Pegylated lipid modified with targeting ligand, related composition and use thereof

Abstract

Disclosed in the present invention is a PEGylated lipid modified with a targeting ligand as shown in formula (1), wherein each T is independently a residue of a monosaccharide or a derivative thereof, y is an integer from 1 to 4, X is a linking bond or a y+1-valent linking group, Ncore is a trivalent linking group having >N- as a branched core, R is a hydrophobic tail chain, P0 is a polyethylene glycol chain segment, and L0 and M are divalent linking groups. The targeting PEGylated lipid of the present invention significantly improves the in vitro and in vivo targeting effects of lipid nanoparticles, and can achieve more efficient delivery of liver-targeting drugs.

Description

Targeting ligand-modified pegylated lipids, related compositions and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of drug delivery, and particularly relates to a targeting ligand-modified pegylated lipid, a lipid composition comprising the same, a lipid drug composition and uses thereof. BACKGROUND

[0002] In clinical applications, many drugs, including nucleic acid drugs, cannot effectively reach the target site when used directly, thus cannot exert sufficient drug efficacy, and even can produce toxicity to cells or tissues other than the target site. Drugs with targeting property can significantly increase their concentration in target cells or tissues, while reducing systemic toxicity. However, most drugs do not have targeting property by themselves, and thus, targeting modification of drugs that need to exert effects at specific sites is an important means to improve their drug efficacy. In order to make drugs have targeting property, a targeting ligand can be directly covalently connected to a drug molecule, or a drug delivery carrier can be modified with a targeting ligand.

[0003] Liposomes are widely used for delivering nucleic acid drugs, genetic vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs and protein drugs, and especially, nanoscale liposomes (lipid nanoparticles) have become one of the most promising delivery technologies. Lipid nanoparticles without surface modification usually do not have sufficient stability in the body circulation, and especially when containing cationic lipids, they can interfere with or even destroy biological membranes in the body circulation. Adding an appropriate amount of pegylated lipids to the lipid composition can achieve pegylated modification of the lipid nanoparticles, in which the hydrophilic polyethylene glycol segment is located on the surface of the liposome or the lipid nanoparticle to form a hydration layer, which not only prevents particle aggregation and increases stability, but also reduces non-specific cell uptake during drug delivery and achieves stealth effect. However, pegylated modification also reduces the uptake of lipid nanoparticles by cells to some extent, making it difficult to deliver drug-loaded lipid nanoparticles to target cells. To solve this problem, the pegylated lipids can be modified with a targeting ligand, for example, CN117615753A and CN115484932A both report pegylated lipids with liver targeting property, in which the targeting group adopts N-acetylgalactosamine (GalNAc). GalNAc is a high-affinity monosaccharide ligand for asialoglycoprotein receptor (ASGPR), and ASGPR is mainly expressed on the surface of hepatocytes, so targeting modification with GalNAc can improve the targeting property to hepatocytes or liver.

[0004] Most of the researches on liver-targeting PEGylated lipids and related compositions focus on the targeting moieties and the linkers, but the specific structure of the lipid moiety is relatively less investigated. For example, the lipid moieties in the liver-targeting PEGylated lipids reported so far are basically carbon-branched structures. CN115515926A discloses a series of nitrogen-branched PEGylated lipids, but it is not clear whether they can be effective liver-targeting delivery materials by modification with monosaccharide targeting groups.

[0005] To solve the above problems, it is necessary to develop a new targeting ligand modified PEGylated lipid.

[0006] SUMMARY

[0007] The present application provides a new type of targeting ligand modified PEGylated lipid, especially a targeting PEGylated lipid with a nitrogen-branched structure of the lipid moiety, which can be applied to the preparation of lipid nanoparticles and drug delivery.

[0008] The present application provides the following embodiments:

[0009] A targeting ligand modified PEGylated lipid, characterized in that the structure is shown in general formula (1):

[0010] or a salt, tautomer, stereoisomer, deuterated product or solvate thereof;

[0011] wherein,

[0012] each T is independently a residue of a monosaccharide or a derivative thereof; preferably, each T is independently a residue of any one of galactose, galactosamine, glucose, glucosamine, mannose, mannosamine, fucose and fucosamine, or a derivative thereof;

[0013] y is 1, and X is a linking bond; or, y is an integer from 2 to 4, X is a (y+1)-valent linking group, and X is selected from any one of wherein any end of X is connected to M; each G3 is independently >CH-, >N- or a trivalent cyclic group; G4 is a tetravalent carbon atom or a tetravalent cyclic group; each L X is independently -B X -, -B X -Z X -B X - or -B X -Z X -B X -Z X -B X -, wherein each Bx is independently a linking bond or an optionally substituted C 1-6 hydrocarbylene group, and any B XNot simultaneously with two Z X Connected; each Z X Independently constitutes a divalent linker containing heteroatoms;

[0014] When y is an integer from 2 to 4, any two Ts are the same or different from each other, and any two L0s are the same or different from each other;

[0015] N core It is a trivalent linker with >N- as the branching core;

[0016] Each R is independently -B R -(Z R -B R ) r -E R , where r is an integer from 0 to 2; each B R Independently for connecting key or optional replacement of C 1-29 A hydrocarbon group, and any one of the B groups is a linking bond. R Not with two Zs at the same time R Connected; each Z R Independently a divalent linker containing heteroatoms; E R C is an optional replacement 1-30 Hydrocarbon group; two Rs are the same or different from each other; all Bs in each R R With E R The sum of the carbon chain lengths is an integer from 5 to 30 independently;

[0017] P0 is -(OCH2CH2) n - And its oxygen end is connected to M, where n is an integer from 1 to 250;

[0018] Each L0 is independently -B L -(Z L -B L ) j -, where j is an integer from 1 to 6; each B L Independently for connecting key or optional replacement of C 1-12 A hydrocarbon group, and any one of the B groups is a linking bond. L Not with two Zs at the same time L Connected; each Z L Independently a divalent linker containing heteroatoms; all B in each L0 L The sum of the carbon chain lengths is an integer from 2 to 24 independently;

[0019] M is -B M -(Z M -B M ) k -, where k is an integer from 1 to 3; each B M Independently for connecting key or optional replacement of C1-12 alkylene, and any one of B is a bond M are not simultaneously bonded to two Z M are not simultaneously bonded to two Z M are independently a heteroatom-containing divalent linker; all B in M M the sum of the carbon chain lengths of B is an integer from 2 to 24;

[0020] Z X , Z R , Z L , and Z M each occurrence is independently selected from the group consisting of -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and ; wherein each Y is independently O, S, or NR c ; each R c is independently a hydrogen atom or a C 1-12 alkyl group;

[0021] The pegylated lipid is monodisperse or polydisperse.

[0022] A method for preparing the aforementioned targeting ligand-modified pegylated lipid, comprising the following steps: (i) coupling reaction of the functionalized pegylated lipid with the targeting ligand; (ii) optionally, deprotection reaction.

[0023] A lipid composition comprising the aforementioned targeting ligand-modified pegylated lipid.

[0024] A lipid pharmaceutical composition comprising the aforementioned lipid composition, further comprising one or more than one pharmaceutically active ingredient.

[0025] A lipid pharmaceutical composition preparation comprising the aforementioned lipid pharmaceutical composition, further comprising a working solution.

[0026] In the present application, the nitrogen-branched pegylated lipid is covalently coupled with a ligand moiety having liver targeting property, to obtain a targeting pegylated lipid having one, two, three or four monosaccharide targeting groups (especially GalNAc). Compared with the targeting pegylated lipid containing a carbon-branched lipid moiety, the targeting pegylated lipid of the present application can significantly improve the in vitro and in vivo targeting effect of the lipid nanoparticle while achieving excellent biocompatibility and serum stability. In particular, the trivalent targeting pegylated lipid of the present application can achieve more efficient liver-targeted drug delivery. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the1H NMR spectrum of G3PL-01b01-OAc. 1 H NMR spectrum.

[0028] Figure 2 is a1H NMR spectrum of G3PL-01b01. 1 Figure 2 is a1H NMR spectrum of G3PL-01b01.

[0029] Figure 3 is a MALDI-TOF MS test result of G3PL-01b01-OAc.

[0030] Figure 4 is a MALDI-TOF MS test result of G3PL-01b01.

[0031] Figure 5 is a HPLC spectrum of G3PL-01b01-OAc.

[0032] Figure 6 is an in vivo targeting test result of LNP07 group (major organs from left to right are heart, liver, spleen, lung, kidney).

[0033] DETAILED DESCRIPTION

[0034] 1. Terminology

[0035] The disclosures of all patents and other publications cited herein are hereby incorporated by reference in their entireties. In the event of any conflict in terminology between the text herein and any document incorporated by reference herein, the text herein controls.

[0036] “Include,” “contain,” and like terms have a non-limiting meaning.

[0037] For objects that are not unique in specific form, the specific form of each occurrence is not particularly limited. For example, -B-(Z-B) x in which the number of Z and B (i.e., the number of occurrences) is x and x+1, respectively; wherein the specific form of each Z is independent of other Zs, and the specific form of each B is also independent of other Bs.

[0038] Chemical structures include, but are not limited to, compounds, groups, fragments, and the like.

[0039] A “combination” is composed of any two or more recited items, which are the same or different. The nature of the “combination” is subject to the routine understanding of one of skill in the art. A combination of linkers is still a linker; for example, a combination of alkylene and -O- includes, but is not limited to, -CH2CH2O-, -CH2OCH2CH2-, -CH2OCH2CH2O-, and the like; for example, a combination of two -S- is -S-S-. A combination of compounds is a composition, which by default does not change the chemical structure of each compound; for example, two or more lipids can constitute a lipid composition.

[0040] A "linking bond" contains no atoms and serves only to connect. When a linking bond has one end pointing into a ring structure, the linking bond can be drawn from any suitable atom of the ring. The ring structure includes, but is not limited to, monocyclic, bicyclic, and polycyclic structures.

[0041] A "linking group" refers to a group containing at least one atom and capable of connecting to two, three, or more moieties, corresponding to divalent, trivalent, and higher valent linking groups, respectively. Some linking groups correspond to specific types of bonds, e.g., -0- can be referred to as an ether group or ether bond, -OC(=0)- can be referred to as an ester group or ester bond, and -OC(=0)NH- can be referred to as a carbamate group or carbamate bond. A linking group can be connected to any adjacent moiety with either of its connecting ends, as long as the chemical structure is reasonable. For example, when connected between Group A and Group B as -NHC(=0)-, the specific connection can be Group A-C(=0)NH-Group B or Group A-NHC(=0)-Group B. Different connecting ends of a linking group can be distinguished by orientation or notation. For example, when the left end of -NHC(=0)- is connected to Group A and the right end is connected to Group B, the specific connection is Group A-NHC(=0)-Group B. When the right end of -NHC(=0)-* is connected to Group B, the specific connection is -NHC(=0)-Group B. When no connecting end is specified, -C(=0)NH- and -NHC(=0)- are considered equivalent, and this rule applies to all linking groups.

[0042] A group can be connected to other chemical structures by a linking bond. A group can contain one or more connecting ends, where a linking group contains at least two connecting ends. A connecting end and its corresponding linking bond can be represented by a dash, or by a label; for example, represents a group -G or G-, represents a linking group -G-.

[0043] When there is a discrepancy in the description of the same object, e.g., a structure does not match a name, the relevant technical solution should be selected or excluded with the purpose of smooth implementation.

[0044] A moiety between two groups or fragments is referred to as a "spacer". Accordingly, the distance between two groups or fragments can be expressed as "spacer length" (abbreviated as "spacer length"), which refers to the minimum number of atoms calculated along the continuous covalent bonds from one end of the spacer to the other end. For example, when a group A and a group B are connected by as a spacer, the corresponding spacer lengths are 6, 6, 7, respectively.

[0045] Numerical ranges can be expressed or implied using the short hand "from x to y," or by two numerals and a hyphen -, or by two numerals and a tilde ~. Numerical ranges are inclusive of their endpoints. The type of number is not limited, including but not limited to integers, non-integers, percentages, fractions, and the like. When not involving an average, numerical ranges indicating the number of groups of a moiety are by default composed of integers, e.g., -(CH2) 1-4 - represents the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, and the like

[0046] "Hydrocarbon" refers to a compound containing only carbon and hydrogen. Hydrocarbons that do not contain benzene rings or other aromatic rings are collectively referred to as "aliphatic hydrocarbons," and hydrocarbons that contain benzene rings or other aromatic rings are collectively referred to as "aromatic hydrocarbons." Aliphatic hydrocarbons can be further divided into alkanes, alkenes, and alkynes, where alkenes contain at least one carbon-carbon double bond and alkynes contain at least one carbon-carbon triple bond.

[0047] "Hydrocarbon group" refers to a monovalent, divalent, and higher valent group formed by the loss of one or more hydrogen atoms from a hydrocarbon compound. Unless otherwise specified, a hydrocarbon group not otherwise specified is a monovalent hydrocarbon group (i.e., hydrocarbyl group), including but not limited to alkyl, alkenyl, alkynyl, and the like. "Hydrocarbylene group" refers to a divalent hydrocarbon group formed by the loss of two hydrogen atoms from a hydrocarbon compound, including but not limited to alkylene, alkenylene, alkynylene, and the like.

[0048] A "substituted" chemical structure contains one or more substituents. A "substituted" structure is also referred to as a "substitution form" relative to an "unsubstituted" structure. A "substituent" can be a single atom or a polyatomic group, and unless otherwise specified, does not include hydrogen. For hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl) or hydrocarbon derivative residues (e.g., heteroalkyl, alkoxy, alkylacyl), substituents in substitution forms include but are not limited to common substituents in the art, such as =0, -OR", =NR", =N-OR", -NR"R", -SR", -F, -Cl, -Br, -I, -SiR"R"R", -C(=O)R", -OC(=O)R", -C(=O)OR", -OC(=O)OR", -C(=O)NR"R", -OC(=O)NR"R", -NR"C(=O)R", -NR"C(=O)NR"R", -NR"C(=O)OR", -CN, -NO2, and the like; where each R" is independently hydrogen, C 1-10 alkyl, or C 1-10 heteroalkyl; and optionally, any two R" together with the nitrogen atom to which they are both attached form a cyclic structure; for example, -NR"R" includes but is not limited to 1-pyrrolidinyl and 4-morpholinyl.​​

[0049] "Optional" means that the described condition or event can or can not occur. For example, "optionally substituted" is equivalent to "substituted or unsubstituted."

[0050] A numerical range recited herein is intended to include all sub-ranges of the same whole number, e.g., a range of 1 to 5 is intended to include 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 5, 3 to 5, etc. 1-12 "1 to 12 carbon atoms" means that the designated group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a range between any two of these values of carbon atoms in the chain. For example, "C1-12 alkyl" means that the alkyl group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a range between any two of these values of carbon atoms in the chain. 1-3 "Substituted alkyl" means a C1, C2, or C3 alkyl group having at least one substituent; specific examples include, but are not limited to, -CH2Cl, -CH2CH2OH, -CH2CH(OCH3)CH3, which can be considered as a -Cl substituted methyl group, a -OH substituted ethyl group, and a -OCH3 substituted propyl group, respectively.

[0051] A "solvate" of a compound means an aggregate that comprises one or more molecules of the compound with one or more solvent molecules. The solvent can be water, and the corresponding solvate can be referred to as a hydrate, including a mono-hydrate, a di-hydrate, a hemi-hydrate, a sesqui-hydrate, a tri-hydrate, a tetra-hydrate, etc. The solvent can also be an organic solvent.

[0052] A "tautomer" of a compound means an isomer of the compound that is in equilibrium with the compound. The tautomerism refers to the isomerization of a molecule that involves the reversible shifting of a proton between two or more atoms of the molecule, often involving the change of a double bond to a single bond and vice versa. Tautomerism includes, but is not limited to, keto-enol tautomerism, amide-imidic acid tautomerism, lactam-lactim tautomerism, enamine-imine tautomerism, enamine-enamine tautomerism (e.g., phosphopyridoxyl catalyzed enzymatic reaction), proton transfer tautomerism, valence tautomerism, etc.

[0053] A "stereoisomer" of a compound means an isomer of the compound that has the same atomic connectivity but has a different three-dimensional structure. Stereoisomerism can be classified as enantiomeric or diastereomeric, where diastereomeric further includes geometric isomeric and conformational isomeric. Enantiomers are molecules that are mirror images of one another and cannot be superimposed on one another. For a compound having stereoisomers, even if a specific stereochemistry is given, all other stereoisomers are disclosed as well.

[0054] The unit of molecular weight is Dalton (Da) by default when not given.

[0055] Compounds containing one or more polymer chains (e.g., polyethylene glycol, functionalized polyethylene glycol derivatives, pegylated lipids, etc.) are collectively referred to as polymers. The molecular weight of a polymer is by default the number average molecular weight. The length of a polymer chain can be expressed in terms of “degree of polymerization”, which by default refers to the number of repeating units, and is specifically the number average degree of polymerization. The repeating unit of a polyethylene glycol chain is an ethylene oxide group, i.e., -CH2CH2O- or -OCH2CH2-.

[0056] Polydispersity index (PDI) is used to measure the homogeneity of a specific parameter of a system. The PDI of a polymer by default is used to measure the homogeneity of molecular weight, and is defined as the ratio of weight average molecular weight (M w ) and number average molecular weight (M n ), i.e., M w / M n . A polymer is considered monodisperse if its PDI is no more than 1.005, and is considered polydisperse if its PDI is greater than 1.005. The PDI of a polymer is typically characterized by gel permeation chromatography (GPC). The PDI of a nanoparticle by default is used to measure the homogeneity of size, and is defined as (σ / D) 2 , where σ is the standard deviation of size, and D is the average particle diameter. A nanoparticle is considered monodisperse if its PDI is about 0, i.e., all particles have the same size; a nanoparticle is considered highly homogeneous if its PDI is no more than 0.1; and a nanoparticle is considered highly polydisperse if its PDI is greater than 0.3, i.e., the size difference is significant. The PDI of a nanoparticle is typically characterized by dynamic light scattering (DLS).

