Antiviral conjugate and preparation method therefor

Abstract

Disclosed are a long-acting and / or broad-spectrum antiviral conjugate, a preparation method therefor, a composition comprising same, and a method for preventing or inhibiting or treating a viral infection. The viral infection may be, for example, a viral infection caused by influenza virus A, influenza virus B, influenza virus C, or avian influenza virus, etc. Specifically, the conjugate comprises a trimer of a moiety that inhibits influenza virus neuraminidase (e.g., zanamivir, peramivir, oseltamivir, or an analog thereof), and an Fc monomer, an Fc domain, an Fc-binding peptide, albumin, or an albumin-binding peptide. Also provided is an intermediate that can be used to synthesize the conjugate. The intermediate can avoid the use of a copper catalyst during the conjugation process, and avoid oxidation of the Fc protein, thereby reducing the immunogenicity of the conjugate and improving the safety thereof.

Description

An antiviral conjugate and its preparation method Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of drug conjugates, and provides a series of antibody-drug conjugates that can prevent and / or inhibit viral growth or treat viral infections. Background Technology

[0002] Viruses are a significant threat to human health, especially influenza viruses. Influenza is an acute respiratory infection caused by a virus. Based on differences in their core proteins, influenza viruses can be classified into four types: influenza A, influenza B, influenza C, and influenza D. Types A and B influenza viruses can cause seasonal influenza. Compared to influenza B viruses, influenza A viruses have more hosts in nature, are more prone to mutation or reassortment, and can spread rapidly among populations, making them the main source of large-scale influenza outbreaks. Since the late 19th century, a major pandemic of a new influenza virus strain has occurred every 10 to 50 years, posing a serious threat to human health.

[0003] Statistics show that 5% to 15% of the population is infected with influenza each year, resulting in 3 to 5 million severe cases and 650,000 deaths.

[0004] In response to the threat of influenza, a series of antiviral treatments have been developed. For example, neuraminidase inhibitors (NAIs) are first-line treatments for influenza virus infection. They selectively inhibit the activity of neuraminidase on the viral envelope by competing with sialic acid, the natural substrate of neuraminidase, thus blocking the enzyme's active site and preventing the viral particles from detaching from infected host cells, inhibiting viral spread between host cells, and reducing viral replication in the body. Currently approved NAIs include oseltamivir, zanamivir, peramivir, and laninamivir. However, NAIs have drawbacks such as a narrow spectrum of antiviral activity, the need for repeated administration, and the occasional occurrence of viral resistance.

[0005] Chinese patent CN113194983A discloses a conjugate for treating viral infections. This conjugate combines a monomer or dimer that inhibits viral neuraminidase with Fc monomers, Fc domain monomers, Fc-binding peptides, albumin, or albumin-binding peptides, resulting in enhanced antiviral activity and a longer duration of antiviral action. However, this patent uses a copper catalyst in the conjugate synthesis process, which can easily cause Fc protein oxidation, posing risks of reduced efficacy and medication safety.

[0006] Therefore, the development of antiviral products with a broad spectrum and long-lasting effect against influenza viruses, especially influenza viruses, remains a very urgent need. Summary of the Invention

[0007] This invention aims to provide a broad-spectrum and / or long-acting antiviral product, particularly against influenza virus, which can broadly inhibit viral infections caused by, for example, influenza A, influenza B, and influenza C, or other viruses, and / or has a longer-lasting antiviral effect. The inventors have achieved simultaneous inhibition of viral infections caused by influenza A, influenza B, and influenza C, or other viruses, and / or a longer-lasting inhibitory effect by conjugating a trimer containing a portion inhibiting influenza virus neuraminidase (e.g., zanamivir, peramivir, oseltamivir, or analogues thereof) to an Fc monomer, an Fc domain, an Fc-binding peptide, albumin, or an albumin-binding peptide to form an antibody-drug conjugate. Furthermore, the conjugate of this invention avoids Fc protein oxidation during preparation, thereby reducing the immunogenicity of the conjugate and improving its safety.

[0008] In one aspect, the present invention relates to antibody-drug conjugates as shown in formula (I-1), pharmaceutically acceptable salts thereof, prodrugs, stereoisomers, tautomers, hydrates, solvates, crystal forms, isotope labels, or metabolite forms thereof:

[0009] Where A1, A2, and A3 may be the same or different and each can be independently selected from formula (AI), formula (A-II), formula (A-III), or formula (A-IV):

[0010] Among them, R 1 Selected from -OH, -NH2, -NHC(=NH)NH2 or -NHC(=NH)NHR a ;

[0011] R 2 Selected from -COOH, -P(=O)(OH)2, or -SO3H;

[0012] R 3 Selected from -COCH3, -COCF3, or -SO2CH3;

[0013] R 5 Selected from hydrogen or C1-C6 alkyl groups;

[0014] L3 can be attached to any possible site in compounds of formula (A-IV);

[0015] X is selected from -O- or -S-;

[0016] Y is selected from -O-, -S-, -NR b -、-O(C=O)NR b -、-O(C=S)NR b -, -O(C=O)O-, -O(C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR b -, -NH(C=NH)NR b -, -NH(C=S)NR b -, -NH(C=S)-, -OCH2(C=O)NR b -,-NH(SO2)-,-NH(SO2)NR b -、-OR b -、-NHR b -or-SR b -;

[0017] R a Selected from

[0018] R b Selected from H, C1-C 20 Alkyl, C3-C 20 cycloalkyl, C3-C 20 Heterocyclic alkyl, C6-C 12 Aryl or C5-C 12 Mixed aromatics;

[0019] L3 is selected from

[0020] Among them, L a Selected from -(CH2) y -,or y is selected from integers from 1 to 10;

[0021] R 4 Selected from hydrogen or C1-C6 alkyl groups;

[0022] L2 is selected from Where x is selected from 1, 2, 3, 4 or 5;

[0023] Q is selected from:

[0024] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatics;

[0025] L1 is selected from z is selected from 1, 2, 3, 4, 5, 6;

[0026] E includes Fc domain monomers, Fc domain dimers, albumin, albumin-binding peptides, or Fc-binding peptides.

[0027] n is selected from 1 or 2;

[0028] T is an integer from 1 to 20.

[0029] In one aspect, the present invention provides an antibody-drug conjugate as shown in formula (I-2), a pharmaceutically acceptable salt thereof, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystal form, an isotope label, or a metabolite thereof:

[0030] Where A1 and A2 may be the same or different and are each independently selected from equation (AI), equation (A-II), equation (A-III), and equation (A-IV):

[0031] Among them, R 1 Selected from -OH, -NH2, -NHC(=NH)NH2 or -NHC(=NH)NHR a ;

[0032] R 2 Selected from -COOH, -P(=O)(OH)2, or -SO3H;

[0033] R 3 Selected from -COCH3, -COCF3, or -SO2CH3;

[0034] R 5 Selected from hydrogen or C1-C6 alkyl groups;

[0035] L3 can be attached to any possible site in compounds of formula (A-IV);

[0036] X is selected from -O- or -S-;

[0037] Y is selected from -O-, -S-, -NR b -、-O(C=O)NR b -、-O(C=S)NR b-, -O(C=O)O-, -O(C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR b -, -NH(C=NH)NR b -, -NH(C=S)NR b -, -NH(C=S)-, -OCH2(C=O)NR b -,-NH(SO2)-,-NH(SO2)NR b -、-OR b -、-NHR b -or-SR b -;

[0038] R a Selected from

[0039] R b Selected from H, C1-C 20 Alkyl, C3-C 20 cycloalkyl, C3-C 20 Heterocyclic alkyl, C6-C 12 Aryl or C5-C 12 Mixed aromatics;

[0040] L3 is selected from

[0041] Among them, L a Selected from -(CH2) y -,or y is selected from 1, 2, 3, 4, and 5;

[0042] R 4 Selected from hydrogen or C1-C6 alkyl groups;

[0043] L2 is selected from Where x is selected from 1, 2, 3, 4 or 5;

[0044] Q is selected from:

[0045] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C12 heteroaryl;

[0046] L1 is selected from z is selected from 1, 2, 3, 4, 5, 6;

[0047] E includes Fc domain monomers, Fc domain dimers, albumin, albumin-binding peptides, or Fc-binding peptides.

[0048] n is selected from 1 or 2;

[0049] T is an integer from 1 to 20.

[0050] In one aspect, the present invention provides a pharmaceutical composition comprising a conjugate of Formula I-1 or Formula I-2, a pharmaceutically acceptable salt thereof, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystal form, an isotope label, or a metabolite thereof, and a pharmaceutically acceptable excipient.

[0051] In one aspect, the present invention provides a method for preventing and / or inhibiting or treating a subject who has or is presumed to have a viral infection, the method comprising administering to the subject an effective amount of a conjugate or composition of formula I-1 or I-2.

[0052] In one aspect, the present invention relates to a method for preparing a coupling of formula I-1 or formula I-2, comprising:

[0053] a: The amino group on the lysine residue of Fc protein reacts with the N3-PEG4-TFP small molecule in a neutral buffer. By controlling the molar ratio of Fc to N3-PEG4-TFP during the reaction, Fc-PEG4-N3 intermediates with different DAR values ​​are obtained.

[0054] b: By reacting the azide group on the Fc-PEG4-N3 intermediate with the alkyne group on the corresponding small molecule intermediate (Int 1-9), coupling samples with different DAR values ​​were obtained by controlling the Fc-PEG4-N3 intermediates with different DAR values.

[0055] In one aspect, the present invention provides a combination product comprising: a conjugate of Formula I-1 or Formula I-2, a pharmaceutically acceptable salt thereof, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystal form, an isotope label or a metabolite thereof, and any one or more antiviral substances.

[0056] Terminology Definition

[0057] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are all standard procedures widely used in the relevant fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0058] In cases where the compound name used in this article differs from the chemical structural formula, the chemical structural formula shall prevail.

[0059] As used herein, the term "neuraminidase inhibitor" or "viral neuraminidase inhibitor" refers to a compound capable of reducing the activity of the enzyme influenza virus neuraminidase (e.g., from influenza A, B, or C). The viral neuraminidase inhibitors of the present invention include oseltamivir, zanamivir, peramivir, and lanimivir, and analogues thereof.

[0060] In this invention, "combined product" is generally understood to mean: a product in which the conjugate of Formula I-1 or Formula I-2, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof is used in conjunction with any one or more antiviral substances. "Combined use" can generally be understood as: used together in one device, or used together in their respective independent forms; the "combination sequence" can be: simultaneously, sequentially, or intermittently.

[0061] In this invention, "any one or more antiviral substances" is not particularly limited and is usually a known antiviral substance, such as a known anti-influenza or anti-avian influenza substance.

[0062] As used in this article, the term "IC" 50 "or "EC" 50 "Hydrogen concentration" or "half-maximal inhibitory concentration" indicates the concentration of influenza virus neuraminidase inhibitor required for 50% inhibition in vitro.

[0063] As used herein, the term "viral infection" means the pathogenic growth of a virus (e.g., influenza virus) in a host organism (e.g., a human subject). A viral infection can be any situation in which the presence of a viral population is harming the host body. Therefore, a subject is "undergoing" a viral infection when an excessive viral population is present in or on the subject's body, or when the presence of a viral population is harming the subject's cells or other tissues.

[0064] As used herein, the term "Fc domain monomer" refers to a polypeptide chain comprising at least one hinge domain and second and third antibody constant domains (CH2 and CH3) or a functional fragment thereof (e.g., a fragment capable of (i) dimerizing with another Fc domain monomer to form an Fc domain, and (ii) binding to an Fc receptor). The Fc domain monomer may be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Additionally, the Fc domain monomer may be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). The Fc domain monomer does not include any portion of an immunoglobulin capable of functioning as an antigen-recognition region, such as a variable domain or complementarity-determining region (CDR). The Fc domain monomers in the conjugates described herein may contain one or more alterations to the sequence of the wild-type Fc domain monomer (e.g., substitutions, additions, or deletions of amino acids 1-10, 1-8, 1-6, or 1-4), which alter the interaction between the Fc domain and the Fc receptor. Examples of suitable alterations are known in the art. Unless otherwise specified herein, the amino acid residues in the IgG or Fc domain monomers are numbered according to the EU numbering system for antibodies, also known as the Kabat EU index, as described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0065] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers capable of binding to an Fc receptor. In the wild-type Fc domain, the two Fc domain monomers dimerize through interactions between two CH3 antibody constant domains, and in some embodiments, one or more disulfide bonds are formed between the hinge domains of the two dimerized Fc domain monomers.

[0066] The term "covalent attachment" refers to two parts of a coupling that are connected to each other by a covalent bond, which is formed between two atoms in the two parts of the coupling.

[0067] As used herein, the term "Fc-binding peptide" refers to a polypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) amino acid residues, having an affinity for and function in binding to an Fc domain (such as any Fc domain described herein). Fc-binding peptides can have different origins, such as synthetic, human, mouse, or rat. The Fc-binding peptides of the present invention comprise Fc-binding peptides engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine ​​or lysine residues, which provide sites for conjugation to compounds of the present invention (e.g., conjugation to neuraminidase inhibitor monomers, dimers, or trimers, including by means of a linker). Most preferably, the Fc-binding peptide will contain a single solvent-exposed cysteine ​​or lysine, thus enabling site-specific conjugation of compounds of the present invention. Fc-binding peptides may comprise only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. When included, non-naturally occurring amino acid residues (e.g., side chains of non-naturally occurring amino acid residues) may serve as attachment sites for compounds of the present invention (e.g., neuraminidase inhibitor monomers, dimers, or trimers, including those via linkers). The Fc-binding peptides of the present invention may be linear or cyclic. The Fc-binding peptides of the present invention include any Fc-binding peptide known to those skilled in the art.

[0068] As used herein, the term "albumin" refers to a polypeptide comprising an amino acid sequence corresponding to naturally occurring albumin (e.g., human serum albumin) or a variant thereof (such as an engineered variant of naturally occurring albumin). Variants of albumin include polymorphisms, fragments such as domains and subdomains, and fusion proteins (e.g., albumin having C-terminal or N-terminal fusions such as polypeptide linkers). Preferably, the albumin has the amino acid sequence of human serum albumin (HSA) or a variant or fragment thereof, most preferably a functional variant or fragment thereof. The albumin of the present invention comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO: 70-71. The albumin of the present invention comprises an albumin engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine ​​or lysine residues, which provides sites for conjugation to compounds of the present invention (e.g., conjugation to neuraminidase inhibitor monomers, dimers, or trimers, including by means of a linker). Most preferably, the albumin will contain a single solvent-exposed cysteine ​​or lysine, thus enabling site-specific conjugation of the compounds of the present invention. The albumin may contain only naturally occurring amino acid residues, or may contain one or more non-naturally occurring amino acid residues. When included, non-naturally occurring amino acid residues (e.g., side chains of non-naturally occurring amino acid residues) may serve as attachment sites for compounds of the present invention (e.g., neuraminidase inhibitor monomers, dimers, or trimers, including by means of a linker).

[0069] As used herein, the term "albumin-binding peptide" refers to a polypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) amino acid residues, having an affinity for albumin (such as any albumin described herein) and the ability to bind said albumin. Preferably, the albumin-binding peptide binds to naturally occurring serum albumin, most preferably human serum albumin. Albumin-binding peptides can have different origins, such as synthetic, human, mouse, or rat. The albumin-binding peptides of the present invention include albumin-binding peptides engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine ​​or lysine residues, which provide sites for conjugation to compounds of the present invention (e.g., conjugation to neuraminidase inhibitor monomers, dimers, or trimers, including by means of a linker). Most preferably, the albumin-binding peptide will contain a single solvent-exposed cysteine ​​or lysine, thus enabling the compounds of the present invention to site-specifically conjugate. The albumin-binding peptide may contain only naturally occurring amino acid residues, or it may contain one or more non-naturally occurring amino acid residues. When included, non-naturally occurring amino acid residues (e.g., side chains of non-naturally occurring amino acid residues) may serve as attachment sites for compounds of the present invention (e.g., neuraminidase inhibitor monomers, dimers, or trimers, including by means of linkers). The albumin-binding peptides of the present invention may be linear or cyclic. The albumin-binding peptides of the present invention include any albumin-binding peptide known to those skilled in the art.

[0070] As used herein, "surface-exposed amino acid," such as surface-exposed cysteine ​​or surface-exposed lysine, or "solvent-exposed amino acid," refers to an amino acid that is readily accessible to the solvent surrounding the protein. Surface-exposed amino acids may be naturally occurring or engineered variants of the protein (e.g., substituted or inserted). In some embodiments, surface-exposed amino acids are those whose substitution does not substantially alter the three-dimensional structure of the protein.

[0071] As used herein, the term "connector" refers to the covalent bonds and groups connecting two or more components in a conjugate (e.g., between two neuraminidase inhibitors in the conjugate described herein, between a neuraminidase inhibitor and an Fc domain or albumin in the conjugate described herein, and between a dimer of two neuraminidase inhibitors and an Fc domain or albumin in the conjugate described herein). In some embodiments, the compound described in formula (II-1) or (II-2) is a connector compound. In some embodiments, the conjugate described herein may contain a connector having a trivalent structure (e.g., a trivalent connector). The trivalent connector has three arms, each arm being covalently connected to a component of the conjugate (e.g., a first arm conjugated to a first neuraminidase inhibitor, a second arm conjugated to a second neuraminidase inhibitor, and a third arm conjugated to an Fc domain or albumin). In some embodiments, the conjugate described herein may contain a connector having a divalent structure (e.g., a divalent connector). The bivalent connector has two arms, each of which is covalently connected to a component of the conjugate (e.g., a first arm conjugated to a first neuraminidase inhibitor and a second arm conjugated to a second neuraminidase inhibitor).

[0072] As used herein, the term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched alkane containing the corresponding number of carbon atoms, and specific examples include, but are not limited to: methyl, ethyl, propyl, n-butyl, isobutyl, isopropyl, tert-butyl, n-pentyl, n-hexyl. In this invention, C1-C6 alkyl groups are preferred.

[0073] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond and a corresponding prefix number of carbon atoms. Specific examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, and 1,4-hexadienyl. In this invention, C2-C6 alkenyl groups are preferred.

[0074] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one triple bond and a corresponding prefix number of carbon atoms. Specific examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butyrynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and 1,4-hexadiynyl. In this invention, the preferred C2-C6 alkynyl group is the C2-C4 alkynyl group.

[0075] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine. In this invention, preferred halogens are fluorine, chlorine, and bromine, with fluorine being more preferred.

[0076] As used herein, the term "halogenated" refers to the substitution of hydrogen atoms in a group or compound by one or more halogen atoms, including full halogenation and partial halogenation.

[0077] As used herein, the term "alkoxy" refers to a group formed in an alkyl-O- configuration.

[0078] As used herein, the term "heteroaryl" refers to a monocyclic group having aromaticity with a corresponding number of carbon atoms in a ring that is at least one heteroatom, said heteroatom being a nitrogen atom, an oxygen atom, and / or a sulfur atom. Specific embodiments include, but are not limited to: furanyl, pyrroleyl, thiopheneyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyrazinyl.

[0079] As used herein, the term "bicyclic heteroaryl" refers to a bicyclic group containing the corresponding prefix number of carbon atoms, in which two rings (at least one of which is aromatic) are fused together and at least one ring atom is a heteroatom, wherein the heteroatom is a nitrogen atom, an oxygen atom and / or a sulfur atom, wherein the nitrogen and / or sulfur is optionally oxidized. Specific embodiments include, but are not limited to: benzopyrrole, benzofuran, benzothiophene, benzoxazole, benzoisoxazole, benzimidazole, benzotriazole, indazole, purine, indole, quinoline, pteridine, isoquinoline, quinazoline, quinoxaline, 1,8-naphthidine, 1,5-naphthidine, 9H-purine, 7H-purine, indole, indoline, 2,3-dihydrobenzofuran, 1,2,3,4-tetrahydronaphthalene, 1,2,3,4-tetrahydroquinoline, 3,4-dihydro-1H-benzopyran, 1,4-benzodioxane, benzomorpholine, and 1,2,3,4-tetrahydroquinoxaline.

[0080] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated monocyclic alkyl group that is not aromatic, preferably having 3-6 ring atoms, i.e., 3-6 membered saturated cycloalkyl and 3-6 membered partially saturated cycloalkyl. Specific examples of 3-6 membered saturated cycloalkyl groups include, but are not limited to, cyclopropane (cyclopropyl), cyclobutane (cyclobutyl), cyclopentane (cyclopentyl), and cyclohexane (cyclohexyl). Specific examples of 3-8 membered partially saturated cycloalkyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohex-1,3-dienyl, cyclohex-1,4-dienyl, cycloheptenyl, cycloheptenyl-1,3-dienyl, cycloheptenyl-1,4-dienyl, cycloheptenyl-1,3,5-trienyl, cyclooctenyl, cyclooctyl-1,3-dienyl, cyclooctyl-1,4-dienyl, cyclooctyl-1,5-dienyl, cyclooctyl-1,3,5-trienyl, and cyclooctatetraenyl.

[0081] As used herein, the term "heterocyclic group" refers to a group obtained by removing a hydrogen atom from a saturated or partially saturated monocyclic or fused-ring compound containing at least one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms), the heteroatom being selected from N, O, or S. The term "3-6 membered nitrogen-containing heterocyclic group" refers to a heterocyclic group containing 3, 4, 5, or 6 ring atoms, of which 1, 2, or 3 are nitrogen atoms; optionally, the heterocyclic group also contains 1 or 2 oxygen or sulfur atoms.

[0082] As used herein, the term "optional substitution" refers to having 0, 1, or more substituents, such as 0-25, 0-20, 0-10, or 0-5 substituents. Generally, the optional substituents are non-interfering substituents. "Non-interfering substituents" refers to the ability of the conjugates described herein (e.g., conjugates of any of formula (I-1) or (I-2)) to bind to viral neuraminidase or inhibit the proliferation of influenza virus.

