Multi-arm polyethylene glycol derivative and bio-related substance modified therewith

Abstract

Disclosed in the present application is a heterofunctional four-arm polyethylene glycol derivative as shown in formula (1), containing at least two different terminal reactive groups or protected forms thereof. Also disclosed in the present application is a conjugate formed by coupling the four-arm polyethylene glycol derivative with a targeting moiety (e.g., folic acid) and a bio-related substance (e.g., irinotecan). The modified bio-related substance can be located not only at the ends of the polyethylene glycol chains but also in a portion between two two-arm structures. The heterofunctional four-arm structure is obtained on the basis of pairing and combining two-arm polyethylene glycol derivatives, possessing multiple functional group combinations, and allowing coupling with targeting groups and drug molecules at different ratios, thereby achieving precise, flexible, and controllable targeting and pharmacological effects.

Description

A multi-arm polyethylene glycol derivative and its modified bio-related substances Technical Field

[0001] This invention relates to the fields of polymer synthesis and biochemical modification, and particularly to a multi-arm polyethylene glycol derivative and its modified biologically relevant substances. Background Technology

[0002] Polyethylene glycol (PEG) modification plays a crucial role in the field of biochemical modification. By utilizing the coupling reaction between the active groups of functionalized polyethylene glycol derivatives and biorelated substances, PEGylation of biorelated substances can be achieved. The modified biorelated substances (including drug molecules) possess many of the excellent properties of polyethylene glycol, such as hydrophilicity, low toxicity, and anticoagulant properties. For example, Greenwald et al. (J. Org. Chem. 1995, 331-336) overcame the poor water solubility of paclitaxel by modifying it with polyethylene glycol.

[0003] Monfardini et al. (Bioconjugate Chem. 1995, 6, 62-69) coupled linear polyethylene glycol segments to the two amino groups of lysine, and then activated the carboxyl group of lysine to obtain monofunctional two-armed V-type polyethylene glycol derivatives for protein modification. This method has been widely used to prepare monofunctional branched polyethylene glycol derivatives and their drug conjugates, significantly improving drug stability in systemic circulation. Furthermore, multi-arm polyethylene glycols, such as three-armed, four-armed, six-armed, and eight-armed polyethylene glycols, are increasingly being used in drug modification. Compared to linear and two-armed polyethylene glycol structures, multi-armed polyethylene glycol systems have significantly lower viscosity, which is beneficial for obtaining better pharmacokinetics. In addition, multi-armed polyethylene glycol structures can contain more active end groups, exhibiting higher drug loading potential.

[0004] Functionalized multi-arm polyethylene glycol (PEG) derivatives can theoretically contain one or more different active groups for the modification of biorelated substances. For PEGylated drugs that also contain targeting groups, two different PEG terminal active groups are typically required to precisely control the ratio of the targeting moiety to the drug molecule. Dai et al. (J. Mater. Chem. B, 2015, 3, 3754-3766) used a homofunctionalized eight-arm PEG carboxylic acid derivative to couple folic acid and betulinic acid, resulting in conjugates containing 1 to 2 folic acid residues and 6 to 7 betulinic acid residues, indicating that the ratio of the targeting moiety to the drug molecule was only approximate. In contrast, heterofunctionalized multi-arm PEG derivatives are more suitable for controlling the ratio of different species coupled to them (e.g., combinations of targeting groups and drug molecules, or combinations of different drug molecules), thus leading to more uniform and stable efficacy.

[0005] Simultaneously, the practical application of heterofunctionalized multi-arm polyethylene glycol derivatives that couple the target moiety and drug molecule is relatively limited, and there is a lack of designs that allow for flexible control of drug efficacy. WO2017089890A1 discloses a method for coupling di-lysine as the central structure with four linear polyethylene glycol segments to obtain terminally heterofunctionalized four-arm polyethylene glycol derivatives. In this method, the three amino groups and one carboxyl group of di-lysine react with different functionalized linear polyethylene glycol derivatives, thus limiting the ratio of different terminal functional groups in the resulting four-arm polyethylene glycol derivative to 3:1. CN107375288A discloses a method for controlling the ratio of the target moiety to drug molecule using branched-terminal four-arm polyethylene glycol derivatives, but this ratio is limited by the specific structure of the branched-terminal structures (e.g., when each branched-terminal contains a pair of heterofunctional groups, the ratio of the coupled target moiety to drug molecule is limited to 1:1).

[0006] Therefore, it is necessary to develop a structural design for heterofunctionalized multi-arm polyethylene glycol derivatives that is easy to implement and allows for flexible control of the ratio of terminal functional groups, in order to further construct conjugates with uniform and controllable pharmacodynamic effects.

[0007] Invention Overview

[0008] This application provides a novel heterofunctionalized four-arm polyethylene glycol derivative, constructed based on a pair of two-arm polyethylene glycol derivatives, which can be used to prepare polyethylene glycol-based drugs containing targeting groups and improve drug efficacy, wherein the content of the targeting groups and drug molecules is flexibly controllable.

[0009] This application relates to a four-armed polyethylene glycol derivative, the structure of which is shown in formula (1):

[0010] Or its salts, tautomers, stereoisomers, isotopic substitutes or solvates;

[0011] in,

[0012] w is 1 or 2;

[0013] X a and X b Each can be independently classified as >CH- or >N-;

[0014] Each L can be independently a divalent or trivalent linker;

[0015] Each R 3 Independently does not exist or is -L x -R;

[0016] L x It is a linker bond or a divalent linker base;

[0017] When at least one R exists 3At that time, -[L(R) 3 )] w - Contains at least one biodegradable component;

[0018] P a1 P a2 P b1 and P b2 Each independently is -L 0 -(CH2CH2O) n -R;

[0019] Each L 0 Independently, it can be a linker or a divalent linker;

[0020] Each n is an independent integer from 5 to 500;

[0021] R 3 P a1 P a2 P b1 and P b2 Each R in the equation is independently C 1-6 alkyl, benzyl or

[0022] Each F 0 Independently a reactive group or its protected form;

[0023] E represents a linker, a divalent linker, or a trivalent linker;

[0024] k is 1 or 2;

[0025] The four-armed polyethylene glycol derivative contains two, three, or more different F-type structures. 0 At least including F 01 and F 02 The F 01 and F 02 For two different structures of F 0 ;

[0026] The four-arm polyethylene glycol derivative is monodisperse or polydisperse.

[0027] This application also relates to a method for preparing a four-armed polyethylene glycol derivative, comprising reaction route 1 or reaction route 2; wherein,

[0028] Reaction route 1: Two-arm polyethylene glycol derivative 2arm-1 and two-arm polyethylene glycol derivative 2arm-2 are directly coupled to obtain the four-arm polyethylene glycol derivative shown in formula (1); or, 2arm-1, 2arm-2 and one or more bifunctional, trifunctional or tetrafunctional small molecules are coupled in one or more steps to obtain the four-arm polyethylene glycol derivative shown in formula (1).

[0029] Reaction route 2: Two-arm polyethylene glycol derivative 2arm-1 and two-arm polyethylene glycol derivative 2arm-2 are directly coupled to obtain INT; or, 2arm-1, 2arm-2 and one or more bifunctional, trifunctional or tetrafunctional small molecules are coupled through one or more steps to obtain INT; the INT is coupled with 1 to 4 identical compounds SM to obtain the tetrafunctional polyethylene glycol derivative shown in formula (1); wherein, the INT is an intermediate containing 1 to 4 identical terminal reactive groups; the compound SM is a trifunctional structure containing a trivalent branched core and at least one unprotected reactive group;

[0030] In any of the above routes, the structures of 2arm-1 and 2arm-2 are as follows:

[0031] Among them, X a and X b Each is independently >CH- or >N-; L a and L b Each is an independent linker or a divalent linker; F a and F b Each is an independent, unprotected reactive group; P 01 P 02 P 03 and P 04 Each is an independent linear polyethylene glycol chain, and P 01 P 02 P 03 and P 04 The ends of each are independently C 1-6 Any of the following: alkyl, benzyl, reactive group or its protected form; 2arm-1 and 2arm-2 may have the same or different structures.

[0032] This application also relates to a polyethylene glycol-modified bio-related substance, the structure of which is shown in formula (1D).

[0033] in,

[0034] M is the four-arm polyethylene glycol derivative shown in formula (1) that has lost all F 0 The residues formed;

[0035] t1 and t2 are each independent integers from 1 to 4;

[0036] Each L M Independently, it can be a linker or a divalent linker;

[0037] Each T is an independent targeting group;

[0038] Each D represents a residue of a biologically relevant substance.

[0039] This application also relates to a method for preparing a polyethylene glycol-modified biorelated substance, comprising coupling the biorelated substance or its derivative with a four-arm polyethylene glycol derivative of formula (1) to obtain a polyethylene glycol-modified biorelated substance of formula (1D).

[0040] Unlike traditional designs that couple multiple polyethylene glycol (PEG) components based on a central core structure, the heterofunctionalized four-arm PEG derivatives involved in this application are based on a modular design of paired two-arm PEG derivatives. By coupling two two-arm modules with different terminal functional groups, a series of heterofunctionalized four-arm PEG derivatives can be flexibly and efficiently constructed. Any one of the two-arm PEG derivatives can be a structure capped with an inert group (such as an alkoxy group) or a structure with one or more reactive groups (or their protected forms) at the PEG chain end, wherein the PEG chain end can be branched or unbranched functionalized.

[0041] The heterofunctionalized four-arm polyethylene glycol derivative of this application can be further coupled with a targeting moiety (e.g., folic acid) and a biorelated substance (e.g., irinotecan) to form a conjugate. First, the ratio of the targeting moiety to the biorelated substance in the conjugate includes, but is not limited to, 1:1, 2:2, 1:3, 3:1, and 4:4, thereby achieving precise and flexibly controllable targeting and efficacy. Second, the modified biorelated substance can be located not only at the end of the polyethylene glycol chain but also in the portion between two two-arm structures; the latter can be achieved by introducing multifunctionalized small molecules. Furthermore, the heterofunctionalized four-arm polyethylene glycol derivative containing intracellularly degradable groups (e.g., pH-sensitive acylhydrazone bonds or reduction-sensitive disulfide bonds) can reduce the shielding effect of polyethylene glycol through degradation of the four-arm structure after cellular uptake, thereby fully exposing the modified biorelated substance and allowing it to exert its efficacy.

[0042] Invention Details

[0043] 1. Terminology Explanation

[0044] All patents and other publications cited herein are incorporated herein in their entirety. In the event of any conflict between the terminology described herein and the cited content, the terminology herein shall prevail.

[0045] The word “includes” and similar expressions have the meanings of openness and inclusion.

[0046] Numerical ranges can be represented using, but are not limited to, hyphens, tildes, and "to," and by default include the endpoints of the range. For example, 1-6, 1~6, and "1 to 6" all represent groups consisting of 1, 6, and all values ​​in between. The type of the numerical values ​​is not particularly limited, and includes, but is not limited to, integers, non-integers, percentages, and fractions. Numerical ranges representing the number of functional groups are by default composed of integers; for example, -(CH2). 1-4 - indicates a group consisting of -CH2-, -(CH2)2-, -(CH2)3-, and -(CH2)4-; for example, express The group that makes up the group.

[0047] Polyethylene glycol or its derivatives are considered monodisperse when the polydispersity index (PDI) is no more than 1.005 (denoted as PDI=1), and polydisperse when the PDI is greater than 1.005.

[0048] Molecular weights without specified units are expressed in Daltons (Da) by default.

[0049] The molecular weight of polyethylene glycol or its derivatives is assumed to be the number average molecular weight (M). n The length of a polyethylene glycol chain can be expressed as the "degree of polymerization," which is the number of repeating units (-CH2CH2O- or -OCH2CH2-). The degree of polymerization is the number-average degree of polymerization by default and can be calculated based on the number-average molecular weight.

[0050] The molecular weight and degree of polymerization of polydisperse polyethylene glycol (PEG) or its derivatives are by default allowed to be within ±10% of a given value, and in some cases may be increased to ±15%, but not exceeding ±20%. For example, when the molecular weight of polydisperse PEG or its derivative is 5 kDa, its molecular weight is by default selected from the range of 4.5-5.5 kDa. For example, when the degree of polymerization of a PEG chain in polydisperse PEG or its derivative is 100, the degree of polymerization of that PEG chain is by default selected from the range of 90-110. The polydisperse product and the PEG chains in the corresponding raw materials may not have strictly the same degree of polymerization value, as long as they are within the aforementioned allowable range. For example, when synthesizing a product containing two PEG segments from PEG raw materials with a degree of polymerization of approximately 112 (n≈112) and another with a degree of polymerization of approximately 113 (n≈113), the degree of polymerization of both segments in the product can be expressed as 112, 113, or other values ​​within the allowable range.

[0051] The molecular weight and degree of polymerization of monodisperse polyethylene glycol or its derivatives are generally strictly equal to a given value, but in some cases can be increased to ±5%.

[0052] For reactions that do not involve the formation or destruction of polyethylene glycol segments, the degree of polymerization of each segment in the product is consistent with that in the corresponding raw materials. In such cases, the degree of polymerization of the product is considered to be explicitly disclosed, even if it is not stated.

[0053] The terms "linker group" and "linker" are used interchangeably, referring to a group containing at least one atom and connected to two or more other parts, including but not limited to divalent, trivalent, and higher valence linkers. Some linkers can also be called "bonds," for example, -O- can be called an ether group or ether bond, -OC(=O)- can be called an ester group or ester bond, and -OC(=O)NH- can be called a carbamate group or carbamate bond. A linker can be connected to other parts using any of its connecting ends. For example, when Group A and Group B are connected by -NHC(=O)-, the specific connection method can be GroupA-C(=O)NH-GroupB or GroupA-NHC(=O)-GroupB. For example, when the connecting end is not specified, -C(=O)NH- and -NHC(=O)- are considered equivalent. They are considered equivalent. It should be noted that the linker base does not include the linker bond.

[0054] A "linking bond" contains no atoms and only serves a connecting function. It can be represented by a short hyphen (such as the "-" at both ends of -C(=O)NH-), or by using... Tags (such as) (Indicates the group -G or G-).

[0055] The numerical range indicated by the C subscript position represents the number of carbon atoms in the group, excluding the contribution of substituents by default. For example, "C 1-3 "Alkyl" refers to an alkyl group having 1, 2, or 3 carbon atoms.

[0056] When the stereoconfiguration of a chemical structure is not specified, any stereoisomer of the chemical structure is permitted, including but not limited to cis / trans isomers, E- / Z- isomers, levorotatory / dextral isomers, etc. Even if a specific stereoconfiguration is given, all other stereoisomers are considered to be disclosed together.

[0057] "Degradable" means that a group, compound, or fragment can undergo irreversible structural changes through the breaking of chemical bonds in its environment. The environment is not particularly limited, but includes, but is not limited to, conditions such as light, heat, low temperature, enzymes, redox reactions, acidity, alkalinity, and physiological environments. The physiological environment is not particularly limited, but includes, but is not limited to, the intracellular or extracellular matrix of normal or diseased serum, heart, liver, spleen, lungs, kidneys, bones, muscles, fat, brain, lymph nodes, small intestine, and gonads. The physiological environment includes in vivo physiological environments or in vitro simulated physiological environments. The in vivo physiological environment also includes physiological conditions during treatment, such as ultraviolet irradiation and hyperthermia. The in vitro simulated physiological environment includes, but is not limited to, physiological saline, buffer solutions, culture media, organoids, and organ-on-a-chip. Degradation can be rapid, slow, or at any other rate. Degradation can occur under conditions such as in vivo metabolic processes (e.g., physiological processes, enzymatic reactions, redox reactions), stimulation of the microenvironment at specific sites in the body (e.g., acidity, alkalinity), and stimulation of clinical treatment (e.g., light, heat, low temperature). It should be noted that linkers that can only break under extreme conditions (such as strong acids, strong bases, and extreme temperatures) are not included in the scope of "degradable" in this application. For example, although ether bonds can break under strong acid conditions such as hydrobromic acid, this application considers them as stable linkers.

[0058] A "pH-sensitive linker" refers to a linker that is stable under one pH condition but degradable under another pH condition. The pH condition can be a specific pH value or a specific pH range. Changes in the pH condition include, but are not limited to, changes from acidic to alkaline, from alkaline to acidic, from weakly acidic to strongly acidic, and from weakly alkaline to strongly alkaline. Exemplary pH-sensitive linkers include hydrazone bonds, acylhydrazone bonds, imine bonds, oxime bonds, orthoester bonds, acetal bonds, ketal bonds, carbamate bonds, and carbonate bonds.

[0059] "Redox-sensitive linkers" are linkers that can degrade under changing redox environments, and typically contain chemical bonds that can be reduced or oxidized and broken. Exemplary redox-sensitive linkers include disulfide bonds, diselenide bonds, etc., whose cleavage can be induced by intracellular glutathione (GSH) or reactive oxygen species (ROS).

[0060] "Enzyme-cleavable linkers" refer to linkers that can be recognized and degraded by specific enzymes. Exemplary enzyme-cleavable linkers include peptide bonds, ester bonds, or glycosidic bonds, which can be broken in the presence of target enzymes (such as proteases, lipases, glycosidases, phosphatases, etc.).

[0061] "Nuclease-sensitive linkers" refer to linkers containing nucleic acid structures or nucleotide bonds (such as phosphodiester bonds) that can be degraded by nucleases (such as deoxynucleases and ribonucleases).

[0062] "Lipase-sensitive linkers" refer to linkers containing chemical bonds (such as ester bonds, amide bonds, etc.) that can be recognized by lipases and can undergo hydrolytic cleavage under the catalysis of lipases.

[0063] "Glycosidase-sensitive linkers" refer to linkers containing glycosidic bonds or glycosidic structures that can be hydrolyzed and broken by specific glycosidases (such as β-glucosidase, β-galactosidase, etc.).

[0064] "Hypoxia-sensitive linkers" refer to linkers that can undergo chemical reduction or structural breakage under hypoxic conditions. These linkers typically contain structural units such as nitroaryl, azo, quinoline oxide, or nitroimidazole, which are broken or structurally altered after enzymatic reduction in a hypoxic environment.

[0065] "Photocuttable linkers" refer to linkers that can undergo photochemical reactions (such as photolysis, photoisomerization, or photoinduced breakage) under illumination of light of a specific wavelength. These linkers typically contain photosensitive structural units, such as o-nitrophenylethyl, azo, coumarin, or porphyrin-derived structures.

[0066] "Thermosensitive linkers" are linkers that are prone to chemical breakage, rearrangement, or degradation when the temperature rises. These linkers typically contain heat-sensitive bonds, such as reversible Diels-Alder bonds, carbonate bonds, urea bonds, or thermosensitive polymer segments.

[0067] "Ultrasound-sensitive linkers" refer to linkers that can undergo chemical bond breakage or structural changes under ultrasonic irradiation or cavitation. These linkers typically rely on acoustic mechanical forces or localized thermal effects to break and can be used in ultrasound-triggered drug delivery systems.

[0068] "X-ray cleavable linkers" refer to linkers that can undergo chemical breakage or dissociation under X-ray irradiation. These linkers typically contain structural units that can be induced by radiation to generate free radicals or ionization reactions, such as aromatic acyl groups, haloaryl groups, or photosensitive thioether bonds, to achieve radiation-triggered structural dissociation or drug release.

[0069] A "salt" of a compound can be an acid addition salt formed by the compound with an inorganic and / or organic acid, a base addition salt formed by the compound with an inorganic and / or organic base, or a combination of any two or more of the aforementioned acid addition salts and / or base addition salts. When a compound contains a basic moiety (e.g., pyridine or imidazole) and an acidic moiety (e.g., carboxylic acid), it can form a zwitterion ("internal salt") and be included in the category of "salt". A salt of a compound can be a pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salt or other salts. A salt of a compound can be formed by reacting the compound with a certain amount (e.g., equivalent) of an acid or base in a medium such as a salt precipitation medium or in an aqueous medium, followed by freeze-drying. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gluconate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, etc. Exemplary base addition salts include ammonium salts, alkali metal salts (e.g., sodium, lithium, and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), salts of organic bases (e.g., organic amines), and salts of amino acids (e.g., arginine or lysine). The basic nitrogen-containing group can be quaternized using reagents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and dipentyl sulfates), long-chain halides (e.g., decyl, lauryl, tetradecyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0070] An "isotope-substituted derivative" of a compound refers to a derivative form obtained by substituting one or more atoms in the molecule of the compound with their respective isotopes.

[0071] The term "solvate" of a compound refers to an aggregate comprising one or more molecules of the compound and one or more solvent molecules. The solvent may be water, and the corresponding solvate may be called a hydrate, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. The solvent may also be an organic solvent.

[0072] The term "tautomer" of a compound refers to the isomers obtained by tautomerism of the compound. Tautomerism refers to isomerism resulting from reversible rearrangement of chemical bonds within a molecule, often involving proton transfer and changes in double bond positions. Exemplary tautomerisms include, but are not limited to, keto-enol tautomerism, amide-imine tautomerism, lactam-lactamimide tautomerism, enamine-imide tautomerism, enamine-enamine tautomerism (e.g., the enzyme reaction catalyzed by pyridoxal phosphate), proton transfer tautomerism, and valence tautomerism.

[0073] A "stereoisomer" of a compound refers to an isomer that has the same atomic sequence as the compound but has a different three-dimensional structure. Stereoisomers can be divided into enantiomers and diastereomers, with diastereomers including cis-trans isomers and conformational isomers. The molecular structures of enantiomers are mirror images of each other and cannot be superimposed.

[0074] "Heteroatoms" refer to atoms other than carbon and hydrogen, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0075] In contrast to compounds, groups formed after the loss of some atoms or groups are also called "residues".

[0076] A "substituent" refers to a group that occupies one or more hydrogen atom positions originally present in the structure of a compound or functional group. For example, -Cl in CH3Cl can be considered a substituent, and the corresponding unsubstituted structure is CH4. Similarly, -OH in -CH2CH(OH)CH2- can be considered a substituent, and the corresponding unsubstituted structure is -CH2CH2CH2-. Likewise, =O in -CH2C(=O)CH2- can be considered a substituent, and the corresponding unsubstituted structure is -CH2CH2CH2-. When a compound or functional group is "substituted," it means that the compound or functional group contains one or more substituents. A "substitution form" refers to the form obtained after one or more hydrogen atoms of a functional group or compound are replaced by substituents. There are no particular restrictions on the types of substituents.

[0077] "Optional" means that the situation or event described may or may not occur. For example, "optionally substituted" is equivalent to "substituted or unsubstituted". As another example, "optionally substituted hydrocarbon group" means that the hydrocarbon group may contain no substituents or may contain one, two, or more substituents.

