Preparation method of nitrogen-branched two-arm polyethylene glycol derivative

Abstract

The invention discloses a preparation method of a nitrogen-branched two-arm polyethylene glycol derivative. The nitrogen-branched two-arm polyethylene glycol derivative is shown as a general formula (1), and the method comprises a reaction between a compound shown as a general formula (P-amide) and a compound shown as a general formula (BSM). Wherein the compound as shown in the general formula (P-amide) is a linear polyethylene glycol derivative containing an amido bond, and the compound as shown in the general formula (BSM) is a bifunctional small molecule compound. The definition of each symbol is described in the text. Through reasonable design of a reaction route and reaction conditions, the problems of difficulty in separation and purification and the like in synthesis of similar target products are solved, and meanwhile, the method has the advantages of simplicity in operation, high product purity and high terminal modification rate.

Description

Technical Field

[0001] This invention relates to the field of polymer synthetic chemistry, specifically to a method for preparing nitrogen-branched two-arm polyethylene glycol derivatives, particularly a method for preparing nitrogen-branched two-arm polyethylene glycol derivatives with a simple process and high product purity. Background Technology

[0002] Polyethylene glycol (PEG) derivatives can be covalently coupled with drug molecules (including protein drugs and small organic molecule drugs), peptides, carbohydrates, lipids, oligonucleotides, affinity ligands, cofactors, liposomes, and biomaterials through their active groups, achieving PEGylation modification of drugs and other biorelated substances. Modified drug molecules can possess many excellent properties of PEG, such as hydrophilicity, flexibility, and anticoagulant properties. Compared to linear structures, PEG derivatives containing branched structures can achieve a unique "umbrella-like" protective effect, resulting in better modification effects.

[0003] Branched two-arm polyethylene glycol derivatives have been widely used in the field of drug modification, including those with the following structural types. The existing technology CN108659227A discloses a method for preparing this derivative using two linear monofunctional polyethylene glycol derivatives as raw materials. This is achieved through the reaction of a secondary amine group (-NH-) in one raw material with an activated carboxyl group (such as -C(=O)NHS) in the other. After the reaction, an acid anhydride is added to continue the reaction before separation and purification using an ion column. This preparation method requires the use of an acid anhydride to reduce the interference caused by the secondary amine group in the separation and purification steps, which not only increases the number of steps but also makes it unsuitable for the preparation of compounds sensitive to anhydrides. Furthermore, this method involves not only two-arm polyethylene glycol acids but also linear polyethylene glycol acids as raw materials, resulting in both the raw materials and the final product carrying negative charges, making ion column separation and purification difficult.

[0004] Therefore, it is necessary to develop new preparation methods to obtain nitrogen-branched two-arm polyethylene glycol derivatives with simple processes and easy product separation. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following implementation scheme:

[0006] A method for preparing a nitrogen-branched two-arm polyethylene glycol derivative, characterized in that the nitrogen-branched two-arm polyethylene glycol derivative is as shown in general formula (1), and the method comprises the following reactions:

[0007]

[0008] Among them, the compound represented by the general formula (P-amide) is a linear polyethylene glycol derivative containing an amide bond, and the compound represented by the general formula (BSM) is a bifunctional small molecule compound.

[0009] T1 and T2 are each independently C 1-20 Hydroxyl groups;

[0010] n1 and n2 are the degrees of polymerization of the polyethylene glycol chain, selected from integers from 8 to 1000;

[0011] L1, L2, and L3 are each independently an alkylene group or a divalent linker formed by combining an alkylene group with a heteroatom-containing group;

[0012] W is a group that can react with the amide bond in the general formula (P-amide) to form a trivalent nitrogen branched core;

[0013] L0 is a linking bond or divalent linker formed by the reaction of W with the amide bond in the general formula (P-amide);

[0014] F1 is -COOH, -COONa, or -COOK;

[0015] The reaction is carried out under strongly alkaline or moderately alkaline conditions;

[0016] The compounds represented by general formula (P-amide) and general formula (1) are both monodisperse or polydisperse.

[0017] The present invention also provides another embodiment:

[0018] A method for preparing a nitrogen-branched two-arm polyethylene glycol derivative, characterized in that the nitrogen-branched two-arm polyethylene glycol derivative is as shown in general formula (2), and the method includes the following two steps:

[0019] Step 1: Obtain the compound shown in general formula (1) by the preparation method described in the aforementioned implementation scheme;

[0020] Step 2: Perform one or more terminal functionalization modifications on the compound shown in general formula (1) to obtain the compound shown in general formula (2):

[0021]

[0022] The compounds shown in general formula (2) have the same T1, T2, n1, n2, L1, L2, L3, L0 and F1 as those shown in general formula (1);

[0023] L4 is a linker formed after the terminal functionalization modification, connecting the trivalent nitrogen branched nucleus and F2;

[0024] k is an integer between 1 and 100;

[0025] F2 is a hydrogen atom or a functional group that can react with biologically relevant substances, or its protected form.

[0026] The present invention also provides another embodiment:

[0027] A method for preparing a conjugate of a nitrogen-branched two-armed polyethylene glycol derivative and a biorelated substance, characterized in that the preparation method of the conjugate includes preparing the nitrogen-branched two-armed polyethylene glycol derivative using any of the preparation methods in the foregoing embodiments.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Beneficial Effect 1: This invention prepares a nitrogen-branched two-arm polyethylene glycol derivative as shown in general formula (1) by reacting a linear polyethylene glycol derivative (P-amide) containing no terminal reactive groups but an intermediate amide bond with a bifunctional small molecule compound (BSM). The amide bond itself has low reactivity, and the intermediate amide bond of the compound represented by general formula (P-amide) is partially embedded by the two PEG segments, further reducing its reactivity. This invention overcomes this problem through the rational design of the reaction conditions, enabling the coupling reaction based on the amide bond to proceed smoothly.

[0030] Beneficial Effect 2: The raw material used in this invention is a polyethylene glycol derivative (P-amide) without terminal polar groups, and the other raw material is a small molecule compound (BSM). Neither of them will have a significant impact on the separation and purification of the product. Therefore, after the reaction, no other additional operations are required to carry out separation and purification to obtain a product with high purity and high terminal modification rate.

[0031] Beneficial effect 3: In this invention, the linear polyethylene glycol derivative (P-amide) containing amide bonds reacts with small molecules to generate the final product, two-arm polyethylene glycol acid. Since only the final product contains charged carboxyl groups, it can be efficiently separated and purified using an ion column.

[0032] Beneficial Effect 4: In this invention, alkylation based on the secondary amide group contained in the general formula (P-amide) can be carried out not only under strongly alkaline conditions, but also under moderately alkaline conditions at relatively low temperatures and without the need for transition metal catalysis, thus expanding the range of compounds represented by the general formula (BSM). Attached Figure Description

[0033] Figure 1 The compound (S1-3) of the general formula (P-amide) prepared in Example 1.1 1 H NMR spectrum.

[0034] Figure 2 The nitrogen-branched two-arm polyethylene glycol acetic acid derivative (E1-1) prepared in Example 1.1 1 H NMR spectrum.

[0035] Figure 3 The nitrogen-branched two-arm polyethylene glycol aldehyde derivative (E2-1) prepared in Example 2 1 H NMR spectrum.

[0036] Figure 4 The nitrogen-branched two-arm polyethylene glycol succinimide ester derivative (E3-1) prepared in Example 3 1 H NMR spectrum.

[0037] Figure 5 The GPC spectrum for E1-1 is shown.