[0083] As used herein, the term “N-protecting group” refers to those groups intended to protect amino groups from unwanted reactions during the synthetic procedure. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 5th edition (John Wiley & Sons, New York, 2014), which is incorporated herein by reference.

[0084] As used in this article, the term "amino acid" refers to both naturally occurring and non-naturally occurring amino acids.

[0085] As used herein, the term “naturally occurring amino acids” means amino acids including Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0086] As used in this article, the term "non-naturally occurring amino acid" means an α-amino acid that is not naturally occurring or found in mammals.

[0087] As used herein, the term "percentage (%) identity" refers to the percentage of amino acid residues in a candidate sequence (e.g., Fc-IgG or a fragment thereof) that are identical to those in a reference sequence after alignment of the sequence, with gaps introduced if necessary to achieve maximum percentage identity (i.e., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences may be ignored for comparative purposes). Alignments used to achieve the determination of percentage identity can be performed in various ways within the skill of the art, such as using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0088] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. Biol. Chem, 243, p3558 (1968).

[0089] As used herein, the term "treatment" or "to be treated" refers to therapeutic treatment of a subject's viral infection (e.g., a viral infection such as influenza). In some embodiments, therapeutic treatment may slow the progression of the viral infection, improve the subject's prognosis, and / or eliminate the infection. In some embodiments, therapeutic treatment of a subject's viral infection may alleviate or improve one or more symptoms or signs associated with the viral infection, reduce the severity of the virus, stabilize (i.e., not worsen) the state of the viral infection, prevent the spread of the viral infection, and / or delay or slow the progression of the viral infection, as compared to the state and / or symptoms of the viral infection in the absence of the therapeutic treatment.

[0090] As used herein, the term "T" or "average of T" refers to the average number of neuraminidase inhibitor monomers, neuraminidase inhibitor dimers, or neuraminidase inhibitor trimers conjugated to the Fc domain or albumin within the conjugate population, and can be from 1 to 20 (e.g., average values ​​of T are 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to 7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5). In some embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0091] As used herein, the term “subject” can refer to a human, a non-human primate or other mammal, such as, but not limited to, dogs, cats, horses, cattle, pigs, turkeys, goats, fish, monkeys, chickens, rats, mice and sheep.

[0092] As used herein, the term “therapeutic effective amount” means the amount that effectively induces the desired effect in a subject or treats a subject suffering from the symptoms or conditions described herein (e.g., viral infection, such as influenza infection), such as a drug dose. It should also be understood herein that “therapeutic effective amount” can be interpreted as the amount administered to give the desired therapeutic and / or preventative effect, at one or more doses or in any dose or route, and / or alone or in combination with other therapeutic agents (e.g., antiviral agents described herein).

[0093] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or pharmaceutical preparation containing at least one active ingredient (e.g., a conjugate of any one of formula (I-1) or (I-2)) and one or more excipients and diluents that enable said active ingredient to be adapted for a method of administration. The pharmaceutical compositions disclosed herein comprise pharmaceutically acceptable components compatible with the conjugates described herein (e.g., conjugates of any one of formula (I-1) or (I-2)).

[0094] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient or diluent in a pharmaceutical composition. For example, a pharmaceutically acceptable carrier may be a medium capable of suspending or dissolving an active conjugate (e.g., a conjugate of either formula (I-1) or (I-2)). A pharmaceutically acceptable carrier must be compatible with the other components of the formulation and harmless to the recipient. In this disclosure, a pharmaceutically acceptable carrier must provide sufficient pharmaceutical stability to the conjugate described herein. The properties of the carrier vary depending on the route of administration. For example, solid carriers are preferred for oral administration; for intravenous administration, aqueous carriers (e.g., WFI and / or buffer solutions) are generally used.

[0095] As used herein, the term "pharmaceuticalally acceptable salt" means a salt of the conjugates described herein (e.g., conjugates of any of formula (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formula (II-1) or (II-2)) that, to the extent of reasonable medical judgment, are suitable for use in the methods described herein without undue toxicity, irritation, and / or anaphylactic reactions. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Pharmaceutical Salts: Properties, Selection, and Use (edited by PHStahl and CGWermuth), Wiley-VCH, 2008. The salt may be prepared on-site during the final isolation and purification of the conjugates or intermediates described herein, or may be prepared separately by reacting the free bases with a suitable organic acid.

[0096] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed by the -COOH group present in the conjugate described herein (e.g., a conjugate of any of formula (I-1) or (I-2)) or intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) and a suitable alcohol, or an ester formed by the -OH group present in the conjugate described herein (e.g., a conjugate of any of formula (I-1) or (I-2)) or intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) and a suitable acid (e.g., a carboxylic acid or an oxy-containing inorganic acid). Suitable ester groups include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, ethyl succinate, stearate, or palmitate. Esters can undergo hydrolysis in the presence of acid or base to yield the corresponding acid or alcohol.

[0097] As used herein, the term "pharmaceutically acceptable amide" means an amide formed by -COOH present in the conjugate (e.g., a conjugate of any of formula (I-1) or (I-2)) or intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) with a suitable amine, or an amide formed by -NH- or -NH2 present in the conjugate (e.g., a conjugate of any of formula (I-1) or (I-2)) or intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) with a suitable acid (e.g., a carboxylic acid, such as acetic acid).

[0098] As used herein, the term "solvate" refers to a substance formed by the association of a conjugate (e.g., a conjugate of any of formula (I-1) or (I-2)) or an intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) with a solvent molecule. The solvent may be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.) or water.

[0099] As used herein, the term "crystal form" refers to the crystal structure of a substance. During crystallization, various factors alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms in the crystal lattice, thus forming different crystal structures. The couplings described herein (e.g., couplings of any of formulas (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formulas (II-1) or (II-2)) may exist in one crystal structure or multiple crystal structures, i.e., they possess "polymorphism." The couplings described herein (e.g., couplings of any of formulas (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formulas (II-1) or (II-2)) may exist in different crystal forms.

[0100] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers primarily include cis-trans isomers and optical isomers. The conjugates described herein (e.g., conjugates of any of formula (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formula (II-1) or (II-2)) may exist in stereoisomer form and therefore encompass all possible stereoisomeric forms, any combination thereof, or any mixture thereof. Examples include a single enantiomer, a single diastereomer, or a mixture of more than one. When the conjugates described herein (e.g., conjugates of any of formula (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formula (II-1) or (II-2)) contain an olefinic double bond, unless otherwise specified, they include cis and trans isomers, and any combination thereof.

[0101] As used herein, the term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom in a molecule to two positions, such as enol and ketone tautomers. The conjugates (e.g., conjugates of any of formula (I-1) or (I-2)) or intermediates (e.g., intermediates of any of formula (II-1) or (II-2)) described herein, if tautomers are present, may exist as a single tautomer or a mixture thereof, preferably in the form of a more stable tautomer predominantly.

[0102] As used herein, the term "isotope label" means that in the conjugates of the present invention, atoms can be replaced by their corresponding isotopes, for example, hydrogen can be replaced by deuterium or tritium.

[0103] As used herein, the term "prodrug" refers to a conjugate (e.g., a conjugate of any of formula (I-1) or (I-2)) or intermediate (e.g., an intermediate of any of formula (II-1) or (II-2)) that can be converted in the body of a subject through reactions such as oxidation, reduction, or hydrolysis. A prodrug may or may not possess the biological activity of a compound of formula (I) (e.g., activity modulating glucose and lipid metabolism, anti-inflammatory activity, antioxidant activity). For example, a compound of formula (I) including a hydroxyl or carboxyl group may be administered in the form of an ester, which is hydrolyzed in vivo to form a hydroxyl or carboxyl compound. Similarly, a compound of formula (I) including an amino group may be acylated, alkylated, or phosphorylated to form a compound such as eicosanoylamino, alanineamino, or neopentanoyloxymethylamino for administration. Further information on the use of prodrugs can be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (Th Higuchi and W Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. EB Roche, American Pharmaceutical Association).

[0104] The term "drug:antibody ratio" or "DAR" refers to the average number of small molecule drug portions conjugated to antibodies, Fc domains, or albumins described herein (e.g., the average number of small molecule drug monomers, dimers, or trimers). In some embodiments described herein, DAR is represented by "T" (e.g., in formula (I-1) or (I-2)). DAR can also be calculated as the average DAR of a population of molecules (such as a population of Fc domains, antibodies, or albumins). DAR values ​​can affect the efficacy, potency, pharmacokinetics, or toxicity of a drug.

[0105] As used in this article, the term “about” indicates a deviation of up to ±5%.

[0106] Any value provided as a range includes the upper and lower limits, and any value contained within the upper and lower limits.

[0107] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0108] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., a metabolic disease or symptoms associated with a metabolic disease) means an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., a metabolic disease or symptoms associated with a metabolic disease); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0109] In some implementations, A1, A2, and A3 in Formula I-1 may be the same or different and are each independently selected from Formulas (A1), (A-II), (A-III), and (A-IV):

[0110] In some implementations, A1 and A2 may be the same or different and each may be independently selected from formulas (A1), (A-II), (A-III), and (A-IV):

[0111] In some embodiments, L3 can be linked to any possible site of the (A-IV) compound;

[0112] In some implementations, R 1 Selected from -OH, -NH2, or -NHC(=NH)NH2.

[0113] In some implementations, R 1 Selected from -NHC(=NH)NH2.

[0114] In some implementations, R 2 Selected from -COOH.

[0115] In some implementations, R 3 Selected from -COCH3 or -COCF3.

[0116] In some implementations, R 3 Selected from -COCH3.

[0117] In some implementations, R 5 It is selected from hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0118] In some implementations, R 5 It is selected from hydrogen, methyl, ethyl, propyl, isopropyl, or tert-butyl.

[0119] In some implementations, R 5 Selected from hydrogen, methyl, or ethyl.

[0120] In some implementations, R 5 Selected from ethyl.

[0121] In some implementations, X is selected from -O-.

[0122] In some implementations, Y is selected from -O-, -S-, and -NR. b -、-O(C=O)NR b -、-O(C=S)NR b -, -O(C=O)O-, -O(C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR b -, -NH(C=NH)NR b -, -NH(C=S)NR b -, -NH(C=S)-, -OCH2(C=O)NR b -,-NH(SO2)-,-NH(SO2)NR b -、-OR b - or -NHR b -and-SR b -

[0123] In some implementations, R b Selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, or C6-C 10 Aryl.

[0124] In some implementations, Y is selected from -O-, -S-, or -NR. b -;R b Selected from H or C1-C6 alkyl groups.

[0125] In some implementations, Y is selected from -O-, -S-, -NH-, or -N(CH3)-.

[0126] In some implementations, Y is selected from -O-, -S-, or -NH-.

[0127] In some implementations, Y is selected from -O- or -NH-.

[0128] In some implementations, A1, A2, and A3 are each independently selected.

[0129] In some implementations, A1, A2, and A3 are each independently selected.

[0130] In some implementation schemes, A1 and A2 are each independently selected.

[0131] In some implementations, A1, A2, and A3 are all selected from...

[0132] In some implementations, A1, A2, and A3 are all selected from...

[0133] In some implementations, A1 and A2 are both selected from...

[0134] In some implementations, A3 is selected from

[0135] In some implementations, A1, A2, and A3 are all selected from...

[0136] In some implementations, A1 and A2 are both selected from...

[0137] In some implementations, A3 is selected from

[0138] In some implementations, y is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0139] In some implementations, y is selected from 1, 2, 3, 4, 5, 6, 7.

[0140] In some implementations, y is selected from 1, 2, 3, 4, and 5.

[0141] In some implementations, L a Selected from -(CH2)2-, -(CH2)3-, -(CH2)4-, or

[0142] In some implementations, L a Selected from -CH2-, -(CH2)2-, -(CH2)3-, or

[0143] In some implementations, L a Selected from -CH2-, or

[0144] In some implementations, R 4 Selected from hydrogen, methyl, ethyl, propyl, or isopropyl;

[0145] In some implementations, R 4 Selected from hydrogen or methyl;

[0146] In some implementations, L3 is selected from:

[0147] In some implementations, L3 is selected from:

[0148] In some implementations, L3 is selected from

[0149] In some implementations, x is selected from 3, 4, and 5.

[0150] In some implementations, x is selected from 3.

[0151] In some implementations, L2 is selected from

[0152] In some implementations, Q is selected from:

[0153] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl.

[0154] In some implementations, the The benzene ring in the sample is optionally surrounded by one or more elements selected from R c The group is replaced by the group.

[0155] In some implementations, the The cyclooctane moiety in R is optionally occupied by one or more reasonable sites selected from R c The group is replaced by the group.

[0156] In some implementations, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -SO2, -NH2, -NO2, methyl, ethyl, cyclopropyl, or phenyl.

[0157] In some implementations, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, or -I.

[0158] In some implementations, the R c Each is independently selected from hydrogen or -F.

[0159] In some implementations, Q is selected from...

[0160] In some implementations, z is selected from 3, 4, and 5.

[0161] In some implementations, z is selected from 4.

[0162] In some implementations, L1 is selected from

[0163] In some implementations, T is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0164] In some implementations, T is selected from 1, 2, 3, 4, and 5.

[0165] In some implementations, E includes Fc domain monomers and Fc domain dimers.

[0166] In some implementations, n is selected from 1 or 2.

[0167] In some embodiments, E is an Fc domain monomer or dimer, each Fc domain monomer independently having the sequence of any one of SEQ ID NO:1-69.

[0168] In some embodiments, E is an Fc domain monomer or dimer, each Fc domain monomer independently having a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to any of the sequences in SEQ ID NO:1-69.

[0169] In some embodiments, E is an Fc domain monomer or dimer, each Fc domain monomer independently having the sequence of SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:19.

[0170] In some embodiments, E is an Fc domain monomer having a sequence of SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:19.

[0171] In some implementations, E is an Fc domain dimer, each Fc domain monomer independently having the sequence of SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:19.

[0172] In some implementations, E is albumin, albumin-binding peptide, or Fc-binding peptide.

[0173] In some implementations, n is 1.

[0174] In some embodiments, E is albumin having the sequence of any one of SEQ ID NO:70-71.

[0175] In some embodiments, E is a protein having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO:70-71.

[0176] In some embodiments, the coupling agent is selected from:

[0177] intermediate

[0178] In one aspect, the present invention provides an intermediate M for synthesizing an antibody-drug conjugate of any embodiment, a pharmaceutically acceptable salt thereof, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystal form, an isotope label, or a metabolite thereof, wherein the intermediate M is formed by linking a cyclooctyne derivative and a small drug molecule via a linkage bond.

[0179] Cyclooctyne derivatives - linking bonds - small drug molecules (monomers, dimers, or trimers)

[0180] intermediate M

[0181] Among them, cyclooctyne derivatives include cyclooctyne structures or derivatives thereof, wherein the C atom in the cyclooctyne structure or derivatives thereof may optionally be replaced by one or more atoms selected from N and O;

[0182] The small molecule portion of a drug can be in the form of a monomer, dimer, or trimer. Each part of the small molecule monomer, dimer, or trimer is independently selected from formula (A1), (A-II), (A-III), or (A-IV):

[0183] L3 can be linked to any possible site in compounds of formula (A-IV); R 1 R 2 R 3 R 5 X, Y are as defined in any of the implementation schemes;

[0184] Connecting bonds can be single, double, or triple bonds, including carbon-carbon bonds, carbon-oxygen bonds, carbon-nitrogen bonds, sulfur bonds, or other covalent bonds. Atoms in a connecting bond can form a ring structure through covalent bonds.

[0185] In some embodiments, the cyclooctyne derivative is selected from formula (MI), formula (M-II), and formula (M-III).

[0186] G1, G2, and G3 are each independently selected from C3-C8 cycloalkyl, C3-C8 heterocyclic, and C6-C 12 Aryl, or C5-C 12 Mixed aromatics;

[0187] W is selected from carbon atoms and nitrogen atoms;

[0188] L4 is selected from -(C=O)CH2CH2-, -(C=O)CH2CH2CH2-, -(C=O)CH2CH2CH2CH2-, -CH2-O-, -CH2CH2-O-, -CH2CH2CH2-O-, -O-, -NH-, -N(CH3)-, -CH2-NH-, -CH2CH2-NH-,

[0189] Any reasonable site of formulas (MI), (M-II), and (M-III) can be optionally occupied by one or more R c Replace, R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatic compounds.

[0190] In some embodiments, G1, G2, and G3 are each independently selected from C3-C8 cycloalkyl or C6-C8 aryl.

[0191] In some embodiments, G1, G2, and G3 are each independently selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl.

[0192] In some embodiments, G1, G2, and G3 are each independently selected from cyclopropyl or phenyl.

[0193] In some implementations, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, -(C=O)CH2CH2CH2-, -(C=O)CH2CH2CH2CH2-, -CH2CH2-O-, -CH2CH2CH2-O-,

[0194] In some implementations, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, -(C=O)CH2CH2CH2-, -CH2CH2-O-, or

[0195] In some implementations, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, or

[0196] In some implementations, R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl.

[0197] In some implementations, R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -NH2, methyl, or ethyl.

[0198] In some implementations, R c Each is independently selected from hydrogen, -F, -Cl, -Br, or methyl.

[0199] In some implementations, R c Each is independently selected from hydrogen or -F.

[0200] In some embodiments, the cyclooctyne derivative is selected from...

[0201] In some implementation schemes, the various portions of the drug small molecule monomer or dimer or trimer are each independently selected from...

[0202] In some embodiments, each part of the drug small molecule monomer or dimer or trimer is selected from...

[0203] In some embodiments, each part of the drug small molecule trimer is selected from...

[0204] In some embodiments, each part of the drug small molecule dimer is selected from...

[0205] In some implementations, the drug small molecule monomer is selected from...

[0206] In some embodiments, each part of the drug small molecule trimer is selected from...

[0207] In some embodiments, each part of the drug small molecule dimer is selected from...

[0208] In some implementations, the drug small molecule monomer is selected from...

[0209] In some implementations, the connection key is selected from -L2-L3-, where L2 and L3 are as defined in any implementation.

[0210] In some implementations, L2 is selected from

[0211] In some implementations, L3 is selected from:

[0212] In some implementations, -L2-L3- is selected from:

[0213] In some implementations, -L2-L3- is selected from:

[0214] In some implementations, -L2-L3- is selected from:

[0215] In one aspect, the present invention provides an intermediate, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite thereof, as shown in formula (II-1) for synthesizing an antibody-drug conjugate of any embodiment.

[0216] Wherein, T, A1, A2, A3, L3, and L2 are as defined in any of the implementation schemes;

[0217] Q' is selected from:

[0218] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatic compounds.

[0219] In one aspect, the present invention provides an intermediate, pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite thereof for synthesizing an antibody-drug conjugate of any embodiment, as shown in formula (II-2).

[0220] Wherein, T, A1, A2, L3, and L2 are as defined in any of the implementation schemes;

[0221] Q' is selected from:

[0222] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatic compounds.

[0223] In some implementations, Q' is selected from:

[0224] The Any reasonable site is optionally selected by one or more sites chosen from R c The group replaced by R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl.

[0225] In some implementations, the The benzene ring in the sample is optionally surrounded by one or more elements selected from Rc The group is replaced by the group.

[0226] In some implementations, the The α-position of the alkyne bond in the α-position is optionally occupied by one or more bonds selected from R. c The group is replaced by the group.

[0227] In some implementations, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -SO2, -NH2, -NO2, methyl, ethyl, cyclopropyl, or phenyl.

[0228] In some implementations, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, or -I.

[0229] In some implementations, the R c Each is independently selected from hydrogen or -F.

[0230] In some implementations, Q' is selected from:

[0231] In some implementations, Q' is selected from:

[0232] In some implementations, Q' is selected from...

[0233] In some implementations, Q' is selected from:

[0234] In some implementations, Q' is selected from:

[0235] In some implementations, the intermediate is selected from:

[0236] In some implementations, the viral infection may be caused by influenza virus, parainfluenza virus, or avian influenza virus.

[0237] In some implementations, the viral infection is influenza A, B, or C virus or parainfluenza virus.

[0238] In some implementations, the subject is immunocompromised.

[0239] In some implementations, the subject has been diagnosed with humoral immune deficiency, T-cell deficiency, neutropenia, asplenia, or complement deficiency.

[0240] In some implementations, the subject is receiving or is about to receive immunosuppressive therapy.

[0241] In some implementations, the subject has been diagnosed with a disease that causes immunosuppression.

[0242] In some implementations, the disease is cancer or acquired immunodeficiency syndrome.

[0243] In some implementations, the cancer is leukemia, lymphoma, or multiple myeloma.

[0244] In some implementations, the subject has already received or is about to receive a hematopoietic stem cell transplant.

[0245] In some implementations, the subject has received or is about to receive an organ transplant.

[0246] In some embodiments, the conjugate or composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, via the surface, intratumorally, via the peritoneum, subcutaneously, subconjunctivally, intracysticly, via the mucosa, intraperitoneally, intraumbilically, intraocularly, orally, via local, by inhalation, by injection, or by infusion.

[0247] In some embodiments, the conjugate of the present invention may be administered to a subject in combination with a second therapeutic agent.

[0248] In some implementations, the second therapeutic agent is an antiviral agent.

[0249] In some embodiments, the antiviral agent is selected from oseltamivir, zanamivir, peramivir, lanimivir, amantadine, rimantadine, or mabaloxavir.

[0250] In some implementations, the second therapeutic agent is an antiviral vaccine.

[0251] In some implementations, the antiviral vaccine induces an immune response against influenza A, B, or C virus or parainfluenza virus in the subject.