[0078] "Reactive groups" refer to groups capable of participating in reactions, including but not limited to various common functional groups in organic chemistry. The reaction can be either a target reaction or a non-target reaction. When two or more reactive groups are present in the reactants, the reactive groups that may participate in non-target reactions are usually protected. "Protection" of reactive groups refers to the strategy of converting the reactive group into a "protected form" using specific reagents. The protected form is not reactive under the conditions of the target reaction and can be deprotected under specific conditions to obtain the unprotected form. The portion of the protected form of a reactive group that differs from the unprotected form is called the "protecting group." For example, -OTBS is a protected form of the hydroxyl group (-OH), where TBS is the protecting group of the hydroxyl group. The protected forms of a compound include those in which one, two, or more reactive groups are protected. The unprotected form is also called the free form.

[0079] Hydrocarbons refer to compounds containing only carbon and hydrogen. Hydrocarbons without benzene rings or other aromatic rings are collectively called "aliphatic hydrocarbons," while those containing benzene rings or other aromatic rings are collectively called "aromatic hydrocarbons." Aliphatic hydrocarbons can be divided into open-chain hydrocarbons and alicyclic hydrocarbons, with open-chain hydrocarbons further divided into straight-chain hydrocarbons and branched-chain hydrocarbons. Based on the number and type of unsaturated bonds, aliphatic hydrocarbons can be divided into alkanes, alkenes, and alkynes; alkanes do not contain unsaturated bonds, alkenes contain at least one carbon-carbon double bond, and alkynes contain at least one carbon-carbon triple bond.

[0080] A hydrocarbon group refers to a monovalent, divalent, or higher-valent group formed by the loss of one or more hydrogen atoms from a hydrocarbon compound. Hydrocarbon groups without a specified valence are assumed to be monovalent hydrocarbon groups (i.e., hydrocarbyl group), including but not limited to alkyl, alkenyl, and ynyl groups. A hydrocarbylene group refers to a divalent hydrocarbon group formed by the loss of two hydrogen atoms from a hydrocarbon compound, including but not limited to alkylene, alkenylene, and ynylene groups. In this application, hydrocarbon groups, hydrocarbylene groups, and higher-valent hydrocarbon groups can be optionally substituted, linear, or branched, and optionally contain cyclic structures.

[0081] A “targeting group” is a group that provides a strong affinity for a specific target (e.g., a molecule, cell, or cell type, such as a hepatocyte), compartment (e.g., a cellular or organ compartment), tissue, organ, or body region. Exemplary targeting groups include, but are not limited to, residues of the following substances: antibodies, antigens, peptides, vitamins, carbohydrates and their derivatives (including but not limited to monosaccharides, such as N-acetylgalactosamine (GalNAc)), folic acid, aptamers, receptor ligands, transferrin, biotin, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. A “liver-targeting group” is a group that targets the liver and / or hepatocytes.

[0082] "Hydroxy protecting groups" include, but are not limited to, hydroxy protecting groups commonly found in the art, such as alkyl acyl groups (e.g., acetyl, tert-butyryl), aralkyl acyl groups (e.g., benzyl), benzyl, triphenylmethyl, trimethylsilyl, tert-butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), allyl, acetal, ketal, etc. The removal of acetyl groups is generally carried out under alkaline conditions, most commonly by ammonolysis with NH3 / MeOH and methanololysis catalyzed by methanol anion. Benzyl groups are easily removed by palladium-catalyzed hydrogenolysis in neutral solution at room temperature, or by reduction cleavage with metallic sodium in ethanol or liquid ammonia. Triphenylmethyl groups are generally removed by catalytic hydrogenolysis. Trimethylsilyl groups are usually removed using reagents containing fluoride ions (e.g., tetrabutylamine fluoride / anhydrous THF). Tert-butyldimethylsilyl ether can be removed using compounds containing fluoride ions (e.g., Bu4N). + F - The diol can be removed in tetrahydrofuran solution or at room temperature with aqueous acetic acid. Protection of the diol includes, but is not limited to, the formation of dioxolane, dioxane, cyclic carbonates, cyclic borates, etc.

[0083] "Carboxyl protecting group" includes, but is not limited to, carboxyl protecting groups commonly found in the art, such as alkyl and aralkyl groups, specifically tert-butyl (tBu), methyl (Me), ethyl (Et), and benzyl (Bn). Protected forms of the carboxyl group include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. Carboxyl protecting groups can be removed by hydrolysis under acid or base catalysis, and occasionally by pyrolysis. Tert-butyl can be removed under mild acidic conditions. Benzyl can be removed by hydrogenolysis.

[0084] "Amino protecting group" includes, but is not limited to, amino / amine protecting groups commonly found in the art, such as aryl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6 Alkylsulfonyl, arylsulfonyl, silyl, etc., specifically such as Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), Fmoc (9-fluorenemethoxycarbonyl), and Cbz (benzyloxycarbonyl). TFA is preferred as the reagent for removing Boc protection. A solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine is preferred as the reagent for removing Fmoc protection. Catalytic hydrogenolysis is preferred for removing Cbz protection.

[0085] "Active ester group" refers to an ester group with high nucleophilic substitution reactivity, which readily reacts with nucleophiles such as amines, alcohols, and thiols by providing a favorable leaving group or reducing the electron density of the carbonyl group. Active ester groups include, but are not limited to, active carboxylic acid ester groups and active carbonate groups. Exemplary active carboxylic acid ester groups include N-hydroxysuccinimide ester (NHS ester), N-hydroxythiosuccinimide ester (sulfo-NHS ester), 1-hydroxybenzotriazole ester (HOBt ester), 1-hydroxy-7-azabenzotriazole ester (HOAt ester), pentafluorophenyl ester (PFP ​​ester), p-nitrophenol ester (p-NP ester), and other active carboxylic acid ester groups that can be obtained by reacting the carboxyl group with activating agents commonly used in the art. Exemplary active carbonates include benzotriazole carbonate, succinimide carbonate, and other active carbonate groups that can be obtained by reacting the hydroxyl group with activating agents commonly used in the art.

[0086] "Biorelated substances" include, but are not limited to, substances described, listed, and cited in documents CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A, and all cited documents. In general, biorelated substances include, but are not limited to, the following substances: drugs, proteins, polypeptides, oligopeptides, enzymes, antigens, antibodies, receptors, small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroidal compounds, lipid compounds, hormones, vitamins, phospholipids, glycolipids, dyes, fluorescent substances, targeting factors, targeting molecules, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, vesicles, liposomes, micelles, etc. The biologically related substances also include their fragments, precursors, activated states, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, fusion proteins, chemically modified substances, recombinant substances, etc., and may also be corresponding agonists, activators, inhibitors, antagonists, regulators, receptors, ligands, antibodies, acting enzymes (such as kinases, hydrolases, lyases, oxygen reductases, isomerases, transferases, deaminases, deiminases, invertases, synthases, etc.), enzyme substrates (such as coagulation cascade protease substrates, etc.). Derivatives of the biologically related substances include, but are not limited to, glycosides, nucleosides, amino acids, and polypeptide derivatives. Chemically modified substances of the biologically related substances include, but are not limited to, products that have undergone changes in functional group types or the introduction of additional functional groups or compound residues (e.g., residues of amino acids, polypeptides, and their derivatives). Before or after binding to a functionalized compound (including polyethylene glycol derivatives), the bioassociated substance is permitted to form a modified or complex bioassociated substance with the bound target molecule, appendage, or delivery carrier. The pharmaceutically acceptable salt can be an inorganic salt, such as hydrochloride, sulfate, or phosphate, or an organic salt, such as oxalate, malate, or citrate.

[0087] "Drug" includes any agent, compound, composition, or mixture that provides physiological or pharmacological effects, either in vivo or in vitro, and often provides beneficial effects. There are no particular limitations on the types of drugs, including but not limited to vaccines, antibodies, vitamins, foods, food additives, nutritional supplements, nutritional health products, and other agents that provide beneficial effects. There are no particular limitations on the scope of the physiological or pharmacological effects of drugs in vivo; they can be systemic or local. There are no particular limitations on the activity of drugs; they are primarily active substances capable of interacting with other substances, but can also be inert substances that do not interact. Inert drugs can be converted into their active form through in vivo action or certain stimulation. The molecular weight of small molecule drugs is generally no more than 1000 Da, and in some cases can be amplified but not exceeding 1500 Da.

[0088] The drug may be delivered to one or more of the following sites in the patient: liver or liver cells, kidney or kidney cells, tumor or tumor cells, CNS (central nervous system, such as brain and / or spinal cord) or CNS cells, PNS (peripheral nervous system) or PNS cells, lung or lung cells, blood vessels or blood vessel cells, skin (such as dermis, follicles) or skin cells, eye (such as macula, follicle, cornea, retina) or eye cells, ear (such as inner ear, middle ear, outer ear) or ear cells.

[0089] The drug can act systemically and / or locally, and can be administered via suitable routes, such as injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal, oral, sublingual, nasal, transmucosal, topical, ophthalmic preparations, inhalation, etc. The drug can be administered in suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0090] "Treatment" refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom, with the aim of delaying the progression of the disease, obstacle, or symptom, alleviating or mitigating symptoms and complications, and / or curing or eliminating the disease, obstacle, or symptom. Patients to be treated are preferably mammals, especially humans.

[0091] 2. Four-arm polyethylene glycol derivatives

[0092] One embodiment of this application is as follows:

[0093] A four-armed polyethylene glycol derivative, the structure of which is shown in formula (1):

[0094] Or its salts, tautomers, stereoisomers, isotopic substitutes or solvates;

[0095] in,

[0096] w is 1 or 2;

[0097] X a and X b Each can be independently classified as >CH- or >N-;

[0098] Each L can be independently a divalent or trivalent linker;

[0099] Each R 3 Independently does not exist or is -L x -R;

[0100] L x It is a linker bond or a divalent linker base;

[0101] When at least one R exists 3 At that time, -[L(R) 3 )] w - Contains at least one biodegradable component;

[0102] P a1 P a2 P b1 and P b2 Each independently is -L 0 -(CH2CH2O) n -R;

[0103] Each L 0 Independently, it can be a linker or a divalent linker;

[0104] Each n is an independent integer from 5 to 500;

[0105] R 3 P a1 P a2 P b1 and P b2 Each R in the equation is independently C 1-6 alkyl, benzyl or

[0106] Each F 0 Independently a reactive group or its protected form;

[0107] E represents a linker, a divalent linker, or a trivalent linker;

[0108] k is 1 or 2;

[0109] The four-armed polyethylene glycol derivative contains two, three, or more different F-type structures. 0 At least including F 01 and F 02 The F 01and F 02 For two different structures of F 0 ;

[0110] The four-arm polyethylene glycol derivative is monodisperse or polydisperse.

[0111] In some specific implementations, the salt of the four-armed polyethylene glycol derivative is a pharmaceutically acceptable salt.

[0112] In some specific implementation plans, X a and X b All are >CH-.

[0113] In some specific implementation plans, X a and X b All are >N-.

[0114] In some specific implementation plans, X a For >CH-, X b It is greater than N-.

[0115] In some specific implementation plans, X a For >N-, X b For >CH-.

[0116] In some specific implementations, the degradable portion is degradable under physiological conditions and is selected from any of the following: pH-sensitive linkers, redox-sensitive linkers, enzyme-cleavable linkers, nuclease-sensitive linkers, lipase-sensitive linkers, glycosidase-sensitive linkers, hypoxia-sensitive linkers, light-cleavable linkers, thermally unstable linkers, ultrasound-sensitive linkers, and X-ray-cleavable linkers.

[0117] In some specific implementations, the degradable portion is a pH-sensitive linker, a redox-sensitive linker, or an enzyme-cleavable linker.

[0118] In some specific embodiments, the degradable portion is selected from any one of ester bonds, disulfide bonds, hydrazone bonds, acylhydrazone bonds, carbamate bonds, and carbonate bonds.

[0119] In some specific embodiments, the four-armed polyethylene glycol derivative is monodisperse, and each n is an independent integer from 5 to 35, preferably an integer from 5 to 25, and more preferably any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

[0120] In some specific embodiments, the four-armed polyethylene glycol derivative is polydisperse, and each n is an independent integer from 20 to 250, preferably an integer from 100 to 125, more preferably an integer from 106 to 118, and even more preferably any one of 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, and 118.

[0121] In some specific implementation plans, P a1 P a2 P b1 and P b2 n has the same value.

[0122] In some specific implementations, the aforementioned n is determined by MALDI-TOF testing.

[0123] In some specific implementation schemes, each F 0 It is independently selected from any one of the amino, secondary amino, hydroxy, mercapto, aldehyde, carboxyl, azide, alkenyl, alkynyl, olefinic ester, succinimide, maleimide, isocyanate, isothiocyanate, halogen, acyl halide, acyl hydrazine, sulfonate, active ester, acid anhydride, and epoxy groups or their protected forms.

[0124] In some specific implementation schemes, two F connected to the same E 0 They have the same structure.

[0125] In some specific implementation schemes, two F connected to the same E 0 They have different structures.

[0126] In some specific implementation plans, F 01 and F 02 They are the protected forms of the same reactive group, but contain different protecting groups.

[0127] In some specific implementation plans, F 01 and F 02 One of them is -NHCbz, and the other is -NHBoc.

[0128] In some specific implementation plans, F 01 and F 02 One of them is -COOBn, and the other is -COOtBu.

[0129] In some specific implementation plans, F 01 and F 02 These are different types of reactive groups or their protected forms.

[0130] In some specific implementation plans, F 01 and F02 One of them is -NH2 or its protected form, and the other is -OH or its protected form.

[0131] In some specific implementation plans, F 01 and F 02 One of them is -NH2 or its protected form, and the other is -COOH or its protected form.

[0132] In some specific implementation plans, F 01 and F 02 One of them is -OH or its protected form, and the other is -COOH or its protected form.

[0133] In some specific implementation schemes, each L 0 Independently for the connector key, C 1-6 Alkylene or -B 0 -(Z 0 -B 0 ) j -; where L 0 In this context, j is an integer from 1 to 3; each B 0 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 0 Not with two Zs at the same time 0 Connected; each Z 0 Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z 0 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl.

[0134] In some specific implementation schemes, each Z 0 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH- and -NHC(=O)NH-.

[0135] In some specific implementation schemes, each L 0Independently selected from any one of -O-, -OCH2-*, -O(CH2)2-*, -OCH2C(=O)-*, -OC(=O)NH-*, -OC(=O)NH(CH2)4-*, -OCH2C(=O)O-*, -OCH2C(=O)OCH2-*, -OCH2C(=O)NH-*, -OCH2C(=O)NH(CH2)2-*, -OCH2C(=O)NH(CH2)3-*, -OCH2C(=O)NH(CH2)4-*, -O(CH2)2NHC(=O)-*, -O(CH2)2NHC(=O)O-*, and -O(CH2)2NHC(=O)OCH2-*, wherein the * terminus is associated with X. a or X b Connected.

[0136] In some specific implementations, each E is independently selected from any of the following:

[0137] (i) Connect key;

[0138] (ii) A divalent linker, preferably C 1-6 Alkylene, more preferably any one of -CH2-, -(CH2)2-, -(CH2)3- and -(CH2)4-;

[0139] (iii) A trivalent linker, preferably any of the following structures: Where m1 is an integer from 1 to 10; m2, m3, m4, and m5 are each an integer from 0 to 3 independently; the * and ** ends are respectively connected to two F with different structures. 0 Connected; more preferably, the trivalent connecting base is

[0140] In some specific implementations, each E in the four-armed polyethylene glycol derivative is independently selected from the aforementioned cases (i) or (ii).

[0141] In some specific implementations, one, two, three, four, or five E's in the four-armed polyethylene glycol derivative are each independently selected from case (iii).

[0142] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (1) has a structure as shown in formula (2):

[0143] In some specific implementation schemes, L in equation (2) 2 For divalent linker -B 2 -(Z 2 -B 2 ) j-; where L 2 j in B is an integer from 1 to 6, preferably 1, 2, 3 or 4; 2 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 2 Not with two Zs at the same time 2 Connected; each Z 2 Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z 2 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0144] Preferably, each Z 2 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH- and -NHC(=O)NH-.

[0145] In some specific implementation schemes, L in equation (2) 2 It contains at least one C 1-6 Alkylene.

[0146] In some specific implementation schemes, L in equation (2) 2 Selected from Any one of them, and either end thereof is connected to X a or X b Connected.

[0147] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (2) has a structure as shown in formulas (2-1), (2-2), (2-3), (2-4), or (2-5):

[0148] Among them, any two L 0 The structures of each group are the same or different, and the values ​​of any two k are the same or different. Each R 1 Independently for C 1-6 Alkyl or benzyl;

[0149] Preferably, all R in the same structural formula 1 It has the same structure and is further preferably methyl.

[0150] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (2-1) has a structure as shown in formula (2-1a):

[0151] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (2-2) has a structure as shown in (2-2a) or (2-2b):

[0152] In some specific embodiments, the four-arm polyethylene glycol derivatives shown in formula (2-3) have structures as shown in (2-3a) or (2-3b):

[0153] In some specific embodiments, the four-arm polyethylene glycol derivatives represented by formula (2-4) have structures as shown in (2-4a), (2-4b), or (2-4c):

[0154] In some specific embodiments, the four-arm polyethylene glycol derivatives represented by formula (2-5) have structures as shown in (2-5a), (2-5b), (2-5c), or (2-5d):

[0155] In some specific implementations, the four-armed polyethylene glycol derivative shown in formula (2) is selected from any of the following: P 4 -A1B1、P 4 -A1B2、P 4 -A3B3、P 4 -A2B1、P 4 -A2B2、P 4 -C1D1、P 4 -E1B1、P 4 -E2D2、P 4 -C1B1、P 4 -A1J1(m), P 4 -A4D2(m), P 4 -C2D1(m); Preferably, n1, n2, n3 and n4 in the aforementioned structure are each an integer from 5 to 250, and more preferably an integer from 5 to 100.

[0156] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (1) has a structure as shown in formula (3):

[0157] In some specific implementation schemes, L in equation (3) 3 For trivalent linker-B 3 -(Z 3 -B 3 )j -G-(B 3 -Z 3 ) j -B 3 -; G is a trivalent branched nucleus and also interacts with L x Connected; L 3 Each j in the set is an independent integer from 1 to 6, preferably 1, 2, 3, or 4; each B 3 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 3 Not with two Zs at the same time 3 Connected; each Z 3 Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z 3 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0158] Preferably, each Z 3 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-;

[0159] Preferably, G is >CH-, >N-, Any one of them, and either end thereof is connected to L x Connected.

[0160] In some specific implementation schemes, L in equation (3) x For connection key, C 1-6 Alkylene or -B x -(Z x -B x ) j -, and L x In this context, j is an integer from 1 to 6; each B x Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. x Not with two Zs at the same time x Connected; each Z xIndependently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z x Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0161] Preferably, each Z x It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-.

[0162] In some specific implementation schemes, L in equation (3) 3 It contains at least one -SS-, -CH=N-NHC(=O)- or -C(=O)NH-N=CH-.

[0163] In some specific implementation schemes, L in *equation (3) 3 Choose from any of the following structures:

[0164] Where L 3 The * end is connected to L x Either of the other two ends is connected to X. a or X b .

[0165] In some specific implementation schemes, L in equation (3) x Selected from link key, C 1-6 Any one of alkylene, *-NHC(=O)(CH2)2-, *-C(=O)NHCH2-, *-(CH2)3NHC(=O)(CH2)2- and *-(CH2)4NHC(=O)(CH2)2-, and L x The * end is connected to L 3 .

[0166] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (3) has a structure as shown in formulas (3-0), (3-1), (3-2), (3-3), (3-4), or (3-5):

[0167] Among them, any two L 0The structures of each group are the same or different, and the values ​​of any two k are the same or different. Each R 1 Independently for C 1-6 Alkyl or benzyl;

[0168] Preferably, all R in the same structural formula 1 It has the same structure and is further preferably methyl.

[0169] In some specific implementations, the four-armed polyethylene glycol derivative shown in formula (3) is selected from any of the following: P 4 -H1F2(Glu), P 4 -G2D1(Glu), P 4 -H2D1(Glu), P 4 -G1C4(Lys).

[0170] In some specific embodiments, the four-arm polyethylene glycol derivative shown in formula (1) has a structure as shown in formula (4):

[0171] In some specific implementations, each L in equation (4) 3 Independently a trivalent linker -B 3 -(Z 3 -B 3 ) j -G-(B 3 -Z 3 ) j -B 3 -; Each G is a trivalent branched nucleus and is also associated with the adjacent L x Connected; L 3 Each j in the set is an independent integer from 1 to 6; each B 3 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 3 Not with two Zs at the same time 3 Connected; each Z 3 Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z 3 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0172] Preferably, each Z 3It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-;

[0173] Preferably, each G is independently >CH- or >N-.

[0174] In some specific implementations, each L in equation (4) x Independently for the connector key, C 1-6 Alkylene or -B x -(Z x -B x ) j -, and L x In this context, j is an integer from 1 to 6; each B x Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. x Not with two Zs at the same time x Connected; each Z x Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z x Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0175] Preferably, each Z x It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-.

[0176] In some specific implementations, at least one L is in equation (4). 3 It contains at least one -SS-, -CH=N-NHC(=O)- or -C(=O)NH-N=CH-.

[0177] In some specific implementation schemes, in equation (4) It has the following structure:

[0178] Each of the two * ends is connected to the adjacent L. x Either of the other two ends is connected to X. a or X b .

[0179] In some specific implementation schemes, the two L's in equation (4) x The same and selected from any one of the following: linking bonds, *-NHC(=O)(CH2)2- and *-C(=O)NHCH2-, wherein each L x The * end is connected to the adjacent L 3 .

[0180] In some specific implementations, the four-armed polyethylene glycol derivative shown in formula (4) is P 4 -C4C4(Cys-Cys).

[0181] 3. Preparation method of four-arm polyethylene glycol derivatives

[0182] In some specific embodiments, the four-armed polyethylene glycol derivative represented by formula (1) can be prepared via any of the following reaction routes:

[0183] Reaction route 1: Two-arm polyethylene glycol derivative 2arm-1 and two-arm polyethylene glycol derivative 2arm-2 are directly coupled to obtain the four-arm polyethylene glycol derivative shown in formula (1); or, 2arm-1, 2arm-2 and one or more bifunctional, trifunctional or tetrafunctional small molecules are coupled in one or more steps to obtain the four-arm polyethylene glycol derivative shown in formula (1).

