A triarm polyethylene glycol derivative containing an amino acid residue

Abstract

This invention discloses an amino acid-containing three-arm polyethylene glycol derivative as shown in formula (1), wherein n i R2 represents the degree of polymerization of the polyethylene glycol chain, selected from an integer between 30 and 1000; each occurrence of R2 is independently represented by C. 1‑6 Alkyl; L 1A L 1B L1 and L2 are each independently divalent linkers; Lys is a lysine residue; AA1 and AA2 are each independently amino acid or amino acid derivative residues; p is selected from integers from 1 to 5, -(AA1) p - indicates a divalent residue formed by the combination of p AA1 residues; q is 0 or 1, indicating that AA2 is absent or that one AA2 residue is present; L3 is a linker or a divalent linker; R 01 The radical is H or a functional group capable of reacting with biorelevant substances, or its protected form; m is 1 or 2. The three-armed polyethylene glycol derivatives of this invention can impart intracellular degradation properties and, even at higher molecular weights, can inhibit cellular vacuolation. The three-armed polyethylene glycol derivatives of this invention can also modify drugs or biorelevant substances, achieving advantages such as high biocompatibility, low toxicity, and high retention of drug activity.

Description

Technical Field

[0001] This invention relates to polyethylene glycol derivatives, and more particularly to a three-arm polyethylene glycol derivative containing amino acid residues that is stable in vivo and degradable in cells for use in modifying pharmaceuticals or biologically related substances. Background Technology

[0002] Polyethylene glycol (PEG) is widely used for surface modification of drugs or drug carriers. Under physiological conditions, the hydration film formed by PEG can effectively protect the modified substance, preventing its rapid degradation or removal, effectively reducing its immunogenicity and systemic toxicity. Furthermore, PEG modification can overcome the problem of poor water solubility in some drugs. PEG-modified drugs or bio-related substances are often obtained by covalently coupling the active groups contained in functionalized PEG derivatives with drug molecules (including protein drugs and small organic molecule drugs), peptides, carbohydrates, lipids, oligonucleotides, affinity ligands, cofactors, liposomes, and biomaterials.

[0003] In 1990, Enzon's first PEGylated protein drug, Adagen, was approved by the FDA. Since then, several PEGylated drugs have been approved for marketing or are in clinical trials. Overall, the safety of PEGylated drugs has been clinically validated. However, higher molecular weight PEG can cause cellular vacuolation, and most PEG-related vacuolation occurs with PEG molecules ≥30 kDa (Toxicol. Pathol. 2015, 43, 959-983). Research data suggests that PEG-related vacuolation is reversible; however, the recovery period in most non-clinical toxicology studies is often too short to fully demonstrate this reversibility. During treatment-free periods, larger or multiple vacuoles may take longer to disappear. The vacuolation of phagocytes caused by PEG or PEGylated drugs is considered a normal physiological response. PEG taken up by phagocytes may eventually be cleared from the body through organs such as the liver, kidneys, or spleen, or it may persist throughout its lifespan. Kupffer cells, for example, have a lifespan of 5 weeks to 14 months, meaning that cellular vacuolation caused by high molecular weight PEG may persist in the human body for a long time, and its long-term effects are not fully understood. While reducing the molecular weight of PEG can effectively prevent cellular vacuolation, it also weakens the protective effect of PEG on the modified drugs.

[0004] In recent years, multi-arm polyethylene glycols (PEGs), such as three-arm, four-arm, six-arm, and eight-arm PEGs, have begun to gain a foothold in the market due to their advantages in structure and drug loading capacity. Traditional linear structures, whether monofunctionalized or bifunctionalized, while improving solubility and reducing toxicity, often result in significantly reduced drug activity due to the encapsulation of drug molecules by PEG chains. Compared to linear structures, multi-arm branched PEG derivatives can simultaneously achieve high solubility, low toxicity, low antigenicity, and high retention of drug activity. Furthermore, the significantly reduced viscosity of multi-arm structures facilitates better pharmacokinetics. Branched PEGs can form an umbrella-shaped protective layer on the drug surface, further effectively improving drug stability during systemic circulation. However, with the higher molecular weight of multi-arm PEGs, cellular vacuolation can be exacerbated.

[0005] To address the aforementioned issues, it is necessary to develop a monofunctionalized and difunctionalized multi-arm polyethylene glycol derivative that is stable in vivo and degradable in cells. Summary of the Invention

[0006] This invention provides novel three-armed monofunctional and bifunctional polyethylene glycol derivatives for the modification of pharmaceuticals or biologically related substances. The three-armed polyethylene glycol derivatives of this invention simultaneously possess systemic circulation stability and intracellular degradability, providing excellent protection to the modified substance, a longer systemic circulation time, low toxicity, good water solubility, intracellular degradability, and high retention of drug activity.

[0007] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0008] One embodiment of the present invention:

[0009] A three-armed polyethylene glycol derivative, the structure of which is shown in general formula (1):

[0010]

[0011] in,

[0012] n i Ri represents the degree of polymerization of the polyethylene glycol chain, where i is selected from any one of 1, 2, and 3, and n1, n2, and n3 are each independently selected from integers between 30 and 1000; R2 is independently C2 each time it appears. 1-6 alkyl;

[0013] L 1A L 1B L1 and L2 are each independently a divalent linker;

[0014] Lys represents lysine residues;

[0015] AA1 and AA2 are each independently an amino acid or amino acid derivative residue; p is selected from an integer from 1 to 5, -(AA1) p - indicates a divalent residue formed by the combination of p AA1 residues; q is 0 or 1, indicating that AA2 is absent or that one AA2 residue exists;

[0016] L3 is a linker bond or a divalent linker group;

[0017] R 01 It is H or a functional group that can react with biologically relevant substances or its protected form; m is 1 or 2; when m is 2, the two L3 structures are the same or different, and the two R... 01 The structures are the same or different;

[0018] The polyethylene glycol derivative is monodisperse or polydisperse;

[0019] Or its salts, tautomers, stereoisomers or solvates.

[0020] The present invention also provides another embodiment:

[0021] A bio-related substance modified with a three-armed polyethylene glycol derivative, characterized in that its structure is shown in general formula (5).

[0022] PLD (5)

[0024] Wherein, P is any of the aforementioned three-armed polyethylene glycol derivatives, D is a biorelated substance residue, and L is a divalent linker formed after the reactive group of the three-armed polyethylene glycol derivative reacts with the biorelated substance.

[0025] The biologically related substances are selected from any one of the following: drugs, proteins, polypeptides, oligopeptides, protein mimics, fragments, enzymes, antigens, antibodies and their fragments, receptors, gene-related aptamers, polysaccharides, proteoglycans, glycoproteins, lipid compounds, hormones, vitamins, vesicles, liposomes, dyes, fluorescent substances, targeting factors, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, micelles; more preferably, any one of the following: small molecule drugs, nucleic acids, steroids, phospholipids, glycolipids; more preferably, any one of the following: small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, steroid compounds; further, the small molecule drugs are preferably selected from any one of flavonoids, terpenoids, carotenoids, saponins, steroids, quinones, anthraquinones, fluoroquinones, coumarins, alkaloids, porphyrins, polyphenols, macrolides, monocyclic alkyl groups, phenylpropanoids, anthracyclines, and aminoglycosides.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides novel three-armed polyethylene glycol derivatives with asymmetric three-armed PEG structures that are monofunctionalized or bifunctionalized, and simultaneously possess both carbo-branched and nitrogen-branched structures, offering more flexible options for the modification of drugs or biorelated substances. The three-armed PEG of this invention not only improves the biocompatibility and in vivo stability of drugs or biorelated substances, but also, compared to existing nitrogen-branched V-type PEGs and carbo-branched multi-arm PEGs that do not contain amino acid residues, contains oligopeptide residues composed of amino acid residues, which endows them with intracellular degradation properties and, at higher molecular weights (30 kDa and above), can inhibit cellular vacuolation. The three-armed polyethylene glycol derivatives of this invention can also modify drugs or biorelated substances, achieving advantages such as excellent protective effects, longer systemic circulation time, high biocompatibility, low toxicity, and high retention of drug activity.

[0028] 1. Detailed Description of the Invention

[0029] This invention provides a detailed description of specific embodiments; however, it should be understood that it is given in an illustrative manner only and not in a restrictive manner, and various variations and modifications within the scope of this invention will be readily apparent to those skilled in the art.

[0030] Where the description in the references cited in this invention differs from the description in this invention, the description in this invention shall prevail; this principle applies to all references cited in the entire specification.

[0031] 1.1 Terminology Explanation

[0032] In this invention, unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents and other publications cited herein are incorporated herein in their entirety by reference. In the event of any conflict between any description or interpretation of terms herein and any document incorporated herein by reference, the following description and interpretation shall prevail. Unless otherwise specified, each term has the following meaning.

[0033] In this invention, unless otherwise specified, the "selected from" / "preferred" status of any two objects is independent. When there are multiple levels of selected from / preferred status, the selected from / preferred status of any two objects can be at the same level or different levels. For example, "L" A L B Each is independently selected from A, B, and C, and can be L. A L B Both can be A, or L. A For A and L B For example, "L" APreferably, it is A (Level 1 preferred), more preferably A1 to A3 (Level 2 preferred), and most preferably A. 11 ~A 13 (Level 3 preferred), L B Preferably B (Level 1 preferred), more preferably B1 to B3 (Level 2 preferred), and most preferably B. 11 ~B 13 (Level 3 Preferred) "Preferred can be A as A1~A3 (Level 2 Preferred) and B as B 11 ~B 13 (Level 3 preferred) or both A and B are Level 3 preferred.

[0034] In this invention, "each independently is / is selected from / is preferred" can not only refer to different categories being independently / selected from / is preferred options in the definition, but can also be further extended to include "each time it appears" to indicate that the same category is independently / is selected from / is preferred options in the definition each time it appears in different positions or at different times. For example, "the bivalent linkage is -NR" c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S-、-C(R c )=N-NR c -or-NR c -N=C(R c )-; where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 The description such as "alkyl" indicates that in "-NR" c -N=C(R c In )-”, there are two Rs c The groups can be the same or different (e.g., an R group can be different from an R group). c For methyl, another R c (is a hydrogen atom or an ethyl group), and "-NR" c C(=O)NR c - any of the R c Can be combined with "-NR" c R in C(=O)O-” c Same or different.

[0035] In this invention, when at least two items are listed, the “combination” of the listed items refers to any two or more combinations of the aforementioned listed items; and the number of items is not limited, the number of any item can be zero, one or more, and when the number of the same item is greater than 1, it can be the same or different specific forms that satisfy the item. For example, “L is a linking bond or is selected from any one or more of the following: alkylene group, divalent alkylene group containing heteroatoms, -O-, -S-, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NH-”. L can be any one of the listed items, or any two or more of the listed items. The combination can be -CH2-O-CH2- (i.e., the combination of -O- and alkylene group in the listed items, wherein the alkylene group is specifically in the form of 2 methylene groups), or -CH2-NH-CH2CH2- (i.e., the combination of -NH- and alkylene group in the listed items, wherein the alkylene group is specifically in the form of 1 methylene group and 1 ethylene group).

[0036] In this invention, unless otherwise specified, the terms “comprising,” “including,” and “containing,” as well as similar expressions, shall be interpreted in an open and inclusive sense in this specification and claims as “including but not limited to” or “non-restrictive inclusion.”

[0037] In this invention, "including but not limited to" a certain range means that items within the range are optional, but not limited to items within the range, and not all structures within the range are applicable. In particular, items explicitly excluded by this invention are not among the candidates, and the successful implementation of this invention is the selection criterion.

[0038] In this invention, the interpretation of numerical intervals includes not only intervals marked with a hyphen (e.g., 1-6), but also intervals marked with a wavy line (e.g., 1 to 6), and intervals marked with "to / to" (e.g., 1 to 6, 1 to 6). Unless otherwise specified, intervals marked in interval form can represent any group consisting of all integers and non-integers within that interval, and the range includes both endpoints. For example, the EO unit average is selected from 22 to 100, and its selection range is not limited to integers within this interval; it can also be any non-integer. As another example, "integers in 1-3" represents the group consisting of 1, 2, and 3. And as yet another 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.

[0039] The numerical ranges in this invention include, but are not limited to, ranges of integers, non-integers, percentages, and fractions, and unless otherwise specified, all ranges include two endpoints.

[0040] In this invention, for polymer molecular weight, "approximately" or "around" generally refers to a numerical range of ±10%, which may be increased to ±15% in some cases, but not exceeding ±20%. A preset value is used as the base. For example, the deviations between 11kDa, 12kDa, and 10kDa are 10% and 20%, respectively, and "approximately 10kDa" includes, but is not limited to, 11kDa and 12kDa. Furthermore, when specifying that the molecular weight of a certain PEG component in the general formula is approximately 5kDa, the corresponding molecular weight or number-average molecular weight is allowed to vary within the range of 5kDa ±10%, that is, 4500 to 5500 Da.

[0041] In this invention, for percentages, when the given value (excluding the percentage sign) is accurate to N (including 1, 0.1, 0.01, 0.001, 0.0001, etc.), "approximately" or "around" generally refers to a numerical range of ±0.5*N. For example, approximately 1% refers to 1 ± 0.5%, that is, the range of 0.5% to 1.5%, and approximately 2.2% refers to 2.2 ± 0.05%, that is, the range of 2.15% to 2.25%.

[0042] In this invention, the degree of polymerization of the polyethylene glycol chain is represented by n. i Represents (i is a natural number selected from 1, 2, 3, ...), where n takes the same value in the same structure. i They can be used interchangeably. For example, when n2 ≈ n3, n2 can be represented by n3, and n3 can be represented by n2.

[0043] In the present invention, the divalent linker can be selected from either of the two linking ends when it is linked to other groups, unless otherwise specified. For example, when an amide bond is used as the divalent linker between Group A and Group B, it can be Group A-C(=O)NH-Group B or Group A-NHC(=O)-Group B.

[0044] In the structural formula of this invention, when the atom assignment of the connected groups is involved, the following is adopted: To mark the connection key, such as Indicates the group structure, specifically, Represented as -CCH3(CH2CH2CH3)2; It is represented as -C(CH2CH2CH3)2-, rather than in the form of a functional group. It is represented as (CH3)2C(CH2CH2CH3)2.

[0045] In this invention, for connecting bonds or groups derived from a cyclic structure, when they are not labeled with a specific cyclic atom but point to the inside of the ring, it indicates that the connecting bond can be derived from any suitable cyclic atom. Furthermore, when the labeled ring is part of a fused or fused ring structure, the connecting bond can be derived from any suitable cyclic atom within that fused or fused ring structure. For example... In the middle, by The labeled linker and group Q can be derived from any suitable position among the eight cyclic carbon atoms excluding the nitrogen atom.