[0038] Figure 6 The GPC spectrum for E2-1.

[0039] Figure 7 The GPC spectrum for E3-1.

[0040] Figure 8 HPLC chromatogram for testing the terminal substitution rate of E1-1.

[0041] Figure 9 HPLC chromatogram for testing the terminal substitution rate of E2-1.

[0042] Figure 10 HPLC chromatogram for testing the terminal substitution rate of E3-1. Invention Details

[0044] 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.

[0045] 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 throughout the entire specification.

[0046] 1. Terminology Explanation

[0047] Unless otherwise stated, all technical and scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art. All disclosures of patents and other publications cited are incorporated herein by reference in their entirety. In the event of any conflict between any description or interpretation of terms herein and any document incorporated herein by reference, the description and interpretation of the terms below shall prevail.

[0048] In this invention, the term "independently" means that the described objects are not limited by each other. For example, "T1 and T2 are each independently C..." 1-20 The description "hydroxyl group" means that the specific forms of T1 and T2 can be the same or different and are not restricted to each other. For example, T1 and T2 can both be methoxyl groups, or T1 can be methoxyl groups while T2 can be ethoxyl groups.

[0049] In this invention, the term "combination" refers to a combination of any two or more of the listed elements. Any element in the combination can be one or more identical or different forms. For example, the divalent linker that satisfies the condition "combination of alkylene groups and heteroatom-containing groups" can be -CH2C(=O)-, -CH2C(=O)CH2-, -CH2OCH2CH2-, etc.

[0050] In this invention, unless otherwise specified, terms such as "comprising" and "including" have an open and inclusive meaning and can be interpreted as "including but not limited to" or "non-restrictive inclusion".

[0051] In this invention, numerical intervals can be marked with a hyphen (e.g., 1-6), a wavy line (e.g., 1~6), or "to / to" (e.g., 1 to 6). Unless otherwise specified, regardless of whether the endpoints of a numerical interval are represented by integers, non-integers, percentages, fractions, or other types of numerical values, the numerical interval itself is a group consisting of two endpoints and any type of numerical value between the endpoints. For example, "1-3" represents a group consisting of 1, 3, and any value greater than 1 and less than 3, while "integers in 1-3" represents a group consisting of 1, 2, and 3. Unless otherwise specified, the average can be an integer or a non-integer. Unless otherwise specified, the number of functional groups is assumed to be an integer; for example, -(CH2). 1-4 - indicates a group consisting of -CH2-, -(CH2)2-, -(CH2)3-, and -(CH2)4-; for example, express The group that makes up the group.

[0052] In this invention, for polymer molecular weight, "about" or "around" generally refers to a numerical range of ±10%, which may be increased to ±15% in some cases, but not exceeding ±20%. For example, "about 10 kDa" generally refers to 9–11 kDa, and in some cases it may refer to 8.5–11.5 kDa, but not exceeding 8–12 kDa. As another example, when the molecular weight of a certain PEG component is about 5 kDa, its molecular weight is allowed to be selected from 5 kDa ±10%, i.e., 4500–5500 Da.

[0053] 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%, i.e., the range of 0.5% to 1.5%, and approximately 2.2% refers to 2.2 ± 0.05%, i.e., the range of 2.15% to 2.25% (where 2.2 is accurate to 0.1, and "approximately" refers to ±0.5*0.1, i.e., ±0.05).

[0054] In this invention, "heteroatoms" refers to atoms other than carbon and hydrogen in an organic compound, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0055] In this invention, the term "residue" refers to a group formed after a compound loses some atoms or groups.

[0056] In this invention, the term "linking group" refers to a group having two or more connecting ends. Unless otherwise specified, a linking group may be connected to other groups using any of its connecting ends, subject to the successful implementation of this invention. For example, when L in -CH2-L-CH2CH2- is -C(=O)O-, the specific cases can be -CH2-C(=O)O-CH2CH2- or -CH2-OC(=O)-CH2CH2-. For certain specific divalent linking groups, the term "group" in their names can be used interchangeably with "bond," such as divalent amide groups and amide bonds, and divalent ether groups and ether bonds.

[0057] In this invention, the term "linking bond" does not contain any atoms; it only serves a connecting function. Linking bonds in chemical structures can be... To mark, such as This represents a monovalent group -(CH2)4CH3. Specifically, when the definition of a group includes "linking bond," it means that the group may not exist and only serves a linking function.

[0058] In this invention, the range of carbon atoms in a group can be indicated by the subscript "C", 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 1-10 carbon atoms, i.e., C1, C2, C3, C4, C5, C6, C7, C8, C9 ... 10 Any of the alkylene groups, unless otherwise specified, may have a straight-chain structure, a branched structure, or a cyclic structure.

[0059] In this invention, the term "substituted" is used to describe a group or compound in the presence of at least one hydrogen atom replaced by a substituent, and unless otherwise specified, there are no particular limitations on the type, number, and atomic number of the substituent.

[0060] In this invention, the term "functional group" refers to a substituent or part that determines the chemical properties of an organic compound and can initiate a characteristic chemical reaction of the molecule. In this invention, "functionalization" refers to the process of obtaining a compound with a specific functional group through a chemical reaction. Compounds modified by functionalization are also called "functionalized derivatives." Compounds containing functional groups are also called "functionalized compounds." Multifunctional compounds contain at least three functional groups.

[0061] In this invention, the term "amide" refers to a compound with the general formula RC(=O)-N(R')2, where R is an organic group, each R' is independently an H or an organic group, and when R or R' is an organic group, its carbon atom is attached to (C=O) or N in the general formula. Based on the different R's, amides can be classified into primary amides, secondary amides, and tertiary amides. In primary amides, both R's are H; in secondary amides, one R' is H and the other R' is an organic group; and in tertiary amides, both R's are organic groups. Secondary amides are also called N-substituted amides, and tertiary amides are also called N,N-disubstituted amides.

[0062] In this invention, the term "amide group" refers to a monovalent, divalent, or trivalent group formed by the loss of some atoms or groups from an amide. For example, -C(=O)NH- is a divalent amide group, and -C(=O)N< is a trivalent amide group. Unless otherwise specified, the "amide bond" in this invention refers to -C(=O)NH- by default.

[0063] In this invention, the term "sulfonate group" refers to a group having the general formula RS(=O)2O-, where R is an organic group and its carbon atom is attached to S in the general formula. The sulfonate group in this invention includes, but is not limited to, methanesulfonate group, benzenesulfonate group, and p-toluenesulfonate group.

[0064] In this invention, the term "active ester group" refers to an ester group with high reactivity, including but not limited to the active ester group disclosed in paragraph

[0355] of CN110591079B.

[0065] In this invention, polyethylene glycol (PEG) and its derivatives refer to those containing -(CH2CH2O). n - A compound consisting of a chain segment; where n is the degree of polymerization, referring to the number of repeating units (-CH2CH2O-). The molecular weight of the polymer defaults to the average molecular weight, and the default exponential average molecular weight (M0.05) is the exponential molecular weight. nThe molecular weight of polyethylene glycol chains can also be measured by the degree of polymerization. Unless otherwise specified, the degree of polymerization refers to the average degree of polymerization, which is the number-average degree of polymerization by default.

[0066] In this invention, unless otherwise specified, any compound represented by a general formula includes its salt (preferably a pharmaceutically acceptable salt), tautomer, stereoisomer, and solvate.