[0184] Reaction route 2: Two-arm polyethylene glycol derivative 2arm-1 and two-arm polyethylene glycol derivative 2arm-2 are directly coupled to obtain INT; or, 2arm-1, 2arm-2 and one or more bifunctional, trifunctional or tetrafunctional small molecules are coupled through one or more steps to obtain INT; the INT is coupled with 1 to 4 identical compounds SM to obtain the tetrafunctional polyethylene glycol derivative shown in formula (1); wherein, the INT is an intermediate containing 1 to 4 identical terminal reactive groups; the compound SM is a trifunctional structure containing a trivalent branched core and at least one unprotected reactive group;

[0185] In any of the above scenarios, the structures of 2arm-1 and 2arm-2 are as follows:

[0186] Among them, X a and X b Each is independently >CH- or >N-, L a and L b Each is an independent linker or a divalent linker, F aand F b Each is an independent, unprotected reactive group, P 01 P 02 P 03 and P 04 Each is an independent linear polyethylene glycol chain, and P 01 P 02 P 03 and P 04 The ends of each are independently C 1-6 Any one of alkyl, benzyl, reactive groups or their protected forms; 2arm-1 and 2arm-2 may have the same or different structures;

[0187] Preferably, the bifunctionalized small molecule is selected from any one of hydrazine, ethylenediamine, ethylene glycol, succinic acid, ethanolamine, glycine, and 3-hydroxypropionic acid;

[0188] Preferably, the trifunctionalized small molecule is selected from any one of glycerol, lysine, ornithine, glutamic acid, glutamine, aspartic acid, asparagine, cysteine, serine, threonine, and tyrosine.

[0189] Preferably, the tetrafunctionalized small molecule is cystine or a dipeptide, wherein the dipeptide is composed of any one or two monomers selected from lysine, ornithine, glutamic acid, glutamine, aspartic acid, asparagine, cysteine, serine, threonine, and tyrosine; more preferably, the tetrafunctionalized small molecule is any one selected from cystine, lysine-lysine, ornithine-ornithine, glutamic acid-glutamic acid, and aspartic acid-aspartic acid.

[0190] Preferably, the compound SM is selected from any of the following structures:

[0191] Where m1 is an integer from 1 to 10, and m3 is an integer from 0 to 3; each R 01 Each R is independently protected by a hydrogen atom or a carboxyl group. 02 Independently protected by a hydrogen atom or an amino group, each R 03 Independently protected by a hydrogen atom or a hydroxyl group; R in each SM 01 R 02 or R 03 At least one of them is a hydrogen atom;

[0192] More preferably, the compound SM is selected from any of the following structures:

[0193] In some specific implementations, 2arm-1 and 2arm-2 are each independently selected from any one of the following series A to series J:

[0194] Series A: Two-arm polyethylene glycol derivatives containing one active ester group, wherein the ends of both polyethylene glycol chains are alkoxy or benzyloxy groups, preferably any one of the following:

[0195] Series B: Two-arm polyethylene glycol derivatives containing one amino group, wherein both polyethylene glycol chain ends are reactive groups or their protected forms and have different structures, preferably any one of the following:

[0196] Series C: Two-arm polyethylene glycol derivatives containing one active ester group, wherein both polyethylene glycol chains are terminated by reactive groups or their protected forms and have the same structure, preferably any one of the following:

[0197] Series D: Two-arm polyethylene glycol derivatives containing one amino group, wherein both polyethylene glycol chain ends are reactive groups or their protected forms and have the same structure, preferably any one of the following:

[0198] Series E: Two-arm polyethylene glycol derivatives containing one active ester group, wherein both polyethylene glycol chain ends are reactive groups or their protected forms and have different structures, preferably any one of the following:

[0199] Series F: Two-arm polyethylene glycol derivatives containing one amino group, wherein both polyethylene glycol chains are terminal to an alkoxy or benzyloxy group, preferably any one of the following:

[0200] Series G: Two-arm polyethylene glycol derivatives containing one aldehyde group, wherein both polyethylene glycol chains are terminal to an alkoxy or benzyloxy group, preferably any one of the following:

[0201] Series H: Two-arm polyethylene glycol derivatives containing one aldehyde group, wherein both polyethylene glycol chains are terminated by reactive groups or their protected forms and have the same structure, preferably any one of the following:

[0202] Series J: Two-arm polyethylene glycol derivatives containing one amino group, wherein one polyethylene glycol chain terminal is a reactive group or its protected form and the other polyethylene glycol chain terminal is an alkoxy or benzyloxy group, preferably any one of the following:

[0203] In any of the above series, each n is an integer from 5 to 500, preferably an integer from 5 to 250, and more preferably an integer from 5 to 100;

[0204] Preferably, 2arm-1 and 2arm-2 are as follows:

[0205] (1) 2arm-1 is selected from series A and 2arm-2 is selected from series B;

[0206] (2) 2arm-1 is selected from series C and 2arm-2 is selected from series D;

[0207] (3) 2arm-1 is selected from series E and 2arm-2 is selected from series B;

[0208] (4) 2arm-1 is selected from series E and 2arm-2 is selected from series D;

[0209] (5) 2arm-1 is selected from series C and 2arm-2 is selected from series B;

[0210] (6) 2arm-1 is selected from series A and 2arm-2 is selected from series J;

[0211] (7) 2arm-1 is selected from series A and 2arm-2 is selected from series D;

[0212] (8) 2arm-1 is selected from series H and 2arm-2 is selected from series F;

[0213] (9) 2arm-1 is selected from series G and 2arm-2 is selected from series D;

[0214] (10) 2arm-1 is selected from series H and 2arm-2 is selected from series D;

[0215] (11) 2arm-1 is selected from series G and 2arm-2 is selected from series C;

[0216] (12) 2arm-1 and 2arm-2 are both selected from series C.

[0217] 4. Polyglycolated bio-related substances

[0218] One embodiment of this application is as follows:

[0219] A polyethylene glycol-modified bio-related substance, the structure of which is shown in formula (1D).

[0220] in,

[0221] M is the four-arm polyethylene glycol derivative shown in formula (1) that has lost all F 0 The residues formed;

[0222] t1 and t2 are each independent integers from 1 to 4;

[0223] Each LM Independently, it can be a linker or a divalent linker;

[0224] Each T is an independent targeting group;

[0225] Each D represents a residue of a biologically relevant substance.

[0226] In some specific implementation schemes, the t1 Ts in equation (1D) are all the same.

[0227] In some specific implementation schemes, the t2 Ds in equation (1D) are all the same.

[0228] In some specific implementation plans, L M It is a divalent linker and is F. 0 Linking groups generated by reactions with target molecules and their derivatives, or F 0 Linking groups formed with biologically related substances and their derivatives.

[0229] In some specific implementations, each L in equation (1D) M Independently for the connector key, C 1-6 Alkylene or -B M -(Z M -B M ) j -, and L M In this context, j is an integer from 1 to 6; each B M Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. M Not with two Zs at the same time M Connected; each Z M Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z M Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl;

[0230] Preferably, each Z M It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH- and -NHC(=O)NH-.

[0231] In some specific implementations, each L in equation (1D) MIndependently selected from any one of *-OC(=O)(CH2)2C(=O)O-, *-NHC(=O)(CH2)2C(=O)O-, *-OC(=O)-, *-NHC(=O)-, *-OC(=O)NHCH2C(=O)O- and *-OCH2C(=O)NHCH2C(=O)O-, wherein L M The * end is connected to M.

[0232] In some specific implementations, each T in formula (1D) is independently selected from residues of any of the following targeting substances or derivatives: glycosyl compounds, monoclonal antibodies, peptides, proteins, vitamins, neurotransmitters, and aptamers.

[0233] In some specific embodiments, each T in formula (1D) is independently selected from residues of any of the following substances: N-acetylgalactosamine, anti-HER2 monoclonal antibody, anti-EGFR monoclonal antibody, RGD peptide, tLyP-1 peptide, LyP-1 peptide, RPARPAR peptide, Angiopep-2 peptide, GE11 peptide, A6 peptide, CD44BP peptide, RP-1 peptide, apoE pseudopeptide, transferrin, low-density lipoprotein, high-density lipoprotein, lectin, lysozyme, streptavidin, folic acid, biotin, m-iodobenzylguanidine, DNA aptamer, and RNA aptamer.

[0234] In some specific implementation schemes, T in equation (1D) is

[0235] In some specific implementations, the biologically related substance is a pharmaceutically active agent, preferably any one of the following: sedative-hypnotic drugs, psychostimulants, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, anti-Parkinson's drugs, analgesics, anti-inflammatory drugs, anti-anxiety drugs, appetite suppressants, anti-migraine drugs, muscle contraction agents, antibiotics, antiviral agents, antifungal agents, antiarthritis drugs, antimalarial drugs, antiemetics, epilepsy drugs, bronchodilators, cytokines, growth factors, antithrombotic drugs, antihypertensive drugs, cardiovascular drugs, antiarrhythmic drugs, antioxidants, antiasthmatics, contraceptives, sympathomimetic drugs, diuretics, lipid regulators, antiandrogens, antiparasitic drugs, anticoagulants, antitumor drugs, hypoglycemic drugs, nutritional supplements, growth supplements, anti-enteritis drugs, vaccines, antibodies, diagnostic agents, and contrast agents.

[0236] In some specific implementations, the biologically related substance is a small molecule or macromolecule drug.

[0237] In some specific embodiments, the biologically related substances are any one of the following or their derivatives: flavonoids, terpenoids, stilbenes, carotenoids, saponins, quinones, coumarins, alkaloids, porphyrins, polyphenols, macrolides, β-lactams, phenylpropanoids, anthracyclines, aminoglycosides, artemisinin, lipids, steroids, amino acids, dipeptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, nucleic acids, antisense nucleic acids, monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0238] In some specific implementations, the biorelated substance is selected from any one of camptothecin, resveratrol, pterostilbene, dihydroartemisinin, betulinic acid, paclitaxel, docetaxel, lidocaine, bupivacaine, paliperidone, propofol, dezocine, ropivacaine, gefitinib, rotigotine, thigaben, catechol, dihydroporphyrin, triptolide, oleanolic acid, capecitabine, cantharidin, and cyclobuforine or its derivatives.

[0239] In some specific implementations, the biorelated substance is camptothecin or its derivatives.

[0240] In some specific embodiments, the biorelated substance is selected from any one of camptothecin, topotecan, irinotecan, SN38, 10-hydroxycamptothecin, belotetane, ixenonotecan, delutecan, rubitecan, 9-aminocamptothecin, letopotecan, gemmatotecan, gemiminotecan, BNP1350, and BN80915, specifically any one of the following structures:

[0241] In some specific implementation schemes, D in equation (1D) is

[0242] In some specific implementations, the polyethylene glycolated bio-related substance represented by formula (1D) is selected from any of the following: P 4 -A1B1-T1Y1、P 4 -A1B2-T1Y1、P 4 -A3B3-T1Y2、P 4 -A2B1-T1Y1、P 4 -A2B2-T1Y1、P 4 -C1D1-T1Y1、P 4 -E1B1-T1Y1、P 4 -E2D2-T1Y2、P 4 -C1B1-T1Y2、P 4 -A1J1(m)-T1Y2、P 4 -A4D2(m)-T1Y2、P 4 -C2D1(m)-T1Y2、P4 -H1F2(Glu)-T1Y1、P 4 -G2D1(Glu)-T1Y1、P 4 -H2D1(Glu)-T1Y1、P 4 -G1C4(Lys)-T1Y1、P 4 -C4C4(Cys-Cys)-T1Y2. Preferably, n1, n2, n3 and n4 in the aforementioned structure are each independently an integer from 5 to 250; more preferably, they are integers from 5 to 100.

[0243] In this application, the group The structure is

[0244] In this application, the group The structure is

[0245] 5. Preparation method of polyethylene glycolated biorelated substances

[0246] In some specific implementations, the polyethylene glycolated biorelated substance represented by formula (1D) can be prepared by any of the following methods:

[0247] Method 1: The bio-related substance is coupled with the four-armed polyethylene glycol derivative shown in formula (1) to obtain the polyethylene glycolated bio-related substance shown in formula (1D);

[0248] Method 2: The bio-related substance is coupled with a two-armed polyethylene glycol derivative, and the resulting intermediate is coupled with another two-armed polyethylene glycol derivative to obtain the PEGylated bio-related substance shown in formula (1D).

[0249] In some specific embodiments, the biorelated substance is an irinotecan derivative, preferably Y1 or Y2: Detailed Implementation

[0250] The preparation methods involved in this application include any protection and deprotection processes for specific functional groups as required by practical needs. Intermediates and final products can be purified by methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction, and column chromatography (including gel columns, ion columns, silica gel columns, etc.). Characterization of the structure and molecular weight of intermediates and final products can be achieved using methods including but not limited to... 1 Methods include 1H NMR, electrophoresis, UV-Vis spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF MS, and circular dichroism spectroscopy. The structures of intermediates and final products are assumed to be determined by... 1Determined by ¹H NMR. The molecular weight of the polymer (default is number-average molecular weight M). n The polydispersity index (PDI) is determined by gel permeation chromatography (GPC) by default. The degree of polymerization of the product (default number-average degree of polymerization) is calculated based on its molecular weight. The following examples are for further detailed description of the invention and are not intended to limit the scope of protection of the invention. The feed amounts in each example can be appropriately scaled up or down to suit actual needs.

[0251] Example 1: Synthesis of two-arm polyethylene glycol derivatives

[0252] Example 1.1: Two-arm polyethylene glycol derivative (Series A)

[0253] (1) Synthesis of A1:

[0254] Step 1: Add methoxy polyethylene glycol succinimide carbonate (S1, 5.10 g, 1.0 mmol; M... n ≈5.1 kDa, PDI=1.02, n≈112) and lysine (S2, 2.19 g, 15.0 mmol) were dissolved in 40 mL of anhydrous dichloromethane (DCM), and N,N-diisopropylethylamine (DIPEA, 3.87 g, 30.0 mmol) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was washed successively with 1 M HCl (20 mL * 2) and purified water (20 mL * 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain S3 (4.30 g; M n ≈5.1kDa, PDI=1.02, n≈112).

[0255] Step 2: Dissolve S1 (2.04 g, 0.4 mmol) and S3 (2.04 g, 0.4 mmol) in 20 mL of anhydrous dichloromethane, add DIPEA (1.55 g, 12.0 mmol), and stir overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (10 mL * 2) and purified water (10 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain S4 (2.66 g; M n ≈10.1kDa, PDI=1.02, n≈112).

[0256] Step 3: Dissolve S4 (1.01 g, 0.1 mmol) in 5 mL of anhydrous dichloromethane, add 5 mL of anhydrous dichloromethane solution containing N-hydroxysuccinimide (NHS, 0.17 g, 1.5 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI HCl, 0.29 g, 1.5 mmol), and then add DIPEA (0.39 g, 3.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain Al (0.54 g; M n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.46-4.42(m,1H,>CHC(=O)O-),4.10-3.90(m,4H,-CH2OC(=O)NH-),3.66-3.40(m,PEG ),3.23(s,6H,CH3O-),3.15-2.97(m,2H,-OC(=O)NHCH2-),2.80(s,4H,NHS),1.85-1.30(m,6H,-CH2(CH2)3CH<).

[0257] (2) Synthesis of A2:

[0258] Step 1: Under an argon atmosphere, methoxylated polyethylene glycolamine (S5, 5.00 g, 1.0 mmol; M...) n ≈5.0kDa, PDI=1.01, n≈112), methoxy polyethylene glycol acetic acid (S6, 5.00g, 1.0mmol; M n ≈5.0 kDa, PDI=1.02, n≈112) and 4-dimethylaminopyridine (DMAP, 0.24 g, 2.0 mmol) were dissolved sequentially in 50 mL of anhydrous dichloromethane. A dichloromethane solution (30 mL) of N,N'-dicyclohexylcarbodiimide (DCC, 3.09 g, 15.0 mmol) was slowly added dropwise under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain S7 (7.22 g; M n ≈10.0kDa, PDI=1.02, n≈112).

[0259] Step 2: S7 (4.00 g, 0.4 mmol), sodium bromoacetate (S8, 1.29 g, 8.0 mmol), sodium hydroxide (NaOH, 0.80 g, 20.0 mmol), and tetrabutylammonium bromide (TBAB, 1.29 g, 4.0 mmol) were added sequentially to a container containing 20 mL of toluene (Tol). The mixture was stirred overnight at 80 °C. After the reaction was complete, the reaction solution was cooled to room temperature, acidified with hydrochloric acid to pH 3, and then extracted with dichloromethane (10 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S9 (1.59 g; M n ≈10.1kDa, PDI=1.02, n≈112).

[0260] Step 3: Dissolve S9 (1.01 g, 0.1 mmol) in 5 mL of anhydrous dichloromethane, add 5 mL of anhydrous dichloromethane solution containing NHS (0.17 g, 1.5 mmol) and EDCI HCl (0.29 g, 1.5 mmol), and then add DIPEA (0.39 g, 3.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain A2 (0.31 g; M n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR (400MHz, (CD3)2SO) δ: 4.42-4.13(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-), 3.70-3.38(m,PEG,-O(CH2)2N<), 3.24(s,6H,CH3O-), 2.80(s,4H,NHS).

[0261] (3) Synthesis of A3:

[0262] Step 1: Under an argon atmosphere, S6 (5.00 g, 1.0 mmol), S2 (2.19 g, 15.0 mmol), and DMAP (0.24 g, 2.0 mmol) were dissolved sequentially in 25 mL of anhydrous dichloromethane. A dichloromethane solution (30 mL) of DCC (3.09 g, 15.0 mmol) was slowly added dropwise under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain S10 (3.98 g; M... n ≈5.1kDa, PDI=1.02, n≈112).

[0263] Step 2: Under an argon atmosphere, S6 (2.00 g, 0.4 mmol), S10 (2.04 g, 0.4 mmol), and DMAP (0.10 g, 0.8 mmol) were dissolved sequentially in 20 mL of anhydrous dichloromethane. A dichloromethane solution (15 mL) of DCC (1.24 g, 6.0 mmol) was slowly added dropwise under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain S11 (2.84 g; M n ≈10.1kDa, PDI=1.02, n≈112).

[0264] Step 3: Dissolve S11 (1.01 g, 0.1 mmol) in 5 mL of anhydrous dichloromethane, add 5 mL of anhydrous dichloromethane solution containing NHS (0.17 g, 1.5 mmol) and EDCI HCl (0.29 g, 1.5 mmol), and then add DIPEA (0.39 g, 3.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain A3 (0.37 g; M n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.70-4.68(m,1H,>CHC(=O)O-),3.86(s,4H,-OCH2C(=O)NH-),3.65-3.39(m,PEG), 3.23(s,6H,CH3O-),3.22-3.17(m,2H,-C(=O)NHCH2-),2.79(s,4H,NHS),1.88-1.31(m,6H,-CH2(CH2)3CH<).

[0265] (4) Synthesis of A4:

[0266] Step 1: Add A3 (10.2g, 1.0mmol; M) n S12 (approximately 10.2 kDa, PDI = 1.02, n ≈ 112) and ethylene glycol (S12, 0.93 g, 15.0 mmol) were dissolved in 50 mL of anhydrous dichloromethane. DIPEA (3.87 g, 30.0 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was washed successively with 1 M HCl (25 mL x 2) and purified water (25 mL x 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain S13 (5.12 g; M...). n≈10.2kDa, PDI=1.02, n≈112).

[0267] Step 2: S13 (4.08 g, 0.4 mmol) and triethylamine (TEA, 0.81 g, 8.0 mmol) were dissolved in acetonitrile (MeCN, 30 mL), and N,N'-succinimide carbonate (DSC, 1.54 g, 6.0 mmol) was added. The reaction was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL), and then washed successively with saturated sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain A4 (1.30 g; M n ≈10.3kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.49-4.43(m,2H,-CH2OC(=O)O-),4.26-4.22(m,1H,>CH-),3.89(s,4H,-OCH2C(=O)NH-),3.72-3.66(m,2H,-OC( =O)CH2-),3.66-3.38(m,PEG),3.24(s,6H,-OCH3),3.23-3.16(m,2H,-C(=O)NHCH2-),2.82(s,4H,NHS),1.89-1.33(m,6H,-CH2(CH2)3CH<).

[0268] Example 1.2: Two-arm polyethylene glycol derivative (Series B)

[0269] (1) Synthesis of B1:

[0270] Step 1: Under an argon atmosphere, α,ω-diamino polyethylene glycol (S14, 5.20 g, 1.0 mmol; M) with one end protected by Cbz was... n ≈5.2kDa, PDI=1.01, n≈113), α-carboxy-ω-hydroxy polyethylene glycol (S15, 5.10g, 1.0mmol; M) protected at one end by TBS. n ≈5.1 kDa, PDI=1.02, n≈112) and DMAP (0.24 g, 2.0 mmol) were dissolved sequentially in 50 mL of anhydrous dichloromethane. A dichloromethane solution (30 mL) of DCC (3.09 g, 15.0 mmol) was slowly added dropwise under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain S16 (6.93 g; M n ≈10.3kDa, PDI=1.02, n≈112).

[0271] Step 2: S16 (4.12 g, 0.4 mmol), S8 (1.29 g, 8.0 mmol), NaOH (0.80 g, 20.0 mmol), and TBAB (1.29 g, 4.0 mmol) were added sequentially to a 20 mL container of toluene. The mixture was stirred overnight at 80 °C. After the reaction was complete, the reaction solution was cooled to room temperature, acidified with hydrochloric acid to pH 3, and then extracted with dichloromethane (10 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S17 (1.28 g; M n ≈10.3kDa, PDI=1.02, n≈112).

[0272] Step 3: Dissolve S17 (1.03 g, 0.1 mmol) in anhydrous dichloromethane (5 mL), add NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol), then add a dichloromethane solution (2 mL) containing DMAP (24 mg, 0.2 mmol) and S18 (0.12 g, 2.0 mmol). Stir the reaction at room temperature for 24 hours. After the reaction is complete, filter to remove insoluble matter, concentrate, and purify by column chromatography to obtain B1 (0.21 g; M n ≈10.4kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.35-7.22(m,5H,Ar-H),5.04(s,2H,Ar-CH2-),4.19-3.88(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.69-3.39(m,PEG,-O( CH2)2N<,-C(=O)NHCH2CH2NH2),3.30-3.26(m,2H,-CH2NHCbz),2.84(t,2H,-C(=O)NHCH2CH2NH2),0.89(s,9H,-C(CH3)3),0.07(s,6H,>Si(CH3)2).

[0273] (2) Synthesis of B2:

[0274] Step 1: Under an argon atmosphere, S15 (2.04 g, 0.4 mmol; M) nS19 (2.02 g, 6.0 mmol) and DMAP (0.10 g, 0.8 mmol) were dissolved sequentially in 20 mL of anhydrous dichloromethane. A dichloromethane solution of DCC (1.24 g, 6.0 mmol) was slowly added dropwise in 15 mL under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was detert-butylated with a TFA / DCM mixture (1:1 v / v), followed by washing with purified water. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S20 (1.77 g; M... n ≈5.4kDa, PDI=1.02, n≈112).