[0252] In some embodiments, in either formula (I-1) or (I-2), the Fc domain-containing composition may replace the Fc domain, and the Fc domain-containing monomer composition may replace the Fc domain monomer. In either formula (I-1) or (I-2) described herein, when n is 1, E is an Fc domain-containing monomer composition. In either formula (I-1) or (I-2) described herein, when n is 2, E is an Fc domain-containing composition.

[0253] In some embodiments, the Fc domain-containing composition is an antibody or an antibody fragment. Antibodies may include any form of immunoglobulin, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein backbones with antibody-like properties, and any other immune-binding motif known in the art, including antibody fragments (e.g., Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv, or SMIP). The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art. Antibody fragments may include binding portions comprising portions derived from or significantly homologous to the antibody, such as the antigenic determinant region of the antibody. Exemplary antibody fragments include Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, scFv, and SMIP.

[0254] In some embodiments, the antibody or antibody fragment is a human, mouse, camelid (e.g., llama, alpaca, or camel), goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat antibody or antibody fragment. In certain embodiments, the antibody is IgG, IgA, IgD, IgE, IgM, or an endosome. In some embodiments, the antibody fragment includes scFv, sdAb, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, Feb, or SMIP.

[0255] In some embodiments, the Fc domain-containing composition (e.g., an antibody or antibody fragment) imparts binding specificity to one or more targets (e.g., an antigen).

[0256] In some embodiments, the one or more targets (e.g., antigens) bound by the Fc domain-containing composition (e.g., antibody or antibody fragment) are viral (e.g., influenza) proteins, such as neuraminidase or hemagglutinin. In some embodiments, the antibody or antibody fragment recognizes a viral surface antigen. In some embodiments, the antibody or antibody fragment targets hemagglutinin.

[0257] In some embodiments, the Fc domain-containing composition (e.g., an antibody or antibody fragment) comprises one or more single-domain antibodies (sdAbs).

[0258] In some embodiments, the Fc domain-containing composition is a multi-domain antibody (MDAb) or multi-domain antibody fragment comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) sdAbs.

[0259] In one aspect, the invention includes conjugates described by either formula (I-1) or (I-2), wherein E is an antibody or antibody fragment. In a preferred embodiment in which E is an antibody or antibody fragment, n is 1. In some embodiments, the antibody or antibody fragment includes any antibody or antibody fragment described herein.

[0260] In some embodiments, the Fc domain monomer in the conjugate as described herein may have a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any of the SEQ ID NOs:1-69 shown below.

[0261] SEQ ID NO:1: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics)

[0262] SEQ ID NO:2: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics)

[0263] SEQ ID NO:3: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics)

[0264] SEQ ID NO:4: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), allotype G1m (fa) (bold italics)

[0265] SEQ ID NO:5: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics)

[0266] SEQ ID NO:6: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), allotype G1m (fa) (bold italics)

[0267] SEQ ID NO:7: Mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m (fa) (bold italics)

[0268] SEQ ID NO:8: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), and allotype G1m(fa) (bold italic).

[0269] SEQ ID NO:9: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined) and allotype G1m(fa) (bold italic).

[0270] SEQ ID NO:10: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0271] SEQ ID NO:11: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0272] SEQ ID NO:12: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0273] SEQ ID NO:13: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0274] SEQ ID NO:14: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0275] SEQ ID NO:15: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0276] SEQ ID NO:16: Mature human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0277] SEQ ID NO:17: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), YTE triple mutation (bold and underlined), and allotype G1m(fa) (bold italic).

[0278] SEQ ID NO:18: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), YTE triple mutation (bold and underlined), and allotype G1m(fa) (bold italic).

[0279] SEQ ID NO:19: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0280] SEQ ID NO:20: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0281] SEQ ID NO:21: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0282] SEQ ID NO:22: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0283] SEQ ID NO:23: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0284] SEQ ID NO:24: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0285] SEQ ID NO:25: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), allotype G1m(fa) (bold italic)

[0286] SEQ ID NO:26: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), LS double mutation (bold and underlined), and allotype G1m(fa) (bold italic).

[0287] SEQ ID NO:27: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), LS double mutation (bold and underlined), and allotype G1m(fa) (bold italic).

[0288] SEQ ID NO:28: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0289] SEQ ID NO:29: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0290] SEQ ID NO:30: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0291] SEQ ID NO:31: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0292] SEQ ID NO:32: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0293] SEQ ID NO:33: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0294] SEQ ID NO:34: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0295] SEQ ID NO:35: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), LS double mutation (bold and underlined), DE double mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0296] SEQ ID NO:36: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), LS double mutation (bold and underlined), DE double mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0297] SEQ ID NO:37: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0298] SEQ ID NO:38: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0299] SEQ ID NO:39: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0300] SEQ ID NO:40: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0301] SEQ ID NO:41: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0302] SEQ ID NO:42: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0303] SEQ ID NO:43: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0304] SEQ ID NO:44: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0305] SEQ ID NO:45: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), LS double mutation (bold and underlined), VLPLL quintuplet mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0306] SEQ ID NO:46: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0307] SEQ ID NO:47: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0308] SEQ ID NO:48: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0309] SEQ ID NO:49: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0310] SEQ ID NO:50: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0311] SEQ ID NO:51: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0312] SEQ ID NO:52: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0313] SEQ ID NO:53: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0314] SEQ ID NO:54: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), LS double mutation (bold and underlined), ALE triple mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0315] SEQ ID NO:55: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0316] SEQ ID NO:56: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0317] SEQ ID NO:57: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0318] SEQ ID NO:58: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0319] SEQ ID NO:59: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0320] SEQ ID NO:60: Mature human IgG1 Fc, Cys to Ser substitution (#), Asn to Asp substitution (*), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0321] SEQ ID NO:61: Mature human IgG1 Fc, Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), allotype G1m(fa) (bold italic)

[0322] SEQ ID NO:62: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), Cys to Ser substitution (#), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0323] SEQ ID NO:63: Mature human IgG1 Fc, with added N-terminal ISAMVRS amino acid residues (underlined), LS double mutation (bold and underlined), ADLE quadruple mutation (italic and underlined), and allotype G1m(fa) (bold italic).

[0324] SEQ ID NO:64: Mature human IgG2 Fc, allotype G2m (bold italic)

[0325] SEQ ID NO:65: Mature human IgG2 Fc, LS double mutation (bold and underlined), allotype G2m (bold italic)

[0326] SEQ ID NO:66: Mature human IgG2 Fc, YTE triple mutation (bold and underlined), allotype G2m (bold italic)

[0327] SEQ ID NO:67: Mature human IgG4 Fc, Ser to Pro substitution (#), allotype G4m(a) (bold italics)

[0328] SEQ ID NO:68: Mature human IgG4 Fc, Ser to Pro substitution (#), LS double mutation (bold and underlined), allotype G4m(a) (bold italic)

[0329] SEQ ID NO:69: Mature human IgG4 Fc, Ser to Pro substitution (#), YTE triple mutation (bold and underlined), allotype G4m(a) (bold italic)

[0330] In some embodiments, the albumin in the conjugate as described herein comprises a protein having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO:70-71. In some embodiments, the albumin in the conjugate as described herein may have the sequence of any of SEQ ID NO:70-71 shown below.

[0331] SEQ ID NO:70 (Human serum albumin (HSA), variant 1)

[0332] SEQ ID NO:71 (Human serum albumin (HSA), variant 2)

[0333] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:1.

[0334] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:2.

[0335] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:3.

[0336] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:4.

[0337] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:5.

[0338] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:6.

[0339] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:7.

[0340] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:8.

[0341] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:9.

[0342] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:10.

[0343] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:11.

[0344] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:12.

[0345] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:13.

[0346] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:14.

[0347] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:15. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:15.

[0348] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:16.

[0349] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:17.

[0350] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:18. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:18.

[0351] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:19. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:19.

[0352] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:20.

[0353] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:21.

[0354] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:22.

[0355] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:23.

[0356] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:24.

[0357] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:25.

[0358] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:26.

[0359] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:27.

[0360] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:28. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:28.

[0361] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:29.

[0362] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:30.

[0363] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:31. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:31.

[0364] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:32. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:32.

[0365] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:33. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:33.

[0366] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:34.

[0367] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:35.

[0368] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:36.

[0369] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:37.

[0370] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:38. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:38.

[0371] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:39.

[0372] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:40. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:40.

[0373] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:41.

[0374] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:42. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:42.

[0375] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:43. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:43.

[0376] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:44. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:45.

[0377] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:46.

[0378] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:47. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:47.

[0379] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:48.

[0380] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:49. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:49.

[0381] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:50. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:50.

[0382] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:51.

[0383] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:52.

[0384] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:53. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:53.

[0385] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:54.

[0386] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:55.

[0387] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:56. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:56.

[0388] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:57.

[0389] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:58.

[0390] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:59. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:59.

[0391] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:60. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:60.

[0392] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:61. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:61.

[0393] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:62. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:62.

[0394] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:63. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:63.

[0395] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:64. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:64.

[0396] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:65. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:65.

[0397] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:66. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:66.

[0398] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:67. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:67.

[0399] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:68. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:68.

[0400] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:69. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:69.

[0401] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:70. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:70.

[0402] In some embodiments of any aspect described herein, E (e.g., each E) comprises the amino acid sequence of SEQ ID NO:71. In some embodiments, E comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO:71.

[0403] In some embodiments where E includes an Fc domain monomer of any aspect described herein, the Fc domain monomer (e.g., an Fc domain monomer having the sequence of any of SEQ ID NO: 1-69) includes a triple mutation corresponding to M252Y / S254T / T256E (YTE). As used herein, it should be understood that "corresponding to" a specific amino acid residue (e.g., of a specific SEQ ID NO.) includes any amino acid residue that a person skilled in the art would understand to be compared to (e.g., of the specific sequence) said specific residue. For example, any of SEQ ID NO: 1-69 may be mutated to include a YTE mutation.

[0404] In some embodiments where E includes an Fc domain monomer of any aspect described herein, the Fc domain monomer (e.g., an Fc domain monomer having the sequence of any of SEQ ID NO: 1-69) includes a double mutant corresponding to M428L / N434S(LS). As used herein, it should be understood that "corresponding to" (e.g., a specific SEQ ID NO.) a specific amino acid residue includes any amino acid residue that a person skilled in the art would understand to be compared with (e.g., the specific residue of the specific sequence). For example, any of SEQ ID NO: 1-69 may be mutated to include an LS mutation.

[0405] In some embodiments where E includes an Fc domain monomer of any aspect described herein, the Fc domain monomer (e.g., an Fc domain monomer having the sequence of any of SEQ ID NO: 1-69) includes a mutant corresponding to N434H. As used herein, it should be understood that "corresponding to" (e.g., a specific SEQ ID NO.) a specific amino acid residue includes any amino acid residue that a person skilled in the art would understand to be compared with (e.g., the specific residue of the specific sequence). For example, any of SEQ ID NO: 1-69 may be mutated to include the N434H mutation.

[0406] In some embodiments where E includes an Fc domain monomer of any aspect described herein, the Fc domain monomer (e.g., an Fc domain monomer having the sequence of any of SEQ ID NO: 1-69) includes a mutant corresponding to C220S. As used herein, it should be understood that "corresponding to" (e.g., of a specific SEQ ID NO.) a specific amino acid residue includes any amino acid residue that a person skilled in the art would understand to be compared to (e.g., of the specific sequence) said specific residue. For example, any of SEQ ID NO: 1-69 may be mutated to include a C220S mutation.

[0407] In some embodiments where E includes an Fc domain monomer in any aspect described herein, the Fc domain monomer (e.g., an Fc domain monomer having a sequence of any of SEQ ID NO: 1-69) is a fragment of the Fc domain monomer (e.g., at least 25 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more) or at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60) from SEQ ID NO: 1-69. Fragments of at least 75 (e.g., 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more) consecutive amino acid lengths.

[0408] In some embodiments of any aspect described herein (e.g., conjugates of either formula (I-1) or (I-2), one or more nitrogen atoms of lysine residues exposed on one or more surfaces of E, or one or more sulfur atoms of cysteine ​​residues exposed on one or more surfaces of E, are covalently conjugated to the linker. The linker compound as shown in formula (II-1) or (II-2) forms the conjugates described in formula (I-1) or (I-2) of the present invention via a SPAAC reaction.

[0409] In some embodiments, the conjugate is conjugate 1 or any regional isomer thereof, and the drug:antibody ratio (DAR) (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4 6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0410] In some embodiments, the conjugate is conjugate 2 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0411] In some embodiments, the conjugate is conjugate 3 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0412] In some embodiments, the conjugate is conjugate 4 or any of its regional isomers, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0413] In some embodiments, the conjugate is conjugate 5 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0414] In some embodiments, the conjugate is conjugate 6 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0415] In some embodiments, the conjugate is conjugate 7 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0416] In some embodiments, the conjugate is conjugate 8 or any of its regional isomers, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0417] In some embodiments, the conjugate is conjugate 9 or any regional isomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some implementations, the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0 and 10.0.

[0418] In this paper, couplings are uniformly represented by “AVC + number”, which may be written in different forms such as “AVC-number”, “AVC(number)” or “AVC number”, and all of the above different representations refer to the same molecule (for example, AVC(022), AVC-022 and AVC022 all represent the same molecule). Attached Figure Description

[0419] Figure 1 shows the SEC-HPLC chromatogram of Fc protein.

[0420] Figure 2 shows the chromatogram of N3-PEG4-TFP SEC-HPLC.

[0421] Figure 3 shows the LC-MS spectrum of N3-PEG4-TFP.

[0422] Figure 4 shows the chromatogram of AVC(022) by SEC-HPLC.

[0423] Figure 5 shows the LC-MS spectrum of AVC(022).

[0424] Figure 6 shows the SEC-HPLC chromatogram of AVC(025).

[0425] Figure 7 shows the LC-MS spectrum of AVC(025).

[0426] Figure 8 shows the NA binding activity of AVC(022) and AVC(025). EC of AVC(022)(DAR=4.1) molecules 50 The value was 13.97 ng / mL. EC50 of AVC(025)(DAR=4.2) molecule 50 The value was 20.39 ng / mL.

[0427] Figure 9 shows the binding activity of AVC(025) with NA at different DAR values. Among them: EC50 of AVC(025)(DAR=2.1) molecule 50 The value was 21.21 ng / mL; EC50 of AVC(025)(DAR=4.2) molecule 50 The value was 17.22 ng / mL; EC50 of AVC(025)(DAR=6.2) molecule 50 The value was 16.40 ng / mL; EC50 of AVC(025)(DAR=9.1) molecule 50 The value was 16.29 ng / mL; EC50 of AVC(025) (DAR = 10.9) molecule 50 The value was 16.74 ng / mL.

[0428] Figure 10 shows the ADCC activities of AVC(022) and AVC(025). EC of AVC(022)(DAR=4.1) molecules 50 The value was 0.03 μg / mL. EC50 of AVC(025)(DAR=4.2) molecule 50 The value was 0.04 μg / mL.

[0429] Figure 11 shows the ADCC activity of AVC(025) with different DAR values. EC50 of AVC(025)(DAR=4.2) molecule 50 The value was 0.04 μg / mL. EC50 of AVC(025) (DAR = 10.9) molecule 50 The value was 0.54 μg / mL.

[0430] Figure 12 shows the inhibitory effect of AVC(022) on the activity of NA enzyme.

[0431] Figure 13 shows the inhibitory effect of AVC(022) on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain, where EC 50 = 4.718 ng / mL.

[0432] Figure 14 shows the inhibitory effect of AVC(022) on the A / Victoria / 2570 / 2019(H1N1) strain, where EC 50 = 92.76 ng / mL.

[0433] Figure 15 shows the inhibitory effect of AVC(022) on the A / Darwin / 9 / 2021(H3N2) strain, where EC 50 = 9.235 ng / mL.

[0434] Figure 16 shows the inhibitory effect of AVC(022) on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain.

[0435] Figure 17 shows the inhibitory effect of oseltamivir on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain.

[0436] Figure 18 shows the inhibitory effect of oseltamivir on the A / Victoria / 2570 / 2019(H1N1) strain.

[0437] Figure 19 shows the inhibitory effect of oseltamivir on the A / Darwin / 9 / 2021(H3N2) strain.

[0438] Figure 20 shows the inhibitory effect of oseltamivir on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain.

[0439] Figure 21 shows the inhibitory effect of AVC(025) on NA enzyme activity.

[0440] Figure 22 shows the inhibitory effect of AVC-025 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro, where EC 50 = 3.438 ng / mL.

[0441] Figure 23 shows the inhibitory effect of AVC-025 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro, where EC...50 = 90.76 ng / mL.

[0442] Figure 24 shows the inhibitory effect of AVC-025 on the A / Darwin / 9 / 2021(H3N2) strain in vitro, where EC... 50 = 3.599 ng / mL.

[0443] Figure 25 shows the inhibitory effect of AVC-025 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro, where EC... 50 =1.035ng / mL.

[0444] Figure 26-1 shows the inhibitory effect of mabaloxavir on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0445] Figure 26-2 In vitro inhibitory effect of peramivir on Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain.

[0446] Figure 26-3 shows the inhibitory effect of AVC-025-DAR2 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0447] Figure 26-4 shows the inhibitory effect of AVC-025-DAR4 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0448] Figure 26-5 shows the inhibitory effect of AVC-025-DAR6 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0449] Figure 26-6 shows the inhibitory effect of AVC-025-DAR9 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0450] Figure 26-7 shows the inhibitory effect of AVC-025-DAR11 on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain in vitro.

[0451] Figure 27-1 shows the inhibitory effect of mabaloxavir on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0452] Figure 27-2 In vitro inhibitory effect of peramivir on the A / Victoria / 2570 / 2019(H1N1) strain.

[0453] Figure 27-3 shows the inhibitory effect of AVC-025-DAR2 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0454] Figure 27-4 shows the inhibitory effect of AVC-025-DAR4 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0455] Figure 27-5 shows the inhibitory effect of AVC-025-DAR6 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0456] Figure 27-6 shows the inhibitory effect of AVC-025-DAR9 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0457] Figure 27-7 shows the inhibitory effect of AVC-025-DAR11 on the A / Victoria / 2570 / 2019(H1N1) strain in vitro.

[0458] Figure 28-1 shows the inhibitory effect of mabaloxavir on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0459] Figure 28-2 In vitro inhibitory effect of peramivir on the A / Darwin / 9 / 2021(H3N2) strain.

[0460] Figure 28-3 shows the inhibitory effect of AVC-025-DAR2 on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0461] Figure 28-4 shows the inhibitory effect of AVC-025-DAR4 on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0462] Figure 28-5 shows the inhibitory effect of AVC-025-DAR6 on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0463] Figure 28-6 shows the inhibitory effect of AVC-025-DAR9 on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0464] Figure 28-7 shows the inhibitory effect of AVC-025-DAR11 on the A / Darwin / 9 / 2021(H3N2) strain in vitro.

[0465] Figure 29-1 shows the inhibitory effect of mabaloxavir on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0466] Figure 29-2 In vitro inhibitory effect of peramivir on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain.

[0467] Figure 29-3 shows the inhibitory effect of AVC-025-DAR2 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0468] Figure 29-4 shows the inhibitory effect of AVC-025-DAR4 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0469] Figure 29-5 shows the inhibitory effect of AVC-025-DAR6 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0470] Figure 29-6 shows the inhibitory effect of AVC-025-DAR9 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0471] Figure 29-7 shows the inhibitory effect of AVC-025-DAR11 on the B / Austria / 1359417 / 2021 (B / Victoria Lineage) strain in vitro.

[0472] Figure 30-1 shows the body weight curves for evaluating the in vivo efficacy of AVC-025. The body weight of the model group decreased significantly, while the body weight rebounded significantly after administration of 0.15 and 0.05 mpk AVC-025. There was no change in body weight after administration of 5 and 0.5 mpk AVC-025.

[0473] Figure 30-2 shows the survival curves for evaluating the in vivo efficacy of AVC-025. At 15 days post-infection, the survival rate in the model group was 0%, the survival rate after administration of 0.05 mpk AVC-025 was approximately 83%, and the survival rate after administration of 5, 0.5, and 0.15 mpk AVC-025 was 100%.

[0474] Figure 30-3 shows the viral load in bronchoalveolar lavage fluid for in vivo efficacy evaluation of AVC-025. Six days after infection, the viral load was significantly reduced after administration of AVC-025 at doses of 5, 0.5, and 0.15 mpk.

[0475] Figure 30-4 shows the body weight curves for evaluating the in vivo efficacy of AVC-022. The model group showed a significant decrease in body weight, while the body weight significantly increased after administration of 0.05 mpk of AVC-022.

[0476] Figure 30-5 shows the survival curves for evaluating the in vivo efficacy of AVC-022. At 15 days post-infection, the survival rate in the model group was 0%, while the survival rate after administration of 0.05 mpk AVC-022 was approximately 83%.

[0477] Figure III-6-1 shows the changes in body weight and survival rate of mice from D15 to D75 after challenge with the virus.

[0478] Figure III-6-2 shows the changes in body weight and survival rate of mice from D30 to D75 after challenge with the virus.

[0479] Figure III-6-3 shows the changes in body weight and survival rate of mice from D45 challenge to D75.

[0480] Figure III-6-4 shows the changes in body weight and survival rate of mice from D60 to D75 after challenge with the virus.

[0481] Figure III-6-5 shows the BLA virus titer in mice.

[0482] Figure III-7-1 shows the in vivo antiviral evaluation of AVC-022.

[0483] Figure III-9-1 shows the blood drug concentration curve of AVC-022 in animal plasma.

[0484] Figure IV-1 shows the SEC-HPLC chromatogram of (Fc-PEG4-N3).

[0485] Figure IV-2 shows the mass spectrometry detection of (Fc-PEG4-N3).

[0486] Figure IV-3 shows the chromatogram of AVC(042) by SEC-HPLC.