[0057] The molecular weight and degree of polymerization of a polydisperse polymer is generally allowed to be within ±10% of the given value, in some cases up to ±15%, but no more than ±20%. For example, for the molecular weight of a polydisperse polymer, “5 kDa” is equivalent to a range of 4.5-5 kDa. For example, for the degree of polymerization of a polydisperse polymer, “100” is equivalent to a range of 90-110. The molecular weight and degree of polymerization of a monodisperse polymer is generally strictly equal to the given value, in some cases up to ±5%.

[0058] For the preparation of monodisperse polymers, the macroscopic product can contain components deviating from the target polymerization degree due to the limitations of the preparation method and purification method. Any component deviating from the target polymerization degree by no more than ±5% (provided that the target polymerization degree is ≥10) or no more than ±0.5 (provided that the target polymerization degree is <10) can be considered as a target component. When the total content of all target components 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%, and most preferably 100%, it is considered that the target monodisperse macroscopic product is obtained. Even if the above content ratio is not reached, as long as the product obtained by using the same or substantially the same preparation method as the present application or the components appearing in the form of main products, associated products or by-products, whether or not separated and purified, are within the scope of the present application.

[0059] "Salts" of a compound include an addition salt of said compound with any one, two or more of an inorganic acid, an organic acid, an inorganic base, and an organic base. When a compound contains both a basic moiety, such as a pyridine, an imidazole, and an acidic moiety, such as a carboxylic acid, zwitterions can be formed and are included within the term "salt." Salts can be pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts. Salts of the compounds can be formed by reacting the compound with an amount of acid or base, such as an equivalent amount, in a medium such as an organic solvent, or in an aqueous medium, and isolating the salt thus formed. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Exemplary base addition salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as amino organic bases, and salts with amino acids such as arginine, lysine and the like. Quaternization reagents of basic nitrogen-containing groups include, but are not limited to, lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides, dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aryl alkyl halides, such as benzyl and phenethyl bromides, and others. The acid and base addition salts are preferably pharmaceutically acceptable salts.

[0060] Heteroatom refers to an atom other than carbon and hydrogen, including but not limited to O, S, N, P, Si, F, Cl, Br, I, B, and the like.

[0061] "Residue" refers to a group formed by the loss of part of an atom or group from a compound. A residue of a specified type of compound generally retains or substantially retains the characteristic backbone of that class of compound. For example, a hydrocarbon derivative residue contains a hydrocarbon or heterohydrocarbon backbone. A "residue" can also be referred to simply as a "group."

[0062] A "functional group" (also "functional moiety") includes, but is not limited to, a reactive group, a protected reactive group, a precursor to a reactive group, and the like. A functional compound can contain one or more functional groups.

[0063] A "reactive group" is a group capable of participating in a reaction, including, but not limited to, various common functional groups in organic chemistry. The reaction can be a desired reaction or an undesired reaction. When there are two or more reactive groups in a reactant, the reactive groups that can undergo undesired reactions are typically protected. "Protection" of a reactive group refers to the strategy of converting the reactive group into a "protected form" by a specific reagent. The protected form is not reactive under the conditions of the desired reaction, and can be deprotected under specific conditions to obtain the unprotected form. In the protected form of a reactive group, the moiety that is different from the unprotected form is called a "protecting group". For example, -OTBS is a protected form of a hydroxyl group (-OH), where TBS is a protecting group for the hydroxyl group. The protected form of a compound includes the protected form of any one, two or more reactive groups contained therein. The unprotected form is also called the free form.

[0064] The "skeleton" of a compound or group refers to its core or basic framework, which constitutes the main topology of the compound or group. The skeleton can be linear or non-linear, cyclic or acyclic. The skeleton is formed by a series of atom bonds, and by default does not include hydrogen atoms or substituents attached to the skeleton. The atoms constituting the skeleton are also called "skeletal atoms". For example, the skeleton of a hydrocarbon is composed of only carbon atoms, and the skeleton of a heterohydrocarbon also contains heteroatoms. The bonding mode of the skeletal atoms is by default covalent bond, including but not limited to localized bond, delocalized bond, semi-localized bond, saturated bond, unsaturated bond, single bond, double bond, triple bond, multiple bond, multicenter bond, and the like. The skeleton of a divalent linking group refers to the structure formed by passing through the atoms in a single direction from one linking end to the other linking end. When the skeleton contains a cyclic structure, all ring-forming atoms are by default considered as skeletal atoms.

[0065] A "carbon chain" refers to a skeletal structure composed of one or more consecutive carbon atoms. The skeleton of a compound or group can not contain a carbon chain, can be composed of only a carbon chain, or can be composed of one or more carbon chains and one or more non-carbon chains. The "carbon chain length" refers to the number of atoms contained in the carbon chain. Part of a cyclic structure can also participate in the formation of a carbon chain. For example, the divalent group A carbon chain along either path contains 4 ring-forming atoms and 2 non-ring-forming atoms, and has a carbon chain length of 6. Ring-forming moieties not counted as part of the carbon chain can be considered as substituents on the carbon chain. A carbon chain can contain one or more fragments from ring-forming moieties, depending on the number of ring-forming moieties involved. The number and identity of substituents on the carbon chain are not particularly limited; for example, -CH2CH2C(=0)CH2- contains one =0 substituent, and has a carbon chain length of 4.

[0066] "Acyl" refers to a monovalent radical of an organic or inorganic oxygen- containing acid, with loss of the hydroxyl group, including but not limited to carbonyl, sulfonyl, and phosphonyl groups. Exemplary acyl groups include, but are not limited to, formyl (i.e., aldehyde, -C(=0)H or -CHO) based on formic acid, acetyl (Ac, -C(=0)CH3) based on acetic acid, benzoyl (Bz, -C(=0)C6H5) based on benzoic acid, mesyl ( -S(=0)2CH3) based on methanesulfonic acid, phosphoryl (-P(=0)(OH)2) based on phosphoric acid, and the like.

[0067] "Targeting group" refers to a group that provides a strong affinity for a particular target (e.g., a molecule, a cell or cell type, such as a hepatocyte), a compartment (e.g., a cellular or organ compartment), a tissue, an organ, or a region of the body. Exemplary targeting groups include, but are not limited to, residues of antibodies, antigens, peptides, vitamins, carbohydrates and derivatives thereof (including but not limited to monosaccharides such as N-acetylgalactosamine (GalNAc)), folate, aptamers, receptor ligands, transferrin, biotin, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. "Liver targeting group" refers to a group that targets the liver and or hepatocytes.

[0068] "Targeting ligand" refers to a compound that contains one or more targeting groups. Targeting ligands that contain reactive groups can be used to chemically modify and confer the same or similar targeting properties to the modified compound.

[0069] A compound containing a targeting group is also referred to as a "targeted compound" and includes, but is not limited to, a compound modified with a targeting ligand, such as a targeted PEGylated lipid. Targeted compounds can be monovalent, divalent, trivalent, tetravalent, and higher valent, containing one, two, three, four, and more targeting groups, respectively.

[0070] The number of carbon atoms of a monosaccharide is not particularly limited, including but not limited to trioses, tetroses, pentoses, hexoses, and heptoses. Monosaccharides include, but are not limited to, allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, fucitol, fucosamine, fucose, fuculose, galactosamine, galactosaminitol, N-acetylgalactosamine, galactose, glucosamine, N-acetylglucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose glyceraldehyde, L-glycero-D-manno-heptose, glycerol, glyceraldehyde, dihydroxyacetone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, mannoheptulose, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, deoxyribose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, xylulose, and derivatives of any of the foregoing. A monosaccharide can be in the D- or L- configuration, cyclic or acyclic structure, and optionally substituted.

[0071] The term "monosaccharide" includes monosaccharides and derivatives thereof. Forms of monosaccharide derivatives include, but are not limited to, deoxy sugars (in which one of the alcohol hydroxyl groups is replaced by a hydrogen atom; e.g., deoxyribose, fucose, fuculose, rhamnose, quinose (epi-rhamnose), etc.), dideoxy sugars (in which two of the alcohol hydroxyl groups are replaced by hydrogen atoms; e.g., 2,3-dideoxyribose, colitose, abequose, paratose, tyvelose, levrotose, etc.), amino sugars (in which at least one of the alcohol hydroxyl groups is replaced by an amino group; e.g., galactosamine, glucosamine, mannosamine, fucosamine, neuraminic acid, muramic acid, lactosamine, etc.), iminosugars (in which an oxygen atom in the ring skeleton is replaced by an imino group), N-substituted (in which the hydrogen atom of at least one of the amino or imino groups of an amino sugar or imino sugar is replaced by a substituent, which can be an alkyl group or an acyl group, etc.; e.g., N-methylglucosamine, N-methylgalactosamine, N-acetylgalactosamine, etc.), thio sugars (in which one or more of the oxygen atoms at any position is replaced by a sulfur atom), seleno sugars (in which one or more of the oxygen atoms at any position is replaced by a selenium atom), telluro sugars (in which one or more of the oxygen atoms at any position is replaced by a tellurium atom), C-substituted (in which the hydrogen atom of at least one of the non-terminal carbon atoms is replaced by a substituent), O-substituted (in which the hydrogen atom of at least one of the alcohol hydroxyl groups is replaced by a substituent, which can be an alkyl group or an acyl group, etc.; or, two hydroxyl groups lose a molecule of water to form an internal ether; or, a diol reacts with an aldehyde or a ketone to form a cyclic acetal), halogenated sugars (in which at least one of the hydroxyl groups or hydrogen atoms at any position is replaced by a halogen atom), unsaturated monosaccharides (in which at least one double bond or triple bond is formed between two adjacent carbon atoms in the carbon skeleton; e.g., enoses), sugar alcohols (in which at least one carbonyl group is reduced to >CHOH), aldonic acids (in which the aldehyde group of an aldose is oxidized to a carboxyl group), ketonic acids (in which the 1 -position hydroxyl group of a ketose is oxidized to a carboxyl group), uronic acids (in which a primary hydroxyl group is oxidized to a carboxyl group), aldaric acids (in which both of the terminal hydroxyl groups are oxidized to carboxyl groups), glycosides (in which at least one non-sugar moiety is linked to a monosaccharide via a glycosidic bond), and combinations of two or more of any of the foregoing. For example, galactosamine is a derivative of the amino sugar form based on galactose; N-methylgalactosamine is a derivative that can be considered as both an N-substituted form based on galactosamine and a derivative of the form based on galactose with both amino sugar and N-substitution; galactose, galactosamine, and N-methylgalactosamine all fall within the scope of "monosaccharide" in the present application.

[0072] A residue of a monosaccharide or a derivative thereof includes, but is not limited to, a group that still has the skeleton of a monosaccharide after one or more of the functional groups such as hydrogen atoms, hydroxyl groups, amino groups, carboxyl groups, and / or thiol groups, etc. of a monosaccharide or a derivative thereof are lost.

[0073] "Hydroxyl protecting groups" include, but are not limited to, those commonly found in the art, such as alkanoyl (e.g., acetyl, tert-butyryl), aralkanoyl (e.g., benzoyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl (TBS), triisopropylsilyl (TIPS), allyl, acetal, ketal, and the like. Removal of acetyl groups is typically carried out under basic conditions, most commonly aminolysis with NH3 / MeOH and methanol anion catalyzed methanolysis. Benzyl groups can be removed using palladium catalyzed hydrogenolysis or by reduction with metallic sodium in ethanol or liquid ammonia. Trityl groups are typically removed by catalytic hydrogenolysis. Trimethylsilyl groups are typically removed using a fluoride ion containing reagent (e.g., tetrabutylammonium fluoride / anhydrous THF, etc.). Tert-butyldimethylsilyl ethers can be removed using a fluoride ion containing reagent or aqueous acetic acid. Protected forms of diols include, but are not limited to, formation of dioxolanes, dioxanes, cyclic carbonates, cyclic boronates, and the like.

[0074] "Carboxyl protecting groups" include, but are not limited to, those commonly found in the art, such as alkyl (e.g., methyl, ethyl, tert-butyl) and aralkyl (e.g., benzyl), preferably tert-butyl (tBu), methyl (Me), ethyl (Et), or benzyl (Bn). Protected forms of carboxyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl. Carboxyl protecting groups can be removed by hydrolysis under catalysis by acid or base, or occasionally by thermolysis. For example, tert-butyl groups can be removed under mild acidic conditions; benzyl groups can be removed by hydrogenolysis.

[0075] "Amino protecting groups" include, but are not limited to, those commonly found in the art, such as arylC 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, C 1-6 alkylsulfonyl, arylsulfonyl, silyl, and the like. Exemplary amino protecting groups include Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), Cbz (benzyloxycarbonyl), and the like. Removal of the Boc protecting group is preferably accomplished using trifluoroacetic acid (TFA). Removal of the Fmoc protecting group is preferably accomplished using a 20% piperidine in N,N-dimethylformamide (DMF) solution. Removal of the Cbz protecting group is preferably accomplished using catalytic hydrogenolysis.

[0076] "Active ester groups" refer to ester groups having a high reactivity, including those commonly found in the art that readily react with nucleophiles (e.g., amines). Exemplary active ester groups include N-hydroxysuccinimidyl ester (NHS ester) groups, p-nitrophenol ester (p-NP ester) groups, o-nitrophenol ester (o-NP ester) groups, and 1-hydroxybenzotriazolyl ester (HOBt ester) groups.

[0077] "Small molecule" refers to a compound having a molecular weight of no more than 1000 Da, and in some cases the upper limit of the molecular weight can be extended to 1500 Da.

[0078] "Lipid" is a broad group of organic compounds that includes fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, and other hydrophobic or amphiphilic compounds. Lipids include simple esters of long-chain aliphatic acids and alcohols, which are further divided into the classes of fats, oils, and waxes; complex esters, also known as "lipoids", including the phospholipids, sphingolipids, glycolipids, sterol lipids, and lipoproteins; and derived lipids, including simple and complex lipid derivatives, which have the general properties of lipids. Lipids can be synthetic or derived (isolated or modified) from natural sources.

[0079] Lipids can be involved in forming various types of pharmaceutical compositions, including but not limited to lipid nanoparticles (LNP), lipopolyplex (LPP), polypeptide nanoparticle (PNP), etc. Lipid nanoparticles refer to nanoparticles (e.g., 1 nm to 1000 nm) comprising one or more lipids.

[0080] "Cationic lipid" can be a lipid that is positively charged at any pH or hydrogen ion activity, or can be an ionizable lipid (i.e., "cationizable lipid") that can be positively charged in response to the pH or hydrogen ion activity in its intended use environment. The latter also includes zwitterionic lipids that satisfy the characteristic. In some cases, the positive charge in a cationic lipid is derived from the presence of a quaternary nitrogen atom. A tertiary amine group is a common cationizable group. For a lipid nanoparticle containing a cationic lipid, it is preferred that about 1% to 100% of the cationic lipids are positively charged at the endosomal pH (e.g., about 5.5 to 6.5).

[0081] "PEGylated lipid" refers to a lipid modified with polyethylene glycol, the structure of which comprises a lipid moiety and a polyethylene glycol moiety.

[0082] "Phospholipid" refers to a lipid containing at least one phosphate group.

[0083] "Sterol lipid" refers to a lipid containing a steroid nucleus. The steroid nucleus is composed of three six-membered carbon rings and one five-membered carbon ring, and the bonding manner of the atoms in the nucleus is not particularly limited, including but not limited to single bond, double bond, etc. The steroid nucleus can be connected to any suitable substituent group. Sterol lipids include but are not limited to cationic lipids based on steroids and neutral lipids based on steroids (e.g., cholesterol), etc.

[0084] Nucleic acids are composed of nucleotide units, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and derivatives thereof. RNA can be naturally occurring or non-naturally occurring ribonucleic acids, including but not limited to small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), single guide RNA (sgRNA), and cas9 mRNA. An "antagomir" is also known as "anti-miR," is a class of chemically engineered oligonucleotides designed to silence endogenous miRNAs.

[0085] "Transfect" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur in vitro, ex vivo, or in vivo.

[0086] "Deliver" refers to providing an entity to a target. For example, delivering a drug and / or therapeutic and / or prophylactic agent to an organ and / or tissue and / or cell of a subject, including but not limited to a human and / or other animal.

[0087] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered, and which is physiologically innocuous, i.e., which does not exhibit excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and is accordingly suitable for use in contact with the tissues of humans and / or other animals. Exemplary diluents include, but are not limited to, water, normal saline, ethanol, propylene glycol, glycerin, and vegetable oils (e.g., soybean oil, sesame oil). Exemplary adjuvants include, but are not limited to, antioxidants (e.g., vitamin E, BHT, BHA), preservatives (e.g., sodium benzoate, parabens), buffers (e.g., phosphate, citrate), and solubilizers (e.g., poloxamer, Tween 80). Exemplary excipients include, but are not limited to, thickening agents (e.g., hypromellose, carbomer), disintegrants (e.g., cross-linked polyvinylpyrrolidone, starch), binding agents (e.g., microcrystalline cellulose, hydroxypropyl cellulose), and lubricants (e.g., magnesium stearate, talc). Exemplary vehicles include, but are not limited to, solvents (e.g., dimethyl sulfoxide, ethanol), emulsifiers (e.g., lecithin, Span), suspending agents (e.g., gelatin, methylcellulose), and oil bases (e.g., cocoa butter, stearate).