[0275] Step 2: Under an argon atmosphere, S20 (1.08 g, 0.2 mmol), S21 (1.04 g, 0.2 mmol), and M... n (≈5.2kDa, PDI=1.01, n≈113) and DMAP (0.05g, 2.0mmol) were dissolved sequentially in 10mL of anhydrous dichloromethane. A dichloromethane solution (10mL) of DCC (0.62g, 3.0mmol) was slowly added dropwise under ice bath conditions. The reaction was stirred overnight at room temperature. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was dissolved in methanol (20mL). A Pd / C catalyst was added, and the mixture was bubbled with hydrogen at room temperature for 14h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was purified by column chromatography to obtain B2 (1.41g; M n ≈10.4kDa, PDI=1.02, n≈112). 1 HNMR(400MHz,(CD3)2SO)δ:4.46-4.39(m,1H,>CH-),3.84(s,2H,-OCH2C(=O)NH-),3.70-3.30(m,PEG,-O(CH2)2NHC(=O) -),2.65(t,2H,-CH2NH2),1.84-1.31(m,15H,-(CH2)3CH2NH2,Boc-tBu),0.88(s,9H,TBS-tBu),0.06(s,6H,>Si(CH3)2).

[0276] (3) Synthesis of B3:

[0277] Referring to the aforementioned synthesis method for B1, the raw material S15 was replaced with α,ω-dicarboxylated polyethylene glycol (S22,M) protected at one end by a tert-butyl group. nWith the reaction conditions and feed ratio remaining constant, B3(M) was obtained (approximately 5.1 kDa, PDI = 1.02, n ≈ 112). n ≈10.4kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.36-7.21(m,5H,Ar-H),5.02(s,2H,Ar-CH2-),4.27-3.90(m,6H,-OCH2C(=O)N<,>NCH2C(=O)-,-OC(= O)CH2O-),3.70-3.37(m,PEG,-O(CH2)2N<,-CH2CH2NH2),3.31-3.27(m,2H,-CH2NHCbz),2.82(t,2H,-CH2NH2),1.46(s,9H,tBu).

[0278] The structure of S22 is as follows:

[0279] Example 1.3: Two-arm polyethylene glycol derivative (Series C)

[0280] (1) Synthesis of C1:

[0281] Step 1: α-amino-ω-hydroxy polyethylene glycol (S23, 5.10 g, 1.0 mmol; M) with one end protected by Cbz was added. n S2 (approximately 5.1 kDa, PDI = 1.01, n ≈ 112), DSC (3.84 g, 15.0 mmol), and TEA (2.02 g, 20.0 mmol) were dissolved in 50 mL of anhydrous dichloromethane and stirred overnight at room temperature. S2 (2.19 g, 15.0 mmol) was added, and the reaction was continued with stirring at room temperature for 12 hours. After the reaction was complete, the mixture was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S24 (4.73 g; M...). n ≈5.3kDa, PDI=1.01, n≈112).

[0282] Step 2: S23 (2.04 g, 0.4 mmol), DSC (1.54 g, 6.0 mmol), and TEA (0.81 g, 8.0 mmol) were dissolved in 30 mL of anhydrous dichloromethane and stirred overnight at room temperature. S24 (2.12 g, 0.4 mmol) was added, and the reaction was continued to be stirred at room temperature for 12 hours. After the reaction was complete, the mixture was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S25 (2.99 g; M... n≈10.4kDa, PDI=1.02, n≈112).

[0283] Step 3: Dissolve S25 (1.04 g, 0.1 mmol) in 5 mL of anhydrous dichloromethane, add 5 mL of anhydrous dichloromethane solution containing NHS (0.17 g, 1.5 mmol) and EDCI HCl (0.29 g, 1.5 mmol), and then add DIPEA (0.39 g, 3.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain Cl (0.44 g; M n ≈10.5kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.38-7.24(m,10H,Ar-H),5.03(s,4H,Ar-CH2-),4.48-4.43(m,1H,>CH-),4.11-3.88(m,4H,-CH2OC(=O)NH-),3.68-3.40 (m,PEG,CbzNHCH2CH2O-),3.33-3.28(m,4H,-CH2NHCbz),3.18-2.99(m,2H,-OC(=O)NHCH2-),2.81(s,4H,NHS),1.86-1.29(m,6H,-CH2(CH2)3CH<).

[0284] (2) Synthesis of C2:

[0285] Referring to the aforementioned synthesis method for C1, the raw material S23 was replaced with polyethylene glycol (S26, M) protected at one end by TBS. n With the reaction conditions and feed ratio remaining constant, C2(M) was obtained (approximately 5.1 kDa, PDI = 1.01, n ≈ 112). n ≈10.5kDa, PDI=1.01, n≈112). 1H NMR(400MHz,(CD3)2SO)δ:4.45-4.40(m,1H,>CHC(=O)O-),4.10-3.86(m,4H,-CH2OC(=O)NH-),3.68-3.37(m,PEG,TBS(CH2)2O-),3.15 -2.96(m,2H,-OC(=O)NHCH2-),2.79(s,4H,NHS),1.83-1.27(m,6H,-CH2(CH2)3CH<),0.88(s,18H,TBS-tBu),0.06(s,12H,>Si(CH3)2).

[0286] The structure of S26 is as follows:

[0287] (3) Synthesis of C3:

[0288] Step 1: Succinic acid (S27, 1.74 g, 10.0 mmol) with one end protected by a tert-butyl group was dissolved in anhydrous dichloromethane (30 mL). NHS (1.73 g, 15.0 mmol) and DCC (3.09 g, 15.0 mmol) were added, followed by a dichloromethane solution (30 mL) containing DMAP (0.24 g, 2.0 mmol) and 3,3'-diaminodipropylamine (S28, 3.19 g, 8.0 mmol) with both ends protected by Cbz. The reaction was stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was dissolved in methanol (50 mL). A Pd / C catalyst was added, and the mixture was bubbled with hydrogen at room temperature for 14 hours to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was purified by column chromatography to obtain S29 (2.60 g).

[0289] Step 2: α,ω-dicarboxylated polyethylene glycol (S30, 5.10 g, 1.0 mmol; M) with one end protected by a benzyl group was added. n (Approximately 5.1 kDa, PDI = 1.02, n ≈ 112) was dissolved in anhydrous dichloromethane (30 mL), and NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol) were added. Then, a dichloromethane solution containing DMAP (24 mg, 0.2 mmol) and S29 (0.11 g, 0.4 mmol) (2 mL) was added. The reaction was stirred at room temperature for 24 hours. After the reaction was complete, the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was used to remove tBu with a TFA / DCM mixed solution (1:1 v / v), followed by washing with purified water. The aqueous phase was extracted with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S31 (3.05 g; Mn ≈10.5kDa, PDI=1.03, n≈112).

[0290] Step 3: Dissolve S31 (1.05 g, 0.1 mmol) in 5 mL of anhydrous dichloromethane, add 5 mL of anhydrous dichloromethane solution containing NHS (0.17 g, 1.5 mmol) and EDCI HCl (0.29 g, 1.5 mmol), and then add DIPEA (0.39 g, 3.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, wash successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify by column chromatography to obtain C3 (0.19 g; M n ≈10.6kDa, PDI=1.03, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.37-7.32(m,10H,Ar-H),5.19-5.09(m,4H,Ar-CH2-),4.01(s,4H,-OC(=O)CH2O-),3.85(s,4H,-OCH2C(=O)NH-),3.68-3.36(m ,PEG,-CH2N<),3.22-3.16(m,4H,-C(=O)NHCH2-),2.96(t,2H,-CH2CONHS),2.81(s,4H,NHS),2.73(t,2H,>NC(=O)CH2-),1.83-1.62(m,4H,-CH2CH2CH2-).

[0291] (4) Synthesis of C4:

[0292] Referring to the aforementioned synthesis method for A2, the raw material S5 was replaced with α-amino-ω-hydroxy polyethylene glycol (S32, M) protected at one end by TBS. n ≈5.1kDa, PDI=1.01, n≈112), replace raw material S6 with S15 (M n With the reaction conditions and feed ratio remaining constant, C4(M) was obtained (approximately 5.1 kDa, PDI = 1.02, n ≈ 112). n ≈10.4kDa, PDI=1.02, n≈112). 1H NMR(400MHz,(CD3)2SO)δ:4.38-4.12(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.69-3.38(m ,PEG,-O(CH2)2N<),2.79(s,4H,NHS),0.89(s,18H,-C(CH3)3),0.07(s,12H,>Si(CH3)2).

[0293] The structure of S32 is as follows:

[0294] Example 1.4: Two-arm polyethylene glycol derivative (Series D)

[0295] (1) Synthesis of D1:

[0296] Referring to the aforementioned synthesis method for B1, the raw material S14 is replaced with S32 (M n With the reaction conditions and feed ratio remaining constant, D1(M) was obtained (approximately 5.1 kDa, PDI = 1.01, n ≈ 112). n ≈10.3kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.24-3.89(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.71-3.35(m,PEG,-O(CH2)2N<, -C(=O)NHCH2CH2NH2),2.80(t,2H,-C(=O)NHCH2CH2NH2),0.89(s,18H,-C(CH3)3),0.07(s,12H,>Si(CH3)2).

[0297] (2) Synthesis of D2:

[0298] Step 1: Under ice bath conditions, dissolve 3-amino-1,2-propanediol (S33, 1.35 g, 6.0 mmol) and S22 (2.04 g, 0.4 mmol; M) containing a Cbz-protected amino group. n DCC (1.24 g, 6.0 mmol) was added in a single batch to a 20 mL solution of dichloromethane containing ≈5.1 kDa, PDI=1.02, n≈112 and DMAP (0.10 g, 0.8 mmol). The mixture was stirred in an ice bath for 30 minutes, then stirred overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (20 mL), and the aqueous phase was extracted with dichloromethane (10 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give S34 (1.57 g; M n≈5.3kDa, PDI=1.02, n≈112).

[0299] Step 2: Under ice bath conditions, DCC (0.31 g, 1.5 mmol) was added in a single batch to a 5 mL solution of dichloromethane containing S34 (0.53 g, 0.1 mmol), S22 (0.51 g, 0.1 mmol), and DMAP (0.02 g, 0.2 mmol). The mixture was stirred under ice bath conditions for 30 minutes, then stirred overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (5 mL), and the aqueous phase was extracted with dichloromethane (2.5 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain D2-Cbz (36.40 g; M n ≈10.4kDa, PDI=1.03, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.37-7.22(m,5H,Ar-H),5.04(s,2H,Ar-CH2-),5.01-4.98(m,1H,>CH-),4. 26-3.95(m,10H,-C(=O)OCH2CH<,-OC(=O)CH2O-), 3.69-3.35(m,PEG,-CH2NHCbz), 1.45(s,18H,tBu).

[0300] Step 3: Dissolve D2-Cbz in methanol, add Pd / C catalyst, and react with hydrogen gas at room temperature for 14 hours to remove Cbz. Filter with diatomaceous earth to remove the catalyst, and distill off the methanol to obtain a two-arm polyethylene glycol derivative D2 containing exposed amino groups, which is used for subsequent reactions without purification.

[0301] (3)Synthesis of D3:

[0302] Step 1: Under anhydrous and oxygen-free conditions, dissolve sodium hydride (NaH, 60%; 0.40 g, 10.0 mmol) and S33 (2.25 g, 10.0 mmol) in toluene (30 mL), then add α-tert-butoxycarbonylamino-ω-methanesulfonate polyethylene glycol (S35, 2.60 g, 0.5 mmol; M) with one end protected by Boc. n (≈5.2kDa, PDI=1.03, n≈112), reacted overnight at 60℃. After the reaction was complete, the reaction solution was concentrated, the residue was dissolved in dichloromethane (30mL), and then washed with water (15mL*2). The organic phase was concentrated, dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain S36 (2.22g; M n≈5.3kDa, PDI=1.03, n≈112).

[0303] Step 2: Under anhydrous and oxygen-free conditions, NaH (60%; 0.16 g, 4.0 mmol) and S36 (1.06 g, 0.2 mmol) were dissolved in toluene (10 mL), and then S35 (1.04 g, 0.2 mmol) was added. The reaction was carried out overnight at 60 °C. After the reaction was completed, the reaction solution was concentrated, and the residue was dissolved in dichloromethane (10 mL) and washed with water (5 mL * 2). The organic phase was concentrated, and the residue was dissolved in methanol (20 mL). Pd / C catalyst was added, and the reaction was carried out by bubbling with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was purified by column chromatography to obtain D3 (1.36 g; M n ≈10.2kDa, PDI=1.03, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:3.68-3.31(m,PEG,-OCH2CH<,-CH2NH2),1.41(s,18H,-Boc).

[0304] Example 1.5: Two-arm polyethylene glycol derivative (Series E)

[0305] (1) Synthesis of E1:

[0306] S17 (1.03g, 0.1mmol; M) n (Approximately 10.3 kDa, PDI = 1.02, n ≈ 112) was dissolved in 5 mL of anhydrous dichloromethane, and an anhydrous dichloromethane solution containing NHS (0.17 g, 1.5 mmol) and EDCI HCl (0.29 g, 1.5 mmol) (approximately 10.3 kDa) was added, followed by DIPEA (0.39 g, 3.0 mmol). The reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was washed successively with 1 M HCl (5 mL * 2) and purified water (5 mL * 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain E1 (0.65 g; M... n ≈10.4kDa, PDI=1.02, n≈112). 1H NMR(400MHz,(CD3)2SO)δ:7.36-7.20(m,5H,Ar-H),5.01(s,2H,Ar-CH2-),4.37-4.12(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.6 9-3.34(m,PEG,-O(CH2)2N<),3.32-3.26(m,2H,-CH2NHCbz),2.79(s,4H,NHS),0.89(s,9H,-C(CH3)3),0.07(s,6H,>Si(CH3)2).

[0307] (2) Synthesis of E2:

[0308] Referring to the aforementioned synthesis method for E1, S17 is replaced with S37 (M n With the reaction conditions and feed ratio remaining constant, E2(M ≈10.3kDa, PDI=1.02, n≈112) was obtained. n ≈10.4kDa, PDI=1.02, n≈112). 1 H NMR(400MHz, (CD3)2SO)δ:7.35-7.21(m,5H,Ar-H),5.01(s,2H,Ar-CH2-),4.39-4.13(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),4. 02(s,2H,-OC(=O)CH2O-),3.67-3.35(m,PEG,-O(CH2)2N<),3.31-3.26(m,2H,-CH2NHCbz),2.79(s,4H,NHS),1.43(s,9H,tBu).

[0309] S37 is an intermediate in the synthesis of B3, and its structure is as follows:

[0310] Example 1.6: Two-arm polyethylene glycol derivatives (Series F)

[0311] (1) Synthesis of F1:

[0312] Referring to the aforementioned synthesis method of B1 (step 3), replace S17 with S9 (M n With the reaction conditions and feed ratio remaining constant, F1(M) was obtained (approximately 10.1 kDa, PDI = 1.02, n ≈ 112). n ≈10.1kDa, PDI=1.02, n≈112). 1H NMR(400MHz,(CD3)2SO)δ:4.20-3.91(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.69-3.36(m,PE G,-O(CH2)2N<,-C(=O)NHCH2CH2NH2),3.24(s,6H,-OCH3),2.82(t,2H,-C(=O)NHCH2CH2NH2).

[0313] (2) Synthesis of F2:

[0314] Referring to the aforementioned synthesis method for B2, S15 is replaced with S6 (M n ≈5.0kDa, PDI=1.02, n≈112), replace S21 with S5(M n With the reaction conditions and feed ratio remaining constant, F2 (M ≈5.0 kDa, PDI=1.01, n≈112) was obtained. n ≈10.1kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.45-4.39(m,1H,>CH-),3.84(s,2H,-OCH2C(=O)NH-),3.70-3.36(m,PEG, -O(CH2)2NHC(=O)-),3.24(s,6H,-OCH3),2.67(t,2H,-CH2NH2),1.85-1.33(m,6H,-(CH2)3CH2NH2).

[0315] Example 1.7: Two-arm polyethylene glycol derivative (Series G)

[0316] (1) Synthesis of G1:

[0317] Under an argon atmosphere, S4 (1.01 g, 0.1 mmol; M) was... n≈10.1 kDa, PDI=1.02, n≈112), 3-aminopropionaldehyde diethyl acetal (S38, 0.22 g, 1.5 mmol), and DMAP (0.02 g, 0.2 mmol) were dissolved sequentially in 5 mL of dichloromethane. A solution of DCC (0.31 g, 1.5 mmol) in dichloromethane (5 mL) was slowly added dropwise under ice bath conditions. The mixture was then brought to room temperature and stirred for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was concentrated, and the residue was added to 10 mL of deionized water and stirred until completely dissolved. The pH was adjusted to 1.0 with 1 mol / L HCl under ice bath conditions. After reacting at room temperature for 4 hours, the mixture was extracted twice with dichloromethane (5 mL * 3). The organic phases were combined, washed with saturated brine, dried, filtered, concentrated, and recrystallized from anhydrous isopropanol to obtain G1 (0.23 g; M...). n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.25-4.16(m,1H,>CHC(=O)NH-),4.13-3.92(m,4H,-CH2OC(=O)NH-),3.72-3.36(m,PEG,-CH2CH2C HO),3.24(s,6H,-OCH3),3.14-2.96(m,2H,-OC(=O)NHCH2-),2.70-2.66(m,2H,-CH2CHO),1.80-1.31(m,6H,-CH2(CH2)3CH<).

[0318] (2) Synthesis of G2:

[0319] Referring to the aforementioned synthesis method for G1, S4 is replaced with S9 (M n With the reaction conditions and feed ratio remaining constant, G2(M ≈10.1kDa, PDI=1.02, n≈112) was obtained. n ≈10.1kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.22-3.92(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.70-3.3 4(m,PEG,-O(CH2)2N<,-CH2CH2CHO),3.25(s,6H,-OCH3),2.73-2.68(m,2H,-CH2CHO).

[0320] (3) Synthesis of G3:

[0321] Referring to the aforementioned synthesis method for G1, S4 is replaced with S11 (M nWith the reaction conditions and feed ratio remaining constant, G3(M) was obtained (approximately 10.1 kDa, PDI = 1.02, n ≈ 112). n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.42-4.33(m,1H,>CH-),3.84(s,4H,-OCH2C(=O)NH-),3.68-3.38(m,PEG,-CH2CH2CHO),3.24( s,6H,-OCH3),3.22-3.16(m,2H,-C(=O)NHCH2(CH2)3-),2.70-2.65(m,2H,-CH2CHO),1.91-1.30(m,6H,-CH2(CH2)3CH<).

[0322] Example 1.8: Two-arm polyethylene glycol derivatives (Series H)

[0323] (1) Synthesis of H1:

[0324] Referring to the aforementioned synthesis method for G1, S4 is replaced with S25 (M n With the reaction conditions and feed ratio remaining constant, H1 (M ≈10.4 kDa, PDI=1.02, n≈112) was obtained. n ≈10.5kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:7.38-7.24(m,10H,Ar-H),5.03(s,4H,Ar-CH2-),4.2 6-4.18(m,1H,>CH-),4.11-3.88(m,4H,-CH2OC(=O)NH-),3.68-3.40(m,PEG,-CH 2CH2NHCbz-,-CH2CH2CHO),3.33-3.28(m,4H,-CH2NHCbz),3.16-2.99(m,2H,-O C(=O)NHCH2-),2.71-2.67(m,2H,-CH2CHO),1.79-1.33(m,6H,-CH2(CH2)3CH<).

[0325] (2) Synthesis of H2:

[0326] Referring to the aforementioned synthesis method for G1, S4 is replaced with S39 (M n With the reaction conditions and feed ratio remaining constant, H2 (M ≈10.3 kDa, PDI=1.02, n≈112) was obtained. n≈10.3kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.21-3.90(m,4H,-OCH2C(=O)N<,>NCH2C(=O)-),3.70-3.32(m,PEG,-O(CH 2)2N<,-CH2CH2CHO),2.72-2.66(m,2H,-CH2CHO),0.88(s,18H,-C(CH3)3),0.06(s,12H,>Si(CH3)2).

[0327] S39 is an intermediate in the C4 synthesis process, and its structure is as follows:

[0328] Example 1.9: Two-arm polyethylene glycol derivatives (Series J)

[0329] (1) Synthesis of J1:

[0330] Referring to the aforementioned synthesis method for B1, S14 is replaced with S5 (M n ≈5.0kDa, PDI=1.01, n≈112), replace S15 with S22 (M n With the reaction conditions and feed ratio remaining constant, J1(M ≈5.1kDa, PDI=1.02, n≈112) was obtained. n ≈10.2kDa, PDI=1.02, n≈112). 1 H NMR(400MHz,(CD3)2SO)δ:4.19-3.94(m,6H,-OCH2C(=O)N<,>NCH2C(=O)-,-OC(=O)CH2O-),3.71-3. 35(m,PEG,-O(CH2)2N<,-CH2CH2NH2),3.25(s,3H,-OCH3),2.84(t,2H,-CH2NH2),1.43(s,9H,tBu).

[0331] (2) Synthesis of J2:

[0332] Referring to the aforementioned synthesis method for B2, S21 is replaced with S5(M) n ≈5.0kDa, PDI=1.01, n≈112), replace S15 with S22 (M n With the reaction conditions and feed ratio remaining constant, J2(M ≈5.1kDa, PDI=1.02, n≈112) was obtained. n ≈10.2kDa, PDI=1.02, n≈112). 1H NMR(400MHz,(CD3)2SO)δ:4.43-4.38(m,1H,>CH-),4.01(s,2H,-OC(=O)CH2O-),3.83(s,2H,-OCH2C(=O)NH-),3.67-3 .34(m,PEG,-O(CH2)2NHC(=O)-),3.23(s,3H,-OCH3),2.65(t,2H,-CH2NH2),1.80-1.30(m,15H,-(CH2)3CH2NH2,tBu).

[0333] Example 2: Four-arm polyethylene glycol derivative P 4 -A1B1 and its conjugates

[0334] Example 2.1: P 4 Synthesis of -A1B1

[0335] Add A1 (10.20g, 1.0mmol; M) n ≈10.2kDa, PDI=1.02, n≈112) and EDCI HCl (2.88g, 15.0mmol) were dissolved in 100mL of dichloromethane, and B1 (10.40g, 1.0mmol; M) was added. n The organic phase was reacted with DMAP (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) and DMAP (0.24 g, 2.0 mmol), followed by DIPEA (3.87 g, 30.0 mmol). The reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with 100 mL of dichloromethane and washed successively with water (100 mL * 2), 1 M HCl solution (100 mL * 2), saturated sodium bicarbonate solution (100 mL * 2), and saturated sodium chloride solution (100 mL * 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -A1B1(7.27g; M n ≈20.5kDa, PDI=1.03, n1+n2+n3+n4≈449). 1H NMR(400MHz,(CD3)2SO)δ:7.34-7.20(m,5H,Ar-H),5.01(s,2H,Ar-CH2-),4.26-3.90(m,9H,>CHC (=O)NH-,-OCH2C(=O)N<,>NCH2C(=O)-,-CH2OC(=O)NH-),3.69-3.36(m,PEG,-O(CH2)2N<),3.33- 3.26(m,6H,-C(=O)NH(CH2)2NHC(=O)-,-CH2NHCbz),3.24(s,6H,-OCH3),3.14-2.95(m,2H,-OC(= O)NHCH2-),1.79-1.30(m,6H,-CH2(CH2)3CH<),0.89(s,9H,-C(CH3)3),0.07(s,6H,>Si(CH3)2).

