] In some embodiments, the light chain variable region VL has the amino acid sequence set forth as SEQ ID NO: 12, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0047] In some embodiments, the light chain variable region VL has the amino acid sequence set forth as SEQ ID NO: 22, or a sequence having at least 70% identity or at most 15 mutations thereto.

[0048] In some embodiments, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 mutations is at most 3, 2, 1, or 0 mutations; the at most 15 mutations is at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations.

[0049] In some embodiments, the mutation is an insertion, a deletion, or a substitution, preferably a conservative amino acid substitution, preferably a back mutation or a hot spot mutation.

[0050] In some embodiments, the antibody unit Ab further has a heavy chain constant region CH1-Fc and / or a light chain constant region CL, wherein the heavy chain constant region can be selected from IgG, such as IgG1, IgG2, IgG3, or IgG4; the IgG can be selected from human IgG, such as human IgG1 or human IgG4; the light chain constant region can be selected from a kappa chain or a lambda chain.

[0051] In some embodiments, the heavy chain constant region CH1-Fc has an amino acid sequence as set forth in SEQ ID NO: 3.

[0052] In some embodiments, the heavy chain constant region CH1-Fc has an amino acid sequence as set forth in SEQ ID NO: 13.

[0053] In some embodiments, the heavy chain constant region CH1-Fc has an amino acid sequence as set forth in SEQ ID NO: 23.

[0054] In some embodiments, the light chain constant region CL has an amino acid sequence as set forth in SEQ ID NO: 4.

[0055] In some embodiments, the light chain constant region CL has an amino acid sequence as set forth in SEQ ID NO: 14.

[0056] In some embodiments, the light chain constant region CL has an amino acid sequence as set forth in SEQ ID NO: 24.

[0057] In some embodiments, the antibody unit Ab comprises:

[0058] (1) a chimeric antibody or a fragment thereof; and / or

[0059] (2) a humanized antibody or a fragment thereof; and / or,

[0060] (3) a fully human antibody or a fragment thereof;

[0061] Preferably, the antibody unit Ab is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fd, a Fv, a scFv, a diabody, or a single domain antibody.

[0062] In some embodiments, the antigen binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, diabody, nanobody, and antibody minimal recognition unit.

[0063] In some embodiments, y is selected from real numbers from 1 to 2, for example, from 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0.

[0064] In some embodiments, y is selected from 1 or 2.

[0065] In some embodiments, the glycoengineered antibody of Formula V is selected from the following glycoengineered antibodies:

[0066] wherein Ab, y and R are as defined above.

[0067] In a third aspect, the present disclosure provides an antibody-drug conjugate of Formula X or a pharmaceutically acceptable salt thereof:

[0068] wherein,

[0069] ring A, Y, m and n are as defined above in the first aspect;

[0070] Ab, y and R are as defined above in the second aspect;

[0071] Z ’ a linker unit formed by reacting Z as defined in the first aspect above with an active group;

[0072] L is a linker unit;

[0073] D is a drug unit.

[0074] In some embodiments, the antibody-drug conjugate of Formula X or a pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugate of Formula X-1 or a pharmaceutically acceptable salt thereof:

[0075] wherein,

[0076] ring A, Y and n are as defined above in the first aspect;

[0077] Ab, R and y are as defined above in the second aspect;

[0078] Z ’ a linker unit formed by reacting Z as defined in the first aspect above with an active group;

[0079] L is a linker unit;

[0080] D is a drug unit.

[0081] In some embodiments, each linker unit Z ’ is independently selected from wherein the c-terminal end is covalently attached to Y.

[0082] In some embodiments, the linker unit L is selected from wherein:

[0083] L a is selected from a chemical bond, j1 is an integer from 0 to 20;

[0084] L b is selected from a chemical bond, (W-CH2CH2) j2 -(OCH2CH2) j2 *, (W-CH2CH2) j2 -(OCH2CH2) j2 -C(=O)*, (W-CH2CH2) j2 -(OCH2CH2) j2 -NHC(=O)*, W is selected from a chemical bond or NH, each j2 is independently selected from an integer from 0 to 20, and the *-end is covalently attached to L c ;

[0085] L c is selected from a chemical bond or a peptide residue consisting of 2 to 7 amino acid residues, said amino acid residues being residues formed from phenylalanine, alanine, proline, isoleucine, glycine, valine, lysine, citrulline, serine, glutamic acid or aspartic acid, said amino acid residues being optionally substituted with one or more R c1 selected from c1 is selected from

[0086] L d is selected from wherein the d-terminal end is covalently attached to the drug unit D.

[0087] In some embodiments, L a is selected from a chemical bond or

[0088] In some embodiments, L b is selected from a chemical bond, (NHCH2CH2) j2 -(OCH2CH2) j2 -NHC(=O)*, (NHCH2CH2) j2-(OCH2CH2) j2 -C(=O)*, each j2 is independently selected from an integer between 0 and 10, the * end is attached to L c is connected.

[0089] In some embodiments, L c is selected from a chemical bond or a peptide residue consisting of 2, 3, 4, or 5 amino acid residues, said amino acid residues being residues formed from phenylalanine, alanine, glycine, valine, or citrulline, said amino acid residues being optionally substituted with one or more R c1 , said R c1 is selected from

[0090] In some embodiments, L c is selected from a chemical bond or a peptide residue consisting of 2, 3, or 4 amino acid residues, said amino acid residues being residues formed from alanine, glycine, valine, or citrulline, said amino acid residues being optionally substituted with one or more R c1 , said R c1 is selected from

[0091] In some embodiments, L c is selected from a chemical bond, wherein the c-terminal end is attached to L d .

[0092] In some embodiments, L c is selected from a chemical bond, wherein the c-terminal end is attached to L d .

[0093] In some embodiments, L d is selected from wherein the d-terminal end is covalently attached to a drug unit D.

[0094] In some embodiments, the linker unit L is selected from wherein the d-terminal end is covalently attached to a drug unit D.

[0095] In some embodiments, the linker unit L is selected from wherein the d-terminal end is covalently attached to a drug unit D.

[0096] In some embodiments, the drug unit D is selected from a cytotoxic drug.

[0097] In some embodiments, the cytotoxic drug is selected from a microtubulin inhibitor including but not limited to dolastatin, auristatin, maytansine, Tubulysins, and cryptomycins, a DNA damaging agent including but not limited to PBD, duocarmycin, and calicheamicin, and a topoisomerase inhibitor including but not limited to camptothecin.

[0098] In some embodiments, the drug unit D is selected from a topoisomerase I inhibitor, auristatin, and PBD.

[0099] In some embodiments, the drug unit D is selected from

[0100] In some embodiments, the structural unit L-D is selected from one of the following structures:

[0101] In some embodiments, the structural unit L-D is selected from one of the following structures:

[0102] In some embodiments, each structural unit Z’-L-D is independently selected from wherein L and D are each as defined above.

[0103] In some embodiments, the antibody-drug conjugate of Formula X or a pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugates or a pharmaceutically acceptable salt thereof:

[0104] wherein L and D are each as defined above, and Ab, R, and y are each as defined in the second aspect above.

[0105] In some embodiments, the antibody-drug conjugate of Formula X or a pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugates or a pharmaceutically acceptable salt thereof:

[0106] Group 1 (ADC-1)

[0107] Group 2 (ADC-2)

[0108] Group 3 (ADC-3)

[0109] Group 4 (ADC-4)

[0110] Group 5 (ADC-5)

[0111] Group 6 (ADC-6)

[0112] Group 7 (ADC-7)

[0113] Group 8 (ADC-8)

[0114] Group 9 (ADC-9)

[0115] Group 10 (ADC-10)

[0116] Group 11 (ADC-11)

[0117] Group 12 (ADC-12)

[0118] Group 13 (ADC-13)

[0119] Group 14 (ADC-14)

[0120] Group 15 (ADC-15)

[0121] Group 16 (ADC-16)

[0122] Group 17 (ADC-17)

[0123] Group 18 (ADC-18)

[0124] Group 19 (ADC-19)

[0125] wherein Ab, R and y are defined as in the second aspect above.

[0126] In a fourth aspect, the present disclosure provides a preparation method of an antibody-drug conjugate shown in formula X, the preparation method comprising steps 1, 2 and 3:

[0127] Step 1: mixing and reacting an antibody and a glycosidase in a buffer solution to obtain a deglycosylated antibody Ab-GlcNAc(Fucα1,6) through separation and purification;

[0128] Step 2: mixing and reacting the deglycosylated antibody Ab-GlcNAc(Fucα1,6), a disaccharide compound shown in formula I and a glycosidase in a buffer solution to obtain a glycoengineered antibody shown in formula V through separation and purification;

[0129] Preferably, the buffer solution in step 1 and / or step 2 is a PBS buffer solution;

[0130] Preferably, the reaction temperature in step 1 and / or step 2 is 10-40°C, more preferably 20-30°C;

[0131] Preferably, the separation and purification method in step 1 and / or step 2 is separation and purification through a protein column;

[0132] Preferably, the glycosidase in step 1 and / or step 2 is at least one selected from Endo-S, Endo-S2, Endo-A, Endo-D, Endo-M, Endo-H, Endo-F2, Endo-F3, Endo-CC1, Endo-CC2, Endo-Om, Endo-CE, Endo-BH, Endo-Sd, Endo-Se and Endo-Rp;

[0133] More preferably, the glycosidase in step 1 and / or step 2 is Endo-S2 (Endoglycosidase-S2, UniProt ID: T1WGN1);

[0134] More preferably, the glycosidase in step 1 and / or step 2 is Streptococcus pyogenes endoglycosidase Endo-S2 (38-819 aa, from UniProt ID: T1WGN1) or Streptococcus pyogenes endoglycosidase Endo-S2 (38-843 aa, from UniProt ID: T1WGN1).

[0135] Step 3: reacting the glycoengineered antibody of formula V with X’-L-D to obtain the antibody-drug conjugate of formula X, wherein X’-L-D is selected from wherein L and D are linker unit and drug unit, respectively, as defined in the third aspect above;

[0136] Preferably, the X’-L-D in step 3 is selected from

[0137] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of formula X or a pharmaceutically acceptable salt thereof as described above, and a pharmaceutically acceptable excipient.

[0138] In another aspect, the present disclosure provides a method of treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of the antibody-drug conjugate of formula X or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as described above.

[0139] In another aspect, the present disclosure provides the use of the antibody-drug conjugate of formula X or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as described above, in the manufacture of a medicament for treating a tumor.

[0140] In another aspect, the present disclosure provides the use of the antibody-drug conjugate of formula X or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as described above, in the treatment of a tumor.

[0141] In another aspect, the present disclosure provides the antibody-drug conjugate of formula X or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as described above, for use in treating a tumor.

[0142] In another aspect, the present disclosure provides the use of the disaccharide compound of formula I or a pharmaceutically acceptable salt thereof as described above, in the manufacture of a glycoengineered antibody or an antibody-drug conjugate.

[0143] The antibody-drug conjugate provided by the present disclosure has significant anti-tumor activity and / or reduced toxic side effects and / or improved product uniformity and / or higher stability.

[0144] Terminology definitions and explanations

[0145] Unless otherwise indicated herein, the terms used in the present disclosure have the following meanings, the definitions of the groups and terms recited in the present disclosure, including the definitions as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in examples, etc., can be combined and incorporated with each other arbitrarily. A particular term should not be considered indefinite or unclear without a specific definition, but should be understood according to the ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0146] In addition, unless otherwise indicated herein, the singular form of a term herein shall include the plural form and the plural form of a term shall include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless expressly stated to the contrary.

[0147] The terms "comprise", "contain" and "have" are used interchangeably herein and are intended to mean the inclusion of the elements recited in the scheme, meaning that the scheme can have other elements in addition to the listed elements. It should also be understood that the use of "comprise", "contain" and "have" is described herein also provides a "consisting of" scheme.

[0148] The term "and / or" is used herein to include the meanings of "and", "or" and "all or any other combination of the elements linked by the term".

[0149] The term "and / or" is used herein to include the meanings of "and", "or" and "all or any other combination of the elements linked by the term".

[0150] The term "HER2" also known as "human epidermal growth factor receptor 2" or ErbB2 herein is a cell-derived oncogene, which is a transmembrane glycoprotein with tyrosine protein kinase activity, and can activate the intracellular tyrosine kinase regulated signal transduction system. The amplification of HER2 gene copy number and protein overexpression will lead to excessive activation of the signal, and the biological function of this signal is closely related to the formation, malignant proliferation, invasion and metastasis, and radiotherapy and chemotherapy resistance of various cancers. The oncogene and its protein product (P185) are overexpressed and amplified in various tumors. Positive expression can be found in various cancers, such as breast cancer, ovarian cancer, gastric cancer, esophageal cancer, salivary gland tumor, lung cancer, bile duct cancer, bladder cancer, prostate cancer, colorectal cancer, etc.

[0151] The term "TROP2" herein refers to human trophoblast cell-surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which is a cell surface receptor expressed by many human tumor cells (e.g., breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer), but Trop-2 has limited expression in normal human tissues. Trop-2 is a single-pass transmembrane surface glycoprotein with a molecular weight of 45 KD, which is a cell membrane calcium channel-related protein related to the regulation of intracellular calcium ion concentration. Trop-2 is related to cyclin D1 and phosphokinase C, and has the function of regulating tumor cell growth, promoting tumor cell invasion and metastasis. Although the specific details of the ligand and signal transduction pathway of Trop-2 are not clear, it is believed that its tumorigenicity and promotion of invasion and metastasis are related to its promotion of intracellular calcium ion concentration.

[0152] The term "B7-H3" herein also refers to CD276, B7RP-2, and B7-H3, which is a member of the B7 family and has 20%-27% amino acid sequence homology with other members of the family. Its transcript is widely expressed, with limited and low expression on normal tissues and immune cells, but overexpressed in various malignant tumors such as melanoma and breast cancer, and can be expressed in cancer cell membranes, cytoplasm, nuclei, and tumor-related vascular systems. In immune cells, T cells and NK cells have no constitutive expression, and some APCs such as DCs have constitutive low expression, and GM-CSF, IFNγ, etc. can induce expression on APCs. It has diverse functions and can regulate immunity, such as stimulating or inhibiting T cell proliferation, inhibiting NK cell function, and is also associated with poor cancer prognosis, and can enhance the malignant ability of tumor cells. As a type I transmembrane glycoprotein, human B7H3 has two subtypes, 2IgB7H3 and 4IgB7H3, and the latter is the main expression subtype, which may play a unique important role in tumor development and immunity.

[0153] The term "linker" or "linker unit" herein refers to a chemical structure fragment that is connected to a linker unit at one end and to a drug unit at the other end.

[0154] The term "drug" or "drug unit" herein refers to a substance with biological activity, such as a cytotoxic agent or an immunomodulatory agent. A cytotoxic agent is a chemical molecule that can have a strong destructive effect on the normal growth of tumor cells. In some embodiments of the present disclosure, the drug or drug unit can be represented as D.

[0155] The term "linker" or "linker unit" herein refers to a chemical structure fragment that covalently links a glycoengineered antibody and a drug-linker. In some embodiments of the present disclosure, the "linker" or "linker unit" is formed by a chemical reaction between Z in a glycoengineered antibody of Formula V and an active group, which refers to a group that has the ability to chemically react with Z under certain conditions, including but not limited to an amino group, a dibenzocyclooctyne group (DBCO), an ethynyl group, a maleimide group.

[0156] The term "antibody-drug conjugate" herein refers to a monoclonal antibody or antibody fragment linked to a biologically active drug through a stable linker unit.

[0157] The term "DAR" or "drug-antibody ratio" herein refers to the average number of drugs linked to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n", which can be an integer or a decimal number.

[0158] The term "antibody unit" or "antibody unit Ab" herein refers to an antibody or antigen binding fragment that can specifically bind to a target antigen, such as a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a Fab'-SH, a F(ab')2, a Fv, a VHH, and a scFv, etc.

[0159] The term "antibody" is used herein in the broadest sense, and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to specifically bind to an antigen. "Antibody" herein encompasses various forms and various structures as long as they exhibit the desired antigen binding activity. For example, in some embodiments, "antibody", "antibody unit", or "Ab" herein is an antibody that does not include a heavy chain N297-linked glycosyl group. The terms "full-length antibody", "intact antibody", and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Antibody" herein includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0160] The term "antibody fragment" or "antigen binding fragment" herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, and the like, regardless of structure, that binds the same antigen recognized by the whole antibody. "Antibody fragment" or "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a particular antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions of the light chains, "Fv" fragments consisting of the variable regions of the light and heavy chains, recombinant single chain polypeptide molecules (scFv) in which light and heavy chain variable regions are connected by a peptide linker, and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. "Antigen binding fragment" and "antibody fragment" herein are used interchangeably herein and do not possess the full structure of a whole antibody, but only contain a portion or a local variant of a whole antibody that possesses the ability to bind to an antigen. "Antigen binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0161] "Antibody" herein also includes surrogate protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and all-synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds can also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0162] An "antibody" herein includes a typical "four-chain antibody" which belongs to an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); a heavy chain refers to a polypeptide chain composed of, in the direction from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain, and, when the full-length antibody is of an IgE isotype, a heavy chain constant region CH4 domain; a light chain is a polypeptide chain composed of, in the direction from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL); the heavy chains and the heavy chains, and the heavy chains and the light chains are connected by disulfide bonds, forming a "Y" shape structure. Due to the difference in the amino acid composition and the arrangement order of the immunoglobulin heavy chain constant region, the antigenicity is also different. Accordingly, the "immunoglobulin" herein can be divided into five categories, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same category of Ig can be divided into different subcategories according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, IgA can be divided into IgA1 and IgA2. The light chain is divided into κ chain or λ chain through the constant region. Each of the five categories of Ig can have κ chain or λ chain.