[0067] In this invention, "tautomer" refers to the isomers obtained by tautomerism of a compound. Tautomerism refers to the process by which the structure of a compound changes due to the transfer of hydrogen atoms or protons within the molecule, often involving the conversion of single and double bonds. The tautomers of this invention include, but are not limited to, keto-enol tautomerism, amide-imine tautomerism, lactam-lactamimide tautomerism, enamine-imide tautomerism, enamine-enamine tautomerism (e.g., the enzyme reaction catalyzed by pyridoxal phosphate), proton transfer tautomerism, and valence tautomerism.

[0068] In this invention, the term "salt" 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 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 may be formed by reacting the compound itself with a certain amount (such as equivalents) of an acid or base in a medium such as salt precipitation or in an aqueous medium, followed by freeze-drying. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gluconate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, etc. Exemplary base addition salts include ammonium salts, alkali metal salts (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 using reagents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and dipentyl sulfates), long-chain halides (e.g., decyl, lauryl, tetradecyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others. In this invention, both acid addition salts and base addition salts of compounds represented by any of the general formulas are preferably pharmaceutically acceptable salts and, for the purposes of this disclosure, are considered equivalent to the free form of the compound.

[0069] In this invention, "stereoisomer" refers to isomers with the same atomic connection sequence but different three-dimensional structures. Stereoisomers can be divided into enantiomers and diastereomers, among which diastereomers include cis-trans isomers and conformational isomers. Enantiomers are molecules that are mirror images of each other and cannot be superimposed.

[0070] In this invention, a "solvent" refers to an aggregate formed by the combination of one or more compound molecules and one or more solvent molecules. The solvent can be water, and the corresponding solvate can be called a hydrate, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. The solvent can also be an organic solvent.

[0071] In this invention, the source of amino acids is not particularly limited unless otherwise specified; they can be natural, non-natural, or a mixture of both. The structural type of amino acids is also not particularly limited unless otherwise specified; they can be L-type, D-type, or a mixture of both.

[0072] In this invention, the definitions and examples of the skeletons of amino acids and their derivatives 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 residues.

[0073] In this invention, the term "biologically related substances" includes, but is 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, 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 also include their precursors, activated states, derivatives, isomers, mutants, analogs, mimics, polymorphs, pharmaceutically acceptable salts, fusion proteins, chemically modified substances, recombinant substances, etc., and may also be corresponding agonists, activators, inhibitors, antagonists, regulators, receptors, ligands 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, including chemically modified products obtained by modifying reactive groups to change their type, and chemically modified products generated by introducing additional functional groups, reactive groups, amino acids or amino acid derivatives, polypeptides, etc., are all considered chemically modified substances of biorelated substances. Before or after binding to a functionalized compound (including polyethylene glycol derivatives), the bioassociated substance is permitted to form a modified or complex bioassociated substance with the bound target molecule, appendage, or delivery carrier. The pharmaceutically acceptable salt can be an inorganic salt, such as hydrochloride, sulfate, or phosphate, or an organic salt, such as oxalate, malate, or citrate.

[0074] In this invention, the preparation process typically requires selectively reacting only the target reactive groups. To prevent unrelated reactive groups from affecting the target reaction, these unrelated reactive groups are usually protected. "Protection" of a reactive group refers to a 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 of the hydroxyl group. The protecting group not only remains stable during the target reaction but can also be removed as needed using conventional techniques in the art. In this invention, the term "deprotection" refers to the process of converting a protected group into its unprotected form.

[0075] In this invention, for polydisperse polymers, the molecular weight is allowed to be within ±10% of a given value, and in some cases may be increased to ±15% but not exceeding ±20%. For example, if the molecular weight of a polydisperse polymer is 5 kDa, then the molecular weight of its individual molecules and macroscopic components is allowed to be in the range of 4500-5500 Da, and the content or purity of the polymer is calculated based on the components with an average molecular weight between 4500-5500 Da.

[0076] In this invention, for monodisperse polymers, when the degree of polymerization is greater than or equal to 10, the allowable deviation of its molecular weight or degree of polymerization is no more than ±5%; when the degree of polymerization is less than 10, the allowable deviation of its degree of polymerization is no more than ±0.5%. When the content of the component meeting the aforementioned requirements reaches a certain percentage (preferably greater than or equal to 90%, more preferably greater than 95%, more preferably greater than 96%, more preferably greater than 98%, more preferably 99-100%), it is considered that a monodisperse macroscopic product of the target component has been obtained. Even if the above-mentioned content ratio is not reached, as long as the preparation method of this invention or a similar method with a basically the same preparation idea is used, the resulting product with insufficient content or the component appearing in the form of a main product, co-product, or by-product, regardless of whether separation and purification are performed, is within the scope of this invention.

[0077] In this invention, a polymer with a polydispersity index (PDI) of no more than 1.005 is considered to have monodispersity, which can be denoted as PDI = 1; a polymer with a PDI of more than 1.005 is considered to have polydispersity.

[0078] In this invention, the default unit for molecular weight is Dalton (Da).

[0079] In this invention, the term "drug" 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 the types of "drugs," including but not limited to drugs, vaccines, antibodies, vitamins, foods, food additives, nutritional supplements, nutritional health products, and other agents that provide beneficial effects. There are no particular limitations on the scope of the physiological or pharmacological effects produced by the "drug" in vivo; it can be systemic or local. There are no particular limitations on the activity of the "drug," which is primarily an active substance capable of interacting with other substances, but can also be an inert substance that does not interact; wherein, an inert drug can be converted into its active form through in vivo action or certain stimulation. "Small molecule drug" refers to a biologically relevant substance or its small molecule mimic or active fragment with a molecular weight not exceeding 1000 Da.

[0080] 2. Compounds of general formula (1)

[0081] In this invention, the preparation method of the compound represented by general formula (1) includes the following reaction:

[0082]

[0083] Among them, the compound represented by the general formula (P-amide) is a linear polyethylene glycol derivative containing an amide bond, and the compound represented by the general formula (BSM) is a bifunctional small molecule compound.

[0084] T1 and T2 are each independently C 1-20 Hydroxyl groups;

[0085] n1 and n2 are the degrees of polymerization of the polyethylene glycol chain, selected from integers from 8 to 1000;

[0086] L1, L2, and L3 are each independently an alkylene group or a divalent linker formed by combining an alkylene group with a heteroatom-containing group;

[0087] W is a group that can react with the amide bond in the general formula (P-amide) to form a trivalent nitrogen branched core;

[0088] L0 is a linking bond or divalent linker formed by the reaction of W with the amide bond in the general formula (P-amide);

[0089] F1 is -COOH, -COONa, or -COOK;

[0090] The reaction is carried out under strongly alkaline or moderately alkaline conditions;

[0091] The compounds represented by general formula (P-amide) and general formula (1) are both monodisperse or polydisperse.

[0092] In one specific embodiment of the present invention, T1 and T2 are each independently C 1-10 The hydrocarbon oxy group is preferably any one of methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and benzyloxy, and more preferably methoxy.

[0093] In one specific embodiment of the present invention, n1 and n2 are each independently an integer from 20 to 700, preferably an integer from 50 to 500.

[0094] In one specific embodiment of the present invention, L1 and L2 are each independently a-(CH2). p -Z-(CH2) q -b or aZ-(CH2) q -b, L3 is a-(CH2) p -Z-(CH2) q -b; where a and b are both linkage bonds; in the general formula (P-amide), a of L1 is linked to a polyethylene glycol segment, b of L1 is linked to -NH-, a of L2 is linked to a polyethylene glycol segment, b of L2 is linked to -C(=O)-, a of L3 is linked to W, and b of L3 is linked to F1; where p and q are each independently selected from integers from 1 to 10; Z is a linkage bond or a divalent linkage group that does not participate in the reaction; when Z is not a linkage bond, it is preferably any one of -C(=O)-, -C(=S), -O-, -S-, -C(=O)O-, -OC(=O)-, -SC(=O)- and -C(=O)S-; preferably, L1 is -CH2CH2-; preferably, L2 is -CH2-; preferably, L3 is -CH2-, -CH2CH2- or -CH2CH2CH2-.