[0487] Figure IV-4 shows the mass spectrometry detection of AVC(042).

[0488] Figure IV-5 shows the chromatogram of AVC(044) SEC-HPLC.

[0489] Figure IV-6 shows the mass spectrometry detection of AVC(044).

[0490] Figure IV-7 shows the chromatogram of AVC(048) by SEC-HPLC.

[0491] Figure IV-8 shows the mass spectrometry detection of AVC(048).

[0492] Figure IV-9 shows the chromatogram of AVC(049) SEC-HPLC.

[0493] Figure IV-10 shows the mass spectrometry detection of AVC(049).

[0494] Figure IV-11 shows the chromatogram of AVC(050) SEC-HPLC.

[0495] Figure IV-12 shows the AVC(050) mass spectrometry detection pattern.

[0496] Figure V-1-1 shows the inhibition curve of AVC(042) against H1N1-PR8 virus.

[0497] Figure V-1-2 shows the inhibition curve of AVC(042) against H3N2-Aichi virus.

[0498] Figure V-2-1 shows the inhibition curve of AVC(044) against H1N1-PR8 virus.

[0499] Figure V-2-2 shows the inhibition curve of AVC(044) against H3N2-Aichi virus.

[0500] Figure V-3-1 shows the inhibition curve of AVC(048) against H1N1-PR8 virus.

[0501] Figure V-3-2 shows the inhibition curve of AVC(048) against H3N2-Aichi virus.

[0502] Figure V-4-1 shows the inhibition curve of AVC(049) against H1N1-PR8 virus.

[0503] Figure V-4-2 shows the inhibition curve of AVC(049) against H3N2-Aichi virus.

[0504] Figure V-5-1 shows the inhibition curve of AVC(050) against H1N1-PR8 virus.

[0505] Figure V-5-2 shows the inhibition curve of AVC(050) against H3N2-Aichi virus.

[0506] Figure V-6-1 shows the inhibition curve of RD2358-042-0(int-8) against H1N1-PR8 virus.

[0507] Figure V-6-2 shows the inhibition curve of RD2358-042-0(int-8) against H3N2-Aichi virus.

[0508] Figure V-7-1 shows the inhibition curve of RD2358-044-0(int-9) against H1N1-PR8 virus.

[0509] Figure V-7-2 shows the inhibition curve of RD2358-044-0(int-9) against H3N2-Aichi virus.

[0510] Figure V-8-1 shows the inhibition curve of RD2358-048-0(int-5) against H1N1-PR8 virus.

[0511] Figure V-8-2 shows the inhibition curve of RD2358-048-0(int-5) against H3N2-Aichi virus.

[0512] Figure V-9-1 shows the inhibition curve of RD2358-049-0(int-6) against H1N1-PR8 virus.

[0513] Figure V-9-2 shows the inhibition curve of RD2358-049-0(int-6) against H3N2-Aichi virus.

[0514] Figure V-10-1 shows the inhibition curve of RD2358-050(int-7) against H1N1-PR8 virus.

[0515] Figure V-10-2 shows the inhibition curve of RD2358-050(int-7) against H3N2-Aichi virus. Detailed Implementation

[0516] In the following examples, the structures of the compounds were determined using nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS). NMR chemical shifts (δ) were expressed in terms of 10⁻⁶. -6 The measurements are given in ppm. NMR measurements were performed using a Bruker AVANCE III 500MHz NMR instrument. The solvents used for the measurements were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), etc., and the internal standard was tetramethylsilane (TMS).

[0517] Analytical liquid chromatography (HPLC) is used for monitoring reaction systems and analyzing the purity of important intermediates and target products. The analytical liquid chromatography system is an Agilent 1260 Infinity II (equipped with a 1260 Binary Pump, Vial Sampler, MCT column oven, and DAD WR detector); the column is a Waters XBridge BEH C18 3.5µm, 4.6 x 150 mm.

[0518] Preparative liquid chromatography (HPLC) is used for the separation and purification of small amounts of important intermediates and target products. The preparative HPLC system is an Agilent 1290 Infinity II (equipped with a 1290 Prep Bin Pump, Prep ALS / FC injector / collector, and a 1260 VWD detector); a C18 reversed-phase silica column.

[0519] LC-MS was used for routine reaction system analysis and determination of the molecular weight signal of the target product. LC-MS system: Waters UPLC H-Class ultra-high performance liquid chromatography-QDa system; Column: Waters XBridge BEH C18 1.7um, 2.1*75mm.

[0520] Liquid mobile phase ratios: Acidic conditions: A: 0.05% formic acid aqueous solution, B: acetonitrile; Alkaline conditions: A: 0.05% ammonia aqueous solution, B: acetonitrile; Neutral conditions: A: 5mM NH4HCO3 aqueous solution, B: acetonitrile. The above data are default mobile phase ratios and can be adjusted appropriately based on the properties of the actual samples during specific analytical testing.

[0521] A rapid purification system was used for the rough preparation and purification of compounds in conventional reaction systems. The instrument model was Biotage Isolera One, and the normal-phase and reverse-phase columns used with it were purchased from Santai Tech. Common specifications for normal-phase silica gel columns are 12g, 25g, 40g, 80g, 120g, and 330g, with specific parameters as follows: 40-63μm. Commonly used specifications for reversed-phase C18 columns are 40g, 80g, 120g, and 330g, with specific parameters as follows: 20-45μm.

[0522] Thin-layer chromatography (TLC) uses silica gel plates for qualitative analysis of the reaction system or for the separation and purification of small samples. The Merck silica gel plates used for qualitative analysis in TLC are as follows: GF 254 20*20cm, glass plate, 0.25mm coating; the product used for separation and purification by thin-layer chromatography is a silica gel plate prepared by Leyan, with the following specifications: GF 254 20*20cm, glass plate, 1mm coating.

[0523] The known starting materials of this invention can be synthesized using or according to methods known in the art, such as literature and patents, or can be purchased from chemical reagent companies such as Alfa Aesar, Acros, Sigma-Aldrich, TCI, Acon, Amex, Aladdin, J&K, Bidex Pharmaceuticals, Leyan, McLean, Accela, Adamas, Hines, and Innocare.

[0524] Example I-1: Synthesis of a trivalent skeleton (compound 5)

[0525] Step A: At room temperature, a solution of tert-butanol containing di-tert-butyl dicarbonate (47 g, 215.349 mmol) was added to a mixture of methanol (100 mL) and tert-butanol (50 mL) containing tris(hydroxymethyl)aminomethane (20 g, 165.098 mmol). The mixture was stirred at room temperature for 16 hours, during which a white precipitate gradually formed. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then added to ethyl acetate (50 mL). The mixture was then placed at 0 °C for 2 hours, filtered to obtain a white solid, and dried under vacuum. This reaction yielded a white solid, compound 2 (35.9 g, 162.260 mmol, 98.28%). 1 HNMR (500MHz, DMSO-d6) δppm 5.78 (br s, 1H), 4.51 (br t, J = 5.5Hz, 3H), 3.51 (d, J = 5.6Hz, 6H), 1.37 (s, 9H).

[0526] Step B: At 0°C, potassium hydroxide (12.5 g, 222.777 mmol) was ground and dispersed in N,N-dimethylformamide (50 mL). A mixed solution of tetrahydrofuran (50 mL) and N,N-dimethylformamide (20 mL) containing compound 2 (7 g, 31.638 mmol) was slowly added. The mixture was stirred at room temperature for 2 hours, and the reaction solution gradually became viscous. Then, 3-bromopropyne (22.7 g, 190.820 mmol) was added, and the reaction solution turned brown. The mixture was then stirred at room temperature for 6 hours. Water (100 mL) was added, and the mixture was extracted with dichloromethane (100 mL * 2). The organic phases were combined, washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the brown crude compound 3 (10.4 g).

[0527] Step C: At room temperature, crude compound 3 (10.4 g) was dissolved in ethyl acetate solution of hydrochloric acid (50 mL, 100.000 mmol). The mixture was then stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a brown crude product, which was used directly in the next step of the reaction.

[0528] Step D: At room temperature, the brown crude product obtained in the previous step was dissolved in methanol (7 mL), and then saturated sodium bicarbonate aqueous solution (50 mL) was added. The mixture was stirred at room temperature for 2 hours, and the pH of the solution was measured to be 7. Then, water (50 mL) was added, and the mixture was extracted with dichloromethane (50 mL * 2). The organic phases were combined, washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a brown residue, which was then separated by silica gel column chromatography (SiO2, PE:EA = 30:70). This reaction yielded a yellow oily product, compound 5 (1.96 g, 8.330 mmol, 26.32%). 1 H NMR (500MHz, CDCl3-d) δppm 4.12 (d, J = 2.3Hz, 6H), 3.40-3.46 (m, 6H), 2.40 (t, J = 2.4Hz, 3H), 1.72 (br s, 2H).

[0529] Example I-2: Synthesis of trivalent peramivir intermediate (compound 11)

[0530] Step E: At room temperature, add 150 mL of ethyl acetate solution of hydrochloric acid (300.000 mmol) to compound 6 (30.0 g, 74.903 mmol), and then stir the mixture at room temperature for 16 hours. The reaction mixture may clump initially; increase the stirring speed or manually break up the clumps to ensure continued normal stirring. Concentrate the reaction solution under reduced pressure to obtain a white solid crude compound 7 (33.6 g). 1 H NMR (500MHz, DMSO-d6): δppm 8.16 (br s,3H),7.96(d,J=10.5Hz,1H),4.27(td,J=10.0,3.4Hz,1H),4.21(dd,J=5.7, 3.7Hz,1H),3.61-3.66(m,3H),3.23-3.37(m,1H),2.70-2.78(m,1H),2.40(dt, J=14.0,8.7Hz,1H),2.30(dt,J=9.2,6.8Hz,1H),1.91(s,3H),1.76-1.84(m,1H ),1.45-1.56(m,1H),1.24-1.40(m,2H),0.97-1.08(m,2H),0.79-0.90(m,6H).

[0531] Step F: At room temperature, N,N-diisopropylethylamine (53 mL, 304.263 mmol) was added to 250 mL of dichloromethane containing 33.6 g of the crude white solid compound 7 from the previous step, and the mixture was stirred for 0.5 h. Then, dimethylaminopyridine (750 mg, 6.139 mmol) and N,N'-bis(tert-butyloxycarbonyl)-1H-pyrazole-1-formamidinium (42 g, 135.331 mmol) were added. The mixture was stirred at room temperature for 16 h, washed with water (200 mL x 2), and the organic phase was washed once more with saturated brine (200 mL). After drying with anhydrous sodium sulfate, the solution was filtered and concentrated under reduced pressure to obtain a colorless oil, which was then separated by silica gel column chromatography (SiO2, PE:EA = 75:25). This reaction yielded a white, foamy product compound 8 (40.0 g, 67.076 mmol, 59.97%). ESI-MS m / z = 543.5 [M+H] + ;Calcu.=542.3.

[0532] Step G: Under an ice-water bath and nitrogen protection, phenyl p-nitrochloroformate (17.8 g, 88.557 mmol) was added to 300 mL of dichloromethane containing compound 8 (32.0 g, 58.968 mmol), followed by dimethylaminopyridine (14.4 g, 117.869 mmol). The mixture was then stirred at room temperature for 6 hours, followed by the addition of phenyl p-nitrochloroformate (11.9 g, 59.005 mmol), and the mixture was stirred at room temperature for another 6 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then separated by silica gel column chromatography (SiO2, PE:EA = 25:75). This reaction yielded a white, foamy product, compound 9 (17.6 g, 24.866 mmol, 42.17%). ESI-MS m / z = 708.5 [M+H] + ;Calcu. = 707.3.

[0533] Step H: Under nitrogen protection, in 200 mL of dichloromethane containing compound 9 (17.6 g, 24.866 mmol), azidotriethylene glycol amine (8.1 g, 37.112 mmol), 1-hydroxybenzotriazole (3.4 g, 24.865 mmol), and N,N-diisopropylethylamine (6.4 g, 49.671 mmol) were added sequentially. The mixture was then stirred at room temperature for 8 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, which was separated by silica gel column chromatography (SiO2, DCM:MeOH = 96:4). This reaction yielded a yellow oily product, compound 10 (8.5 g, 10.802 mmol, 43.44%). ESI-MS m / z = 787.4 [M+H] + ;Calcu. = 786.5.

[0534] Step I: At room temperature, in a solution of 20 mL tetrahydrofuran containing compound 10 (5.2 g, 6.608 mmol), add another 20 mL tetrahydrofuran containing compound 5 (560 mg, 2.380 mmol), followed by 10 mL water solutions containing copper sulfate pentahydrate (257 mg, 1.029 mmol) and sodium ascorbate (410 mg, 2.070 mmol). The mixture is stirred at room temperature for 2 hours. Then, 30 mL water is added, and the mixture is extracted with dichloromethane (50 mL x 3). The organic phases are combined, washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a brown oil. The crude product is dissolved in N,N-dimethylformamide (3 mL) and subjected to reversed-phase separation. Column: Santai SepaFlash C18 (Spherical C18, 20-45 μm). The reaction yielded a white, foamy product, compound 11 (5.8 g, 1.609 mmol, 67.58%). The mobile phase consisted of water (containing 0.05% TFA) and acetonitrile. The flow rate was 60 mL / min. The gradient was acetonitrile from 0% to 100% over 30 minutes. The detection wavelength was 254 nm. ESI-MS m / z = 866.0 [M / 3+H] + ;Calcu.=2594.5.

[0535] Example I-3: Synthesis of the trivalent target product of peramivir (int-2)

[0536] Step J: Under nitrogen protection, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg, 0.522 mmol), 1-hydroxybenzotriazole (444 mg, 3.286 mmol), 4-dimethylaminopyridine (312 mg, 2.554 mmol), and N,N-diisopropylethylamine (48 mg, 0.371 mmol) were added to N,N-dimethylformamide (24 mL) containing 5,8,11-trioxa-2-azatridecanoic acid-1-tert-butyl ester (222 mg, 0.579 mmol) at room temperature and nitrogen protection. The mixture was stirred at room temperature for 0.5 hours, then compound 11 (2.5 g, 0.770 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a residue. The crude product was dissolved in N,N-dimethylformamide (5 mL) and subjected to reversed-phase separation. The chromatographic column was Santak. SepaFlash C18(Spherical C18,20-45μm, The reaction yielded a yellow, foamy product compound 12 (830 mg, 0.280 mmol, 36.38%). The mobile phase consisted of water (containing 0.05% TFA) and acetonitrile. The flow rate was 60 mL / min. The gradient was acetonitrile from 0% to 100% over 30 minutes. The detection wavelength was 254 nm. ESI-MS m / z = 1443.4 [M / 2+H] + ;Calcu.=2883.6.

[0537] Step K: At room temperature, compound 12 (830 mg, 0.280 mmol) was added to a mixed solution of trifluoroacetic acid (2 mL) and dichloromethane (1 mL). The mixture was then stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a residue. The crude product was dissolved in N,N-dimethylformamide (1 mL) and subjected to reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 40-75 μm). 40 g); mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 0% to 100% over 30 minutes; detection wavelength: 210 nm. The reaction yielded a yellow, foamy product compound 13 (540 mg, 0.247 mmol, 85.83%). ESI-MS m / z = 1092.8 [M / 2+H] + ;Calcu.=2183.3.

[0538] Step L: At room temperature, sodium bicarbonate (42 mg, 0.500 mmol) was added to 10 mL of dimethyl sulfoxide (540 mg, 0.247 mmol) containing compound 13. The mixture was stirred at room temperature for 20 minutes. Then, ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidone-1-yl)carbonate (100 mg, 0.500 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a residue. The crude product was dissolved in N,N-dimethylformamide (1 mL) and subjected to reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 40-75 μm). 40 g); mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 210 nm. The reaction yielded a yellow, foamy product compound 14 (560 mg, 0.213 mmol, 86.37%). ESI-MS m / z = 1181.3 [M / 2+H] +;Calcu.=2359.3.

[0539] Step M: At room temperature, 25 mL of 5% sodium hydroxide aqueous solution was added to 25 mL of pure water containing compound 14 (530 mg, 0.225 mmol), and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then separated by HPLC under the following conditions: Welch Ultimate C18 (30 mm × 250 mm, 5 μm); A: 0.2% formic acid in water; B: MeCN; 40 mL / min 0–3 min, 10% B; 3–15 min, 10%–35% B; detector, UV 210 nm. The reaction yielded a white solid product, compound 15, i.e., int-2 (116.64 mg, 0.047 mmol, 20.90%). 1 H NMR(500MHz,DMSO-d6)δppm 8.77-9.07(m,3H),8.01(s,3H),7.59-7.83(m,3H),7.26-7.43(m,2H),6.95-7.18(m,7H),6.87(s,1H),6.68-6.81(m,3H),5.19(br s,3H),4.51(br t,J=5.1Hz,6H),4.47(s,6H),4.16-4.29(m,3H),3.98-4.06(m,2H),3.79-3.85(m,6H),3.78(br s,2H),3.66(br s,8H),3.43-3.57(m,38H),2.95-3.25(m,14H),2.52-2.68(m,6H),2.37(br dd,J=4.0,2.1Hz,3H),2.09-2.26(m,6H),1.81(s,9H),1.43-1.67(m,9H),1.18-1.35(m,5H),1.07(br s,6H),0.89-1.00(m,4H),0.81-0.88(m,18H). ESI-HRMS m / z=1159.6563[M / 2+H] + ;Calcu.=2317.2893.

[0540] Example I-4: Synthesis of peramivir trimer derivative (int-1)

[0541] Step A: At room temperature, triethylamine (0.35 mL, 2.485 mmol) was added to 8.0 mL of N,N-dimethylformamide containing dibenzocyclooctyne-N-hydroxysuccinimino ester (500 mg, 1.243 mmol) and aminotriethylene glycol acetic acid (386 mg, 1.864 mmol). After the addition, the mixture was stirred overnight at room temperature. The reaction solution was then directly subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 80 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 60 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a yellow oily product, compound 16 (580 mg, 1.174 mmol, 94.45%). ESI-MS m / z = 495.4 [M+H] + ;Calcu.=494.2.

[0542] Step B: At room temperature, compound 11 (2.0 g, 0.770 mmol) was added to 20.0 mL of N,N-dimethylformamide containing compound 16 (0.57 g, 1.156 mmol), N,N-diisopropylethylamine (0.4 g, 3.082 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.9 g, 2.311 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (10.0 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 330 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 100 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a white solid product, compound 17 (1.1 g, 0.365 mmol, 47.31%). ESI-MS m / z = 1024.9 [M / 3+H] + ;Calcu.=3070.7.

[0543] Step C: At room temperature, trifluoroacetic acid (6.0 mL) was added to dichloromethane (2.0 mL) containing compound 17 (990 mg, 0.322 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction solution was then directly subjected to reversed-phase separation using a Santai SepaFlash C18 (Spherical C18, 20-45 μm) column. 120 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 80 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a white solid product, compound 18 (330 mg, 0.134 mmol, 41.47%). ESI-MS m / z = 1236.7 [M / 2+H] + ;Calcu.=2470.3.

[0544] Step D: At room temperature, 0.05 mol / L lithium hydroxide solution (16.0 mL, 0.801 mmol) was added to 16.0 mL of water (330 mg, 0.134 mmol) containing dissolved compound 18. After the addition was complete, the mixture was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to neutral with 1N hydrogen chloride aqueous solution, and the solution was concentrated under reduced pressure to obtain the crude product. The crude product was sent for preparative separation under the following conditions: Waters Xbridge CSH C18 (10 mm × 150 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 5 mL / min; 0–0.4 min, 10% B; 0.4–7.5 min, 10%–35% B, 7.5–11.5 min, 35%–100% B, detector, UV 254 nm). Freeze-drying under reduced pressure yielded a white solid product, compound 19, namely int-1 (140.68 mg, 0.054 mmol, 40.30%). 1H NMR (500MHz, DMSO-d6) δppm 8.67-9.02(m,3H),8.00(s,3H),7.73-7.79(m,1H),7.67(br d,J=7.63Hz,1H),7.61(br d J=7.32Hz,3H),7.41-7.52(m,4H),7.26-7.39(m,5H),6.95-7.10(m,5H),6.84-6.91(m,1H),6.70-6.81(m,3H),5.20(br s,3H),4.99-5.06(m,1H),4.48-4.54(m,6H),4.43-4.48(m,6H),4.19-4.29(m,3H),3.78-3.83(m,6H),3.77(br s,2H),3.65(br s,8H),3.58-3.63(m,2H),3.49-3.53(m,9H),3.45(br s,21H),3.28(br d,J=5.95Hz,5H),3.13-3.21(m,6H),3.08(dt,J=11.10,5.51Hz,5H),2.5 3-2.62(m,6H),2.36-2.43(m,4H),2.23(dt,J=15.45,7.76Hz,1H),1.96-2 .04(m,1H),1.78-1.85(m,8H),1.70-1.78(m,2H),1.49-1.64(m,6H),1.1 8-1.33(m,5H),1.01-1.14(m,7H),0.90-1.00(m,4H),0.69-0.90(m,20H). ESI-HRMS m / z=1215.1584[M / 2+H] + ;Calcu.=2428.3002.

[0545] Example I-5: Synthesis of a divalent skeleton (compound 23)

[0546] Step A: At room temperature, add 100 mL of anhydrous ethanol containing 36.0 g (164.5 mmol) of di-tert-butyl dicarbonate to 250 mL of anhydrous ethanol containing 15.0 g (164.6 mmol). After the addition, stir the mixture overnight at room temperature. Filter the reaction mixture, slurry the product with methyl tert-butyl ether, and dry to give a white solid product, compound 20 (30.0 g, crude). The crude product can be used directly in the next reaction without purification. ESI-MS m / z = 192.2 [M+H] + ;Calcu. = 191.1.