[0163] The "antibody" herein also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Vicugna pacos, etc., and immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fishes such as sharks.

[0164] The "antibody" herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as Camelidae, Lama glama, Lama guanicoe, Vicugna pacos, sheep, rabbits, mice, rats or cartilaginous fishes (such as sharks).

[0165] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the antibody, such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0166] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques of genetic engineering, antibody engineering, and the like. Exemplarily, "engineered antibodies" include humanized antibodies, antibody fragments (e.g., scFv, sdAb, and the like), bispecific antibodies, and the like.

[0167] The term "monospecific" means having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.

[0168] The term "multispecific antibody" refers to an antibody having at least two antigen binding sites, each of which binds a different epitope of the same antigen or different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes to which the antibody / antigen binding molecule can bind.

[0169] The term "valency" denotes the presence of a specified number of binding sites in an antibody / antigen binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen binding molecule.

[0170] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single variable domain and one constant domain of the light chain, and the first constant domain of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising a VL domain and a CL constant domain of a light chain, and a VH domain and a CH1 domain of a heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains carry a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is the product of oxidation of the CH1 hinge region of two Fab' fragments.

[0171] An "Fv" fragment is the minimum fragment of IgG and IgM that contains the complete antigen-binding site, Fv fragments have the same binding characteristics as Fab and have a similar three-dimensional structure, the VH and VL chains of Fv fragments are held together by non-covalent interactions.

[0172] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond between the VH and VL of the scFv can also be present, forming a disulfide-bond linked Fv (dsFv).

[0173] The term "diabody" has VH and VL domains on a single polypeptide chain, but uses a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0174] The term "chimeric antibody" refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody, which can be derived from a particular species or belong to a particular antibody class or subclass, and the other portion of the light or / and heavy chain is derived from another antibody, which can be derived from the same or different species or belong to the same or different antibody class or subclass, but retains the binding activity of the target antigen (U.S.P 4,816,567, Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 6855 (1984)). For example, the term "chimeric antibody" can include an antibody (e.g., human murine chimeric antibody) in which the variable regions of both the heavy and light chains are from a first antibody (e.g., a murine antibody), and the constant regions of both the heavy and light chains are from a second antibody (e.g., a human antibody).

[0175] The term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., FRs in the variable region and / or constant region) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like.

[0176] The term "fully human antibody" refers to an antibody that has variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. "Fully human antibodies" herein can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0177] The term "naked antibody" herein refers to an antibody that is not linked to, fused to, or conjugated to another agent or molecule (e.g., a label or a drug), a peptide or polypeptide. In particular embodiments, a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylation enzymes) of the host cell. In certain embodiments, a naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylation enzymes). In certain embodiments, a naked antibody is a whole antibody, while in other embodiments, a naked antibody is an antigen-binding fragment of a whole antibody, such as a Fab antibody.

[0178] The term "variable region" refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen, "variable region of the heavy chain" and "VH" or "HCVR" are used interchangeably, and "variable region of the light chain" and "VL" or "LCVR" are used interchangeably. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th Ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain can be sufficient to confer antigen-binding specificity. The term "complementarity determining region" and "CDR" are used interchangeably herein to refer to the hypervariable region of a heavy chain variable region (VH) or light chain variable region (VL), which is also referred to as a hypervariable loop (HVR) because it forms loops that are believed to be involved in binding to an antigen, where the heavy chain variable region CDRs can be abbreviated as HCDRs and the light chain variable region CDRs can be abbreviated as LCDRs. The term "framework region" or "FR region" is used interchangeably herein to refer to those amino acid residues in a variable region of a heavy or light chain of an antibody that are outside the CDRs. Typically, a canonical antibody variable region is composed of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0179] The "CDRs" herein can be annotated and defined in ways known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tools websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0180] The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding the antibody to an antigen but exhibits effector functions such as interaction with Fc receptors, which has a more conserved amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes both "full length heavy chain constant region" and "heavy chain constant region fragment", the former has substantially similar structure as the native antibody constant region, while the latter only includes "a portion of the full length heavy chain constant region". Exemplarily, a typical "full length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it further includes CH4 domain; when the antibody is heavy chain antibody, it does not include CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domain.

[0181] The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which can be selected from constant kappa domain or constant lambda domain.

[0182] The term "Fc" herein refers to the carboxy-terminal portion of an intact antibody that is generated upon papain cleavage, which typically includes the CH3 and CH2 domains of an antibody. The Fc region includes, e.g., native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary slightly, the Fc region of a human IgG heavy chain typically extends from the amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. The C-terminal lysine (residue 447 according to the Kabat numbering system) of the Fc region can be removed, e.g., during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain, and therefore the Fc region can or can not include Lys447.

[0183] The term "epitope" herein includes any protein determinant capable of specific binding to an immunoglobulin, scFv or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an antibody can be raised against an N-terminal or C-terminal peptide of a polypeptide.

[0184] The terms "specific binding," "immunobinding," and "immunobinding properties" herein refer to the type of noncovalent interaction that forms between an immunoglobulin molecule and an antigen for which the immunoglobulin has specificity. The strength or affinity of an immunobinding interaction can be expressed as the dissociation constant (KD) of the interaction, where a smaller KD indicates a greater affinity. The immunobinding properties of a selected polypeptide can be quantitatively determined using methods well known in the art. One such method requires the determination of the rates of association and dissociation of antigen binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that influence the rates equally in both directions. Thus, a "binding rate constant" (Kon) and a "dissociation rate constant" (Koff) can be determined by calculating the concentration and actual rates of association and dissociation. (See Nature 361 : 186-87 (1993)). The ratio Koff / Kon enables all parameters unrelated to affinity to be eliminated, and is equal to the dissociation constant KD.

[0185] The term "mutation" herein includes genetic mutation and amino acid mutation, wherein the genetic mutation refers to a deletion, insertion of heterologous nucleic acid, inversion or substitution, which can lead to a change in the amino acid sequence in the corresponding protein product; the amino acid mutation, also known as non-synonymous single nucleotide mutation, is due to the change of some single base, resulting in the change of amino acid sequence in the protein product. The change of amino acid affects the stability, interaction and enzyme activity of the protein, thereby leading to the occurrence of diseases.

[0186] The term "amino acid substitution" herein refers to those in which at least one amino acid residue in the natural or starting sequence is removed and a different amino acid is inserted in its place. The substitutions can be single, in which only one amino acid in the molecule has been substituted, or they can be multiple, in which two or more amino acids in the same molecule have been substituted.

[0187] The term "conservative amino acid substitution" herein refers to the replacement of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitutions include the substitution of non-polar (hydrophobic) residues such as isoleucine, valine, and leucine; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a non-polar residue.

[0188] The term "mutant" herein refers to a "variant" of the protein or peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity to the amino acid sequence of the protein or peptide.

[0189] The term "amino acid" as used herein refers to the basic unit that constitutes a protein, confers a specific molecular structure to the protein, and imparts biochemical activity to the molecule. In chemistry, amino acids refer to organic compounds that contain an amino group (-NH2) and a carboxyl group (-COOH) in their structure. Depending on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified as alpha, beta, gamma, delta, and so on. In alpha amino acids, the amino and carboxyl groups are attached to the same carbon atom, while in beta amino acids, the amino and carboxyl groups are attached to adjacent carbon atoms, and so on. In biology, amino acids usually refer specifically to alpha amino acids, i.e., amino acids in which the amino and carboxyl groups are directly attached to the same -CH- structure, and have the general formula H2NCHRCOOH (R represents an organic substituent). Exemplary, the common 20 amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartate, histidine, asparagine, glutamate, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline.

[0190] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually denoted 5' to 3'. In the present context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. A nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both the sense and the antisense strand, as well as single- and double-stranded forms. Also, the nucleic acid molecules described herein can contain naturally-occurring or non-naturally-occurring nucleotides. Examples of non-naturally-occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as vectors for the direct expression of an antibody of the present disclosure in vitro and / or in vivo, e.g. in a host or patient. Such DNA (e.g. cDNA) or RNA (e.g. mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce the antibody in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0191] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of the present disclosure contain polynucleotide sequences as well as additional sequence elements, e.g., for expressing proteins and / or for integrating these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used to express the antibodies and antibody fragments of the present disclosure include plasmids containing regulatory sequences that direct transcription of the gene, such as promoter and enhancer regions. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA produced from transcription of the genes. These sequence elements include, e.g., 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites, in order to direct efficient transcription of the genes carried on the expression vectors. The expression vectors of the present disclosure can also contain a polynucleotide that encodes a marker for selection of cells containing such vectors. Examples of suitable markers include genes that encode antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or neomycin) resistance.

[0192] The term "host cell" herein refers to a cell into which foreign nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny of the original transformant that have a mutation not present in the original cell, regardless of the number of passages. The progeny can not necessarily be identical to the parent cell either because they have undergone some mutations as a result of replication or they have undergone a differentiation event. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0193] The term "identity" herein can be calculated as follows: to determine the percent "identity" of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., matrices can be used for comparison purposes, gaps can be introduced in the sequence of either the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence then the molecules are identical at that position.

[0194] The percent identity between two sequences is varied as a function of the number of identical positions shared by the sequences and the length of the shorter of the two sequences, taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap.

[0195] "Real numbers x~y" herein means n is any real number greater than or equal to x and less than or equal to y.

[0196] The phrase "an integer of x to y" herein means both the case where each integer in the range (including x and y) is included, and the case where a range value with any two of the above integers as end points is included. For example, "n is an integer of 1 to 10" means that n includes the case where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the case where n is selected from a range value with any two of the above 10 integers as end points, for example, n is selected from 2 to 9, 3 to 9, 3 to 8, 4 to 6, and the like.

[0197] The phrase "an integer of x to y" herein means both the case where each integer in the range (including x and y) is included, and the case where a range value with any two of the above integers as end points is included. For example, "n is an integer of 1 to 10" means that n includes the case where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the case where n is selected from a range value with any two of the above 10 integers as end points, for example, n is selected from 2 to 9, 3 to 9, 3 to 8, 4 to 6, and the like. represents a connection site.

[0198] The graphical representation of a racemic or enantiomerically pure compound herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, a wedge and hashed wedge represents the absolute configuration of a stereocenter, with a solid and hashed wedge represents the relative configuration of a stereocenter (e.g., the cis-trans configuration of an alicyclic compound).

[0199] The term "tautomer" refers to isomers of a molecule which differ in the location of a proton. Compounds of the disclosure can exhibit tautomerism. A tautomeric compound can exist in two or more interconvertible forms. Tautomers generally exist in equilibrium and attempts to isolate a single tautomer usually result in a mixture whose physical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates; in phenols, the enol form predominates. The disclosure includes all tautomeric forms of the compounds.

[0200] The term "stereoisomer" refers to isomers having the same molecular formula but differing in the arrangement of atoms in space. Stereoisomers include enantiomers and diastereomers.

[0201] The compounds of the present disclosure can have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or asymmetric double bonds, and thus the compounds of the present disclosure can exist in particular geometric or stereoisomeric forms. The particular geometric or stereoisomeric forms can be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as racemic mixtures or other mixtures thereof, such as those that contain one enantiomeric or diastereomeric excess, all of which are within the scope of the compounds of the present disclosure. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms can be present in a substituent group, and all such isomers and mixtures thereof are included within the definition of the compounds of the present disclosure. The compounds of the present disclosure containing an asymmetric atom can be isolated in optically active form or as racemic mixtures, by separation of the racemic mixture into its component enantiomers, or by synthesis from an optically active starting material or an optically active reagent.

[0202] The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent group, provided that the valence of the particular atom is not exceeded, and that the substituted compound is stable. When the substituent is oxo (i.e., =0), it means that two hydrogen atoms are replaced by the oxo group. Oxos cannot be on an aromatic group.

[0203] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and this description includes instances where said event or circumstance occurs and instances where it does not. For example, an ethyl group "optionally" substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), mono-substituted (CH2CH2F, CH2CH2C1, etc.), poly-substituted (CHFCH2F, CH2CHF2, CHFCH2C1, CH2CHC12, etc.), or fully substituted (CF2CF3, CF2CC13, CC12CC13, etc.). One skilled in the art will appreciate that for any given group containing one or more substituents, no substitution or substitution pattern is introduced that is not spatially possible and / or synthetically feasible.

[0204] When any variable (e.g., R a , R b ) occurs more than one time in a compound, its definition in each occurrence is independent of its definition at every other occurrence. For example, if a group is substituted with 2 R b groups, then each R b is selected independently from the others.

[0205] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0206] When one of the variables is selected from a chemical bond, it is meant that the two groups to which it is attached are directly connected, such as L represents a chemical bond in A-L-Z means that the structure is actually A-Z.

[0207] When a linking group is referred to herein without specifying the direction of attachment, the direction of attachment is arbitrary.

[0208] C m -C n , means having an integer number of carbon atoms in the range m-n.

[0209] The term "alkyl" means a monovalent hydrocarbon group of formula C n H 2n+1 . The alkyl group can be straight-chained or branched. The term "C1-C 10 alkyl" means a straight-chained or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of said alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl-, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc. The term "C1-C6alkyl" means a straight-chained or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The "C1-C 10 alkyl" as described herein can encompass the range of "C1-C6alkyl", "C1-C4alkyl", or "C1-C3alkyl", etc. The "C1-C6alkyl" can further encompass "C1-C4alkyl" or "C1-C3alkyl".

[0210] The term "alkylene" means a saturated divalent hydrocarbon group having straight-chained or branched chains. "C1-C 10 alkylene" means a saturated divalent hydrocarbon group having straight-chained or branched chains having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, including but not limited to methylene, CH2CH2, or CH2CH2CH2.

[0211] The term "cycloalkyl" means a monovalent carbocyclic ring that is fully saturated and exists as a monocyclic, fused ring, bridged ring, or spiro ring, etc. The term "C3-C6cycloalkyl" is understood to mean a saturated monocyclic, fused ring, spiro ring, or bridged ring having 3, 4, 5, or 6 carbon atoms, specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0212] The term "C3-C10cycloalkylene" refers to a fully saturated and bivalent carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring, and the like, having 3, 4, 5, 6, 7, 8, 9, or 10 ring carbon atoms. The "C3-C10cycloalkylene" described herein can include "C3-C6cycloalkylene." 10 The term "C3-C10cycloalkylene" refers to a fully saturated and bivalent carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring, and the like, having 3, 4, 5, 6, 7, 8, 9, or 10 ring carbon atoms. The "C3-C10cycloalkylene" described herein can include "C3-C6cycloalkylene." 10 The term "C3-C10cycloalkylene" refers to a fully saturated and bivalent carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring, and the like, having 3, 4, 5, 6, 7, 8, 9, or 10 ring carbon atoms. The "C3-C10cycloalkylene" described herein can include "C3-C6cycloalkylene."

[0213] The term "heterocyclyl" refers to a monovalent radical that is a fully saturated or partially saturated monocyclic, fused, spiro, or bridged ring, and the like, which is overall non-aromatic, having 1 to 5 heteroatoms or heteroatom groups (i.e., an atom group containing a heteroatom) in the ring atoms, which "heteroatoms or heteroatom groups" include, but are not limited to, a nitrogen atom (N), an oxygen atom (O), a sulfur atom (S), a phosphorus atom (P), a boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, and the like. The term "4-7 membered heterocyclyl" refers to a heterocyclyl having 4, 5, 6, or 7 ring atoms, and having 1 to 3 ring atoms independently selected from the above-mentioned heteroatoms or heteroatom groups. Examples of 4-membered heterocyclyl include, but are not limited to, azetidinyl, oxetanyl; examples of 5-membered heterocyclyl include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclyl include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclyl include, but are not limited to, diazepanyl. The "4-7 membered heterocyclyl" can include "4-7 membered heterocycloalkyl", "5-6 membered heterocyclyl", "5-6 membered heterocycloalkyl", and the like.

[0214] The term "heterocyclyl" refers to a fully saturated or partially saturated, monocyclic, bicyclic, spiro, or bridged ring radical, which is not aromatic overall, having from 1 to 5 ring heteroatoms or heteroatom groups, including but not limited to nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=0)2-, -S(=0)-, -P(=0)2-, -P(=0)-, -NH-, -S(=0)(=NH)-, -C(=0)NH-, or -NHC(=0)NH-, etc. The term "4-14 membered heterocyclyl" refers to a heterocyclyl radical having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, and having from 1 to 5 ring heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups, and the term "4-14 membered heterocyclyl" can include "4-7 membered heterocyclyl."