[0095] In one specific embodiment of the present invention, W is a halogen group or a sulfonate group, selected from any one of -F, -Cl, -Br, -I, alkyl sulfonate group and aryl sulfonate group, preferably -Br.

[0096] In one specific embodiment of the present invention, L0 is a connecting key or C 1-6 Alkylene, preferably a linking bond or a methylene bond, more preferably a linking bond.

[0097] In one specific embodiment of the present invention, the base used in the aforementioned strongly alkaline or moderately alkaline conditions is an organic or inorganic base, selected from any one of sodium tert-butoxide, potassium tert-butoxide, triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium monohydrogen phosphate, and potassium monohydrogen phosphate; wherein, sodium hydride, potassium hydride, sodium hydroxide, or potassium hydroxide is preferably used in strongly alkaline conditions, and triethylamine, trisodium phosphate, or tripotassium phosphate is preferably used in moderately alkaline conditions.

[0098] In one specific embodiment of the present invention, the solvent used in the aforementioned reaction for preparing the compound of general formula (1) is selected from any one of water, methanol, ethanol, benzene, toluene, xylene, diethyl ether, petroleum ether, tetrahydrofuran, dioxane, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, 1,4-dioxane, acetonitrile, dimethyl sulfoxide, and ethyl acetate, or a mixed solvent containing any two or three of the aforementioned solvents;

[0099] When the reaction is carried out under strongly alkaline conditions, the solvent used is preferably toluene, dichloromethane or tetrahydrofuran, more preferably toluene or dichloromethane, and most preferably toluene;

[0100] When the reaction is carried out under moderately alkaline conditions, the solvent used is preferably toluene, dichloromethane, tetrahydrofuran or acetonitrile, more preferably dichloromethane or tetrahydrofuran, and most preferably tetrahydrofuran.

[0101] In one specific embodiment of the present invention, the reaction temperature for preparing the compound represented by general formula (1) is selected from 20-130°C; when the reaction is carried out under strongly alkaline conditions, the reaction temperature is preferably 60-100°C, more preferably 80°C; when the reaction is carried out under moderately alkaline conditions, the reaction temperature is preferably 30-70°C, more preferably 50°C.

[0102] In one specific embodiment of the present invention, the reaction time for preparing the compound represented by general formula (1) is 4-24 hours, preferably 10-24 hours.

[0103] In one specific embodiment of the present invention, the aforementioned reaction for preparing the compound represented by general formula (1) further utilizes a phase transfer catalyst; the phase transfer catalyst is a quaternary ammonium salt, a quaternary phosphonium salt, a sulfonium salt, or an arsenic salt, preferably a quaternary ammonium salt, more preferably tetrabutylammonium fluoride or tetrabutylammonium bromide; in a more specific embodiment of the present invention, when the reaction is carried out under strongly alkaline conditions, the molar ratio of the compound represented by general formula (P-amide), the compound represented by general formula (BSM), the base, and the phase transfer catalyst is 1:(10-30):(20-70):(5-15), preferably 1:20:(40-50):10; when the reaction is carried out under moderately alkaline conditions, the molar ratio of the compound represented by general formula (P-amide), the compound represented by general formula (BSM), the base, and the phase transfer catalyst is 1:(10-30):(20-70):(30-50), preferably 1:20:(40-50):40.

[0104] In one specific embodiment of the present invention, the method for preparing the compound represented by the aforementioned general formula (1) further includes reacting a linear polyethylene glycol amine derivative and a linear polyethylene glycol carboxylic acid derivative to obtain the compound represented by the general formula (P-amide), wherein the reaction is as follows:

[0105]

[0106] The linear polyethylene glycol amine derivatives and linear polyethylene glycol carboxylic acid derivatives in this reaction are both monodisperse or polydisperse.

[0107] In one specific embodiment of the present invention, the number average molecular weight of the compound represented by general formula (1) is 1-100 kDa, preferably 2-50 kDa, more preferably 10-50 kDa, and most preferably 10 kDa, 20 kDa, 30 kDa, 40 kDa or 50 kDa.

[0108] In one specific embodiment of the present invention, the structure of the compound represented by general formula (1) is selected from any of the following:

[0109]

[0110]

[0111] 3. Compounds of general formula (2)

[0112] In this invention, the preparation method of the compound represented by general formula (2) includes the following two steps:

[0113] Step 1: Obtain the compound shown in general formula (1) by any of the aforementioned methods;

[0114] Step 2: Perform one or more terminal functionalization modifications on the compound shown in general formula (1) to obtain the compound shown in general formula (2):

[0115]

[0116] In general formula (2), T1, T2, n1, n2, L1, L2, L3, L0, and F1 are the same as those in general formula (1);

[0117] L4 is a linker formed after the terminal functionalization modification, connecting the trivalent nitrogen branched nucleus and F2;

[0118] k is an integer between 1 and 100;

[0119] F2 is a hydrogen atom or a functional group that can react with biologically relevant substances, or its protected form.

[0120] In one specific embodiment of the present invention, F2 is any one of the following groups or their protected forms: epoxy group, 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; F2 is preferably any one of the following structures:

[0121]

[0122]

[0123] In one specific embodiment of the present invention, L4 is a divalent or trivalent linker, selected from -CH2C(=O)-Y- or -CH2C(=O)-Y-(CH2). j -、-CH2C(=O)-Y-(CH2) j -M-、-CH2C(=O)-Y-(CH2) j -M-(CH2) j -or-CH2C(=O)-NH-A<; where,

[0124] Y is either -O- or -NH-;

[0125] M can be any one of -O-, -S-, -SS-, -C(=O)O-, -OC(=O-, and -OC(=O)O-;

[0126] Each j is an independent integer from 1 to 10, preferably 1, 2, 3, 4 or 5;

[0127] A represents a trivalent residue of an amino acid, which is the residue obtained after the amino acid loses all its amino, carboxyl, hydroxyl, and thiol groups.

[0128] Preferably, L4 is -CH2C(=O)O-, -CH2C(=O)OCH2CH2-, -CH2C(=O)NHCH2CH2-, -CH2C(=O)OCH2CH2OC(=O)CH2CH2-, Any one of them.

[0129] In one specific embodiment of the present invention, the aforementioned k is 1 or 2, preferably 1.

[0130] In one specific embodiment of the present invention, the aforementioned terminal functionalization modification is performed once, twice consecutively, or three times consecutively.

[0131] In one specific embodiment of the present invention, each terminal functionalization modification is a micro-modification or a small molecule modification; the micro-modification is selected from any one of protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, acidification, and basification; the small molecule modification is selected from alkylation reaction, amidation reaction, or esterification reaction, and the small molecule is a bifunctional or multifunctional small molecule.

[0132] In one specific embodiment of the present invention, the structure of the compound represented by general formula (2) is selected from any of the following:

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] 4. Compounds of nitrogen-branched two-arm polyethylene glycol derivatives with biorelated substances

[0139] In this invention, the method for preparing the combination of nitrogen-branched two-arm polyethylene glycol derivative and biologically related substances includes preparing the nitrogen-branched two-arm polyethylene glycol derivative by any of the aforementioned methods.