[0088] "Therapeutic agent" and "drug" are used interchangeably and include any agent, compound, composition, or mixture that provides physiological or pharmacological effects, either in vivo or in vitro, and often provides beneficial effects. There is no particular limitation on the types of "therapeutic agents," including but not limited to vaccines, antibodies, vitamins, foods, food additives, nutritional supplements, nutritional health products, and other agents that provide beneficial effects. Therapeutic agents can act systemically and / or locally. Suitable routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, intratracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal, ocular administration, as well as infusion, inhalation, and nebulization. Suitable dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0089] "Active pharmaceutical ingredients" are mainly active substances that can interact with other substances, but they can also be inert substances that do not interact; among them, inert substances can be transformed into active forms through in vivo action or certain stimuli.

[0090] A "vaccine" is a preventive or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0091] The effective amount of a drug is generally understood as the amount of drug sufficient to produce a therapeutic effect when administered to a subject. Preferred effective amounts can be determined by those skilled in the art based on various factors, including but not limited to pharmacokinetic parameters (such as bioavailability, metabolism, half-life, etc.), the severity of the disease, disorder, or symptom, the subject's weight, the subject's immune status, the route of administration, and the desired biological endpoint.

[0092] "Liver disease" or "liver disorder" includes any condition that is associated with or causes changes or damage to the structure of liver tissue, such as liver fibrosis, cirrhosis, and liver cancer. Specifically, "liver disease" includes, but is not limited to, infectious diseases (such as hepatitis B, hepatitis C, and hepatitis D), autoimmune diseases (such as primary biliary cirrhosis and autoimmune hepatitis), genetic or hereditary diseases (hereditary hemochromatosis, Wilson's disease, cystic fibrosis, and diabetes), metabolic and / or diet-related diseases (obesity, diabetes, alcohol abuse, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH)), cancer or neoplastic diseases (such as hepatocellular carcinoma (HCC)), and other diseases (such as gallstones, Budd-Chiari syndrome, and primary sclerosing cholangitis).

[0093] “Treatment” refers to the handling and care of a subject in need for the purpose of obtaining a desired pharmacological and / or physiologic effect, including administration of a drug to a subject in need. The effect can be prophylactic, in terms of completely or partially preventing a disease, disorder or condition, and / or can be therapeutic, in terms of a partial or complete cure for a disease, disorder or condition. In particular, types of treatment include, but are not limited to: (a) preventing the disease, disorder or condition from occurring in an individual that is predisposed to the disease, disorder or condition but has not yet been diagnosed as having it; (b) inhibiting the disease, disorder or condition; (c) alleviating the symptoms of the disease, disorder or condition. “Subject in need” and “subject” are interchangeable, preferably a mammal, especially a human.

[0094] 2. A target ligand-modified polyethylene glycolized lipid

[0095] One embodiment of the present application:

[0096] A target ligand-modified polyethylene glycolized lipid, characterized by the structure of Formula (1):

[0097] or a salt, tautomer, stereoisomer, deuterated form, or solvate thereof;

[0098] wherein,

[0099] each T is independently a residue of a monosaccharide or a derivative thereof; preferably, each T is independently a residue of any one of galactose, galactosamine, glucose, glucosamine, mannose, mannosamine, fucose, and fucosamine, or a derivative thereof;

[0100] y is 1 and X is a linking bond; or, y is an integer from 2 to 4, X is a y+1 valent linking group, and X is selected from any one of wherein either end of X is connected to M; each G3 is independently >CH-, >N-, or a trivalent cyclic group; G4 is a tetravalent carbon atom or a tetravalent cyclic group; each L X is independently -B X -, -B X -Z X -B X -, or -B X -Z X -B X -Z X -B X -, wherein each Bx is independently a linking bond or an optionally substituted C 1-6 hydrocarbylene group, and no more than one B X is a linking bond at the same time as two Z X are connected; each Z Xindependently a divalent linking group comprising a heteroatom;

[0101] y is an integer from 2 to 4, any two T are the same or different from each other, any two L0are the same or different from each other;

[0102] N core is a trivalent linking group with a >N- as branching core;

[0103] each R is independently -B R -(Z R -B R ) r -E R wherein r is an integer from 0 to 2; each B R is independently a bond or an optionally substituted C 1-29 hydrocarbylene group, and any B R that is a bond is not simultaneously connected to two Z R ; each Z R is independently a divalent linking group comprising a heteroatom; E R is an optionally substituted C 1-30 hydrocarbyl group; two R are the same or different from each other; all B R in each R are the same or different from each other; the sum of the carbon chain lengths of E R is independently an integer from 5 to 30;

[0104] P0is -(OCH2CH2) n - and its oxygen end is connected to M, wherein n is an integer from 1 to 250;

[0105] each L0is independently -B L -(Z L -B L ) j - wherein j is an integer from 1 to 6; each B L is independently a bond or an optionally substituted C 1-12 hydrocarbylene group, and any B L that is a bond is not simultaneously connected to two Z L ; each Z L is independently a divalent linking group comprising a heteroatom; the sum of the carbon chain lengths of all B L in each L0is independently an integer from 2 to 24;

[0106] M is -B M -(Z M -B M ) k - wherein k is an integer from 1 to 3; each B M is independently a bond or an optionally substituted C 1-12 hydrocarbylene group, and any B Mtwo Zs are not simultaneously attached to two Zs M each Z is independently a heteroatom-containing divalent linker; all B M in M are independently selected from the group consisting of -C(R M the sum of the carbon chain lengths of B

[0107] Z X , Z R , Z L , and Z M each occurrence is independently selected from the group consisting of -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and ; wherein each Y is independently O, S, or NR c ; each R c is independently a hydrogen atom or a C 1-12 alkyl group;

[0108] The pegylated lipid is monodisperse or polydisperse.

[0109] In some specific embodiments, each T is independently a targeting group, preferably a liver targeting group.

[0110] In some specific embodiments, the form of the monosaccharide derivative is preferably N-substituted, O-substituted, or a combination thereof, and each substituent for N-substitution or O-substitution is independently selected from the group consisting of R T , -C(=O)R T , -C(=O)NH2, -C(=O)N(R T )2, -S(=O)2OH, -S(=O)2R T , -P(=O)(OH)2, and -P(=O)(R T )2; wherein each R T is independently an optionally substituted C 1-3 alkyl, phenyl, or benzyl group.

[0111] In some specific embodiments, each R T has a number of substituents independently ranging from 0 to 5.

[0112] In some specific embodiments, each substituent in each R T is independently selected from the group consisting of -OH, -F, -Cl, -Br, -I, and -N3.

[0113] In some specific embodiments, each T is independently a residue of any one of galactose, galactosamine, and derivatives thereof; the galactose derivative is preferably in the O-substituted form; the galactosamine derivative is preferably in the N-substituted and / or O-substituted form;

[0114] Preferably, each T is independently wherein R a is selected from is any one of R b is -H or -CH3, preferably -H; each R d is independently -H, -C(=0)CH3,

[0115] More preferably, each T is independently is any one of

[0116] Most preferably, each of the y T is

[0117] In some specific embodiments, each Z R is independently selected from any one of -0-, -S-S-, -C(=0)-, -OC(=0)-, -C(=0)0-, -OC(=0)0-, -NHC(=0)-, -C(=0)NH-, -NHC(=0)0-, -OC(=0)NH-, and -NHC(=0)NH-;

[0118] In some specific embodiments, each Z R is independently -0-, -OC(=0)-, -C(=0)0-, -NHC(=0)-, or -C(=0)NH-.

[0119] In some specific embodiments, each E R is independently optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl.

[0120] In some specific embodiments, each of the two R in general formula (1) is independently selected from any one of -E R , -B R -Z R -E R , -B R -Z R -B R -Z R -E R , wherein each B R is independently optionally substituted C 1-29 alkylene, E R is optionally substituted C 1-30 alkyl, or optionally substituted C 2-30alkenyl.

[0121] In some specific embodiments, each of the two R in general formula (1) is independently selected from any one of the following structures:

[0122] wherein each t R is independently an integer from 0 to 10.

[0123] In some specific embodiments, each of the two R in general formula (1) is independently selected from any one of the following structures:

[0124] In some specific embodiments, N core has the following structure:

[0125] wherein each B N is independently a bond or an optionally substituted C 1-12 alkylene; each Z N is independently a heteroatom-containing divalent linker, preferably -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and ; each Y is independently O, S, or NR c ; each R c is independently a hydrogen atom or a C 1-12 alkyl group; m1, m2, and m3 are each independently 0, 1, 2, or 3; N core has one end connected to P0 and the other two ends each connected to one R.

[0126] In some specific embodiments, each B N is independently a bond or an unsubstituted C 1-12 alkylene.

[0127] In some specific embodiments, each B N is independently a bond, methylene, ethylene, propylene, butylene, pentylene, or hexylene.

[0128] In some specific embodiments, each Z N is independently -O-, -S-S-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and .

[0129] In some specific implementations, m1 and m2 are each independently 0 or 1.

[0130] In some specific implementation schemes, m3 is 1, 2, or 3.

[0131] In some specific implementation plans, N core The structure is Any one of the following; wherein each tn is an independent integer from 1 to 4, preferably 1 or 2; preferably, N core Choose from any of the following structures:

[0132] In some specific implementation schemes, each Z M Independently selected from -O-, -SS-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH- and Any one of them.

[0133] In some specific implementation plans, each B M Independently for connecting key or optional replacement of C 1-12 Alkylene; any B M To replace C 1-12 When alkylene is used, it is preferable that the number of substituents is 1 and that it is -OH, -CH2OH or...

[0134] In some specific implementation schemes, M is -B M -Z M -B M -and selected from-B M -NHC(=O)-B M -、-B M -C(=O)NH-B M -、-B M -OC(=O)-B M -、-B M -C(=O)OB M -、-B M -OC(=O)OB M -、-B M -NHC(=O)OB M -and-B M -OC(=O)NH-B M - any one of the following or its alternative forms, and its left end is connected to X; or,

[0135] M is -B M -(Z M -B M )2- and is selected from -B M -NHC(=O)-B M -C(=O)NH-B M -, M -NHC(=O)-B M -C(=O)O-B M -, M -OC(=O)-B M -C(=O)NH-B M -, M -OC(=O)-B M -C(=O)O-B M -, M -C(=O)NH-B M -C(=O)NH-B M -, M -C(=O)NH-B M -C(=O)O-B M -, M -C(=O)O-B M -C(=O)NH-B M -, M -C(=O)O-B M -C(=O)O-B M -, M -NHC(=O)-B M -C(=O)-B M -, M -OC(=O)-B M -C(=O)-B M -, M -C(=O)NH-B M -C(=O)-B M -, M -C(=O)O-B M -C(=O)-B M -, M -C(=O)-B M -NHC(=O)-B M -, M -C(=O)-B M -OC(=O)-B M -, M -C(=O)-B M -C(=O)NH-B M -, M -C(=O)-BM -C(=O)OB M -、-B M -C(=O)-B M -C(=O)-B M -、-B M -C(=O)NH-B M -NHC(=O)OB M -、-B M -C(=O)OB M -NHC(=O)OB M -、-B M -C(=O)NH-B M -NHC(=O)NH-B M -、-B M -C(=O)OB M -NHC(=O)NH-B M -、-B M -NHC(=O)-B M -OC(=O)NH-B M -、-B M -OC(=O)NH-B M -NHC(=O)OB M -、-B M -OC(=O)NH-B M -OC(=O)NH-B M -、-B M -NHC(=O)OB M -NHC(=O)OB M -、-B M -NHC(=O)OB M -OC(=O)NH-B M -、-B M -NHC(=O)NH-B M -NHC(=O)OB M -、-B M -OC(=O)NH-B M -NHC(=O)NH-B M -and-B M -NHC(=O)NH-B M -NHC(=O)NH-B M - any one of the following or its alternative forms, and its left end is connected to X; or,

[0136] M is -B M -(Z M -B M )3-and selected from-B M-NHC(=O)-B M -NHC(=O)-B M -C(=O)-B M -,-B M -NHC(=O)-B M -OC(=O)-B M -C(=O)-B M -,-B M -OC(=O)-B M -NHC(=O)-B M -C(=O)-B M -,-B M -OC(=O)-B M -OC(=O)-B M -C(=O)-B M -,-B M -C(=O)NH-B M -NHC(=O)-B M -C(=O)-B M -,-B M -C(=O)NH-B M -OC(=O)-B M -C(=O)-B M -,-B M -C(=O)O-B M -NHC(=O)-B M -C(=O)-B M -,-B M -C(=O)O-B M -OC(=O)-B M -C(=O)-B M -,-B M -OC(=O)NH-B M -NHC(=O)-B M -C(=O)-B M -,-B M -C(=O)O-B M -S-S-B M -OC(=O)-B M -,-B M -C(=O)O-B M -S-S-B M -OC(=O)NH-B M -,-B M -NHC(=O)O-B M -S-S-B M -OC(=O)-B M -and -B M-NHC(=O)O-B M -S-S-B M -OC(=O)NH-B M - of any one of the foregoing or a substituted form thereof, and the left end of X is attached to M;

[0137] In any of the foregoing cases, the substituted form of M is preferably a form in which one hydrogen atom on the carbon chain of M is replaced by -OH or -CH2OH.

[0138] In some particular embodiments, each B X is independently a bond or optionally substituted C 1-6 alkylene, preferably a bond or unsubstituted C 1-6 alkylene.

[0139] In some particular embodiments, each Z X is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, and -OC(=O)NH-.

[0140] In some particular embodiments, each L X is independently

[0141] In some particular embodiments, X is is preferably of any one of the foregoing, wherein the left end of X is attached to M.

[0142] In some particular embodiments, X is is preferably of any one of the foregoing, wherein the left end of X is attached to M; tx is an integer from 0 to 6; the number of R X is independently a hydroxyl or C 1-3 alkoxy group attached to a ring atom; more preferably, X is selected from of any one of the foregoing, wherein the left end of X is attached to M.

[0143] In some particular embodiments, X is is preferably of any one of the foregoing, wherein the left end of X is attached to M.

[0144] In some specific embodiments, X is a residue of a dimeric amino acid or a residue of a multimeric amino acid; preferably, each amino acid unit in X is independently selected from any one of glycine, alanine, beta-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine, and citrulline.

[0145] In some specific embodiments, X is a residue of a multifunctional small molecule, or X is connected by residues of two or more multifunctional small molecules; the multifunctional small molecule contains at least two identical or different functional groups; each multifunctional small molecule is independently preferably a polyol, a polythiol, a polycarboxylic acid, a primary polyamine, a secondary polyamine, a polyol containing a hetero-functional group, a polythiol containing a hetero-functional group, a polycarboxylic acid containing a hetero-functional group, a primary polyamine containing a hetero-functional group, a secondary polyamine containing a hetero-functional group, any one of glycine, alanine, beta-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine, and citrulline; wherein the hetero-functional group refers to another functional group that is different from the type of other functional groups in the molecule.

[0146] In some specific embodiments, X is selected from any one of the trivalent, tetravalent, or pentavalent linkers disclosed in CN110591079A.

[0147] In some specific embodiments, each Z L is independently selected from -O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and is any one of.

[0148] In some specific embodiments, L0is selected from -(CH2) ta -C(=O)NH-(CH2) ta -, -(CH2) ta -O-(CH2) tb -O-(CH2) tb -NHC(=O)-(CH2) ta -, -(CH2) ta -C(=O)NH-(CH2) tb -NHC(=O)-(CH2) tb -O-(CH2) ta -, -(CH2)ta -O-(CH2) tb -O-(CH2) tb -NHC(=O)-(CH2) tb -O-(CH2) ta - and -(CH2) ta -O-(CH2) tb -O-(CH2) tb -O-(CH2) tb -C(=O)NH-(CH2) ta - any one of the following, and either end of L0 is connected to X; wherein each ta is independently an integer from 0 to 12, preferably an integer from 1 to 6; each tb is independently an integer from 1 to 12, preferably an integer from 0 to 6.

[0149] In some specific implementations, the interval length between any T and M is 5 to 30 atoms; when y is 1, 2 or 3, the interval length is preferably 7 to 17 atoms; when y is 4, the interval length is preferably 19 to 26 atoms.

[0150] In some specific implementation schemes, (T-L0) y -X- is selected from any of the following structures:

[0151] In some specific implementation schemes, N core The nitrogen-branched lipid moiety consisting of two Rs -N core (R)2 is selected from any of the following structures:

[0152] In some specific implementations, the polyethylene glycol-modified lipids represented by general formula (1) are monodisperse; wherein n is an integer from 1 to 100, preferably an integer from 3 to 60, and more preferably an integer from 10 to 47.

[0153] In some specific implementations, the polyethylene glycol-modified lipids represented by general formula (1) are monodisperse; wherein n is any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47.

[0154] In some specific implementations, the polyethylene glycol-modified lipids represented by general formula (1) are polydisperse; wherein n is an integer from 10 to 250, preferably an integer from 20 to 60.

[0155] In some embodiments, the polyethylene glycolylated lipid of Formula (1) is polydisperse; wherein n is any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60.

[0156] In some embodiments, the polyethylene glycolylated lipid of Formula (1) has a number average molecular weight selected from the group consisting of 2000 Da to 5000 Da.

[0157] In some embodiments, the polyethylene glycolylated lipid of Formula (1) is selected from the group consisting of any one of GPL-0102, GPL-0202, GPL-0301, GPL-01N01, GPL-03N02, GPL-01N09, GPL-03N03, G2PL-0102, G2PL-0201, G2PL-0301, G2PL-03N02, G3PL-01b01, G3PL-02b01, G3PL-03b01, G3PL-04b01, G3PL-05b01, G3PL-06b01, G3PL-07b01, G3PL-01b10, G3PL-01b11, G3PL-01b12, G3PL-01c02, G3PL-02c02, G3PL-03c02, G3PL-04c02, G3PL-05c02, G3PL-06c02, G3PL-07c02, G3PL-01a02, G3PL-02a02, G3PL-06a02, G3PL-07a02, G3PL-03a01, G3PL-04a01, G3PL-05a02, G3PL-01b03, G3PL-01b04, G3PL-01b05, G3PL-01b06, G3PL-01b07, G3PL-01b08, G3PL-01b09, G3PL-03e02, G3PL-03d03, G3PL-05d03, G3PL-01bN02, G3PL-01bN04, G3PL-02bN03, G3PL-07bN04, G3PL-01bN05, G3PL-01bN06, G3PL-01bN07, G3PL-01bN08, G4PL-01a02, G4PL-01b01, G4PL-02b01, G4PL-03b01, G4PL-01c01, G4PL-01bN01.