[0215] The term "C6-Ci0arylene" refers to a carbocyclic, aromatic, divalent radical of a single ring or fused rings having a conjugated pi-electron system, said aromatic ring having 5, 6, 7, 8, 9, or 10 ring carbon atoms, for example a ring having 6 carbon atoms ("C6arylene"), or a ring having 9 carbon atoms ("C9arylene"). 10 The term "C6-Ci0arylene" refers to a carbocyclic, aromatic, divalent radical of a single ring or fused rings having a conjugated pi-electron system, said aromatic ring having 5, 6, 7, 8, 9, or 10 ring carbon atoms, for example a ring having 6 carbon atoms ("C6arylene"), or a ring having 9 carbon atoms ("C9arylene").

[0216] The term "5-10 membered heteroarylene" refers to an aromatic, divalent radical of a monocyclic or fused ring system having 5, 6, 7, 8, 9, or 10 ring atoms, containing at least one ring atom selected from N, O, S, and the remaining ring atoms being C.

[0217] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0218] The term "treatment" refers to surgical or pharmacological interventions, whose purpose is to prevent, slow down (reduce), or halt the progression of an unwanted physiological change or pathological condition, such as cancer, autoimmune disease, and viral infection, in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those who have a condition or disease, those who are predisposed to having a condition or disease, or those in which a condition or disease is to be prevented. When referring to slowing down, reducing, diminishing, ameliorating, palliating, and the like, the terms also include eliminating, disappearing, not occurring, and the like.

[0219] The term "effective amount" means the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent, which prevents, slows or halts the progression of a disease condition or the disease. An "effective amount" also means the amount of a compound which alleviates a symptom, e.g., treats, cures, prevents or slows the progression of, or increases the speed of treatment, cure, prevention or slowing of, the relevant medical condition. When the active ingredient is administered individually to an individual, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to combined amounts of the active ingredients that will achieve the therapeutic result, regardless of whether administration is combined, sequential or simultaneous.

[0220] The term "subject" refers to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys) or non-primate mammals that receive treatment for a disease or condition.

[0221] The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art considering the knowledge in the art and the disclosure.

[0222] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0223] The term "pharmaceutically acceptable salt" refers to salts of a compound that are pharmaceutically acceptable, including salts of inorganic acids or organic acids, and salts of inorganic bases or organic bases.

[0224] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with a pharmaceutically acceptable excipient. The goal of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism.

[0225] The term "pharmaceutically acceptable excipient" refers to an excipient that is not biologically or otherwise undesirable, i.e., the excipient can be administered to an organism without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The presence of an excipient in a composition does not imply that the excipient is intended to be part of the dosage form. Suitable excipients are well known to those skilled in the art.

[0226] The words "comprise" or "comprising" and variations thereof such as "comprises" or "comprising", when used in this document, can be understood to encompass the terms "consisting of" or "consisting of" and variations thereof, e.g., "consists" or "consisting of".

[0227] The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl.

[0228] Certain isotopically-labeled compounds of the present disclosure (for example, those 3 H and 14 C) can be used in compound and / or substrate tissue distribution analysis. Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present disclosure can generally be prepared by

[0229] The pharmaceutical compositions of the present disclosure can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage form. For example, the pharmaceutical compositions of the present disclosure can be in the form of sterile injectable aqueous solutions. Other solvents or solvents systems can be used for the pharmaceutical compositions of the present disclosure at the time of use, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0230] In all methods of administration of the compounds described herein, the daily dose is from 0.001 mg / kg to 600 mg / kg of body weight, preferably from 0.05 mg / kg to 200 mg / kg of body weight, more preferably from 0.1 mg / kg to 100 mg / kg of body weight, in single or divided doses.

[0231] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0232] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0233] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety. Detailed Implementation

[0234] The present disclosure is described in detail below with reference to embodiments, but this does not imply any adverse limitation thereof. The present disclosure has been described in detail herein, including specific embodiments thereof. It will be apparent to those skilled in the art that various changes can be made to the specific embodiments of the present disclosure without departing from the spirit and scope thereof. All reagents used in this disclosure are commercially available and can be used without further purification.

[0235] Unless otherwise stated, the proportions expressed for mixed solvents are volume-based.

[0236] Unless otherwise stated, % refers to wt%.

[0237] Compounds are processed manually or Software naming conventions are used; commercially available compounds use supplier catalog names.

[0238] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are measured in units of 10⁻¹⁰. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., with tetramethylsilane (TMS) as the internal standard; "IC 50 "Hardest effective concentration (HIC)" refers to the concentration at which half of the maximum inhibitory effect is achieved. 50EC50 refers to the concentration that produces 50% of the maximal effect.

[0239] Example 1, Synthesis of disaccharide compound

[0240] Example 1.1, 4-(3-oxobutyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-hydroxymethyl-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl]oxy)-tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy]ethyl)-benzamide (Compound II)

[0241] Step 1: Synthesis of 4-(3-oxobutyl)benzoic acid (Intermediate 1-1)

[0242] To a solution of 3-buten-2-ol (11 g), 4-bromobenzoic acid (15 g) and triethylamine (12 g) in N,N-dimethylformamide (150 mL) was added palladium acetate (837 mg) and tri(o-tolyl)phosphine (2 g) under nitrogen atmosphere. The reaction mixture was stirred at 100 °C for 3 hr. The reaction mixture was then added directly to water (100 mL) and extracted with ethyl acetate (100 mL) for three times. The combined organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography (0-100% EA / PE) to give the title compound (2.2 g).

[0243] MS m / z (ESI): 193.1 [M+H] +

[0244] Step 2: Synthesis of 4-(3-oxobutyl)benzoic acid (1-hydroxy-2,5-dioxopyrrolidine) ester (Intermediate 1-2)

[0245] To a solution of Intermediate 1-1 (1.6 g) and 1-hydroxyphthalimide (958 mg) in dichloromethane (50 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.9 g) under nitrogen atmosphere at 0 °C. The reaction mixture was stirred at 20 °C for 16 hr. After completion of the reaction, the reaction mixture was washed with 1 N aqueous hydrochloric acid solution (80 mL) for three times and saturated sodium bicarbonate solution (80 mL) for once. The organic phase was dried over magnesium sulfate, filtered and concentrated to give the title compound (1.3 g).

[0246] MS m / z (ESI): 312.1 [M+Na] +

[0247] Step 3: Synthesis of N-((3R,4R,5S,6R)-5-(((2S,3S,4S,5S,6R)-6-((2-(2-(2- aminoethoxy)ethoxy)ethoxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)- 2,4-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide hydrochloride (Intermediate 1-4)

[0248] To a mixture of compound 1-3 (56 mg, synthesized according to the literature method in ACS Chemical Biology, 2021, 16, 2502) and 10% palladium on carbon (30 mg) was added methanol (5 mL) and 3M hydrogen chloride / cyclopentyl methyl ether solution (0.1 mL) at room temperature. The resulting mixture was stirred under hydrogen atmosphere at room temperature for 16 h. The resulting mixture was then filtered, and the filtrate was concentrated and dried under vacuum to give the title compound (28 mg).

[0249] LC-MS: m / z (ESI): 515.3 [M+H] + .

[0250] Step 4: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5- acetylamino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl]oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy]ethyl)-4-(3-oxobutyl)benzamide (Intermediate 1-5)

[0251] Intermediate 1-4 (10 mg) and Intermediate 1-2 (5 mg) were dissolved in N,N- dimethylformamide (100 uL), and DIEA (4 mg) was added under a nitrogen atmosphere. The reaction solution was stirred at 20 °C for 3 hr. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Xtimate C18 150*40mm*10um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 10%-30%, 9 min) to give the title compound (2.3 mg).

[0252] MS m / z (ESI): 689.3 [M+H] +

[0253] Step 5: Synthesis of 4-(3-oxobutyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5- trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-hydroxymethyl-2-methyl- 3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl]oxy)tetrahydro-2H-pyran-2- yl)methoxy)ethoxy)ethoxy]ethyl)-benzamide (Compound II)

[0254] Intermediate 1-5 (2.3 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3- chloride (20 mg), triethylamine (32 mg) were added into water (0.5 mL), replaced by nitrogen for 3 times, the reaction was stirred at 25 °C for 16 hr. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: C18 150 x 40 mm; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 0%-40%, 20 min) to give the title compound (1.3 mg).

[0255] LC-MS: m / z (ESI): 671.4 [M+H] + .

[0256] 1 H NMR (400 MHz, D2O) δ = 7.70 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 5.99 (d, J = 7.3 Hz, 1H), 4.65-4.62 (m, 1H), 4.33-4.29 (m, 1H), 4.10-4.05 (m, 1H), 3.94 (d, J = 2.4 Hz, 1H), 3.84-3.78 (m, 1H), 3.74-3.64 (m, 13H), 3.62-3.53 (m, 5H), 3.47-3.33 (m, 2H), 2.93 (s, 4H), 2.18 (s, 3H), 2.02 (s, 3H).

[0257] Example 1.2, 7-(3-azidopropanoyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5- trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl- 3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2- yl)methoxy)ethoxy)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (Compound I2)

[0258] Step 1: Synthesis of benzyl 7-tert-butoxycarbonyl-7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 2-1)

[0259] To a solution of 7-tert-butoxycarbonyl-7-azaspiro[3.5]nonane-2-carboxylic acid (500 mg) in DMF (5 mL) was added cesium carbonate (1.21 g) and benzyl bromide (317.51 mg) at room temperature. The reaction was stirred at 50 °C for 16 h. Then the reaction was quenched with water (20 mL) and extracted with ethyl acetate (5 mL x 3). The organic phase was washed with water and brine, and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give the title compound (667.3 mg).

[0260] MS m / z (ESI): 382.2 [M+Na] + .

[0261] Step 2: Synthesis of benzyl 7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 2-2)

[0262] To a solution of Intermediate 2-1 (667.3 mg) in 1,4-dioxane (1 mL) was added hydrogen chloride-1,4-dioxane solution (2 M, 1.86 mL) at room temperature. The reaction was stirred at room temperature for 1 hr. Then the reaction was directly concentrated to dryness under reduced pressure to give the title compound (549.13 mg).

[0263] MS m / z (ESI): 260.2 (M+H] + .

[0264] Step 3: Synthesis of benzyl 7-(3-azidopropionyl)-7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 2-3)

[0265] To a solution of Intermediate 2-2 (489 mg) in DMF (3.77 mL) was added 3-azidopropanoic acid (285.39 mg), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (935.51 mg) and N,N-diisopropylethylamine (2.14 g) at room temperature. The reaction was stirred at 25 °C for 2 hr. Then the reaction was diluted with ethyl acetate (100 mL) and washed with water (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by flash silica gel column (20 g) with eluent of 0-40% ethyl acetate / petroleum ether at 50 mL / min to give the title compound (493 mg). 20 g flash silica gel column, eluent 0-40% ethyl acetate / petroleum ether, gradient @ 50 mL / min

[0266] MS m / z (ESI): 357.2 (M+H] + .

[0267] Step 4: Synthesis of 7-(3-azidopropanoyl)-7-azaspiro[3.5]nonane-2-carboxylic acid (Intermediate 2-4)

[0268] To a solution of Intermediate 2-3 (433 mg) in methanol (13 mL) was added lithium hydroxide (58.19 mg) in water (13 mL) at room temperature. The reaction was stirred at 20 °C for 1 hr. The reaction was then adjusted to pH 7 with 1 M aqueous hydrochloric acid solution. The mixture was directly purified by high performance liquid chromatography (column: C18 150*30 mm*5 um; mobile phase: [A: water (0.05% trifluoroacetic acid), B: acetonitrile]; B%: 10%-50%, 11 min) to give the title compound (212 mg).

[0269] MS m / z (ESI): 267.2 (M+H] +

[0270] Step 5: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5- acetylamino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-(3-azidopropanoyl)-7- azaspiro[3.5]nonane-2-carboxamide (Intermediate 2-5)

[0271] Intermediate 1-4 (30 mg), Intermediate 2-4 (17 mg), N,N-diisopropylethylamine (31 mg), 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (27 mg) were added into N,N-dimethylformamide (1 mL), which was replaced with nitrogen for 3 times. The reaction was stirred at 25 °C for 2 hr. The reaction was directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 13%-33%, 11 min) to give the title compound (10 mg).

[0272] MS m / z (ESI): 763.2 [M+H] + .

[0273] Step 6: Synthesis of 7-(3-azidopropanoyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (Compound 12)

[0274] Intermediate 2-5 (8 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3- chloride (115 mg), triethylamine (53 mg) were added to water (1 mL), sparged with nitrogen 3 times, and the reaction was stirred at 25 °C for 16 hr. After the reaction was completed, the reaction was purified directly by high performance liquid chromatography (column: C18 150 x 40 mm; mobile phase: [A: water (0.1% triethylamine), B: acetonitrile]; B%: 0% - 30%, 20 min), and lyophilized after adding an aqueous solution containing 21 micrograms of sodium hydroxide to obtain the title compound (3.3 mg).

[0275] 1 H NMR (400 MHz, D20) d = 6.06 (d, J = 7.3 Hz, 1H), 4.37 - 4.30 (m, 1H), 4.18 - 4.11 (m, 1H), 3.95 (d, J = 2.6 Hz, 1H), 3.90 - 3.84 (m, 1H), 3.80 - 3.63 (m, 12H), 3.63 - 3.52 (m, 7H), 3.52 - 3.46 (m, 3H), 3.46 - 3.34 (m, 5H), 3.20 - 3.07 (m, 1H), 2.70 (q, J = 6.1 Hz, 2H), 2.14 - 2.06 (m, 2H), 2.04 (d, J = 1.6 Hz, 3H), 2.00 - 1.92 (m, 2H), 1.72 - 1.67 (m, 1H), 1.66 - 1.61 (m, 1H), 1.60 - 1.55 (m, 1H), 1.54 - 1.48 (m, 1H).

[0276] MS m / z (ESI): 745.5 (M+H] + .

[0277] Example 1.3, 7-(3-azidopropionyl)-N-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (Compound I3)

[0278] Step 1: Synthesis of p-toluenesulfonic acid (2-azidoethanol) ester (Intermediate 3-1)

[0279] 2-azidoethanol (1 g), triethylamine (2.4 mL) and DMAP (70.15 mg) were dissolved in dichloromethane (30 mL) under nitrogen atmosphere, 4-methylbenzenesulfonyl chloride (3.28 g) was added at 0 °C, the reaction solution was stirred at 20 °C for 3 hr. Then the reaction solution was washed with water (40 mL) for three times, the organic phase was combined and dried over anhydrous magnesium sulfate, filtered and concentrated, the residue was purified by column chromatography to give the title compound (2.2 g).

[0280] MS m / z (ESI): 242.3 [M+H] +

[0281] Step 2: Synthesis of N-((2R,3R,4R,5S,6R)-5-(((2S,3S,4S,5R,6R)-6-((2-azidoethoxy)methyl)-3,5- di(benzyloxy)-4-((4-methoxybenzyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-2,4- di(benzyloxy)-6-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)acetamide (Intermediate 3-3)

[0282] Intermediate 3-2 (0.5 g, synthesized according to the method in ACS Chemical Biology, 2021, 16, 2502) was dissolved in N,N-dimethylformamide (0.5 mL), NaH (86.56 mg) was added at 0 °C under nitrogen atmosphere, the reaction solution was stirred at 20 °C for 0.5 hr, Intermediate 3-1 (913.81 mg) was added to the system, the reaction solution was stirred at 60 °C for 16 hr. Then the reaction solution was purified by reverse phase column to give the title compound (60 mg).

[0283] MS m / z (ESI): 1023.6 [M+H] +

[0284] Step 3: Synthesis of N-((3R,4R,5S,6R)-5-(((2S,3S,4S,5S,6R)-6-((2- aminoethoxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-2,4- dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)acetamide hydrochloride (Intermediate 3-4)

[0285] To a mixture of compound 3-3 (60 mg) and 10% palladium on carbon (10 mg) was added methanol (5 mL) and 2 M hydrogen chloride / dioxane solution (0.5 mL) at room temperature. The resulting mixture was stirred under hydrogen atmosphere at room temperature for 16 h. The resulting mixture was then filtered, and the filtrate was concentrated to dryness under reduced pressure to give the title compound (50 mg).

[0286] LC-MS: m / z (ESI): 427.2 [M+H] + .

[0287] Step 4: Synthesis of N-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5- acetylamino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methoxy)ethyl)-7-(3-azidopropionyl)-7- azaspiro[3.5]nonane-2-carboxamide (Intermediate 3-5)

[0288] Intermediate 3-4 (40 mg) and Intermediate 2-4 (12.49 mg) were dissolved in pyridine (0.5 mL), and EDCI (13.49 mg) was added under nitrogen atmosphere. The reaction was stirred at 25 °C for 2 hr. The reaction was then purified directly by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 0%-40%, 11 min) to give the title compound (5 mg).