[0046] In this invention, the range of carbon atoms in a group is indicated by a subscript at the subscript position of C, representing the number of carbon atoms in the group. Unless otherwise specified, the number of carbon atoms does not include the contribution of substituents. For example, C 1-12 This indicates that it "has 1 to 12 carbon atoms". For example, C 1-10 Alkylene refers to any alkylene with a carbon number within the range indicated by the subscript, i.e., C1, C2, C3, C4, C5, C6, C7, C8, C9 ... 10 Any of the alkylene groups, including but not limited to straight-chain C 1-10 Alkylenes (e.g., -(CH2)6-) and branched C 1-10 Alkylenes (e.g., -(CH2)3-CH(CH3)-(CH2)3-). Another example is "substituted C". 1-12 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9 ... 10 C 11 Or C 12 An alkyl group is obtained by replacing at least one hydrogen atom of the alkyl group with a substituent, wherein there is no particular limitation on the number of carbon atoms and heteroatoms in the substituent.

[0047] In this invention, when the structure involved has isomers, it can be any one of them unless otherwise specified. For example, for a structure with cis-trans isomers, it can be either the cis or trans structure; for a structure with E / Z isomers, it can be either the E or Z structure; and when it is optically active, it can be either levorotatory or dextrorotatory.

[0048] In this invention, if there is a difference between the structure described herein and the name of the structure, the described structure shall have greater weight.

[0049] In this invention, the molecular weight of polyethylene glycol and its derivatives refers to the average molecular weight by default, and unless otherwise specified, "average molecular weight" generally refers to "number-average molecular weight" M. nThe number-average molecular weight can refer to the molecular weight of either polydisperse blocks or monodisperse blocks or substances. Unless otherwise specified, the unit of measurement for molecular weight is Daltons (Da). The "degree of polymerization" can also be used to characterize the molecular weight of a polyethylene glycol chain, specifically referring to the number of repeating units (vinyl oxide units). Accordingly, "average degree of polymerization" and "number-average degree of polymerization" are preferred to characterize the average or number-mean value of the number of repeating units.

[0050] In this invention, "any suitable" in phrases such as "any suitable linker" and "any suitable reactive group" refers to a structure that conforms to the basic principles of chemical structure and enables the preparation method of this invention to be successfully implemented. Chemical structures described in this way can be considered to have a clear and definite scope.

[0051] In this invention, the terms "stable existence" and "degradable" of a functional group are relative concepts.

[0052] In this invention, "degradable" refers to the breaking of chemical bonds, specifically the breaking of at least two independent residues. If the structure is altered after a chemical change, but the linker remains a single, intact linker, it still falls under the category of "stable existence." The conditions for degradation are not particularly limited; they can be in vivo physiological conditions, in vitro simulated physiological environments, or other conditions, preferably in vivo physiological conditions and in vitro simulated physiological conditions. The physiological conditions are not particularly limited and include, but are not limited to, sites such as serum, heart, liver, spleen, lungs, kidneys, bones, muscles, fat, brain, lymph nodes, small intestine, and gonads. These can refer to intracellular or extracellular matrix conditions, and can refer to normal physiological tissues or diseased physiological tissues (such as tumors, inflammation, etc.). The in vitro simulated environment is not particularly limited and includes, but is not limited to, physiological saline, buffer solutions, and culture media. The rate of degradation is not particularly limited; for example, it can refer to rapid degradation under enzymatic action or slow hydrolysis under physiological conditions. The in vivo physiological conditions include physiological conditions during treatment, such as ultraviolet irradiation and thermotherapy. The degradation is not limited to conditions such as light, heat, low temperature, enzymes, redox reactions, acidity, alkalinity, physiological conditions, and in vitro simulated environments, with a preference for degradation under these conditions. Degradability refers to degradation occurring under any of the above-mentioned conditions. Light conditions include, but are not limited to, visible light, ultraviolet light, infrared light, near-infrared light, and mid-infrared light. Thermal conditions refer to temperatures above normal physiological temperature, typically above 37°C and usually below 45°C, preferably below 42°C. Low temperature conditions refer to temperatures below human physiological temperature, preferably below 25°C, more preferably ≤10°C, specifically examples include refrigeration temperatures, freezing temperatures, liquid nitrogen therapy temperatures, 2–10°C, 4–8°C, 4°C, 0°C, and -20±5°C. Enzymatic conditions are not particularly limited; all enzymes that can be generated under physiological conditions are included, such as peptidases, proteases, and lyases. Redox conditions are not particularly limited, such as redox transitions between thiol groups and disulfide bonds, and hydrogenation-reduction transitions. The acidic and alkaline conditions mentioned mainly refer to the pH conditions of internal sites such as normal tissues, diseased tissues, and organs or tissues under treatment. For example, the stomach is acidic, and tumor sites are often acidic as well. Here, "degradable" refers to degradation through metabolic processes in the body (such as physiological processes, enzymes, redox reactions, etc.), degradation due to microenvironmental stimulation at specific sites in the body (such as acidity or alkalinity), or degradation under clinical treatment stimuli (such as light, heat, low temperature). It should be noted that bond breaking under extreme conditions relative to living organisms in organic chemistry, such as strong acids, strong bases, and high temperatures (such as above 100°C), is not included in the scope of degradable conditions in this invention. For example, although ether bonds can break under strong acid conditions such as hydrobromic acid, they are consistently classified as stable linkers in this invention.

[0053] In this invention, "stable existence" refers to the ability of a linker to remain as a complete linker (a linker is stably covalently connected to its adjacent groups). This allows for chemical changes that maintain the integrity of the linker. The chemical changes are not particularly limited and include, but are not limited to, isomerization, oxidation, reduction, ionization, protonation, deprotonation, and substitution reactions. The conditions for stable existence are not particularly limited and include, but are not limited to, light, heat, low temperature, enzymes, redox reactions, neutral, acidic, alkaline, physiological conditions, and in vitro simulated environments. Stability is preferably achieved under light, heat, enzymes, redox reactions, acidic conditions, and alkaline conditions. Stable existence here means maintaining a stable connection during in vivo metabolic circulation without special stimulation (such as pH conditions at specific sites, light, heat, or low temperatures during treatment), and without molecular weight reduction due to chain breakage (as long as the overall integrity is maintained).

[0054] In this invention, "stable existence" is not an absolute concept for the same linker. For example, amide bonds are much more stable than ester bonds under acidic or alkaline conditions, and the "stable existence" linkers in this invention include amide bonds. However, peptide bonds, which are amide bonds formed by the dehydration condensation of the α-carboxyl group and the α-amino group of an amino acid molecule, can be broken when exposed to specific enzymes, and are therefore also included in the "degradable" linkers. Similarly, urethane groups, thiourethane groups, etc., can be both stable and degradable linkers. More generally, urethane groups, thiourethane groups, etc., tend to undergo slow degradation, while non-peptide amide bonds are stable during in vivo circulation. Furthermore, common ester bonds can degrade under acidic or alkaline conditions, while ester bonds contained in special structures can also degrade under ultraviolet light. For example, even if certain chemical bonds can be degraded under the action of specific enzymes, if the circulation pathway does not pass through or basically does not pass through the specific enzyme environment during clinical use (such as in the case of targeted drug administration), the corresponding chemical bonds can still be considered to be stable.

[0055] In this invention, compounds of formula (1) should be understood to include salts of compounds of formula (1). The term "salt" as used is selected from any one, any two, or any combination of two or more of acid addition salts formed with inorganic and / or organic acids and base addition salts formed with inorganic and / or organic bases. When a compound of formula (1) contains a basic moiety (e.g., but not limited to pyridine or imidazole) and an acidic moiety (e.g., but not limited to carboxylic acids), an amphoteric ion ("internal salt") may be formed and included in the term "salt" as used. A "salt" may be a pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salt or other salts. Salts of compounds of formula (1) may be formed by reacting a compound of formula (1) with a certain amount (such as an equivalent) of an acid or base in a medium such as a salt precipitation medium or in an aqueous medium and then freeze-drying. Exemplary acid addition salts include acetate, adipic acid salt, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gluconate, glyceryl phosphate, hemisulfate, heptanate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, and 2-naphthalene. Sulfonates, nicotinates, nitrates, oxalates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, neopentanoates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc.; exemplary base addition salts include ammonium salts, alkali metal salts (such as sodium, lithium, and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts containing organic bases (e.g., organic amines), and salts containing amino acids (such as arginine or lysine). The basic nitrogen-containing group can be quaternized with 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. Both the acid addition salt and the base addition salt are preferably pharmaceutically acceptable salts and, for the purposes of this disclosure, are considered equivalent to the free form of the corresponding compound of general formula (1).

[0056] The heteroatoms used in this invention are not particularly limited, and include, but are not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0057] In this invention, in contrast to compounds, groups formed after the loss of some atoms or groups are also referred to as residues.

[0058] In this invention, groups with a valence state of 2 or greater are collectively referred to as "linking groups". Linking groups may contain only one atom, such as ether groups (-O-) and thioether groups (-S-). In particular, when the definition of a group includes "linking bond", it means that the group may not exist and only serves a linking function.

[0059] In this invention, regarding the valence state of a group, "multivalent" means that the valence state is at least 3.

[0060] In this invention, unless otherwise specified, "linkage bond" refers to a bond that only serves a connecting function and does not contain any atoms.

[0061] In this invention, the "group" in the divalent linker can be replaced with "bond" without changing the meaning. For example, a divalent ether group can also be called an ether bond (-O-), a divalent ester group can also be called an ester bond (-OC(=O)- or -C(=O)O-), and a divalent carbamate group can also be called a carbamate bond (-OC(=O)NH- or -NHC(=O)O-).

[0062] In this invention, when the number of atoms contained in a substituent is 1, it can also be referred to as a "substituent atom".

[0063] In this invention, when the compound or group is "substituted", it means that the compound or group contains one or more substituents.

[0064] In this invention, unless otherwise specified, "amino" and "amine group" have the same meaning, including monovalent, divalent, trivalent, and tetravalent neutral or cationic groups, substituted or unsubstituted. For example, -NH2 in CH3-NH2 can be called "amino," "amine group," or "primary amine group." Similarly, -NH- in CH3-NH-CH3 can be called "secondary amine group" or "secondary amino group," and -NH-CH3 can also be understood as a methyl-substituted amine group.

[0065] In this invention, "amine group" includes, but is not limited to, primary amine groups, secondary amine groups, tertiary amine groups, and quaternary ammonium ions. For example, -NR t R t and -N + R t R t R t , where each R t Each can be independently composed of a hydrogen atom or any hydrocarbon group.

[0066] In this invention, when the valence state is not specified, "hydrocarbon group" can be a monovalent hydrocarbon group, a divalent hydrocarbon group, a trivalent hydrocarbon group, ..., an n-valent hydrocarbon group, where n is the highest valence state that the hydrocarbon group can possess.

[0067] In this invention, "hydroalkyl group" is a divalent hydrocarbon group.

[0068] In this invention, secondary amine bonds and hydrazine bonds refer to "-NH-" or "-NH-NH-" with both ends capped by hydrocarbon groups, such as -CH2-NH-CH2- and -CH2-NH-NH-CH2-; while -C(=O)-NH- is called an amide bond and is not considered to contain secondary amine bonds.

[0069] In this invention, "functional group" also refers to "functional group," preferably a reactive group, a protected reactive group, a precursor of a reactive group, etc. "Multi-functional" means that the number of functional groups is at least three, such as a polyol being a compound containing at least three hydroxyl groups, a polythiol being a compound containing at least three mercapto groups, etc. It should be noted that other types of functional groups are permissible, such as tris(hydroxymethyl)aminomethane being a triol containing one amino group, and citric acid being a tricarboxylic acid containing one hydroxyl group.

[0070] Unless otherwise specified, the reactive groups in the preparation method of the present invention also include their protected forms, which can be deprotected at any suitable step in the actual preparation process to obtain the corresponding active forms.

[0071] In this invention, the ring-forming atoms are the atoms that together constitute the ring framework.

[0072] The source of amino acids in this invention is not particularly limited unless otherwise specified; they can be from natural sources, non-natural sources, or a mixture of both. The structural type of amino acids in this invention is not particularly limited unless otherwise specified; they can refer to L-type, D-type, or a mixture of both.

[0073] In this invention, the definitions and examples of amino acid skeletons, amino acid derivative skeletons, and cyclic monosaccharide skeletons in references CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A, and the cited references are also incorporated herein by reference. Unless otherwise specified, the skeleton refers to the residue. Amino acid residues include amino acids with a hydrogen atom removed from the amino group and / or a hydroxyl group removed from the carboxyl group and / or a hydrogen atom removed from the thiol group and / or with the amino group protected and / or with the carboxyl group protected and / or with the thiol group protected. Loosely speaking, amino acid residues may be referred to as amino acids. Specifically, residues formed by losing a carboxyl hydroxyl group (including all C-terminal carboxyl hydroxyl groups, as well as the carboxyl hydroxyl groups on the side groups of aspartic acid and glutamic acid), a hydrogen atom on a hydroxyl group, a hydrogen atom on a phenolic hydroxyl group (tyrosine), a hydrogen atom on a thiol group (such as cysteine), a hydrogen atom on a nitrogen atom (including all N-terminal hydrogen atoms, as well as hydrogen atoms in the amino groups of side groups such as lysine and ornithine, and hydrogen atoms in the amino groups of histidine and tryptophan), an amino group on an amide (such as aspartamide and glutamine), or an amino group or hydrogen atom in a guanidine side group. Amino acid derivative residues refer to atomic or group portions that, in addition to possessing the basic characteristics of amino acids, also possess non-amino acid basic characteristics.

[0074] In this invention, "biologically related substances" include, but are not limited to, the substances described, listed, and cited in documents CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A, and all cited documents. In general, biologically related substances include, but are not limited to, the following substances: drugs, proteins, polypeptides, oligopeptides, protein mimics, fragments and analogs, enzymes, antigens, antibodies and their fragments, receptors, small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroids, 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 biorelated substances mentioned can be the biorelated substances themselves, or their precursors, activated states, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, fusion proteins, chemically modified substances, recombinant substances, etc. They can also be corresponding agonists, activators, inhibitors, antagonists, regulators, receptors, ligands or ligands, antibodies and their fragments, 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.). The derivatives include, but are not limited to, glycosides, nucleosides, amino acids, and polypeptide derivatives. Chemically modified products that form new reactive groups, i.e., modified products generated by altering the type of reactive groups, introducing additional functional groups, reactive groups, amino acids or amino acid derivatives, polypeptides, etc., are all considered chemically modified biorelated substances. Before or after binding with functionalized polyethylene glycol, biorelated substances may also have target molecules, appendages, or delivery carriers bound to them, forming modified or complex biorelated substances. 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. The term "drug" in this invention includes any agent, compound, composition, or mixture that provides physiological or pharmacological effects in vivo or in vitro, and often provides beneficial effects. There are no particular limitations on its types, including but not limited to drugs, vaccines, antibodies, vitamins, foods, food additives, nutrients, nutritional supplements, and other agents that provide beneficial effects. The scope of the physiological or pharmacological effects produced by the "drug" in vivo is not particularly limited; it may have systemic effects or only local effects.The activity of the "drug" is not particularly limited, mainly referring to an active substance that can interact with other substances, but it can also be an inert substance that does not interact; however, an inert drug can be transformed into an active form through in vivo action or certain stimulation. Among them, "small molecule drug" refers to a biologically related substance with a molecular weight not exceeding 1000 Da, or a small molecule mimic or active fragment of any biologically related substance.