[0140] In one specific embodiment of the present invention, the biologically related substance is 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, and micelles; preferably, the biologically related substance is selected from any one of small molecule drugs, nucleic acids, steroids, phospholipids, and glycolipids; more preferably, the biologically related substance is selected from any one of small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, and steroidal compounds; wherein, the small molecule drug is preferably selected from any one of flavonoids, terpenoids, carotenoids, saponins, steroids, quinones, anthraquinones, fluoroquinones, coumarins, alkaloids, porphyrins, polyphenols, macrolides, monocyclic alkyl groups, phenylpropionic acids, anthracyclines, and aminoglycosides.

[0141] In one specific embodiment of the present invention, the biologically related substance is a genetically engineered drug selected from any one of antibodies, antibody fragments, interleukins, lysozymes, interferons, auxins, erythropoietin, and granulocyte colony-stimulating factors; the interferon is preferably α-, β-, or γ-interferon.

[0142] In one specific embodiment of the present invention, the method for preparing the aforementioned combination of nitrogen-branched two-arm polyethylene glycol derivative and biorelated substances includes the step of reacting the terminal reactive groups of the nitrogen-branched two-arm polyethylene glycol derivative with biorelated substances to generate covalent linkages. Detailed Implementation

[0143] The preparation method of this invention includes any protection and deprotection processes required for the reaction. 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, thin-film dialysis, supercritical extraction, and column chromatography. The structure and molecular weight of the final product can be characterized using methods including but not limited to NMR, electrophoresis, UV-Vis spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, and circular dichroism spectroscopy. In this invention, it is preferred to use... 1 The structure was confirmed by 1H-NMR, and the number-average molecular weight (Mn) was measured by gel permeation chromatography (GPC). n The polydispersity index (PDI) and the terminal substitution rate were determined by high-performance liquid chromatography (HPLC). The preparation of nitrogen-branched two-arm polyethylene glycol derivatives is described below with reference to specific examples. These examples are for further detailed explanation of the invention and are not intended to limit the scope of protection of the invention.

[0144] Example 1: Preparation of nitrogen-branched two-arm polyethylene glycol carboxylic acid derivatives

[0145] Example 1.1: Preparation of nitrogen-branched two-arm polyethylene glycol acetic acid derivatives

[0146] The preparation method of nitrogen-branched two-arm polyethylene glycol acetic acid derivative E1-1 is as follows:

[0147] Method 1:

[0148]

[0149] Step a: Under a nitrogen atmosphere, methoxy polyethylene glycol ethylamine derivative S1-1 (20.00 g, 1.0 mmol, M) was added. n =20kDa, PDI=1.01), methoxy polyethylene glycol acetic acid derivative S1-2 (20.00g, 1.0mmol, M n =20 kDa, PDI=1.01), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl, 0.96 g, 5.0 mmol), 1-hydroxybenzotriazole (HOBt, 0.68 g, 5.0 mmol), and triethylamine (TEA, 0.81 g, 8.0 mmol) were sequentially dissolved in 200 mL of dichloromethane (DCM) under ice bath conditions. The mixture was then brought to room temperature and stirred for 24 h. After the reaction was complete, the solution was concentrated, and 200 mL of water was added. The solution was washed with ethyl acetate (100 mL x 2). The aqueous phase was extracted with dichloromethane (100 mL x 2). The organic phases were combined, concentrated, and recrystallized to obtain S1-3 (30.88 g). 1 H NMR (400MHz, (CD3)2SO) δ: 7.63(t,1H,-C(=O)NH-), 3.87(s,2H,-OCH2C(=O)NH-), 3.74-3.37(m,PEG,-O(CH2)2NHC(=O)-), 3.24(s,6H,-OCH3).

[0150] Step b: The above-mentioned S1-3 (20.00 g, 0.5 mmol), sodium bromoacetate (S1-4, 1.61 g, 10.0 mmol), sodium hydride (NaH, 60%; 1.00 g, 25.0 mmol), and tetrabutylammonium bromide (TBAB, 1.61 g, 5.0 mmol) were added to a three-necked flask containing 200 mL of toluene (Tol). The three-necked flask was equipped with a mechanical stirrer, a thermometer, and a reflux condenser. The mixture was heated to 80 °C and stirred under reflux to carry out the reaction. After reacting overnight, the reaction solution was cooled to room temperature, acidified with hydrochloric acid (6N) to pH = 3, and then extracted twice with dichloromethane (100 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by ion exchange chromatography to obtain E1-1 (6.91 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 4.26–3.94 (m, 4H, -OCH₂C(=O)N<, >NCH₂COOH), 3.70–3.36 (m, PEG, -OCH₂CH₂N<), 3.24 (s, 6H, -OCH₃). Molecular weight M was determined by GPC. n =40.1 kDa, PDI = 1.01. Terminal modification rate is 100%.

[0151] Method 2:

[0152]

[0153] Add S1-3 (20.00 g, 0.5 mmol, M) from Method 1 above to a reaction flask equipped with a magnetic stir bar and 200 mL tetrahydrofuran (THF). n =40.0 kDa, PDI = 1.01), bromoacetic acid (S1-5, 1.39 g, 10.0 mmol), potassium phosphate (K3PO4, 4.24 g, 20.0 mmol), and tetrabutylammonium bromide (TBAB, 6.44 g, 20.0 mmol). The reaction flask was capped and sealed with film, and the reaction was stirred at 50 °C for 24 hours. After the reaction was completed, the mixture was concentrated, 200 mL of water was added, and the mixture was washed with ethyl acetate (100 mL * 2). The aqueous phase was extracted with dichloromethane (100 mL * 2). The organic phases were combined, concentrated under reduced pressure, and purified by ion exchange chromatography to obtain the nitrogen-branched two-arm polyethylene glycol acetic acid derivative E1-1 (6.17 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 4.29–3.96 (m, 4H, -OCH₂C(=O)N<, >NCH₂COOH), 3.72–3.35 (m, PEG, -OCH₂CH₂N<), 3.23 (s, 6H, -OCH₃). Molecular weight M was determined by GPC. n=40.1 kDa, PDI = 1.01. Terminal modification rate is 100%.

[0154] Example 1.2: Preparation of nitrogen-branched two-arm polyethylene glycol acetate derivatives

[0155] Using method 1 of Example 1.1, the acidification treatment in step b was replaced with extraction under alkaline conditions to obtain the sodium salt form E1-2 of E1-1, with the following structure:

[0156]

[0157] Example 1.3: Preparation of nitrogen-branched two-arm polyethylene glycol acetic acid derivatives

[0158] Using either method one or method two of Example 1.1, the linear monofunctional polyethylene glycol derivative raw materials S1-1 and S1-2 were simultaneously replaced with corresponding structures having a number average molecular weight of approximately 5 kDa or 10 kDa, while keeping other conditions unchanged. This yielded nitrogen-branched two-arm polyethylene glycol carboxylic acid derivatives with the same structural formula as E1-1 and number average molecular weights of 10.1 kDa and 20.1 kDa, respectively, with an end-modification rate of 100% in both cases.

[0159] Example 2: Preparation of nitrogen-branched two-arm polyethylene glycol aldehyde derivatives

[0160] The compound of general formula (1) can be modified at the end to obtain the compound of general formula (2) with an aldehyde group as the terminal functional group. The end modification can be achieved by bifunctionalizing small molecules, such as S2-1 containing an amino group and an acetal group (the protected form of the aldehyde group).