[0547] Step B: At 0°C, potassium hydroxide (46.0 g, 821.4 mmol) was dispersed in ultra-dry N,N-dimethylformamide (200 mL) to obtain an N,N-dimethylformamide solution containing potassium hydroxide. Compound 20 (30 g, 157.0 mmol) was dissolved in tetrahydrofuran (200 mL) and slowly added to the N,N-dimethylformamide solution containing potassium hydroxide. The mixture was reacted at room temperature for half an hour. Then, 3-bromopropyne (120.1 g, 627.8 mmol) was added to the reaction solution. After the addition was complete, the mixture was reacted at 0°C for 7 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (300 mL * 3). The organic phase was washed once with water (400 mL), once with saturated brine (400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow solid product, compound 21 (45.2 g, crude product). The crude product can be used directly in the next reaction without purification.

[0548] Step C: At room temperature, 150 mL of 2N ethyl hydrochloride solution was added to compound 21 (45.2 g, crude). After the addition was complete, the mixture was left at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain a brown oily liquid compound 22 (60.0 g, crude). The crude product was used directly in the next reaction without purification.

[0549] Step D: At room temperature, saturated sodium bicarbonate aqueous solution (150 mL) was added to compound 22 (60.0 g, crude product in methanol (15 mL). After the addition was complete, the mixture was left at room temperature for 2 hours. The reaction solution was extracted three times with dichloromethane (150 mL * 3), washed once with water (200 mL), washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and dried under vacuum to obtain the yellow oily product compound 23 (3.0 g, 17.9 mmol, 10.90%). 1 H NMR (500MHz, DMSO-d6) δppm 4.10-4.16(m,4H),3.42-3.44(m,2H),3.36-3.40(m,2H),3.27-3.31(m,2H),2.94-3.01(m,1H),1.37-1.59(m,2H).

[0550] Example I-6: Synthesis of divalent peramivir intermediate (compound 24)

[0551] Step E: At room temperature, in 200 mL of tetrahydrofuran containing compound 10 (7.3 g, 9.277 mmol) and compound 23 (0.70 g, 4.175 mmol), an aqueous solution of copper sulfate pentahydrate (1.2 g, 4.638 mmol) and sodium ascorbate (1.8 g, 9.277 mmol) was added. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in N,N-dimethylformamide (20 mL) for reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 330 g); mobile phase: water (containing 0.1% FA) and acetonitrile; flow rate: 100 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a white solid product, compound 24 (2.8 g, 1.608 mmol, 17.34%). ESI-MS m / z = 871.2 [M / 2+H] + ;Calcu.MW=1740.0.

[0552] Example I-7: Synthesis of the divalent target product of peramivir (int-3)

[0553] Step F: At room temperature, compound 24 (1.2 g, 0.689 mmol) was added to N,N-dimethylformamide (15.0 mL) containing N-tert-butoxycarbonyl-polyethylene glycol-acetic acid (0.32 g, 1.034 mmol), N,N-diisopropylethylamine (0.36 g, 2.757 mmol), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.8 g, 2.068 mmol). After the addition, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (10.0 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 330 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 80 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a yellow solid product, compound 25 (550 mg, 0.271 mmol, 39.30%). ESI-MS m / z = 1015.8 [M / 2+H] + ;Calcu.MW=2029.1.

[0554] Step G: Reaction Feeding: At room temperature, trifluoroacetic acid (4.5 mL) was added to 1.5 mL of dichloromethane containing compound 25 (500 mg, 0.246 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (3.0 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 80 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 60 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm. Lyophilization under reduced pressure yielded a yellow solid product, compound 26 (320 mg, 0.210 mmol, 85.15%). ESI-MS m / z = 765.6 [M / 2+H] + ;Calcu.MW=1528.9.

[0555] Step H: At room temperature, 15.6 mL of 0.05 mol / L lithium hydroxide aqueous solution (0.784 mmol) was added to 8.0 mL of water (300 mg, 0.196 mmol) containing dissolved compound 26. After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (5.0 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 80 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 60 mL / min; gradient: acetonitrile from 0% to 100% over 30 minutes; detection wavelength: 254 nm. Lyophilized under reduced pressure, yielding a white solid product, compound 27 (200 mg, 0.133 mmol, 67.97%). ESI-MS m / z = 751.5 [M / 2+H] + ;Calcu.MW=1500.9.

[0556] Step I: At room temperature, triethylamine (25 mg, 0.247 mmol) was added to N,N-dimethylformamide (2.0 mL) containing compound 27 (190 mg, 0.127 mmol) and ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (56 mg, 0.192 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction solution was directly sent to a preparative separator under the following conditions: Waters Xbridge CSH C18 (10 mm × 150 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 5 mL / min; 0–0.4 min, 25% B; 0.4–7.5 min, 25%–75% B; 7.5–10.0 min, 75%–100% B; detector, UV 254 nm. The solution was lyophilized under reduced pressure to obtain a white solid product, compound 28, i.e., int-3 (55.35 mg, 0.031 mmol, 24.40%). 1 H NMR(500MHz,DMSO-d6)δppm 8.85-9.02(m,1H),7.97-8.07(m,2H),7.62-7.80(m,2H),7.42-7.49(m,1H),6.91-7.15(m,5H),6.66-6.82(m,2H),5.12-5.23(m ,2H),4.46-4.53(m,7H),4.17-4.28(m,2H),4.06-4.14(m,1H),3.99-4.04(m,1H)\,3.85-3.90(m,2H),3.78-3.83(m,4H),3.60-3 .73(m,3H),3.44-3.58(m,27H),3.35-3.43(m,20H),3.01-3.20(m,7H),2.63(m,2H),2.53-2.59(m,2H),2.34-2.42(m,2H),2.09- 2.26(m,3H),1.77-1.86(m,6H),1.46-1.65(m,5H),1.21-1.33(m,3H),1.01-1.12(m,4H),0.91-0.99(m,3H),0.77-0.91(m,12H). ESI-HRMS m / z=839.4745[M / 2+H] + ;Calcu.=1676.9349.

[0557] Example I-8: Synthesis of key intermediate RD2358-A8

[0558] Step A: This step was carried out in parallel in two groups. 450g of RD2358-022-A0 (total 900g, 2247mmol) solid was added to each of two 5L three-necked flasks. Then, 1.5L of ethyl acetate (total 3L, the reaction solution was a suspension) was slowly added to each flask. The entire system was cooled in an ice bath and mechanically stirred (200rpm). Then, 2L of cold ethyl hydrochloride solution (total 4L, 4N HCl in EA) was slowly added. The system temperature was gradually raised from the ice bath to room temperature and stirred overnight at room temperature. After the reaction was complete, the reaction solution was filtered, and the filter cake solid was the crude product. The filter cake was washed three times with ethyl acetate (1L*3), and then vacuum dried to obtain the crude product RD2358-022-A1 (800g, crude). No purification was required for this product, and it was used directly in the next reaction.

[0559] Step B: This step was carried out in five parallel groups. In five 5L three-necked flasks, 160g of RD2358-022-A1 (800g crude) and 214g of (Z)-((tert-butyloxycarbonyl)imino)(1H-pyrazol-1-yl)methyl)carbamate tert-butyl ester (1070g, 3450mmol) were added, followed by the slow addition of 3L of dichloromethane (15L total). Subsequently, 4.4g of 4-dimethylaminopyridine (22g, 180mmol) solid and 210g of N,N-diisopropylethylamine (1050g, 8123mmol) liquid starting material were added. The system temperature was gradually raised from an ice bath to room temperature and maintained at room temperature with stirring overnight. After the reaction was complete, the reaction system was sequentially washed with water, saturated sodium bicarbonate solution, and saturated brine. The treated organic phase was dried over anhydrous sodium sulfate, mixed with silica gel for column chromatography, and concentrated under reduced pressure. The crude product was then purified by normal-phase column chromatography (column chromatography, SiO2, PE:EA = 80:20-30:70). Concentration under reduced pressure yielded a white solid product RD2358-022-A2 (1079 g, 1988 mmol, 80.75%). ESI-MS m / z = 543.5 [M+H] + ;Calcu.=542.3.

[0560] Step C: This step was carried out in two parallel groups. 412.5 g of RD2358-022-A2 (total 825 g, 1520 mmol) and 3 L of tetrahydrofuran (total 6 L) were added to two 5 L three-necked flasks, respectively. After the starting materials were completely dissolved, the entire system was cooled in an ice bath with mechanical stirring. Then, 313 g of N,N-carbonyldiimidazole (total 616 g, 3798 mmol) solid was added to each flask. The system temperature was gradually raised from the ice bath to room temperature and maintained at room temperature with stirring overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure and then redissolved with ethyl acetate. The organic phase was then washed with water and saturated brine. The treated organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product RD2358-022-A3 (970 g, crude). The crude product was used directly in the next reaction without purification. ESI-MS m / z = 637.6 [M+H] + ;Calcu. = 636.3.

[0561] Step D: This step was carried out in parallel in four groups. In four 5L three-necked flasks, 175g of RD2358-022-A3 (total 700g, 1099mmol), 90g of 2-(2-(2-azidooxy)ethoxy)ethyl-1-amine (total 360g, 1649mmol), 2L of acetonitrile (total 8L), and 155mL of triethylamine (total 620mL, 4460mmol) were added respectively, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure and then redissolved with ethyl acetate. The organic phase was then washed with water and saturated brine. The treated organic phase was dried over anhydrous sodium sulfate, mixed with silica gel for column chromatography, and concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography (column chromatography, SiO2, PE:EA = 30:70-50:50). Concentration under reduced pressure yielded a pale yellow, foamy solid product, RD2358-022-A4 (553 g, 702 mmol, 63.92% in 2 steps). ESI-MS m / z = 787.7 [M+H] + ;Calcu. = 786.4.

[0562] Step E: This step was carried out in three parallel groups. In three 5L three-necked flasks, 182.7g of RD2358-022-A4 (total 548g, 696mmol), 18.2g of 1,3-bis(prop-2-yn-1-yloxy)-2-((prop-2-en-1-yloxy)methyl)prop-2-amine (total 54.6g, 232mmol), and 1.8L of tetrahydrofuran (total 5.4L) were added respectively. After the raw materials were completely dissolved, 0.6L of copper sulfate pentahydrate aqueous solution (29g, 116mmol dissolved in 1.8L water) was slowly added, and after stirring evenly, 0.6L of sodium ascorbate aqueous solution (46g, 232mmol dissolved in 1.8L water) was slowly added. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to remove the tetrahydrofuran. After discarding a large amount of remaining water, tetrahydrofuran was added to redissolve the insoluble matter. Then, silica gel was added for column chromatography, followed by concentration under reduced pressure. The crude product was purified by normal-phase column chromatography (column chromatography, SiO2, DCM:MeOH = 96:4-91:9). Concentration under reduced pressure yielded a yellow, foamy solid product RD2358-022-A5 (456 g, 175 mmol, 75.69%). ESI-MS m / z = 1299.2 [M / 2+H] + ;Calcu.=2594.5.

[0563] Step F: In a 5L three-necked flask, add 38g (123mmol) of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid, 95g (249mmol) of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate, and 2.4L of N,N-dimethylformamide, followed by 43g (332mmol) of N,N-diisopropylethylamine. After the addition of materials is complete, place the entire system at room temperature and start a magnetic stirrer, stirring at room temperature for 1 hour. Then, slowly add RD2358-022-A5 (211g, 83mmol) to the system. At this point, the system is a clear, brownish-yellow color. Stir the entire mixture at room temperature overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was dissolved in methanol (200 mL) and purified twice using a reverse-phase column chromatography system. Reverse-phase purification conditions: Column: Santai SepaFlash C18 (Spherical C18, 20-45 μm). 800 g); mobile phase: water (containing 0.1% FA) and methanol; flow rate: 100 mL / min; gradient: methanol from 0% to 100% over 60 minutes; detection wavelength: 210 nm; 90% methanol elution product. Concentration under reduced pressure yielded a yellow solid product RD2358-022-A6 (223 g, 78 mmol, 94.86%). ESI-MS m / z = 962.9 [M / 3+H] + ;Calcu.=2883.6.

[0564] Step G: In a 2L round-bottom flask, add RD2358-022-A6 (160g, 55mmol) and dichloromethane (550mL) sequentially. After the raw materials dissolve, start magnetic stirring (400rpm), place the round-bottom flask in an ice bath to cool, and slowly add trifluoroacetic acid (1100mL, added over 10 minutes) dropwise. After the addition is complete, the system will be a clear, brownish-yellow color. Stir the entire system at room temperature for 1 hour. After the reaction is complete, concentrate the reaction solution under reduced pressure, dissolve the crude product in methanol (150mL), and perform reverse-phase purification (column chromatography) twice. Reverse-phase purification conditions: Column: Santai SepaFlash C18 (Spherical C18, 20-45μm, 800 g); Mobile phase: water (containing 0.1% FA) and acetonitrile; Flow rate: 100 mL / min; Gradient: acetonitrile from 0% to 100% over 30 minutes; Detection wavelength: 210 nm, 30% acetonitrile precipitates as product. Concentration under reduced pressure yielded a yellow solid product RD2358-022-A7 (110.5 g, 50 mmol, 91.22%). ESI-MS m / z = 729.3 [M / 3+H] + ;Calcu.=2183.2.

[0565] Step H: In a 10L three-necked flask, add RD2358-022-A7 (99g, 45mmol) and cool the entire system in an ice bath. Then, slowly add 0.05mol / L lithium hydroxide solution (4622mL, 231mmol). After the addition is complete, start magnetic stirring (400rpm). The system will become a clear, pale yellow color. Gradually raise the temperature from the ice bath to room temperature and stir at room temperature for 2 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure. Dissolve the crude product in methanol (150mL) and perform reverse-phase purification (column chromatography) twice. Reverse-phase purification conditions: Column: Santai SepaFlash C18 (Spherical C18, 20-45μm, 800 g); Mobile phase: water (containing 0.1% FA) and acetonitrile; Flow rate: 60 mL / min; Gradient: acetonitrile from 0% to 100% over 30 minutes; Detection wavelength: 210 nm, 25% acetonitrile precipitates the product. Concentration under reduced pressure yielded a yellow solid product RD2358-022-A8 (48.2 g, 22 mmol, 49.64%). ESI-MS m / z = 715.3 [M / 3+H] + ;Calcu.=2142.2.

[0566] Example I-9: Synthesis of RD2358-042-0(int-8)

[0567] Step A: At room temperature, bis(4-nitrophenyl) carbonate (138.1 mg, 0.454 mmol) was added to 1.0 mL of N,N-dimethylformamide containing dissolved 5,6-dihydro-11,12-dihydrodibenzo[A,E]cyclooctene-5-ol (50.0 mg, 0.227 mmol) and N,N-diisopropylethylamine (44.0 mg, 0.340 mmol). After addition, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in N,N-dimethylformamide (10 mL) for reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 80 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 30 minutes, with 70% fraction collected; Detection wavelength: 210 nm. A colorless oily liquid product RD2358-042-A1 (70.2 mg, 0.182 mmol, 80.24%) was obtained. ESI-MS m / z = 386.2 [M+H] + ;Calcu.MW=385.1.

[0568] Step B: At room temperature, triethylamine (0.1 mL, 0.046 mmol) was added to 1.0 mL of acetonitrile containing RD2358-022-A8 (50.0 mg, 0.023 mmol) and RD2358-042-B1 (13.5 mg, 0.035 mmol). After addition, the mixture was stirred overnight at room temperature. The reaction solution was directly sent to a preparative separator under the following conditions: ChromCore HILIC-Amide (10 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0–3.5 min, 30% B; 3.5–15 min, 30%–35% B; 15–20 min, 35%–50% B; detector, UV 254 nm). Freeze-drying under reduced pressure yielded a white solid product RD2358-042-0 (13.6 mg, 0.006 mmol, 24.47%). ESI-MS m / z = 797.4 [M+H]+; Calcu.MW = 2387.3.

[0569] Example I-10: Synthesis of RD2358-044-0(int-9)

[0570] Step A: At room temperature, cesium carbonate (33.06 g, 101.458 mmol) was added to acetonitrile (300.0 mL) containing cycloheptan-1,3-dione (8.00 g, 63.411 mmol). After the addition was complete, the mixture was stirred at room temperature for 30 min. The reaction solution was cooled to 0 °C, and Selectfluor (40.43 g, 114.140 mmol) was added. After the addition was complete, the mixture was reacted at 25 °C for 2 h. The reaction solution was filtered through diatomaceous earth, and the crude product was concentrated under reduced pressure. The crude product was separated by silica gel column chromatography (SiO2, PE:EA = 65:35). The reaction yielded a colorless oily product RD2358-044-C1 (5.69 g, 35.092 mmol, 55.34%). 1 H NMR(500MHz,CHLOROFORM-d)δppm 1.69-1.77(m,1H)1.87-1.94(m,1H)2.01-2.06(m,2H)2.07-2.11(m,1H)2.64-2.70(m,1H)2.72-2.80(m,2H).

[0571] Step B: At room temperature, (4-methoxycarbonylbenzyl)triphenylphosphine bromide (23.04 g, 46.890 mmol) was added to tetrahydrofuran (300.0 mL) containing dissolved RD2358-044-C1 (5.69 g, 45.086 mmol). After the addition, the mixture was cooled to 0 °C, and then 1,8-diazobisspirocyclic [5.4.0]undecyl-7-ene (8.24 g, 54.104 mmol) was added. After the addition, the mixture was reacted at 0 °C for 2 h. The reaction solution was filtered through diatomaceous earth and concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (SiO2, PE:EA = 50:50). The reaction yielded a colorless oily product RD2358-044-C2 (4.11 g, 13.931 mmol, 30.90%). ESI-MS m / z = 295.1 [M+H] + ;Calcu.MW=294.1.

[0572] Step C: At room temperature, palladium on carbon (410.0 mg) was added to 50.0 mL of ethanol containing RD2358-044-C2 (4.10 g, 13.931 mmol). After the addition was complete, the entire mixture was purged with hydrogen gas and the mixture was stirred at room temperature for 2 h. The reaction solution was filtered through diatomaceous earth, and the filtration was concentrated under reduced pressure to obtain the crude product RD2358-044-C3 (3.34 g, crude). ESI-MS m / z = 297.1 [M+H] + ;Calcu.MW=296.1.

[0573] Step D: At room temperature, RD2358-044-C3 (3.00 g, 10.124 mmol) was dissolved in dichloromethane (20.0 mL), and the solution was cooled to -78 °C. Trimethylaluminum (6.1 mL, 12.149 mmol) and trimethylsilyldiazomethane (8.1 mL, 12.149 mmol) were added to the reaction solution. After the addition was complete, the mixture was stirred at -78 °C for 30 min. The reaction solution was quenched with methanol, brought to room temperature, and then saturated potassium sodium tartrate solution (30.0 mL) was added. After the addition was complete, the mixture was extracted with dichloromethane (20.0 mL * 3), washed once with saturated brine, and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in ethyl acetate (2.5 mL) and methanol (1.5 mL), and then 3N hydrochloric acid (3.0 mL) was added. After the addition was complete, the reaction solution was stirred at room temperature for 8 h. The reaction solution was adjusted to neutral with saturated sodium bicarbonate, extracted with ethyl acetate (10.0 mL * 3), washed once with saturated brine (10.0 mL), and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography (SiO2, PE:EA = 1:0-10:1). This reaction yielded a colorless oily product RD2358-044-C4 (1.53 g, 4.930 mmol, 48.70%). ESI-MS m / z = 311.1 [M+H] + ;Calcu.MW=310.1.

[0574] Step E: At room temperature, 0.5N bis(trimethylsilyl)amino potassium solution (8.7 mL, 4.360 mmol) was added to tetrahydrofuran (20.0 mL), and the mixture was cooled to -78 °C. Then, RD2358-044-C4 (1.23 g, 3.963 mmol) was added. After the addition was complete, the mixture was reacted at -78 °C for 1 h. Next, N,N-bis(trifluoromethanesulfonyl)aniline (1.56 g, 4.360 mmol) was added. After the addition was complete, the reaction mixture was slowly brought back to room temperature and stirred overnight at room temperature. The reaction mixture was quenched with methanol and concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography (SiO2, PE:EA = 1:0-10:1). This reaction yielded a colorless oily product RD2358-044-C5 (1.15 g, 2.599 mmol, 65.58%). ESI-MS m / z = 443.2 [M+H] + ;Calcu.MW=442.1.

[0575] Step F: At -78°C, 2.5N n-butyllithium (1.8 mL) was added to 0.75 mL of diisopropylamine in 20.0 mL of tetrahydrofuran to obtain a 0.198 M solution of diisopropylaminolithium. At room temperature, RD2358-044-C5 (600.0 mg, 1.356 mmol) was added to 10.0 mL of tetrahydrofuran. After the addition was complete, the mixture was cooled to -20°C, and then the prepared LDA solution (10.3 mL, 2.034 mmol) was added to the reaction solution. After the addition was complete, the reaction solution was slowly brought back to room temperature and stirred at room temperature for 1 h. The reaction solution was quenched with methanol and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography (SiO2, PE:EA = 90:10). The reaction yielded a colorless oily product RD2358-044-C6 (98.2 mg, 0.336 mmol, 24.78%). ESI-MS m / z = 293.1 [M+H] + ;Calcu.MW=292.1.

[0576] Step G: At room temperature, lithium hydroxide monohydrate (160.9 mg, 6.718 mmol) was added to 4.0 mL of 1,4-dioxane and 1.0 mL of water in RD2358-044-C6 (98.2 mg, 0.336 mmol). After the addition was complete, the mixture was heated to 55 °C and reacted for 3 h. The pH of the reaction solution was adjusted to weakly acidic with 1 N dilute hydrochloric acid, and a solid precipitated. After filtration and concentration under reduced pressure, a white solid product RD2358-044-C7 (78.6 mg, 0.282 mmol, 84.06%) was obtained. ESI-MS m / z = 279.1 [M+H] + ;Calcu.MW=278.1.