[0289] MS m / z (ESI): 675.3 [M+H] +

[0290] Step 5: Synthesis of 7-(3-azidopropionyl)-N-(2-(((2R,3S,4S,5S,6S)-3,4,5- trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethyl)-7- azaspiro[3.5]nonane-2-carboxamide (Compound I3)

[0291] Intermediate 3-5 (5 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3-carboxylate (16 mg), triethylamine (25.84 μL) were added to water (0.5 mL), sparged with nitrogen 3 times, the reaction was stirred at 25 °C for 2 hr. The reaction was then purified directly by high performance liquid chromatography (column: YMC-Actus Triart C18 150*30mm*5um; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 5%-35%, 30 min), lyophilized after adding an aqueous solution containing 14 micrograms of sodium hydroxide to give the title compound (1 mg).

[0292] LC-MS: m / z (ESI): 657.4 [M+H] + .

[0293] 1 H NMR (400 MHz, D2O) δ = 5.97 (d, J = 7.3 Hz, 1H), 4.23 (s, 1H), 4.07 (s, 1H), 3.87-3.86 (m, 1H), 3.75 (d, J = 10.4 Hz, 1H), 3.69-3.50 (m, 7H), 3.50-3.43 (m, 4H), 3.43-3.36 (m, 3H), 3.33-3.29 (m, 5H), 3.12-3.01 (m, 1H), 2.61 (q, J = 6.2 Hz, 2H), 2.05-1.97 (m, 2H), 1.95 (s, 3H), 1.92-1.84 (m, 2H), 1.61 (s, 1H), 1.54 (s, 1H), 1.49 (s, 1H), 1.43 (s, 1H).

[0294] Example 1.4, 7-(3-azidopropionyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine-2-carboxamide (Compound I4)

[0295] Step 1: Synthesis of ethyl 7-benzyloxycarbonyl-5,6,7,8-tetrahydroimidazo[1,2- a]pyrazine-2-carboxylate (Intermediate 4-2)

[0296] Benzyl chloroformate (1.75 g) and triethylamine (3.11 g) were added to a solution of compound 4-1 (2 g) in dichloromethane (40 mL). The reaction was stirred at 20 °C for 16 hr. The reaction was then directly added to water (80 mL), extracted with dichloromethane (50 mL) for three times, the organic phase was combined and dried over anhydrous magnesium sulfate, filtered and concentrated to give the title compound (2.66 g).

[0297] MS m / z (ESI): 330.2 [M+H] +

[0298] Step 2: Synthesis of 7-benzyloxycarbonyl-5,6,7,8-tetrahydroimidazo[l,2- a]pyrazine-2-carboxylic acid (intermediate 4-3)

[0299] Sodium hydroxide (1.09 g) in water (10 mL) was added to a solution of intermediate 4-2 (2.5 g) in tetrahydrofuran (10 mL) and methanol (10 mL). The reaction was stirred at 20 °C for 1 hr. The reaction was then directly added to water (20 mL), the pH was adjusted to 6 with 1 M hydrochloric acid solution, extracted with ethyl acetate (20 mL) for three times, the organic phase was combined and dried over anhydrous magnesium sulfate, filtered and concentrated to give the title compound (1 g).

[0300] MS m / z (ESI): 302.2 [M+H] +

[0301] Step 3: Synthesis of tert-butyl 7-benzyloxycarbonyl-5,6,7,8-tetrahydroimidazo[l,2- a]pyrazine-2-carboxylate (intermediate 4-4)

[0302] O,N,N'-triisopropylisourea (897.56 mg) was added to a solution of intermediate 4-3 (900 mg) in dichloromethane (20 mL). The reaction was stirred at 20 °C for 16 hr. The reaction was then directly concentrated and purified by normal phase column (tetrahydrofuran / petroleum ether = 0-50%) to give the title compound (678 mg).

[0303] MS m / z (ESI): 358.1 [M+H] +

[0304] Step 4: Synthesis of tert-butyl 5,6,7,8-tetrahydroimidazo[l,2-a]pyrazine-2- carboxylate (intermediate 4-5)

[0305] Palladium on carbon (10% wt, 0.1 g) was added to a solution of intermediate 4-4 (678 mg) in tetrahydrofuran (20 mL) under nitrogen protection. The reaction was purged with hydrogen gas for three times, the reaction was stirred at 25 °C under hydrogen atmosphere for 16 hr. The reaction was then filtered and directly concentrated to give the title compound (538 mg).

[0306] MS m / z (ESI): 224.2 [M+H] +

[0307] Step 5: Synthesis of tert-butyl 7-(3-azidopropanoyl)-5,6,7,8-tetrahydroimidazo[l,2- a]pyrazine-2-carboxylate (Intermediate 4-6)

[0308] To a solution of Intermediate 4-5 (448 mg) and 3-azidopropanoic acid (277 mg) in DMF (5 mL) was added 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (908 mg) and DIEA (778 mg). The reaction was stirred at 20 °C for 1 hr. The reaction was then purified directly by high performance liquid chromatography (column: C18 150 x 30 mm; mobile phase: [A: 0.05% trifluoroacetic acid, B: acetonitrile]; B%: 8% - 48% over 9 min) to give the title compound (330 mg).

[0309] MS m / z (ESI): 321.2 [M+H] +

[0310] Step 6: Synthesis of 7-(3-azidopropanoyl)-5,6,7,8-tetrahydroimidazo[l,2-a]pyrazine-2- carboxylic acid (Intermediate 4-7)

[0311] To a solution of Intermediate 4-6 (330 mg) in dioxane (10 mL) was added trifluoroacetic acid (563 mg). The reaction was stirred at 20 °C for 2 hr. The reaction was then concentrated under reduced pressure and the crude product was purified by high performance liquid chromatography (column: C18 150 x 30 mm; mobile phase: [A: 0.05% hydrochloric acid, B: acetonitrile]; B%: 0% - 25% over 9 min) to give the title compound (170 mg).

[0312] MS m / z (ESI): 265.0 [M+H] +

[0313] Step 7: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5-acetamido-4,6- dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-(3-azidopropanoyl)-5,6,7,8- tetrahydroimidazo[l,2-a]pyrazine-2-carboxamide (Intermediate 4-8)

[0314] Intermediate 1-4 (30 mg) and intermediate 4-7 (17.26 mg) were dissolved in N,N- dimethylformamide (0.5 mL), HATU (24.65 mg) and DIEA (37.94 μL) were added, and the reaction solution was stirred at 25 °C for 1 hr. The reaction solution was then directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 0%-20%, 11 min) to give the title compound (14 mg).

[0315] MS m / z (ESI): 761.4 [M+H] +

[0316] Step 8: Synthesis of 7-(3-azidopropanoyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5- trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy) ethyl)-5,6,7,8-tetrahydroimidazo[l,2-a]pyrazine-2-carboxamide (Compound I4)

[0317] Intermediate 4-8 (13 mg), triethylamine (178.76 μL), 2-chloro-l,3-dimethyl-lH- benzimidazole-3-carbonitrile (92.75 mg) were added to water (1 mL), replaced with nitrogen for 3 times, and the reaction solution was stirred at 0 °C for 1 hr. The reaction solution was then directly purified by high performance liquid chromatography (column: C18-3 100*30mm*5um; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 0%-30%, 20 min), and freeze-dried after adding an aqueous solution containing 40 micrograms of sodium hydroxide to give the title compound (1.9 mg).

[0318] LC-MS: m / z (ESI): 743.5 [M+H] + .

[0319] 1H NMR (400 MHz, D20) d = 7.56 (s, 1H), 5.93 (d, J = 7.3 Hz, 1H), 4.24 (s, 1H), 4.11 (d, J = 4.9 Hz, 1H), 4.04 (d, J = 5.4 Hz, 2H), 3.93 (d, J = 5.4 Hz, 2H), 3.87-3.85 (m, 1H), 3.75 (d, J = 10.3 Hz, 1H), 3.68-3.41 (m, 23H), 3.37 (d, J = 6.2 Hz, 1H), 3.29 (d, J = 7.5 Hz, 1H), 2.79-2.70 (m, 2H), 1.94 (s, 3H).

[0320] Example 1.5, 7-((4-(4-azidobutanoyl)-l-oxo-l-thioxomorpholin-l-ylidene)amino)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)benzo[d][l,3]dioxol-4-formamide (Compound I5)

[0321] Step 1: Synthesis of tert-butyl l-((7-(ethoxycarbonyl)benzo[d][l,3]dioxol-4-yl)-l- iminothiomorpholine-4-carboxylate 1-oxide (Intermediate 5-1)

[0322] To tert-butyl l-iminothiomorpholine-4-carboxylate 1-oxide (180 mg), Intermediate 1A (209.78 mg) was dissolved in anhydrous toluene (5 mL) and palladium acetate (8.62 mg), 2,2'-bis(di-p-tolylphosphino)-l,l'-binaphthalene (39.22 mg) and cesium carbonate (500.59 mg) were added under nitrogen atmosphere. The reaction was stirred at 110 °C for 48 hr. After filtration, the residue was concentrated and purified by column chromatography (eluent: 0-100% EA / PE) to give the title compound (220 mg).

[0323] MS m / z (ESI): 427.1 [M+H] +

[0324] Step 2: Synthesis of ethyl 7-((l-oxido-l-thiomorpholin-l-ylidene)amino)benzo[d][l,3]dioxole-4-carboxylate (Intermediate 5-2)

[0325] Intermediate 5-1 (220 mg) was dissolved in 2M hydrochloric acid in dioxane (5 mL), the reaction was stirred at 25 °C for 16 hr. After that, the reaction was filtered and concentrated to give the title compound (180 mg).

[0326] MS m / z (ESI): 327.0 [M+H] +

[0327] Step 3: Synthesis of ethyl 7-((4-(4-azidobutyryl)-1-oxido-1-thiomorpholine-1- ylidene)amino)benzo[d][1,3]dioxol-4-carboxylate (Intermediate 5-3)

[0328] Intermediate 5-2 (60 mg) and 4-azidobutyric acid (23.74 mg) were dissolved in pyridine (1 mL), to the system was added EDCI (70.49 mg), the reaction was stirred at 25 °C for 1 hr. After that, the reaction was filtered and concentrated, the residue was purified by column chromatography to give the title compound (72 mg).

[0329] MS m / z (ESI): 438.2 [M+H] +

[0330] Step 4: Synthesis of 7-((4-(4-azidobutyryl)-1-oxido-1-thiomorpholine-1- ylidene)amino)benzo[d][1,3]dioxol-4-carboxylic acid (Intermediate 5-4)

[0331] Intermediate 5-3 (62 mg) and lithium hydroxide (13.58 mg) were dissolved in methanol (1 mL) and water (1 mL), the reaction was stirred at 25 °C for 1 hr. After that, the reaction was filtered and purified by high performance liquid chromatography (column: C18 150*30mm; mobile phase: [A: water (0.08% ammonium bicarbonate), B: acetonitrile]; B%: 0%-36%, 9min) to give the title compound (35 mg).

[0332] LC-MS: m / z (ESI): 410.1 [M+H] + .

[0333] Step 5: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5-acetamido-4,6- dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-((4-(4-azidobutyryl)-1-oxo-1- thiomorpholine-1-ylidene)amino)benzo[d][1,3]dioxol-4-carboxamide (Intermediate 5-5)

[0334] Intermediate 1-4 (40 mg) and intermediate 5-4 (31.83 mg) were dissolved in N,N- dimethylformamide (0.5 mL), HATU (35.20 mg) and DIEA (135.41 μL) were added, and the reaction solution was stirred at 25 °C for 1 hr. The reaction solution was then directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 10%-30%, 11 min) to give the title compound (11 mg).

[0335] MS m / z (ESI): 906.4 [M+H] +

[0336] Step 6: Synthesis of 7-((4-(4-azidobutanoyl)-l-oxo-l-thioxymorpholin-l- ylidene)amino)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7- hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy) tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)benzo[d][l,3]dioxol-4- formamide (Compound I5)

[0337] Intermediate 5-5 (11 mg), triethylamine (127 μL), 2-chloro-l,3-dimethyl-lH- benzimidazole-3-carbonitrile (76.97 mg) were added to water (0.5 mL), and the reaction solution was stirred at 0 °C for 1 hr under a nitrogen atmosphere. The reaction solution was then directly purified by high performance liquid chromatography (column: C18 150*40mm; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 5%-35%, 20 min), and the title compound (4.3 mg) was obtained by lyophilization after adding a 40 microgram sodium hydroxide aqueous solution.

[0338] LC-MS: m / z (ESI): 888.5 [M+H] + .

[0339] 1H NMR (400 MHz, D20) δ = 7.20-7.14 (m, 1H), 6.73 (d, J = 8.8 Hz, 1H), 6.04 (s, 2H), 5.90 (d, J = 7.3 Hz, 1H), 4.33-4.21 (m, 2H), 4.16-3.97 (m, 2H), 3.93-3.81 (m, 2H), 3.77-3.71 (m, 1H), 3.69-3.44 (m, 24H), 3.34-3.26 (m, 4H), 2.55-2.45 (m, 2H), 1.97-1.90 (m, 3H), 1.83-1.75 (m, 2H)

[0340] Example 1.6, N 1 -(3-azidopropyl)-N 4 -(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5- (hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy]ethyl)-cubane-1,4-dicarboxamide (Compound 16)

[0341] Step 1: Synthesis of 4-methoxycarbonyl-cubane-1-carboxylic acid (Intermediate 6-1)

[0342] Cubane-1,4-dicarboxylic acid dimethyl ester (1 g) was dissolved in tetrahydrofuran (5 mL) and methanol (5 mL), under nitrogen atmosphere, aqueous sodium hydroxide solution (2.5 M, 1.91 mL) was added, the reaction was stirred at 25 °C for 16 hr. The reaction was then directly concentrated to remove tetrahydrofuran and methanol, water (20 mL) was added to the reaction, extracted with dichloromethane (30 mL) once, the aqueous phase was adjusted to pH 2 with 5 M aqueous hydrochloric acid solution, extracted with dichloromethane (60 mL) three times, the combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound (0.5 g).

[0343] MS m / z (ESI): 207.1 [M+H] +

[0344] Step 2: Synthesis of 4-methoxycarbonyl-N-(3-azidopropyl)-cubane-1-carboxamide (Intermediate 6-2)

[0345] To a solution of intermediate 6-1 (250 mg) and 3-azidopropylamine (127.46 mg) in N,N-dimethylformamide (3 mL) was added HATU (686.13 mg) and DIEA (470.10 mg) under nitrogen atmosphere. The reaction mixture was stirred at 25 °C for 2 hr. The reaction mixture was then diluted with ethyl acetate (30 mL) and washed with water (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (eluent: 0-30% tetrahydrofuran / petroleum ether, gradient @ 50 mL / min) to give the title compound (220 mg). 12g Flash column chromatography on silica gel (eluent: 0-30% tetrahydrofuran / petroleum ether, gradient @ 50 mL / min) was used to purify the title compound (220 mg).

[0346] MS m / z (ESI): 289.0 (M+H] + .

[0347] Step 3: Synthesis of 4-carboxy-N-(3-azidopropyl)-cubane-1-carboxamide (intermediate 6-3)

[0348] To a solution of intermediate 6-2 (200 mg) in methanol (2 mL) was added lithium hydroxide (58.22 mg) in water (0.5 mL) at room temperature. The reaction mixture was stirred at 20 °C for 1 hr. The reaction mixture was then adjusted to pH 2 with 5 M aqueous hydrochloric acid solution and extracted with ethyl acetate (15 mL x 3). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give the title compound (150 mg).

[0349] MS m / z (ESI): 275.1 [M+H] +

[0350] Step 4: N 1 -(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5-acetamido-4,6-dihydroxy-2- (hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- yl)methoxy)ethoxy)ethoxy)ethyl)-N 4 Synthesis of N-(3-azidopropyl)cubane-1,4-dicarboxamide (intermediate 6-4)

[0351] Intermediate 1-4 (30 mg) and intermediate 6-3 (23.55 mg) were dissolved in DMF (0.5 mL), HATU (30.81 mg) and DIEA (21.11 mg) were added under nitrogen atmosphere, the reaction was stirred at 25 °C for 3 hr. The reaction was directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*50um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 3%-23%, 11 min) to give the title compound (18 mg).

[0352] MS m / z (ESI): 771.5 [M+H] +

[0353] Step 5: N 1 -(3-azidopropyl)-N 4 Synthesis of (2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7- hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy) tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy]ethyl)-cubane-1,4-dicarboxamide (Compound I6)

[0354] Intermediate 6-4 (16 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3-ium chloride (90 mg), triethylamine (63 mg) were added to water (0.5 mL), replaced with nitrogen for 3 times, the reaction was stirred at 25 °C for 16 hr. The reaction was directly purified by high performance liquid chromatography (column: C18 150*40mm; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 0%-40%, 20 min), lyophilized after adding water solution containing 40 micrograms of sodium hydroxide to give the title compound (10.6 mg).