[0161] The preparation method of nitrogen-branched two-arm polyethylene glycol propionaldehyde derivative E2-1 is as follows:

[0162]

[0163] Under a nitrogen atmosphere, E1-1 (20.05 g, 0.5 mmol), 3-aminopropionaldehyde diethyl acetal (S2-1, 1.47 g, 10.0 mmol), EDC HCl (5.76 g, 30.0 mmol), HOBt (4.05 g, 30.0 mmol), and TEA (4.55 g, 45.0 mmol) were sequentially dissolved in 200 mL of dichloromethane under an ice bath. The mixture was then brought to room temperature and stirred for 24 h. After the reaction was complete, the solution was concentrated, and 200 mL of water was added. The solution was washed with ethyl acetate (100 mL x 2). The pH of the aqueous phase was adjusted to 1.0 with 1 mol / L HCl under an ice bath. After reacting at room temperature for 4 hours, the solution was extracted twice with dichloromethane (100 mL x 2). The organic phases were combined, washed with saturated brine, dried, filtered, concentrated, and recrystallized from anhydrous isopropanol to give E2-1 (5.73 g).1 ¹H NMR (400 MHz, (CD₃)₂SO) δ: 4.26–3.85 (m, 4H, -OCH₂C(=O)N<, >NCH₂C(=O)NH⁻), 3.74–3.35 (m, PEG, -OCH₂CH₂N<, -C(=O)NHCH₂CH₂CHO), 3.23 (s, 6H, -OCH₃), 2.69–2.65 (m, 2H, -C(=O)NHCH₂CH₂CHO). Molecular weight M was determined by GPC. n =40.2kDa, PDI=1.01. Terminal modification rate: 99.1%.

[0164] Example 3: Preparation of nitrogen-branched two-arm polyethylene glycol succinimide ester derivatives

[0165] The compound of general formula (1) can be modified once to obtain the compound of general formula (2) with an active ester group at the end. The end modification can be achieved by a carboxyl activator, such as N-hydroxysuccinimide (NHS).

[0166] The preparation method of nitrogen-branched two-arm polyethylene glycol succinimide ester derivative E3-1 is as follows:

[0167]

[0168] E1-1 (4.01 g, 0.1 mmol) was dissolved in 40 mL of dichloromethane, and NHS (0.35 g, 3.0 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 0.62 g, 3.0 mmol) were added. 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 then recrystallized to obtain E3-1 (3.65 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 4.78–4.13 (m, 4H, -OCH₂C(=O)N<, >NCH₂CONHS), 3.70–3.38 (m, PEG, -OCH₂CH₂N<), 3.24 (s, 6H, -OCH₃), 2.86–2.78 (m, 4H, NHS). Molecular weight M was determined by GPC. n =40.2kDa, PDI=1.01. End-modification rate: 99.5%.

[0169] Example 4: Preparation of nitrogen-branched two-arm polyethylene glycol p-nitrobenzene ester derivatives

[0170] The compound shown in general formula (1) can be modified at the end twice to obtain two compounds shown in general formula (2), for example, by using a bifunctionalized small molecule to carry out two coupling reactions in succession.

[0171] The preparation method of nitrogen-branched two-arm polyethylene glycol p-nitrobenzene ester derivative E4-2 is as follows:

[0172]

[0173] Step a: Under a nitrogen atmosphere, E1-1 (8.02 g, 0.2 mmol), ethylene glycol (S4-1, 0.62 g, 10.0 mmol), 4-dimethylaminopyridine (DMAP, 0.24 g, 2.0 mmol), and TEA (0.81 g, 8.0 mmol) were dissolved sequentially in 80 mL of dichloromethane. A dichloromethane solution (20 mL) of EDC HCl (1.54 g, 8.0 mmol) was slowly added dropwise under an ice bath. The mixture was then brought to room temperature and stirred for 24 h. After the reaction was complete, the solution was concentrated, and 200 mL of water was added. The mixture was washed with ethyl acetate (100 mL * 2). The aqueous phase was extracted with dichloromethane (100 mL * 2). The organic phases were combined, concentrated, and recrystallized to obtain E4-1 (7.51 g), which also satisfies general formula (2). The molecular weight M was determined by GPC. n =40.2kDa, PDI=1.01. Terminal modification rate: 99.9%.

[0174] Step b: Under ice bath conditions, 10 mL of a dichloromethane solution containing p-nitrophenyl chloroformate (S4-2, 1.01 g, 5.0 mmol) was slowly added dropwise to a dichloromethane solution (40 mL) containing E4-1 (4.02 g, 0.1 mmol) and TEA (0.51 g, 5.0 mmol). After stirring for one hour, the mixture was brought to room temperature and the reaction was continued for 24 hours. After the reaction was completed, the mixture was extracted, concentrated, and recrystallized to obtain E4-2 (3.49 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 8.28 (d, 4H, Ar), 7.38 (d, 4H, Ar), 4.47–4.38 (m, 4H, -C(=O)OCH₂CH₂OC(=O)O-), 4.27–3.92 (m, 4H, -OCH₂C(=O)N<, >NCH₂COO-), 3.74–3.36 (m, PEG, -OCH₂CH₂N<), 3.24 (s, 6H, -OCH₃). Molecular weight M was determined by GPC. n =40.3kDa, PDI=1.01. End-modification rate: 99.8%.

[0175] Example 5: Preparation of nitrogen-branched two-arm polyethylene glycol amine derivatives

[0176] The preparation method of nitrogen-branched two-arm polyethylene glycol propylamine derivative E5-1 is as follows:

[0177] First, a nitrogen-branched two-arm polyethylene glycol derivative was synthesized, and then the end was modified to obtain another nitrogen-branched two-arm polyethylene glycol derivative with a different end group structure, corresponding to the preparation method of the compound shown in general formula (2).

[0178]

[0179] E1-1 (4.01 g, 0.1 mmol) and ethylenediamine containing a Boc-protected amino group (S5-1, 0.48 g, 3.0 mmol) were dissolved in 40 mL of dichloromethane. N,N-diisopropylethylamine (DIPEA, 0.65 g, 5.0 mmol), HOBt (0.41 g, 3.0 mmol), and EDC HCl (0.58 g, 3.0 mmol) were added sequentially under ice bath conditions. The mixture was brought to room temperature and stirred overnight. After the reaction was complete, the sample was washed sequentially with saturated sodium bicarbonate solution (10 mL x 2), water (10 mL x 2), and brine (10 mL x 2). The solution was concentrated under reduced pressure, and the Boc protection was removed with a TFA / DCM mixture (1:1 v / v). The sample was then washed with purified water and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and recrystallized to obtain E5-1 (3.33 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 4.30–3.86 (m, 4H, -OCH₂C(=O)N<, >NCH₂C(=O)NH₃), 3.74–3.33 (m, PEG, -OCH₂CH₂N<, -C(=O)CH₂CH₂NH₂), 3.24 (s, 6H, -OCH₃), 2.84 (t, 2H, -C(=O)CH₂CH₂NH₂). GPC determined the molecular weight M. n =40.2kDa, PDI=1.01. Terminal modification rate: 99.9%.