[0336] Example 2.2: P 4 Synthesis of -A1B1-T1Y1

[0337] Step 1: Place P 4 -A1B1 (4.10 g, 0.2 mmol) was dissolved in tetrabutylammonium fluoride in tetrahydrofuran solution (1 MTBAF / THF; 50 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (50 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. Irinotecan-succinic acid derivative (Y1, 1.37 g, 2.0 mmol, irinotecan residues are indicated as Irinotecan), DMAP (0.05 g, 0.4 mmol), and DCC (0.41 g, 2.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (50 mL), and the aqueous phase was extracted with dichloromethane (25 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain P. 4 -A1B1-Y1(3.56g; M n ≈21.0kDa, PDI=1.03, n1+n2+n3+n4≈449);

[0338] The structure of irinotecan-succinic acid derivative Y1 is as follows:

[0339] Step 2: Place P 4-A1B1-Y1 (2.10 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (20 mL). NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol) were added, followed by a dichloromethane solution (10 mL) containing DMAP (24 mg, 0.2 mmol) and folic acid (T1, 0.66 g, 1.5 mmol, folic acid residues are represented as FA). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A1B1-T1Y1(1.50g; M n ≈21.3kDa, PDI=1.03, n1+n2+n3+n4≈449; FA: Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.71(s,1H),8.18(d,1H),7.83(s,1H),7.65-7.63(m,2H),7.58(dd,1H),7.15(s, 1H),6.68-6.66(m,2H),5.73(d,1H),5.32(d,1H),5.24(s,2H),4.49-4.46(m,2H),4.32-3.92(m,12H),3.71 -3.35(m,PEG),3.33-3.29(m,4H),3.24(s,6H),3.18-2.96(m,8H),2.95-2.88(m,1H),2.80-2.70(m,4H),2. 67-2.61(m,4H),2.53-2.44(m,2H),2.34-2.27(m,2H),2.05-1.93(m,2H),1.82-1.32(m,19H),0.96(t,3H).

[0340] Example 3: Four-arm polyethylene glycol derivative P 4 -A1B2 and its conjugates

[0341] Example 3.1: P 4 Synthesis of -A1B2

[0342] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative A1(M) was... n (≈10.2kDa, PDI=1.02, n≈112) and B2(M nThe product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -A1B2(M n ≈20.5kDa, PDI=1.04, n1+n2+n3+n4≈449). 1 H NMR(400MHz,(CD3)2SO)δ:4.45-4.37(m,1H,>CH-),4.25-4.17(m,1H,>CH-),4.14-3.94(m,4 H,-CH2OC(=O)NH-),3.82(s,2H,-OCH2C(=O)NH-),3.72-3.32(m,PEG,-O(CH2)2NHC(=O)-),3 .25(s,6H,-OCH3),3.23-3.17(m,2H,-C(=O)NHCH2-),3.16-2.98(m,2H,-OC(=O)NHCH2-),1. 93-1.31(m,21H,-CH2(CH2)3CH<,Boc-tBu),0.89(s,9H,TBS-tBu),0.07(s,6H,>Si(CH3)2).

[0343] Example 3.2: P 4 Synthesis of -A1B2-T1Y1

[0344] Step 1: Referring to the method in Example 2.2, P 4 -A1B2 undergoes TBS removal and then esterification with Y1 to obtain P. 4 -A1B2-Y1(3.33g, M n ≈21.0kDa, PDI=1.04, n1+n2+n3+n4≈449).

[0345] Step 2: Place P 4-A1B2-Y1 (2.10 g, 0.1 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction, the mixture was washed with purified water (10 mL * 2), and the aqueous phase was extracted with dichloromethane (10 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol) were added to the filtrate, followed by a dichloromethane solution (10 mL) containing DMAP (24 mg, 0.2 mmol) and T1 (0.66 g, 1.5 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A1B2-T1Y1(1.64g; M n ≈21.4kDa, PDI=1.04, n1+n2+n3+n4≈449; FA: Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.72(s,1H),8.16(d,1H),7.82(s,1H),7.64-7.62(m,2H),7.58(dd,1H),7.13(s,1H ),6.68-6.64(m,2H),5.74(d,1H),5.32(d,1H),5.23(s,2H),4.50-4.45(m,2H),4.45-4.36(m,1H),4.31-4.18( m,8H),3.83(s,2H),3.70-3.32(m,PEG),3.24(s,6H),3.23-3.05(m,10H),2.94-2.85(m,1H),2.81-2.73(m,4H) ,2.66-2.60(m,4H),2.54-2.43(m,2H),2.36-2.28(m,2H),2.06-1.92(m,2H),1.91-1.29(m,25H),0.95(t,3H).

[0346] Example 4: Four-arm polyethylene glycol derivative P 4 -A3B3 and its conjugates

[0347] Example 4.1: P 4 Synthesis of -A3B3

[0348] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative A3(M) was... n (≈10.2kDa, PDI=1.02, n≈112) and B3(M nThe product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -A3B3(M n ≈20.5kDa, PDI=1.03, n1+n2+n3+n4≈449). 1 H NMR(400MHz,(CD3)2SO)δ:7.37-7.23(m,5H,Ar-H),5.04(s,2H,Ar-CH2-),4.43-4.32(m,1H,>CHC(=O )O-),4.29-3.91(m,6H,-OCH2C(=O)N<,>NCH2C(=O)-,-OC(=O)CH2O-),3.83(s,4H,-OCH2C(=O)NH-), 3.70-3.37(m,PEG,-O(CH2)2N<),3.34-3.27(m,6H,-C(=O)NH(CH2)2NHC(=O)-,-CH2NHCbz),3.24(s, 6H,-OCH3),3.23-3.18(m,2H,-C(=O)NHCH2-),1.92-1.33(m,6H,-CH2(CH2)3CH<),1.45(s,9H,tBu).

[0349] Example 4.2: P 4 Synthesis of -A3B3-T1Y2

[0350] Step 1: Place P 4 -A3B3 (4.10 g, 0.2 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 40 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (20 mL x 2), and the aqueous phase was extracted with dichloromethane (20 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.35 g, 3.0 mmol) and DCC (0.62 g, 3.0 mmol) were added to the filtrate, followed by a dichloromethane solution (20 mL) containing DMAP (0.05 g, 0.4 mmol), DIPEA (0.52 g, 4.0 mmol), and irinotecan-glycine hydrochloride derivative (Y2, 2.04 g, 3.0 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -A3B2-Y2(3.71g; M n ≈21.1kDa, PDI=1.03, n1+n2+n3+n4≈449);

[0351] The structure of irinotecan-glycine hydrochloride derivative Y2 is as follows:

[0352] Step 2: Referring to the method in Example 2.2, P 4 -A3B2-Y2 removes Cbz, and then undergoes an amidation reaction with T1 to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A3B2-T1Y2(1.77g; M n ≈21.4kDa, PDI=1.03, n1+n2+n3+n4≈449; FA: Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.73(s,1H),8.16(d,1H),7.84(s,1H),7.66-7.63(m,2H),7.57(dd,1H),7.14(s,1H),6 .68-6.63(m,2H),5.75(d,1H),5.34(d,1H),5.23(s,2H),4.52-4.48(m,2H),4.43-4.09(m,6H),4.05(d,2H),3.82 (s,6H),3.69-3.36(m,PEG),3.33-3.29(m,4H),3.25(s,6H),3.23-3.17(m,2H),3.18-3.05(m,6H),2.96-2.86(m, 1H),2.81-2.72(m,4H),2.55-2.44(m,2H),2.34-2.25(m,2H),2.06-1.92(m,2H),1.90-1.30(m,19H),0.96(t,3H).

[0353] Example 5: Four-arm polyethylene glycol derivative P 4 -A2B1 and its conjugates

[0354] Example 5.1: P 4 Synthesis of -A2B1

[0355] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative A2 (M) was... n (≈10.2kDa, PDI=1.02, n≈112) and B1(M n The product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -A2B1(M n ≈20.4kDa, PDI=1.02, n1+n2+n3+n4≈449). 1H NMR(400MHz,(CD3)2SO)δ:7.35-7.23(m,5H,Ar-H),5.02(s,2H,Ar-CH2-),4.21-3.89(m,8H,-OCH2C(=O)N<,>NCH2C(=O)-),3.68-3.34(m,PEG, -O(CH2)2N<),3.34-3.25(m,6H,-C(=O)NH(CH2)2NHC(=O)-,-CH2NHCbz),3.24(s,6H,-OCH3),0.89(s,9H,-C(CH3)3),0.07(s,6H,>Si(CH3)2).

[0356] Example 5.2: P 4 Synthesis of -A2B1-T1Y1

[0357] Referring to the method in Example 2.2, P 4 -A2B1, after TBS removal, undergoes esterification with Y1 to obtain an intermediate containing one irinotecan residue; subsequently, after Cbz removal, the intermediate undergoes amidation with T1 to obtain a folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A2B1-T1Y1 (21.3kDa, PDI=1.02, n1+n2+n3+n4≈449; FA: Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.74(s,1H),8.17(d,1H),7.83(s,1H),7.66-7.63(m,2H),7.58(dd,1H),7.15(s,1H ),6.69-6.63(m,2H),5.76(d,1H),5.35(d,1H),5.24(s,2H),4.50-4.42(m,2H),4.29-3.91(m,11H),3.72-3.36 (m,PEG),3.35-3.29(m,4H),3.25(s,6H),3.20-3.05(m,6H),2.95-2.87(m,1H),2.81-2.72(m,4H),2.69-2.62( m,4H),2.55-2.43(m,2H),2.37-2.30(m,2H),2.09-1.95(m,2H),1.87-1.41(m,10H),1.39(t,3H),0.97(t,3H).

[0358] Example 6: Four-arm polyethylene glycol derivative P 4 -A2B2 and its conjugates

[0359] Example 6.1: P 4 Synthesis of -A2B2

[0360] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative A2 (M) was... n (≈10.2kDa, PDI=1.02, n≈112) and B2(M n The product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -A2B2(M n ≈20.4kDa, PDI=1.02, n1+n2+n3+n4≈449). 1 H NMR(400MHz,(CD3)2SO)δ:4.45-4.36(m,1H,>CH-),4.19-3.90(m,4H,-OCH2C( =O)N<,>NCH2C(=O)-),3.81(s,2H,-OCH2C(=O)NH-),3.69-3.31(m,PEG,-O(CH2 )2N<),3.23(s,6H,-OCH3),3.23-3.16(m,2H,-C(=O)NHCH2-),1.90-1.28(m,1 5H,-CH2(CH2)3CH<,Boc-tBu),0.87(s,9H,TBS-tBu),0.05(s,6H,>Si(CH3)2).

[0361] Example 6.2: P 4 Synthesis of -A2B2-T1Y1

[0362] Referring to the method in Example 3.2, P 4 -A2B2, after TBS removal, undergoes esterification with Y1 to obtain an intermediate containing one irinotecan residue; subsequently, after Boc removal from the intermediate, it undergoes amidation with T1 to obtain a folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A2B2-T1Y1(M n ≈21.3kDa, PDI=1.02, n1+n2+n3+n4≈449; FA: Irinotecan=1:1). 1H NMR(400MHz,(CD3)2SO)δ:8.71(s,1H),8.16(d,1H),7.84(s,1H),7.64-7.61(m,2H),7.56(dd,1H),7.11( s,1H),6.69-6.64(m,2H),5.74(d,1H),5.33(d,1H),5.24(s,2H),4.53-4.47(m,2H),4.46-3.92(m,8H),3. 84(s,2H),3.69-3.35(m,PEG),3.24(s,6H),3.22-3.08(m,8H),2.97-2.88(m,1H),2.83-2.75(m,4H),2.6 5-2.60(m,4H),2.53-2.41(m,2H),2.37-2.28(m,2H),2.08-1.93(m,2H),1.88-1.29(m,19H),0.96(t,3H).

[0363] Example 7: Four-arm polyethylene glycol derivative P 4 -C1D1 and its conjugates

[0364] Example 7.1: P 4 Synthesis of -C1D1

[0365] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative C1(M) was... n (≈10.5kDa, PDI=1.02, n≈112) and D1(M n The product (approximately 10.3 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -C1D1(M n ≈20.7kDa, PDI=1.03, n1+n2+n3+n4≈448). 1H NMR(400MHz,(CD3)2SO)δ:7.35-7.22(m,10H,Ar-H),5.02(s,4H,Ar-CH2-),4.24-3.87(m,9H,> CH-,-OCH2C(=O)N<,>NCH2C(=O)-,-CH2OC(=O)NH-),3.66-3.37(m,PEG,-CH2CH2NHCbz,-O(CH2 )2N<),3.32-3.26(m,8H,-CH2NHCbz,-C(=O)NH(CH2)2NHC(=O)-),3.18-3.00(m,2H,-OC(=O)NH CH2-),1.80-1.32(m,6H,-CH2(CH2)3CH<),0.89(s,18H,-C(CH3)3),0.07(s,12H,>Si(CH3)2).

[0366] Example 7.2: P 4 Synthesis of -C1D1-T1Y1

[0367] Step 1: Place P 4 -C1D1 (4.14 g, 0.2 mmol) was dissolved in 1 M TBAF / THF (50 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (50 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. Y1 (2.75 g, 4.0 mmol), DMAP (0.10 g, 0.8 mmol), and DCC (0.82 g, 4.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (50 mL), and the aqueous phase was extracted with dichloromethane (25 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain P. 4 -C1D1-Y1(2.80g; M n ≈21.9kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0368] Step 2: Place P 4-C1D1-Y1 (2.19 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (25 mL). NHS (0.35 g, 3.0 mmol) and DCC (0.62 g, 3.0 mmol) were added, followed by a dichloromethane solution (15 mL) containing DMAP (0.05 g, 0.4 mmol) and T1 (1.32 g, 3.0 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -C1D1-T1Y1(1.03g; M n ≈22.4kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=2:2). 1 H NMR(400MHz,(CD3)2SO)δ:8.72(s,2H),8.15(d,2H),7.87(s,2H),7.65-7.62(m,4H),7.58(dd,2H),7.14 (s,2H),6.70-6.65(m,4H),5.75(d,2H),5.34(d,2H),5.22(s,4H),4.51-4.44(m,4H),4.29-3.83(m,15H) ,3.69-3.34(m,PEG),3.33-3.30(m,4H),3.17-3.00(m,14H),2.95-2.87(m,2H),2.86-2.79(m,8H),2.66 -2.60(m,8H),2.53-2.42(m,4H),2.36-2.26(m,4H),2.05-1.91(m,4H),1.87-1.30(m,32H),0.97(t,6H).

[0369] Example 8: Four-arm polyethylene glycol derivative P 4 -E1B1 and its conjugates

[0370] Example 8.1: P 4 Synthesis of -E1B1

[0371] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative E1(M) was... n (≈10.4kDa, PDI=1.02, n≈112) and B1(M n The product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P.4 -E1B1(M n ≈20.7kDa, PDI=1.02, n1+n2+n3+n4≈450). 1 H NMR(400MHz,(CD3)2SO)δ:7.36-7.23(m,10H,Ar-H),5.03(s,4H,Ar-CH2-),4.20-3.89(m,8H,-OCH2C(=O)N<,>NCH2C(=O)-),3.69-3.3 6(m,PEG,-O(CH2)2N<),3.31-3.26(m,8H,-CH2NHCbz,-C(=O)NH(CH2)2NHC(=O)-),0.88(s,18H,-C(CH3)3),0.06(s,12H,>Si(CH3)2).

[0372] Example 8.2: P 4 Synthesis of -E1B1-T1Y1

[0373] Referring to the method in Example 7.2, P 4 -E1B1, after TBS removal, undergoes esterification with Y1 to obtain an intermediate containing two irinotecan residues; then, after Cbz removal, the intermediate undergoes amidation with T1 to obtain a folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A2B2-T1Y1(M n ≈22.4kDa, PDI=1.02, n1+n2+n3+n4≈450; FA: Irinotecan=2:2). 1 H NMR(400MHz,(CD3)2SO)δ:8.74(s,2H),8.18(d,2H),7.88(s,2H),7.66-7.61(m,4H),7.59(dd,2H),7.15(s, 2H),6.73-6.69(m,4H),5.76(d,2H),5.35(d,2H),5.24(s,4H),4.53-4.45(m,4H),4.32-3.88(m,14H),3.73 -3.36(m,PEG),3.32-3.26(m,4H),3.19-3.07(m,12H),2.95-2.87(m,2H),2.85-2.78(m,8H),2.68-2.63(m, 8H),2.53-2.40(m,4H),2.35-2.23(m,4H),2.06-1.90(m,4H),1.83-1.41(m,20H),1.38(t,6H),0.96(t,6H).

[0374] Example 9: Four-arm polyethylene glycol derivative P 4 -E2D2 and its conjugates

[0375] Example 9.1: P 4 Synthesis of -E2D2

[0376] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative E2(M) was... n (≈10.4kDa, PDI=1.02, n≈112) and D2(M n The product (approximately 10.3 kDa, PDI = 1.03, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -E2D2(M n ≈20.6kDa, PDI=1.04, n1+n2+n3+n4≈449). 1 H NMR(400MHz,(CD3)2SO)δ:7.36-7.21(m,5H,Ar-H),5.10-5.05(m,1H,-CH<),5.02(s,2H,Ar-CH2-),4.27-3.90(m,16H,-OCH2C(=O)N<,>NCH2C (=O)-,-OC(=O)CH2O-,-C(=O)CH2CH<),3.70-3.37(m,PEG,-O(CH2)2N<,>CHCH2NHC(=O)-),3.31-3.27(m,2H,-CH2NHCbz),1.46(s,27H,tBu).

[0377] Example 9.2: P 4 Synthesis of -E2D2-T1Y2

[0378] Step 1: Place P 4 -E2D2 (6.18 g, 0.3 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 60 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (30 mL * 2), and the aqueous phase was extracted with dichloromethane (30 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (1.55 g, 13.5 mmol) and DCC (2.78 g, 13.5 mmol) were added to the filtrate, followed by a dichloromethane solution (100 mL) containing DMAP (0.22 g, 1.8 mmol), DIPEA (2.32 g, 18.0 mmol), and Y2 (9.18 g, 13.5 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4-E2D2-Y2(2.89g; M n ≈22.3kDa, PDI=1.04, n1+n2+n3+n4≈449).

[0379] Step 2: Referring to the method in Example 2.2, P 4 -E2D2-Y2 removes Cbz, then undergoes an amidation reaction with T1 to give the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -E2D2-T1Y2(1.53g; M n ≈22.6kDa, PDI=1.04, n1+n2+n3+n4≈449; FA: Irinotecan=1:3). 1 H NMR(400MHz,(CD3)2SO)δ:8.74(s,1H),8.18(d,3H),7.83(s,3H),7.65-7.61(m,2H),7.55(dd,3H),7.14(s ,3H),6.67-6.61(m,2H),5.76(d,3H),5.34(d,3H),5.23(s,6H),5.12-5.07(m,1H),4.53-4.48(m,2H),4.3 2-3.91(m,17H),3.83(s,6H),3.74-3.36(m,PEG),3.17-3.05(m,18H),2.97-2.86(m,3H),2.83-2.73(m,12 H),2.56-2.42(m,6H),2.35-2.27(m,2H),2.06-1.93(m,2H),1.83-1.43(m,30H),1.38(t,9H),0.96(t,9H).

[0380] Example 10: Four-arm polyethylene glycol derivative P 4 -C1B1 and its conjugates

[0381] Example 10.1: P 4 Synthesis of -C1B1

[0382] Referring to the method in Example 2.1, the two-arm polyethylene glycol derivative C1(M) was... n (≈10.5kDa, PDI=1.02, n≈112) and B1(M n The product (approximately 10.4 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -C1B1(M n≈20.8kDa, PDI=1.02, n1+n2+n3+n4≈449). 1 H NMR(400MHz,(CD3)2SO)δ:7.39-7.26(m,15H,Ar-H),5.04(s,6H,Ar-CH2-),4.25-3.86(m,9H,> CH-,-CH2OC(=O)NH-,-OCH2C(=O)N<,>NCH2C(=O)-),3.68-3.38(m,PEG,-CH2CH2NHCbz-,-O(CH 2)2N<),3.32-3.27(m,10H,-CH2NHCbz,-C(=O)NH(CH2)2NHC(=O)-),3.17-2.98(m,2H,-OC(=O) NHCH2-),1.81-1.32(m,6H,-CH2(CH2)3CH<),0.89(s,9H,-C(CH3)3),0.07(s,6H,>Si(CH3)2).

[0383] Example 10.2: P 4 Synthesis of -C1B1-T1Y2

[0384] Step 1: Place P 4 -C1B1 (4.16 g, 0.2 mmol) was dissolved in 1 M TBAF / THF (50 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (50 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and filtered. DSC (0.77 g, 3.0 mmol) and TEA (0.40 g, 4.0 mmol) were added to the filtrate, and the mixture was stirred overnight at room temperature. Y2 (2.04 g, 3.0 mmol) was then added, and the reaction was continued to be stirred at room temperature for 12 hours. After the reaction was complete, the mixture was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain P. 4 -C1B1-Y2(2.96g; M n ≈21.4kDa, PDI=1.02, n1+n2+n3+n4≈449).

[0385] Step 2: Place P 4-C1B1-Y2 (2.14 g, 0.1 mmol) was dissolved in methanol (30 mL), and a Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (20 mL). NHS (0.52 g, 4.5 mmol) and DCC (0.93 g, 4.5 mmol) were added, followed by a dichloromethane solution (20 mL) containing DMAP (0.07 g, 0.6 mmol) and T1 (1.98 g, 4.5 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -C1B1-T1Y2(1.15g; M n ≈22.2kDa, PDI=1.02, n1+n2+n3+n4≈449; FA: Irinotecan=3:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.71(s,3H),8.16(d,1H),7.82(s,1H),7.64-7.60(m,6H),7.53(dd,1H), 7.13(s,1H),6.65-6.58(m,6H),5.77(d,1H),5.33(d,1H),5.23(s,2H),4.54-4.47(m,6H),4.24-3. 86(m,16H),3.70-3.35(m,PEG),3.33-3.28(m,4H),3.19-2.97(m,8H),2.96-2.84(m,1H),2.82-2.7 1(m,4H),2.56-2.41(m,2H),2.33-2.25(m,6H),2.05-1.92(m,6H),1.84-1.33(m,19H),0.97(t,3H).