[0577] Step H: At room temperature, N-hydroxysuccinimide (24.9 mg, 0.216 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (41.4 mg, 0.216 mmol) were added to dichloromethane (2.0 mL) containing RD2358-044-C7 (40.0 mg, 0.144 mmol). After the addition was complete, the mixture was reacted at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography (SiO2, PE:EA = 75:25). This reaction yielded a colorless oily product RD2358-044-C8 (45.2 mg, 0.120 mmol, 83.33%). ESI-MS m / z = 295.1 [M+H] + ;Calcu.MW=294.1.

[0578] Step I: At room temperature, add RD2358-044-C8 (6.8 mg, 0.018 mmol) and triethylamine (2.7 mg, 0.027 mmol) to 1.0 mL of dimethyl sulfoxide (DMSO) containing RD2358-022-A8 (58.2 mg, 0.027 mmol). After the addition is complete, let the mixture react overnight at room temperature. The reaction solution is directly sent to the preparative separator. Preparative conditions: ChromCore HILIC-Amide (10 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0–3 min, 30% B; 3–3.5 min, 30%–35% B; 3.5–15 min, 35%–50% B; detector, UV 254 nm). Freeze-drying under reduced pressure yielded a white solid product RD2358-044-0 (18.96 mg, 0.008 mmol, 43.55%). ESI-MS m / z = 1202.1 [M+H]+; Calcu.MW = 2401.3.

[0579] Example I-11: Synthesis of RD2358-048-0(int-5)

[0580] Step A: At room temperature, 4-azidobutyl-1-amine (1.9 g, 16.490 mmol) was added to acetonitrile (2.0 mL) containing dissolved RD2358-011-A3 (7.0 g, 10.993 mmol) and triethylamine (2.4 mL, 17.589 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (SiO2, PE:EA = 1:0-2:1). The reaction yielded a white solid product RD2358-048-A1 (5.8 g, 8.494 mmol, 77.27%). ESI-MS m / z = 683.6 [M+H] + ;Calcu.MW=682.4.

[0581] Step B: At room temperature, in 50.0 mL of tetrahydrofuran containing RD2358-048-A1 (5757.8 mg, 8.432 mmol) and 1,3-bis(2-propyn-1-yloxy)-2-(2-propyn-1-yloxymethyl)propane-2-amine (670.0 mg, 2.848 mmol), 12.5 mL each of sodium ascorbate (564.1 mg, 2.848 mmol) and copper sulfate pentahydrate (355.5 mg, 1.424 mmol) were added. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (SiO2, DCM:MeOH = 95:5). The reaction yielded a yellow solid product RD2358-048-A2 (4600.0 mg, 2.014 mmol, 70.73%). ESI-MS m / z = 762.2 [M+H] + ;Calcu.MW=2282.3.

[0582] Step C: At room temperature, N,N-dimethylformamide (RD2358-048-A2) (2.50 g, 1.095 mmol) was added to 25.0 mL of N,N-dimethylformamide containing 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid (0.50 g, 1.642 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate (1.25 g, 3.284 mmol), and N,N-diisopropylethylamine (0.57 g, 4.379 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (10 mL) and subjected to reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 80 g); Mobile phase: water (containing 0.05% FA) and acetonitrile; Flow rate: 60 mL / min; Gradient: acetonitrile from 0% to 100% over 30 min; Detection wavelength: 254 nm, 95% ACN. The product was lyophilized under reduced pressure to give a white solid product RD2358-048-A3 (1.9 g, 0.738 mmol, 67.45%). ESI-MS m / z = 858.6 [M+H] + ;Calcu.MW=2571.5.

[0583] Step D: At room temperature, trifluoroacetic acid (14.0 mL) was added to dichloromethane (7.0 mL) containing RD2358-048-A3 (2.0 g, 0.777 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol (5 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 80 g); mobile phase: water (containing 0.05% FA) and acetonitrile; flow rate: 60 mL / min; gradient: acetonitrile from 0% to 100% over 30 min; detection wavelength: 254 nm, 35% ACN. The product was lyophilized under reduced pressure to give a white solid product RD2358-048-A4 (1.4 g, 0.748 mmol, 96.20%). ESI-MS m / z = 936.9 [M+H] + ;Calcu.MW=1871.1.

[0584] Step E: At room temperature, add 0.05 mol / L lithium hydroxide aqueous solution (89.7 mL, 4.487 mmol) to RD2358-048-A4 (1.40 g, 0.748 mmol). After the addition is complete, stir the mixture at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure to obtain the crude product. Dissolve the crude product in methanol (10 mL) and perform reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 330 g); Mobile phase: water (containing 0.05% FA) and acetonitrile; Flow rate: 90 mL / min; Gradient: acetonitrile from 0% to 100% over 30 min; Detection wavelength: 254 nm, 36% ACN. The product was lyophilized under reduced pressure to give a white solid product RD2358-048-A5 (684.0 mg, 0.374 mmol, 49.98%). ESI-MS m / z = 611.0 [M+H] + ;Calcu.MW=1829.1.

[0585] Step F: At room temperature, triethylamine (0.04 mL, 0.273 mmol) was added to N,N-dimethylformamide (5.0 mL) containing RD2358-048-A5 (250.0 mg, 0.137 mmol) and ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (59.7 mg, 0.205 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was sent for preparative separation under the following conditions: Shim-pack GIST-Col-Prep-027 (19mm × 250mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 12mL / min; 0–3min, 10% B; 3–3.2min, 10%–15% B; 3.2–15min, 15%–50% B; detector, UV 254nm). The product was lyophilized under reduced pressure to obtain a white solid product RD2358-048-0 (124.12mg, 0.062mmol, 45.29%). ESI-MS m / z = 1004.2 [M+H]+; Calcu.MW = 2005.1.

[0586] Example I-12: Synthesis of RD2358-049-0(int-6)

[0587] Step A: At room temperature, triethylamine (1.2 mL, 8.633 mmol) was added to dioxane (50 mL) containing 1,3-bis(propen-2-en-1-yloxy)-2-((propen-2-yn-1-yloxy)methyl)prop-2-amine (1 g, 4.250 mmol) and fluorenemethyloxycarbonyl chloride (1.3 g, 5.025 mmol). After the addition was complete, the mixture was stirred at room temperature for 3 hours. After the reaction was complete, water was added to quench the reaction mixture, and the mixture was extracted with EA. The organic phases were combined, washed with water and saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a solid residue. The residue was separated by silica gel column chromatography (SiO2, PE:EA = 30:70). A colorless oily liquid product RD2358-049-A1 (1.8 g, 3.934 mmol, 92.57%) was obtained. ESI-MS m / z = 458.3 [M+H] + ;Calcu.MW=457.2.

[0588] Step B: At room temperature, in a solution of tetrahydrofuran (250 mL) and water (80 mL) containing RD2358-049-A1 (1300 mg, 2.841 mmol), tert-butyl(2-(2-azidoethoxy)ethyl)(methyl)carbamate (3364 mg, 13.770 mmol), copper sulfate pentahydrate (491 mg, 1.967 mmol) in water (10 mL) and sodium ascorbate (779 mg, 3.932 mmol) in water (10 mL) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was dissolved in water (10 mL) and methanol (10 mL) for reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 330 g); mobile phase: water (containing 0.1% FA) and methanol; flow rate: 100 mL / min; gradient: acetonitrile from 0% to 100% over 30 minutes, with 80% fraction collected; detection wavelength: 254 nm. A yellow foamy solid product RD2358-049-A2 (3.47 g, 2.915 mmol, 74.10%) was obtained. ESI-MS m / z = 1191.1 [M+H] + ;Calcu.MW=1189.6.

[0589] Step C: At room temperature, add 40 mL of trifluoroacetic acid to 20 mL of dichloromethane containing 2 g (1.680 mmol) of RD2358-049-A2. After the addition is complete, stir the mixture at room temperature for 2 hours. After the reaction is complete, evaporate the reaction solution to dryness to obtain a yellow oily crude product RD2358-049-A3, which can be directly used for the next reaction. ESI-MS m / z = 890.8 [M+H] + ;Calcu.MW=889.5.

[0590] Step D: At room temperature, triethylamine (2.802 mL, 20.160 mmol) was added to 100 mL of acetonitrile containing dissolved RD2358-049-A3 and RD2358-012-A3 (3.57 g, 5.040 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in N,N-dimethylformamide (10 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 30 minutes, with 75% fraction collected; Detection wavelength: 210 nm. The product RD2358-049-A4 (1.76 g, 0.678 mmol, 40.35%) was given as a white solid. ESI-MS m / z = 1188.2 [M / 2+H] + ;Calcu.MW=2372.4.

[0591] Step E: At room temperature, N,N-diisopropylethylamine (0.37 g, 2.865 mmol) was added to N,N-dimethylformamide (30 mL) containing 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid (0.33 g, 1.074 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.82 g, 2.148 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. Then, RD2358-049-A4 (1.7 g, 0.716 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The crude product was dissolved in N,N-dimethylformamide (10 mL). Chromatographic column: Santai SepaFlash C18 (Spherical C18, 20-45 μm). 220 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 60 minutes, with 90% of the fraction collected; Detection wavelength: 210 nm. A yellow foamy solid product RD2358-049-A5 (1.72 g, 0.646 mmol, 90.18%) was obtained. ESI-MS m / z = 888.7 [M / 3+H] + ;Calcu.MW=2661.5.

[0592] Step F: At room temperature, trifluoroacetic acid (20 mL) was added to 10 mL of dichloromethane containing RD2358-049-A5 (1.72 g, 0.646 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was dissolved in N,N-dimethylformamide (5 mL) and subjected to reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 70 mL / min; Gradient: methanol increased from 0% to 100% over 100 minutes, with 45% fraction collected; Detection wavelength: 210 nm. The product RD2358-049-A6 (1.23 g, 0.627 mmol, 97.05%) was given as a pale yellow solid. ESI-MS m / z = 982.1 [M / 2+H] + ;Calcu.MW=1961.1.

[0593] Step G: At 0°C, a solution of lithium hydroxide (0.08 g, 3.134 mmol) in water was slowly added to 200 mL of water containing RD2358-049-A6 (1.23 g, 0.627 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the pH was adjusted to neutral with hydrochloric acid, and the reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in N,N-dimethylformamide (5 mL) and subjected to reversed-phase separation using a Santai SepaFlash C18 column (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 70 mL / min; Gradient: methanol increased from 0% to 100% over 100 minutes, with 50% fraction collected; Detection wavelength: 210 nm. The product RD2358-049-A7 (720 mg, 0.375 mmol, 59.82%) was given as a pale yellow solid. ESI-MS m / z = 961.1 [M / 2+H] + ;Calcu.MW=1919.1.

[0594] Step H: At room temperature, in N,N-dimethylformamide (0.5 mL) containing N,N-dimethylformamide with RD2358-049-A7 (100 mg, 0.052 mmol) and ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (16 mg, 0.055 mmol) was added. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction, the product was directly separated by HPLC without further treatment. Preparation conditions: Waters Xbridge Prep C18 (19mm × 250mm, 5μm); A: 0.1% TFA in water, B: MeCN; 18mL / min; 0–3min, 10% B; 3–3.5min, 10–20% B; 3.5–15min, 20–65% B; 15–15.5min, 65–100% B; detector, UV 210nm. The product was lyophilized under reduced pressure to obtain a white solid product RD2358-049-0 (67.30mg, 0.029mmol, 56.53%). ESI-MS m / z = 1049.1 [M / 2+H] + ;Calcu.MW=2095.2.

[0595] Example I-13: Synthesis of RD2358-050(int-7)

[0596] Step A: Under nitrogen protection, a tetrabutylammonium fluoride solution in tetrahydrofuran (1 M in THF, 51 mL, 51.000 mmol) was added to a mixture of tert-butyl 4-(2-bromoethyl)piperazine-1-carboxylate (10 g, 34.106 mmol) and trimethylsilyl azide (5.9 g, 51.211 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was diluted with ethyl acetate, filtered through silica gel, washed with ethyl acetate, and evaporated to dryness to give a pale yellow oily liquid RD2358-050-B1 (8.5 g, 33.291 mmol, 97.61%). ESI-MS m / z = 256.2 [M+H] + ;Calcu.MW=255.2.

[0597] Step B: At room temperature, trifluoroacetic acid (70 mL) was added to dichloromethane (70 mL) containing RD2358-050-B1 (9.3 g, 36.425 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain crude RD2358-050-B2, which was directly used for the next reaction. ESI-MS m / z = 156.2 [M+H] + ;Calcu.MW=155.1.

[0598] Step C: At room temperature, 11.2 g (44.081 mmol) of 2-(2-bromoethyl)isoindene-1,3-dione and 9.5 mL (68.347 mmol) were added to 150 mL of ethanol containing crude RD2358-050-B2. After the addition was complete, the mixture was heated to 85 °C and stirred overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, washed twice with water, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain crude RD2358-050-B3, which was directly used for the next reaction. ESI-MS m / z = 329.2 [M+H] + ;Calcu.MW=328.2.

[0599] Step D: At room temperature, hydrazine hydrate (10 mL, 164.922 mmol) was added to 150 mL of ethanol containing crude RD2358-050-B3. After the addition was complete, the mixture was heated to 100 °C and stirred for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (30 mL) and stirred at room temperature for 1 hour. The filtrate was filtered and evaporated to dryness to obtain a pale yellow oily liquid RD2358-050-B4 (3.3 g, 16.644 mmol, 48.95% in three steps). ESI-MS m / z = 329.2 [M+H] + ;Calcu.MW=328.2.

[0600] Step E: At room temperature, triethylamine (4.5 mL, 32.375 mmol) was added to 200 mL of acetonitrile containing dissolved RD2358-022-A3 (5 g, 7.852 mmol) and RD2358-050-B4 (2.5 g, 12.609 mmol). After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was dissolved in N,N-dimethylformamide (20 mL) for reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 330 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 30 minutes, with 75% of the fraction collected; Detection wavelength: 210 nm. The product RD2358-050-A4 (3.59 g, 4.681 mmol, 59.61%) was given as a white solid. ESI-MS m / z = 767.7 [M+H] + ;Calcu.MW=766.5.

[0601] Step F: At room temperature, in tetrahydrofuran (50 mL) containing RD2358-050-A4 (4.3 g, 5.607 mmol) and 1,3-bis(prop-2-en-1-yloxy)-2-((prop-2-yn-1-yloxy)methyl)prop-2-amine (0.43 g, 1.828 mmol), copper sulfate pentahydrate (0.23 g, 0.934 mmol) in water (12.5 mL) and sodium ascorbate (0.37 g, 1.869 mmol) in water (12.5 mL) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in N,N-dimethylformamide (20 mL) for reversed-phase separation. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 330 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 30 minutes, with 85% of the fraction collected; Detection wavelength: 210 nm. The product RD2358-050-A5 (1.79 g, 0.706 mmol, 37.77%) was given as a white solid. ESI-MS m / z = 1268.8 [M / 2+H] + ;Calcu.MW=2534.5.

[0602] Step G: At room temperature, N,N-diisopropylethylamine (0.36 g, 2.823 mmol) was added to N,N-dimethylformamide (25 mL) containing 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid (0.33 g, 1.059 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (0.81 g, 2.117 mmol). After the addition was complete, the mixture was stirred at room temperature for 1 hour. Then, RD2358-050-A5 (1.79 g, 0.706 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The crude product was dissolved in N,N-dimethylformamide (10 mL) and separated twice by reversed-phase chromatography. The chromatographic column was a Santai SepaFlash C18 (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: methanol increased from 0% to 100% over 30 minutes, with 80% of the fraction collected; Detection wavelength: 210 nm. A yellow foamy solid product RD2358-050-A6 (1.78 g, 0.630 mmol, 89.26%) was obtained. ESI-MS m / z = 1413.5 [M / 2+H] + ;Calcu.MW=2823.7.

[0603] Step H: At room temperature, add 20 mL of anhydrous lithium hydroxide (70 mg, 2.923 mmol) in water to 10 mL of acetonitrile containing RD2358-050-A6 (1780 mg, 0.630 mmol). After the addition is complete, stir the mixture at room temperature for 8 hours, and the reaction gradually becomes clear. After the reaction is complete, adjust the pH to approximately 7 using hydrochloric acid (1 M), concentrate the reaction solution under reduced pressure, dissolve the crude product in N,N-dimethylformamide (10 mL), and perform reversed-phase separation. Chromatographic column: Santai SepaFlash C18 (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: acetonitrile from 0% to 100% over 30 minutes, with 80% fraction collected; Detection wavelength: 254 nm. A yellow foaming solid product RD2358-050-A7 (700 mg, 0.251 mmol, 39.92%) was obtained. ESI-MS m / z = 1413.5 [M / 2+H] + ;Calcu.MW=2823.7.

[0604] Step I: At room temperature, add 20 mL of trifluoroacetic acid to 10 mL of dichloromethane containing RD2358-050-A7 (700 mg, 0.251 mmol). After the addition is complete, stir the mixture at room temperature for 2 hours. After the reaction is complete, concentrate the reaction solution under reduced pressure, dissolve the crude product in N,N-dimethylformamide (10 mL), and perform reversed-phase separation. Chromatographic column: Santai SepaFlash C18 (Spherical C18, 20-45 μm). 120 g); Mobile phase: water (containing 0.1% FA) and methanol; Flow rate: 100 mL / min; Gradient: acetonitrile from 0% to 100% over 30 minutes, with 80% fraction collected; Detection wavelength: 254 nm. The product RD2358-050-A8 (490 mg, 0.235 mmol, 93.56%) was given as a pale yellow solid. ESI-MS m / z = 695.3 [M / 3+H] + ;Calcu.MW=2081.3.

[0605] Step J: At room temperature, triethylamine (0.026 mL, 0.188 mmol) was added to N,N-dimethylformamide (5 mL) containing RD2358-050-A8 (190 mg, 0.091 mmol) and ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidone-1-yl) carbonate (40 mg, 0.137 mmol). After the addition was complete, the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was directly separated by HPLC without further treatment. Preparation conditions: ChromCore 120C18 (21mm × 250mm, 5μm); A: 0.1% TFA in water, B: 0.1% TFA in MeCN; 18mL / min; 0–3min, 30% B; 3–15min, 30% B; 15–16min, 30–100% B; detector, UV 210nm). Lyophilization under reduced pressure yielded a white solid product RD2358-050-0 (61.92mg, 0.026mmol, 28.37%). ESI-MS m / z = 754.0 [M / 3+H] + ;Calcu.MW=2257.4.

[0606] Example II-1: Preparation of Fc protein (SEQ ID NO:1)

[0607] The nucleotide sequence of the IgG1 Fc protein was obtained through gene synthesis. This nucleotide sequence, carrying a mouse heavy chain antibody signal peptide, was cloned into a eukaryotic expression vector. The plasmid was delivered to Expi293F cells using PEI MAX transfection reagent (Polysciences). After fermentation culture, the cells were centrifuged and filtered to obtain the supernatant. Affinity chromatography was performed using MabSelect PrismA (GE Healthcare, Sweden) affinity chromatography media. Proteins were eluted with 50 mM citrate buffer, and then... Proteins were collected using EMD TMAE Hicap(M) (Millipore, Switzerland) anion exchange chromatography medium in flow-through mode, followed by further impurity removal using Eshmuno CPX (Merck) cation exchange chromatography medium. Finally, HiPrep was used. M The protein was replaced with 20 mM PB (pH 6.8) buffer using a 26 / 10 desalting column (Cytiva), and the final SEC-HPLC purity of the protein was 99.8% (Figure 1).

[0608] Example II-2: Preparation of Fc-PEG4-N3

[0609] Fc protein was displaced into PBS solution (containing 0.5 mM EDTA) using a 30 kD ultrafiltration centrifuge tube (Millipore). A stock solution of 5 mg / mL N3-PEG4-TFP (custom-made by Hunan Huateng Pharmaceutical Co., Ltd.) was prepared using DMSO. Fc protein (5 mg / mL, 120 mg, 2.15 μmol) was added to 8.5 eq of N3-PEG4-TFP (5 mg / mL, 8.04 mg, 18.3 μmol), and the mixture was allowed to react overnight at room temperature with shaking. The sample was then displaced into PBS solution using a 30 kD ultrafiltration centrifuge tube (Millipore) to obtain 100 mg of Fc-PEG4-N3. SEC-HPLC results showed a sample purity of 99.4% (Figure 2); non-reducing LC-MS results showed an average DAR value of 4.3 (Figure 3).

[0610] Example II-3: Preparation of Coupling 2 (i.e., AVC(022) / AVC-022 / AVC022)

[0611] A 25 mM int-2 stock solution was prepared using DMSO. The Fc-PEG4-N3 protein synthesized in the previous step (5 mg / mL, 45 mg, 0.81 μmol) was added to 10 eq of the int-2 stock solution (25 mM, 18.78 mg, 8.1 μmol), followed by the addition of DMSO (305.8 μL). The DMSO content was approximately 6.5% of the total system. The mixture was shaken at room temperature and reacted overnight. The sample was then replaced with 20 mM histidine buffer (pH 5.0) using a 30 kDa ultrafiltration centrifuge tube (Millipore) to obtain 15 mg of AVC(022). SEC-HPLC results showed a sample purity of 99.5% (Figure 4); reducing LC-MS results showed an average DAR value of 3.7 (Figure 5).