[0075] In this invention, "oligopeptide" refers to a peptide molecule formed by two or more amino acids, preferably having 2 to 20 amino acid residues constituting the oligopeptide. The "oligopeptide residues" can be monovalent, divalent, trivalent, or higher valence states.

[0076] In this invention, unless otherwise specified, "monosaccharide" refers to a monosaccharide residue, that is, a monosaccharide backbone, including open-chain monosaccharides and cyclic monosaccharides (such as furanose rings and pyranose rings).

[0077] The monosaccharide group in this invention can be selected from residues of any compound including, but not limited to, monosaccharides, sugar alcohols, deoxysugars, amino sugars, amino sugar derivatives (such as amide derivatives), sugar acids, and glycosides, and can have an open-chain structure or a cyclic structure. Examples include amino residues formed by removing an amino hydrogen atom from an amino sugar, and acyl groups formed by removing a carboxyl hydroxyl group from a sugar acid. The monosaccharide can include, but is not limited to, aldoses (polyhydroxy aldehydes) and ketoses (polyhydroxy ketones). Examples include alkyl ether derivatives and methyl ether derivatives, such as malachite alcohol. The number of carbon atoms in the monosaccharide group in this invention is not particularly limited, and includes, but is not limited to, tetroses, pentoses, hexoses, and heptoses. Pentoses and hexoses are preferred. Examples of tetroses, pentoses, hexoses, heptoses, sugar alcohols, deoxysugars, amino sugars, amide derivatives of amino sugars, sugar acids, and glycosides include, but are not limited to, the structures disclosed in CN106967213A.

[0078] The polyethylene glycol derivatives of the present invention can be used to deliver bioactive ingredients to a patient in one or more of the following ways: liver or liver cells (e.g., hepatocytes), kidney or kidney cells, tumor or tumor cells, CNS or CNS cells (central nervous system, e.g., brain and / or spinal cord), PNS or PNS cells (peripheral nervous system), lung or lung cells, blood vessels or blood vessel cells, skin or skin cells (e.g., dermal cells and / or follicular cells), eye or eye cells (e.g., macula, fovea, cornea, retina), ear or ear cells (e.g., inner ear, middle ear and / or outer ear cells).

[0079] In this invention, "micro-modification" refers to a chemical modification process that can be completed through a simple chemical reaction. This simple chemical reaction process mainly includes processes such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, or alteration of leaving groups.

[0080] In this invention, "micro-modification" corresponds to "micro-transformation," referring to a structural form that can form the target reactive group after undergoing simple chemical reaction processes such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group. The alteration of the leaving group refers to the transformation of the leaving group, such as, but not limited to, the transformation from an ester form to an acyl chloride form.

[0081] In some specific embodiments of this invention, the reaction process also involves the "protection" and "deprotection" of relevant functional groups. To prevent a reactive group from affecting the reaction, it is typically protected. In some specific embodiments of this invention, when there are two or more reactive groups, only the target reactive group is selectively reacted, thus protecting the other reactive groups. The protecting group not only remains stable during the target reaction but can also be removed as needed using conventional techniques in the art.

[0082] In this invention, "protection" of reactive groups refers to the strategy of reversibly converting the reactive group to be protected into an inert group (non-reactive group) using a specific reagent. The portion of the protected group that differs from its unprotected form is called a "protecting group." For example, -OTBS is a protected form of the hydroxyl group (-OH), where the -TBS group is the protecting group for the hydroxyl group.

[0083] In this invention, "deprotection" and "unprotection" have the same meaning, both referring to the process of changing the protected group from a protected form to an unprotected form.

[0084] In this invention, the "hydroxyl protecting group" includes all groups that can be used as protecting groups for the common hydroxyl group. The hydroxyl protecting group is preferably an alkyl acyl (e.g., acetyl, tert-butyryl), aralkyl acyl (e.g., benzyl), benzyl, triphenylmethyl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal, or ketal. The removal of the acetyl group is generally carried out under alkaline conditions, most commonly by ammonolysis of NH3 / MeOH and methanololysis catalyzed by methanol anion; benzyl is 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 is generally removed by catalytic hydrogenolysis; trimethylsilyl is usually removed using reagents containing fluoride ions (e.g., tetrabutylamine fluoride / anhydrous THF); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (e.g., Zn / CH3OH), and can be removed by fluoride ions (e.g., Bu4N). + F - The diol is removed in a tetrahydrofuran solution, or at room temperature with aqueous acetic acid. Protection of the diol is preferably achieved by forming dioxolane, dioxane, cyclic carbonates, or cyclic borates.

[0085] In this invention, the "thiol protecting group" includes all groups that can be used as protecting groups for the common thiol group. Similar to hydroxyl groups, thiol groups can be protected in the form of thioethers and thioesters. Preferably, the thiol protecting group is tert-butyl, benzyl, substituted benzyl, diphenylmethyl, substituted diphenylmethyl, triphenylmethyl, acetyl, benzoyl, tert-butyloxycarbonyl, benzyloxycarbonyl, thioacetal, or thioketal. Deprotection of thioethers can be achieved by acid-catalyzed reduction with Na / NH3 or by heavy metal ions such as Ag. + Hg + The reaction can be completed by treatment with hydrogen sulfide. Some hemisulfuric acetals, including those with S-diphenylmethyl, S-triphenylmethyl sulfide, S-2-tetrahydropyranyl, and S-isobutyromethyl groups, can be oxidized to disulfides with (SCN)₂, iodine, or thionyl chloride, and then reduced to thiols. The formation and deprotection methods for thioesters are the same as those for carboxylic acid esters.

[0086] In this invention, a "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or a deprotection reaction. The carboxyl protecting group is preferably alkyl (e.g., methyl, ethyl, tert-butyl) or aralkyl (e.g., benzyl), more preferably tert-butyl (tBu), methyl (Me), or ethyl (Et). In this invention, a "protected carboxyl group" refers to the group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, or benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under acid or base catalysis, and occasionally by thermal decomposition. For example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0087] In this invention, "amino protecting group" is equivalent to "amine protecting group" and includes all groups that can be used as protecting groups for ordinary amino / amine groups, such as aryl C. 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6Alkyl sulfonyl, aryl sulfonyl, or silyl groups are preferred. The amino protecting group is preferably Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), or Fmoc (9-fluorenemethoxycarbonyl). The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably combinations of TFA and H2O, LiOH and MeOH, or LiOH and EtOH. The reagent for removing Boc protection is TFA or HCl / EA; TFA is preferred. The deprotecting agent used for removing Fmoc protection is a solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine.

[0088] In this invention, the "alkynyl protecting group" includes all groups that can be used as common alkynyl protecting groups, preferably trimethylsilyl (TMS), triethylsilyl, tert-butyldimethylsilyl (TBS), or biphenyldimethylsilyl. TMS-protected alkynyl groups are readily deprotected under alkaline conditions, such as K₂CO₃ / MeOH or KOH / MeOH. TBS-protected alkynyl groups can be deprotected in a tetrahydrofuran solution of tetrabutylammonium fluoride (TBAF / THF).

[0089] In this invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, an alcohol hydroxyl group, a phenolic hydroxyl group, etc.; the amino / amine group protected by the amino protecting group is not particularly limited, and can be, for example, derived from primary amines, secondary amines, hydrazines, amides, etc. The amino group in this invention is not particularly limited, and includes, but is not limited to, primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium ions.

[0090] In this invention, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. The type of hydroxyl protecting group is not particularly limited; taking benzyl, silyl ether, acetal, and tert-butyl groups for protecting terminal hydroxyl groups as examples, the corresponding deprotection methods include, but are not limited to:

[0091] A: Deprotection of benzyl groups

[0092] Benzyl deprotection can be achieved by hydrogenation of a hydrogen reducing agent and a hydrogen donor. The water content in this reaction system should be less than 1% for the reaction to proceed smoothly.

[0093] The hydrogenation reduction catalyst is not limited, but palladium and nickel are preferred. The support is not limited, but alumina or carbon is preferred, and carbon is more preferred. The amount of palladium used is 1 to 100 wt% of the protected hydroxyl compound, preferably 1 to 20 wt% of the protected hydroxyl compound.

[0094] The reaction solvent is not particularly limited, as long as both the raw materials and products can be used as solvents, but methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is more preferred. The hydrogen donor is not particularly limited, but hydrogen, cyclohexene, 2-propanol, and ammonium formate are preferred. The reaction temperature is preferably 25 to 40°C. The reaction time is not particularly limited, and the reaction time is negatively correlated with the amount of catalyst used, preferably 1 to 5 hours.

[0095] B: Deprotection of acetals and ketals

[0096] The preferred acetals or ketals for this type of hydroxyl protection are ethyl vinyl ethers, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, etc. Deprotection of these acetals and ketals is achieved under acidic conditions, with the solution pH preferably between 0 and 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products; water is preferred. The reaction temperature is preferably between 0 and 30°C.

[0097] C: Deprotection of silyl ether

[0098] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, and tert-butyldiphenylsilyl ether. Deprotection of these silyl ethers is achieved using fluoride-containing compounds, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, and potassium fluoride, more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of fluoride-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the amount of fluoride is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will occur; if the amount of deprotecting reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, excess reagent or compound will cause purification problems, may be introduced into subsequent steps, and thus cause side reactions. There are no particular restrictions on the reaction solvent, as long as it can dissolve the reactants and products. Aprotic solvents are preferred, more preferably tetrahydrofuran and dichloromethane. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed.

[0099] D: Deprotection of tert-butyl

[0100] The deprotection of the tert-butyl group is carried out under acidic conditions, preferably with a solution pH of 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve both the reactants and products; water is preferred. The reaction temperature is preferably 0 to 30°C.

[0101] In this invention, "carboxyl activation" refers to the activation treatment of carboxyl groups with a carboxyl activator. Activated carboxyl groups can promote better condensation reactions, such as inhibiting the generation of racemic impurities in the condensation reaction and accelerating the reaction rate. "Carboxyl activating group" refers to a residue of the carboxyl activator. The carboxyl activator is one or more of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N-dicyclohexylcarbodiimide (DCC), preferably a combination of NHS / EDCI, NHS / DCC, and HONb / DCC, with the most preferred combination being NHS / EDCI.

[0102] In this invention, the condensing agent used in the reaction is not limited, but N,N'-dicyclohexylcarbonyldiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) are preferred, with DCC being the most preferred. The amount of condensing agent used is generally 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 to 10 times. A suitable catalyst (such as 4-dimethylaminopyridine) can be added to this reaction.

[0103] In this invention, the obtained product can be purified by methods such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction.

[0104] In this invention, the solvent for the reaction can be a solvent-free solvent or an aprotic solvent. The aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or dimethylformamide.

[0105] In this invention, the base used in the reaction can be an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine, preferably triethylamine or pyridine) or an inorganic base (e.g. K2CO3).

[0106] In this invention, the repeating unit of the polyethylene glycol component is an ethylene oxide unit, namely -CH2CH2O- or -OCH2CH2-, also referred to as an EO unit. The number of repeating units is also referred to as the number of EO units, and the average number of repeating units is also referred to as the average number of EO units, preferably the number average.

[0107] In this invention, for polydisperse cases, the “equality” or “identity” or “equality” (including other forms of equivalent expression) of the molecular weight / degree of polymerization of a single molecule of a compound and the number-average molecular weight / degree of polymerization of the compound components in the macroscopic substance are not limited to being strictly equal in numerical value unless otherwise specified. Rather, they are exponentially close or approximately equal, and the deviation of such closeness or approximation is preferably no more than ±10%, usually based on a preset value.

[0108] In this invention, for monodisperse compounds, the same or equal number of vinyl oxide units in a single compound molecule and its general formula refers to strict numerical equality. For example, if the number of EO units in a certain PEG component is set to 11, then a set value of 12 is not within the set range. However, for macroscopic products obtained by a certain preparation method to obtain a compound component containing a set number of EO units, due to limitations in the preparation and purification methods, the macroscopic product may contain other EO unit arrays besides the target EO unit array. In this case, when the average number of EO units deviates from the preset number of EO units by no more than ±5% (base ≥ 10) or no more than ±0.5% (base < 1), the macroscopic product is considered to have other EO unit arrays besides the target EO unit array. When the content of a component that meets the range of the number of EO units or the average number of EO units reaches a certain percentage (preferably ≥90%, more preferably >95%, more preferably greater than 96%, more preferably greater than 98%, more preferably 99% to 100%), it is also considered that a monodisperse macroscopic product containing the target component has been obtained. Even if the above-mentioned content ratio is not reached, as long as the preparation method of the present invention or a similar method with the same preparation idea is used, the product with insufficient content, the component appearing in the form of a main product, co-product or by-product, regardless of whether separation and purification are performed, are all within the scope of the present invention.

[0109] In this invention, when the molecular weight of a compound formula of a polydisperse component is described using Da, kDa, number of repeating units, and number of EO units, the molecular weight value for a single compound molecule is allowed to fall within a certain range of a given value (including the endpoints, preferably within ±10%). When the molecular weight of a compound formula of a monodisperse component is described using the number of vinyl oxide units, there is no range fluctuation, and it is a discrete point. However, the average number of EO units in the prepared product may fluctuate within a certain range due to the non-uniformity of molecular weight (not exceeding ±10% or ±1, preferably not exceeding ±5% or ±0.5). For example, the molecular weight of mPEG (methoxy polyethylene glycol unit) is 5 kDa, which means that the molecular weight of a single molecule in the general formula is between 4500 and 5500 Da. The average molecular weight of the corresponding component in the prepared product is 5 kDa. That is, the product with an average molecular weight between 4500 and 5500 Da is the target product, and only the component with a molecular weight in this range contributes to the content of the target component. For example, if mPEG is designed to have 22 vinyl oxide units, then the number of EO units in all compound molecules in the general formula should be strictly 22. However, the prepared product may be a mixture of compounds with 20, 21, 22, 23, or 24 EO units. In this case, when the average number of EO units falls within the range of 22 ± 2.2 (preferably within the range of 22 ± 1.1), it is considered to have obtained the target component, and the component with a molecular weight in this range can be considered as the target component for calculating purity.

[0110] In this invention, unless otherwise specified, "mPEG" refers to a methoxy-terminated polyethylene glycol segment with the following structural formula: Where, n i The degree of polymerization of the polyethylene glycol chain is an integer selected from 1 to 1000.