[0355] LC-MS: m / z (ESI): 753.5 [M+H] + .

[0356] 1H NMR (400 MHz, D20) δ = 5.99 (d, J = 7.3 Hz, 1H), 4.26 (s, 1H), 4.09 (s, 6H), 3.89-3.87 (m, 1H), 3.79 (s, 1H), 3.68-3.64 (m, 4H), 3.65-3.58 (m, 8H), 3.56-3.52 (m, 5H), 3.49-3.45 (m, 1H), 3.43-3.38 (m, 1H), 3.34 (t, J = 5.3 Hz, 3H), 3.28 (t, J = 6.5 Hz, 2H), 3.22 (t, J = 6.6 Hz, 2H), 1.97 (s, 3H), 1.78-1.65 (m, 2H)

[0357] Example 1.7, 7-((2-(4-azidobutanamido)ethyl)sulfonyl)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (Compound I7)

[0358] Step 1: Synthesis of di-tert-butyl 7-azaspiro[3.5]nonane-2,7-dicarboxylate (Intermediate 7-1)

[0359] To a solution of 7-tert-butoxycarbonyl-7-azaspiro[3.5]nonane-2-carboxylic acid (500 mg) and Intermediate 1A (1.1 g) in dichloromethane (10 mL) was stirred at 40 °C for 16 hr. The reaction mixture was directly concentrated and the residue was purified by column chromatography to give the title compound (450 mg).

[0360] 1 H NMR (400 MHz, DMSO) δ = 3.28-3.22 (m, 2H), 3.20-3.14 (m, 2H), 3.07-2.94 (m, 1H), 2.02-1.93 (m, 2H), 1.91-1.81 (m, 2H), 1.52-1.45 (m, 2H), 1.40 (s, 9H), 1.38 (s, 9H).

[0361] Step 2: Synthesis of tert-butyl 7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 7-2)

[0362] Intermediate 7-1 (450 mg), zinc bromide (620 mg) were added into dichloromethane (10 mL), the reaction was stirred at 35 °C for 16 hr, then the reaction was directly filtered and concentrated to give the title compound (270 mg).

[0363] MS m / z (ESI): 226.2 (M+H] + .

[0364] Step 3: Synthesis of tert-butyl 7-((2-(4-azidobutanamido)ethyl)sulfonyl)-7- azaspiro[3.5]nonane-2-carboxylate (Intermediate 7-5)

[0365] Intermediate 7-2 (270 mg), triethylamine (363 mg) were added into dichloromethane (5 mL), 2-chloroethanesulfonyl chloride (234 mg) was added at 0 °C under nitrogen atmosphere, the reaction was stirred at 25 °C for 2 hr. Then the reaction was directly concentrated, the residue was purified by column chromatography to give the title compound (120 mg).

[0366] MS m / z (ESI): 316.2 (M+H] + .

[0367] Step 4: Synthesis of tert-butyl 7-((2-aminoethyl)sulfonyl)-7-azaspiro[3.5]nonane-2- carboxylate (Intermediate 7-4)

[0368] Intermediate 7-3 (120 mg) was added into 7M ammonia-methanol solution (5 mL), the reaction was stirred at 40 °C for 16 hr, then the reaction was directly concentrated to give the title compound (100 mg).

[0369] MS m / z (ESI): 333.0 (M+H] + .

[0370] Step 5: Synthesis of tert-butyl 7-((2-(4-azidobutanamido)ethyl)sulfonyl)-7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 7-5)

[0371] Intermediate 7-4 (100 mg), 4-azidobutyric acid (43 mg), HATU (136 mg), DIEA (195 mg) were added into DMF (2 mL), the reaction was stirred at 25 °C for 3 hr, then the reaction was directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 40%-60%, 11 min) to give the title compound (30 mg).

[0372] MS m / z (ESI): 444.1 (M+H] +.

[0373] Step 6: Synthesis of 7-((2-(4-azidobutanamido)ethyl)sulfonyl)-7- azaspiro[3.5]nonane-2-carboxylic acid (Intermediate 7-6)

[0374] Intermediate 7-5 (30 mg), trifluoroacetic acid (1 mL) was added into dichloromethane (5 mL), the reaction was stirred at 25 °C for 3 hr, then the reaction was directly concentrated to give the title compound (26 mg).

[0375] MS m / z (ESI): 388.0 (M+H] + .

[0376] Step 7: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5- acetylamino-4,6-dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-((2-(4- azidobutanamido)ethyl)sulfonyl)-7-azaspiro[3.5]nonane-2-carboxamide (Intermediate 7-7)

[0377] Intermediate 1-4 (35 mg), Intermediate 7-6 (25 mg), HATU (29 mg), DIEA (16 mg) was added into DMF (1 mL), the reaction was stirred at 25 °C for 3 hr, then the reaction was directly purified by high performance liquid chromatography (column: Boston Prime CI 8 150*30 mm*5 um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 8%-28%, 11 min) to give the title compound (17 mg).

[0378] MS m / z (ESI): 884.5 (M+H] + .

[0379] Step 8: Synthesis of 7-((2-(4-azidobutanamido)ethyl)sulfonyl)-N-(2-(2-(2- (((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5- (hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (Compound I7)

[0380] Intermediate 7-7 (17 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3-chloride (105 mg), and triethylamine (49 mg) were added to water (2 mL). The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25 °C for 16 h. The reaction solution was then purified directly by high performance liquid chromatography (column: C18 150×40 mm; mobile phase: [A: water (0.0125% ammonia), B: acetonitrile]; B%: 5%-35%, 20 min). After adding an aqueous solution containing 38 μg of sodium hydroxide, the solution was lyophilized to obtain the title compound (7 mg).

[0381] 1 H NMR (400MHz, D2O) δ = 6.00 (d, J = 7.4Hz, 1H), 4.27 (s, 1H), 4.13-4.05 (m, 1H), 3.93-3.87 (m, 1H), 3.84-3 .79(m,1H),3.71-3.66(m,4H),3.65-3.58(m,8H),3.56-3.51(m,6H),3.50-3.46(m,1H),3.44-3.38(m ,1H),3.33-3.29(m,3H),3.28-3.22(m,4H),3.19-3.13(m,2H),3.11-3.05(m,3H),2.26(t,J=7.3Hz,2 H),2.05-1.96(m,5H),1.92-1.85(m,2H),1.79(t,J=6.9Hz,2H),1.69-1.64(m,2H),1.58-1.51(m,2H)

[0382] MS m / z (ESI): 866.6 (M+H) + .

[0383] Example 1.8, N 1 -(4-azidobutyl)-N 1 -(2-(2-(2-azidoethoxy)ethoxy)ethyl)-N 5 -(2-((2-((2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)carbamoyl)-7-azaspiro[3,5]nonane-7-yl)sulfonyl)ethyl)glutaramide (compound I8)

[0384] Step 1: Synthesis of tert-butyl (4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)carbamate (Intermediate 8-2)

[0385] Intermediate 8-1A (1.18 g) was dissolved in tetrahydrofuran (20 mL) and sodium hydride (567.73 mg, 60% active content) was added under nitrogen atmosphere. The reaction was stirred at 25 °C for 0.25 h. Then intermediate 8-1 (2 g) was dissolved in tetrahydrofuran (20 mL) and added slowly to the reaction. The reaction was stirred at 50 °C for 16 h. The reaction was then directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*50um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 53%-73%, 10 min) to give the title compound (500 mg).

[0386] LC-MS: m / z (ESI): 372.2 [M+H] +

[0387] Step 2: Synthesis of 4-azido-N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)butan-1-amine (Intermediate 8-3)

[0388] To intermediate 8-2 (600 mg) was added hydrogen chloride / 1,4-dioxane solution (2 M, 5 mL) at room temperature. The reaction was stirred at 25 °C for 3 h. The reaction was then directly concentrated to dryness under reduced pressure to give the title compound (430 mg).

[0389] LC-MS: m / z (ESI): 272.2 [M+H] +

[0390] Step 3: Synthesis of 5-((4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-5- oxopentanoic acid (Intermediate 8-4)

[0391] To a solution of intermediate 8-3 (430 mg) in dichloromethane (2 mL) and pyridine (0.5 mL) was added glutaric anhydride (271.25 mg) at room temperature. The reaction was stirred at 25 °C for 16 h. The reaction was then directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*50um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 30%-50%, 10 min) to give the title compound (550 mg).

[0392] LC-MS: m / z (ESI): 386.3 [M+H] +

[0393] Step 4: Synthesis of tert-butyl 7-((l-azido-9-(4-azidobutyl)-10,14-dioxo- 3,6-dioxa-9,15-diazahexadecan-17-yl)sulfonyl)-7-azaspiro[3.5]nonane-2-carboxylate (Intermediate 8-5)

[0394] Intermediate 8-4 (50 mg) and Intermediate 7-4 (43.13 mg) were dissolved in DMF (1 mL), HATU (73.41 mg) and DIEA (50.3 mg) were added under nitrogen atmosphere, the reaction was stirred at 25 °C for 3 h. The reaction was directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*50um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 50%-70%, 10 min) to give the title compound (55 mg).

[0395] LC-MS: m / z (ESI): 700.5 [M+H] +

[0396] Step 5: Synthesis of 7-((l-azido-9-(4-azidobutyl)-10,14-dioxo-3,6-dioxa-9,15- diazahexadecan-17-yl)sulfonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid (Intermediate 8-6)

[0397] To a solution of Intermediate 8-5 (55 mg) in dichloromethane (1 mL) was added trifluoroacetic acid (1 mL) at room temperature, the reaction was stirred at 25 °C for 1 h. The reaction was directly concentrated to dryness under reduced pressure to give the title compound (50 mg).

[0398] LC-MS: m / z (ESI): 644.3 [M+H] +

[0399] Step 6: N 1 -(2-((2-((2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5-acetamido-4,6- dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)amino carbonyl)-7-azaspiro[3.5]nonan-7- yl)sulfonyl)ethyl)-N 5 -(4-azidobutyl)-N 5 Synthesis of (2-(2-(2-azidoethoxy)ethoxy)ethyl)pentanediamide (Intermediate 8-7)

[0400] Intermediate 8-6 (40 mg) and intermediate 1-4 (34.24 mg) were dissolved in DMF (1 mL), HATU (35.16 mg) and DIEA (24.09 mg) were added under nitrogen atmosphere, the reaction solution was stirred at 25 °C for 1 h. The reaction solution was then directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*50um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 23%-43%, 10 min) to give the title compound (23 mg).

[0401] LC-MS: m / z (ESI): 1140.5 [M+H] +

[0402] Step 7: N 1 -(4-azidobutyl)-N 1 -(2-(2-(2-azidoethoxy)ethoxy)ethyl)-N 5 -(2-((2-((2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)carbamoyl)-7-azaspiro[3.5]non-7-yl)sulfonyl)ethyl)pentanediamide (Compound I8)

[0403] Intermediate 8-7 (23 mg), 2-chloro-1,3-dimethyl-1H-benzimidazole-3- chloride (54.96 mg), triethylamine (61.23 mg) were added to water (1 mL), the reaction solution was stirred at 25 °C for 16 h under nitrogen atmosphere. The reaction solution was then directly purified by high performance liquid chromatography (column: C18 150*40mm; mobile phase: [A: 0.0125% ammonia water, B: acetonitrile]; B%: 20%-50%, 20 min), after adding an aqueous solution (1 mL) containing 40 micrograms of sodium hydroxide to the eluent containing the target product, drying gave the title compound (8 mg).

[0404] LC-MS: m / z (ESI): 1122.8 [M+H] + .

[0405] 1H NMR (400 MHz, D20) δ = 5.99 (d, J = 7.3 Hz, 1H), 4.30-4.24 (m, 1H), 4.12-4.05 (m, 1H), 3.91-3.87 (m, 1H), 3.80 (s, 1H), 3.70-3.66 (m, 4H), 3.65-3.57 (m, 16H), 3.56-3.51 (m, 6H), 3.50-3.43 (m, 3H), 3.42-3.38 (m, 3H), 3.35-3.26 (m, 6H), 3.26-3.20 (m, 3H), 3.19-3.13 (m, 2H), 3.08 (s, 3H), 2.30-2.35 (m, 2H), 2.25-2.20 (m, 2H), 2.00-1.95 (m, 5H), 1.92-1.84 (m, 2H), 1.82-1.74 (m, 2H), 1.69-1.64 (m, 2H), 1.60-1.46 (m, 6H).

[0406] Example 1.9, 7-((4-(5-((4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-5- oxopentanoyl)-l-oxo-l-thioxomorpholin-1-ylidene)amino)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)- 3,4,5-trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy) ethyl)benzo[d][l,3]dioxol-4-formamide (Compound I9)

[0407] Step 1: Synthesis of ethyl 7-((4-(5-((4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)- 5-oxopentanoyl)-l-oxo-l-thioxomorpholin-1-ylidene)amino)benzo[d][l,3]dioxole-4-carboxylate (Intermediate 9-2)

[0408] Intermediate 8-4 (120 mg) and Intermediate 5-2 (102 mg) were dissolved in DMF (2 mL), to the solution was added HATU (141 mg) and DIEA (542 μί), the reaction was stirred at 25 °C for 1 h. Then water (10 mL) was added to the reaction, washed with ethyl acetate (10 mL) for three times, the organic phase was combined and dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure, purified by silica gel column chromatography (THF / PE, 0-100%) to give the title compound (200 mg).

[0409] MS m / z (ESI): 694.2 [M+H] +

[0410] Step 2: Synthesis of 7-((4-(5-((4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-5- oxopentanoyl)-l-oxo-l-thioxymorpholin-3-yl)methyl)benzo[d][l,3]dioxol-4-yl azide (Intermediate 9-2)

[0411] Intermediate 9-2 (200 mg) and lithium hydroxide (27.6 mg) were dissolved in methanol (1.5 mL) and water (1.5 mL), and the reaction was stirred at 25 °C for 16 h. After that, the reaction was concentrated to dryness under reduced pressure to give the title compound (230 mg).

[0412] LC-MS: m / z (ESI): 666.2 [M+H] + .

[0413] Step 3: Synthesis of N-(2-(2-(2-(((2R,3S,4S,5S,6S)-6-(((2R,3S,4R,5R)-5-acetamido-4,6- dihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-yl)methoxy)ethoxy)ethoxy)ethyl)-7-((4-(5-((4-azidobutyl)(2-(2-(2- azidoethoxy)ethoxy)ethyl)amino)-5-oxopentanoyl)-l-oxo-l-thioxymorpholin-3- yl)methyl)benzo[d][l,3]dioxol-4-yl azide (Intermediate 9-4)

[0414] Intermediate 1-4 (50 mg) and Intermediate 9-3 (64.7 mg) were dissolved in DMF (0.5 mL), and HATU (44 mg) and DIEA (169 μί) were added, and the reaction was stirred at 25 °C for 1 h. After that, the reaction was directly purified by high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-45%, 10 min) to give the title compound (16 mg).

[0415] LC-MS: m / z (ESI): 1162.3 [M+H] + .

[0416] Step 4: Synthesis of 7-((4-(5-((4-azidobutyl)(2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-5- oxopentanoyl)-l-oxo-l-thioxymorpholin- 1 -ylidene)amino)-N-(2-(2-(2-(((2R,3S,4S,5S,6S)-3,4,5- trihydroxy-6-(((3aR,5R,6S,7R,7aR)-7-hydroxy-5-(hydroxymethyl)-2-methyl-3a,6,7,7a- tetrahydro-5H-pyrano[3,2-d]oxazol-6-yl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)ethoxy)ethoxy) ethyl)benzo[d][l,3]dioxol-4-formamide (Compound 19)

[0417] Intermediate 9-4 (15 mg), triethylamine (173 μL), 2-chloro-l,3-dimethyl-lH- benzimidazole-3-carbonitrile (89.9 mg) were added to water (1 mL) and the reaction stirred at 0 °C under a nitrogen atmosphere for 1 h. The reaction was then purified directly by high performance liquid chromatography (column: C18 150 x 40 mm; mobile phase: [A: 0.0125% ammonia in water, B: acetonitrile]; B%: 15% - 45% over 20 min) and the eluent containing the target product was lyophilized after addition of an aqueous solution containing 35 micrograms of sodium hydroxide (1 mL) to give the title compound (3.8 mg).

[0418] LC-MS: m / z (ESI): 1144.7 [M+H] + .

[0419] 1 H NMR (400 MHz, D20) δ = 7.19 (d, J = 8.7 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.05 (s, 2H), 5.91 (d, J = 7.5 Hz, 1H), 4.34 - 4.25 (m, 1H), 4.26 - 4.22 (m, 1H), 4.15 - 4.07 (m, 1H), 4.03 - 3.99 (m, 1H), 3.88 - 3.80 (m, 2H), 3.71 - 3.45 (m, 34H), 3.40 - 3.21 (m, 9H), 2.51 - 2.38 (m, 4H), 1.94 (s, 3H), 1.83 - 1.76 (m, 2H), 1.62 - 1.45 (m, 4H).