[0158] In some specific embodiments, all T in the PEGylated lipid of Formula (1) are the same.

[0159] In some specific embodiments, the PEGylated lipid of Formula (1) contains at least two different T.

[0160] In some specific embodiments, all L0 in the PEGylated lipid of Formula (1) are the same.

[0161] In some specific embodiments, the PEGylated lipid of Formula (1) contains at least two different L0.

[0162] In some specific embodiments, two R in the PEGylated lipid of Formula (1) are the same.

[0163] In some specific embodiments, two R in the PEGylated lipid of Formula (1) are different.

[0164] 3. Methods for preparing PEGylated lipids

[0165] The starting materials used in each of the methods can be obtained by purchase or self-synthesis.

[0166] When no polymerization reaction is involved, the degree of polymerization of the polymer chains in the product is by default the same as that of the corresponding starting material.

[0167] Monodisperse starting materials and polydisperse starting materials can be replaced by each other to obtain the corresponding monodisperse or polydisperse products.

[0168] The reactive groups involved in the methods also include their protected forms, and the protected forms can be deprotected at any suitable step in the actual preparation process to obtain the corresponding active forms.

[0169] The actual preparation process can include any necessary micro-modification, intermediate preparation, post-treatment or purification process familiar to those skilled in the art, whether mentioned in the present application or not. The micro-modification includes but is not limited to any one of protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, acidification, and basification.

[0170] The alternative range of the coupling reaction is not particularly limited, as long as two identical or different reactive groups can form a covalent linkage through the reaction. The covalent linkage can be a linkage based on a linkage or a linker. The coupling reaction in the same preparation process can contain a single step or a stepwise coupling reaction, and preferably each step of the coupling reaction is independently any one of an alkylation reaction, a condensation reaction, an amidation reaction, an esterification reaction, a thioesterification reaction, a ring-opening reaction, a ring-closing condensation reaction, an addition reaction, a cycloaddition reaction, an α, β-unsaturated bond addition reaction, an alkynyl addition reaction, a Schiff base reaction combined with a reduction reaction, a click reaction, an azide-alkyne addition reaction, a 1,3-dipolar cycloaddition reaction, a Diels-Alder addition reaction, a thiol-yne reaction, a thiol-ene reaction, a thiol-vinyl reaction, and a condensation reaction. The reaction conditions of the coupling reaction are related to the type of covalent linkage generated by the reaction, and the existing published technology can be used. The coupling reaction can generate a linkage, a stable linker or a cleavable linker.

[0171] In some specific embodiments, the method for preparing a polyethylene glycolylated lipid represented by general formula (1) comprises the following steps: (i) coupling a functionalized polyethylene glycolylated lipid with a targeting ligand; (ii) optionally, performing a deprotection reaction;

[0172] The functionalized polyethylene glycolylated lipid includes but is not limited to:

[0173] and the polyethylene glycolylated lipid with a terminal functional group disclosed in CN115515926A, wherein the polymerization degree of the polyethylene glycol chain and the polydispersity index of the polyethylene glycolylated lipid are not particularly limited, including but not limited to any optional value disclosed in the aforementioned documents;

[0174] The targeting ligand is preferably a compound containing two to four N-acetylgalactosamine groups, wherein the hydroxyl group of the N-acetylgalactosamine moiety is preferably protected by an acetyl group or a benzoyl group; the targeting ligand also contains any one of the functional groups capable of reacting with the functionalized polyethylene glycolylated lipid, such as an amino group, a secondary amine group, a hydroxyl group, a thiol group, a carboxyl group, an azido group, an alkenyl group, an alkynyl group, an isocyanate group, an isothiocyanate group, a halogen group, an acyl halide group, a sulfonate group, an active ester group, an acid anhydride group and an epoxy group;

[0175] The deprotection reaction converts the protected targeting group into an unprotected form.

[0176] In this application, the monovalent, divalent, trivalent and quadrivalent targeting polyethylene glycolylated lipids are optionally prepared by any one of the following general preparation methods:

[0177] General preparation method one:

[0178] The O-acetylated targeting ligand (10 eq; e.g. GalNAc-01, (GalNAc)2-01, (GalNAc)3-01a, (GalNAc)4-01a, etc.) is optionally deprotected from other specific protecting groups (e.g. tBu or Boc under TFA / DCM conditions, Cbz or Bn under H2, Pd / C, MeOH conditions, TBS or TIPS under TBAF / THF conditions) to give an intermediate containing a free carboxyl, amino or hydroxyl group. The intermediate is dissolved in dichloromethane, and functionalized polyethyleneglycolated lipid (1 eq; e.g. PEG-L-01~PEG-L-20), 4-dimethylaminopyridine (DMAP, 2 eq) and N,N'-dicyclohexylcarbodiimide (DCC, 15 eq) are added sequentially. After stirring at room temperature for 24 h, the precipitate is removed by filtration. The filtrate is dried over anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography to give an O-acetylated intermediate (e.g. GPL-0102-OAc).

[0179] Deacetylation (method one): The aforementioned O-acetylated intermediate is dissolved in anhydrous methanol, and a catalytic amount of metallic sodium is added. The mixture is stirred at room temperature for 3 h. After the reaction is completed, the reaction mixture is acidified to pH = 7.4 using an acidic resin. The solid is washed with anhydrous methanol. The organic phases are combined, dried over anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography to give the targeting polyethyleneglycolated lipid.

[0180] Deacetylation (method two): The aforementioned O-acetylated intermediate is dissolved in ethanol at room temperature, and aqueous ammonia is added. After heating at 40 °C for 48 h, the reaction mixture is concentrated under reduced pressure, and purified by column chromatography to give the targeting polyethyleneglycolated lipid.

[0181] General preparation method two:

[0182] The polyethyleneglycolated lipid containing a free hydroxyl group (1 eq; e.g. PEG-L-03), N,N'-succinimidyl carbonate (DSC, 12 eq) and triethylamine (TEA, 30 eq) are dissolved in anhydrous dichloromethane, and the reaction is stirred at room temperature overnight. A dichloromethane solution of the targeting ligand containing a free amino group and an O-acetyl group (12 eq; e.g. the amine compound obtained by removing the Boc protection of (GalNAc)3-03d) is added, and the reaction is stirred at room temperature for another 24 h. After the reaction is completed, the reaction mixture is washed with saturated sodium chloride solution. The organic phase is dried over anhydrous magnesium sulfate, filtered, concentrated and the residue is purified by column chromatography to give an O-acetylated intermediate (e.g. G3PL-03d03-OAc).

[0183] The removal of the O-acetyl group is the same as in General Preparation 1.

[0184] General Preparation 3:

[0185] The targeting ligand is coupled to a bifunctional polyethylene glycol derivative to give a targeting ligand-modified polyethylene glycol intermediate. The intermediate is coupled to a reactive group-containing lipid compound to give a targeting ligand-modified pegylated lipid. The preparation process also includes any necessary protection / deprotection. The reactive group-containing lipid compound includes, but is not limited to, the following structures:

[0186] In the present application, any one of the polydisperse PEG starting materials referred to in the preparation methods or examples can be replaced by a corresponding monodisperse PEG starting material having the same chemical structure, and a monodisperse targeting pegylated lipid is thus obtained, and vice versa. The degree of polymerization before and after the replacement can be the same or different. For example, based on the preparation methods of GPL-0102 (Example 6.1), GPL-03N02 (Example 6.5), and G3PL-01b01 (Example 8.1), monodisperse products GP(45)L-0102, GP(45)L-03N02, G3P(43)L-01b01, and G3P(45)L-01b01 having the following structures can be obtained by the aforementioned method:

[0187] 4. Lipid compositions, lipid pharmaceutical compositions, and formulations thereof

[0188] One embodiment of the present application:

[0189] A lipid composition comprising a targeting pegylated lipid; the targeting pegylated lipid is a targeting ligand-modified pegylated lipid represented by formula (1).

[0190] In some specific embodiments, the lipid composition further comprises one or more non-targeting pegylated lipids and / or one or more cationic lipids and / or one or more phospholipids and / or one or more sterol lipids and / or one or more anionic lipids.

[0191] In some specific embodiments, the non-targeting pegylated lipid is selected from the group consisting of polyethylene glycol-dipalmitoyl phosphatidylcholine (PEG-DPPC), polyethylene glycol-dimyristoleate (PEG-DMG), polyethylene glycol-distearyl phosphatidyl ethanolamine (PEG-DSPE), polyethylene glycol-dioleoyl phosphatidyl ethanolamine (PEG-DOPE), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-diacyl glycerol (PEG-DAG), and polyethylene glycol-dialkyloxypropyl (PEG-DAA).

[0192] In some particular embodiments, the non-targeted pegylated lipid is selected from the group consisting of PEG 500-dipalmitoyl phosphatidyl choline, PEG 2000-dipalmitoyl phosphatidyl choline, PEG 500-distearyl phosphatidyl ethanolamine, PEG 2000-distearyl phosphatidyl ethanolamine, PEG 500-dioleoyl phosphatidyl ethanolamine, PEG 2000-dioleoyl phosphatidyl ethanolamine, PEG 500-dimyristoyl glycerol, and PEG 2000-dimyristoyl glycerol (PEG2k-DMG).

[0193] In some particular embodiments, the non-targeted pegylated lipid is selected from the group consisting of the following structures:

[0194] wherein n1 is an integer from 10 to 250.

[0195] In some particular embodiments, the phospholipid is selected from 1,2-dilinoleoyl- sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- ditridecanyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl- sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoyl- phosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl- phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1- stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl- phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0196] In some particular embodiments, the sterol lipid is selected from cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, soysterol, rapeseed sterol, tomatidine, and ursolic acid.

[0197] In some specific embodiments, the cationic lipid is selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate) salt (DOTAP), 1,2-dioleyloxy-3-trimethylammonium propane chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazoline chloride (DOTIM), 1,2-dioleoyl-3-dimethylamino-propane (DODMA), 2,3-bis(tetradecanoyloxy)propyltrimethylammonium chloride (DMTAP), didecyldimethylammonium chloride (DDAC), didecyldimethylammonium bromide (DDAB), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester (DLin-MC3-DMA), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), ((4-hydroxybutyl)azanediyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102),

[0198] In some specific embodiments, the anionic lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (18:1 PA), 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt (14:0 PA), bis(monooleoylglycero)phosphate ammonium salt (18:1 BMP), and cardiolipin (CL).

[0199] In some specific embodiments, the lipid composition consists of one cationic lipid, one phospholipid, one steroloid lipid, and one targeted PEGylated lipid, optionally further comprising one non-targeted PEGylated lipid.

[0200] In some specific embodiments, the molar percentage of the cationic lipid in the total lipid of the lipid composition is preferably from 30% to 65%, more preferably from 35% to 55%.

[0201] In some specific embodiments, the molar percentage of the phospholipid in the total lipid in the lipid composition is preferably 2% to 15%, more preferably 5% to 12%.

[0202] In some specific embodiments, the molar percentage of the sterol lipid in the total lipid in the lipid composition is preferably 25% to 50%, more preferably 38% to 50%.

[0203] In some specific embodiments, the molar percentage of the total polyethylene glycolated lipid in the total lipid in the lipid composition is preferably 0.5% to 10%, more preferably 1% to 3%, more preferably 1.2% to 2.1%; the total polyethylene glycolated lipid consists of non-targeted polyethylene glycolated lipid and targeted polyethylene glycolated lipid.

[0204] In some specific embodiments, the molar percentage of the targeted polyethylene glycolated lipid in the total lipid in the lipid composition is preferably 0.001% to 10%, more preferably 0.005% to 1.5%, more preferably 0.15% to 0.65%, more preferably 0.5%.

[0205] In some specific embodiments, the molar percentage of the cationic lipid, the phospholipid, the sterol lipid, the total polyethylene glycolated lipid in the total lipid in the lipid composition is 60%, 7.5%, 31%, 1.5%, respectively, or is 57.5%, 7.5%, 33.5%, 1.5%, respectively, or is 55%, 7.5%, 36%, 1.5%, respectively, or is 50%, 10%, 38.5%, 1.5%, respectively, or is 48%, 9%, 41.5%, 1.5%, respectively.

[0206] An embodiment of the present application:

[0207] A lipid pharmaceutical composition comprising any one of the aforementioned lipid compositions, and further comprising one or more than one pharmaceutically active ingredient.

[0208] In some specific embodiments, the pharmaceutically active ingredient is selected from any one of nucleic acid, small molecule, polypeptide, and protein.

[0209] In some specific embodiments, the pharmaceutically active ingredient is nucleic acid.

[0210] In some specific embodiments, the pharmaceutically active ingredient is selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, and ribozyme.

[0211] In some specific embodiments, the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA, and siRNA.

[0212] In some specific embodiments, the pharmaceutically active ingredient is selected from any one of DNA, mRNA, miRNA, and siRNA.

[0213] One embodiment of the present application:

[0214] Use of any one of the preceding lipid pharmaceutical compositions in the preparation of a therapeutic agent.

[0215] In some specific embodiments, the therapeutic agent is selected from any one of an antitumor agent, an antibiotic agent, an antiviral agent, an antifungal agent, an antiparasitic agent, and a vaccine.

[0216] In some specific embodiments, the therapeutic agent is selected from therapeutic agents for treating liver diseases.

[0217] In some specific embodiments, the liver disease is selected from any one of liver fibrosis, liver cirrhosis, and liver cancer.

[0218] One embodiment of the present application:

[0219] A lipid pharmaceutical composition preparation comprising any one of the preceding lipid pharmaceutical compositions, and further comprising a working solution.

[0220] In some specific embodiments, the working solution is a pharmaceutically acceptable diluent or excipient.

[0221] In some specific embodiments, the working solution is selected from any one of deionized water, ultrapure water, phosphate buffer, and physiological saline.

[0222] In some specific embodiments, the working solution is a phosphate buffer or physiological saline, preferably physiological saline.

[0223] In some specific embodiments, the lipid pharmaceutical composition in the lipid pharmaceutical composition preparation forms a drug-loaded lipid nanoparticle. DETAILED DESCRIPTION

[0224] The following specific examples are further to illustrate the present application, but not to limit the scope of protection of the present application. Both intermediates and final products can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, counter-precipitation, column chromatography (silica gel column chromatography, ion column chromatography, gel column chromatography, etc.), membrane dialysis, or supercritical extraction, etc. The characterization of structure and molecular weight can be performed by methods including but not limited to nuclear magnetic resonance, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, mass spectrometry, circular dichroism, etc. In the following examples, the molecular weight of the polymer product is determined by MALDI-TOF MS, and the polydispersity index (PDI) is determined by gel permeation chromatography (GPC).

[0225] Example 1: Synthesis of small molecule intermediates

[0226] Example 1.1: Synthesis of compound S-01

[0227] Step 1: D-galactosamine pentaacetate (a1, 2.00 g, 5.15 mmol) was dissolved in 20 mL of anhydrous 1,2-dichloroethane (DCE), and trimethylsilyl trifluoromethanesulfonate (TMSOTf, 1.0 mL, 5.4 mmol) was added. After the mixture was stirred at 60 °C for 4 h, it was cooled to room temperature, and triethylamine (TEA, 2.9 mL, 20.6 mmol) was added dropwise. Stirring was continued for 15 min, and the mixture was diluted with dichloromethane (DCM, 60 mL) and washed with saturated sodium bicarbonate solution (40 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography gave compound a2 (1.26 g).

[0228] Step 2: Compound a2 (0.99 g, 3.0 mmol) was dissolved in 20 mL of anhydrous 1,2-dichloroethane, a small amount of dry molecular sieve powder was added, and 5-hexen-1-ol (a3, 0.33 g, 3.3 mmol) was added. After stirring at room temperature for 30 min, the reaction system was placed in an ice bath, and TMSOTf (0.3 mL, 1.5 mmol) was added under nitrogen protection. The ice bath was removed, and the reaction was stirred overnight. After the reaction was completed, the molecular sieve powder was removed by filtration. The filtrate was diluted with 60 mL of dichloromethane, filtered with celite, washed with saturated sodium bicarbonate solution (40 mL*2) and saturated sodium chloride solution (40 mL*2), and dried over anhydrous magnesium sulfate. After filtration, the solution was concentrated under reduced pressure, and purification by column chromatography gave compound a4 (1.13 g).

[0229] Step 3: a4 (0.86 g, 2.0 mmol) was dissolved in a mixture of 10 mL of dichloromethane and 10 mL of acetonitrile, 10 mL of deionized water and sodium periodate (0.47 g, 2.2 mmol) were added, and stirring was performed in an ice bath for 10 min. A catalytic amount of ruthenium trichloride was added, and the reaction was performed at room temperature overnight. After the reaction was completed, 30 mL of water was added to dilute the solution, and the pH was adjusted to about 7.5 with saturated sodium bicarbonate solution. The aqueous phase was extracted with dichloromethane (20 mL*3), and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with citric acid, and the solution was extracted with dichloromethane (20 mL*3). The combined organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to give compound S-01 (0.71 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 1H, H-4Gal), 5.00-4.96 (m, 1H, H-3Gal), 4.48 (d, 1H, H-1Gal), 4.10-3.96 (m, 3H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.89 (m, 1H, H-2Gal), 3.68-3.52 (m, 1H, -OCH a H b CH2-), 3.46-3.33 (m, 1H, -OCH a H b CH2-), 2.31-2.16 (m, 2H, -CH2COOH), 2.09 (s, 3H, -Ac), 2.00 (s, 3H, -Ac), 1.90 (s, 3H, -Ac), 1.81 (s, 3H, -Ac), 1.64-1.50 (m, 4H, -CH2CH2CH2COOH). MS (ESI): m / z = 448.0 ([M+H] + ).