[0111] In this invention, a product with a PDI < 1.005 can be considered monodisperse and can be denoted as PDI = 1.

[0112] In this invention, the number of repeating units in the "single-chain component" is at least 2.

[0113] The polyethylene glycol-based pharmaceutical products of the present invention can act systemically and / or locally. For this purpose, they can be administered via suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulations, or inhalation administration. For these routes of administration, suitable dosage forms can be used to administer the pharmaceutical compositions of the present invention. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0114] In this invention, treatment refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom, intended to include 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. The patient to be treated is preferably a mammal, especially a human.

[0115] 1.2 Three-arm polyethylene glycol derivatives

[0116] One embodiment of the present invention:

[0117] A three-armed polyethylene glycol derivative, the structure of which is shown in general formula (1):

[0118]

[0119] in,

[0120] n i Ri represents the degree of polymerization of the polyethylene glycol chain, where i is selected from any one of 1, 2, and 3, and n1, n2, and n3 are each independently selected from integers between 30 and 1000; R2 is independently C2 each time it appears. 1-6 alkyl;

[0121] L 1A L 1B L1 and L2 are each independently a divalent linker;

[0122] Lys represents lysine residues;

[0123] AA1 and AA2 are each independently an amino acid or amino acid derivative residue; p is selected from an integer from 1 to 5, -(AA1) p - indicates a divalent residue formed by the combination of p AA1 residues; q is 0 or 1, indicating that AA2 is absent or that one AA2 residue exists;

[0124] L3 is a linker bond or a divalent linker group;

[0125] R 01 It is H or a functional group that can react with biologically relevant substances or its protected form; m is 1 or 2; when m is 2, the two L3 structures are the same or different, and the two R... 01 The structures are the same or different.

[0126] In this invention, compounds of general formula (1) can exist in non-solventized or solvated forms, including hydrated forms. Generally speaking, for the purposes of this disclosure, a solvated form having a pharmaceutically acceptable solvent (such as water, ethanol, etc.) is equivalent to a non-solventized form.

[0127] In this invention, compounds of general formula (1) and their salts and solvates may exist in their tautomeric forms, including ketone-enol tautomerism, amide-imino tautomerism, lactam-lactamimide tautomerism, enamine-imide tautomerism, proton transfer tautomerism, and valence tautomerism.

[0128] In this invention, compounds of general formula (1) should be understood to include their salts, tautomers, stereoisomers or solvates.

[0129] In one specific embodiment of the present invention, the number average molecular weight corresponding to each PEG chain is 1000, 1500, 2000, 2500, 3000, 3350, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 Da.

[0130] In one specific embodiment of the present invention, the PEG chains are all polydisperse, and the degree of polymerization of the PEG chains is selected from about 30 to about 1000, more preferably from about 20 to about 500; more preferably from 100 to about 500.

[0131] 1.2.1. Other general formulas

[0132] One specific embodiment of the present invention:

[0133] A three-armed polyethylene glycol derivative, p=1, AA1 being a glycine residue, has the structure shown in general formula (2):

[0134]

[0135] in,

[0136] L 1A L 1B Each is independently selected from C 1-6 Alkylene or -(CH2) 1-6 C(=O)-, wherein the left end of the linker is connected to the PEG chain; more preferably, each is independently selected from -CH2CH2-, -CH2-, -CH2C(=O)-, -CH2CH2C(=O)-; further preferably L 1A L 1B It is any of the following situations:

[0137] Case (1): L 1A L 1B Each can be independently -CH2CH2- or -CH2-;

[0138] Case (2): L1A L 1B One of them is -CH2CH2- or -CH2-, and the other is -CH2C(=O)- or -CH2CH2C(=O)-;

[0139] The definitions of the remaining parameters are the same as those in general formula (1).

[0140] One specific embodiment of the present invention:

[0141] A three-armed polyethylene glycol derivative, corresponding to q=0 in general formula (2), AA2 is absent, and its structure is as shown in general formula (3):

[0142]

[0143] Among them, L3 is preferably a linker, -O-, or -O(CH2). tp OC(=O)O-、-NH(CH2) tp -、-NH(CH2) tp C(=O)NH(CH2) tp -、-NH(CH2) tp NHC(=O)(CH2) tp - any one of them, and the right end is connected to R 01 Connected; tp is an integer from 1 to 4, preferably 1 or 2;

[0144] R 01 Preferred ions are -OH, -CHO, -COOH, and -NH2.

[0145] The structure of the three-armed polyethylene glycol derivative is more preferably any one of the following:

[0146]

[0147]

[0148]

[0149] The definitions of the remaining parameters are the same as those in general formula (2).

[0150] One specific embodiment of the present invention:

[0151] A three-armed polyethylene glycol derivative, corresponding to q=1 in general formula (2), has an AA2 and its structure is shown in general formula (4):

[0152]

[0153] in,

[0154] R 01Preferably -OH or -CHO;

[0155] L3 is preferably a linker bond or -NH(CH2) tp - and the right end is with R 01 Connected; tp is an integer from 1 to 4, preferably 1 or 2;

[0156] AA2 is preferably a trivalent amino acid residue or an amino acid derivative residue, more preferably a trivalent glutamic acid residue or a trivalent aspartic acid residue;

[0157] The structure of the three-armed polyethylene glycol derivative is more preferably any one of the following:

[0158]

[0159] The definitions of the remaining parameters are the same as those in general formula (2).

[0160] 1.2.2. Bivalent Connecting Base

[0161] In this invention, L 1A L 1B L1 and L2 are each independently a divalent linker; L3 is a linker bond or a divalent linker.

[0162] In one specific embodiment of the present invention, L2 is preferably -(CH2). tq -、-(CH2) tq C(=O)-、-C(=O)(CH2) tq C(=O)-、-(CH2) tq NHC(=O)(CH2) tq C(=O)-、-(CH2) tq C(=O)NH(CH2) tqAny of C(=O)-, wherein tq is an independent integer from 0 to 4 each time it appears, and the right end of the linker is connected to the lysine residue in formula (1); L2 is more preferably -C(=O)-, -CH2C(=O)-, -CH2CH2C(=O)-, -CH2CH2CH2C(=O)-, -C(=O)CH2CH2C(=O)-, -C(=O)CH2CH2CH2C(=O)-, -CH2CH2NHC(=O)CH2CH2C (=O)-, -CH2CH2CH2NHC(=O)CH2CH2C(=O)-, -CH2CH2NHC(=O)CH2CH2CH2C(=O)-, -CH2CH2CH2NHC(=O)CH2C Any of H2CH2C(=O)-, -CH2C(=O)NHCH2C(=O)-, -CH2CH2C(=O)NHCH2C(=O)-, -CH2CH2CH2C(=O)NHCH2C(=O)-.

[0163] In one specific embodiment of the present invention, L2 is more preferably -(CH2). tq -or-(CH2) tq C(=O)-, tq is an integer from 0 to 4; L2 is preferably -C(=O)-, -CH2CH2C(=O)- or -CH2CH2CH2C(=O)-.

[0164] In one specific embodiment of the present invention, L3 is preferably a linking bond, alkylene group, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, or -NR. c -、-NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c C(=O)S- any one, any two, or any combination of two or more;

[0165] R c For H, C 1-6 Any one of alkyl, carbocyclic, and heterocyclic groups; preferably R c For H;

[0166] The alkylene group is preferably -(CR) a R b ) t- where t is an independent integer from 1 to 12 each time it appears, and R a R b Each independently represents H and C. 1-6 Any one of alkyl, carbocyclic, or heterocyclic groups, t R a and t R b Selected from the same structure, a combination of two different structures, or a combination of two or more different structures; R is preferred. a R b Each occurrence is independently -CH3 or -H, more preferably R. a R b All are H; preferably t is an integer from 1 to 4, more preferably t is 1 or 2.

[0167] In one specific embodiment of the present invention, L is preferred. 1A L 1B Each is independently selected from C 1-6 Alkylene, preferably -CH2CH2- or -CH2-.

[0168] In one specific embodiment of the present invention, the above-mentioned divalent linker L 1A L 1B There are no particular limitations on the stability of L3. Each of the divalent linkers or any divalent linker formed with an adjacent heteroatom group is independently a linker, a stable linker, or a degradable linker. The stable linker can exist stably under in vivo physiological conditions and in vitro simulated physiological conditions. It is preferably any one of linker, alkylene, amide, ether, carbamate, secondary amino, and carbonyl, or any one or more of amide, ether, carbamate, secondary amino, and carbonyl and a combination of one or more alkylene groups.

[0169] 1.2.3. Amino acids / oligopeptides

[0170] In this invention, AA1 and AA2 are each independently an amino acid or amino acid derivative residue.

[0171] In one specific embodiment of the present invention, each of the p AA1 residues and q AA2 residues is independently selected from any one of the following residues or derivative residues: glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine, and citrulline.

[0172] When p=1, -AA1- is preferably a glycine residue;

[0173] When p≥2, -(AA1) p- is an oligopeptide residue or an oligopeptide derivative residue; the oligopeptide is a dipeptide, tripeptide, tetrapeptide or pentapeptide, preferably glycine-phenylalanine, histidine-β-alanine, glycine-glycine, lysine-glycine, lysine-glutamic acid, glycine-glycine-glycine, glycine-glycine-phenylalanine, glycine-phenylalanine-glycine, glycine-lysine-glycine, glycine-histidine-lysine, glycine-cysteine-glutamic acid, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, arginine-glycine-glycine. The following is a selection of aspartic acid, glycine-histidine-proline, glycine-leucine-phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, glycine-lysine-glycine-lysine, glycine-phenylalanine-glutamic acid-phenylalanine-glycine, arginine-lysine-aspartic acid-valine-tyrosine, more preferably glycine-phenylalanine, histidine-β-alanine, glycine-glycine, lysine-glycine, glycine-glycine-phenylalanine, glycine-cysteine-glutamic acid, and glycine-leucine-phenylalanine-glycine;

[0174] When q is 1, AA2 is preferably a glutamic acid residue or an aspartic acid residue.

[0175] 1.2.4.R 01

[0176] In this invention, R 01 It is H or a functional group that can react with biologically relevant substances, or its protected form.

[0177] In one specific embodiment of the present invention, R is preferred. 01 The protected form is any one or any one of the following groups: epoxy group, alcohol hydroxyl group, thiol group, carboxyl group, amino group, aldehyde group, active ester group, active carbonate group, carbamate group, isocyanate group, isothiocyanate group, succinimide group, maleimide group, alkenyl group, alkynyl group, olefinic group, azide group, cyano group, dithiopyridyl group, α-haloacetylalkynyl group, folic acid group, rhodamine group, biotinyl group, monosaccharide group, and polysaccharide group; preferably any one of the following structures:

[0178]

[0179] 1.2.6. Preparation method

[0180] In one specific embodiment of the present invention, the three-arm polyethylene glycol derivative is obtained through a preparation process involving coupling reaction and / or polymerization reaction;

[0181] The coupling reaction is not particularly limited in its selection range, as long as two identical or different reactive groups can form a covalent linker through the reaction; the same preparation process may contain single-step or stepwise coupling reactions, preferably each coupling reaction is independently one of the following: alkylation, condensation, amidation, esterification, thioesterification, ring-opening, ring-closing condensation, addition, cycloaddition, α,β-unsaturated bond addition, alkynyl addition, Schiff base reaction combined with reduction, click reaction, azide-alkynyl addition, 1,3-dipolar cycloaddition, Diels-Alder addition, thiol-yne reaction, thiol-ene reaction, thiol-vinyl reaction, and condensation reaction; the reaction conditions of the coupling reaction are related to the type of covalent linker generated by the reaction, and existing publicly available technologies can be used; the valence state of the covalent linker generated by the coupling reaction can be divalent or trivalent, preferably divalent; the coupling reaction can generate stable groups or degradable groups.

[0182] The polymerization reaction involves at least the following two steps: deprotonation of a small molecule initiator and polymerization of ethylene oxide; the small molecule initiator can be a directly obtained raw material or an intermediate in the preparation process.

[0183] In the preparation process, when coupling reaction and polymerization reaction exist simultaneously, there is no restriction on the order of coupling reaction and polymerization reaction.

[0184] In this invention, the raw materials used in each preparation method can be purchased or synthesized by the user.

[0185] In this invention, the method for preparing monodisperse polyethylene glycol derivatives can replace the monodisperse raw material containing polyethylene glycol components with a polydisperse raw material of the same components to obtain the corresponding polydisperse product; similarly, the method for preparing polydisperse polyethylene glycol derivatives can replace the polydisperse raw material containing polyethylene glycol components with a monodisperse raw material of the same components to obtain the corresponding monodisperse product. The structures provided in this invention, and their corresponding monodisperse and polydisperse polyethylene glycol derivatives, are all within the scope of this invention.

[0186] In some specific embodiments of this invention, polyethylene glycol derivative raw materials, including but not limited to linear polyethylene glycol derivatives and nonlinear polyethylene glycol derivatives, can be prepared by referring to the methods in CN108530637B, CN110591079A, CN108659227A, CN108530617B or CN1243779C.

[0187] The intermediates and final products prepared in this invention can be purified by methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction. The structure and molecular weight of the final products can be characterized using methods including but not limited to NMR, electrophoresis, UV-Vis spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, and circular dichroism.

[0188] 1.2.6.1. Preparation of three-arm polyethylene glycol derivatives

[0189] In this invention, the reaction route described in the preparation method does not represent the actual complete preparation process. Those skilled in the art are aware that any necessary micro-modifications (e.g., protection or deprotection), intermediate preparation, post-processing, or purification processes can be performed in actual operation to obtain the polyethylene glycol derivative of this invention, which is still within the scope of this invention.

[0190] In this invention, the preparation method of the three-arm polyethylene glycol derivative represented by general formula (1) can employ one or more of the following processes, and the order of implementation of each process is not particularly limited provided that it can be implemented smoothly; wherein, RPEG is The process includes:

[0191] -(AA1) P - Introduction: Using the two-arm polyethylene glycol derivative 2arm-PEG as a raw material, a small molecule of amino acid or oligopeptide H2N-(AA1) is coupled. k -COO-PG1, where k is selected from integers from 1 to 5. When k is less than p, two or more amino acids or oligopeptide molecules need to be coupled until the sum of all k equals p, to obtain intermediate IM-1.

[0192] PG1 is a carboxyl protecting group, preferably tBu (tert-butyl).

[0193]

[0194] A specific example is the preparation of S1-3 in Example 1.1:

[0195]

[0196] Introduction of lysine branching structure: Intermediate IM-2 is obtained by coupling IM-1 with a small molecule N-1 containing Lys residues. Here, T is a linear or nonlinear structure without polyethylene glycol components, and its terminal contains a reactive group or its slightly modified form (e.g., -COOH or -COOtBu, aldehyde or acetal). T may have the same or different structure each time it appears.