[0420] Example 2, Preparation / Source of Drug-Linker (X’-L-D) Compounds

[0421] The structure of the drug-linker LD1 compound is shown below, which was purchased from MedChemExpress (MCE) with the catalog number HY-136314,

[0422] The structure of the Drug-Linker LD2 compound is shown below,

[0423] The structure of the Drug-Linker LD3 compound is shown below,

[0424] The structure of the Drug-Linker LD4 compound is shown below, which is prepared according to patent document WO2018182341A1,

[0425] Example 2.1, N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11- dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxepino[4,5-g]pyrano[3',4':6,7]indolizino[1,2- b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan- 18-yl)-4-((Z)-11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamide (Compound LD5)

[0426] Step 1: Synthesis of (2S,10S)-16-amino-10-benzyl-2-cyclopropyl-6,9,12,15-tetraoxo-3- oxapenta-8,11,14-triazadecanoic acid (Intermediate LD5-2)

[0427] Intermediate LD5-1 (synthesized according to the method reported in reference patent WO2023217227, total weight of resin plus substrate 50 mg, substrate amount about 60 umol) was dissolved in dichloromethane (4 mL) and hexafluoroisopropanol (1 mL). The resulting mixture was shaken on a shaker at 25 °C for 0.5 hr. After that, the reaction solution was filtered to remove the resin, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain the title compound (10 mg).

[0428] LC-MS: m / z (ESI): 464.3 [M+H] +

[0429] Step 2: Synthesis of (2S,10S)-10-benzyl-2-cyclopropyl-21-((Z)-11,12-didehydrodibenzo[b,f] azocin-5(6H)-yl)-6,9,12,15,18,21-hexaoxo-3-oxa-5,8,11,14,17-pentaazaheneicosanoic acid (Intermediate LD5-4)

[0430] Compound LD5-3 (50 mg) and compound LD5-2 (51 mg) were added into DMF (1 mL), the reaction was stirred at 25 °C for 2 h, then the reaction was used directly for the next step.

[0431] MS m / z (ESI): 773.2 [M+Na] + .

[0432] Step 3: Synthesis of N-((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11- dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b] quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)-4- ((Z)-11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamide (Compound LD5)

[0433] Intermediate LD5-4 (reaction solution of previous step), LD5-5 (synthesized by following the method reported in patent WO2023217227, 26 mg), 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (25 mg), 1-hydroxy-7-azabenzotriazole (17 mg) and pyridine (25 mg) were dissolved in DMF (4 mL), the reaction was stirred at 25 °C under nitrogen protection for 1 h. Then the reaction was directly purified by high performance liquid chromatography (column: 2_Phenomenex Gemini C18 75*40mm*3um; mobile phase: [A: water (0.008% ammonium bicarbonate), B: acetonitrile]; B%: 34%-58%, 9 min) to give the title compound (30.2 mg).

[0434] 1H NMR (400 MHz, DMSO-d6) δ = 8.68-8.64 (m, 1H), 8.61-8.54 (m, 1H), 8.35-8.22 (m, 1H), 8.20-7.90 (m, 3H), 7.76 (s, 1H), 7.67-7.59 (m, 1H), 7.57-7.54 (m, 1H), 7.49 (s, 1H), 7.47-7.37 (m, 3H), 7.35-7.29 (m, 2H), 7.27-7.23 (m, 2H), 7.23-7.17 (m, 4H), 7.16-7.14 (m, 1H), 6.51 (s, 1H), 5.52-5.29 (m, 4H), 5.03-4.91 (m, 1H), 4.86-4.58 (m, 3H), 4.51-4.32 (m, 2H), 3.68-3.64 (m, 2H), 3.53 (s, 6H), 3.14-2.91 (m, 2H), 2.82-2.70 (m, 1H), 2.25 (s, 1H), 2.13-1.99 (m, 1H), 1.93-1.70 (m, 3H), 0.97-0.93 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H), 0.40-0.24 (m, 4H)

[0435] MS m / z (ESI): 1178.4 [M+Na] + .

[0436] Example LD6, (S)-N 4 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa- 2,7,10,13,16-pentaazaoctadec-18-yl)-2-(4-((Z)-11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamido)- N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa- 2,7,10,13,16-pentaazaoctadec-18-yl)-2-(4-((Z)-11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamido)- N 4 -methyl succinamide (Compound LD6)

[0437] Step 1 : Synthesis of (S)-5-allyloxycarbonyl-l-(9H-fluoren-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxa-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatetratriacontane-34-carboxylic acid (Intermediate LD6-1)

[0438] This compound was synthesized by solid phase synthesis of polypeptides according to the following steps:

[0439] 1) To a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g) was added dichloromethane under nitrogen protection;

[0440] 2) Diisopropylethylamine DIEA (6.2 g) was added dropwise and mixed for 2 h;

[0441] 3) Methanol (13 mL) was added and mixed for 0.5 h;

[0442] 4) Filtered and washed with DMF three times;

[0443] 5) Added 20% piperidine in DMF and reacted for 30 min;

[0444] 6) Filtered and washed with DMF five times;

[0445] 7) Added the material in the "Starting material" column of the table below and mixed for 30 s, then added the material in the "Reagent" column of the table below. The reaction was carried out under nitrogen bubbling for 1 h;

[0446] 8) Steps 4-7 were repeated to complete the desired polypeptide synthesis.

[0447] After the solid phase preparation was complete, 20% hexafluoroisopropanol in dichloromethane was added to the resulting mixture and stirred for 1.5 h. After filtration, the filtrate was collected and concentrated, and the resulting mixture was purified by preparative liquid chromatography (A: 0.075% trifluoroacetic acid in water, B: acetonitrile) to give the title compound (2.15 g).

[0448] MS m / z (ESI): 1035.6 [M+H] + .

[0449] Step 2: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 1 -amino-3, 6, 9, 12, 15, 18, 21, 24, 27-nonamethyl-1,4,7,10,13,16,19,22,25,28- decaoxo-3,6,9,12,15,18,21,24,27-nonazatritriacontane-31 -carboxylic acid allyl ester (Intermediate LD6-2)

[0450] Intermediate LD6-1 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), HATU (728.95 mg) were added into DMF (5 mL), the reaction was stirred at 25 °C for 3 h under nitrogen atmosphere. After the reaction was completed, the reaction was concentrated to dryness under reduced pressure, the residue was purified by reverse phase high performance liquid preparative chromatography (Waters XBridge® 80g C 18 Column, mobile phase gradient 0~40% acetonitrile / water, flow rate 40 mL / min) to give the title compound (800 mg).

[0451] MS m / z (ESI): 1034.6 [M+H] + .

[0452] Step 3: Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 1 -amino-3, 6, 9, 12, 15, 18, 21, 24, 27-nonamethyl-1,4,7,10,13,16,19,22,25,28- decaoxo-3,6,9,12,15,18,21,24,27-nonazatritriacontane-31 -carboxylic acid (Intermediate LD6-3)

[0453] Intermediate LD6-2 (800 mg), tetrakis(triphenylphosphine)palladium (201.84 mg) and 1.3-dimethylbarbituric acid (108.71 mg) were dissolved in DMF (5 mL), the reaction was stirred at 25 °C for 4 h under nitrogen atmosphere. After the reaction was completed, the crude product was purified by reverse phase high performance liquid preparative chromatography (Waters XBridge® 80g C 18 Column, mobile phase gradient 0~40% acetonitrile / water, flow rate 40 mL / min) to give the title compound (700 mg).

[0454] MS m / z (ESI): 994.9 [M+H] + .

[0455] Step 4: Synthesis of resin-supported (2S, 10S, 19S)-19-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecazatetraconta-18,21,24,27,30,33,36,39,42,45,48-undecaene (Intermediate LD6-5)

[0456] Intermediate LD6-3 (700 mg) was added to DMF (10 mL), then intermediate LD6-4 (reference patent WO2023217227 reported method for synthesis, total weight of resin plus substrate 400 mg, substrate amount about ~ 500 umol), HBTU (336.58 mg) and diisopropylethylamine (205.62 mg) were added. The reaction was shaken on a shaker under a nitrogen atmosphere at 25°C for 1 h. After the reaction was completed, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) in turn, and repeated 3 times. The resin was filtered to dryness, and dried under vacuum to obtain the title compound (420 mg).

[0457] MS m / z (ESI): 1461.4 [M+Na] + .

[0458] Step 5: Synthesis of resin-supported (2S, 10S, 19S)-19,48-diamino-10-benzyl-2- cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecazatetraconta-18,21,24,27,30,33,36,39,42,45,48-undecane (Intermediate LD6-6)

[0459] Intermediate LD6-5 (420 mg) was added to DMF (5 mL), then piperidine (1.25 mL) was added. The reaction was placed on a shaker at 25°C for 1 h. After the reaction was completed, the resin was washed with methanol (10 mL) and dichloromethane (10 mL) in turn, and repeated 3 times. The resin was filtered to dryness, and dried under vacuum to obtain the title compound (400 mg).

[0460] MS m / z (ESI): 1217.4 [M+H] + .

[0461] Step 6: Synthesis of (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediate LD6-7)

[0462] Intermediate LD6-6 (500 mg) was added to a mixed solvent of dichloromethane (8 mL) and hexafluoroisopropanol (2 mL) and shaken at 25 °C for 0.5 h. The reaction solution was filtered to remove resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (10 mM ammonium bicarbonate + 0.05% ammonia), B: acetonitrile]; B%: 0%-26%, 9 min) to obtain the title compound (94 mg).

[0463] MS m / z (ESI): 1217.7 [M+H] + .

[0464] Step 7: Synthesis of (2S,10S,19S)-48-amino-10-benzyl-2-cyclopropyl-19-(4-((Z)-11,12-disdehydrodibenzo[b,f]azaoctyl-5(6H)-yl)-4-oxobutyrylamino)-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediate LD6-8)

[0465] Intermediate LD6-7 (67 mg) and compound LD5-3 (25 mg) were added to N,N-dimethylformamide (1 mL), and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was then used directly in the next step, containing the title compound (92 mg).

[0466] Step 8: Synthesis of (S)-N4-(26-amino-3,6,9,12,15,18,21,24- octamethyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosyl)-N1-((4S,12S)-12- benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13- tetrahydro-10H-[1,3]dioxepino[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 14-yl-2,2-d2)-3,8,11,14,17-pentaoxo-5-oxa-2,7,10,13,16-pentaazaoctadecan-18-yl)- 2-(4-((Z)-11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanamido)-N4- methylsuccinamide (Compound LD6)

[0467] Intermediate LD6-8 (92 mg), Compound LD5-5 (26 mg), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (25 mg), 1- hydroxy-7-azabenzotriazole (17 mg) and pyridine (20 mg) were dissolved in DMF (4 mL), the reaction was stirred at 25 °C under nitrogen protection for 1 h. After the reaction was completed, the reaction was directly purified by high performance liquid chromatography (column: 2_Phenomenex Gemini C18 75*40mm*3um; mobile phase: [A: water (0.0125% ammonia water), B: acetonitrile]; B%: 20%-45%, 15 min) to give the title compound (10.8 mg).

[0468] 1H NMR (400 MHz, DMSO-d6) d = 8.68-8.64 (m, 1H), 8.61-8.52 (m, 1H), 8.32-8.23 (m, 1H), 8.20-8.16 (m, 1H), 8.09-7.87 (m, 3H), 7.76 (s, 1H), 7.65-7.54 (m, 2H), 7.52-7.40 (m, 4H), 7.38-7.28 (m, 3H), 7.26-7.06 (m, 8H), 6.53 (s, 1H), 5.42-5.40 (m, 4H), 5.03-4.93 (m, 1H), 4.85-4.71 (m, 2H), 4.68-4.62 (m, 1H), 4.52-4.44 (m, 2H), 4.42-4.19 (m, 10H), 4.14-3.90 (m, 17H), 2.98-2.69 (m, 31H), 2.28-2.24 (m, 1H), 2.13 (s, 1H), 2.04-1.94 (m, 1H), 1.93-1.70 (m, 3H), 1.00-0.92 (m, 1H), 0.86 (t, J = 7.1 Hz, 3H), 0.40-0.25 (m, 4H)

[0469] MS m / z (ESI): 955.9 [(M+2H) / 2] + .

[0470] The structure of the drug-linker LD7 compound is shown below, which was purchased from MedChemExpress (MCE) with the catalog number HY-147363,

[0471] The drug-linker LD8 was prepared according to the patent document CN111065621A,

[0472] The drug-linker LD9 was purchased from MCE (catalog number: HY-15575),

[0473] Example 3: Construction and production of antibodies

[0474] 3.1 Construction and expression of antibodies

[0475] 3.1.1 Construction and production of anti-human HER2 monoclonal antibodies

[0476] The sequences of the anti-human HER2 monoclonal antibodies are shown in Table 3-1 below. The heavy chain and light chain nucleotide sequences were cloned into the pTT5 vector (purchased from Yobio) respectively, and plasmids were prepared according to the established standard molecular biology methods. For details, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The expression vector and transfection reagent PEI (Polysciences, item number: 24765-1) were mixed in OPTI-MEM (Gibco, item number: 11058021) and incubated for 15 min, then added to Expi293F cells (manufacturer: Thermofisher, item number: A14527) and cultured in a 5% CO2, 120 rpm, 37°C shaker. On the second day after transfection, OPM-293 ProFeed (Shanghai Aupma, item number: F081918-001) and 6 g / L glucose (manufacturer: Sigma, item number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0477] Table 3-1 Sequence information of anti-human HER2 antibody and CDR analysis (according to Kabat division)

[0478] 3.1.2 The sequences of the anti-human TROP2 monoclonal antibodies are shown in Table 3-2 below:

[0479] Table 3-2 Sequence information of anti-human TROP2 antibody and CDR analysis (according to Kabat division)

[0480] 3.1.3 The sequences of the anti-human B7H3 monoclonal antibodies are shown in Table 3 below:

[0481] Table 3-3 Sequence information of anti-human B7H3 antibody and CDR analysis (according to Kabat division)

[0482] 3.2 Antibody purification

[0483] The above-mentioned antibodies are purified from cell culture supernatant by Protein A affinity chromatography (Cytiva, item number 17549802). The Protein A affinity column is washed with 0.5M NaOH for 3-5 column volumes, and then washed with pure water for 3-5 column volumes. The chromatography column is equilibrated with 1x PBS (pH 7.4) buffer system for 3-5 column volumes. The cell supernatant is loaded for binding at a low flow rate, and after completion of binding, the chromatography column is washed with 1x PBS (pH 7.4) for 3-5 column volumes until the ultraviolet absorption falls to the baseline. The sample is eluted with eluent (50mM acetic acid buffer, pH 3.5), and the elution peak is collected according to ultraviolet monitoring, and the eluted product is temporarily stored by rapidly adjusting the pH to 5-6 with 1M Tris-HCl pH 8.0. The buffer system is replaced by ultrafiltration, dialysis or desalting column, or the polymeric components in the eluted product are removed by using, for example, a molecular sieve to improve the purity of the sample. After purification, the protein that meets the purity requirements is sterile filtered with a 0.22μm filter (Millipore, item number SLGVR13SL), and after SEC-HPLC purity detection, it is divided and stored at -80℃ for standby.

[0484] Example 4: Preparation method of antibody-drug conjugate

[0485] Preparation method 1 of antibody-drug conjugate

[0486] Step 1: Preparation of deglycosylated antibody Ab-GlcNAc(Fucα1,6)

[0487] Endo-S2 glycosidase and the antibody prepared in Example 3 are mixed in PBS buffer according to a mass ratio of 1:100-200, and reacted at 30℃ for 1-4 hours. After the reaction is completed, the deglycosylated antibody Ab-GlcNAc(Fucα1,6) is purified by a protein A purification column, and the obtained deglycosylated antibody is desalted by a G25 column or replaced into a phosphate (PBS) buffer by an ultrafiltration tube.

[0488] Step 2: Preparation of glycoengineered antibody

[0489] The deglycosylated antibody Ab-GlcNAc(Fucα1,6) prepared in Example 1 is mixed with the disaccharide compound prepared in Example 1 in a proper molar ratio (1:10 to 1:50) in PBS buffer, and then Endo-S2 glycosidase is added (mass ratio 1:100-200), and reacted at 25-32℃ for 1-8 hours. After the reaction is completed, the glycoengineered antibody shown in formula V is obtained by purifying by a protein A purification column, and the reaction product is desalted by a G25 column or replaced into an acetic acid-sodium acetate (pH 5.2) or phosphate (PBS, pH 7.3) buffer by an ultrafiltration tube to remove the unreacted glycosylation compound.

[0490] Step 3: Preparation of antibody-drug conjugate

[0491] 3.1 Preparation of conjugate by formation of oxime bond

[0492] To the above glycoengineered antibody solution containing 50 mM sodium acetate (pH 5.2), 8-20 molar equivalents of drug-linker compound (e.g. compounds LD4-LD7 described in Example 2) were added and mixed at 30°C for 16 hours. After the reaction was completed, the residual compound in the solution was removed by G25 desalting column or ultrafiltration tube exchange, etc. The prepared ADC product was stored in 20 mM acetic acid-sodium acetate (pH 5.5) buffer. The structure of the prepared ADC and the corresponding number are shown as follows:

[0493] wherein y is a real number of 1-2.