[0386] Example 11: Four-arm polyethylene glycol derivative P 4 -A1J1(m) and its conjugates

[0387] Example 11.1: Synthesis of compound SM-1

[0388] Step 1: Dissolve Boc-1-tert-butyl glutamate (S40, 4.55 g, 15.0 mmol) in anhydrous dichloromethane (50 mL), add NHS (2.59 g, 22.5 mmol) and DCC (4.64 g, 22.5 mmol), then add a dichloromethane solution (100 mL) containing DMAP (0.37 g, 3.0 mmol) and 3,6,9-trioxaundecan-1,11-diamine (S41, 7.34 g, 22.5 mmol) protected at one end by Cbz. Stir the reaction at room temperature for 24 hours. After the reaction is complete, filter to remove insoluble matter. Concentrate the filtrate under reduced pressure, and remove Boc and tBu from the crude product using a TFA / DCM mixture (1:1 v / v), then wash with purified water. Extract the aqueous phase with dichloromethane. The organic phases were combined, dried with anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S42 (5.77 g).

[0389] Step 2: S42 (4.56 g, 10.0 mmol) was dissolved in tert-butyl acetate (50 mL), and perchloric acid (HClO4, 1.50 g, 15.0 mmol) was slowly added dropwise with stirring in an ice bath. After the addition was complete, the reaction mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was diluted with water (50 mL). The aqueous phase was separated and extracted with ethyl acetate (25 mL * 2). The organic phases were combined and washed successively with 10% sodium carbonate solution (50 mL * 2) and water (50 mL * 2), dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain SM-1 (4.49 g). 1 H NMR(400MHz,(CD3)2SO)δ:7.34-7.21(m,5H,Ar-H),5.01(s,2H,Ar-CH2-),3.71-3.37(m,15H,-(CH2CH2O )3CH2-,-CH<),3.32-3.27(m,2H,-CH2NHCbz),2.41-2.28(m,2H,-CH2C(=O)NH-),2.17-1.95(m,1H,>CHH a H b -), 1.83-1.69(m, 1H, > CHH a H b -), 1.45(s, 9H, tBu).

[0390] Example 11.2: P 4 Synthesis of -A1J1(m)

[0391] Step 1: Add A1 (10.2g, 1.0mmol; M) n≈10.2kDa, PDI=1.02, n≈112) and EDCI HCl (2.88g, 15.0mmol) were dissolved in 100mL of dichloromethane, and J1 (10.2g, 1.0mmol; M) was added. n The organic phase was reacted with DMAP (approximately 10.2 kDa, PDI = 1.02, n ≈ 112) and DMAP (0.24 g, 2.0 mmol), followed by DIPEA (3.87 g, 30.0 mmol). The reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with 100 mL of dichloromethane and washed successively with water (100 mL * 2), 1 M HCl solution (100 mL * 2), saturated sodium bicarbonate solution (100 mL * 2), and saturated sodium chloride solution (100 mL * 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -A1J1(10.35g; M n ≈20.3kDa, PDI=1.02, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:4.22-3.93(m,11H,-CH2OC(=O)NH-),3.70-3.36(m,PEG,-O(CH2)2N<),3.34-3.30(m,4H,-C(=O)NH(C H2)2NHC(=O)-),3.24(s,9H,-OCH3),3.15-2.98(m,2H,-OC(=O)NHCH2-),1.82-1.33(m,6H,-CH2(CH2)3CH<),1.44(s,9H,tBu).

[0392] Step 2: Place P 4 -A1J1 (10.15 g, 0.5 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 100 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (50 mL * 2), and the aqueous phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.86 g, 7.5 mmol) and DCC (1.55 g, 7.5 mmol) were added to the filtrate, followed by a dichloromethane solution (50 mL) containing DMAP (0.12 g, 1.0 mmol) and SM-1 (3.84 g, 7.5 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -A1J1(m)(7.88g; M n≈20.8kDa, PDI=1.02, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:7.35-7.23(m,5H,Ar-H),5.02(s,2H,Ar-CH2-),4.64-4.57(m,1H,>CHCOOtBu),4.24-3.9 5(m,9H,>CHC(=O)NH-,-OCH2C(=O)N<,>NCH2C(=O)-,-CH2OC(=O)NH-),3.83(s,2H,-NHC(=O)CH2O-),3.74-3.37(m,P EG,-O(CH2)2N<),3.34-3.27(m,6H,-C(=O)NH(CH2)2NHC(=O)-,-CH2NHCbz),3.25(s,9H,-OCH3),3.17-2.99(m,2H,- OC(=O)NHCH2-),2.42-2.17(m,4H,Glu-γ-CH2-,Glu-β-CH2-),1.82-1.30(m,6H,-CH2(CH2)3CH<),1.45(s,9H,tBu).

[0393] Example 11.3: P 4 Synthesis of -A1J1(m)-T1Y2

[0394] Step 1: Place P 4 -A1J1(m) (6.24 g, 0.3 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 60 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (30 mL * 2), and the aqueous phase was extracted with dichloromethane (30 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.52 g, 4.5 mmol) and DCC (0.93 g, 4.5 mmol) were added to the filtrate, followed by a dichloromethane solution (30 mL) containing DMAP (0.07 g, 0.6 mmol), DIPEA (0.77 g, 6.0 mmol), and Y2 (3.06 g, 4.5 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -A1J1(m)-Y2(4.63g; M n ≈21.3kDa, PDI=1.02, n1+n2+n3+n4≈448).

[0395] Step 2: Place P 4-A1J1(m)-Y2 (2.13 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (20 mL). NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol) were added, followed by a dichloromethane solution (10 mL) containing DMAP (24 mg, 0.2 mmol) and T1 (0.66 g, 1.5 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A1J1(m)-T1Y2(1.44g; M n ≈21.6kDa, PDI=1.02, n1+n2+n3+n4≈448; FA: Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.74(s,1H),8.14(d,1H),7.83(s,1H),7.65-7.60(m,2H),7.56(dd,1H),7.11(s,1H ),6.67-6.62(m,2H),5.76(d,1H),5.34(d,1H),5.24(s,2H),4.54-4.48(m,2H),4.29-3.93(m,13H),3.80(s,2H ),3.70-3.35(m,PEG),3.31-3.28(m,4H),3.23(s,9H),3.16-2.98(m,8H),2.95-2.86(m,1H),2.82-2.70(m,4H) ,2.53-2.43(m,2H),2.34-2.23(m,5H),2.15-2.11(m,1H),2.07-1.93(m,2H),1.83-1.28(m,19H),0.95(t,3H).

[0396] Example 12: Four-arm polyethylene glycol derivative P 4 -A4D2(m) and its conjugates

[0397] Example 12.1: P 4 Synthesis of -A4D2(m)

[0398] Step 1: Add D2 (10.30g, 1.0mmol; M) n(Approximately 10.3 kDa, PDI = 1.03, n ≈ 112) DIPEA (3.87 g, 30.0 mmol) was dissolved in 100 mL of dichloromethane, and A4 (10.30 g, 1.0 mmol; M) was added with stirring. n (≈10.3 kDa, PDI=1.02, n≈112), the reaction was carried out overnight at room temperature. After the reaction was completed, the reaction mixture was poured into 200 mL of water, and then extracted three times with dichloromethane (100 mL*3). The organic phases were collected and combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -A4D2(12.66g; M n ≈20.5kDa, PDI=1.03, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:5.03-4.99(m,1H,-CHCH2NHC(=O)O-),4.30-3.96(m,15H,>C HC(=O)O-,-C(=O)OCH2CH<,-OC(=O)CH2O-,-C(=O)O(CH2)2OC(=O)NH-),3.87(s,4H,-O CH2C(=O)NH-),3.69-3.36(m,PEG,-CHCH2NHC(=O)O-),3.24(s,6H,-OCH3),3.22-3.15 (m,2H,-C(=O)NHCH2(CH2)3-),1.88-1.30(m,6H,-CH2(CH2)3CH<),1.44(s,18H,tBu).

[0399] Step 2: Place P 4 -A4D2 (10.25 g, 0.5 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 100 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (50 mL * 2), and the aqueous phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (1.73 g, 15.0 mmol) and DCC (3.09 g, 15.0 mmol) were added to the filtrate, followed by a dichloromethane solution (100 mL) containing DMAP (0.24 g, 2.0 mmol) and SM-1 (7.68 g, 15.0 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -A4D2(m)(6.90g; M n≈21.4kDa, PDI=1.03, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:7.34-7.21(m,10H,Ar-H),5.00(s,4H,Ar-CH2-),5.01-4.97(m,1H,-CHCH2NHC(=O)O-),4.65-4.55 (m,2H,>CHCOOtBu),4.33-3.97(m,11H,>CHC(=O)O-,-C(=O)OCH2CH<,-OC(=O)CH2O-,-C(=O)O(CH2)2OC(=O)NH-),3.85(s,8H, -OCH2C(=O)NH-),3.71-3.35(m,PEG,-CHCH2NHC(=O)O-),3.32-3.26(m,4H,-CH2NHCbz),3.24(s,6H,-OCH3),3.20-3.16(m,2H ,-C(=O)NHCH2(CH2)3-),2.43-2.16(m,8H,Glu-γ-CH2-,Glu-β-CH2-),1.87-1.28(m,6H,-CH2(CH2)3CH<),1.44(s,18H,tBu).

[0400] Example 12.2: P 4 Synthesis of -A4D2(m)-T1Y2

[0401] Step 1: Place P 4 -A4D2(m) (6.42 g, 0.3 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 70 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (35 mL * 2), and the aqueous phase was extracted with dichloromethane (35 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (1.04 g, 9.0 mmol) and DCC (1.85 g, 9.0 mmol) were added to the filtrate, followed by a dichloromethane solution (60 mL) containing DMAP (0.15 g, 1.2 mmol), DIPEA (1.55 g, 12.0 mmol), and Y2 (6.12 g, 9.0 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -A4D2(m)-Y2(3.72g; M n ≈22.5kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0402] Step 2: Place P4 -A4D2(m)-Y2 (2.25 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (25 mL). NHS (0.35 g, 3.0 mmol) and DCC (0.62 g, 3.0 mmol) were added, followed by a dichloromethane solution (15 mL) containing DMAP (0.05 g, 0.4 mmol) and T1 (1.32 g, 3.0 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -A4D2(m)-T1Y2(0.96g; M n ≈23.1kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=2:2). 1 H NMR(400MHz,(CD3)2SO)δ:8.75(s,2H),8.15(d,2H),7.82(s,2H),7.65-7.59(m,4H),7.58(dd,2H),7.12(s ,2H),6.66-6.61(m,4H),5.74(d,2H),5.33(d,2H),5.24(s,4H),5.02-4.96(m,1H),4.56-4.49(m,4H),4.3 2-3.95(m,19H),3.84(s,8H),3.73-3.36(m,PEG),3.25(s,6H),3.19-3.08(m,14H),2.95-2.87(m,2H),2.8 1-2.70(m,8H),2.56-2.44(m,4H),2.36-2.13(m,12H),2.08-1.94(m,4H),1.89-1.29(m,32H),0.96(t,6H).

[0403] Example 13: Four-arm polyethylene glycol derivative P 4 -C2D1(m) and its conjugates

[0404] Example 13.1: P 4 Synthesis of -C2D1(m)

[0405] Step 1: Referring to the method in Example 11.2, the two-arm polyethylene glycol derivative C2(M) was... n (≈10.5kDa, PDI=1.01, n≈112) and D1(M) nThe product (approximately 10.3 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -C2D1(M n ≈20.7kDa, PDI=1.02, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:4.22-3.91(m,5H,>CHC(=O)NH-,-OCH2C(=O)N<,>NCH2C (=O)-),4.14-3.93(m,4H,-CH2OC(=O)NH-),3.70-3.36(m,PEG,-O(CH2)2N<),3.33 -3.30(m,4H,-C(=O)NH(CH2)2NHC(=O)-),3.15-2.95(m,2H,-OC(=O)NHCH2-),1.82 -1.33(m,6H,-CH2(CH2)3CH<),0.88(s,36H,-C(CH3)3),0.06(s,24H,>Si(CH3)2).

[0406] Step 2: Place P 4 -C2D1 (10.35 g, 0.5 mmol) was dissolved in 1 M TBAF / THF (100 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (150 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and filtered. DSC (7.68 g, 30.0 mmol) and TEA (4.04 g, 40.0 mmol) were added to the filtrate, and the mixture was stirred overnight at room temperature. Then SM-1 (15.36 g, 30.0 mmol) was added, and the reaction was continued to be stirred at room temperature for 12 hours. After the reaction was complete, the mixture was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -C2D1(m)(7.74g; M n ≈22.4kDa, PDI=1.02, n1+n2+n3+n4≈448). 1H NMR(400MHz,(CD3)2SO)δ:7.36-7.23(m,20H,Ar-H),5.04(s,8H,Ar-CH2-),4.25-3.9 0(m,21H,>CHC(=O)NH-,-OCH2C(=O)N<,>NCH2C(=O)-,>CHCOOtBu,-CH2OC(=O)NH-),3 .75-3.38(m,PEG,-O(CH2)2N<),3.32-3.24(m,12H,-C(=O)NH(CH2)2NHC(=O)-,-CH2N HCbz),3.16-2.95(m,2H,-OC(=O)NHCH2-),2.28-2.10(m,12H,Glu-γ-CH2-,Glu-β-CH a H b -), 1.98-1.90(m,4H,Glu-β-CH a H b -),1.84-1.32(m,6H,-CH2(CH2)3CH<),1.45(s,36H,tBu).

[0407] Example 13.2: P 4 Synthesis of -C2D1(m)-T1Y2

[0408] Step 1: Place P 4 -C2D1(m) (6.72 g, 0.3 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 70 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (35 mL * 2), and the aqueous phase was extracted with dichloromethane (35 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (2.07 g, 18.0 mmol) and DCC (3.71 g, 18.0 mmol) were added to the filtrate, followed by a dichloromethane solution (120 mL) containing DMAP (0.29 g, 2.4 mmol), DIPEA (3.10 g, 24.0 mmol), and Y2 (12.24 g, 18.0 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -C2D1(m)-Y2(2.85g; M n ≈24.7kDa, PDI=1.02, n1+n2+n3+n4≈448).

[0409] Step 2: Place P 4-C2D1(m)-Y2 (2.47 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (25 mL). NHS (0.69 g, 6.0 mmol) and DCC (1.24 g, 6.0 mmol) were added, followed by a dichloromethane solution (30 mL) containing DMAP (0.10 g, 0.8 mmol) and T1 (2.65 g, 6.0 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -C2D1(m)-T1Y2(0.67g; M n ≈25.8kDa, PDI=1.02, n1+n2+n3+n4≈448; FA: Irinotecan=4:4). 1 H NMR(400MHz,(CD3)2SO)δ:8.72(s,4H),8.14(d,4H),7.82(s,4H),7.63-7.58(m,8H),7.55(dd,4H),7.10( s,4H),6.65-6.60(m,8H),5.73(d,4H),5.34(d,4H),5.22(s,8H),4.53-4.47(m,8H),4.32-3.91(m,33H),3 .70-3.36(m,PEG),3.33-3.22(m,4H),3.20-2.94(m,26H),2.94-2.85(m,4H),2.80-2.68(m,16H),2.55-2 .45(m,8H),2.39-2.29(m,16H),2.14-2.12(m,4H),2.06-1.93(m,12H),1.83-1.28(m,58H),0.95(t,12H).

[0410] Example 14: Four-arm polyethylene glycol derivative P 4 -H1F2(Glu) and its conjugates

[0411] Example 14.1: P 4 Synthesis of -H1F2(Glu)

[0412] Step 1: Dissolve N-Boc-glutamic acid-1-benzyl ester (triSM-1, 10.11 g, 30.0 mmol) in anhydrous dichloromethane (200 mL), then add NHS (3.45 g, 30.0 mmol), DCC (6.18 g, 30.0 mmol), DMAP (0.49 g, 4.0 mmol), and F2 (20.20 g, 2.0 mmol) in sequence. n ≈10.1kDa, PDI=1.02, n≈112). The reaction was stirred at room temperature for 24 hours. After the reaction was complete, the insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, dissolved in methanol (200mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 hours to remove Bn. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was purified by column chromatography to obtain F2(Glu)-COOH (17.16g; M). n ≈10.4kDa, PDI=1.02, n≈112).

[0413] Step 2: Dissolve F2(Glu)-COOH (15.60 g, 1.5 mmol) in anhydrous dichloromethane (150 mL), and then add benzyl hydrazide carboxylate (CbzNHNH2, 3.74 g, 22.5 mmol), EDCI HCl (4.32 g, 22.5 mmol), 1-hydroxybenzotriazole (HOBt, 3.04 g, 22.5 mmol), and DIPEA (5.81 g, 45.0 mmol) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (150 mL), and then wash sequentially with 1M HCl (100 mL x 3), saturated brine (100 mL x 3), and saturated sodium bicarbonate solution (100 mL x 3). Dry the organic phase with anhydrous magnesium sulfate and filter. Concentrate the filtrate under reduced pressure, dissolve it in methanol (150 mL), add Pd / C catalyst, and bubble with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, methanol was distilled off, and the residue was purified by column chromatography to obtain S43 (14.33 g; M n ≈10.4kDa, PDI=1.02, n≈112).

[0414] Step 3: Dissolve S43 (10.40 g, 1.0 mmol) in ethanol (EtOH, 200 mL), and add H1 (10.50 g, 1.0 mmol; M n The reaction mixture contained approximately 10.5 kDa (PDI = 1.02, n ≈ 112) and a catalytic amount of glacial acetic acid. The mixture was stirred at 70 °C for 6 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography to give a four-armed polyethylene glycol derivative, P0. 4-H1F2(Glu)(8.49g; M n ≈20.9kDa, PDI=1.03, n1+n2+n3+n4≈448). 1 H NMR(400MHz, (CD3)2SO)δ:7.48(t,1H,-CH=N-),7.35-7.22(m,10H,Ar-H),5.01(s,4H,Ar-CH2-),4.41-4.30(m,1H,>CHC(=O)NH-),4.25- 4.16(m,2H,>CHNHC(=O)O-,>CHNHBoc),4.13-3.87(m,4H,-CH2OC(=O)NH-),3.81(s,2H,-OCH2C(=O)NH-),3.67-3.37(m,PEG,-O(CH2)2NHC (=O)-,-C(=O)NHCH2CH2CH=N-),3.31-3.26(m,4H,-CH2NHCbz),3.23(s,6H,-OCH3),3.22-3.17(m,2H,-C(=O)NHCH2(CH2)3-),3.15-2.98( m,2H,-OC(=O)NHCH2-),2.57-2.43(m,2H,-CH2CH=N-),2.34-1.95(m,4H,-NHC(=O)(CH2)2CH<),1.87-1.30(m,21H,-CH2(CH2)3CH<,Boc).

[0415] Example 14.2: P 4 Synthesis of -H1F2(Glu)-T1Y1 (Method 1)

[0416] Step 1: Place P 4 -H1F2(Glu) (8.36 g, 0.4 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 100 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (50 mL * 2), and the aqueous phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.69 g, 6.0 mmol), DCC (1.24 g, 6.0 mmol), DMAP (0.10 g, 0.8 mmol), and Y1 (4.12 g, 6.0 mmol) were added to the filtrate sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction, insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -H1F2(Glu)-Y1(2.56g; M n ≈21.4kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0417] Step 2: Place P 4 -H1F2(Glu)-Y1 (2.14 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, and the methanol was distilled off. The residue was dissolved in anhydrous dichloromethane (25 mL), and NHS (0.35 g, 3.0 mmol), DCC (0.62 g, 3.0 mmol), DMAP (0.05 g, 0.4 mmol), and T1 (1.32 g, 3.0 mmol) were added sequentially. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, and the mixture was concentrated and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -H1F2(Glu)-T1Y1(1.13g; M n ≈22.0kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=2:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.73(s,2H),8.14(d,1H),7.82(s,1H),7.65-7.62(m,4H),7.55(dd,1H),7.47(t,1H),7 .12(s,1H),6.67-6.61(m,4H),5.75(d,1H),5.33(d,1H),5.23(s,2H),4.52-4.46(m,4H),4.36-4.13(m,5H),4.13- 3.86(m,4H),3.80(s,2H),3.69-3.35(m,PEG),3.24(s,6H),3.23-3.08(m,10H),2.93-2.83(m,1H),2.80-2.72(m, 4H),2.55-2.41(m,8H),2.35-2.22(m,7H),2.15-2.12(m,1H),2.08-1.95(m,4H),1.89-1.33(m,25H),0.96(t,3H).

[0418] Example 14.3: P 4 Synthesis of -H1F2(Glu)-T1Y1 (Method 2)

[0419] Step 1: Dissolve triSM-1 (10.11 g, 30.0 mmol) in anhydrous dichloromethane (200 mL), then add NHS (3.45 g, 30.0 mmol), DCC (6.18 g, 30.0 mmol), DMAP (0.49 g, 4.0 mmol), and F2 (20.20 g, 2.0 mmol) in sequence. n ≈10.1 kDa, PDI=1.02, n≈112). The reaction was stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was treated with a TFA / DCM mixture (1:1 v / v) to remove Boc, followed by washing with purified water. The aqueous phase was extracted with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain F2(Glu)-NH2 (16.54 g; M n ≈10.4kDa, PDI=1.02, n≈112).

[0420] Step 2: Dissolve Y1 (15.46 g, 22.5 mmol) in anhydrous dichloromethane (200 mL), then add NHS (2.59 g, 22.5 mmol), DCC (4.64 g, 22.5 mmol), DMAP (0.37 g, 3.0 mmol), and F2(Glu)-NH2 (15.60 g, 1.5 mmol) sequentially. Stir the reaction at room temperature for 24 hours. After the reaction is complete, filter to remove insoluble matter. Concentrate the filtrate under reduced pressure, dissolve the residue in methanol (200 mL), add Pd / C catalyst, and react with hydrogen bubbling at room temperature for 14 hours to remove Bn. Filter with diatomaceous earth to remove the catalyst, distill off the methanol, and purify the residue by column chromatography to obtain F2(Glu)-Y1 (14.59 g; M n ≈11.0kDa, PDI=1.02, n≈112).