[0197]

[0198] A specific example is the preparation of S1-9 in Example 1.2:

[0199]

[0200] The introduction of PEG single-chain components: There are two cases: (1) when two-arm PEG is used as raw material, a third PEG single-chain component is introduced; (2) only polyethylene glycol derivatives containing single-chain components are used as PEG source.

[0201] Case (1): Intermediate IM-2 and polyethylene glycol derivative RPEG-L containing a single-chain component F -F G Coupling was performed to obtain intermediate IM-3, in which L F It is a divalent linker, preferably a linker bond, -OCH2CH2- or -OCH2CH2CH2; F G The reactive group is selected from, but is not limited to, any one of -COOH, -F, -Cl, -Br, -OMs, -OTs, -CHO, -COCl, -CONHS, and active ester groups, preferably -COOH or The definition of T is the same as that described above.

[0202]

[0203] A specific example is the preparation of E1-1 in Example 1.1 (before removing tBu protection):

[0204]

[0205] Case (2): One amino group of small molecule N-2 reacts with a polyethylene glycol derivative RPEG-L containing a PEG single-chain component. F The reactive group W undergoes a single-step or stepwise reaction to obtain the two-armed polyethylene glycol intermediate IM-4. W is preferably a leaving group, more preferably -Cl, -Br, -OMs, or -OTs. RPEG, L... F Same as in case (1); PG2 is an amino protecting group. After removing PG2, the third PEG single-chain component is introduced as in case (1).

[0206]

[0207] A specific example is the preparation of E9-1 in Example 9, where S9-5 corresponds to IM-4:

[0208]

[0209] Terminal functionalization: achieved through terminal micro-modification and / or further coupling with small molecules. The structure corresponds to the transformation of T. The terminal micro-modification is selected from one or more of the following chemical reaction processes: protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, and modification of the leaving group. A complete preparation example may include multiple continuous or discontinuous transformations of T. When q is 1, i.e., AA2 is present, the transformation of T involves coupling a small molecule containing an AA2 residue, preferably a glutamic acid residue. Specific examples include Example 1.1, where T undergoes a transformation from -COOtBu to -COOH; and Example 1.4, where T undergoes a transformation from -COOH to -C(=O)O(CH2)2OH. The transformation; for example, in Example 7, T undergoes a change from -COOH to The transformation.

[0210] Unless otherwise specified, the above parameters are defined in the same way as general formula (1).

[0211] R01-based terminal functionalization: R01-based functionalization of polyethylene glycol derivatives satisfying general formula (1) 01 The terminal functionalization results in a structure that still satisfies the general formula (1). The method of terminal functionalization is not particularly limited and is related to the type of the final functional group or its protected form, mainly including the functionalization of terminal hydroxyl groups and the conversion from reactive groups to target functional groups or their protected forms. In this invention, the specific preparation methods for the functionalization of terminal hydroxyl groups include, but are not limited to, those described in paragraphs

[0960] to

[1205] of document CN104530417A. In this invention, the conversion from reactive groups to target functional groups or their protected forms can be achieved through any of the following methods:

[0212] Method 1: Direct modification, based on the direct modification of reactive groups, to obtain the target functional group or its protected form. Examples include the transformation of carboxyl groups into acyl halides, acyl hydrazides, esters, thioesters, and dithioesters; and the transformation of hydroxyl, mercapto, alkynyl, amino, and carboxyl groups into their corresponding protected structures. Another example is the modification of hydroxyl and amino groups by acid anhydrides.

[0213] Method 2: Coupling reaction between two reactive groups. Using a heterofunctionalizing agent containing one reactive group and a target functional group or its protected form as raw materials, the target functional group or its protected form is introduced through the reaction between the reactive group and the terminal reactive group of a polyethylene glycol derivative. There are no particular restrictions on the reaction mode or method between the two reactive groups; the reaction conditions are related to the type of divalent linker generated and existing publicly available techniques can be used. Examples include alkylation, alkenyl addition reactions, alkynyl addition reactions, Schiff base reactions combined with reduction reactions, and condensation reactions. Alkylation reactions are preferably based on thiol or amino alkylation, corresponding sequentially to the formation of thioether bonds, secondary amino groups, or tertiary amino groups. Condensation reactions include, but are not limited to, condensation reactions that generate ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, and carbamate groups. For example, using isofunctionalizing reagents containing groups such as azide, alkynyl, alkenyl, trithioester, mercapto, dienyl, furanyl, 1,2,4,5-tetraazine, and cyanate, along with the target functional group or its protected form, as raw materials, the target functional group or its protected form is introduced through a click reaction. The reaction between two reactive groups is accompanied by the formation of a new bond. Typical examples of the newly formed divalent linker include amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, and triazole groups.

[0214] Method 3: Obtain the target functional group or its protected form through a combination of direct modification and coupling reaction.

[0215] 1.2.7. Structural Examples

[0216] In one specific embodiment of the present invention, the three-arm polyethylene glycol derivative has a structure selected from any one of the following structural formulas:

[0217]

[0218]

[0219]

[0220] 1.2.8. Bio-related substances modified with three-arm polyethylene glycol derivatives

[0221] One embodiment of the present invention:

[0222] A bio-related substance modified with a three-armed polyethylene glycol derivative, characterized in that its structure is shown in general formula (5).

[0223] PLD (5)

[0225] Wherein, P is any of the three-armed polyethylene glycol derivatives mentioned above, D is a biorelated substance residue, and L is a divalent linker formed after the reactive group of the three-armed polyethylene glycol derivative reacts with the biorelated substance.

[0226] The biologically related substances are selected from any one of the following: drugs, proteins, polypeptides, oligopeptides, protein mimics, fragments, enzymes, antigens, antibodies and their fragments, receptors, gene-related aptamers, polysaccharides, proteoglycans, glycoproteins, lipid compounds, hormones, vitamins, vesicles, liposomes, dyes, fluorescent substances, targeting factors, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, micelles; more preferably, any one of the following: small molecule drugs, nucleic acids, steroids, phospholipids, glycolipids; more preferably, any one of the following: small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, steroid compounds; further, the small molecule drugs are preferably selected from any one of flavonoids, terpenoids, carotenoids, saponins, steroids, quinones, anthraquinones, fluoroquinones, coumarins, alkaloids, porphyrins, polyphenols, macrolides, monocyclic alkyl groups, phenylpropanoids, anthracyclines, and aminoglycosides.

[0227] In one specific embodiment of the present invention, the biologically related substance is preferably a genetically engineered drug, selected from any one of antibodies, antibody fragments, interleukins, lysozymes, interferons, auxins, EPO, and GCSF, wherein the interferon is preferably α-, β-, or γ-interferon. 2. Detailed Implementation

[0228] The preparation of three-arm polyethylene glycol derivatives, the preparation of drugs modified with three-arm polyethylene glycol derivatives, and bioactivity testing are further described below with reference to specific embodiments. These specific embodiments are for further detailed explanation of the invention and are not intended to limit the scope of protection of the invention.

[0229] Example 1: Three-armed monofunctional polyethylene glycol derivatives (E1-1, E1-2)

[0230] Example 1.1: Preparation of three-armed monofunctional polyethylene glycol carboxylic acid derivative E1-1

[0231]

[0232] In the corresponding general formula (3-2), E1-1, L3 is the connecting key, R 01 The design molecular weight is -OH. The designed total molecular weight is approximately 40.2 kDa, n1≈226, n2≈226, n3≈450.

[0233] The preparation method is as follows:

[0234] Step a: The two-arm polyethylene glycol acetic acid derivative S1-1 (30.15 g, 1.5 mmol, M...) n (Approximately 20.1 kDa, n1≈n2≈226, PDI=1.04) was dissolved in 150 mL of anhydrous dichloromethane, and N-hydroxysuccinimide (NHS, 0.26 g, 2.3 mmol) was added, followed by dicyclohexylcarbodiimide (DCC, 0.46 g, 2.3 mmol). 4-(dimethylamino)pyridine (DMAP, 0.04 g, 0.3 mmol) was added to 30 mL of dichloromethane solution containing glycine (S1-2, 1.97 g, 15.0 mmol) with a tBu-protected carboxyl group. The two solutions were mixed and reacted with stirring at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The tBu protection was removed using a TFA / DCM mixed solution (1:1 v / v), followed by washing with purified water, extraction with dichloromethane, drying of the extract with anhydrous sodium sulfate, filtration, concentration, and purification by column chromatography to obtain S1-3 (18.27 g).

[0235] Step b: Add S1-3 (16.16g, 0.8mmol, M) n (approximately 20.2 kDa, n1≈n2≈226, PDI=1.04) was dissolved in 100 mL of anhydrous dichloromethane, and NHS (0.14 g, 1.2 mmol) and DCC (0.25 g, 1.2 mmol) were added. DMAP (0.02 g, 0.2 mmol) was added to 40 mL of dichloromethane solution containing lysine (S1-4, 3.40 g, 8.0 mmol) with the Fmoc-protected amino group. The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The Fmoc protecting group was removed with 20% piperidine / DMF solution, the solvent was removed by rotary evaporation, and the solution was purified by column chromatography to obtain S1-5 (8.43 g).

[0236] Step c: The methoxy polyethylene glycol propionic acid derivative S1-6 (7.96 g, 0.4 mmol, M) n (≈19.9kDa, n3≈450, PDI=1.04) was dissolved in 50mL of anhydrous dichloromethane, and NHS (0.07g, 0.6mmol) was added, followed by DCC (0.12g, 0.6mmol). DMAP (0.01g, 0.1mmol) was added to S1-5 (8.12g, 0.4mmol, M) nThe two solutions were mixed and stirred at room temperature for 24 hours to remove insoluble matter by filtration. The solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), washed with purified water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain E1-1 (5.44 g). 1 H NMR(400MHz, CDCl3)δ:4.27-4.21(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<) ,3.74-3.43(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2C H2C(=O)NH-),3.37(s,9H,-OCH3),3.23-3.16(m,2H,Lys-ε-CH2-),2.52-2.47(t,2 H,-OCH2CH2C(=O)NH-),1.87-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). The molecular weight, as determined by GPC analysis, is approximately 40.2 kDa, and the PDI is 1.05.

[0237]

[0238] Example 1.2: Preparation of three-armed monofunctionalized polyethylene glycol aldehyde derivative E1-2

[0239]

[0240] In the corresponding general formula (3-2), E1-2, L3 is -NH(CH2)2-, R 01 The design molecular weight is -CHO. The designed total molecular weight is approximately 40.3 kDa, n1≈226, n2≈226, n3≈450.

[0241] Step a: Lysine S1-7 (5.03 g, 10.0 mmol) containing Fmoc and Cbz protected amino groups was dissolved in 80 mL of anhydrous dichloromethane, and NHS (1.27 g, 11.0 mmol) and DCC (2.27 g, 11.0 mmol) were added. DMAP (0.24 g, 2.0 mmol) was added to 30 mL of a dichloromethane solution of 3-aminopropionaldehyde diethyl acetal S1-8 (1.76 g, 12.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The Fmoc protecting group was removed with 20% piperidine / DMF solution, the solvent was removed by rotary evaporation, and the solution was purified by column chromatography to obtain M-1 (3.73 g).

[0242]

[0243] Step b: Add S1-3 (16.16g, 0.8mmol, M) n M-1 (3.28 g, 8.0 mmol) was dissolved in 100 mL of anhydrous dichloromethane. NHS (0.14 g, 1.2 mmol) and DCC (0.25 g, 1.2 mmol) were added. DMAP (0.02 g, 0.2 mmol) was added to 50 mL of dichloromethane solution containing M-1 (3.28 g, 8.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The crude product was dissolved in methanol and reduced with Pd / C. After the reaction was complete, the product was purified to obtain S1-9 (8.70 g) containing exposed amino groups.

[0244] Step c: Add S1-6 (7.96g, 0.4mmol, M) n (Approximately 19.9 kDa, n3 ≈ 450, PDI = 1.04) was dissolved in 50 mL of anhydrous dichloromethane, and NHS (0.07 g, 0.6 mmol) was added, followed by DCC (0.12 g, 0.6 mmol). DMAP (0.01 g, 0.1 mmol) was added to S1-9 (8.16 g, 0.4 mmol, M... n The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, the solution was concentrated, the crude product was dissolved in water, the pH of the solution was adjusted to 2.0 with hydrochloric acid, and the reaction was stirred for 16 hours. After the reaction was completed, 10% sodium chloride was added, the pH of the solution was adjusted to 6.4±0.2 with sodium bicarbonate, the solution was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and recrystallized from anhydrous isopropanol to give E1-2 (5.96 g). 1H NMR(400MHz, CDCl3)δ:4.26-4.19(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.75-3.38( m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH-;2H,-CH2CH2 The molecular weight is 3.36 (s, 9H, -OCH3), 3.22-3.13 (m, 2H, Lys-ε-CH2-), 2.74-2.64 (m, 2H, -CH2CH2CHO), 2.52-2.47 (t, 2H, -OCH2CH2C(=O)NH-), 1.80-1.35 (m, 6H, Lys-β-CH2-, Lys-γ-CH2-, Lys-σ-CH2-). The GPC-analyzed molecular weight is approximately 40.3 kDa, and the PDI is 1.06.

[0245]

[0246] Example 1.3: Preparation of three-arm monofunctional polyethylene glycol succinimide active ester derivative E1-3

[0247]

[0248] In the corresponding general formula (3-2), E1-3, L3 is -O-, R 01 It is a succinimide group. The designed total molecular weight is approximately 40.3 kDa, n1≈226, n2≈226, n3≈450.

[0249] The preparation method is as follows:

[0250] E1-1 (8.04g, 0.2mmol, M) n E1-3 (7.70 g) was dissolved in 100 mL of dichloromethane. NHS (0.12 g, 1.0 mmol) and DCC (0.21 g, 1.0 mmol) were added, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the insoluble matter was removed by filtration, the mixture was concentrated under reduced pressure, and purified by column chromatography to obtain E1-3 (7.70 g). 1HNMR(400MHz, CDCl3)δ:4.47-4.40(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.75 -3.43(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH- The molecular weight is 3.37 (s, 9H, -OCH3), 3.19-3.13 (m, 2H, Lys-ε-CH2-), 2.77 (s, 4H, NHS-CH2-), 2.52-2.47 (t, 2H, -OCH2CH2C(=O)NH-), and 1.92-1.31 (m, 6H, Lys-β-CH2-, Lys-γ-CH2-, Lys-σ-CH2-). The GPC-analyzed molecular weight is approximately 40.3 kDa, and the PDI is 1.05.

[0251]

[0252] Example 1.4: Preparation of three-arm monofunctionalized polyethylene glycol p-nitrobenzene reactive ester derivative E1-4

[0253]

[0254] In the corresponding general formula (3-2), E1-4, L3 is -O(CH2)2OC(=O)O-, R 01 The desired molecular weight is p-nitrophenyl. The designed total molecular weight is approximately 40.4 kDa, n1≈226, n2≈226, n3≈450.