[0494] 3.2 Preparation of conjugate by click chemistry method

[0495] To the above glycoengineered antibody solution containing PBS buffer, 8-20 molar equivalents of drug-linker compound (e.g. compounds LD1-LD6 described in Example 2) were added and mixed at 25°C for 16 hours. After the reaction was completed, the residual compound in the solution was removed by G25 desalting column or ultrafiltration tube exchange, etc. The prepared ADC product was stored in 20 mM acetic acid (pH 5.5) buffer. The structure of the prepared ADC and the corresponding number are shown as follows:

[0496] wherein y is a real number of 1-2;

[0497] and

[0498] wherein y is a real number of 1-2.

[0499] The purity, structure and DAR value of the ADC product were analyzed and confirmed by SEC, HIC-HPLC and LC-MS methods.

[0500] Antibody-drug conjugate preparation method 2

[0501] Add 10 times molar equivalent of TCEP (tris (2-carboxyethyl) phosphine hydrochloride) to 2-10 mg / ml antibody solution (1xPBS, 1 mM EDTA, pH 7.4), mix and incubate at 37°C for 2 hours, then add 15 times molar equivalent of drug-linker compound (compound LD8 described in Example 2), mix and react at 25°C for 1-2 hours. After the reaction is completed, use 20 mM acetic acid-sodium acetate buffer, pH 5.5 to ultrafiltrate and replace the solution to remove residual unreacted free small molecules, use SEC-HPLC to detect sample purity, and use LC-MS mass spectrum to detect coupling.

[0502] Antibody-drug conjugate preparation method 3

[0503] Add 2.5 times molar equivalent of TCEP (tris (2-carboxyethyl) phosphine hydrochloride) to 2-10 mg / ml antibody solution (1xPBS, 1 mM EDTA, pH 7.4), mix and incubate at 37°C for 2 hours, then add 8 times molar equivalent of drug-linker compound (compound LD9 described in Example 2), mix and react at 25°C for 1-2 hours. After the reaction is completed, use 20 mM acetic acid-sodium acetate buffer, pH 5.5 to ultrafiltrate and replace the solution to remove residual unreacted free small molecules, use SEC-HPLC to detect sample purity, and use LC-MS mass spectrum to detect coupling.

[0504] Antibody-drug conjugate preparation method 4

[0505] According to the patent CN109081871B, add 2.5 times molar equivalent of TCEP (tris (2-carboxyethyl) phosphine hydrochloride) to 2-10 mg / ml antibody solution (1xPBS, 1 mM EDTA, pH 7.4), mix and incubate at 4°C for 16 hours, then add 8 times molar equivalent of drug-linker compound (compound LD8 described in Example 2), mix and react at 4°C for 6 hours. After the reaction is completed, use 20 mM acetic acid-sodium acetate buffer, pH 5.5 to ultrafiltrate and replace the solution to remove residual unreacted free small molecules, use SEC-HPLC to detect sample purity, and use LC-MS mass spectrum to detect coupling.

[0506] Antibody-drug conjugate purity detection (SEC-HPLC)

[0507] SEC-HPLC method was used to analyze ADC samples, to characterize the sample's molecular size homogeneity, and to determine the sample's purity. The HPLC used in this method was Agilent 1260, the column was TSKgel G3000SWXL from Tosoh Bioscience, the mobile phase was 200 mM phosphate buffer pH 7.0, the detection temperature was 25 °C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the loading amount was 50 μg, and the analysis time was 40 minutes. For SEC-HPLC data, the chromatogram was analyzed by manual integration method, and the protein purity was calculated according to the area normalization method. The main peak was considered as monomer, the chromatographic peak before the main peak was called aggregate, and the chromatographic peak after the main peak was called fragment. The test results are shown in Table 4-1.

[0508] Determination of DAR value of antibody-drug conjugate (HIC-HPLC)

[0509] HIC-HPLC method was used to analyze the sample to be tested, to characterize its DAR value. The HPLC used in this method was Agilent 1260, the column was TSKgel Butyl-NPR from Tosoh Bioscience, the mobile phase A was 1.5 M ammonium sulfate, 50 mM phosphate buffer pH 7.0, the mobile phase B was 25 mM phosphate buffer pH 7.0 containing 20% isopropanol, the detection temperature was 25 °C, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm and 250 nm. The sample to be tested was first concentrated to 1 mg / mL, then 2 M ammonium sulfate, 100 mM phosphate buffer pH 7.0 solution was added, the final concentration of ammonium sulfate was 0.8 M, mixed evenly, the supernatant was taken, the loading amount was 40 μg, the mobile phase gradient was that the mobile phase B was increased from 0% to 100% in 50 minutes, and the analysis time was 70 minutes. Based on the different absorption signals of antibody and compound at 280 nm and 250 nm, and the principle that the compound content is positively correlated with the DAR value, the DAR value of each HIC chromatographic peak was estimated. For HIC-HPLC data, the chromatogram was analyzed by manual integration method, and the area percentage of each peak under the condition of 280 nm absorption was calculated according to the area normalization method. The area percentage of each chromatographic peak was multiplied by its estimated DAR value, and the sum was the average DAR. The test results are shown in Table 4-1.

[0510] Determination of antibody-drug conjugate by mass spectrometry (LC-MS)

[0511] Detection method 1 (for detecting ADC compounds prepared according to the antibody-drug conjugate preparation method 1 in Example 4):

[0512] The ADC sample was diluted to 1 mg / mL with 50 mM Tris buffer, mixed well, centrifuged, and loaded onto the HPLC system. The HPLC system used in this method employed a Thermo Vanquish, with a loading volume of 1 μg. The chromatographic column was a Protein BEH SEC column from Waters (specifications: [not specified]). The mass spectrometer used in this method was QExactive Plus (1.7 μm, 4.6 mm x 300 mm). The mobile phase was 25% acetonitrile solution (containing 0.1% formic acid and 0.05% TFA), the flow rate was 0.2 mL / min, the detection wavelengths were 280 nm and 220 nm, and the analysis time was 30 minutes. The raw mass spectrometry data was analyzed using the Biopharma Finder 4.1 software. The Respect algorithm was used for deconvolution processing, and the molecular weight information of each mass peak was calculated. Based on this, the DAR value of the ADC sample was calculated. The detection results are shown in Tables 4-1 and 4-2.

[0513] Detection Method 2 (for detecting ADC compounds prepared according to methods 2-4 of Example 4):

[0514] The sample was diluted halved with 100 mM Tris buffer (pH = 8.0), and 1 M DTT was added to a final concentration of 20 mM. The sample was incubated at 37°C for 1 hour. The sample was then centrifuged for analysis. The HPLC method used in this study employed a Thermo Vanquish HPLC system. The column used was a Protein BEH SEC column from Waters (specifications: [specifications missing]). The mass spectrometer used in this method was Q Exactive Plus (1.7 μm, 4.6 mm x 300 mm). The mobile phase was 25% acetonitrile solution (containing 0.1% formic acid and 0.05% TFA), the flow rate was 0.2 mL / min, the detection wavelengths were 280 nm and 390 nm, the protein loading was 1 μg, and the analysis time was 30 min. The mass spectrometer scan time was 4–15 min after sample injection. The main mass spectrometry parameters were spray voltage 3.8 kV, capillary heating temperature 300 °C, sheath gas flow rate 35 arb, and precursor ion scan range 900–4500 m / z. Finally, the mass spectrometry data analysis software Biopharma Finder 4.1 was used to deconvolve the data using the Respect algorithm to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component. The DAR value of the ADC sample was then calculated based on these results, which are shown in Table 4-1.

[0515] Table 4-1 Antibody-drug conjugates, their DAR values ​​and SEC purity

[0516] " / " means that the reaction substrate is not present.

[0517] Table 4-2 Antibody-drug conjugate mass spectrometry confirmation results

[0518] * ADC1-I1 refers to the glycoengineered antibody prepared from Trastuzumab-GlcNAc(Fucal,6) and sugar chain I1 by the method described in step 2 of the fourth aspect above.

[0519] Example 5: Biological activity and related property tests

[0520] 5.1 Antibody-drug conjugate anti-proliferative activity test on tumor cells

[0521] 5.1.1 Anti-Her2-ADC anti-proliferative activity test on tumor cells

[0522] Cells and materials: Human breast cancer cell line SKBR3 with high expression of Her2 was purchased from ATCC, McCoy's 5a medium (Gibco #16600-082), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA), 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0523] Cell culture: SKBR3 cells were cultured in McCoy's 5a medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0524] Cell proliferation activity detection: Cell-Titer Glo reagent was used to detect the inhibitory activity of ADC on the proliferation of SKBR3 cell line. SKBR3 cells were digested and dispersed from cell culture bottles, resuspended with the corresponding fresh culture medium, and the cell density was adjusted. SKBR3 cells were 5000 cells / 90 μL / well, inoculated in a 96-well plate, and incubated at 37°C, 5% CO2 overnight. ADC concentration was diluted to 100 nM with complete culture medium, and 3-fold gradient dilution was performed, a total of 8 concentration gradients, then 10 μL ADC dilution solution was transferred to the 96-well plate, the initial concentration of ADC was 10 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 3 days. Cell-Titer Glo reagent was added to detect cell activity.

[0525] Another negative control group and positive control group are set as Bottom and Top respectively. The negative control group is without cells, only with the same volume of medium, and other operations are consistent with the experimental group; the positive control group is without the test antibody, and other operations are consistent with the experimental group.

[0526] Data analysis:

[0527] Calculate the inhibition percentage (% Inhibition) and fit the IC 50 .

[0528] Inhibition percentage (% Inhibition) = 1-100% * (Signal-Bottom) / (Top-Bottom).

[0529] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0530] Experimental results:

[0531] Under the conditions of this experiment, the ADC of the present disclosure exhibits strong proliferation inhibition activity on the human breast cancer cell line SKBR3 with high expression of Her2.

[0532] Table 5-1 ADC anti-tumor cell proliferation activity (SKBR3 cell line)

[0533] 5.1.2, Anti-Her2-ADC anti-proliferation activity test on tumor cells NCI-N87

[0534] Cells and materials: The human gastric cancer cell line NCI-N87 with high expression of Her2 was purchased from the Chinese Academy of Sciences Cell Bank, 1640 culture medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-056) were purchased from GIBCO (USA), 96-well plates (Greiner Bio-one #655098) were purchased from Corning (USA), and Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0535] Cell culture: NCI-N87 cells were cultured in 1640 culture medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Cells were cultured at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0536] Cell proliferation activity detection: The Cell-Titer Glo reagent was used to detect the inhibition activity of ADC on the proliferation of NCI-N87 cell strain. NCI-N87 cells were digested and dispersed from cell culture bottles, resuspended with the corresponding fresh culture medium, and the cell density was adjusted. NCI-N87 cells were 3000 cells / 90 μL / well, inoculated in a 96-well plate, and incubated at 37°C, 5% CO2 for overnight. The ADC concentration was diluted to 250nM with complete culture medium, 10-fold gradient dilution was performed for ADC1-I1-LD4, and 4-fold gradient dilution was performed for other ADCs, a total of 8 concentration gradients for each ADC, then 10 μL ADC dilution solution was transferred to the 96-well plate, and the initial concentration of ADC was 25nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell-Titer Glo reagent was added to detect cell activity.

[0537] A negative control group and a positive control group were set up as Bottom and Top, respectively. The negative control group did not add cells, but added the same volume of culture medium, and the other operations were consistent with the experimental group; the positive control group did not add the test antibody, and the other operations were consistent with the experimental group.

[0538] Data analysis:

[0539] The inhibition percentage (%) was calculated and fitted to obtain the IC 50 .

[0540] The inhibition percentage (%) was calculated and fitted to obtain the IC 50 .

[0541] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0542] Graphpad curve fitting, the drug concentration when the inhibition score is 50% is calculated as the absolute IC 50 .

[0543] Experimental results:

[0544] Under the conditions of this experiment, the ADC of the present disclosure showed strong proliferation inhibition activity on the human gastric cancer cell line NCI-N87 with high expression of Her2.

[0545] Table 5-2 ADC anti-tumor cell proliferation activity (NCI-N87 cell strain)

[0546] 5.1.3 Anti-Trop2-ADC Anti-proliferation Activity Test on Tumor Cells

[0547] Cells and materials: Human pancreatic cancer cell line BxPC-3 with high expression of Trop2 was purchased from Nanjing Kebai Biotechnology Co., Ltd. RPMI 1640 medium (Gibco # 2240-0089), penicillin-streptomycin (Gibco # 15140-122), and 0.25% Trypsin-EDTA (Gibco # 25200-072) were purchased from Gibco (USA), 96-well plates (Xinyou # 062096) were purchased from Hangzhou Xinyou Biotechnology Co., Ltd., Cell Viability Detection reagent (VKEY-BIO Technologies # A2010005N) was purchased from Yaoke Yuan Biotechnology Co., Ltd. (China), and serum (ExCell # FSP500) was purchased from Yikaisai Biotechnology Co., Ltd.

[0548] Cell culture: BxPC3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0549] Cell proliferation activity detection: Cell Viability Detection reagent was used to detect the inhibitory activity of ADC on BxPC-3 cell line proliferation. BxPC3 cells were digested and dispersed from cell culture bottles, resuspended with corresponding fresh culture medium, and the cell density was adjusted. BxPC3 cells were 2000 cells / 80 μL / well, inoculated in 96-well plates, and incubated at 37°C, 5% CO2 for overnight. ADC concentration was diluted to 2500 nM with complete culture medium, and 5-fold gradient dilution was performed, with a total of 9 concentration gradients. Then 20 μL of ADC dilution solution was transferred to the 96-well plate, and the initial concentration of ADC was 500 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 7 days. Cell Viability Detection reagent was added to detect cell activity.

[0550] A negative control group and a positive control group were set up as Bottom and Top, respectively. The negative control group did not add cells, but added the same volume of culture medium, and the other operations were consistent with the experimental group. The positive control group did not add the test antibody, and the other operations were consistent with the experimental group.

[0551] Data analysis:

[0552] The inhibition percentage (%) was calculated and the IC 50 .

[0553] % Inhibition = 100% * (Top - Signal) / (Top - Bottom).

[0554] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0555] Experimental results:

[0556] Under the experimental conditions, the ADC of the present disclosure showed strong proliferation inhibition activity on the human pancreatic cancer cell line BxPC3 with high expression of Trop2.

[0557] Table 5-3 Anti-tumor cell proliferation activity of ADC (BxPC3 cell line)

[0558] 5.1.4 Anti-Trop2-ADC anti-proliferation activity test on tumor cells MDA-MB-468

[0559] Cells and materials: The human breast cancer cell line MDA-MB-468 with high expression of Trop2 was purchased from Meilunbio, DMEM medium (Meilunbio #PWL003), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-072) were purchased from Gibco (USA), 96-well plates (Xinyou #062096) were purchased from Hangzhou Xinyou Biotechnology Co., Ltd., Cell Viability Detection reagent (VKEY-BIO Technologies #A2010005N) was purchased from Yaoke Yuan Biotechnology Co., Ltd. (China), and serum (ExCell #FSP500) was purchased from ExCell Biotech Co., Ltd.

[0560] Cell culture: MDA-MB-468 cells were cultured in DMEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Cells were cultured at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0561] Cell Viability Detection reagent was used to detect the inhibitory activity of ADC on the proliferation of MDA-MB-468 cell line. MDA-MB-468 cells were digested and dispersed from cell culture bottles, resuspended with the corresponding fresh culture medium, and the cell density was adjusted. MDA-MB-468 cells were 1000 cells / 80 μL / well, inoculated in a 96-well plate, and incubated at 37°C, 5% CO2 overnight. The ADC concentration was diluted to 2500nM with complete culture medium, and the ADC was diluted by 5 times gradient, a total of 9 concentration gradients, then 20 μL ADC dilution solution was transferred to the 96-well plate, and the initial concentration of ADC was 500nM. The 96-well plate was incubated at 37°C, 5% CO2 for 7 days. Cell viability detection reagent was added to detect cell activity.

[0562] A negative control group and a positive control group were set up as Bottom and Top, respectively. The negative control group did not add cells, but added the same volume of culture medium, and the other operations were consistent with the experimental group; the positive control group did not add the test antibody, and the other operations were consistent with the experimental group.

[0563] Data analysis:

[0564] The percentage of inhibition (%Inhibition) was calculated and fitted to obtain the IC 50 .

[0565] The percentage of inhibition (%Inhibition) = 100% * (Top-Signal) / (Top-Bottom).

[0566] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0567] Experimental results:

[0568] Under the conditions of this experiment, the ADC of the present disclosure showed strong proliferation inhibitory activity on the human breast cancer cell line MDA-MB-468 with high expression of Trop2.