[0612] Example II-4: Preparation of Coupling 1 (i.e., AVC(025) / AVC-025 / AVC025)

[0613] A 25 mM int-1 small molecule stock solution was prepared using DMSO. The Fc-PEG4-N3 protein synthesized in the previous step (5 mg / mL, 42.5 mg, 0.77 μmol) was added to 10 eq of the int-1 small molecule stock solution (25 mM, 18.59 mg, 7.7 μmol), followed by the addition of DMSO (288.8 μL). The DMSO content was approximately 6.5% of the total system. The mixture was shaken at room temperature and reacted overnight. The sample was then replaced with 20 mM histidine buffer (pH 5.0) using a 30 kDa ultrafiltration centrifuge tube (Millipore) to obtain 16 mg of AVC(025). SEC-HPLC results showed a sample purity of 99.4% (Figure 6); LC-MS results showed an average DAR value of 3.9 (Figure 7).

[0614] Example II-5: Preparation of AVC(025) with different DAR values

[0615] Fc protein was displaced into PBS solution using 30kD ultrafiltration centrifuge tubes (Millipore). A stock solution of 5 mg / mL N3-PEG4-TFP (custom-made by Hunan Huateng Pharmaceutical Co., Ltd.) was prepared using DMSO. Different molar equivalents of N3-PEG4-TFP (5 mg / mL) were added to each Fc protein (5 mg / mL), and the mixture was shaken at room temperature and reacted overnight. The samples were then displaced into PBS solution using 30kD ultrafiltration centrifuge tubes (Millipore) to obtain Fc-PEG4-N3 with different DAR values.

[0616] A 25 mM int-1 small molecule stock solution was prepared using DMSO. Fc-PEG4-N3 protein (5 mg / mL) with different DAR values ​​synthesized in the previous step was taken and added to different molar equivalents of the int-1 small molecule stock solution (25 mM), followed by the addition of DMSO, with the DMSO content accounting for approximately 6.5% of the total system. The mixture was shaken at room temperature and reacted overnight. The samples were then replaced with 20 mM histidine buffer (pH 5.0) using 30 kDa ultrafiltration centrifuge tubes (Millipore) to obtain AVC(025) with different DAR values. The feed amounts and DAR values ​​are shown in Table II-1.

[0617] Table II-1 Correspondence between different DAR values ​​of Fc-PEG4-N3 and AVC(025) and feed ratio

[0618] Example II-6: Analysis of the binding activity of the conjugate to ceramidinase

[0619] To verify the binding activity of the conjugate to neuraminidase (NA), the half-maximal effective concentration (EC50) of the conjugate to NA was determined using enzyme-linked immunosorbent assay (ELISA). 50 NA protein (Influenza A H1N1, purchased from Sinocare, Cat#11058-VNAHC) was coated onto 96-well plates. Serially diluted conjugates were added and incubated at 37°C for 1 hour. Then, biotin-labeled GAH Fc protein (Goat Anti-Human IgG Fc, Multi-Species SP ads-UNLB, purchased from Southern Biotech, Cat#2014-01) was added and incubated at 37°C for 1 hour. Finally, streptavidin-HRP (purchased from Thermo, Cat#21130) was added and incubated at 37°C for 1 hour. The absorbance values ​​of different concentrations of conjugates were then measured using a microplate reader (MD, SpectraMax190).

[0620] The binding activities of the conjugates AVC(022) and AVC(025) and NA were comparable, with no significant difference (Figure 8). The binding activities of AVC(025) and NA with different DAR values ​​were comparable, with no significant difference (Figure 9).

[0621] Example II-7: ADCC Effect Analysis of Couplings

[0622] To verify whether the conjugate has an ADCC effect, the Jurkat / NFAT-Lucia / FcγRIIIa V158 transgenic cell line stably expressing the Fcγ receptor was used as effector cells (purchased from InvivoGen, catalog number: jktl-nfat-cd16), and HEK293T cells overexpressing neuraminidase (NA enzyme, H5N1) (HEK293T-NA, laboratory-constructed) were used as target cells. The ADCC effect was verified using the QUANTI-Luca assay reagent. TM (Purchased from InvivoGen, catalog number: rep-qlc2) The activity of Lucia luciferase in cell culture supernatant was quantitatively detected to evaluate the ADCC activity of the conjugates. The ADCC activity of the conjugates AVC(022) and AVC(025) was evaluated using this method. Peramivir and Fc protein were used as controls. For ADCC activity evaluation, the conjugates and control molecules were serially diluted. The initial concentration in each well was 100 μg / ml (peramivir was 30 μM, with a mass concentration of approximately 118 μg / ml), followed by 3-fold dilutions, for a total of 12 gradients, including one zero concentration point. The ratio of effector cells to target cells was 1:1. After incubation at 37°C for 18 h, the luciferase activity (RLU) in the cell expression supernatant was detected using a microplate reader (brand: MD, model: iD5).

[0623] The control molecules peramivir and Fc protein showed no ADCC activity signal, while the conjugates AVC(022) and AVC(025) exhibited strong ADCC activity. Under the same DAR value, the EC50 of AVC(022) and AVC(025) was significantly higher. 50 The values ​​are close, indicating that the ADCC activities of the two conjugates are comparable (Figure 10). Furthermore, the ADCC activity of AVC(025) with different DAR values ​​was measured. The results showed that there were significant differences in ADCC activity between AVC(025) with different DAR values; the conjugate with a DAR value of 10.9 had weaker ADCC activity than the conjugate with a DAR value of 4.2 (Figure 11).

[0624] Activity test example:

[0625] The MDCK cells used in this invention were purchased from ATCC; the Neuraminidase enzyme was purchased from Sinocare (catalog number: 11058-VNAHC); 4MuNANA was purchased from MCE (catalog number: HY-W019823); and MES was purchased from BIOFROX (catalog number: 145224-94-8).

[0626] Example III-1: Inhibition of NA enzyme activity by AVC-022

[0627] (1) Take a 96-well plate and add reagents in the following order:

[0628] Reagent A: NA enzyme fluorescent substrate working solution;

[0629] Reagent B: MES buffer;

[0630] Drugs to be tested: AVC-022 at different concentrations;

[0631] Incubate at room temperature for 30 minutes;

[0632] (2) Add 50 μL of NA enzyme fluorescent substrate working solution to each well and incubate at 37°C for 1 hour;

[0633] (3) Excitation light was 355nm and emission light was 460nm. Fluorescence intensity was detected.

[0634] Data processing:

[0635] As shown in Figure 12, AVC-022 has a significant inhibitory effect on NA enzyme activity, and its EC50... 50 It is 1.233 nM.

[0636] Example III-2: In vitro antiviral effect of AVC-022

[0637] (1) Cell preparation: Seed MDCK cells in a 96-well black transparent bottom cell plate and culture them in a cell culture incubator until the density reaches 70-80% before use;

[0638] (2) Drug dilution: The dissolved candidate drug was diluted to eight concentration gradients using 2 μg / mL TPCK opti-MEM medium. (The final working concentration was halved.)

[0639] Oseltamivir concentration gradient: 0.00006, 0.0006, 0.006, 0.06, 0.3, 0.6, 6, 60 μg / mL

[0640] Drug concentration gradient: 0.0006, 0.006, 0.06, 0.6, 3, 6, 60, 600 μg / mL

[0641] (3) Virus dilution: Dilute the stock virus solution to 30 TCID50 / 50 μl using 2 μg / mL TPCK opti-MEM medium. (The final working concentration is 30 TCID50 / 50 μl)

[0642] (4) Viral infection and addition of test drugs: The drug dilutions and virus dilutions of different concentration gradients in 2) were mixed at a ratio of 1:1 and incubated at 37°C for 60 min. 100 μl of the mixture was added to each cell well and 3 replicates were set up. Cell control (CC) and virus control (VC) were set up and cultured in a CO2 incubator for 72 h.

[0643] (5) Results detection: Cell viability was detected in an ELISA reader using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit.

[0644] (6) Results Analysis: The inhibition rate was calculated using Microsoft Office: Inhibition rate (%) = (OD experimental group - OD virus control) / (OD cell control - OD virus control) × 100%; the viral inhibition curve was fitted using GraphPad Prism 10, and the drug EC50 was calculated. 50 .

[0645] Data processing:

[0646] The results are shown in Figures 13-20. Figures 13-16 show the inhibitory effects of AVC-022 on four strains of Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1), A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria Lineage), respectively, and their EC50 values ​​are also shown. 50 The effective concentrations were 4.718 ng / mL (0.0716 nM), 92.76 ng / mL (1.408 nM), 9.235 ng / mL (0.140 nM), and 2.270 ng / mL (0.0344 nM), respectively. Figures 17-20 show the inhibition curves of oseltamivir against the four strains, with their EC50 values... 50 The effective concentrations were 2.442 μg / mL (8588 nM), 0.1323 μg / mL (465 nM), 0.002564 μg / mL (9.02 nM), and 0.3244 μg / mL (1140 nM), respectively. The results showed that AVC-022 had a significantly better antiviral effect than oseltamivir.

[0647] Example III-3: Inhibition of NA enzyme activity by AVC-025

[0648] The effect of AVC-025 on NA enzyme activity was verified according to Example III-1. The results are shown in Figure 21. AVC-025 has a significant inhibitory effect on NA enzyme activity, with an EC50 of 1.299 nM.

[0649] Example III-4: In vitro antiviral effect of AVC-025

[0650] The inhibitory effect of AVC-025 on four influenza virus strains was verified according to Example III-2. The results are shown in Figures 22-25. AVC-025 showed the EC50 of four influenza virus strains: Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1), A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria Lineage). 50 The effective concentrations were 3.438 ng / mL (0.0516 nM), 90.76 ng / mL (1.362 nM), 3.599 ng / mL (0.054 nM), and 1.035 ng / mL (0.0155 nM), respectively. These were significantly superior to the inhibitory effects of oseltamivir on the four strains (Figures 17-20).

[0651] Example III-5: In vitro antiviral effects of AVC-025 with different DAR values

[0652] The inhibitory effect of AVC-025 with different DAR values ​​on four influenza virus strains was verified according to Example III-2. The results are shown in Annexes 26-1—26-7, 27-1—27-7, 28-1—28-7, and 29-1—29-7. AVC-025 with DAR values ​​of 2.1, 4.2, 6.2, 9.1, and 10.9 showed inhibitory effects on the four strains of Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1), A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria Lineage). 50 As shown in Table III-1, its antiviral effect is significantly better than that of peramivir and mabaloxavir.

[0653] Table III-1

[0654] Example III-6: In vivo viral prevention experiment of AVC

[0655] The preventive efficacy of the AVC drug of this invention against influenza viruses such as H1N1, H3N2, H5N1, and influenza B was evaluated in BALB / c, DBA / 2, and SCID mouse models. The challenge virus was a strain capable of causing lethal infection in mice. Both the model group and the experimental group of Balb / c mice were administered a negative control and the AVC drug. The negative control in the model group was saline, and the AVC drug in the experimental group was AVC-022. Each experimental group consisted of 12 animals. On Day 0, each group received a single intravenous injection of 3 mpk of AVC-022 via the tail vein, while the model group received an equal volume of saline via the tail vein on Day 0. Influenza virus strain A / California / 04 / 2009 was administered intranasally on Days 15, 30, 45, and 60 (nasal challenge), with a challenge titer of 4.33 lg TCID50 / 50 μL / animal. Mice were monitored daily for body weight and survival rate after drug administration. Animals with more than 25% weight loss or those near death were considered dead. Observation continued for 75 days. Six mice were sacrificed on days 21, 36, 51, and 66, and their bronchoalveolar lavage fluid and lung tissue were collected for viral load detection and histopathological examination. The specific experimental results are as follows:

[0656] D15 challenge test

[0657] The G1 control group (no virus modeling), G2 model group, and G3 experimental group 1 were challenged with intranasal drops on day 15 after drug administration. Weight change rate and survival rate were monitored from day 15 to day 75. Results showed that the weight of the control group fluctuated within the normal range and generally increased steadily, with a survival rate of 100%; experimental group 1 experienced only a slight weight loss 2-3 days after challenge, followed by recovery, with a survival rate of 100%; all members of the model group died after challenge, with a survival rate of 0% (see Figure III-6-1).

[0658] D30 Virus Challenge Test

[0659] In experimental group G4, mice were challenged with intranasal administration of the virus on day 30 (d30) after drug administration. The rate of change in body weight and survival rate were monitored from day 30 to day 75. The results showed that some mice experienced a slight decrease in body weight 2-6 days after challenge, which then gradually recovered to normal levels. Overall, the body weight showed an increasing trend, and the survival rate was 100% (Figure III-6-2).

[0660] D45 challenge test

[0661] In experimental group G5, mice were challenged with intranasal administration of the virus on day 45 (d45) after drug administration. The rate of weight change and survival rate were monitored from day 45 to day 75. The results showed that some mice experienced a slight decrease in weight 2-6 days after challenge, which then gradually recovered to normal levels. Overall, the weight showed an increasing trend, and the survival rate was 67% (Figure III-6-3).

[0662] D60 challenge test

[0663] Both the G2 model group and the G6 experimental group were challenged with intranasal drops on day 60 (d60) after drug administration. Body weight change and survival rates were monitored from day 60 to day 75. Results showed that the model group experienced a body weight loss of approximately 20%-40% and a survival rate of 0%; the G6 experimental group experienced body weight loss 2-7 days after challenge, with weight gradually recovering to pre-challenge levels from approximately day 8, and a survival rate of 83% (Figure III-6-4).

[0664] Virus titer detection in mice after prophylactic administration

[0665] The mice were divided into three groups: G1 (blank group), G2 (model group), and G3–6 (experimental groups) (challenged on days 15, 30, 45, and 60, respectively). Six mice were challenged with the A / California / 04 / 2009 strain (4.33lg TCID50 / 50μL / mouse) for six days. BLA (bronchoalveolar lavage fluid) was collected from each group, and viral titer was detected using the TCID50 method. Results showed that no live virus was detected in the blank group; high viral titers were detected in all model groups; and viral titers were significantly lower in the experimental groups. Specifically, no virus was detected in mouse samples challenged on days 15 and 30, while viral titers in mouse samples challenged on days 45 and 60 were still significantly lower than in the model group (Figure III-6-5).

[0666] Example III-7: In vivo antiviral therapy for AVC

[0667] The antiviral therapeutic effects of the AVC conjugate of this invention against influenza viruses such as H1N1, H3N2, H5N1, and influenza B were evaluated in mouse models including BALB / c, DBA / 2, and SCID. The challenge viruses were viral strains capable of causing lethal infection in mice. Mice were challenged with influenza virus via nasal drop. After challenge, negative controls, positive controls, and the AVC drug were administered. Mouse weight and survival rate were monitored daily. Animals with more than 25% weight loss or near death were considered dead. Observation continued for 14 days. On day 6, six mice were sacrificed, and their bronchoalveolar lavage fluid and lung tissue were collected for viral load detection and histopathological examination.

[0668] To evaluate the in vivo antiviral efficacy of AVC-022, mice were infected with A / California / 07 / 2009 virus via intranasal instillation, and AVC-022 (0.05 mpk) was administered intravenously in a single dose 2 hours post-infection. The mice were divided into three groups, with six mice in each group. Body weight and survival rate were monitored for 14 days post-infection. Results showed that the AVC-022 group exhibited significant antiviral efficacy with a survival rate of 83%, while the model group had a survival rate of 0%. Furthermore, the body weight loss in the AVC-022 group was significantly less than that in the model group. In conclusion, AVC-022 demonstrates good in vivo antiviral activity even at a relatively low dose (0.05 mpk) (Figure III-7-1).

[0669] Example III-8: In vivo safety evaluation of AVC

[0670] The in vivo safety of the AVC conjugate AVC-022 of this invention was evaluated in animal models including mice, rats, and monkeys. AVC-022 was subcutaneously injected into the animals on days 1, 8, and 15, and the animals' health status, weight, and other indicators were continuously monitored. After monitoring, the animals were sacrificed, and their major organs were collected for morphological, weight, pathological, and other indicator tests. Specific descriptions and results are as follows and are shown in Tables III-8-1 and III-8-2.

[0671] Table III-8-1 Safety Study of Subcutaneous Injection of AVC-022 SD Rats with the AVC Conjugate of the Present Invention

[0672] Following repeated subcutaneous injections of 60 and 200 mg / kg of AVC-022 into SD rats, no abnormalities related to the test substance were observed in clinical observation, body weight, hematology, coagulation, serum biochemistry, CD3 / 4 / 8 cell typing, gross anatomical observation, or histopathology (Table III-8-2). No drug-related abnormalities were observed at the administration site during gross anatomical observation and microscopic examination.

[0673] Table III-8-2 Safety data of the AVC conjugate AVC-022 SD rat subcutaneous injection in this invention.

[0674] III-8-3 Safety Study of AVC-022 Conjugate of the Invention in Rhesus Monkeys via Subcutaneous Injection

[0675] Following repeated subcutaneous injections of 60 and 200 mg / kg of AVC-022 into rhesus monkeys, no abnormalities related to the test substance were observed in clinical observation, body weight, hematology, coagulation, serum biochemistry, CD3 / 4 / 8 cell typing, gross anatomical observation, or histopathology (Ⅲ-8-4). Gross anatomical observation and microscopic examination of the administration site also revealed no drug-related abnormalities.

[0676] Table III-8-4 Safety test data of AVC-022, the AVC conjugate of the present invention, for subcutaneous injection in rhesus monkeys.

[0677] Example III-9: In vivo pharmacokinetic assay of AVC

[0678] The pharmacokinetic data, including the in vivo half-life, of the AVC conjugate of this invention were evaluated in animal models such as mice, rats, and monkeys. In rhesus monkeys, a single intravenous injection of 5 mg / L of the conjugate AVC-022 of this invention was administered, and blood samples were collected at different time points after administration to analyze the half-life, AUC, metabolites, and other indicators of AVC-022.

[0679] AVC-022 was administered at a dose of 5 mg / kg via intravenous injection (IV), with a single dose of 10 mL / kg. Plasma samples were collected at 4 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, 288 h, 360 h, 432 h, 504 h, 576 h, 672 h, and 796 h post-administration for bioanalysis. The results showed that AVC-022 has a relatively long half-life of approximately 319 h (Figure III-9-1).

[0680] Table III-9-1 Metabolic kinetic parameters of AVC-022 in rhesus monkey plasma

[0681] Example III-10a: In vivo therapeutic efficacy evaluation data

[0682] In BALB / c mice, influenza virus (A / California / 07 / 2009) with an LD50 of 10x was administered intranasally. Two hours after challenge, mice were given the conjugate AVC-025 intravenously (0.05, 0.15, 0.5, and 5 mg / kg). The positive control, baloxavir, was administered orally (5 mg / kg). Changes in body weight and survival time were measured, and the viral load in bronchoalveolar lavage fluid was measured on day 6 post-treatment. The results demonstrated that AVC-025 had a superior antiviral effect compared to baloxavir (Figures 30-1–30-3).

[0683] Example III-10b: In vivo therapeutic efficacy evaluation data

[0684] In BALB / c mice, 10xLD50 of influenza virus (A / California / 07 / 2009) was administered intranasally. Two hours after challenge, the conjugate AVC-022 (0.05 mg / kg) was administered intravenously. Changes in body weight and survival time of the mice were then detected (Figures 30-4-30-5).

[0685] Example IV-1 Preparation of Fc-PEG4-N3

[0686] Fc protein was displaced into PBS solution (containing 12 mM PB + 137 mM NaCl, pH 7.4) using a 30 kDa ultrafiltration centrifuge tube (Millipore). A stock solution of 5 mg / mL N3-PEG4-TFP (custom-made by Hunan Huateng Pharmaceutical Co., Ltd.) was prepared using DMSO. Fc protein (5 mg / mL, 343 mg, 6.17 μmol) was added to 9.4 eq of N3-PEG4-TFP (5 mg / mL, 25.49 mg, 58.0 μmol), and the mixture was allowed to react overnight at room temperature with shaking. The sample was then displaced into PBS solution (containing 50 mM PB + 137 mM NaCl, pH 7.4) using a 30 kDa ultrafiltration centrifuge tube (Millipore) to obtain the Fc-PEG4-N3 intermediate. SEC-HPLC results showed that the sample purity was 99.6% (Table IV-1, Figure IV-1); non-reducing LC-MS results showed that the average DAR value was 4.6 (Table IV-1, Figure IV-2).

[0687] Example IV-2 Preparation of conjugates (AVC(042), AVC(044), AVC(049), AVC(050))

[0688] 10 mM stock solutions of int-8, int-9, int-6, and int-7 were prepared using DMSO. The Fc-PEG4-N3 intermediate synthesized in the previous step (5 mg / mL) was added to 12 eq of the 10 mM stock solutions of int-8, int-9, int-6, and int-7, with DMSO accounting for approximately 10% of the total system. The mixture was shaken at room temperature and reacted overnight. The samples were then replaced with 20 mM histidine buffer (pH 5.0) using 30 kDa ultrafiltration centrifuge tubes (Millipore) to obtain AVC(042), AVC(044), AVC(049), and AVC(050) samples. The SEC-HPLC and LC-MS results are summarized in Table IV-1. SEC-HPLC chromatograms are shown in Figures IV-3, IV-5, IV-9, and IV-11; LC-MS chromatograms are shown in Figures IV-4, IV-6, IV-10, and IV-12.

[0689] Example IV-3 Preparation of the conjugate (AVC(048))

[0690] A 5 mM int-5 small molecule stock solution was prepared using DMSO. The Fc-PEG4-N3 protein synthesized in the previous step (5 mg / mL) was added to 12 eq of the int-5 small molecule stock solution (5 mM), followed by the addition of DMSO, bringing the DMSO content to approximately 20% of the total system. The mixture was shaken at room temperature and reacted overnight. The sample was then replaced with 20 mM histidine buffer (pH 5.0) using a 30 kDa ultrafiltration centrifuge tube (Millipore) to obtain the AVC(048) sample. The SEC-HPLC and LC-MS results are summarized in Table IV-1. The SEC-HPLC chromatogram is shown in Figure IV-7; the LC-MS chromatogram is shown in Figure IV-8.