[0255] The preparation method is as follows:

[0256] Step a: Under an argon atmosphere, E1-1 (20.10g, 0.5mmol, M) n ≈40.2 kDa, n1≈n2≈226, n3≈450, PDI=1.05), ethylene glycol (S1-10, 0.31 g, 5.0 mmol), and DMAP (0.01 g, 0.1 mmol) were dissolved sequentially in dichloromethane (200 mL). A dichloromethane solution of DCC (0.12 g, 0.6 mmol) was added dropwise under ice bath conditions (3 mL). After the addition was complete, the reaction temperature was raised to room temperature, and the reaction was stirred for 24 h. After the reaction was complete, the precipitate was removed by filtration. The solution was concentrated and purified by column chromatography to obtain S1-11 (10.42 g).

[0257] Step b: Under ice bath conditions, slowly add 1 mL of a dichloromethane solution containing p-nitrophenyl chloroformate (S1-12, 0.05 g, 0.2 mmol) dropwise to a solution containing S1-11 (8.06 g, 0.2 mmol, M... nThe solution of triethylamine (TEA, 0.04 g, 0.4 mmol) in dichloromethane (80 mL) was stirred for one hour, then brought to room temperature and reacted for another 24 hours. After the reaction was complete, the mixture was extracted, concentrated, and purified by column chromatography to obtain E1-4 (6.71 g). 1 H NMR(400MHz, CDCl3)δ:8.28(d,4H,Ar),7.38(d,4H,Ar),4.48-4.40(m,4H,-C(=O)O(CH2)2OC(=O)O-), 4.26-4.17(m,1H,Lys-α-CH<),4.13(s,2H,-OCH2C(=O)N<),3.75-3.41(m,PEG;2H,Gly-CH2-;4H,-O(C The molecular weight is approximately 40.4 kDa, and the PDI is 1.05. (H2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH-), 3.37(s,9H,-OCH3), 3.22-3.15(m,2H,Lys-ε-CH2-), 2.52-2.48(t,2H,-OCH2CH2C(=O)NH-), 1.89-1.35(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.4 kDa and a PDI of 1.05.

[0258]

[0259] Example 2: Three-armed monofunctional polyethylene glycol derivatives (E2-1, E2-2)

[0260] Example 2.1: Preparation of three-armed monofunctional polyethylene glycol carboxylic acid derivative E2-1

[0261]

[0262] In the corresponding general formula (3-3), E2-1, L3 is the connecting key, R 01 The molecule is -OH. The designed total molecular weight is approximately 30.4 kDa, n1≈n2≈n3≈226.

[0263] The preparation method is as follows:

[0264] Methoxylated polyethylene glycol butyric acid derivative S2-1 (10.10 g, 1.0 mmol, M) nDissolve ≈10.1 kDa, n3≈226, PDI=1.03) in 50 mL of anhydrous dichloromethane, add NHS (0.17 g, 1.5 mmol), then add DCC (0.31 g, 1.5 mmol). Add DMAP (0.02 g, 0.2 mmol) to S1-5 (20.3 g, 1.0 mmol, M). n The two solutions were mixed and stirred at room temperature for 24 hours to remove insoluble matter by filtration. The solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), washed with purified water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain E2-1 (9.36 g). 1 H NMR(400MHz, CDCl3)δ:4.27-4.21(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.72-3. 40(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2CH2C(=O)NH-), 3.36 (s, 9H, -OCH3), 3.23-3.14 (m, 2H, Lys-ε-CH2-), 2.35-2.21 (m, 2H, -OCH2CH2CH2C(=O)NH-), 1.89-1.32 (m, 6H, Lys-β-CH2-, Lys-γ-CH2-, Lys-σ-CH2-; 2H, -OCH2CH2CH2C(=O)NH-). GPC analysis indicates a molecular weight of approximately 30.4 kDa and a PDI of 1.06.

[0265]

[0266] Example 2.2: Preparation of three-armed monofunctionalized polyethylene glycol aldehyde derivative E2-2

[0267]

[0268] In the corresponding general formula (3-3), E2-2, L3 is -NH(CH2)2-, R 01 The design molecular weight is -CHO. The designed total molecular weight is approximately 30.4 kDa, n1≈n2≈n3≈226.

[0269] The preparation method is as follows:

[0270] S2-1 (10.10g, 1.0mmol, M) nDissolve NHS (approximately 10.1 kDa, n3 ≈ 226, PDI = 1.03) in 50 mL of anhydrous dichloromethane, add NHS (0.17 g, 1.5 mmol), then add DCC (0.31 g, 1.5 mmol). Add DMAP (0.02 g, 0.2 mmol) to S1-9 (20.40 g, 1.0 mmol, M... n The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, the solution was concentrated, the crude product was dissolved in water, the pH of the solution was adjusted to 2.0 with hydrochloric acid, and the reaction was stirred for 16 hours. After the reaction was completed, 10% sodium chloride was added, the pH of the solution was adjusted to 6.4±0.2 with sodium bicarbonate, the solution was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and recrystallized from anhydrous isopropanol to give E2-2 (9.70 g). 1 HNMR(400MHz, CDCl3)δ:4.26-4.19(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.74-3.39(m,PEG;2H ,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2CH2C(=O)NH-;2H,-C(=O)NHCH2CH2CHO;9H, -OCH3), 3.19-3.11(m,2H,Lys-ε-CH2-), 2.73-2.66(m,2H,-C(=O)NHCH2CH2CHO), 2.34-2.22(m,2H,-OCH2CH2CH2C(=O)NH-), 1.82-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-;2H,-OCH2CH2CH2C(=O)NH-). The GPC-analyzed molecular weight is approximately 30.4 kDa, and the PDI is 1.05.

[0271]

[0272] Example 3: Three-armed monofunctional polyethylene glycol derivatives (E3-1, E3-2)

[0273] Example 3.1: Preparation of three-armed monofunctional polyethylene glycol carboxylic acid derivative E3-1

[0274]

[0275] In the corresponding general formula (3-1), E3-1, L3 is the connecting key, and R... 01The design molecular weight is -OH. The designed total molecular weight is approximately 40.2 kDa, n1≈n2≈226, n3≈450.

[0276] The preparation method is as follows:

[0277] Methoxylated polyethylene glycol succinimide carbonate S3-1 (20.00 g, 1.0 mmol, M) n Dissolve ≈20.0 kDa, n3≈450, PDI=1.04) in 100 mL of anhydrous dichloromethane, add NHS (0.17 g, 1.5 mmol), then add DCC (0.31 g, 1.5 mmol). Add DMAP (0.02 g, 0.2 mmol) to S1-5 (20.3 g, 1.0 mmol, M). n The two solutions were mixed and stirred at room temperature for 24 hours to remove insoluble matter by filtration. The solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), washed with purified water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain E3-1 (10.65 g). 1 H NMR (400MHz, CDCl3) δ: 4.30-4.17(m,1H,Lys-α-CH<;2H,-OCH2CH2OC(=O)NH-), 4.12(s,2H,-OCH2C(=O)N<), 3.75-3.44(m,PEG; 2H,Gly-CH2-;4H,-O(CH2)2N<; 2H,>NCH2C(=O)NH-; 2H,-OCH2CH2OC(=O)NH-),3.37(s,9H,-OCH3),3.23-3.12( m,2H,Lys-ε-CH2-),1.88-1.32(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). The GPC test showed a molecular weight of approximately 40.2 kDa and a PDI of 1.06.

[0278]

[0279] Example 3.2: Preparation of three-armed monofunctionalized polyethylene glycol aldehyde derivative E3-2

[0280]

[0281] In the corresponding general formula (3-1), E3-2, L3 is -NH(CH2)2-, R 01The design molecular weight is -CHO. The designed total molecular weight is approximately 40.2 kDa, n1≈n2≈226, n3≈450.

[0282] The preparation method is as follows:

[0283] S3-1 (20.00g, 1.0mmol, M) n Dissolve ≈20.0 kDa, n3≈450, PDI=1.04) in 100 mL of anhydrous dichloromethane, add NHS (0.17 g, 1.5 mmol), then add DCC (0.31 g, 1.5 mmol). Add DMAP (0.02 g, 0.2 mmol) to S1-9 (20.40 g, 1.0 mmol, M). n The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, the solution was concentrated, the crude product was dissolved in water, the pH of the solution was adjusted to 2.0 with hydrochloric acid, and the reaction was stirred for 16 hours. After the reaction was completed, 10% sodium chloride was added, the pH of the solution was adjusted to 6.4±0.2 with sodium bicarbonate, the solution was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and recrystallized from anhydrous isopropanol to give E3-2 (11.66 g). 1 H NMR(400MHz, CDCl3)δ:4.28-4.16(m,1H,Lys-α-CH<;2H,-OCH2CH2OC(=O)NH-),4.12(s,2H,-OC H2C(=O)N<),3.75-3.38(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH 2OC(=O)NH-; 2H,-C(=O)NHCH2CH2CHO), 3.36(s,9H,-OCH3), 3.21-3.11(m,2H,Lys-ε-CH2-), 2.73-2.62(m,2H,-C(=O)NHCH2CH2CHO), 1.78-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.2 kDa and a PDI of 1.06.

[0284]

[0285] Example 4: Three-arm monofunctional polyethylene glycolamine derivative (E4-1)

[0286]

[0287] In the corresponding general formula (3-1), E4-1, L3 is -NH(CH2)2-, R 01 The design molecular weight is -NH2. The designed total molecular weight is approximately 40.2 kDa, n1≈n2≈226, n3≈450.

[0288] The preparation method is as follows:

[0289] E3-1 (20.10g, 0.5mmol, M) n Ethylenediamine (S4-1, 0.30 g, 5.0 mmol) with a concentration of approximately 40.2 kDa, n1≈n2≈226, n3≈450, and PDI=1.06 was dissolved in 200 mL of anhydrous dichloromethane. NHS (0.09 g, 0.8 mmol) was added, followed by DCC (0.15 g, 0.8 mmol). DMAP (0.01 g, 0.1 mmol) was added to 5 mL of dichloromethane solution containing ethylenediamine (S4-1, 0.30 g, 5.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure and purified by column chromatography to obtain E4-1 (7.04 g). 1 H NMR(400MHz, CDCl3)δ:4.30-4.17(m,1H,Lys-α-CH<;2H,-OCH2CH2OC(=O)NH-),4.12(s,2H,-OCH2 C(=O)N<),3.77-3.43(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2OC( =O)NH-), 3.36(s,9H,-OCH3), 3.33-3.25(m,2H,-C(=O)NHCH2CH2NH2), 3.19-3.10(m,2H,Lys-ε-CH2-), 2.84(m,2H,-C(=O)NHCH2CH2NH2), 1.77-1.32(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.2 kDa and a PDI of 1.06.

[0290]

[0291] Example 5: Three-armed monofunctional polyethylene glycol maleimide derivative (E5-1)

[0292]

[0293] In the corresponding general formula (3-1), E5-1, L3 is -NH(CH2)2NHC(=O)(CH2)2-, R 01It is a maleimide group. The designed total molecular weight is approximately 40.4 kDa, n1≈n2≈226, n3≈450.

[0294] The preparation method is as follows:

[0295] E4-1 (20.10g, 0.5mmol, M) n Dissolved 2,6-di-tert-butyl-4-methylphenol (BHT) in 200 mL of anhydrous dichloromethane (n1≈n2≈226, n3≈450, PDI=1.06), NHS (0.09 g, 0.8 mmol) was added, followed by DCC (0.15 g, 0.8 mmol). DMAP (0.01 g, 0.1 mmol) was added to 15 mL of dichloromethane solution containing a furan-protected 3-maleimide propionic acid (S5-1, 1.19 g, 5.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction, insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. 50 mL of toluene (Tol) was added, followed by 0.50 g of 2,6-di-tert-butyl-4-methylphenol (BHT). The mixture was heated to 125 °C and stirred for 5 hours to remove the furan-protected group. Concentrate and recrystallize from anhydrous isopropanol to obtain E5-1 (5.60 g). 1 H NMR (400MHz, CDCl3) δ: 6.72 (s, 2H, MAL), 4.28-4.17 (m, 1H, Lys-α-CH<; 2H, -OCH2CH2OC (=O)NH-), 4.13 (s, 2H, -OCH2C(=O)N<),3.85(t,2H,-CH2CH2-MAL),3.78-3.43(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O )NH-;2H,-OCH2CH2OC(=O)NH-), 3.37(s,9H,-OCH3), 3.30-3.23(m,4H,-C(=O)NH(CH2)2NHC(=O)-), 3.19-3.11(m,2H,Lys-ε-CH2-), 2.57(t,2H,-CH2CH2-MAL), 1.78-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.4 kDa and a PDI of 1.06.

[0296]

[0297] Example 6: Three-arm monofunctional polyethylene glycol derivatives (E6-1, E6-2)

[0298] Example 6.1: Preparation of three-armed monofunctional polyethylene glycol carboxylic acid derivative E6-1

[0299]

[0300] In the corresponding general formula (3-2), E6-1, L3 is -NHCH2-, R 01 The molars are -COOH. The designed total molecular weight is approximately 40.3 kDa, n1≈n2≈226, n3≈450.

[0301] The preparation method is as follows:

[0302] E1-1 (20.10g, 0.5mmol, M) n S1-2 (0.66 g, 5.0 mmol) was dissolved in 200 mL of anhydrous dichloromethane. NHS (0.09 g, 0.8 mmol) and DCC (0.15 g, 0.8 mmol) were added. DMAP (0.01 g, 0.1 mmol) was added to 10 mL of dichloromethane solution containing S1-2 (0.66 g, 5.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), followed by washing with purified water, extraction with dichloromethane, drying the extract with anhydrous sodium sulfate, filtering, concentrating, and purifying by column chromatography to obtain E6-1 (8.28 g). 1 H NMR(400MHz, CDCl3)δ:4.29-4.18(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.74 -3.43(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH- ;2H,-C(=O)NHCH2COOH), 3.36(s,9H,-OCH3), 3.19-3.10(m,2H,Lys-ε-CH2-), 2.53-2.45(t,2H,-OCH2CH2C(=O)NH-), 1.79-1.35(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.3 kDa and a PDI of 1.05.

[0303]

[0304] Example 6.2: Preparation of three-armed monofunctionalized polyethylene glycol aldehyde derivative E6-2

[0305]

[0306] In the corresponding general formula (3-2), E6-2, L3 is -NHCH2C(=O)NH(CH2)2-, R 01 The design molecular weight is -CHO. The designed total molecular weight is approximately 40.3 kDa, n1≈n2≈226, n3≈450.