[0180] Example 6: Preparation of nitrogen-branched two-arm polyethylene glycol maleimide derivatives

[0181] The preparation method of nitrogen-branched two-arm polyethylene glycol maleimide derivative E6-1 is as follows:

[0182]

[0183] E4-1 (4.02 g, 0.1 mmol) was dissolved in 40 mL of anhydrous dichloromethane, and EDC HCl (0.77 g, 4.0 mmol) and TEA (0.40 g, 4.0 mmol) were added. DMAP (0.12 g, 1.0 mmol) was added to 15 mL of dichloromethane solution of 3-maleimide propionic acid (S6-1, 1.19 g, 5.0 mmol) containing a furan protecting group. The two solutions were mixed and reacted at room temperature for 24 hours. After the reaction was complete, 40 mL of water was added, and the mixture was washed with ethyl acetate (20 mL x 2). The aqueous phase was extracted with dichloromethane (20 mL x 2). The organic phases were combined, concentrated under reduced pressure, and 40 mL of toluene was added, followed by 0.40 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 protecting group. Precipitated with anhydrous diethyl ether and recrystallized from anhydrous isopropanol to give E6-1 (3.76 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 6.70 (s, 2H, Mal), 4.24–3.93 (m, 4H, -OCH₂C(=O)N<, >NCH₂COO-), 3.85 (t, 2H, -O(C=O)CH₂CH₂-Mal), 3.76–3.36 (m, PEG, -OCH₂CH₂N<), 3.24 (s, 6H, -OCH₃), 2.69 (t, 4H, -C(=O)OCH₂CH₂OC(=O)-), 2.54 (t, 2H, -O(C=O)CH₂CH₂-Mal). GPC determined the molecular weight M. n =40.3kDa, PDI=1.01. End-modification rate is 100%.

[0184] Example 7: Preparation of nitrogen-branched two-arm polyethylene glycol dicarboxylic acid derivative

[0185] By using trifunctionalized small molecules for terminal modification, compounds with terminal difunctionalized general formula (2) can be obtained.

[0186] The preparation method of nitrogen-branched two-arm polyethylene glycol dicarboxylic acid derivative E7-1 is as follows:

[0187]

[0188] E1-1 (4.01 g, 0.1 mmol) and glutamic acid (S7-1, 0.78 g, 3.0 mmol) containing two tBu protected carboxyl groups were dissolved in 40 mL of dichloromethane. DIPEA (0.65 g, 5.0 mmol) and DMAP (0.12 g, 1.0 mmol) were added. After stirring at room temperature for 5 min, EDC HCl (0.58 g, 3.0 mmol) was added, and the reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was washed successively with saturated sodium bicarbonate solution (10 mL x 2), water (10 mL x 2), and brine (10 mL x 2). The mixture was concentrated under reduced pressure, and the tBu protection was removed with a TFA / DCM mixture (1:1 v / v). The mixture was then washed with purified water and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and recrystallized to obtain E7-1 (3.69 g). 1 ¹H NMR (400MHz, (CD₃)₂SO) δ: 4.45–4.41 (m, 1H, -C(=O)NHCH<), 4.25–3.86 (m, 4H, -OCH₂C(=O)N<, >NCH₂C(=O)NH⁻), 3.75–3.35 (m, PEG, -OCH₂CH₂N<), 3.23 (s, 6H, -OCH₃), 2.41 (t, 2H, >CHCH₂CH₂COOH), 2.20–1.95 (m, 2H, >CHCH₂CH₂COOH). Molecular weight M was determined by GPC. n =40.2kDa, PDI=1.01. End-modification rate: 99.4%.

[0189] Comparative Example 1: Investigating the effect of different reaction solvents on the yield

[0190] The effect of the solvent used on the yield of E1-1 was investigated for the reactions S1-3 and S1-4 in step 2 of method 1 in Example 1, and the reactions S1-3 and S1-5 in method 2. The results are summarized in Table 1.

[0191] Table 1 Effect of solvent on E1-1 yield

[0192]

[0193] 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.

[0194] 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 process for the preparation of a nitrogen branched two-armed polyethylene glycol derivative, characterized in that, The nitrogen branched two-arm polyethylene glycol derivative is shown in general formula (1), and the method comprises the following reactions: The compound shown in general formula (P-amide) is a linear polyethylene glycol derivative containing an amide bond, and the compound shown in general formula (BSM) is a bifunctional small molecule compound; T1, T2are each independently C 1-20 hydrocarbyloxy; n1 and n2 are the polymerization degrees of the polyethylene glycol chain, and are selected from integers from 8 to 1000; L1, L2, and L3 are each independently an alkylene group or a divalent linking group formed by combination of an alkylene group and a heteroatom-containing group; W is a group capable of reacting with the amide bond in general formula (P-amide) and forming a trivalent nitrogen branched core; L0 is a linking bond or a divalent linking group formed after W reacts with the amide bond in general formula (P-amide); F1 is -COOH, -COONa, or -COOK; The reaction is carried out under strong alkaline conditions or moderate alkaline conditions; The compounds shown in general formula (P-amide) and general formula (1) are monodisperse or polydisperse.

2. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, T1, T2are each independently C 1-10 hydroxyl, preferably any one of hydroxyl, methoxyl, ethoxyl, propoxyl, butoxyl, pentoxy, hexyloxy and benzyloxy, more preferably methoxyl.

3. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, n1 and n2 are each independently an integer from 20 to 700, preferably an integer from 50 to 500.

4. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, L1, L2are each independently a-(CH2) p -Z-(CH2) q -b or a-Z-(CH2) q -b, L3is a-(CH2) p -Z-(CH2) q -b; wherein a and b are each a linking bond; in general formula (P-amide), a of L1 is connected to the polyethylene glycol segment, b of L1 is connected to -NH-, a of L2 is connected to the polyethylene glycol segment, b of L2 is connected to -C(=O)-, a of L3 is connected to W, and b of L3 is connected to F1; wherein p and q are each independently selected from integers from 1 to 10; Z is a linking bond or a divalent linking group that does not participate in the reaction; when Z is not a linking bond, it is preferably selected from any one of -C(=O)-, -C(=S)-, -O-, -S-, -C(=O)O-, -OC(=O)-, -SC(=O)-, and -C(=O)S-; Preferably, L1 is -CH2CH2-; preferably, L2 is -CH2-; and preferably, L3 is -CH2-, -CH2CH2-, or -CH2CH2CH2-.

5. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, W is a halogen group or a sulfonate group, and is selected from any one of -F, -Cl, -Br, -I, an alkyl sulfonate group, and an aryl sulfonate group, and is preferably -Br.

6. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, L0is a bond or C 1-6 alkylene, preferably a bond or methylene, more preferably a bond.

7. The method for preparing nitrogen-branched two-arm polyethylene glycol derivatives according to claim 1, characterized by, The base used in the strong alkaline conditions or the moderate alkaline conditions is an organic base or an inorganic base, and is selected from any one of sodium tert-butoxide, potassium tert-butoxide, triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, diisopropylethylamine, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, monosodium hydrogen phosphate, and monopotassium hydrogen phosphate; wherein sodium hydride, potassium hydride, sodium hydroxide, or potassium hydroxide is preferably used in the strong alkaline conditions, and triethylamine, trisodium phosphate, or tripotassium phosphate is preferably used in the moderate alkaline conditions.

8. The method for preparing nitrogen-branched two-arm polyethylene glycol derivatives according to claim 1, characterized by, The solvent used in the reaction is selected from any one of water, methanol, ethanol, benzene, toluene, xylene, diethyl ether, petroleum ether, tetrahydrofuran, dioxane, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, 1,4-dioxane, acetonitrile, dimethyl sulfoxide, ethyl acetate, or a mixed solvent containing any two or three of the aforementioned solvents; When the reaction is carried out under strong alkaline conditions, the solvent used is preferably toluene, dichloromethane or tetrahydrofuran, more preferably toluene or dichloromethane, and most preferably toluene; When the reaction is carried out under moderate alkaline conditions, the solvent used is preferably toluene, dichloromethane, tetrahydrofuran or acetonitrile, more preferably dichloromethane or tetrahydrofuran, and most preferably tetrahydrofuran.

9. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, The reaction temperature is selected from 20-130℃; when the reaction is carried out under strong alkaline conditions, the reaction temperature is preferably 60-100℃, and more preferably 80℃; when the reaction is carried out under moderate alkaline conditions, the reaction temperature is preferably 30-70℃, and more preferably 50℃.

10. The method for preparing nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized by, The reaction time is 4-24 hours, and preferably 10-24 hours.

11. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The reaction also uses a phase transfer catalyst; the phase transfer catalyst is a quaternary ammonium salt, a quaternary phosphonium salt, a sulfonium salt or an arsenic salt, preferably a quaternary ammonium salt, and more preferably tetrabutylammonium fluoride or tetrabutylammonium bromide.

12. The method for preparing a nitrogen-branched two-arm polyethylene glycol derivative according to claim 11, characterized by, When the reaction is carried out under strong alkaline conditions, the molar ratio of the compound represented by general formula (P-amide), the compound represented by general formula (BSM), the base and the phase transfer catalyst is 1:(10-30):(20-70):(5-15), and preferably 1:20:(40-50):10; when the reaction is carried out under moderate alkaline conditions, the molar ratio of the compound represented by general formula (P-amide), the compound represented by general formula (BSM), the base and the phase transfer catalyst is 1:(10-30):(20-70):(30-50), and preferably 1:20:(40-50):

40.

13. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The method further comprises reacting the linear polyethylene glycol amine derivative and the linear polyethylene glycol carboxylic acid derivative to obtain the compound represented by general formula (P-amide), and the reaction is as follows: Both the linear polyethylene glycol amine derivative and the linear polyethylene glycol carboxylic acid derivative in this reaction are monodisperse or polydisperse.

14. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The number average molecular weight of the compound represented by general formula (1) is 1-100kDa, preferably 2-50kDa, more preferably 10-50kDa, and most preferably 10kDa, 20kDa, 30kDa, 40kDa or 50kDa.

15. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 1, characterized in that, The structure of the compound represented by general formula (1) is selected from any one of the following:

16. A method for preparing a nitrogen branched two-armed polyethylene glycol derivative, characterized by, The nitrogen-branching two-arm polyethylene glycol derivative is represented by general formula (2), and the method comprises the following two steps: Step one: obtaining the compound represented by general formula (1) by the preparation method of any one of claims 1-15; Step two: performing one or more terminal functionalization modifications on the compound represented by general formula (1) to obtain the compound represented by general formula (2): T1, T2, n1, n2, L1, L2, L3, L0 and F1 in general formula (2) are the same as in general formula (1); L4 is a linking group formed after the terminal functionalization modification, which links the trivalent nitrogen-branching core and F2; k is an integer from 1 to 100; F2 is a hydrogen atom or a functional group capable of reacting with a biorelated substance or a protected form thereof.

17. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, F2 is any one of an epoxy group, a hydroxyl group, a thiol group, a carboxyl group, an amino group, an aldehyde group, an active ester group, an active carbonate group, a carbamate group, an isocyanate group, an isothiocyanate group, a succinimidyl group, a maleimido group, an alkenyl group, an alkynyl group, an alkenoate group, an azido group, a cyano group, a dithiopyridyl group, an alpha-haloacetyl alkynyl group, a folate group, a rhodamine group, a biotin group, a monosaccharide group, and a polysaccharide group, or a protected form thereof; F2 is preferably any one of the following structures:

18. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, L4 is a divalent or trivalent linker, selected from -CH2C(=O)-Y- and -CH2C(=O)-Y-(CH2). j -、-CH2C(=O)-Y-(CH2) j -M-、-CH2C(=O)-Y-(CH2) j -M-(CH2) j -or-CH2C(=O)-NH-A<; where, Y is -O- or -NH-; M is any one of -O-, -S-, -S-S-, -C(=O)O-, -OC(=O)- and -OC(=O)O-; each j is independently an integer from 1 to 10, preferably 1, 2, 3, 4 or 5; A is a trivalent residue of an amino acid, which is a residue obtained by removing all amino, carboxyl, hydroxyl and mercapto groups from an amino acid; Preferably, L4is -CH2C(=0)0-, -CH2C(=0)OCH2CH2-, -CH2C(=0)NHCH2CH2-, -CH2C(=0)OCH2CH2OC(=0)CH2CH2-, any one of.

19. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, said k is 1 or 2, preferably 1.

20. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, the number of the end-functionalization modification is one, two or three in succession.

21. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, each end-functionalization modification is a micro-modification or a small molecule modification; the micro-modification is selected from any one of protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, acidification, basification; the small molecule modification is selected from an alkylation reaction, an amidation reaction or an esterification reaction, and the small molecule is a bifunctional or a multifunctional small molecule.

22. The method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to claim 16, characterized in that, the structure of the compound represented by general formula (2) is selected from any one of the following:

23. A method for producing a conjugate of a nitrogen- branched two-armed polyethylene glycol derivative and a biorelated substance, characterized by, the method for preparing the conjugate comprises using the method for preparing the nitrogen-branched two-arm polyethylene glycol derivative according to any one of claims 1-22 to prepare the nitrogen-branched two-arm polyethylene glycol derivative.

24. A process for preparing a conjugate of a nitrogen-branched two-arm polyethylene glycol derivative according to claim 23 and a biorelated substance, characterized by, the biorelated substance is selected from any one of a drug, a protein, a polypeptide, an oligopeptide, a protein mimetic, a fragment, an enzyme, an antigen, an antibody and a fragment thereof, a receptor, a gene-related substance aptamer, a polysaccharide, a proteoglycan, a glycoprotein, a lipid compound, a hormone, a vitamin, a vesicle, a liposome, a dye, a fluorescent substance, a targeting factor, a cytokine, a neurotransmitter, an extracellular matrix substance, a plant or animal extract, a virus, a vaccine, a cell, a micelle; preferably, the biorelated substance is selected from any one of a small molecule drug, a nucleic acid, a steroid, a phospholipid, a glycolipid; more preferably, the biorelated substance is selected from any one of a small molecule drug, a nucleoside, a nucleotide, an oligonucleotide, an antisense oligonucleotide, a polynucleotide, a steroid; wherein the small molecule drug is preferably selected from any one of a flavonoid, a terpenoid, a carotenoid, a saponin, a steroid, a steroidal, a quinone, an anthraquinone, a fluoroquinone, a coumarin, an alkaloid, a porphyrin, a polyphenol, a macrolide, a monobactam, a phenylpropanoid, an anthracycline, an aminoglycoside.

25. The method for producing a conjugate of a nitrogen-branched two-arm polyethylene glycol derivative and a biorelated substance according to claim 23, characterized by, the biorelated substance is a genetically engineered drug selected from any one of an antibody, an antibody fragment, an interleukin, a lysozyme, an interferon, a growth hormone, an erythropoietin and a granulocyte colony-stimulating factor; the interferon is preferably an α-, β- or γ-interferon.

26. The method for producing a conjugate of a nitrogen-branched two-arm polyethylene glycol derivative and a biorelated substance according to claim 23, characterized by, the method comprises the step of generating a covalent linkage by reacting the terminal reactive groups of the nitrogen-branched two-arm polyethylene glycol derivative with the biorelated substance.

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