[0230] Example 1.2: Synthesis of compound S-02

[0231] Step 1: Compound a2 (1.97 g, 6.0 mmol) and azido-triglycol (a5, 1.58 g, 9.0 mmol) were dissolved in 1,2-dichloroethane (20 mL). A small amount of dry molecular sieve powder was added, and after stirring at room temperature for 30 min, TMSOTf (0.5 mL, 3.0 mmol) was added, and the reaction was stirred overnight. After the reaction was completed, the mixture was filtered with celite. The filtrate was diluted with 60 mL of dichloromethane and washed with saturated sodium bicarbonate solution (40 mL*2) and water (40 mL*2), and dried over anhydrous magnesium sulfate. After filtration, it was concentrated under reduced pressure and purified by column chromatography to give compound a6 (2.45 g).

[0232] Step 2: Compound a6 (2.02 g, 4.0 mmol) was dissolved in tetrahydrofuran (THF, 40 mL), and triphenylphosphine (PPh3, 1.26 g, 4.8 mmol) was added. The mixture was stirred at room temperature for two days. After TLC analysis showed that the starting material was consumed, water (0.2 mL, 12.0 mmol) was added, and the reaction was stirred for 24 h. Trifluoroacetic acid (TFA, 0.6 mL, 8.0 mmol) and toluene (Tol, 40 mL) were added, and the mixture was concentrated to near dryness, co-evaporated with toluene (40 mL*2), and finally purified by column chromatography to give compound S-02 (1.27 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.21 (d, 1H, H-4Gal), 4.99-4.95 (m, 1H, H-3Gal), 4.46 (d, 1H, H-1Gal), 4.08-3.94 (m, 3H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.87 (m, 1H, H-2Gal), 3.82-3.66 (m, 10H, -OCH2CH2O-, -OCH2CH2NH2), 3.20-3.12 (m, 2H, -OCH2CH2NH2), 2.08 (s, 3H, -Ac), 2.01 (s, 3H, -Ac), 1.92 (s, 3H, -Ac), 1.84 (s, 3H, -Ac). MS (ESI): m / z = 479.0 ([M+H] + ).

[0233] Example 1.3: Synthesis of compounds X-01 and X-07

[0234] Step 1: tris(hydroxymethyl)aminomethane (b1, 1.19 g, 5.0 mmol) was dissolved in 20 mL of dichloromethane, TEA (2.8 mL, 20.2 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the reaction was stirred at 40 °C overnight. After the reaction was completed, the mixture was diluted with 60 mL of dichloromethane, and then washed with saturated sodium bicarbonate solution (40 mL*2) and saturated sodium chloride solution (40 mL*2) in turn. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound b2 (2.12 g).

[0235] Step 2: Compound b2 (1.88 g, 4.0 mmol) was dissolved in 20 mL of N,N- dimethylformamide (DMF), 3-hydroxypropionic acid tert-butyl ester (b3, 1.93 g, 13.2 mmol) and potassium carbonate (K2CO3, 2.21 g, 16.0 mmol) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and poured into 20 mL of dichloromethane. After washing with 10% citric acid (10 mL*2) and saturated sodium chloride solution (10 mL*2) in turn, it was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound X-07 (2.27 g). 1H NMR (400 MHz, (CD3)2SO) d: 7.40-7.27 (m, 5H, Ar-H), 5.03 (s, 2H, Ar-CH2-), 3.66 (s, 6H, (-OCH2)3C-), 3.64 (t, 6H, -OC(=O)CH2CH2O-), 2.44 (t, 6H, -OC(=O)CH2CH2O-), 1.43 (s, 27H, (CH3)3C-). MS (ESI): m / z = 640.1 ([M+H] + ).

[0236] Step 3: Compound X-07 (1.92 g, 3.0 mmol) was dissolved in TFA / DCM mixed solution (1:1 v / v; 20 mL) and stirred for 3 h to remove tBu. After the reaction was completed, it was washed with purified water (10 mL*2), and the aqueous phase was extracted with dichloromethane (10 mL*2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. To the filtrate were sequentially added 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCI, 1.90 g, 9.9 mmol), 1-hydroxybenzotriazole hydrate (HOBt H2O, 1.51 g, 9.9 mmol), N-tert-butoxycarbonyl-1,3-propanediamine (b4, 1.72 g, 9.9 mmol), and N,N-diisopropylethylamine (DIPEA, 1.28 g, 9.9 mmol), and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was sequentially washed with 10% citric acid (20 mL*2), saturated sodium bicarbonate solution (20 mL*2), and saturated sodium chloride solution (20 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound X-01 (2.43 g). 1 H NMR (400 MHz, (CD3)2SO) d: 7.39-7.28 (m, 5H, Ar-H), 5.01 (s, 2H, Ar-CH2-), 3.64 (s, 6H, (-OCH2)3C-), 3.62 (t, 6H, -NHC(=O)CH2CH2O-), 3.26-3.23 (m, 6H, -CH2NHC(=O)-), 3.10-3.08 (m, 6H, BocNHCH2-), 2.41 (t, 6H, -NHC(=O)CH2CH2O-), 1.60-1.58 (m, 6H, -CH2CH2CH2-), 1.41 (s, 27H, (CH3)3C-). MS (ESI): m / z = 940.4 ([M+H] + ).

[0237] Example 1.4: Synthesis of compound X-02

[0238] N-Boc-N'-Cbz-L-lysine (b5, 1.14 g, 3.0 mmol) was dissolved in 15 mL of dichloromethane, followed by the addition of EDC HCI (0.63 g, 3.3 mmol) and HOBt H20 (0.50 g, 3.3 mmol), and then a solution of compound b6 (1.32 g, 3.3 mmol, obtained by reacting 3,3'-diaminodipropylamine with benzyl chloroformate) in dichloromethane (15 mL) was added. DIPEA (0.43 g, 3.3 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed with 10% citric acid (15 mL*2), saturated sodium bicarbonate solution (15 mL*2), and saturated sodium chloride solution (15 mL*2) in sequence. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound X-02 (1.66 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 7.40-7.27 (m, 15H, Ar-H), 5.02 (s, 6H, Ar-CH2-), 4.58-4.43 (m, 1H, >CH-), 3.48-3.35 (m, 4H, -C(=0)N(CH2-)2), 3.08-2.94 (m, 6H, CbzHNCH2-), 1.80-1.25 (m, 19H, CbzHNCH2CH2CH2N<, CbzHNCH2CH2CH2CH<, -C(CH3)3). MS (ESI): m / z = 762.2 ([M+H] + ).

[0239] Example 1.5: Synthesis of compound X-03

[0240] Boc-D-glutamic acid-1-benzyl ester (b7, 1.01 g, 3.0 mmol) was dissolved in 15 mL of dichloromethane, followed by the addition of EDC HCI (0.63 g, 3.3 mmol) and HOBt H20 (0.50 g, 3.3 mmol), and then a solution of 1,7-bis-Boc-1,4,7-triazahcpatane (b8, 1.00 g, 3.3 mmol) in dichloromethane (15 mL) was added. DIPEA (0.43 g, 3.3 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed with 10% citric acid (15 mL*2), saturated sodium bicarbonate solution (15 mL*2), and saturated sodium chloride solution (15 mL*2) in sequence. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound X-03 (1.40 g). 1H NMR (400 MHz, (CD3)2SO) d: 7.36-7.32 (m, 5H, Ar-H), 5.23-5.11 (m, 2H, Ar-CH2-), 4.42-4.36 (m, 1H, >CH-), 3.25-3.17 (m, 8H, Boc HNCH2CH2N<), 2.40-1.98 (m, 4H, >NC(=0)CH2CH2CH<), 1.43 (s, 27H, -C(CH3)3). MS (ESI): m / z = 623.1 ([M+H] + ).

[0241] Example 1.6: Synthesis of compound X-04

[0242] Compound b9 (1.01 g, 3.0 mmol, obtained from lysine reacted with benzyl chloroformate and then reacted with tert-butyl acetate) was dissolved in 15 mL of dichloromethane, and EDC HCI (0.63 g, 3.3 mmol) and HOBt H20 (0.50 g, 3.3 mmol) were added successively, followed by the addition of a dichloromethane solution (15 mL) containing compound b10 (1.24 g, 3.0 mmol, obtained from lysine reacted with benzyl chloroformate). DIPEA (0.43 g, 3.3 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (15 mL*2), saturated sodium bicarbonate solution (15 mL*2), and saturated sodium chloride solution (15 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain compound X-04 (1.57 g). 1 H NMR (400 MHz, (CD3)2SO) d: 7.36-7.32 (m, 5H, Ar-H), 5.23-5.11 (m, 2H, Ar-CH2-), 4.42-4.36 (m, 1H, >CH-), 3.25-3.17 (m, 8H, Boc HNCH2CH2N<), 2.40-1.98 (m, 4H, >NC(=0)CH2CH2CH<), 1.43 (s, 27H, -C(CH3)3). MS (ESI): m / z = 623.1 ([M+H] + ).

[0243] Example 1.7: Synthesis of compound X-05

[0244] Compound bll (1.11 g, 4.0 mmol, obtained from the reaction of 1,5- dehydrate-D-sorbitol with triisopropylchlorosilane) and tert-butyl acrylate (b12, 2.56 g, 20.0 mmol) were dissolved in 30 mL of dichloromethane, and tetrabutylammonium bromide (TBAB, 0.26 g, 0.8 mmol) and 50 wt% NaOH solution (1.60 g, 20 mmol) were added. After stirring the reaction at 20 °C for 3 h, 30 mL of water was added and mixed well. The aqueous phase was separated and extracted with dichloromethane (15 mL*2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give compound X-05 (2.23 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 3.98-3.60 (m, 9H, H-l, H-6, H-6', -OC(=0)CH2CH20-), 3.36-3.22 (m, 4H, H-2, H-3, H-4, H-5), 3.11-3.07 (m, 1H, H-l'), 2.56-2.53 (m, 6H, -OC(=0)CH2CH20-), 1.43 (s, 27H, tBu), 1.14-1.01 (m, 21H, TIPS). MS (ESI): m / z = 705.3 ([M+H] + ).

[0245] Example 1.8: Synthesis of compound X-06

[0246] Compound b13 (1.01 g, 3.0 mmol, obtained from the reaction of L-glutamic acid-5-benzyl ester with di-tert-butyl dicarbonate) was dissolved in 15 mL of dichloromethane, and EDC HCI (0.63 g, 3.3 mmol) and HOBt H20 (0.50 g, 3.3 mmol) were added successively, followed by the addition of a dichloromethane solution (15 mL) of compound b14 (0.98 g, 3.0 mmol, obtained from the reaction of glutamic acid with benzyl alcohol). DIPEA (0.43 g, 3.3 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (15 mL*2), saturated sodium bicarbonate solution (15 mL*2), and saturated sodium chloride solution (15 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give compound X-06 (1.82 g). 1H NMR (400 MHz, (CD3)2SO) d: 7.38-7.33 (m, 15H, Ar-H), 5.20-5.10 (m, 6H, Ar-CH2-), 4.62-4.54 (m, 1H, >CHC(=0)OBn), 4.26-4.17 (m, 1H, >CHNHBoc), 2.48-2.26 (m, 4H, -OC(=0)CH2CH2CH<), 2.24-1.84 (m, 4H, -OC(=0)CH2CH2CH<), 1.43 (s, 9H, Boc). MS (ESI): m / z = 647.1 ([M+H] + ).

[0247] Example 1.9: Synthesis of compound N-01

[0248] Step 1: Dissolve tetradecylamine (b15, 0.85 g, 4.0 mmol) in dichloromethane (10 mL), add 3-chloropropyne (b16, 0.30 g, 4.0 mmol) and TEA (0.8 mL, 6.0 mmol) successively, and stir the reaction at room temperature overnight. After the reaction is completed, remove the solvent under reduced pressure. Partition the residue between ethyl acetate and water. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by column chromatography to obtain compound b17 (0.90 g).

[0249] Step 2: Dissolve myristic acid (b18, 0.68 g, 3.0 mmol) in 10 mL of dichloromethane, add EDC HCI (0.63 g, 3.3 mmol) and HOBt H20 (0.50 g, 3.3 mmol) successively, and then add a dichloromethane solution (10 mL) of compound b17 (0.75 g, 3.0 mmol). Add DIPEA (0.43 g, 3.3 mmol), and stir the reaction at room temperature overnight. After the reaction is completed, wash the reaction liquid successively with 10% citric acid (10 mL*2), saturated sodium bicarbonate solution (10 mL*2), and saturated sodium chloride solution (10 mL*2). Dry the organic phase over anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain compound N-01 (1.12 g). 1 H NMR (400 MHz, (CD3)2SO) d: 4.22 & 4.05 (2d, 2H, -CH2CºCH), 3.40 (dt, 2H, >NCH2-), 2.43-2.22 (m, 3H, -CºCH, >NC(=0)CH2-), 1.58-1.22 (m, 46H, -CH2(CH2) 11 CH3, -CH2(CH2) 12H NMR (400 MHz, (CD3)2SO) δ: 4.21 (d, 2H, -CH2C≡CH), 3.55 (t, 2H, -OCH2CH2N<), 3.35 (t, 2H, -OCH2CH2N<), 3.19 (t, 2H, >NCH2-), 2.42 (t, 1H, -C≡CH), 2.22 (t, 2H, >NC(=O)CH2-), 1.60 - 1.22 (m, 46H, -CH2(CH2) + ).

[0250] Example 1.10: Synthesis of compound N-02

[0251] Step 1: 2-(2-Propynoxy)ethanamine (b19, 0.40 g, 4.0 mmol) was dissolved in dichloromethane (12 mL), and bromotetradecane (b20, 1.11 g, 4.0 mmol) and TEA (0.8 mL, 6.0 mmol) were added successively. The reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed under reduced pressure. The residue was partitioned with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound b21 (1.02 g).

[0252] Step 2: b18 (0.68 g, 3.0 mmol) was dissolved in 10 mL of dichloromethane, and EDC HCI (0.63 g, 3.3 mmol) and HOBt H2O (0.50 g, 3.3 mmol) were added successively, followed by the addition of a dichloromethane solution (10 mL) of compound b21 (0.89 g, 3.0 mmol). DIPEA (0.43 g, 3.3 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (10 mL*2), a saturated sodium bicarbonate solution (10 mL*2), and a saturated sodium chloride solution (10 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give compound N-02 (1.34 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.21 (d, 2H, -CH2C≡CH), 3.55 (t, 2H, -OCH2CH2N<), 3.35 (t, 2H, -OCH2CH2N<), 3.19 (t, 2H, >NCH2-), 2.42 (t, 1H, -C≡CH), 2.22 (t, 2H, >NC(=O)CH2-), 1.60 - 1.22 (m, 46H, -CH2(CH2) 11 CH3,-CH2(CH2) 12 H NMR (400 MHz, (CD3)2SO) δ: 4.21 (d, 2H, -CH2C≡CH), 3.55 (t, 2H, -OCH2CH2N<), 3.35 (t, 2H, -OCH2CH2N<), 3.19 (t, 2H, >NCH2-), 2.42 (t, 1H, -C≡CH), 2.22 (t, 2H, >NC(=O)CH2-), 1.60 - 1.22 (m, 46H, -CH2(CH2) + ).

[0253] Example 1.11: Synthesis of compound N-03

[0254] Compound b21 (0.59 g, 2.0 mmol) was dissolved in acetonitrile (10 mL), and compound b20 (0.55 g, 2.0 mmol), potassium carbonate (0.83 g, 6.0 mmol) and potassium iodide (0.33 g, 2.0 mmol) were added successively. The reaction was stirred at 85 °C overnight. After the reaction was completed, the reaction solution was cooled to room temperature, and then filtered with diatomite. The filter cake was washed with ethyl acetate. The filtrate and washing liquid were combined, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound N-03 (0.77 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.23 (d, 2H, -CH2C≡CH), 3.60 (t, 2H, -OCH2CH2N<), 2.69 (t, 2H, -OCH2CH2N<), 2.55 (t, 4H, >NCH2-), 2.44 (t, 1H, -C≡CH), 1.54-1.19 (m, 48H, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). MS (ESI): m / z = 492.4 ([M+H] + ).

[0255] Example 1.12: Synthesis of compound N-04

[0256] Step 1: Boc-glycine (b23, 1.05 g, 6.0 mmol) was dissolved in 15 mL of dichloromethane, and EDC HCl (1.27 g, 6.6 mmol) and HOBt H2O (1.01 g, 6.6 mmol) were added successively, followed by the addition of a dichloromethane solution (5 mL) containing propargylamine (b22, 0.33 g, 6.0 mmol). DIPEA (0.85 g, 6.6 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (10 mL*2), saturated sodium bicarbonate solution (10 mL*2) and saturated sodium chloride solution (10 mL*2). The organic phase was concentrated, and the residue was dissolved in a TFA / DCM mixed solution (1:1 v:v, 20 mL). The reaction was stirred for 3 h to remove the Boc protecting group. After the deprotection was completed, the reaction solution was washed with purified water (10 mL*2). The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound b24 (0.61 g).

[0257] Step 2: Compound b24 (0.45 g, 4.0 mmol) was dissolved in dichloromethane (12 mL), followed by the addition of b20 (1.11 g, 4.0 mmol) and TEA (0.8 mL, 6.0 mmol). The reaction was stirred at room temperature overnight. After the reaction was completed, the solvent was removed under reduced pressure. The residue was partitioned with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound b25 (0.98 g).