[0307] The preparation method is as follows:

[0308] E6-1 (20.15g, 0.5mmol, M) n S1-8 (0.74 g, 5.0 mmol) was dissolved in 200 mL of anhydrous dichloromethane. NHS (0.09 g, 0.8 mmol) and DCC (0.15 g, 0.8 mmol) were added. DMAP (0.01 g, 0.1 mmol) was added to 10 mL of dichloromethane solution containing S1-8 (0.74 g, 5.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction, insoluble matter was removed by filtration, and the solution was concentrated. The crude product was dissolved in water, and the pH was adjusted to 2.0 with hydrochloric acid. The solution was stirred for 16 hours. After the reaction, 10% sodium chloride was added, and the pH was adjusted to 6.4 ± 0.2 with sodium bicarbonate. The solution was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and recrystallized from anhydrous isopropanol to obtain E6-2 (7.31 g). 1 H NMR(400MHz, CDCl3)δ:4.31-4.20(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C(=O)N<),3.74-3.45(m,PEG; 4H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH-),3.42-3.38(m,2H,-C(=O)N The molecular weight is approximately 40.3 kDa, with a PDI of 1.05. The molecular weight is determined by GPC analysis. The molecular weight is 3.36 (s, 9H, -OCH3), 3.18-3.10 (m, 2H, Lys-ε-CH2-), 2.71-2.63 (m, 2H, -C(=O)NHCH2CH2CHO), 2.53-2.45 (t, 2H, -OCH2CH2C(=O)NH-), and 1.79-1.32 (m, 6H, Lys-β-CH2-, Lys-γ-CH2-, Lys-σ-CH2-).

[0309]

[0310] Example 7: Three-armed monofunctional polyethylene glycol dicarboxylic acid derivative (E7-1)

[0311] The three-armed monofunctional polyethylene glycol carboxylic acid derivatives E1-1, E2-1, and E3-1 can be further coupled with glutamic acid to obtain three-armed monofunctional polyethylene glycol dicarboxylic acid derivatives containing two carboxyl groups. The following example illustrates the preparation of E7-1 by reacting E1-1 with glutamic acid containing a tBu-protected carboxyl group:

[0312]

[0313] In general formula (4), E7-1, L2 is -CH2CH2C(=O)-, and AA2 is a trivalent glutamic acid residue. m is 2, both L3s are connection keys, and both Rs are... 01 All are -OH. The designed total molecular weight is approximately 40.3 kDa, n1≈n2≈226, n3≈450.

[0314] The preparation method is as follows:

[0315] E1-1 (20.10g, 0.5mmol, M) n (Approximately 40.2 kDa, n1≈n2≈226, n3≈450, PDI=1.05) was dissolved in 200 mL of anhydrous dichloromethane, and NHS (0.09 g, 0.8 mmol) and DCC (0.15 g, 0.8 mmol) were added. DMAP (0.01 g, 0.1 mmol) was added to 20 mL of dichloromethane solution containing glutamic acid (S7-1, 1.30 g, 5.0 mmol) with two tBu protected carboxyl groups. The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was completed, the insoluble matter was removed by filtration, and the solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), washed with purified water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain E7-1 (10.46 g). 1H NMR(400MHz, CDCl3)δ:4.49-4.40(m,1H,Glu-α-CH),4.27-4.19(m,1H,Lys-α-CH<),4.12(s,2H,-OCH2C( =O)N<),3.76-3.42(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH-), 3.36(s,9H,-OCH3), 3.22-3.15(m,2H,Lys-ε-CH2-), 2.54-2.47(t,2H,-OCH2CH2C(=O)NH-), 2.42(t,2H,Glu-γ-CH2-), 2.19-1.94(m,2H,Glu-β-CH2-), 1.78-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.3 kDa and a PDI of 1.05.

[0316]

[0317] Example 8: Three-armed monofunctional polyethylene glycol dialdehyde derivative (E8-1)

[0318] The dicarboxylic acid derivatives based on E1-1, E2-1, and E3-1 prepared according to Example 7 can be further used as raw materials to prepare corresponding dialdehyde derivatives. Taking dicarboxylic acid derivative E7-1 as an example, the three-armed monofunctionalized polyethylene glycol dialdehyde derivative E8-1 was prepared:

[0319]

[0320] In the corresponding general formula (4), E8-1, L2 is -CH2CH2C(=O)-, and AA2 is a trivalent glutamic acid residue. m is 2, both L3 are -NH(CH2)2-, and both R... 01 All are -CHO. The designed total molecular weight is approximately 40.4 kDa, n1≈n2≈226, n3≈450.

[0321] The preparation method is as follows:

[0322] E7-1 (20.15g, 0.5mmol, M) nThe crude product (n1≈n2≈226, n3≈450, PDI=1.05) was dissolved in 200 mL of anhydrous dichloromethane, and NHS (0.09 g, 0.8 mmol) and DCC (0.15 g, 0.8 mmol) were added. DMAP (0.01 g, 0.1 mmol) was added to 30 mL of dichloromethane solution of S8-1 (2.03 g, 5.0 mmol, S8-1 is synthesized from glutamic acid with amino groups protected by S1-8 and Cbz). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was complete, insoluble matter was removed by filtration, the solution was concentrated, and the crude product was dissolved in water. The pH of the solution was adjusted to 2.0 with hydrochloric acid, and the reaction was stirred for 16 hours. After the reaction was complete, 10% sodium chloride was added, the pH of the solution was adjusted to 6.4±0.2 with sodium bicarbonate, extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and recrystallized from anhydrous isopropanol to obtain E8-1 (8.04 g). 1 H NMR(400MHz, CDCl3)δ:4.47-4.40(m,1H,Glu-α-CH),4.29-4.20(m,1H,Lys-α-CH<),4.14(s,2H,-OCH2C(=O)N<),3.75-3 .44(m,PEG;2H,Gly-CH2-;4H,-O(CH2)2N<;2H,>NCH2C(=O)NH-;2H,-OCH2CH2C(=O)NH-),3.42-3.38(m,4H,-CH2CH2CHO), 3.36(s,9H,-OCH3), 3.23-3.17(m,2H,Lys-ε-CH2-), 2.74-2.63(m,4H,-CH2CH2CHO), 2.55-2.46(t,2H,-OCH2CH2C(=O)NH-), 2.41(t,2H,Glu-γ-CH2-), 2.20-1.96(m,2H,Glu-β-CH2-), 1.77-1.32(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). GPC analysis indicates a molecular weight of approximately 40.4 kDa and a PDI of 1.05.

[0323]

[0324] Example 9: Three-armed monofunctional polyethylene glycol carboxylic acid derivative (E8-1)

[0325]

[0326] In the corresponding general formula (3-4), E9-1, L3 is the connecting key, R 01The design molecular weight is -OH. The designed total molecular weight is approximately 40.2 kDa, n1≈226, n2≈226, n3≈450.

[0327] The preparation method is as follows:

[0328] Step a: First alkylation reaction: Methoxylated polyethylene glycol sulfonate derivative S9-3 (12.12 g, 1.2 mmol, M n The product (approximately 10.1 kDa, n1 ≈ 226, PDI = 1.03) was dissolved in 60 mL of dichloromethane. Small molecule raw material S9-2 (5.78 g, 12.0 mmol, prepared from glycine with Boc-protected amino group and S1-4) was added. The mixture was reacted at room temperature for 24 hours. After the reaction was completed, the reaction solution was concentrated and precipitated twice in diethyl ether to remove impurities, yielding intermediate S9-4 (10.84 g).

[0329] Step b: Second alkylation reaction: S9-4 (8.40 g, 0.8 mmol, M n S9-3 (12.12 g, 1.2 mmol) with approximately 10.5 kDa, n1 ≈ 226, PDI = 1.03 was dissolved in 100 mL of dichloromethane and reacted at room temperature for 24 hours. After the reaction was completed, the reaction solution was concentrated, and phosphate buffer solution with pH = 7.0 was added and stirred for 16 hours. After column chromatography, intermediate S9-5 (8.61 g) containing a symmetrical nitrogen-branched two-arm polyethylene glycol structure was obtained.

[0330] Step c: Add S1-6 (7.96g, 0.4mmol, M) n Dissolve ≈19.9 kDa, n3≈450, PDI=1.04) in 50 mL of anhydrous dichloromethane, add NHS (0.07 g, 0.6 mmol), then add DCC (0.12 g, 0.6 mmol). Add DMAP (0.01 g, 0.1 mmol) to S9-5 (8.12 g, 0.4 mmol, M). n The two solutions were mixed and stirred at room temperature for 24 hours to remove insoluble matter by filtration. The solution was concentrated under reduced pressure. The tBu protection was removed with a TFA / DCM mixed solution (1:1 v / v), washed with purified water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain E9-1 (5.50 g). 1H NMR(400MHz, CDCl3)δ:4.26-4.18(m,1H,Lys-α-CH<),3.72-3.43(m,PEG;2H,Gly-CH 2-;2H,-OCH2CH2C(=O)NH-;4H,-OCH2CH2N<),3.37(s,9H,-OCH3),3.27(s,2H,>NCH2 C(=O)NH-),3.21-3.11(m,2H,Lys-ε-CH2-),2.80(t,2H,-OCH2CH2N<),2.52-2.44(t ,2H,-OCH2CH2C(=O)NH-),1.90-1.34(m,6H,Lys-β-CH2-,Lys-γ-CH2-,Lys-σ-CH2-). The molecular weight, as determined by GPC testing, is approximately 40.2 kDa, and the PDI is 1.07.

[0331]

[0332] Example 10: Irinotecan derivative modified with three-arm monofunctional polyethylene glycol (E10-1)

[0333]

[0334] The preparation method is as follows:

[0335] E4-1 (20.10g, 0.5mmol, M) n (Approximately 40.2 kDa, n1≈n2≈226, n3≈450, PDI=1.06) was dissolved in 200 mL of anhydrous dichloromethane, and NHS (0.09 g, 0.8 mmol) was added, followed by DCC (0.15 g, 0.8 mmol). DMAP (0.01 g, 0.1 mmol) was added to 50 mL of dichloromethane solution containing irinotecan propionic acid derivative (S10-1, 3.44 g, 5.0 mmol). The two solutions were mixed and stirred at room temperature for 24 hours. After the reaction was complete, the precipitate was removed by filtration. The solution was concentrated and purified by column chromatography to obtain E10-1 (6.93 g). 1H NMR(400MHz, CDCl3)δ:8.19-8.17(d,1H),7.84(s,1H),7.59(dd,1H),7.17(s,1H),5.70 -5.39(m,2H),5.24(s,2H),4.50-4.39(m,2H),4.26-4.23(m,3H),4.13(s,2H),3.76-3.4 3(m,PEG+10H), 3.36(s,9H), 3.30-3.24(m,4H), 3.18-3.06(m,6H), 2.95-2.88(m,1H), 2.82-2.74(m,4H), 2.72-2.56(m,4H), 2.52-2.43(m,2H), 2.08-1.32(m,19H), 0.97(t,3H). GPC analysis showed a molecular weight of approximately 40.9 kDa and a PDI of 1.06.

[0336]

[0337] Example 11: Biological activity test

[0338] (1) Serum stability evaluation

[0339] 1 mL of mouse serum was added to each of 18 1.3 mL microcentrifuge tubes. Fifteen of these tubes contained the polyethylene glycol (PEG) derivative of this invention; one contained a PEG derivative without degradable groups (Comparative Example C-1, 30.2 kDa); one contained a three-arm PEG derivative containing lysine-lysine dipeptide residues (Comparative Example C-2, 40.4 kDa); and one contained a three-arm PEG derivative containing glycine-lysine-lysine tripeptide residues (Comparative Example C-3, 40.5 kDa). The concentration of the PEG derivative in each tube was set to 5.0 mg / mL. After incubation at 37°C for 96 hours, 200 μL of the sample was taken. Protein removal from the serum: Acetonitrile was added, and the mixture was vortexed for 1 minute to precipitate the proteins. The mixture was then centrifuged, and the supernatant was collected. Removal of hydrophobic substances such as fatty acids: Hexane was added to the recovered solution, and the mixture was vortexed for 1 minute and then centrifuged. The lower layer was recovered, and the mixture was concentrated under vacuum to recover the polyethylene glycol derivative. The molecular weight was determined by GPC, and the decomposition rate was calculated using the following formula:

[0340] Decomposition rate = (M before the experiment) n Peak area % - M after experiment n (peak area %) / (M before experiment) n (peak area %) × 100%

[0341] Among them, M n The average molecular weight of a polyethylene glycol derivative, for example, the average molecular weight M of E1-1. nThe value is 40.2 kDa. Comparative examples C-1, C-2, and C-3 were synthesized with reference to CN1243779C, CN1995094A, and CN101168594A, respectively, and their structures are as follows:

[0342]

[0343] The results of the degradation rate determination in mouse serum are shown in Table 1. The results show that the degradation rate of the nitrogen-branched three-arm monofunctional polyethylene glycol derivatives of the present invention in serum does not exceed 5%, and is slightly lower than that of the comparative examples of three-arm polyethylene glycol derivatives with all branched structures having carbon cores (C-2 and C-3 had degradation rates of 9% and 7% in serum, respectively), indicating that the three-arm monofunctional polyethylene glycol derivatives of the present invention have good serum stability.

[0344] Table 1. Degradation rate of polyethylene glycol derivatives in mouse serum

[0345]

[0346] (2) Evaluation of intracellular degradability

[0347] Experimental group: 10 mL of RPMI-1640 (10% FBS Pn / St) culture medium was used to inoculate 10 × 10⁶ cells / mL of culture medium into 100 mm culture dishes. 6 RAW264.7 cells were cultured at 37°C for 24 hours, then transferred to a culture medium containing 10.0 mg / mL of the polyethylene glycol derivative of this invention or a comparative polyethylene glycol derivative. After further culture at 37°C for 96 hours, the cells were dissolved in 1% SDS solution and diluted with PBS. Protein removal: Acetonitrile was added, and the mixture was vortexed for 1 minute to precipitate proteins. The supernatant was then collected by centrifugation. Removal of hydrophobic substances such as fatty acids: Hexane was added to the recovered solution, and the mixture was vortexed for 1 minute and centrifuged. The lower layer was collected and concentrated under vacuum to recover the polyethylene glycol derivative.

[0348] Control group: The culture medium containing 10.0 mg / mL of the polyethylene glycol derivative of the present invention or the comparative polyethylene glycol derivative was kept at 37°C for 96 hours without cell inoculation, and the polyethylene glycol derivative was recovered in the same manner as the experimental group.

[0349] The recovered polyethylene glycol derivatives were subjected to GPC testing, and the decomposition rate was calculated according to the aforementioned formula. The results are summarized in Table 2. The results show that the nitrogen-branched three-arm monofunctional polyethylene glycol derivatives have a high decomposition rate in cells, with E3-1 and E3-2 having a decomposition rate of over 98%, significantly higher than the control group.