[0569] Table 5-4 ADC anti-tumor cell proliferation activity (MDA-MB-468 cell line)

[0570] 5.1.5 Anti-B7H3-ADC anti-proliferative activity test on tumor cells NCI-H146

[0571] Cell and materials: B7H3 high expression human small cell lung cancer cell line NCI-H146 was purchased from ATCC, RPMI-1640 medium (Gibco #2240-0089), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-072) were purchased from Gibco (USA), 96-well plates (Xinyou #062096) were purchased from Hangzhou Xinyou Biotechnology Co., Ltd., Cell Viability Detection reagent (VKEY-BIO Technologies #A2010005N) was purchased from Yuyuan Biotechnology Co., Ltd. (China), serum (ExCell #FSP500) was purchased from ExCell Biotechnology Co., Ltd. (China).

[0572] Cell culture: NCI-H146 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum + 1% penicillin-streptomycin at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0573] Cell proliferation activity detection: Cell Viability Detection reagent was used to detect the inhibition activity of ADC on NCI-H146 cell line proliferation. After centrifugation to collect NCI-H146 cells, blow dispersing treatment was performed, and the cells were resuspended with the corresponding fresh culture medium and the cell density was adjusted. NCI-H146 cells were 10000 cells / 80μL / well, inoculated in 96-well plates. ADC concentration was diluted to 500nM with complete culture medium, and 5-fold gradient dilution was performed, a total of 8 concentration gradients, then 20μL ADC dilution solution was transferred to the 96-well plate, the initial concentration of ADC was 100nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell viability detection reagent was added to detect cell viability.

[0574] A negative control group and a positive control group were set up as Bottom and Top, respectively. The negative control group did not add cells, only added the same volume of culture medium, and the other operations were consistent with the experimental group; the positive control group did not add the test antibody, and the other operations were consistent with the experimental group.

[0575] Data analysis:

[0576] The inhibition percentage (%) was calculated and the IC 50 of the compound was fitted.

[0577] The inhibition percentage (%) = 100% * (Top-Signal) / (Top-Bottom).

[0578] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0579] Experimental results:

[0580] Under the experimental conditions, the ADC of the present disclosure showed strong proliferation inhibition activity on the human small cell lung cancer cell line NCI-H146 with high expression of B7H3.

[0581] Table 5-5 ADC anti-tumor cell proliferation activity (NCI-H146 cell strain)

[0582] 5.1.6 Anti-B7H3-ADC anti-proliferation activity test on tumor cells A375

[0583] Cells and materials: B7H3 high expression human malignant melanoma cell line A375 was purchased from Beina Biological Technology Co., Ltd. DMEM medium (Meilunbio #PWL003), penicillin-streptomycin (Gibco #15140-122), and 0.25% Trypsin-EDTA (Gibco #25200-072) were purchased from Gibco (USA), 96-well plates (Xinyou #062096) were purchased from Hangzhou Xinyou Biological Technology Co., Ltd., Cell Viability Detection reagent (VKEY-BIO Technologies #A2010005N) was purchased from Yaoke Yuan Biological Technology Co., Ltd. (China), and serum (ExCell #FSP500) was purchased from ExCell Biotechnology Co., Ltd. (China).

[0584] Cell culture: A375 cells were cultured in DMEM medium containing 10% fetal bovine serum + 1% penicillin-streptomycin. Cells were cultured at 37°C, 5% CO2, and cells in the logarithmic growth phase were used for experiments.

[0585] Cell Viability Detection reagent was used to detect the inhibition activity of ADC on the proliferation of A375 cell strain. A375 cells were digested and dispersed from cell culture bottles, resuspended with corresponding fresh culture medium, and the cell density was adjusted. A375 cells were 300 cells / 80 μL / well, inoculated in a 96-well plate, and incubated at 37°C, 5% CO2 for overnight. ADC was diluted to 500 nM with complete culture medium, and 5-fold gradient dilution was performed, a total of 8 concentration gradients, and then 20 μL of ADC dilution solution was transferred to the 96-well plate, and the initial concentration of ADC was 100 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell viability detection reagent was added to detect cell activity.

[0586] A negative control group and a positive control group were set as Bottom and Top, respectively. The negative control group did not add cells, but added the same volume of culture medium, and the other operations were consistent with the experimental group; the positive control group did not add the test antibody, and the other operations were consistent with the experimental group.

[0587] Data analysis:

[0588] The inhibition percentage (%) Inhibition was calculated and fitted to obtain the IC 50 .

[0589] The inhibition percentage (%) Inhibition = 100% * (Top-Signal) / (Top-Bottom).

[0590] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0591] Graphpad curve fitting, the drug concentration when the inhibition score is 50% is calculated as the absolute IC 50 .

[0592] Experimental results:

[0593] Under the conditions of this experiment, the ADC of the present disclosure showed strong proliferation inhibition activity on B7H3 high expression human malignant melanoma cells A375.

[0594] Table 5-6 ADC anti-tumor cell proliferation activity (A375 cell strain)

[0595] Example 6: Thermal stability detection of antibody conjugate

[0596] The melting temperature of ADC samples can be detected by differential scanning fluorometry (DSF). Before testing the samples, Applied Biosystems... TM Protein Thermal Shift TM The dye in the Dye Kit (catalog number: 4461146) was diluted from 1000X to 50X using the kit's included buffer. A suitable detection system was prepared and mixed thoroughly, then added to a 96-well plate. Three replicates of the same sample were made, with 20 μl of sample added to each well. The plate was then coated with a membrane, centrifuged for 5–10 seconds, and placed in a real-time quantitative PCR instrument (Applied Biosystems-AB7500) for detection. Melting curve settings: reaction volume 20 μl; temperature mode selected as Continuous; temperature settings as follows:

[0597] The test results are shown below.

[0598] Tm1 (melting temperature 1) typically corresponds to the denaturation temperature of the antibody's CH2 domain. Tm2 (melting temperature 2) corresponds to the denaturation temperature of the Fab (antigen-binding domain) domain. The results show that the ADC disclosed in this paper has a higher melting temperature 1, indicating that the ADC obtained by this method has better structural stability and good drug-like properties.

[0599] Example 7: Plasma stability test of antibody conjugates

[0600] 7.1 Plasma stability test of antibody conjugates

[0601] A certain amount of ADC sample was added to sterile human plasma (IgG removed) to achieve a final ADC concentration of 0.5 mg / ml. The sample was incubated at 37°C for 0, 3, 7, and 14 days, and then stored at -80°C. 50 μL of Protein A packing material (MabSelect SuRe) was added to each tube, and the mixture was shaken for 1 hour for adsorption. After washing, elution, and Tris-HCl neutralization, the incubated ADC sample was obtained. The purity change was detected by SEC-HPLC, and the DAR value change was detected by LC-MS to determine the plasma stability of the sample. The test results are shown in Table 7.

[0602] Table 7-1 In vitro plasma stability of antibody-drug conjugates

[0603] Experimental results show that the DAR value of the ADC disclosed herein did not change significantly during plasma incubation, and the SEC purity changed little, indicating that the ADC disclosed herein has good stability in plasma.

Claims

A disaccharide compound of Formula I or a pharmaceutically acceptable salt thereof: wherein Ring A is selected from C6-C 10 arylene, 5-10 member heteroarylene, 4-14 member heterocyclic or C3-C 10 Cycloalkylene, the C6-C 10 arylene, 5-10 member heteroarylene, 4-14 member heterocyclic or C3-C 10 Cycloalkylene groups are optionally surrounded by one or more R a replace; Each R a It is independently selected from C1-C3 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclic group, halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2 or COOH; Y is selected from a chemical bond, C1-C6alkylene, C=0, S(=0)2, C(=0)NR 10 alkylene, C=0, S(=0)2, C(=0)NR b , S(=0)2NR b , C3-C6cycloalkylene, 4-7 membered heterocyclylene, O, S, NR b , or any combination thereof, each R b independently selected from H or C1-C6alkyl, each m1, m2is independently selected from an integer between 0 and 20, and the b terminus is independently covalently attached to Z; each Z is independently selected from Each R d Independently selected from H, C1-C6 alkyl, C3-C6 cycloalkyl, or 4-7 membered heterocyclic groups; m is an integer from 1 to 5; n is an integer from 1 to 20. A glycoengineered antibody of the formula V, wherein the sugar chain in the glycoengineered antibody is covalently linked to the Asn at position 297 of the heavy chain of the antibody unit Ab: wherein, Ab is an antibody unit; ring A, Y, Z, m and n are each as defined in claim 1; R is selected from H or y is selected from a real number from 1 to 2. An antibody-drug conjugate of Formula X or a pharmaceutically acceptable salt thereof: wherein, Ab is an antibody unit; y is selected from a real number from 1 to 2; R is selected from H or ring A, Y, m and n are each as defined in claim 1; Z' is a linker unit formed by reacting Z as defined in claim 1 with a reactive group; L is a linker unit; D is a drug unit. The disaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1, or the glycoengineered antibody according to claim 2, or the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein the ring A is selected from phenylene, 4-10 membered heterocyclylene, or C3-C 10 cycloalkylene, said phenylene, 4-10 membered heterocyclylene, or C3-C 10 cycloalkylene is optionally substituted with one or more R a each R a is independently selected from C1-C3 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, halogen, CN, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl), N(C1-C3 alkyl)2, C(=O)NH2, or COOH; or said ring A is selected from The disaccharide compound or a pharmaceutically acceptable salt thereof according to claim 1 or 4, or the glycoengineered antibody according to claim 2 or 4, or the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3 or 4, wherein Y is selected from the group consisting of C1-C6 alkylene, C=0, S(=0)2, C(=0)NH, or any combination thereof, said b-terminus is independently covalently linked to Z. The disaccharide compound of any one of claims 1, 4-5 or a pharmaceutically acceptable salt thereof, or the glycoengineered antibody of any one of claims 2, 4-5, or the antibody-drug conjugate of any one of claims 3-5 or a pharmaceutically acceptable salt thereof, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The disaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1, 4-6, or the glycoengineered antibody according to any one of claims 2, 4-6, wherein each Z is independently selected from The disaccharide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1, 4-7, or the glycoengineered antibody according to any one of claims 2, 4-7, wherein the structural unit selected from the group consisting of The disaccharide compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the disaccharide compound or pharmaceutically acceptable salt thereof is selected from the following disaccharide compounds or pharmaceutically acceptable salts thereof: The glycoengineered antibody of any one of claims 2, 4-8 or the antibody-drug conjugate of any one of claims 3-6 or a pharmaceutically acceptable salt thereof, wherein the antibody unit Ab is an antibody or an antigen-binding fragment that can specifically bind to a target antigen. The glycoengineered antibody of any one of claims 2, 4-8, 10 or the antibody-drug conjugate of any one of claims 3-6, 10 or a pharmaceutically acceptable salt thereof, wherein y is selected from 1 or 2. The glycoengineered antibody according to claim 2 is selected from the group consisting of: The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, wherein the antibody-drug conjugate or pharmaceutically acceptable salt thereof is selected from the antibody-drug conjugate or pharmaceutically acceptable salt thereof represented by the following Formula X-1: The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13, wherein each linker unit Z' is independently selected from wherein the c-terminus is covalently linked to Y. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-14, wherein the linker unit L is selected from wherein: L a selected from a chemical bond, j1 is an integer from 0 to 20; L b is selected from a chemical bond, (W-CH2CH2) j2 -(OCH2CH2) j2 *, (W-CH2CH2) j2 -(OCH2CH2) j2 -C(=O)*, (W-CH2CH2) j2 -(OCH2CH2) j2 -NHC(=O)*, W is selected from a chemical bond or NH, each j2 is independently selected from an integer from 0 to 20, the * end is connected to L c ; L c a chemical bond or a peptide residue consisting of 2 to 7 amino acid residues, said amino acid residues being residues formed from phenylalanine, alanine, proline, isoleucine, glycine, valine, lysine, citrulline, serine, glutamic acid or aspartic acid, said amino acid residues being optionally substituted by one or more R c1 substituents, said R c1 substituents being selected from L d selected from wherein the d-terminus is covalently linked to a drug unit D. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein the L a is selected from a chemical bond or The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein L b is selected from a chemical bond, (NHCH2CH2) j2 -(OCH2CH2) j2 -NHC(=O)*, (NHCH2CH2) j2 -(OCH2CH2) j2 -C(=O)*, each j2 is independently selected from an integer of 0 to 10, and the * end is connected to L c . The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein L c is selected from a chemical bond or a peptide residue consisting of 2, 3, 4, or 5 amino acid residues, said amino acid residues being residues formed from phenylalanine, alanine, glycine, valine, or citrulline, said amino acid residues being optionally substituted with one or more R c1 , said R c1 is selected from The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein L c is selected from a chemical bond, wherein the c-terminus is linked to L d connection. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 15, wherein the linker unit L is selected from wherein the d-terminus is covalently linked to a drug unit D. The antibody-drug conjugate of any one of claims 3-6, 10-11, 13-20 or a pharmaceutically acceptable salt thereof, wherein the drug unit D is selected from a cytotoxic drug. The antibody-drug conjugate of claim 21 or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug is selected from a microtubulin inhibitor comprising a dolastatin, an auristatin, a maytansine, a tubulysin, and a cryptomycin drug, a DNA damaging agent comprising a PBD, a duocarmycin, and a calicheamicin drug, and a topoisomerase inhibitor comprising a camptothecin drug; or the cytotoxic drug is selected from a topoisomerase I inhibitor, an auristatin, or a PBD drug; or the cytotoxic drug is selected from The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-22, wherein the structural unit L-D is selected from one of the following structures: The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-23, wherein the antibody-drug conjugate or pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugates or pharmaceutically acceptable salts thereof: The antibody-drug conjugate of any one of claims 3-6, 10-11, 13-24 or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the group consisting of the antibody-drug conjugates or a pharmaceutically acceptable salt thereof shown in the following table: Group 1 (ADC-1) and / or Group 2 (ADC-2) and / or Group 3 (ADC-3) and / or Group 4 (ADC-4) and / or Group 5 (ADC-5) and / or Group 6 (ADC-6) and / or Group 7 (ADC-7) and / or Group 8 (ADC-8) and / or Group 9 (ADC-9) and / or Group 10 (ADC-10) and / or and / or and / or Group 11 (ADC-11) and / or and / or and / or Group 12 (ADC-12) and / or and / or and / or Group 13 (ADC-13) and / or and / or and / or Group 14 (ADC-14) and / or and / or and / or Group 15 (ADC-15) and / or and / or and / or Group 16 (ADC-16) and / or and / or and / or Group 17 (ADC-17) and / or Group 18 (ADC-18) and / or Group 19 (ADC-19) and / or A method of preparing an antibody-drug conjugate as described in any one of claims 3-6, 10-11, 13-25, the method of preparation comprising steps 1, step 2, and step 3: Step 1: mixing an antibody and a glycosidase in a buffer to react, and isolating and purifying to obtain a deglycosylated antibody Ab-GlcNAc(Fucα1,6); Step 2: reacting, in a buffer, the deglycosylated antibody Ab-GlcNAc(Fucα1,6), the disaccharide compound of Formula I according to claim 1 and a glycosidase to obtain a glycoengineered antibody of Formula V by separation and purification; Preferably, the buffer in step 1 and / or step 2 is a PBS buffer; Preferably, the reaction temperature in step 1 and / or step 2 is 10-40°C, more preferably 20-30°C; Preferably, the separation and purification method in step 1 and / or step 2 is separation and purification by protein column; Preferably, the glycosidase in step 1 and / or step 2 is at least one selected from the group consisting of Endo-S, Endo-S2, Endo-A, Endo-D, Endo-M, Endo-H, Endo-F2, Endo-F3, Endo-CC1, Endo-CC2, Endo-Om, Endo-CE, Endo-BH, Endo-Sd, Endo-Se and Endo-Rp; More preferably, the glycosidase in step 1 and / or step 2 is Endo-S2 (Endoglycosidase-S2, UniProt ID: T1WGN1); More preferably, the glycosidase in step 1 and / or step 2 is Streptococcus pyogenes endoglycosidase Endo-S2 (38-819 aa, from UniProt ID: T1WGN1) or Streptococcus pyogenes endoglycosidase Endo-S2 (38-843 aa, from UniProt ID: T1WGN1). Step 3: reacting the glycoengineered antibody of Formula V with X'-L-D, wherein X'-L-D is selected from the group consisting of H2N-L-D, wherein L and D are linker unit and drug unit, respectively; Preferably, said X'-L-D in step 3 is selected from H2N-L-D or A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-25, and a pharmaceutically acceptable excipient. A method for treating a tumor in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-25, or the pharmaceutical composition according to claim 27. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-25, or the pharmaceutical composition according to claim 27 in the manufacture of a medicament for treating a tumor. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-25, or the pharmaceutical composition according to claim 27 in the treatment of a tumor. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 3-6, 10-11, 13-25, or the pharmaceutical composition according to claim 27 for use in the treatment of a tumor. Use of a disaccharide compound of Formula I or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1, 4-9 in the manufacture of a glycoengineered antibody as claimed in any one of claims 2, 4-8, 10-12 or an antibody-drug conjugate as claimed in any one of claims 3-6, 10-11, 13-25.

Images