[0421] Step 3: Dissolve F2(Glu)-Y1 (11.00 g, 1.0 mmol) in anhydrous dichloromethane (150 mL), and then add CbzNHNH2 (2.49 g, 15.0 mmol), EDCI HCl (2.88 g, 15.0 mmol), HOBt (2.03 g, 15.0 mmol), and DIPEA (3.87 g, 30.0 mmol) sequentially. Stir the reaction overnight at room temperature. After the reaction is complete, dilute with dichloromethane (150 mL), and then wash sequentially with 1M HCl (100 mL x 3), saturated brine (100 mL x 3), and saturated sodium bicarbonate solution (100 mL x 3). Dry the organic phase with anhydrous magnesium sulfate and filter. Concentrate the filtrate under reduced pressure, dissolve it in methanol (150 mL), add Pd / C catalyst, and bubble with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, methanol was distilled off, and the residue was purified by column chromatography to obtain S44 (8.98 g; M n ≈11.0kDa, PDI=1.02, n≈112).

[0422] Step 4: Dissolve S44 (4.40 g, 0.4 mmol) in ethanol (EtOH, 100 mL), and add H1 (4.20 g, 0.4 mmol; M n The reaction mixture contained approximately 10.5 kDa (PDI = 1.02, n ≈ 112) and a catalytic amount of glacial acetic acid. The mixture was stirred at 70°C for 6 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography to obtain P... 4 -H1F2(Glu)-Y1(3.25g; M n ≈21.4kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0423] Step 5: Referring to step 2 of Example 14.2, P 4 -H1F2(Glu)-Y1 coupled with T1 yields folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -H1F2(Glu)-T1Y1(1.21g; M n ≈22.0kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=2:1).

[0424] Example 15: Four-arm polyethylene glycol derivative P 4 -G2D1(Glu) and its conjugates

[0425] Example 15.1: P 4 Synthesis of -G2D1(Glu)

[0426] Referring to the method of Example 14.1, H1 is replaced with G2 (M n ≈10.1kDa, PDI=1.02, n≈112), replace F2 with D1(M n With the given conditions remaining unchanged (≈10.3 kDa, PDI=1.02, n≈112), a four-armed polyethylene glycol derivative P was obtained. 4 -G2D1(Glu)(M n ≈20.7kDa, PDI=1.02, n1+n2+n3+n4≈448). 1 H NMR(400MHz, (CD3)2SO)δ:7.45(t,1H,-CH=N-),4.24-3.90(m,9H,-OCH2C(=O)N< ,>NCH2C(=O)-,>CHNHBoc),3.68-3.28(m,PEG,-O(CH2)2N<,-C(=O)NHCH2-),3.2 4(s,6H,-OCH3),2.56-2.43(m,2H,-CH2CH=N-),2.35-1.93(m,4H,-NHC(=O)(CH2 )2CH<),1.43(s,9H,Boc-tBu),0.88(s,18H,TBS-tBu),0.06(s,12H,>Si(CH3)2).

[0427] Example 15.2: P 4 Synthesis of -G2D1(Glu)-T1Y1

[0428] Step 1: Place P 4 -G2D1(Glu) (8.28 g, 0.4 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 100 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction, the mixture was washed with purified water (50 mL * 2), and the aqueous phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.69 g, 6.0 mmol), DCC (1.24 g, 6.0 mmol), DMAP (0.10 g, 0.8 mmol), and Y1 (4.12 g, 6.0 mmol) were added to the filtrate sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction, insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -G2D1(Glu)-Y1(2.21g; M n ≈21.2kDa, PDI=1.02, n1+n2+n3+n4≈448).

[0429] Step 2: Place P 4 -G2D1(Glu)-Y1 (2.12 g, 0.1 mmol) was dissolved in 1 M TBAF / THF solution (30 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. T1 (0.88 g, 2.0 mmol), DMAP (0.05 g, 0.4 mmol), and DCC (0.41 g, 2.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (30 mL), and the aqueous phase was extracted with dichloromethane (15 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -G2D1(Glu)-T1Y1(1.33g; M n ≈21.9kDa, PDI=1.02, n1+n2+n3+n4≈448; FA: Irinotecan=2:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.76(s,2H),8.14(d,1H),7.84(s,1H),7.68-7.64(m,4H),7.56(dd,1H),7.44(t,1H) ,7.15(s,1H),6.70-6.63(m,4H),5.76(d,1H),5.31(d,1H),5.24(s,2H),4.53-4.47(m,4H),4.33-3.90(m,15H) ,3.69-3.27(m,PEG),3.25(s,6H),3.17-3.06(m,6H),2.94-2.85(m,1H),2.82-2.73(m,4H),2.58(t,2H),2.56- 2.44(m,6H),2.43(t,4H),2.36-2.24(m,3H),2.16-2.12(m,1H),2.10-1.42(m,14H),1.39(t,3H),0.97(t,3H).

[0430] Example 16: Four-arm polyethylene glycol derivative P 4 -H2D1(Glu) and its conjugates

[0431] Example 16.1: P 4 Synthesis of -H2D1(Glu)

[0432] Referring to the method in Example 14.1, H1 is replaced with H2 (Mn ≈10.3kDa, PDI=1.02, n≈112), replace F2 with D1(M n With the given conditions remaining unchanged (≈10.3 kDa, PDI=1.02, n≈112), a four-armed polyethylene glycol derivative P was obtained. 4 -H2D1(Glu)(M n ≈20.9kDa, PDI=1.02, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:7.44(t,1H,-CH=N-),4.26-3.91(m,9H,-OCH2C(=O)N<,>N CH2C(=O)-,>CHNHBoc),3.70-3.27(m,PEG,-O(CH2)2N<,-C(=O)NH(CH2)2NHC(=O)-,- C(=O)NHCH2CH2CH=N-),2.55-2.41(m,2H,-CH2CH=N-),2.34-1.91(m,4H,-NHC(=O)( CH2)2CH<),1.43(s,9H,Boc-tBu),0.88(s,36H,TBS-tBu),0.06(s,24H,>Si(CH3)2).

[0433] Example 16.2: P 4 Synthesis of -H2D1(Glu)-T1Y1

[0434] Step 1: Place P 4 -H2D1(Glu) (8.36 g, 0.4 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 100 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction, the mixture was washed with purified water (50 mL * 2), and the aqueous phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (0.69 g, 6.0 mmol), DCC (1.24 g, 6.0 mmol), DMAP (0.10 g, 0.8 mmol), and Y1 (4.12 g, 6.0 mmol) were added to the filtrate sequentially. The mixture was stirred at room temperature for 24 hours. After the reaction, insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -H2D1(Glu)-Y1(2.48g; M n ≈21.4kDa, PDI=1.02, n1+n2+n3+n4≈448).

[0435] Step 2: Place P 4-H2D1(Glu)-Y1 (2.14 g, 0.1 mmol) was dissolved in 1 M TBAF / THF solution (30 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. T1 (1.76 g, 4.0 mmol), DMAP (0.10 g, 0.8 mmol), and DCC (0.82 g, 4.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (30 mL), and the aqueous phase was extracted with dichloromethane (15 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -H2D1(Glu)-T1Y1(0.81g; M n ≈22.7kDa, PDI=1.02, n1+n2+n3+n4≈448; FA: Irinotecan=4:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.75(s,4H),8.12(d,1H),7.81(s,1H),7.65-7.60(m,8H),7.55(dd,1H),7.41 (t,1H),7.13(s,1H),6.68-6.60(m,8H),5.75(d,1H),5.32(d,1H),5.23(s,2H),4.55-4.46(m,8H),4.28- 3.88(m,21H),3.68-3.29(m,PEG),3.16-3.08(m,6H),2.91-2.83(m,1H),2.81-2.72(m,4H),2.56(t,2H), 2.53-2.40(m,14H),2.35-2.23(m,3H),2.17-2.12(m,1H),2.12-1.40(m,18H),1.38(t,3H),0.96(t,3H).

[0436] Example 17: Four-arm polyethylene glycol derivative P 4 -G1C4(Lys) and its conjugates

[0437] Example 17.1: P 4 Synthesis of -G1C4(Lys)

[0438] Step 1: Combine C4 (20.80g, 2.0mmol; M) nDissolve 5.76 g (30.0 mmol) of lysine derivative (triSM-2, 10.08 g, 30.0 mmol) containing one Cbz-protected amino group and one tBu-protected carboxyl group in 200 mL of dichloromethane. Add DMAP (0.49 g, 4.0 mmol) and DIPEA (7.74 g, 60.0 mmol). Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with 100 mL of dichloromethane and wash successively with water (150 mL x 2), 1 M HCl solution (150 mL x 2), saturated sodium bicarbonate solution (150 mL x 2), and saturated sodium chloride solution (150 mL x 2). Dry the organic phase with anhydrous magnesium sulfate and filter. The filtrate was concentrated under reduced pressure, and the crude product was treated with a TFA / DCM mixture (1:1 v / v) to remove tBu, followed by washing with purified water. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain C4(Lys)-COOH (16.81 g; M n ≈10.5kDa, PDI=1.02, n≈112).

[0439] Step 2: C4(Lys)-COOH (15.74 g, 1.5 mmol) was dissolved in anhydrous dichloromethane (150 mL), and tert-butyl hydrazide formate (BocNHNH2, 2.97 g, 22.5 mmol), EDCI HCl (4.32 g, 22.5 mmol), HOBt (3.04 g, 22.5 mmol), and DIPEA (5.81 g, 45.0 mmol) were added sequentially. The reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with dichloromethane (150 mL), and then washed sequentially with 1M HCl (100 mL * 3), saturated saline solution (100 mL * 3), and saturated sodium bicarbonate solution (100 mL * 3). The organic phase was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the crude product was used to remove Boc with a TFA / DCM mixed solution (1:1 v / v), followed by washing with purified water. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S45 (11.80 g; M). n ≈10.5kDa, PDI=1.02, n≈112).

[0440] Step 3: Dissolve S45 (10.50 g, 1.0 mmol) in ethanol (EtOH, 200 mL), and add G1 (10.50 g, 1.0 mmol; M nThe reaction mixture contained approximately 10.2 kDa (PDI = 1.02, n ≈ 112) and a catalytic amount of glacial acetic acid. The mixture was stirred at 70°C for 6 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography to give a four-armed polyethylene glycol derivative, P0. 4 -G1C4(Lys)(9.01g; M n ≈20.7kDa, PDI=1.03, n1+n2+n3+n4≈448). 1 H NMR(400MHz, (CD3)2SO)δ:7.47(t,1H,-CH=N-),7.38-7.24(m,5H,Ar-H),5.00(s,2H,Ar-CH2-),4.44-4.38(m,1H,>C HC(=O)NH-),4.25-3.88(m,5H,>CHNHC(=O)O-,-OCH2C(=O)N<,>NCH2C(=O)-),4.14-3.91(m,4H,-CH2OC(=O)NH-),3. 70-3.35(m,PEG,-O(CH2)2N<,-C(=O)NHCH2-),3.23(s,6H,-OCH3),3.20-2.93(m,4H,-OC(=O)NHCH2-,-CH2NHCbz),2 .53-2.40(m,2H,-CH2CH=N-),1.85-1.26(m,12H,-CH2(CH2)3CH<),0.87(s,18H,TBS-tBu),0.05(s,12H,>Si(CH3)2).

[0441] Example 17.2: P 4 Synthesis of -G1C4(Lys)-T1Y1

[0442] Step 1: Place P 4 -G1C4(Lys) (8.28 g, 0.4 mmol) was dissolved in methanol (150 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth, the methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (100 mL). NHS (0.69 g, 6.0 mmol), DCC (1.24 g, 6.0 mmol), DMAP (0.10 g, 0.8 mmol), and Y1 (4.12 g, 6.0 mmol) were added sequentially. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -G1C4(Lys)-Y1(2.70g; M n ≈21.2kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0443] Step 2: Place P 4 -G1C4(Lys)-Y1 (2.12 g, 0.1 mmol) was dissolved in 1 M TBAF / THF solution (30 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. T1 (0.88 g, 2.0 mmol), DMAP (0.05 g, 0.4 mmol), and DCC (0.41 g, 2.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (30 mL), and the aqueous phase was extracted with dichloromethane (15 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -G1C4(Lys)-T1Y1(1.19g; M n ≈21.9kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=2:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.73(s,2H),8.11(d,1H),7.82(s,1H),7.67-7.62(m,4H),7.55(dd,1H),7.43(t,1 H),7.14(s,1H),6.70-6.61(m,4H),5.77(d,1H),5.30(d,1H),5.23(s,2H),4.52-4.46(m,4H),4.44-3.88(m, 12H),4.16-3.92(m,4H),3.74-3.37(m,PEG),3.24(s,6H),3.18-2.95(m,10H),2.94-2.83(m,1H),2.83-2.71 (m,4H),2.57(t,2H),2.50-2.38(m,10H),1.82-1.28(m,12H),2.07-1.43(m,14H),1.38(t,3H),0.96(t,3H).

[0444] Example 18: Four-arm polyethylene glycol derivative P 4 -C4C4 (Cys-Cys) and its conjugates

[0445] Example 18.1: P 4 Synthesis of -C4C4 (Cys-Cys)

[0446] Step 1: Add C4 (10.40g, 1.0mmol; M) n ≈10.4 kDa, PDI=1.02, n≈112) and EDCI HCl (2.88 g, 15.0 mmol) were dissolved in 100 mL of dichloromethane. Cystine bis(tert-butyl) dihydrochloride (tetraSM-1, 5.96 g, 15.0 mmol) and DMAP (0.24 g, 2.0 mmol) were added, followed by DIPEA (3.87 g, 30.0 mmol). The reaction was stirred overnight at room temperature. After the reaction was complete, the solution was diluted with 100 mL of dichloromethane and washed successively with water (100 mL * 2), 1M HCl solution (100 mL * 2), saturated sodium bicarbonate solution (100 mL * 2), and saturated sodium chloride solution (100 mL * 2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S46 (9.66 g; M n ≈10.6kDa, PDI=1.02, n≈112).

[0447] Step 2: Dissolve C4 (5.20 g, 0.5 mmol) and EDCI HCl (1.44 g, 7.5 mmol) in 100 mL of dichloromethane, add S46 (5.30 g, 0.5 mmol) and DMAP (0.12 g, 1.0 mmol), then add DIPEA (1.94 g, 15.0 mmol). Stir the reaction overnight at room temperature. After the reaction is complete, dilute with 100 mL of dichloromethane, then wash successively with water (100 mL * 2), 1 M HCl solution (100 mL * 2), saturated sodium bicarbonate solution (100 mL * 2), and saturated sodium chloride solution (100 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain the four-armed polyethylene glycol derivative P. 4 -C4C4(Cys-Cys)(8.14g; M n ≈20.9kDa, PDI=1.03, n1+n2+n3+n4≈448). 1 H NMR(400MHz,(CD3)2SO)δ:4.71(dd,2H,Cys-α-CH),4.22-3.92(m,8H,-OCH2C(=O)N<,>NCH2C(=O)-),3.67-3.33(m,P EG,-O(CH2)2N<),3.15(d,4H,Cys-β-CH2),1.44(s,18H,-COOtBu),0.88(s,36H,TBS-tBu),0.06(s,24H,>Si(CH3)2).

[0448] Example 18.2: P4 Synthesis of -C4C4(Cys-Cys)-T1Y2

[0449] Step 1: Place P 4 -C4C4(Cys-Cys) (6.27 g, 0.3 mmol) was dissolved in a TFA / DCM mixture (1:1 v / v; 70 mL), and the mixture was stirred for 3 hours to remove tBu. After the reaction, the mixture was washed with purified water (35 mL * 2), and the aqueous phase was extracted with dichloromethane (35 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. NHS (1.04 g, 9.0 mmol) and DCC (1.85 g, 9.0 mmol) were added to the filtrate, followed by a dichloromethane solution (60 mL) containing DMAP (0.15 g, 1.2 mmol), DIPEA (1.55 g, 12.0 mmol), and Y2 (6.12 g, 9.0 mmol). The mixture was stirred at room temperature for 24 hours. After the reaction, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain P. 4 -C4C4(Cys-Cys)-Y2(4.57g; M n ≈22.0kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0450] Step 2: Place P 4 -C4C4(Cys-Cys)-Y2 (2.20 g, 0.1 mmol) was dissolved in 1 M TBAF / THF solution (30 mL), and the reaction was stirred for 6 hours to remove TBS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (30 mL) and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was placed in an ice bath. T1 (2.65 g, 6.0 mmol), DMAP (0.10 g, 0.8 mmol), and DCC (1.24 g, 6.0 mmol) were added sequentially. After stirring for 30 minutes, the reaction was continued overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (30 mL), and the aqueous phase was extracted with dichloromethane (15 mL * 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain the folic acid-modified four-arm polyethylene glycol-irinotecan conjugate P. 4 -C4C4(Cys-Cys)-T1Y2(0.43g; M n ≈23.2kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=4:2). 1H NMR(400MHz,(CD3)2SO)δ:8.72(s,4H),8.13(d,2H),7.83(s,2H),7.64-7.58(m,8H),7.55(dd,2H ),7.15(s,2H),6.69-6.61(m,8H),5.72(d,2H),5.31(d,2H),5.22(s,4H),4.88-4.80(m,2H),4.5 7-4.47(m,8H),4.24-3.88(m,24H),3.66-3.34(m,PEG),3.15-3.06(m,14H),2.92-2.82(m,2H),2 .80-2.70(m,8H),2.53-2.42(m,4H),2.41(t,8H),2.15-1.41(m,28H),1.39(t,6H),0.96(t,6H).

[0451] Example 19: Preparation of a comparative example

[0452] Example 19.1: P 2 Synthesis of -C1(2K)-T1Y2

[0453] Step 1: Add C1(2K)(8.20g, 0.4mmol; M n ≈20.5kDa, PDI=1.02, n1≈n2≈225; referring to the preparation method of C1, the molecular weight of polyethylene glycol raw materials was adjusted to obtain it; C1(2K) has the same structural formula as C1) and EDCI HCl (1.15g, 6.0mmol) were dissolved in 80mL of dichloromethane, Y2 (4.08g, 6.0mmol) and DMAP (0.10g, 0.8mmol) were added, and then DIPEA (2.58g, 20.0mmol) was added. The reaction was stirred overnight at room temperature. After the reaction was completed, 80mL of dichloromethane was added for dilution, and then the mixture was washed successively with water (80mL*2), 1M HCl solution (80mL*2), saturated sodium bicarbonate solution (80mL*2) and saturated sodium chloride solution (80mL*2). The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain C1(2K)-Y2 (2.97g; M n ≈21.0kDa, PDI=1.02, n1≈n2≈225).

[0454] Step 2: C1(2K)-Y2 (2.10 g, 0.1 mmol) was dissolved in methanol (30 mL), and Pd / C catalyst was added. The mixture was bubbled with hydrogen at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomaceous earth. The methanol was distilled off, and the residue was dissolved in anhydrous dichloromethane (20 mL). NHS (0.35 g, 3.0 mmol) and DCC (0.62 g, 3.0 mmol) were added, followed by a dichloromethane solution (15 mL) containing DMAP (0.05 g, 0.4 mmol) and T1 (1.32 g, 3.0 mmol). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated, and purified by column chromatography to obtain the folic acid-modified two-arm polyethylene glycol-irinotecan conjugate P. 2 -C1(2K)-T1Y2(1.34g; M n ≈21.6kDa, PDI=1.02, n1≈n2≈225; FA:Irinotecan=2:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.74(s,2H),8.14(d,1H),7.83(s,1H),7.68-7.64(m,4H),7.55(dd,1H), 7.12(s,1H),6.67-6.60(m,4H),5.75(d,1H),5.36(d,1H),5.24(s,2H),4.54-4.48(m,4H),4.30-4. 16(m,3H),4.14-3.90(m,6H),3.71-3.36(m,PEG),3.13-2.97(m,8H),2.95-2.84(m,1H),2.80-2.71 (m,4H),2.57-2.44(m,2H),2.32-2.22(m,4H),2.06-1.90(m,4H),1.84-1.27(m,19H),0.95(t,3H).

[0455] Example 19.2: P 2 Synthesis of -B1(2K)-T1Y1

[0456] Step 1: Add B1(2K)(8.12g, 0.4mmol; M n≈20.3kDa, PDI=1.02, n1≈n2≈225; Following the preparation method of B1, the molecular weight of the polyethylene glycol raw material was adjusted to obtain B1(2K)-Boc(7.12g, Mn2 ... n ≈20.4kDa, PDI=1.02, n1≈n2≈225).

[0457] Step 2: Dissolve B1(2K)-Boc (4.08 g, 0.2 mmol) in 1 M TBAF / THF (50 mL) and stir for 6 hours to remove TBS. After the reaction is complete, concentrate under reduced pressure, dissolve the residue in dichloromethane (50 mL), and wash with saturated ammonium chloride solution. Dry the organic phase with anhydrous magnesium sulfate, filter, and place the filtrate in an ice bath. Add Y1 (1.37 g, 2.0 mmol), DMAP (0.05 g, 0.4 mmol), and DCC (0.41 g, 2.0 mmol) sequentially. Stir for 30 minutes, and then continue stirring overnight at room temperature. Quench the reaction with saturated sodium chloride solution (50 mL), and extract the aqueous phase with dichloromethane (25 mL * 2). Combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain B1(2K)-Y1 (3.26 g; M n ≈20.9kDa, PDI=1.02, n1≈n2≈225).

[0458] Step 3: Dissolve B1(2K)-Y1 (2.09 g, 0.1 mmol) in methanol (30 mL), add Pd / C catalyst, and react with hydrogen bubbling at room temperature for 14 h to remove Cbz. Filter with diatomaceous earth to remove the catalyst, distill off the methanol, dissolve the residue in anhydrous dichloromethane (20 mL), add NHS (0.17 g, 1.5 mmol) and DCC (0.31 g, 1.5 mmol), and then add a dichloromethane solution (10 mL) containing DMAP (24 mg, 0.2 mmol) and T1 (0.66 g, 1.5 mmol). Stir the reaction at room temperature for 24 h. After the reaction is complete, filter to remove insoluble matter, concentrate, and purify by column chromatography to obtain the folic acid-modified two-arm polyethylene glycol-irinotecan conjugate P. 2 -B1(2K)-T1Y1(1.77g; M n≈21.2kDa, PDI=1.02, n1≈n2≈225; FA:Irinotecan=1:1). 1 H NMR(400MHz,(CD3)2SO)δ:8.72(s,1H),8.15(d,1H),7.85(s,1H),7.64-7.60(m,2H),7.52(dd,1H),7.12(s,1 H),6.68-6.63(m,2H),5.77(d,1H),5.35(d,1H),5.25(s,2H),4.54-4.49(m,2H),4.30-3.89(m,7H),3.66-3. 37(m,PEG),3.35-3.30(m,2H),3.28-3.25(m,2H),3.16-3.05(m,6H),2.99-2.87(m,1H),2.82-2.71(m,4H),2 .66-2.62(m,4H),2.53-2.41(m,2H),2.34-2.25(m,2H),2.06-1.92(m,2H),1.83-1.30(m,22H),0.96(t,3H).