[0258] Step 3: Compound b25 (0.62 g, 2.0 mmol) was dissolved in acetonitrile (10 mL), followed by the addition of compound b20 (0.55 g, 2.0 mmol), potassium carbonate (0.83 g, 6.0 mmol), and potassium iodide (0.33 g, 2.0 mmol). The reaction was stirred at 85 °C overnight. After the reaction was completed, the reaction solution was cooled to room temperature and filtered with celite. The filter cake was washed with ethyl acetate. The filtrate and the washing solution were combined and concentrated under reduced pressure. The residue was purified by column chromatography to give compound N-04 (0.80 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.04 (dd, 2H, -CH2C≡CH), 3.00 (s, 2H, -NHC(=O)CH2-), 2.50 (t, 4H, >NCH2-), 2.20 (t, 1H, -C≡CH), 1.57-1.20 (m, 48H, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). MS (ESI): m / z = 505.3 ([M+H] + ).

[0259] Example 2: Synthesis of monovalent targeting ligand

[0260] Example 2.1: Synthesis of compound GalNAc-01

[0261] Compound S-01 (1.79 g, 4.0 mmol) and EDC HCI (1.15 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane, followed by the addition of ethylenediamine (SM-D-01, 0.72 g, 12.0 mmol) and 4-dimethylaminopyridine (DMAP, 0.10 g, 0.8 mmol). The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was diluted with 40 mL of dichloromethane and washed with water (30 mL*2), 1 M HCI solution (30 mL*2), saturated sodium bicarbonate solution (30 mL*2), and saturated sodium chloride solution (30 mL*2) in sequence. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give compound GalNAc-01 (1.58 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.22 (d, 1H, H-4Gal), 5.01-4.97 (m, 1H, H-3Gal), 4.48 (d, 1H, H-1Gal), 4.09-3.96 (m, 3H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.87 (m, 1H, H-2Gal), 3.65-3.51 (m, 1H, -OCH a H b CH2-), 3.46-3.32 (m, 3H, -OCH a H b CH2-), 3.46-3.32 (m, 3H, -OCH + .

[0262] Example 2.2: Synthesis of compound GalNAc-02

[0263] Compound S-01 (1.79 g, 4.0 mmol) and EDC HC1 (1.15 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane, ethyl 2-(2-(2-(2-hydroxyethoxy)-ethoxy)ethoxy)carbamate tert-butyl ester (SM-D-01-Boc, 1.49 g, 6.0 mmol) and DMAP (0.10 g, 0.8 mmol) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, 40 mL of dichloromethane was added for dilution, and then sequentially washed with water (30 mL*2), 1M HC1 solution (30 mL*2), saturated sodium bicarbonate solution (30 mL*2), and saturated sodium chloride solution (30 mL*2). The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound GalNAc-02 (2.16 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 1H, H-4Gal), 5.03-4.98 (m, 1H, H-3Gal), 4.45 (d, 1H, H-1Gal), 4.07-3.95 (m, 3H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.86 (m, 1H, H-2Gal), 3.70-3.38 (m, 12H, -OCH2(CH2)3-, -OCH2CH2O-, -OCH2CH2NHBoc), 3.38-3.21 (m, 2H, -C(=O)NHCH2CH2O-, -CH2NHBoc), 2.13 (t, 2H, -CH2C(=O)NH-), 2.10 (s, 3H, -Ac), 2.01 (s, 3H, -Ac), 1.88 (s, 3H, -Ac), 1.85-1.58 (m, 7H, -Ac, -CH2(CH2)2CH2-), 1.43 (s, 9H, Boc). MS (ESI): m / z = 678.1 ([M+H] + ).

[0264] Example 2.3: Synthesis of compound GalNAc-03

[0265] Dissolve 1,5-pentanediol monobenyl ester (SM-A-02-Bn, 0.89 g, 4.0 mmol) and EDC HCI (1.15 g, 6.0 mmol) in 20 mL dichloromethane, add compound S-02 (1.53 g, 3.2 mmol) and DMAP (0.10 g, 0.8 mmol), stir at room temperature overnight. After the reaction is completed, dilute with 40 mL dichloromethane, and then sequentially wash with water (30 mL*2), 1M HCI solution (30 mL*2), saturated sodium bicarbonate solution (30 mL*2) and saturated sodium chloride solution (30 mL*2). Concentrate the organic phase under reduced pressure, dissolve the residue in methanol, add Pd / C catalyst, and bubble with hydrogen gas at room temperature for 14 h to remove the Bn protecting group. Remove the catalyst by filtration with diatomite, evaporate the methanol, and purify the residue by column chromatography to obtain compound GalNAc-03 (1.53 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.18 (d, 1 H, H-4Gal), 4.98-4.93 (m, 1 H, H-3Gal), 4.47 (d, 1 H, H-1 Gal), 4.05-3.93 (m, 3H, H-5Gal, H-6Gal, H-6'Gal), 3.91-3.87 (m, 1 H, H-2Gal), 3.83-3.24 (m, 12H, -OCH2CH2O-, -OCH2CH2NHC(=O)-), 2.28 (t, 2H, -CH2COOH), 2.18 (t, 2H, -NHC(=O)CH2-), 2.08 (s, 3H, -Ac), 2.00 (s, 3H, -Ac), 1.90 (s, 3H, -Ac), 1.83-1.69 (m, 5H, -Ac, -CH2CH2CH2-). MS (ESI): m / z = 593.0 ([M+H] + ).

[0266] Example 3: Synthesis of bivalent targeting ligands

[0267] Example 3.1 : Synthesis of compound (GalNAc)2-01

[0268] Step 1 : Compound c1 (2.90 g, 10.0 mmol, obtained from reaction of 1,3-diamino-2- hydroxypropane with di-tert-butyl dicarbonate), N,N'-succinimidyl carbonate (DSC, 3.07 g, 12.0 mmol) and TEA (4.2 mL, 30.0 mmol) were dissolved in 30 mL of dry dichloromethane and stirred at room temperature overnight. Compound SM-E-04-Cbz (2.83 g, 12.0 mmol, obtained from reaction of 1,5-diaminopentane with benzyl chloroformate) was added and stirring was continued at room temperature for 3 hours. After completion of the reaction, it was washed with saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated. The residue was purified by column chromatography to obtain compound c2 (4.09 g).

[0269] Step 2: Compound c2 (0.88 g, 1.6 mmol) was dissolved in TFA / DCM mixed solution (1:1 v / v; 10 mL) and stirred for 3 h to remove tBu. After the reaction was completed, it was washed with purified water (5 mL*2), and the aqueous phase was extracted with dichloromethane (5 mL*2). The organic phase was combined, dried over anhydrous magnesium sulfate, and filtered. Compound S-01 (1.79 g, 4.0 mmol), EDC HCI (1.15 g, 6.0 mmol), and DMAP (0.10 g, 0.8 mmol) were sequentially added to the filtrate, and the reaction was stirred at room temperature overnight. After the reaction was completed, it was diluted with 20 mL of dichloromethane and sequentially washed with water (20 mL*2), 1 M HCI solution (20 mL*2), saturated sodium bicarbonate solution (20 mL*2), and saturated sodium chloride solution (20 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound (GalNAc)2-01 (1.15 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 7.36-7.23 (m, 5H, Ar-H), 5.25 (d, 2H, H-4Gal), 5.04-5.00 (m, 4H, H-3Gal, Ar-CH2-), 4.69-4.67 (m, 1H, >CH-), 4.50 (d, 2H, H-1Gal), 4.10-3.96 (m, 6H, H-5Gal, H-6Gal, H-6'Gal), 3.91-3.83 (m, 2H, H-2Gal), 3.63-3.15 (m, 12H, -OCH2(CH2)3-, -C(=O)NHCH2CH<-, -OC(=O)NHCH2-), 2.18-2.15 (m, 4H, -CH2C(=O)NH-), 2.14 (s, 6H, -Ac x 2), 2.02 (s, 6H, -Ac x 2), 1.91 (s, 6H, -Ac x 2), 1.84-1.46 (m, 18H, -Ac x 2, -CH2(CH2)2CH2-, -OC(=O)NHCH2CH2-), 1.38-1.30 (m, 2H, -(CH2)2CH2(CH2)2-). MS (ESI): m / z = 1211.4 ([M+H] + ).

[0270] Example 3.2: Synthesis of compound (GalNAc)2-02

[0271] Step 1: Compound SM-A-04-tBu (1.30 g, 6.0 mmol, obtained from heptanedioic acid and tert-butyl acetate) and EDC HCI (1.73 g, 9.0 mmol) were dissolved in 20 mL of dichloromethane, compound b14 (1.57 g, 4.8 mmol) and DMAP (0.15 g, 1.2 mmol) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, it was diluted with 40 mL of dichloromethane and washed sequentially with water (30 mL*2), 1 M HCI solution (30 mL*2), saturated sodium bicarbonate solution (30 mL*2), and saturated sodium chloride solution (30 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound c3 (2.17 g).

[0272] Step 2: Compound c3 (2.10 g, 4.0 mmol) was dissolved in methanol (30 mL), Pd / C catalyst was added, and the reaction was bubbled with hydrogen gas at room temperature for 14 h to remove the Cbz. The catalyst was removed by filtration with celite, and the methanol was evaporated. The residue was dissolved in anhydrous dichloromethane (50 mL). EDC HCI (1.15 g, 6.0 mmol), compound S-02 (4.78 g, 10.0 mmol), and DMAP (0.10 g, 0.8 mmol) were added sequentially, and the reaction was stirred at room temperature overnight. After the reaction was completed, it was diluted with 50 mL of dichloromethane and washed sequentially with water (50 mL*2), 1 M HCI solution (50 mL*2), saturated sodium bicarbonate solution (50 mL*2), and saturated sodium chloride solution (50 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound (GalNAc)2-02 (3.66 g). 1 H NMR (400 MHz, (CD3)2SO) d: 5.19 (d, 2H, H-4Gal), 5.00-4.94 (m, 2H, H-3Gal), 4.49 (d, 2H, H-1Gal), 4.45-4.37 (m, 1H, >CH-), 4.04-3.94 (m, 6H, H-5Gal, H-6Gal, H-6'Gal), 3.89-3.85 (m, 2H, H-2Gal), 3.70-3.24 (m, 24H, -OCH2CH2O-, -OCH2CH2NHC(=O)-), 2.34-2.23 (m, 4H, -NHC(=O)CH2CH2CH<, -CH2C(=O)O-), 2.15-2.11 (m, 3H, -NHC(=O)CH2CH2CH a H bCH<,>CHNHC(=O)CH2-), 2.09 (s, 6H, -Ac x 2), 2.00 (s, 6H, -Ac x 2), 1.97-1.94 (m, 1H, -NHC(=O)CH2CH a H b CH<), 1.90 (s, 6H, -Ac x 2), 1.78 (s, 6H, -Ac x 2), 1.68-1.53 (m, 4H, -CH2CH2CH2CH2CH2-), 1.43 (s, 9H, tBu), 1.38-1.26 (m, 2H, -(CH2)2CH2(CH2)2-). MS (ESI): m / z = 1266.3 ([M+H] + ).

[0273] Example 3.3: Synthesis of compound (GalNAc)2-03

[0274] Step 1: Compound SM-A-02-tBu (0.56 g, 3.0 mmol, obtained from the reaction of glutaric acid and tert-butyl acetate) and EDC HCI (0.86 g, 4.5 mmol) were dissolved in 15 mL of dichloromethane, compound b6 (1.44 g, 3.6 mmol) and DMAP (0.07 g, 0.6 mmol) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, 25 mL of dichloromethane was added for dilution, and then it was washed with water (20 mL*2), 1M HCI solution (20 mL*2), saturated sodium bicarbonate solution (20 mL*2), and saturated sodium chloride solution (20 mL*2) in sequence. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound c4 (1.06 g).

[0275] Step 2: Compound c4 (0.91 g, 1.6 mmol) was dissolved in methanol (15 mL), Pd / C catalyst was added, and the reaction was bubbled with hydrogen gas at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomite, and methanol was evaporated. The residue was dissolved in anhydrous dichloromethane (20 mL). Compound S-01 (1.79 g, 4.0 mmol), and EDC HCI (1.15 g, 6.0 mmol) and DMAP (0.10 g, 0.8 mmol) were added in sequence, and the reaction was stirred at room temperature overnight. After the reaction was completed, 20 mL of dichloromethane was added for dilution, and then it was washed with water (20 mL*2), 1M HCI solution (20 mL*2), saturated sodium bicarbonate solution (20 mL*2), and saturated sodium chloride solution (20 mL*2) in sequence. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound (GalNAc)2-03 (1.21 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.26-5.23 (m, 2H, H-4Gal), 5.00-4.94 (m, 2H, H-3Gal), 4.49 (d, 2H, H-1Gal), 4.10-3.98 (m, 6H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.86 (m, 2H, H-2Gal), 3.68-3.49 (m, 4H, -OCH2CH2-), 3.35-3.15 (m, 8H, -C(=O)NHCH2CH2CH2N<-), 2.44-2.36 (m, 4H, >NC(=O)CH2CH2CH2C(=O)O-), 2.22 (t, 4H, -CH2C(=O)NH-), 2.06 (s, 6H, -Ac x 2), 2.00 (s, 6H, -Ac x 2), 1.93-1.89 (m, 8H, -Ac x 2, >NC(=O)CH2CH2CH2C(=O)O-), 1.83-1.57 (m, 18H, -Ac x 2, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2N<), 1.42 (s, 9H, tBu). MS (ESI): m / z = 1160.4 ([M+H] + ).

[0276] Example 4: Synthesis of trivalent targeting ligands

[0277] Example 4.1: Synthesis of compounds (GalNAc)3-01a~(GalNAc)3-01c

[0278] Compound X-01 (3.76 g, 4.0 mmol) in Example 1 was dissolved in a TFA / DCM mixed solution (1:1 v / v; 40 mL) and stirred for 3 hours to remove tBu. After the reaction was completed, it was washed with purified water (20 mL*2), and the aqueous phase was extracted with dichloromethane (20 mL*3). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. To the filtrate were sequentially added compound S-01 (8.94 g, 20.0 mmol), EDC HCI (4.22 g, 22.0 mmol), HOBt H2O (3.37 g, 22.0 mmol), and DIPEA (2.84 g, 22.0 mmol), and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was sequentially washed with 10% citric acid (50 mL*2), saturated sodium bicarbonate solution (50 mL*2), and saturated sodium chloride solution (50 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound (GalNAc)3-01a (6.48 g). 1H NMR (400 MHz, (CD3)2SO) δ: 7.38-7.26 (m, 5H, Ar-H), 5.00 (s, 2H, Ar-CH2-), 5.26 (d, 3H, H-4Gal), 5.04-4.98 (m, 3H, H-3Gal), 4.45 (d, 3H, H-1Gal), 4.04-3.94 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.88 (m, 3H, H-2Gal), 3.63-3.33 (m, 18H, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, (-OCH2)3C-), 3.30-3.24 (m, 12H, -C(=O)NHCH2CH2CH2NHC(=O)-), 2.26 (t, 6H, -NHC(=O)CH2CH2O-), 2.18-2.13 (m, 6H, -CH2C(=O)NH-), 2.09 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.87 (s, 9H, -Ac x 3), 1.83-1.61 (m, 27H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2NHC(=O)-). MS (ESI): m / z = 1927.6 ([M+H] + ).

[0279] Compound (GalNAc)3-01a (2.57 g, 1.3 mmol) was dissolved in methanol (30 mL), Pd / C catalyst was added, and the reaction was carried out by bubbling with hydrogen gas at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomite, and methanol was evaporated. The residue was dissolved in anhydrous dichloromethane (30 mL). Mono-tert-butyl succinate (SM-A-01-tBu, 0.70 g, 4.0 mmol), EDC HCI (0.84 g, 4.4 mmol), HOBt H2O (0.67 g, 4.4 mmol), and DIPEA (0.57 g, 4.4 mmol) were added in turn, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed with 10% citric acid (15 mL*2), saturated sodium bicarbonate solution (15 mL*2), and saturated sodium chloride solution (15 mL*2) in turn. The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound (GalNAc)3-01b (1.69 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.25 (d, 3H, H-4Gal), 5.06-5.00 (m, 3H, H-3Gal), 4.46 (d, 3H, H-1Gal), 4.07-3.96 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.88 (m, 3H, H-2Gal), 3.67-3.33 (m, 18H, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, (-OCH2)3C-), 3.28-3.23 (m, 12H, -C(=O)NHCH2CH2CH2NHC(=O)-), 2.60 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.45 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.29 (t, 6H, -NHC(=O)CH2CH2O-), 2.18-2.12 (m, 6H, -CH2C(=O)NH-), 2.08 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.88 (s, 9H, -Ac x 3), 1.81-1.57 (m, 27H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2NHC(=O)-), 1.42 (s, 9H, tBu). MS (ESI): m / z = 1949.7 ([M+H] + ).