[0691] Table IV-1 Summary of AVC Sample Results

[0692] Example V-1: In vitro antiviral effect of AVC-042

[0693] (1) Cell preparation: Seed MDCK cells in a 96-well black transparent bottom cell plate and culture them in a cell culture incubator until the density reaches 70-80% before use;

[0694] (2) Drug dilution: The dissolved candidate drug was diluted to six concentration gradients using 2 μg / mL TPCK opti-MEM medium.

[0695] Drug concentration gradient: 0.0000018, 0.00018, 0.018, 1.8, 18, 180 μg / mL

[0696] (3) Virus dilution: Dilute the stock virus solution to 30 TCID50 / 50 μl using 2 μg / mL TPCK opti-MEM medium. (The final working concentration is 30 TCID50 / 50 μl)

[0697] (4) Viral infection and addition of test drugs: The drug dilutions and virus dilutions of different concentration gradients in 2) were mixed at a ratio of 1:1 and incubated at 37°C for 60 min. 50 μl of the mixture was added to each cell well, and 3 replicates were set up. At the same time, cell control (CC) and virus control (VC) were set up and cultured in a CO2 incubator for 72 h. Therefore, the final drug concentration gradient was: 0.0000003, 0.00003, 0.003, 0.3, 3, 30 μg / mL.

[0698] (5) Result detection: using CellTiter-Lumi TM The Plus luminescent cell viability assay kit is used to detect cell viability in an ELISA reader.

[0699] (6) Results Analysis: The inhibition rate was calculated using Microsoft Office: Inhibition rate (%) = (OD experimental group - OD virus control) / (OD cell control - OD virus control) × 100%; the viral inhibition curve was fitted using GraphPad Prism 10, and the drug EC50 was calculated. 50 .

[0700] Data processing:

[0701] The results are shown in Table V-1. AVC-042 showed positive ECGs for two strains: Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 0.001 nM and 0.761 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-1-1 and V-1-2).

[0702] Example V-2: In vitro antiviral effect of AVC-044

[0703] The inhibitory effect of AVC-044 on two influenza virus strains was verified according to Example V-1. The results are shown in Table V-1. AVC-044 showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 0.007 nM and 1.554 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-2-1 and V-2-2).

[0704] Example V-3: In vitro antiviral effect of AVC-048

[0705] The inhibitory effect of AVC-048 on two influenza virus strains was verified according to Example V-1. The results are shown in Table V-1. AVC-048 showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 0.021 nM and 2.277 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-3-1 and V-3-2).

[0706] Example V-4: In vitro antiviral effect of AVC-049

[0707] The inhibitory effect of AVC-049 on two influenza virus strains was verified according to Example V-1. The results are shown in Table V-1. AVC-049 showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The values ​​were 0.065 nM and could not be calculated. Its inhibitory effect on the Influenza Virus Infectious A / Puerto Rico / 8 / 1934(H1N1) strain was significantly better than that of oseltamivir (Figure V-4-1, Figure V-4-2).

[0708] Example V-5: In vitro antiviral effect of AVC-050

[0709] The inhibitory effect of AVC-050 on two influenza virus strains was verified according to Example V-1. The results are shown in Table V-1. AVC-050 showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 8.605 nM and 1.103 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-5-1 and V-5-2).

[0710] Example V-6: In vitro antiviral efficacy of RD2358-042-0(int-8)

[0711] (1) Cell preparation: Seed MDCK cells in a 96-well black transparent bottom cell plate and culture them in a cell culture incubator until the density reaches 70-80% before use;

[0712] (2) Drug dilution: The dissolved candidate drug was diluted to six concentration gradients using 2 μg / mL TPCK opti-MEM medium.

[0713] Drug concentration gradient: 0.00000018, 0.000018, 0.0018, 0.18, 1.8, 18 μg / mL

[0714] (3) Virus dilution: Dilute the stock virus solution to 30 TCID50 / 50 μl using 2 μg / mL TPCK opti-MEM medium. (The final working concentration is 30 TCID50 / 50 μl)

[0715] (4) Viral infection and addition of test drugs: The drug dilutions and virus dilutions of different concentration gradients in 2) were mixed at a ratio of 1:1 and incubated at 37°C for 60 min. 100 μl of the mixture was added to each cell well and 3 replicates were set up. At the same time, cell control (CC) and virus control (VC) were set up and cultured in a CO2 incubator for 72 h. Therefore, the final drug concentration gradient was: 0.00000003, 0.000003, 0.0003, 0.03, 0.3, 3 μg / mL.

[0716] (5) Result detection: using CellTiter-Lumi TM The Plus luminescent cell viability assay kit is used to detect cell viability in an ELISA reader.

[0717] (6) Results Analysis: The inhibition rate was calculated using Microsoft Office: Inhibition rate (%) = (OD experimental group - OD virus control) / (OD cell control - OD virus control) × 100%; the viral inhibition curve was fitted using GraphPad Prism 10, and the drug EC50 was calculated. 50 .

[0718] Data processing:

[0719] The results are shown in Table V-1. RD2358-042-0(int-8) is used for the EC50 analysis of two strains: Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 0.018 nM and 5.439 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-6-1 and V-6-2).

[0720] Example V-7: In vitro antiviral efficacy of RD2358-044-0(int-9)

[0721] The inhibitory effect of RD2358-044-0(int-9) on two influenza virus strains was verified according to Example V-6. The results are shown in Table V-1. RD2358-044-0(int-9) showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 24.73 nM and 10.29 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-7-1 and V-7-2).

[0722] Example V-8: In vitro antiviral efficacy of RD2358-048-0(int-5)

[0723] The inhibitory effect of RD2358-048-0(int-5) on two influenza virus strains was verified according to Example V-6. The results are shown in Table V-1. RD2358-048-0(int-5) showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The effective doses were 1.692 nM and 4.827 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-8-1 and V-8-2).

[0724] Example V-9: In vitro antiviral efficacy of RD2358-049-0(int-6)

[0725] The inhibitory effect of RD2358-049-0(int-6) on two influenza virus strains was verified according to Example V-6. The results are shown in Table V-1. RD2358-049-0(int-6) showed an EC50 of 1,500 against both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The concentrations were 0.072 nM and 6.112 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on both strains (Figures V-9-1 and V-9-2).

[0726] Example V-10: In vitro antiviral efficacy of RD2358-050(int-7)

[0727] The inhibitory effect of RD2358-050(int-7) on two influenza virus strains was verified according to Example V-6. The results are shown in Table V-1. RD2358-050(int-7) showed an EC50 inhibitory effect on both Influenza Virus Infectious A / Puerto Rico / 8 / 1934 (H1N1) and Influenza A virus (A / Aichi / 2 / 1968 (H3N2)). 50 The effective doses were 0.285 nM and 7.121 nM, respectively. These were significantly better than the inhibitory effects of oseltamivir on the two strains (Figure V-10-1, Figure V-10-2).

[0728] Table V-1

Claims

1. An antibody-drug conjugate of formula (I-1), a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite thereof: in, A1, A2, and A3 may be the same or different and each can be independently selected from equations (AI), (A-II), (A-III), and (A-IV): Among them, R 1 Selected from -OH, -NH2, -NHC(=NH)NH2 or -NHC(=NH)NHR a ; R 2 Selected from -COOH, -P(=O)(OH)2, or -SO3H; R 3 Selected from -COCH3, -COCF3, or -SO2CH3; R 5 Selected from hydrogen or C1-C6 alkyl groups; L3 can be attached to any possible site in compounds of formula (A-IV); X is selected from -O- or -S-; Y is selected from -O-, -S-, -NR b -、-O(C=O)NR b -、-O(C=S)NR b -, -O(C=O)O-, -O(C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR b -, -NH(C=NH)NR b -, -NH(C=S)NR b -, -NH(C=S)-, -OCH2(C=O)NR b -,-NH(SO2)-,-NH(SO2)NR b -、-OR b -、-NHR b -or-SR b -; R a Selected from R b Selected from H, C1-C 20 Alkyl, C3-C 20 cycloalkyl, C3-C 20 Heterocyclic alkyl, C6-C 12 Aryl or C5-C 12 Mixed aromatics; L3 is selected from Among them, L a Selected from -(CH2) y -、 y is selected from integers from 1 to 10; R 4 Selected from hydrogen or C1-C6 alkyl groups; L2 is selected from Where x is selected from 1, 2, 3, 4 or 5; Q is selected from: The Any reasonable site is optionally selected by one or more sites chosen from R c The R group is replaced by the group that replaces it. c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatics; L1 is selected from z can be selected from 1, 2, 3, 4, 5, or 6; E includes Fc domain monomers, Fc domain dimers, albumin, albumin-binding peptides, or Fc-binding peptides. n is selected from 1 or 2; T is an integer from 1 to 20.

2. The coupling compound according to claim 1, wherein, A1, A2, and A3 may be the same or different and each can be independently selected from equations (AI), (A-II), (A-III), and (A-IV): Among them, R 1 Selected from -OH, -NH2, or -NHC(=NH)NH2; L3 can be attached to any possible site in compounds of formula (A-IV); Preferably, R 1 Selected from -NHC(=NH)NH2; R 2 Selected from -COOH; R 3 Selected from -COCH3, -COCF3; Preferably, R 3 Selected from -COCH3; R 5 Selected from hydrogen, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; Preferably, R 5 Selected from hydrogen, methyl, ethyl, propyl, isopropyl, or tert-butyl; Preferably, R 5 Selected from hydrogen, methyl, or ethyl; Preferably, R 5 Selected from ethyl; X is selected from -O-; Y is selected from -O-, -S-, -NR b -、-O(C=O)NR b -、-O(C=S)NR b -, -O(C=O)O-, -O(C=O)-, -NH(C=O)O-, -NH(C=O)-, -NH(C=NH)-, -NH(C=O)NR b -, -NH(C=NH)NR b -, -NH(C=S)NR b -, -NH(C=S)-, -OCH2(C=O)NR b -,-NH(SO2)-,-NH(SO2)NR b -、-OR b -、-NHR b -or-SR b -; R b Selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, or C6-C 10 Aryl; Preferably, Y is selected from -O-, -S-, or -NR. b -;R b Selected from H, or C1-C6 alkyl; Preferably, Y is selected from -O-, -S-, -NH-, or -N(CH3)-; Preferably, Y is selected from -O-, -S-, or -NH-; Preferably, Y is selected from -O- or -NH-.

3. The coupling compound according to claims 1-2, wherein, A1, A2, and A3 may be the same or different and each can be selected independently. Preferably, A1, A2, and A3 are each independently selected from... Preferably, A1, A2, and A3 are all selected from... Preferably, A1, A2, and A3 are all selected from... Preferably, A1 and A2 are both selected from... Preferably, A3 is selected from Preferably, A1, A2, and A3 are all selected from... Preferably, A1 and A2 are both selected from... Preferably, A3 is selected from 4. The coupling according to claim 1, wherein, y is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, y is selected from 1, 2, 3, 4, 5, 6, 7; Preferably, y is selected from 1, 2, 3, 4, and 5; Preferably, L a Selected from -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, or Preferably, L a Selected from -CH2-, or R 4 Selected from hydrogen, methyl, ethyl, propyl, or isopropyl; Preferably, R 4 Selected from hydrogen or methyl; Preferably, L3 is selected from: Preferably, L3 is selected from: Preferably, L3 is selected from 5. The coupling according to claim 1, wherein, x is selected from 3, 4, and 5; Preferably, x is selected from 3; L2 is selected from 6. The coupling according to any one of claims 1-5, wherein, Q is selected from: The Any reasonable site is optionally selected by one or more sites chosen from R c The R group is replaced by the group that replaces it. c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl; Preferably, the The benzene ring in the sample is optionally surrounded by one or more elements selected from R c The groups are replaced; Preferably, the The cyclooctane moiety in R is optionally occupied by one or more reasonable sites selected from R c The groups are replaced; Preferably, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -SO2, -NH2, -NO2, methyl, ethyl, cyclopropyl, or phenyl; Preferably, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, or -I; Preferably, the R c Each is independently selected from hydrogen or -F; Preferably, Q is selected from 7. The coupling according to claim 6, wherein, z is selected from 3, 4, and 5; Preferably, z is selected from 4; Preferably, L1 is selected from 8. The coupling according to any one of claims 1-7, wherein, T is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, T is selected from 1, 2, 3, 4, and 5.

9. The coupling according to any one of claims 1-8, wherein, E contains Fc domain monomers or Fc domain dimers; n is selected from 1 or 2; Preferably, E is an Fc domain monomer or dimer, and each Fc domain monomer independently has the sequence of any one of SEQ ID NO:1-69; Preferably, E is an Fc domain monomer or dimer, each Fc domain monomer independently having a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to any of the sequences in SEQ ID NO:1-69; Preferably, E is an Fc domain monomer or dimer, and each Fc domain monomer independently has the sequence of SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:19; Preferably, E is an Fc domain monomer having a sequence of SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:19; Preferably, E is an Fc domain dimer, and each Fc domain monomer independently has the sequence of SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:

19.

10. The coupling according to any one of claims 1-8, wherein, E represents albumin, albumin-binding peptide, or Fc-binding peptide; Preferably, n is 1; Preferably, E is albumin having the sequence of any one of SEQ ID NO:70-71; Preferably, E is a protein having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of SEQ ID NO:70-71.

11. The coupling agent according to any one of claims 1-10, selected from:

12. An intermediate M, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite thereof, for synthesizing the antibody-drug conjugate of any one of claims 1-11, wherein intermediate M is formed by linking a cyclooctyne derivative and a small drug molecule via a linkage bond. Cyclooctyne derivatives - linking bonds - small drug molecules (monomers, dimers, or trimers) intermediate M in, Cyclooctyne derivatives include cyclooctyne structures or derivatives thereof, wherein the C atom in the cyclooctyne structure or derivatives thereof may optionally be replaced by one or more atoms selected from N and O; The small molecule portion of a drug can be in the form of a monomer, dimer, or trimer. Each part of the small molecule monomer, dimer, or trimer is independently selected from formula (A1), (A-II), (A-III), or (A-IV): L3 can be linked to any possible site in compounds of formula (A-IV); R 1 R 2 R 3 R 5 X, Y are as defined in any one of claims 1-10; Connecting bonds can be single, double, or triple bonds, including carbon-carbon bonds, carbon-oxygen bonds, carbon-nitrogen bonds, sulfur bonds, or other covalent bonds. Atoms in a connecting bond can form a ring structure through covalent bonds.

13. The intermediate M of claim 12, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof, in, Cyclooctyne derivatives are selected from formulas (MI), (M-II), and (M-III). G1, G2, and G3 are each independently selected from C3-C8 cycloalkyl, C3-C8 heterocyclic, and C6-C 12 Aryl, or C5-C 12 Mixed aromatics; W is selected from carbon atoms and nitrogen atoms; L4 is selected from -(C=O)CH₂CH₂-, -(C=O)CH₂CH₂CH₂-, -(C=O)CH₂CH₂CH₂CH₂-, -CH₂-O-, -CH₂CH₂-O-, -CH₂CH₂CH₂-O-, -O-, -NH-, -N(CH₃)-, -CH₂-NH-, -CH₂CH₂-NH-, Any reasonable site of formulas (MI), (M-II), and (M-III) can be optionally occupied by one or more R c Replace, R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatics; Preferably, G1, G2, and G3 are each independently selected from C3-C8 cycloalkyl and C6-C8 aryl groups; Preferably, G1, G2, and G3 are each independently selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl; Preferably, G1, G2, and G3 are each independently selected from cyclopropyl or phenyl; Preferably, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, -(C=O)CH2CH2CH2-、-(C=O)CH2CH2CH2CH2-、-CH2CH2-O-、-CH2CH2CH2-O-、 Preferably, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, -(C=O)CH2CH2CH2-, -CH2CH2-O-, or Preferably, L4 is selected from -(C=O)CH2CH2-, -CH2-O-, -O-, or Preferably, R c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl; Preferably, R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -NH2, methyl, or ethyl; Preferably, R c Each is independently selected from hydrogen, -F, -Cl, -Br, or methyl; Preferably, R c Each is independently selected from hydrogen or -F; Preferably, the cyclooctyne derivative is selected from...

14. The intermediate M of claim 12, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof, in, The small molecule portion of a drug can be in the form of a monomer, dimer, or trimer. Preferably, each part of the drug small molecule monomer or dimer or trimer is independently selected from... Preferably, each part of the drug small molecule monomer or dimer or trimer is selected from... Preferably, each part of the drug small molecule trimer is selected from... Preferably, each part of the drug small molecule dimer is selected from... Preferably, the drug small molecule monomer is selected from... Preferably, each part of the drug small molecule trimer is selected from... Preferably, each part of the drug small molecule dimer is selected from... Preferably, the drug small molecule monomer is selected from...

15. The intermediate M of claim 12, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof, in, The connection key is selected from -L2-L3-, where L2 and L3 are defined as in any one of claims 1-10; Preferably, L2 is selected from Preferably, L3 is selected from: Preferably, -L2-L3- is selected from: Preferably, -L2-L3- is selected from: Preferably, -L2-L3- is selected from:

16. The intermediate M of claim 12, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof, can be represented by formula (II-1). in, T, A1, A2, A3, L3, L2 are as defined in any one of claims 1-10; Q' is selected from: The Any reasonable site is optionally selected by one or more sites chosen from R c The R group is replaced by the group that replaces it. c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C 12 Alkyl, C3-C 12 cycloalkyl, C6-C 12 Aryl, or C5-C 12 Mixed aromatic compounds.

17. The intermediate of claim 16, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite form thereof, wherein, Q' is selected from: The Any reasonable site is optionally selected by one or more sites chosen from R c The R group is replaced by the group that replaces it. c Each is independently selected from hydrogen, halogen, -OH, -SO2, -NH2, -NO2, C1-C6 alkyl, C1-C6 alkyl, C3-C8 cycloalkyl, or C6-C 12 Aryl; Preferably, the The benzene ring in the sample is optionally surrounded by one or more elements selected from R c The groups are replaced; Preferably, the The α-position of the alkyne bond in the α-position is optionally occupied by one or more bonds selected from R. c The groups are replaced; Preferably, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, -I, -OH, -SO2, -NH2, -NO2, methyl, ethyl, cyclopropyl, or phenyl; Preferably, the R c Each is independently selected from hydrogen, -F, -Cl, -Br, or -I; Preferably, the R c Each is independently selected from hydrogen or -F; Preferably, Q' is selected from: Preferably, Q' is selected from: Preferably, Q' is selected from Preferably, Q' is selected from: Preferably, Q' is selected from:

18. The intermediate, pharmaceutically acceptable salt thereof, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label, or metabolite thereof as described in any one of claims 12-17, selected from:

19. A method for preparing an antibody-drug conjugate as described in any one of claims 1-11, comprising, a: By reacting the amino group on the lysine residue of the Fc protein with the N3-PEG4-TFP small molecule in a neutral buffer, and controlling the molar ratio of Fc to N3-PEG4-TFP during the reaction, Fc-PEG4-N3 intermediates with different DAR values ​​were obtained. b: By reacting the azide group on the Fc-PEG4-N3 intermediate with the alkyne group on the corresponding small molecule intermediate (Int 1-9), coupling samples with different DAR values ​​were obtained by controlling the Fc-PEG4-N3 intermediates with different DAR values.

20. A pharmaceutical composition comprising the conjugate of any one of claims 1-11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

21. A method for preventing and / or inhibiting or treating a subject who has or is presumed to have a viral infection, the method comprising administering to the subject an effective amount of the conjugate or a combination thereof as described in any one of claims 1-11.

22. The method of claim 21, wherein the viral infection is caused by an influenza virus, a parainfluenza virus, or an avian influenza virus.

23. The method of any one of claims 21-22, wherein the viral infection is influenza A, B, or C virus or parainfluenza virus.

24. The method of any one of claims 21-23, wherein the subject is immunocompromised.

25. The method of any one of claims 21-24, wherein the subject has been diagnosed with humoral immune deficiency, T-cell deficiency, neutropenia, asplenia, or complement deficiency.

26. The method of any one of claims 21-25, wherein the subject is being treated with or is about to be treated with immunosuppressive therapy.

27. The method of any one of claims 21-26, wherein the subject has been diagnosed with a disease that causes immunosuppression.

28. The method of claim 27, wherein the disease is cancer or acquired immunodeficiency syndrome.

29. The method of claim 28, wherein the cancer is leukemia, lymphoma, or multiple myeloma.

30. The method of any one of claims 21-29, wherein the subject has received or is about to receive a hematopoietic stem cell transplant.

31. The method of any one of claims 21-30, wherein the subject has received or is about to receive an organ transplant.

32. The method of any one of claims 21-31, wherein the conjugate or composition is administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, via the surface, intratumorally, via the peritoneum, subcutaneously, subconjunctivally, intracysticly, via the mucosa, intraperitoneally, intraumbilically, intraocularly, via the mouth, via local, by inhalation, by injection, or by infusion.

33. The method of any one of claims 21-32, wherein the subject may be further treated with a second therapeutic agent.

34. The method of claim 33, wherein the second therapeutic agent is an antiviral agent.

35. The method of claim 34, wherein the antiviral agent is selected from oseltamivir, zanamivir, peramivir, lanimivir, amantadine, rimantadine, or mabaloxavir.

36. The method of claim 33, wherein the second therapeutic agent is an antiviral vaccine.

37. The method of claim 36, wherein the antiviral vaccine induces an immune response against influenza A, B, or C virus or parainfluenza virus in the subject.

38. Combined products, including: The conjugates of Formula I-1, their pharmaceutically acceptable salts, prodrugs, stereoisomers, tautomers, hydrates, solvates, crystal forms, isotopic markers or their metabolite forms, and any one or more antiviral substances.

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