[0459] Example 19.3: P 4 Synthesis of -A1J2-Y2

[0460] Step 1: Referring to the method in Example 2.1, the two arms of polyethylene glycol derivative A1 (M) were... n (≈10.2kDa, PDI=1.02, n≈112) and J2(M n The product (approximately 10.2 kDa, PDI = 1.02, n ≈ 112) underwent an amidation reaction to obtain a four-armed polyethylene glycol derivative P. 4 -A1J2(M n ≈20.4kDa, PDI=1.03, n1+n2+n3+n4≈448).

[0461] Step 2: Referring to the method in Example 4.2, P 4 -A1J2, after the removal of tBu, undergoes an amidation reaction with Y2 to obtain a four-arm polyethylene glycol-irinotecan conjugate P without the targeting group. 4 -A1J2-Y2(M n ≈20.9kDa, PDI=1.03, n1+n2+n3+n4≈448; FA: Irinotecan=0:1). 1H NMR(400MHz,(CD3)2SO)δ:8.16(d,1H),7.83(s,1H),7.56(dd,1H),7.14(s,1H),5.7 4(d,1H),5.32(d,1H),5.23(s,2H),4.44-4.35(m,1H),4.24-4.15(m,1H),4.14-3.9 3(m,6H),3.81(s,4H),3.69-3.34(m,PEG),3.24(s,9H),3.22-2.97(m,10H),2.95-2 .83(m,1H),2.80-2.72(m,4H),2.56-2.42(m,2H),1.91-1.28(m,25H),0.95(t,3H).

[0462] Example 20: Biological Activity Test

[0463] (1) Serum stability evaluation

[0464] 1 mL of mouse serum was added to each of 18 1.3 mL microcentrifuge tubes, dividing them into one control group and 17 experimental groups. The experimental groups were respectively given the tetra-armed polyethylene glycol derivative (P) described in Examples 2–18. 4 -A1B1、P 4 -A1B2、P 4 -A3B3、P 4 -A2B1、P 4 -A2B2、P 4 -C1D1、P 4 -E1B1、P 4 -E2D2、P 4 -C1B1、P 4 -A1J1(m), P 4 -A4D2(m), P 4 -C2D1(m), P 4 -H1F2(Glu), P 4 -G2D1(Glu), P 4 -H2D1(Glu), P 4 -G1C4(Lys), P 4 -C4C4(Cys-Cys)), and the control group was treated with polyethylene glycol standard with a molecular weight of 20 kDa, at a concentration of 5.0 mg / mL. After incubation at 37°C for 96 hours, 200 μL of sample was taken. Acetonitrile was added, and the mixture was vortexed for 1 minute to precipitate proteins from the serum. The sample was then centrifuged, and the supernatant was collected. Hexane was added to the recovered solution, and the mixture was vortexed for 1 minute and centrifuged. The lower layer was collected, concentrated under vacuum, and the sample was recovered. The molecular weight was determined by GPC, and the decomposition rate was calculated using the following formula:

[0465] Decomposition rate = (M before the experiment) n Peak area % - M after the experiment n (peak area %) / (M before experiment) n (peak area %) × 100%; where M n The number-average molecular weight is that of the four-arm polyethylene glycol-irinotecan conjugate.

[0466] The results showed that the decomposition rate of the experimental groups did not exceed 3%, indicating that the four-armed polyethylene glycol derivative of the present invention has good serum stability.

[0467] (2) In vitro anticancer efficacy test

[0468] One blank control group, one positive control group, and 17 experimental groups were set up. The blank control group received no drugs, the positive control group received irinotecan, and the experimental groups received four-arm targeted conjugates (including the folic acid-modified four-arm polyethylene glycol-irinotecan conjugates from Examples 2-18, as shown in Table 1). Six cancer cells were selected as in vitro cancer cell models: COLO205 human colon cancer cells, human colon adenocarcinoma cells HT29, human lung adenocarcinoma cells A549, pancreatic cancer cells MiaPaCa-2, human ovarian cancer cells A2780, and human ovarian adenocarcinoma cells OVCAR-3.

[0469] Six replicates were set up for each group. The inoculation density was 1×10⁶. 4 Cells / well: 100 μL of cell suspension was seeded per well into 96-well plates. After seeding, the plates were incubated at 37°C and 4% CO2 for 24 h. The old culture medium was discarded. 100 μL of medium containing 10 nM tetra-armed targeting conjugate was added to each well of the experimental group, 100 μL of medium containing 10 nM irinotecan was added to each well of the positive control group, and 100 μL of fresh culture medium was added to each well of the blank control group. After incubation for another 48 h, 20 μL of 5 mg / mL MTT in PBS buffer was added to each well. After incubating the MTT with cancer cells for 4 h, the mixture of culture medium and MTT buffer was discarded. 150 μL of DMSO was added to each well to dissolve the blue-purple crystalline formazan from the live cells. The plates were gently shaken until fully dissolved, and the absorbance at 490 nm was measured using a microplate reader. The experiment was repeated three times, and the average value was taken.

[0470] Cell viability = (absorbance value of drug group / absorbance value of blank group) × 100%.

[0471] The results showed that both the positive control group and the experimental group had significant inhibitory effects on the proliferation of the aforementioned six types of cancer cells, and the survival rate of cancer cells in some experimental groups was even lower than that in the positive control group, indicating that the four-arm polyethylene glycol derivative of the present invention does not significantly affect the activity of the modified drug.

[0472] Table 1 Results of in vitro anticancer efficacy test

[0473] (3) In vivo anti-tumor effect test

[0474] The control group (irinotecan, P) was set up according to Table 2. 2 -C1(2K)-T1Y2、P 2 -B1(2K)-T1Y1、P 4 -A1J2-Y2) and experimental group (P 4 -G2D1(Glu)-T1Y1、P 4 -A1B1-T1Y1、P 4 -C1D1-T1Y1、P 4 -C1B1-T1Y2、P 4 -E2D2-T1Y2、P 4 Thirty-three mice inoculated with S180 tumors were randomly divided into 11 groups. Inoculation occurred on day 0, followed by tail vein injections at a dose of 30 mg / kg (based on irinotecan) on days 4, 7, and 10. The blank control group received saline, the irinotecan group received irinotecan hydrochloride solution, and the remaining control and experimental groups received the corresponding polyethylene glycol-modified irinotecan saline preparations. On day 13 post-inoculation, mice were euthanized by cervical dislocation, the tumors were removed, and the mice were weighed. The tumor inhibition rate was calculated using the following formula:

[0475] Tumor inhibition rate = [GH / G]*100% (G represents the average tumor weight in the blank group, and H represents the average tumor weight in the control group or experimental group).

[0476] The following conclusions can be drawn from the results shown in Table 2:

[0477] (1) P in the control group 2 -C1(2K)-T1Y2 and the P of the present invention 4 The targeting group to irinotecan ratio (FA:Irinotecan) of -G2D1(Glu)-T1Y1 is 2:1, and the irinotecan is not located at the PEG terminus; however, P 4 -G2D1(Glu)-T1Y1 showed a significantly higher tumor inhibition rate than P. 2 -C1(2K)-T1Y2;

[0478] (2) P in the control group 2 -B1(2K)-T1Y1 and the P of the present invention 4 The targeting group to irinotecan ratio (FA:Irinotecan) of -A1B1-T1Y1 is 1:1, with all irinotecan located at the PEG terminus; however, P4 -A1B1-T1Y1 showed a significantly higher tumor inhibition rate than P. 2 -B1(2K)-T1Y1;

[0479] (3) P in the control group 4 -A1J2-Y2 contains no targeting groups, and its tumor inhibition rate is significantly lower than that of P in this invention. 4 -A1B1-T1Y1(FA:Irinotecan=1:1),P 4 -C1D1-T1Y1(FA:Irinotecan=2:2),P 4 -C1B1-T1Y2(FA:Irinotecan=3:1),P 4 -E2D2-T1Y2(FA:Irinotecan=1:3) and P 4 -A1J1(m)-T1Y2(FA:Irinotecan=1:1).

[0480] Table 2. Control and experimental group settings for in vivo antitumor effect testing.

[0481] The above results indicate that, compared with two-arm structures of the same molecular weight and non-targeted four-arm structures, the four-arm polyethylene glycol derivatives of the present invention enable the modified drug to exert its efficacy more fully in vivo.

Claims

1. A four-armed polyethylene glycol derivative, the structure of which is shown in formula (1): Or its salts, tautomers, stereoisomers, isotopic substitutes or solvates; in, w is 1 or 2; X a and X b Each can be independently classified as >CH- or >N-; Each L can be independently a divalent or trivalent linker; Each R 3 Independently does not exist or is -L x -R; L x It is a linker bond or a divalent linker base; When at least one R exists 3 At that time, -[L(R) 3 )] w - Contains at least one biodegradable component; P a1 P a2 P b1 and P b2 Each independently is -L 0 -(CH2CH2O) n -R; Each L 0 Independently, it can be a linker or a divalent linker; Each n is an independent integer from 5 to 500; R 3 P a1 P a2 P b1 and P b2 Each R in the equation is independently C 1-6 alkyl, benzyl or Each F 0 Independently a reactive group or its protected form; E represents a linker, a divalent linker, or a trivalent linker; k is 1 or 2; The four-armed polyethylene glycol derivative contains two, three, or more different F-type structures. 0 At least including F 01 and F 02 The F 01 and F 02 For two different structures of F 0 ; The four-arm polyethylene glycol derivative is monodisperse or polydisperse.

2. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, X a and X b All are >CH-; or, X a and X b All are >N-; or, X a For >CH-, X b For >N-; or, X a For >N-, X b For >CH-.

3. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, The degradable portion is degradable under physiological conditions and is selected from any of the following: pH-sensitive linker, redox-sensitive linker, enzyme-cleavable linker, nuclease-sensitive linker, lipase-sensitive linker, glycosidase-sensitive linker, hypoxia-sensitive linker, light-cleavable linker, thermally unstable linker, ultrasound-sensitive linker, and X-ray-cleavable linker. The degradable portion is preferably a pH-sensitive linker, more preferably any one of a hydrazone bond, an acylhydrazone bond, a carbamate bond, and a carbonate bond; Alternatively, the degradable portion is preferably a redox-sensitive linker, more preferably a disulfide bond or a diselenide bond; Alternatively, the degradable portion is preferably an enzyme-cleavable linker, more preferably an ester bond.

4. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, The four-armed polyethylene glycol derivative is monodisperse, and each n is an independent integer from 5 to 35, preferably an integer from 5 to 25, and more preferably an integer from 5 to 15. Alternatively, the four-armed polyethylene glycol derivative is polydisperse, with each n being an independent integer from 20 to 250, preferably an integer from 100 to 125, and more preferably an integer from 106 to 118. Preferably, P a1 P a2 P b1 and P b2 n has the same value.

5. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Each F 0 Independently selected from any one of the following groups or their protected forms: amino, secondary amino, hydroxyl, mercapto, aldehyde, carboxyl, azide, alkenyl, alkynyl, olefinic ester, succinimide, maleimide, isocyanate, isothiocyanate, halogen, acyl halide, acyl hydrazine, sulfonate, active ester, acid anhydride, and epoxy; two F groups connected to the same E phase. 0 They have the same or different structures.

6. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, F 01 and F 02 The protected forms are the same reactive group but contain different protecting groups, preferably any of the following: (i)F 01 and F 02 One of them is -NHCbz, and the other is -NHBoc; (ii)F 01 and F 02 One of them is -COOBn, and the other is -COOtBu; Or, F 01 and F 02 The reactive groups or their protected forms are of different kinds, preferably any of the following: (iii)F 01 and F 02 One of them is -NH2 or its protected form, and the other is -OH or its protected form; (iv)F 01 and F 02 One of them is -NH2 or its protected form, and the other is -COOH or its protected form; (v)F 01 and F 02 One of them is -OH or its protected form, and the other is -COOH or its protected form.

7. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Each L 0 Independently for the connector key, C 1-6 Alkylene or -B 0 -(Z 0 -B 0 ) j -; where L 0 In this context, j is an integer from 1 to 3; each B 0 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 0 Not simultaneously with two Z 0 Connected; each Z 0 Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z 0 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z 0 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, and -NHC(=O)NH-; Preferably, each L 0 Independently selected from any one of -O-, -OCH2-*, -O(CH2)2-*, -OCH2C(=O)-*, -OC(=O)NH-*, -OC(=O)NH(CH2)4-*, -OCH2C(=O)O-*, -OCH2C(=O)OCH2-*, -OCH2C(=O)NH-*, -OCH2C(=O)NH(CH2)2-*, -OCH2C(=O)NH(CH2)3-*, -OCH2C(=O)NH(CH2)4-*, -O(CH2)2NHC(=O)-*, -O(CH2)2NHC(=O)O-*, and -O(CH2)2NHC(=O)OCH2-*, wherein the * terminus is associated with X. a or X b Connected.

8. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Each E is independently selected from any of the following cases: (i) Connect key; (ii) A divalent linker, preferably C 1-6 Alkylene, more preferably any one of -CH2-, -(CH2)2-, -(CH2)3- and -(CH2)4-; (iii) A trivalent linker, preferably any of the following structures: Where m1 is an integer from 1 to 10; m2, m3, m4, and m5 are each an integer from 0 to 3; the * and ** terminals are respectively connected to two Fs with different structures. 0 Connected; more preferably, the trivalent connecting base is selected from any of the following structures: Preferably, each E in the four-armed polyethylene glycol derivative is independently selected from case (i) or (ii); or, one, two, three, four or five E in the four-armed polyethylene glycol derivative are each independently selected from case (iii).

9. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Its structure is shown in equation (2): Preferably, L 2 For divalent linker -B 2 -(Z 2 -B 2 ) j -; where L 2 In this context, j is an integer from 1 to 6; each B 2 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 2 Not simultaneously with two Z 2 Connected; each Z 2 Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z 2 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z 2 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, and -NHC(=O)NH-; Preferably, the L 2 It contains at least one C 1-6 Alkylene; more preferably, the L 2 Choose from any of the following structures: Among them, L 2 either end of X a or X b Connected; Preferably, the structure of the four-armed polyethylene glycol derivative is as shown in formula (2-1), (2-2), (2-3), (2-4), or (2-5): Among them, any two L 0 The structures of each group are the same or different, and the values ​​of any two k are the same or different. Each R 1 Independently for C 1-6 Alkyl or benzyl; Preferably, all R in the same structural formula 1 Having the same structure and further preferably methyl; More preferably, the structure of the four-armed polyethylene glycol derivative is as shown in formulas (2-1a), (2-2a), (2-2b), (2-3a), (2-3b), (2-4a), (2-4b), (2-4c), (2-5a), (2-5b), (2-5c), or (2-5d):

10. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Its structure is shown in equation (3): Preferably, L 3 For trivalent linker-B 3 -(Z 3 -B 3 ) j -G-(B 3 -Z 3 ) j -B 3 -; G is a trivalent branched nucleus and also interacts with L x Connected; L 3 Each j in the set is an independent integer from 1 to 6; each B 3 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 3 Not with two Zs at the same time 3 Connected; each Z 3 Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z 3 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z 3 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-; Preferably, G is >CH-, >N-, Any one of them, and either end thereof is connected to L x Connected; Preferably, L x For connection key, C 1-6 Alkylene or -B x -(Z x -B x ) j -, and L x In this context, j is an integer from 1 to 6; each B x Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. x Not with two Zs at the same time x Connected; each Z x Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z x Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z x It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-; Preferably, the L 3 Contains at least one -SS-, -CH=N-NHC(=O)- or -C(=O)NH-N=CH-; more preferably, L 3 Choose from any of the following structures: Where L 3 The * end is connected to L x Either of the other two ends is connected to X. a or X b ; Preferably, the L x Selected from link key, C 1-6 Any one of alkylene, *-NHC(=O)(CH2)2-, *-C(=O)NHCH2-, *-(CH2)3NHC(=O)(CH2)2- and *-(CH2)4NHC(=O)(CH2)2-, and L x The * end is connected to L 3 ; Preferably, the structure of the four-armed polyethylene glycol derivative is as shown in formulas (3-0), (3-1), (3-2), (3-3), (3-4), or (3-5): Among them, any two L 0 The structures of each group are the same or different, and the values ​​of any two k are the same or different. Each R 1 Independently for C 1-6 Alkyl or benzyl; Preferably, all R in the same structural formula 1 It has the same structure and is further preferably methyl.

11. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, Its structure is shown in equation (4): Preferably, each L 3 Independently a trivalent linker -B 3 -(Z 3 -B 3 ) j -G-(B 3 -Z 3 ) j -B 3 -; Each G is a trivalent branched nucleus and is also associated with the adjacent L x Connected; L 3 Each j in the set is an independent integer from 1 to 6; each B 3 Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. 3 Not with two Zs at the same time 3 Connected; each Z 3 Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z 3 Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z 3 It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-; Preferably, each G is independently >CH- or >N-; Preferably, each L x Independently for the connector key, C 1-6 Alkylene or -B x -(Z x -B x ) j -, and L x In this context, j is an integer from 1 to 6; each B x Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. x Not with two Zs at the same time x Connected; each Z x Independently, it is any one of -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q-, -CH=N-NHC(=O)-, and -C(=O)NH-N=CH-; Z x Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z x It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, -SS-, -CH=N-NHC(=O)- and -C(=O)NH-N=CH-; Preferably, at least one L 3 Contains at least one -SS-, -CH=N-NHC(=O)- or -C(=O)NH-N=CH-; Preferably, The structure is as follows: Each of the two * ends is connected to the adjacent L. x Either of the other two ends is connected to X. a or X b ; Preferably, two L x The same and selected from any one of the following: linking bonds, *-NHC(=O)(CH2)2- and *-C(=O)NHCH2-, wherein each L x The * end is connected to the adjacent L 3 .

12. The four-armed polyethylene glycol derivative according to claim 1, characterized in that, The structure of the four-armed polyethylene glycol derivative is as follows: Among them, n1, n2, n3 and n4 are each independent integers from 5 to 100.

13. A polyethylene glycol-modified bio-related substance having the structure shown in Formula (1D), in, M is the four-armed polyethylene glycol derivative of any one of claims 1-12 that loses all F. 0 And the residues formed; t1 and t2 are each independent integers from 1 to 4; Each L M Independently, it can be a linker or a divalent linker; Each T is an independent targeting group; Each D represents a residue of a biologically relevant substance; Preferably, all T values ​​in t1 are the same, and all D values ​​in t2 are the same.

14. The polyethylene glycol-modified bio-related substance according to claim 13, characterized in that, Each L M Independently for the connector key, C 1-6 Alkylene or -B M -(Z M -B M ) j -, and L M In this context, j is an integer from 1 to 6; each B M Independently for the connector key or C 1-6 Alkylene, and any one of the B atoms is a linking bond. M Not simultaneously with two Z M Connected; each Z M Independently defined as -Q-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, -QC(=Q)Q- and any one of them; Z M Each Q in the equation is independently an oxygen atom, a sulfur atom, or an NR atom. c , where R c It is a hydrogen atom or a carbon atom. 1-3 alkyl; Preferably, each Z M It is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, and -NHC(=O)NH-; Preferably, each L M Independently selected from any one of *-OC(=O)(CH2)2C(=O)O-, *-NHC(=O)(CH2)2C(=O)O-, *-OC(=O)-, *-NHC(=O)-, *-OC(=O)NHCH2C(=O)O- and *-OCH2C(=O)NHCH2C(=O)O-, wherein L M The * end is connected to M.

15. The polyethylene glycol-modified bio-related substance according to claim 13, characterized in that, Each T is independently selected from residues of any one of the following targeted substances or their derivatives: carbohydrates, monoclonal antibodies, peptides, proteins, vitamins, neurotransmitters, and aptamers. Preferably, it is a residue from any one of the following: N-acetylgalactosamine, anti-HER2 monoclonal antibody, anti-EGFR monoclonal antibody, RGD peptide, tLyP-1 peptide, LyP-1 peptide, RPARPAR peptide, Angiopep-2 peptide, GE11 peptide, A6 peptide, CD44BP peptide, RP-1 peptide, apoE pseudopeptide, transferrin, low-density lipoprotein, high-density lipoprotein, lectin, lysozyme, streptavidin, folic acid, biotin, m-iodobenzylguanidine, DNA aptamers, and RNA aptamers. Preferably, T is...

16. The polyethylene glycol-modified bio-related substance according to claim 13, characterized in that, The biologically related substance is a pharmaceutically active agent, preferably any one of the following: sedative-hypnotic drugs, psychostimulants, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, anti-Parkinson's drugs, analgesics, anti-inflammatory drugs, anti-anxiety drugs, appetite suppressants, anti-migraine drugs, muscle contraction agents, antibiotics, antiviral agents, antifungal agents, antiarthritis drugs, antimalarial drugs, antiemetics, epilepsy drugs, bronchodilators, cytokines, growth factors, anticancer drugs, antithrombotic drugs, antihypertensive drugs, cardiovascular drugs, antiarrhythmic drugs, antioxidants, antiasthmatics, contraceptives, sympathomimetic drugs, diuretics, lipid regulators, antiandrogens, antiparasitic drugs, anticoagulants, antitumor drugs, hypoglycemic drugs, nutritional supplements, growth supplements, anti-enteritis drugs, vaccines, antibodies, diagnostic agents, and contrast agents.

17. The polyethylene glycol-modified bio-related substance according to claim 13, characterized in that, The biologically related substances are small molecule or macromolecule drugs, preferably any one of the following or their derivatives: flavonoids, terpenoids, stilbenes, carotenoids, saponins, quinones, coumarins, alkaloids, porphyrins, polyphenols, macrolides, β-lactams, phenylpropanoids, anthracyclines, aminoglycosides, artemisinin, lipids, steroids, amino acids, dipeptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, nucleic acids, antisense nucleic acids, monosaccharides, disaccharides, oligosaccharides, and polysaccharides; More preferably, the biorelated substance is selected from any one of camptothecin, resveratrol, pterostilbene, dihydroartemisinin, betulinic acid, paclitaxel, docetaxel, lidocaine, bupivacaine, paliperidone, propofol, dezocine, ropivacaine, gefitinib, rotigotine, tiagabin, catechol, dihydroporphyrin, triptolide, oleanolic acid, capecitabine, cantharidin, and cyclobufotin or its derivatives. More preferably, the biorelated substance is camptothecin or a derivative thereof; Most preferably, the biorelated substance is selected from any one of camptothecin, topotecan, irinotecan, SN38, 10-hydroxycamptothecin, belotetan, ixenonotecan, delutecan, rubitecan, 9-aminocamptothecin, letopotecan, gemmatotecan, gemiminotecan, BNP1350, and BN80915, specifically having any one of the following structures: Preferably, D is 18. The polyethylene glycol-modified bio-related substance according to claim 13, characterized in that, The PEGylated bio-related substance is selected from any one of the following: Where n1, n2, n3 and n4 are each independent integers from 5 to 100; The structure is The structure is

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