[0280] Compound (GalNAc)3-01a (2.57 g, 1.3 mmol) was dissolved in methanol (30 mL), Pd / C catalyst was added, and the reaction was carried out by bubbling with hydrogen gas at room temperature for 14 h to remove Cbz. The catalyst was removed by filtration with diatomite, and methanol was evaporated. The residue was dissolved in anhydrous dichloromethane (30 mL). Compound SM-B-01-TBS (0.82 g, 4.0 mmol, obtained by reacting 3-hydroxypropionic acid with tert-butyldimethylsilyl chloride) was added, followed by EDC HCI (0.84 g, 4.4 mmol), HOBt H2O (0.67 g, 4.4 mmol) and DIPEA (0.57 g, 4.4 mmol), and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed with 10% citric acid (20 mL*2), saturated sodium bicarbonate solution (20 mL*2) and saturated sodium chloride solution (20 mL*2) in sequence. The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography to obtain compound (GalNAc)3-01c (1.80 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 3H, H-4Gal), 5.02-4.95 (m, 3H, H-3Gal), 4.44 (d, 3H, H-1Gal), 4.05-3.93 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.89-3.83 (m, 5H, H-2Gal, -NHC(=O)CH2CH2OTBS), 3.65-3.34 (m, 18H, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, (-OCH2)3C-), 3.30-3.22 (m, 12H, -C(=O)NHCH2CH2CH2NHC(=O)-), 2.39 (t, 2H, -NHC(=O)CH2CH2OTBS), 2.24 (t, 6H, -NHC(=O)CH2CH2O-), 2.20-2.14 (m, 6H, -CH2C(=O)NH-), 2.09 (s, 9H, -Ac x 3), 1.98 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.80-1.57 (m, 27H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2NHC(=O)-), 0.89 (s, 9H, -C(CH3)3), 0.07 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1979.8 ([M+H] + ).

[0281] Example 4.2: Synthesis of compounds (GalNAc)3-02a~(GalNAc)3-02c

[0282] Referring to the preparation method of Example 4.1, the compound X-02 in Example 1 was subjected to deprotection of the Cbz protecting group under the condition of H2, Pd / C, MeOH, and then subjected to an amidation reaction with compound S-01 to obtain compound (GalNAc)3-02a. 1HNMR (400 MHz, (CD3)2SO) δ: 5.27-5.24 (m, 3H, H-4Gal), 5.02-4.96 (m, 3H, H-3Gal), 4.56-4.42 (m, 4H, H-1Gal, >CH-), 4.11-3.98 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.84 (m, 3H, H-2Gal), 3.70-3.46 (m, 6H, -OCH2CH2-), 3.36-3.17 (m, 10H, -C(=O)NHCH2-, -CH2N<), 2.23 (t, 6H, -CH2C(=O)NH-), 2.08 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.88 (s, 9H, -Ac x 3), 1.79-1.23 (m, 40H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2N<, -CH2(CH2)3CH<, -C(CH3)3). MS (ESI): m / z = 1647.6 ([M+H] + ).

[0283] Reference to the preparation method of Example 4.1, the compound (GalNAc)3-02a was removed Boc protecting group under TFA / DCM conditions, and then reacted with compound SM-A-02-Bn to obtain compound (GalNAc)3-02b. 1H NMR (400 MHz, (CD3)2SO) δ: 7.36-7.29 (m, 5H, Ar-H), 5.28-5.24 (m, 3H, H-4Gal), 5.19-5.09 (m, 2H, Ar-CH2-), 5.01-4.94 (m, 3H, H-3Gal), 4.48-4.39 (m, 4H, H-1Gal, >CH-), 4.08-3.96 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.85 (m, 3H, H-2Gal), 3.67-3.44 (m, 6H, -OCH2CH2-), 3.37-3.14 (m, 10H, -CH2C(=O)NHCH2-, -CH2N<), 2.32 (t, 2H, -CH2C(=O)OBn), 2.21 (t, 6H, -CH2C(=O)NHCH2-), 2.16-2.13 (m, 2H, >CHNHC(=O)CH2-), 2.09 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.87 (s, 9H, -Ac x 3), 1.77-1.19 (m, 33H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2N<, -CH2(CH2)3CH<, -NHC(=O)CH2CH2CH2C(=O)OBn). MS (ESI): m / z = 1751.6 ([M+H] + ).

[0284] Reference to the preparation method of Example 4.1, the compound (GalNAc)3-02a was deprotected under TFA / DCM conditions, and then reacted with compound SM-B-01-TBS to obtain compound (GalNAc)3-02c. 1H NMR (400 MHz, (CD3)2SO) δ: 5.28-5.23 (m, 3H, H-4Gal), 4.99-4.92 (m, 3H, H-3Gal), 4.49-4.38 (m, 4H, H-1Gal, >CH-), 4.05-3.94 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.91-3.84 (m, 5H, H-2Gal, -NHC(=O)CH2CH2OTBS), 3.70-3.45 (m, 6H, -OCH2CH2-), 3.34-3.11 (m, 10H, -CH2C(=O)NHCH2-, -CH2N<), 2.41 (t, 2H, -NHC(=O)CH2CH2OTBS), 2.22 (t, 6H, -CH2C(=O)NHCH2-), 2.10 (s, 9H, -Ac x 3), 2.00 (s, 9H, -Ac x 3), 1.88 (s, 9H, -Ac x 3), 1.76-1.15 (m, 31H, -Ac x 3, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2N<, -CH2(CH2)3CH<), 0.89 (s, 9H, -C(CH3)3), 0.07 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1733.5 ([M+H] + ).

[0285] Example 4.3: Synthesis of compounds (GalNAc)3-03a~(GalNAc)3-03e

[0286] According to the preparation method of Reference Example 4.1, the Boc protecting group of compound X-03 in Example 1 was removed under TFA / DCM condition, and then subjected to amidation reaction with compound S-01 to obtain compound (GalNAc)3-03a. 1H NMR(400MHz,(CD3)2SO)δ:7.34-7.30(m,5H,Ar-H),5.25(d,3H,H-4Gal),5.21-5.10(m,2H,Ar-CH2-),5.02-4.98(m, 3H,H-3Gal),4.63-4.56(m,1H,>CHC(=O)OBn),4.49(d,3H,H-1Gal),4.12-3.97(m,9H,H-5Gal,H-6Gal,H-6'Gal),3.9 3-3.87(m,3H,H-2Gal),3.72-3.47(m,14H,-OCH2CH2-,-C(=O)NH(CH2)2N<),2.38-1.95(m,28H,>NC(=O)CH2CH2CH<,- CH2C(=O)NH-,-Ac×6),1.87(s,9H,-Ac×3),1.76-1.20(m,21H,-Ac×3,-CH2(CH2)2CH2-).MS(ESI):m / z=1610.5([M+H] + ).

[0287] Compound (GalNAc)3-03a (2.15 g, 1.3 mmol) was dissolved in methanol (30 mL), and a Pd / C catalyst was added. The reaction mixture was bubbled with hydrogen at room temperature for 14 h to remove Bn. The catalyst was removed by filtration with diatomaceous earth, and the methanol was distilled off. The residue was dissolved in anhydrous dichloromethane (30 mL). Benzyl 3-hydroxypropionate (SM-C-01-Bn, 0.36 g, 2.0 mmol) and DMAP (0.03 g, 0.3 mmol) were added sequentially, followed by a single addition of N,N'-dicyclohexylcarbodiimide (DCC, 0.41 g, 2.0 mmol). The reaction mixture was stirred at 0 °C for 30 min, and then stirred overnight at room temperature. The reaction was quenched with saturated sodium chloride solution (30 mL). The aqueous phase was extracted with dichloromethane (15 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give compound (GalNAc)3-03b (1.61 g). 1H NMR (400 MHz, (CD3)2SO) δ: 7.32-7.27 (m, 5H, Ar-H), 5.22 (d, 3H, H-4Gal), 5.20-5.10 (m, 2H, Ar-CH2-), 5.00-4.95 (m, 3H, H-3Gal), 4.63-4.55 (m, 1H, >CHC(=O)O-), 4.45 (d, 3H, H-1Gal), 4.28 (t, 2H, -C(=O)OCH2CH2C(=O)OBn), 4.14-3.99 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.86 (m, 3H, H-2Gal), 3.70-3.44 (m, 14H, -OCH2CH2-, -C(=O)NH(CH2)2N<), 2.67 (t, 2H, -C(=O)OCH2CH2C(=O)OBn), 2.37-1.96 (m, 28H, >NC(=O)CH2CH2CH<, -CH2C(=O)NH-, -Ac x 6), 1.88 (s, 9H, -Ac x 3), 1.78-1.18 (m, 21H, -Ac x 3, -CH2(CH2)2CH2-). MS (ESI): m / z = 1682.6 ([M+H] + ).

[0288] Referring to the preparation method of (GalNAc)3-03b as described previously, compound (GalNAc)3-03a was deprotected by removing the Bn protecting group under the condition of H2, Pd / C, MeOH, and then esterified with 3-(tert-butyldimethylsilyloxy)propanol (SM-D-02-TBS) to obtain compound (GalNAc)3-03c. 1H NMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 3H, H-4Gal), 5.01-4.96 (m, 3H, H-3Gal), 4.61-4.53 (m, 1H, >CHC(=0)0-), 4.44 (d, 3H, H-1Gal), 4.15 (t, 2H, -C(=0)OCH2CH2CH2OTBS), 4.10-3.96 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.85 (m, 3H, H-2Gal), 3.69 (t, 2H, -C(=0)OCH2CH2CH2OTBS), 3.67-3.40 (m, 14H, -OCH2CH2-, -C(=0)NH(CH2)2N<), 2.34-1.95 (m, 28H, >NC(=0)CH2CH2CH<, -CH2C(=0)NH-, -Ac x 6), 1.87 (s, 9H, -Ac x 3), 1.82-1.16 (m, 23H, -Ac x 3, -CH2(CH2)2CH2-, -C(=0)OCH2CH2CH2OTBS), 0.88 (s, 9H, -C(CH3)3), 0.06 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1692.6 ([M+H] + ).

[0289] According to the preparation method of (GalNAc)3-03b described above, compound (GalNAc)3-03a was deprotected by removing the Bn protecting group under the condition of H2, Pd / C, MeOH, and then esterified with N-Boc-ethanolamine (SM-F-01-Boc) to obtain compound (GalNAc)3-03d. 1H NMR (400 MHz, (CD3)2SO) δ: 5.19 (d, 3H, H-4Gal), 5.01-4.95 (m, 3H, H-3Gal), 4.66-4.57 (m, 1H, >CHC(=0)0-), 4.46 (d, 3H, H-1Gal), 4.11-3.96 (m, 11H, H-5Gal, H-6Gal, H-6'Gal, -C(=0)OCH2CH2NHBoc), 3.92-3.84 (m, 3H, H-2Gal), 3.71-3.41 (m, 14H, -OCH2CH2-, -C(=0)NH(CH2)2N<), 3.35-3.31 (m, 2H, -C(=0)OCH2CH2NHBoc), 2.34-1.91 (m, 28H, >NC(=0)CH2CH2CH<, -CH2C(=0)NH-, -Ac x 6), 1.88 (s, 9H, -Ac x 3), 1.75-1.19 (m, 30H, -Ac x 3, -CH2(CH2)2CH2-, -C(CH3)3). MS (ESI): m / z = 1663.4 ([M+H] + ).

[0290] Reference to the preparation method of Example 4.1, compound (GalNAc)3-03a was deprotected with Bn under the condition of H2, Pd / C, MeOH, and then reacted with N-tert-butoxycarbonyl-1,2-ethanediamine (SM-E-01-Boc) to give compound (GalNAc)3-03e. 1H NMR (400 MHz, (CD3)2SO) δ: 5.17 (d, 3H, H-4Gal), 5.00-4.94 (m, 3H, H-3Gal), 4.48 (d, 3H, H-1Gal), 4.44-4.35 (m, 1H, >CH-), 4.10-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.92-3.83 (m, 3H, H-2Gal), 3.70-3.38 (m, 14H, -OCH2CH2-, -C(=O)NH(CH2)2N<), 3.35-3.31 (m, 2H, -C(=O)NHCH2CH2NHBoc), 3.29-3.24 (m, 2H, -C(=O)NHCH2CH2NHBoc), 2.32-1.90 (m, 28H, >NC(=O)CH2CH2CH<, -CH2C(=O)NH-, -Ac x 6), 1.87 (s, 9H, -Ac x 3), 1.74-1.16 (m, 30H, -Ac x 3, -CH2(CH2)2CH2-, -C(CH3)3). MS (ESI): m / z = 1662.5 ([M+H] + ).

[0291] Example 4.4: Synthesis of compounds (GalNAc)3-04a~(GalNAc)3-04c

[0292] According to the preparation method of Reference Example 4.1, the compound X-04 in Example 1 was subjected to deprotection of Cbz under the condition of H2, Pd / C, MeOH, and then subjected to amidation reaction with compound S-01 to obtain compound (GalNAc)3-04a. 1HNMR (400 MHz, (CD3)2SO) δ: 5.22 (d, 3H, H-4Gal), 5.03-4.94 (m, 3H, H-3Gal), 4.53-4.43 (m, 5H, H-1Gal, >CHC(=0)NH-, >CHC(=0)0-), 4.06-3.97 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.89-3.85 (m, 3H, H-2Gal), 3.66-3.35 (m, 6H, -OCH2(CH2)3-), 3.22-3.18 (m, 4H, -C(=0)NHCH2-), 2.16-2.11 (m, 6H, -CH2C(=0)NH-), 2.09 (s, 9H, -Ac x 3), 1.98 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.88-1.18 (m, 42H, -Ac x 3, -CH2(CH2)2CH2-, -(CH2)3CH<, -C(CH3)3). MS (ESI): m / z = 1617.6 ([M+H] + ).

[0293] Compound (GalNAc)3-04a (2.16 g, 1.3 mmol) was dissolved in TFA / DCM mixed solution (1:1 v / v; 20 mL), and stirred for 3 hours to remove tBu. After the reaction was completed, it was washed with purified water (10 mL*2), and the aqueous phase was extracted with dichloromethane (10 mL*2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. To the filtrate were sequentially added compound SM-C-02-Bn (0.39 g, 2.0 mmol) and DMAP (0.03 g, 0.3 mmol), and then DCC (0.41 g, 2.0 mmol) was added at once. After the reaction mixture was stirred at 0°C for 30 minutes, it was further stirred at room temperature overnight. The reaction was quenched with a saturated sodium chloride solution (40 mL). The aqueous phase was extracted with dichloromethane (20 mL*2). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain compound (GalNAc)3-04b (1.80 g). 1H NMR (400 MHz, (CD3)2SO) δ: 7.36-7.32 (m, 5H, Ar-H), 5.24 (d, 3H, H-4Gal), 5.19-5.09 (m, 2H, Ar-CH2-), 5.02-4.94 (m, 3H, H-3Gal), 4.63-4.41 (m, 5H, H-1Gal, >CHC(=0)NH-, >CHC(=0)0-), 4.08-3.94 (m, 11H, H-5Gal, H-6Gal, H-6'Gal, >CHC(=0)OCH2-), 3.88-3.84 (m, 3H, H-2Gal), 3.69-3.36 (m, 6H, -OCH2(CH2)3-), 3.25-3.20 (m, 4H, -C(=0)NHCH2-), 2.33 (t, 2H, -CH2COOBn), 2.16-2.10 (m, 6H, -CH2C(=0)NH-), 2.09 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.87-1.21 (m, 35H, -Ac x 3, -CH2(CH2)2CH2-, -CH2CH2CH2COOBn, -(CH2)3CH<). MS (ESI): m / z = 1738.7 ([M+H] + ).

[0294] Referring to the preparation method of the aforementioned (GalNAc)3-04b, compound (GalNAc)3-04a was deprotected with tBu under TFA / DCM conditions, and then esterification reaction was carried out with compound SM-D-01-TBS to obtain compound (GalNAc)3-04c. 1H NMR (400 MHz, (CD3)2SO) δ: 5.26 (d, 3H, H-4Gal), 5.00-4.91 (m, 3H, H-3Gal), 4.60-4.39 (m, 5H, H-1Gal, >CHC(=0)NH-, >CHC(=0)0-), 4.17 (t, 2H, >CHC(=0)OCH2-), 4.08-3.96 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.89-3.84 (m, 3H, H-2Gal), 3.81 (t, 2H, -CH2OTBS), 3.68-3.37 (m, 6H, -OCH2(CH2)3-), 3.28-3.21 (m, 4H, -C(=0)NHCH2-), 2.18-2.13 (m, 6H, -CH2C(=0)NH-), 2.10 (s, 9H, -Ac x 3), 2.00 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.85-1.20 (m, 33H, -Ac x 3, -CH2(CH2)2CH2-, -(CH2)3CH<), 0.88 (s, 9H, -C(CH3)3), 0.06 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1720.6 ([M+H] + ).

[0295] Example 4.5: Synthesis of compounds (GalNAc)3-05a~(GalNAc)3-05d

[0296] According to the preparation method of Reference Example 4.1, the tBu protecting group of compound X-05 in Example 1 was removed under TFA / DCM condition, and then subjected to amidation reaction with compound S-02 to obtain compound (GalNAc)3-05a. 1H NMR (400 MHz, (CD3)2SO) d: 5.21 (d, 3H, H-4Gal), 5.00-4.95 (m, 3H, H-3Gal), 4.45 (d, 3H, H-1Gal), 4.06-3.25 (m, 61H, H-5Gal, H-6Gal, H-6'Gal, H-2Gal, H-1, H-6, H-6', H-2, H-3, H-4, H-5, -NHC(=0)CH2CH20-, -OCH2CH20-, -OCH2CH2NHC(=0)-), 3.11-3.05 (m, 1H, H-1'), 2.50-2.46 (m, 6H, -NHC(=0)CH2CH20-), 2.08 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.78 (s, 9H, -Ac x 3), 1.16-1.02 (m, 21H, TIPS). MS (ESI): m / z = 1917.7 ([M+H] + ).