[0350] Table 2. Decomposition rate of polyethylene glycol derivatives in cells / culture medium

[0351]

[0352] (3) Evaluation of cell vacuolation

[0353] Cellular vacuolation was evaluated using three-arm polyethylene glycol derivatives of the present invention, including E3-2 (40.4 kDa) containing glycine-glycine-lysine tripeptide residues, comparative example C-1 (30.2 kDa) without degradable groups, comparative example C-2 (40.4 kDa) containing lysine-lysine dipeptide residues, and comparative example C-3 (40.5 kDa) containing glycine-lysine-lysine tripeptide residues. Balb / c mice (8 weeks old, male) were randomly divided into a blank control group, three control groups (C-1, C-2, C-3), and an experimental group (E3-2), with four mice in each group. Mice in the blank control group were injected with 20 μL of physiological saline via the tail vein, mice in the control group were injected with 20 μL of physiological saline solution (200 mg / mL) containing the comparative polyethylene glycol derivative via the tail vein, and mice in the experimental group were injected with 20 μL of physiological saline solution (200 mg / mL) containing E3-2 via the tail vein. These treatments were administered once every 3 days for 10 consecutive days. After administration, the mice were perfused and fixed with 4% paraformaldehyde solution, and paraffin sections were prepared. HE staining and immunostaining with anti-PEG antibody were performed to assess vacuolar formation in the choroid plexus epithelial cells of the brain. The results showed that significant cellular vacuolar formation was observed in mice injected with C-0 and C-1, while no vacuolar formation was observed in the blank control group, the control group injected with C-2, and the experimental group injected with E3-2. Based on the intracellular degradation assay results, this indicates that the three-armed polyethylene glycol derivative of this invention can significantly inhibit cellular vacuolar formation.

[0354] (4) Biological assays for PEGylated irinotecan

[0355] Cytotoxicity assay:

[0356] The cytotoxicity of PEGylated irinotecan E10-1 (where the polyethylene glycol component is derived from E4-1) was tested using the MTT assay. Three control groups were set up: a blank control group (no drug, only culture medium), a positive control group (with irinotecan), and an experimental group (with PEGylated irinotecan (E10-1)). Drug concentrations of 1 nM, 10 nM, and 100 nM were set in both the positive control and experimental groups, with six replicates per concentration and three repetitions. Six cancer cell lines were used 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.

[0357] With an inoculation density of 1×10 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 1 nM, 10 nM, and 100 nM E10-1 were added to each of the three experimental groups, respectively. 100 μL of medium containing the corresponding concentration of irinotecan was added to each of the three positive control groups, and 100 μL of fresh culture medium was added to 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 MTT with cancer cells for 4 h, the mixture of culture medium and MTT buffer was discarded. 150 μL of DMSO (150 μL / well) was added to dissolve the blue-purple crystals of 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.

[0358] Cell viability = (absorbance value of drug group / absorbance value of blank group) × 100%.

[0359] The results showed that the positive control group (irinotecan) and the experimental group (E10-1) both significantly inhibited the proliferation of the aforementioned six types of cancer cells, and the cell survival rate of the experimental group was basically the same as that of the positive control group. The polyethylene glycol derivative E4-1 of the present invention was used to modify the irinotecan drug and retained the drug activity.

[0360] Anti-tumor effects:

[0361] An animal transplantable tumor assay was conducted. H22 mouse hepatocellular carcinoma cells were inoculated subcutaneously into the right axilla of mice to form solid tumors. The drug was administered via tail vein injection 2 and 7 days after inoculation, using a single-dose administration method. Two weeks after administration, the mice were euthanized by cervical dislocation, the tumors were removed, and weighed. The results showed that, compared to the blank control, both irinotecan and the two polyethylene glycol-modified irinotecan derivatives (E10-1) exhibited significant tumor-suppressing effects, with E10-1 showing a higher tumor inhibition rate than irinotecan alone.

[0362] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

[0363] For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments are given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A three-armed polyethylene glycol derivative, the structure of which is shown in general formula (1): in, n i Ri represents the degree of polymerization of the polyethylene glycol chain, where i is selected from any one of 1, 2, and 3, and n1, n2, and n3 are each independently selected from integers between 30 and 1000; R2 is independently C2 each time it appears. 1-6 alkyl; L 1A L 1B L1 and L2 are each independently a divalent linker; Lys represents lysine residues; AA1 and AA2 are each an independent amino acid residue; p is an integer selected from 1 to 5, -(AA1) p - indicates a divalent residue formed by the combination of p AA1 residues; q is 0 or 1, indicating that AA2 is absent or that one AA2 residue exists; L3 is a linker bond or a divalent linker group; R 01 The protected form is any one or any one of the following groups: epoxy group, alcohol hydroxyl group, thiol group, carboxyl group, amino group, aldehyde group, active ester group, active carbonate group, carbamate group, isocyanate group, isothiocyanate group, succinimide group, maleimide group, alkenyl group, alkynyl group, olefinic group, azide group, cyano group, dithiopyridyl group, α-haloacetylalkynyl group, folic acid group, rhodamine group, monosaccharide group, and polysaccharide group; m is 1 or 2; when m is 2, the two L3 structures are the same or different, and the two R groups are... 01 The structures are the same or different.

2. The three-arm polyethylene glycol derivative according to claim 1, characterized in that, The number average molecular weight of each PEG chain is 1000, 1500, 2000, 2500, 3000, 3350, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 Da.

3. The three-arm polyethylene glycol derivative according to claim 1, characterized in that, The PEG chains corresponding to n1, n2, and n3 are all polydisperse, and each occurrence of n1, n2, and n3 is independently selected from 30 to 1000.

4. The three-arm polyethylene glycol derivative according to claim 3, characterized in that, Each occurrence of n1, n2, and n3 is independently selected from 30 to 500.

5. The three-arm polyethylene glycol derivative according to claim 3, characterized in that, Each of the n1, n2, and n3 is independently selected from 100 to 500 each time it appears.

6. The three-arm polyethylene glycol derivative according to claim 1, characterized in that, R 01 It can be any of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 7. The three-arm polyethylene glycol derivative according to claim 1, characterized in that, L2 is -(CH2) tq -、-C(=O)-、-(CH2) tq C(=O)-、-C(=O)(CH2) tq C(=O)-、-(CH2) tq NHC(=O)(CH2) tq C(=O)-、-(CH2) tq C(=O)NH(CH2) tq Any of C(=O)-, wherein tq is an integer from 1 to 4 each time it appears, and the right end of the linker is connected to the lysine residue in formula (1).

8. The three-arm polyethylene glycol derivative according to claim 7, characterized in that, L2 is -C(=O)-, -CH2C(=O)-, -CH2CH2C(=O)-, -CH2CH2CH2C(=O)-, -C(=O)CH2CH2C(=O)-, -C(=O)CH2CH2CH2C(=O)-, -CH2CH2NHC(=O)CH2CH2C(=O)-, -CH2CH2CH2NHC(=O)CH2CH2C (=O)-, -CH2CH2NHC(=O)CH2CH2CH2C(=O)-, -CH2CH2CH2NHC(=O)CH2CH2CH2C(=O)-, -CH Any of 2C(=O)NHCH2C(=O)-, -CH2CH2C(=O)NHCH2C(=O)-, -CH2CH2CH2C(=O)NHCH2C(=O)-.

9. The three-arm polyethylene glycol derivative according to claim 1, characterized in that, L3 represents a linking bond, alkylene group, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c C(=O)S- any one or any combination of two or more of them; R c For H or C 1-6 Any one of the alkyl groups.

10. The three-armed polyethylene glycol derivative according to claim 9, characterized in that, R c For H.

11. The three-armed polyethylene glycol derivative according to claim 9, characterized in that, The alkylene group is -(CR) a R b ) t - where t is an independent integer from 1 to 12 each time it appears, and R a R b Each independently is H or C 1-6 Any one of the alkyl groups, t R a and t R b Selected from the same structure or a combination of two or more different structures.

12. The three-armed polyethylene glycol derivative according to claim 11, characterized in that, R a R b Each occurrence is independently either -CH3 or -H.

13. The three-armed polyethylene glycol derivative according to claim 11, characterized in that, R a R b Both are H.

14. The three-armed polyethylene glycol derivative according to claim 11, characterized in that, t is an integer from 1 to 4.

15. The three-armed polyethylene glycol derivative according to claim 11, characterized in that, t is 1 or 2.

16. The three-armed polyethylene glycol derivative according to claim 1, characterized in that, Each of the p AA1 residues and q AA2 residues is independently selected from any one of the following: glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine, and citrulline. When p≥2, -(AA1) p - represents an oligopeptide residue; the oligopeptide is a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

17. The three-armed polyethylene glycol derivative according to claim 16, characterized in that, When p=1, -AA1- is a glycine residue.

18. The three-armed polyethylene glycol derivative according to claim 16, characterized in that, The oligopeptide is any one of glycine-phenylalanine, histidine-β-alanine, glycine-glycine, lysine-glycine, lysine-glutamic acid, glycine-glycine-glycine, glycine-glycine-phenylalanine, glycine-phenylalanine-glycine, glycine-lysine-glycine, glycine-histidine-lysine, glycine-cysteine-glutamic acid, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, arginine-glycine-aspartic acid, glycine-histidine-proline, glycine-leucine-phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, glycine-lysine-glycine-lysine, glycine-phenylalanine-glutamic acid-phenylalanine-glycine, and arginine-lysine-aspartic acid-valine-tyrosine.

19. The three-armed polyethylene glycol derivative according to claim 16, characterized in that, The oligopeptide is any one of glycine-phenylalanine, histidine-β-alanine, glycine-glycine, lysine-glycine, glycine-glycine-phenylalanine, glycine-cysteine-glutamic acid, and glycine-leucine-phenylalanine-glycine.

20. The three-armed polyethylene glycol derivative according to claim 16, characterized in that, When q is 1, AA2 is a glutamic acid residue or an aspartic acid residue.

21. The three-arm polyethylene glycol derivative according to any one of claims 16-20, characterized in that, p is 1, AA1 is a glycine residue; the structure of the three-armed polyethylene glycol derivative is shown in general formula (2): in, L 1A L 1B Each is independently selected from C 1-6 Alkylene or -(CH2) 1-6 C(=O)-, where the left end of the linker is connected to the PEG chain.

22. The three-armed polyethylene glycol derivative according to claim 21, characterized in that, L 1A L 1B Each is independently selected from -CH2CH2-, -CH2-, -CH2C(=O)-, -CH2CH2C(=O)-.

23. The three-arm polyethylene glycol derivative according to claim 21, characterized in that, L 1A L 1B It is any of the following situations: Case (1): L 1A L 1B Each can be independently -CH2CH2- or -CH2-; Case (2): L 1A L 1B One of them is -CH2CH2- or -CH2-, and the other is -CH2C(=O)- or -CH2CH2C(=O)-.

24. The three-armed polyethylene glycol derivative according to claim 21, characterized in that, q is 0, AA2 does not exist, and the structure is as shown in general formula (3): 。 25. The three-armed polyethylene glycol derivative according to claim 24, characterized in that, L3 is a connector, -O-, -O(CH2) tp OC(=O)O-、-NH(CH2) tp -、-NH(CH2) tp C(=O)NH(CH2) tp -、-NH(CH2) tp NHC(=O)(CH2) tp - any one of them, and the right end is connected to R 01 Connected; tp is an integer from 1 to 4.

26. The three-armed polyethylene glycol derivative according to claim 25, characterized in that, tp is 1 or 2.

27. The three-armed polyethylene glycol derivative according to claim 24, characterized in that, R 01 -OH, -CHO, -COOH, -NH2, , or .

28. The three-armed polyethylene glycol derivative according to claim 24, characterized in that, The structure of the three-armed polyethylene glycol derivative is any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 29. The three-armed polyethylene glycol derivative according to claim 21, characterized in that, If q is 1, there exists an AA2 with the structure shown in general formula (4): 。 30. The three-armed polyethylene glycol derivative according to claim 29, characterized in that, R 01 It can be -OH or -CHO.

31. The three-armed polyethylene glycol derivative according to claim 29, characterized in that, L3 is a linker bond or -NH(CH2) tp - and the right end is with R 01 Connected; tp is an integer from 1 to 4.

32. The three-armed polyethylene glycol derivative according to claim 31, characterized in that, tp is 1 or 2.

33. The three-armed polyethylene glycol derivative according to claim 29, characterized in that, AA2 is a trivalent amino acid residue.

34. The three-armed polyethylene glycol derivative according to claim 33, characterized in that, AA2 is a trivalent glutamic acid residue or a trivalent aspartic acid residue.

35. The three-armed polyethylene glycol derivative according to claim 29, characterized in that, The structure of the three-armed polyethylene glycol derivative is any one of the following: 、 、 。 36. The three-arm polyethylene glycol derivative according to claim 1, wherein the structure is selected from any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 。 37. A bio-related substance modified with a three-armed polyethylene glycol derivative, characterized in that, The structure is shown in general formula (5). Wherein, P is the three-armed polyethylene glycol derivative according to any one of claims 1-36, D is a biorelated substance residue, and L is a divalent linker formed after the reactive group of the three-armed polyethylene glycol derivative reacts with the biorelated substance. The biologically related substances are selected from any one of the following: drugs, proteins, polypeptides, oligopeptides, protein mimics, antigens, antibodies and their fragments, receptors, gene-related aptamers, polysaccharides, glycoproteins, lipid compounds, hormones, vitamins, vesicles, liposomes, dyes, fluorescent substances, targeting factors, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, vaccines, and micelles.

38. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 37, characterized in that, The biologically related substances are enzymes or proteoglycans.

39. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 37, characterized in that, The biologically related substances are selected from any one of the following: small molecule drugs, nucleic acids, phospholipids, and glycolipids.

40. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 39, characterized in that, The small molecule drug is a steroid.

41. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 37, characterized in that, The biologically related substances are selected from any of the following: small molecule drugs, nucleosides, nucleotides, oligonucleotides, and polynucleotides.

42. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 41, characterized in that, The small molecule drug is a steroid compound.

43. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 41, characterized in that, The biologically related substance is an antisense oligonucleotide.

44. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 41, characterized in that, The small molecule drug is selected from any one of flavonoids, terpenoids, carotenoids, saponins, steroids, steroids, fluoroquinones, alkaloids, porphyrins, polyphenols, macrolides, monocyclic acyl compounds, phenylpropanoids, and anthracyclines.

45. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 41, characterized in that, The small molecule drug is anthraquinone or coumarin.

46. ​​The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 37, characterized in that, The biologically related substance is a genetically engineered drug, selected from any one of antibodies, antibody fragments, interleukins, lysozyme, interferon, auxin, EPO, and GCSF.

47. The bio-related substance modified with a three-arm polyethylene glycol derivative according to claim 46, characterized in that, The interferon is selected from α-, β-, or γ-interferon.

Images