[0297] (GalNAc)3-05a (2.56 g, 1.3 mmol) was dissolved in 1 M TBAF / THF (40 mL) and the reaction was stirred for 6 hours to remove the TIPS protecting group. After the reaction was completed, it was concentrated under reduced pressure, the residue was dissolved with dichloromethane (25 mL) and washed with saturated ammonium chloride solution. The organic phase was dried with anhydrous magnesium sulfate and filtered. To the filtrate, compound SM-A-02-Bn (0.44 g, 2.0 mmol) and DMAP (0.03 g, 0.3 mmol) were sequentially added, and then DCC (0.41 g, 2.0 mmol) was added at once. After the reaction mixture was stirred at 0°C for 30 minutes, it was further stirred at room temperature overnight. The reaction was quenched with saturated sodium chloride solution (25 mL) and extracted with dichloromethane (25 mL*2). The organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound (GalNAc)3-05b (2.10 g). 1 H NMR (400 MHz, (CD3)2SO) d: 7.34-7.26 (m, 5H, Ar-H), 5.23 (d, 3H, H-4Gal), 5.16-5.07 (m, 2H, Ar-CH2-), 5.01-4.95 (m, 3H, H-3Gal), 4.48 (d, 3H, H-1Gal), 4.27 (dd, 1H, >CHCH a H bOC(=0)-), 4.06 - 3.27 (m, 62H, H-5Gal, H-6Gal, H-6'Gal, H-2Gal, H-1, H-2, H-3, H-4, H-5, -NHC(=0)CH2CH20-, -OCH2CH20-, -OCH2CH2NHC(=0)-, -CH2OTBS, >CHCH a H b OC(=0)-), 3.13 - 3.06 (m, 1H, H-1'), 2.47 - 2.43 (m, 6H, -NHC(=0)CH2CH20-), 2.34 - 2.31 (m, 4H, -OC(=0)CH2CH2CH2COOBn), 2.08 (s, 9H, -Ac x 3), 1.98 (s, 9H, -Ac x 3), 1.93 - 1.89 (m, 2H, -CH2CH2CH2-), 1.87 (s, 9H, -Ac x 3), 1.77 (s, 9H, -Ac x 3). MS (ESI): m / z = 1965.5 ([M+H] + ).

[0298] Referring to the preparation method of the aforementioned compound (GalNAc)3-05b, compound (GalNAc)3-05a was deprotected with TIPS under the condition of TBAF / THF, and then esterification reaction was carried out with compound SM-B-01-TBS to obtain compound (GalNAc)3-05c. 1 H NMR (400 MHz, (CD3)2SO) δ: 5.25 (d, 3H, H-4Gal), 5.03 - 4.96 (m, 3H, H-3Gal), 4.46 (d, 3H, H-1Gal), 4.25 (dd, 1H, >CHCH a H b OC(=0)-), 4.06 - 3.27 (m, 62H, H-5Gal, H-6Gal, H-6'Gal, H-2Gal, H-1, H-2, H-3, H-4, H-5, -NHC(=0)CH2CH20-, -OCH2CH20-, -OCH2CH2NHC(=0)-, -CH2OTBS, >CHCH a H bOC(=0)-), 3.16-3.09 (m, 1H, H-1'), 2.53 (t, 2H, -OC(=0)CH2-), 2.47-2.43 (m, 6H, -NHC(=0)CH2CH20-), 2.09 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.88 (s, 9H, -Ac x 3), 1.75 (s, 9H, -Ac x 3), 0.88 (s, 9H, -C(CH3)3), 0.06 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1947.7 ([M+H] + ).

[0299] (GalNAc)3-05a (1.92 g, 1.0 mmol) was dissolved in 1 M TBAF / THF (30 mL) and stirred for 6 hours to remove the TIPS protecting group. After the reaction was completed, it was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (20 mL) and washed with a saturated ammonium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and filtered. To the filtrate was added DSC (0.31 g, 1.2 mmol) and TEA (0.4 mL, 3.0 mmol) sequentially, and stirred at room temperature overnight. After the reaction was completed, compound SM-E-02-Cbz (0.18 g, 1.2 mmol, obtained by reacting 1,3-propanediamine with benzyl chloroformate) was added, and stirring was continued at room temperature for 3 hours. After the reaction was completed, it was washed with a saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound (GalNAc)3-05d (1.63 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 7.35-7.22 (m, 5H, Ar-H), 5.24 (d, 3H, H-4Gal), 5.00-4.95 (m, 5H, H-3Gal, Ar-CH2-), 4.46 (d, 3H, H-1Gal), 4.27 (dd, 1H, >CHCH a H b OC(=0)NH-), 4.21 (dd, 1H, >CHCH a H bHNMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 3H, H-4Gal), 5.03-4.97 (m, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.44-4.36 (m, 1H, >CHNHC(=0)-), 4.28-4.20 (m, 1H, >CHNHBoc), 4.06-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.94-3.25 (m, 39H, H-2Gal, -0(CH2CH20)2CH2CH2NHC(=0)-), 2.37-1.97 (m, 26H, -NHC(=0)CH2CH2CH<, -Ac x 6), 1.88 (s, 9H, -Ac x 3), 1.74 (s, 9H, -Ac x 3), 1.43 (s, 9H, Boc). MS (ESI): m / z = 1757.5 ([M+H] + ).

[0300] Example 4.6: Synthesis of compounds (GalNAc)3-06a~(GalNAc)3-06c

[0301] Referring to the preparation method of Example 4.1, the Bn protecting group of compound X-06 in Example 1 was removed under the condition of H2, Pd / C, MeOH, and then an amide reaction was carried out with compound S-02 to obtain compound (GalNAc)3-06a. 1 HNMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 3H, H-4Gal), 5.03-4.97 (m, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.44-4.36 (m, 1H, >CHNHC(=0)-), 4.28-4.20 (m, 1H, >CHNHBoc), 4.06-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.94-3.25 (m, 39H, H-2Gal, -0(CH2CH20)2CH2CH2NHC(=0)-), 2.37-1.97 (m, 26H, -NHC(=0)CH2CH2CH<, -Ac x 6), 1.88 (s, 9H, -Ac x 3), 1.74 (s, 9H, -Ac x 3), 1.43 (s, 9H, Boc). MS (ESI): m / z = 1757.5 ([M+H] + ).

[0302] Reference to the preparation method of Example 4.1, compound (GalNAc)3-06a was deprotected with Boc under TFA / DCM condition, and then reacted with compound SM-A-02-Bn to obtain compound (GalNAc)3-06b. 1 H NMR (400 MHz, (CD3)2SO) δ: 7.36-7.28 (m, 5H, Ar-H), 5.20 (d, 3H, H-4Gal), 5.17-5.08 (m, 2H, Ar-CH2-), 5.01-4.93 (m, 3H, H-3Gal), 4.46 (d, 3H, H-1Gal), 4.44-4.34 (m, 2H, >CHNHC(=O)-), 4.07-3.97 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.25 (m, 39H, H-2Gal, -O(CH2CH2O)2CH2CH2NHC(=O)-), 2.36-1.97 (m, 30H, -NHC(=O)CH2CH2CH<, -Ac x 6, -CH2CH2CH2C(=O)OBn), 1.88 (s, 9H, -Ac x 3), 1.83-1.70 (m, 11H, -Ac x 3, -CH2CH2CH2-). MS (ESI): m / z = 1861.6 ([M+H] + ).

[0303] Reference to the preparation method of Example 4.1, compound (GalNAc)3-06a was deprotected with Boc under TFA / DCM condition, and then reacted with compound SM-A-02-Bn to obtain compound (GalNAc)3-06b. 1H NMR (400 MHz, (CD3)2SO) δ: 5.21 (d, 3H, H-4Gal), 5.01-4.94 (m, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.45-4.34 (m, 2H, >CHNHC(=0)-), 4.06-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.24 (m, 41H, H-2Gal, -0(CH2CH20)2CH2CH2NHC(=0)-, -CH20TBS), 2.38-1.98 (m, 28H, -NHC(=0)CH2-, -CH2CH<, -Ac x 6), 1.89 (s, 9H, -Ac x 3), 1.75 (s, 9H, -Ac x 3), 1.65-1.32 (m, 6H, -CH2(CH2)3CH2-), 0.87 (s, 9H, -C(CH3)3), 0.05 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1885.7 ([M+H] + ).

[0304] Example 4.7: Synthesis of compounds (GalNAc)3-07a~(GalNAc)3-07c

[0305] According to the preparation method in Reference Example 4.1, the tBu protecting group of compound X-07 was removed under TFA / DCM condition, and then subjected to amidation reaction with compound S-02 to obtain compound (GalNAc)3-07a. 1 H NMR (400 MHz, (CD3)2SO) δ: 5.21 (d, 3H, H-4Gal), 5.01-4.94 (m, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.45-4.34 (m, 2H, >CHNHC(=0)-), 4.06-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.93-3.24 (m, 41H, H-2Gal, -0(CH2CH20)2CH2CH2NHC(=0)-, -CH20TBS), 2.38-1.98 (m, 28H, -NHC(=0)CH2-, -CH2CH<, -Ac x 6), 1.89 (s, 9H, -Ac x 3), 1.75 (s, 9H, -Ac x 3), 1.65-1.32 (m, 6H, -CH2(CH2)3CH2-), 0.87 (s, 9H, -C(CH3)3), 0.05 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1885.7 ([M+H] + ).

[0306] Reference to the preparation method in Example 4.1, compound (GalNAc)3-07a was deprotected with H2, Pd / C, MeOH, and then subjected to amidation reaction with compound SM-A-03-Bn to obtain compound (GalNAc)3-07b. 1 H NMR (400 MHz, (CD3)2SO) δ: 7.41-7.27 (m, 5H, Ar-H), 5.20 (d, 3H, H-4Gal), 5.02 (s, 2H, Ar-CH2), 5.00-4.91 (m, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.07-3.96 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.88-3.83 (m, 3H, H-2Gal), 3.80-3.24 (m, 48H, -O(CH2CH2O)2CH2CH2NHC(=O)-, -NHC(=O)CH2CH2O-, (-OCH2)3C-), 2.49-2.43 (m, 6H, -NHC(=O)CH2CH2O-), 2.30 (t, 2H, -CH2COOBn), 2.25-2.18 (m, 2H, -NHC(=O)CH2(CH2)3-), 2.09 (s, 9H, -Ac x 3), 1.99 (s, 9H, -Ac x 3), 1.89 (s, 9H, -Ac x 3), 1.77 (s, 9H, -Ac x 3), 1.68-1.57 (m, 4H, -CH2(CH2)2CH2-). MS (ESI): m / z = 1936.6 ([M+H] + ).

[0307] Reference to the preparation method in Example 4.1, compound (GalNAc)3-07a was deprotected with H2, Pd / C, MeOH, and then subjected to amidation reaction with compound SM-B-01-TBS to obtain compound (GalNAc)3-07c. 1H NMR (400 MHz, (CD3)2SO) δ: 5.23 (d, 3H, H-4Gal), 5.02-4.92 (m, 3H, H-3Gal), 4.48 (d, 3H, H-1Gal), 4.06-3.94 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.87-3.82 (m, 5H, H-2Gal, -NHC(=O)CH2CH2OTBS), 3.78-3.23 (m, 48H, -O(CH2CH2O)2CH2CH2NHC(=O)-, -NHC(=O)CH2CH2O-, (-OCH2)3C-), 2.48-2.43 (m, 6H, -NHC(=O)CH2CH2O-), 2.37 (t, 2H, -NHC(=O)CH2CH2OTBS), 2.08 (s, 9H, -Ac x 3), 1.98 (s, 9H, -Ac x 3), 1.88 (s, 9H, -Ac x 3), 1.75 (s, 9H, -Ac x 3), 0.88 (s, 9H, -C(CH3)3), 0.06 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 1904.7 ([M+H] + ).

[0308] Example 5: Synthesis of tetravalent targeting ligands

[0309] Example 5.1: Synthesis of compounds (GalNAc)4-01a~(GalNAc)4-01c

[0310] Compound (GalNAc)2-01 (2.42 g, 2.0 mmol) was dissolved in methanol (30 mL), Pd / C catalyst was added, and the reaction was carried out at room temperature for 14 h with hydrogen gas bubbling to remove Bn. The catalyst was removed by filtration with diatomite, and the methanol was evaporated, and the residue was dissolved in anhydrous dichloromethane (25 mL). N-Boc-D-glutamic acid (dl, 0.25 g, 1.0 mmol), EDC HCI (0.58 g, 3.0 mmol) and DMAP (0.05 g, 0.4 mmol) were added in turn, and the reaction was stirred at room temperature overnight. After the reaction was completed, 25 mL of dichloromethane was added for dilution, and then washed with water (25 mL*2), 1M HCI solution (25 mL*2), saturated sodium bicarbonate solution (25 mL*2) and saturated sodium chloride solution (25 mL*2) in turn. The organic phase was dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound (GalNAc)4-01a (1.48 g). 1H NMR (400 MHz, (CD3)2SO) δ: 5.22 (d, 4H, H-4Gal), 5.03-4.98 (m, 4H, H-3Gal), 4.67-4.64 (m, 2H, >CHOC(=0)NH-), 4.48 (d, 4H, H-1Gal), 4.28-4.20 (m, 1H, >CHNHBoc), 4.07-3.94 (m, 12H, H-5Gal, H-6Gal, H-6'Gal), 3.90-3.85 (m, 4H, H-2Gal), 3.66-3.17 (m, 24H, -OCH2(CH2)3-, -C(=0)NHCH2-, -OC(=0)NHCH2-), 2.37-1.97 (m, 36H, -NHC(=0)CH2CH2CH2-, -Ac x 8, -CH2C(=0)NH-), 1.89 (s, 12H, -Ac x 4), 1.82-1.27 (m, 49H, -Ac x 4, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -C(CH3)3). MS (ESI): m / z = 2364.9 ([M+H] + ).

[0311] Reference to the preparation method in Example 4.1, compound (GalNAc)4-01a was deprotected with TFA / DCM to remove Boc, and then reacted with compound SM-A-04-Bn to obtain compound (GalNAc)4-01b. 1HNMR (400 MHz, (CD3)2SO) δ: 7.36-7.29 (m, 5H, Ar-H), 5.20 (d, 4H, H-4Gal), 5.15-5.05 (m, 2H, Ar-CH2-), 5.00-4.96 (m, 4H, H-3Gal), 4.66-4.62 (m, 2H, >CHOC(=0)NH-), 4.46 (d, 4H, H-1Gal), 4.43-4.36 (m, 1H, >CHNHC(=0)-), 4.04-3.92 (m, 12H, H-5Gal, H-6Gal, H-6'Gal), 3.88-3.84 (m, 4H, H-2Gal), 3.69-3.19 (m, 24H, -OCH2(CH2)3-, -C(=0)NHCH2-, -OC(=0)NHCH2-), 2.37-1.94 (m, 40H, -NHC(=0)CH2CH2CH2-, -CH2C(=0)NH-, -NHC(=0)CH2(CH2)3CH2COOBn, -Ac x 8), 1.88 (s, 12H, -Ac x 4), 1.83-1.25 (m, 46H, -Ac x 4, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-). MS (ESI): m / z = 2497.0 ([M+H] + ).

[0312] Reference to the preparation method in Example 4.1, compound (GalNAc)4-01a was deprotected with TFA / DCM to remove Boc, and then reacted with compound SM-B-09-TBS to obtain compound (GalNAc)4-01c. 1H NMR (400 MHz, (CD3)2SO) δ: 5.21 (d, 4H, H-4Gal), 5.01-4.96 (m, 4H, H-3Gal), 4.64-4.60 (m, 2H, >CHOC(=O)NH-), 4.47 (d, 4H, H-1Gal), 4.43-4.35 (m, 1H, >CHNHC(=O)-), 4.05-3.93 (m, 12H, H-5Gal, H-6Gal, H-6'Gal), 3.89-3.84 (m, 4H, H-2Gal), 3.67-3.16 (m, 26H, -OCH2(CH2)3-, -C(=O)NHCH2-, -OC(=O)NHCH2-, -CH2OTBS), 2.36-1.91 (m, 38H, -NHC(=O)CH2CH2CH<-, -Ac x 8, -CH2C(=O)NH-), 1.87 (s, 12H, -Ac x 4), 1.82-1.26 (m, 56H, -Ac x 4, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)8CH2-), 0.87 (s, 9H, -C(CH3)3), 0.05 (s, 6H, >Si(CH3)2). MS (ESI): m / z = 2563.1 ([M+H] + ).

[0313] Example 5.2: Synthesis of compound (GalNAc)4-02b

[0314] Step 1: Compound SM-A-04-Bn (2.00 g, 8.0 mmol, obtained from the reaction of heptanedioic acid and benzyl alcohol) and EDC HCI (2.30 g, 12.0 mmol) were dissolved in 30 mL of dichloromethane, 1,9-bis-Boc-1,5,9-triazononane (d2, 3.18 g, 9.6 mmol) and DMAP (0.20 g, 1.6 mmol) were added, and the reaction was stirred at room temperature overnight. After the reaction was completed, 60 mL of dichloromethane was added for dilution, and then sequentially washed with water (50 mL*2), 1 M HCI solution (50 mL*2), saturated sodium bicarbonate solution (50 mL*2), and saturated sodium chloride solution (50 mL*2). The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound d3 (2.57 g).

[0315] Step 2: Compound d3 (0.54 g, 1.0 mmol) was dissolved in TFA / DCM mixed solution (1:1 v / v; 10 mL) and stirred for 3 h to remove the Boc protecting group. After the reaction was completed, it was washed with purified water (5 mL*2), and the aqueous phase was extracted with dichloromethane (5 mL*3). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. Compound (GalNAc)2-02 (2.42 g, 2.0 mmol), EDC HCI (0.58 g, 3.0 mmol), and DMAP (0.05 g, 0.4 mmol) were sequentially added to the filtrate, which was stirred at room temperature overnight. After the reaction was completed, it was diluted with 35 mL of dichloromethane and sequentially washed with water (30 mL*2), 1 M HCI solution (30 mL*2), saturated sodium bicarbonate solution (30 mL*2), and saturated sodium chloride solution (30 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound (GalNAc)4-02b (1.54 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 7.34-7.27 (m, 5H, Ar-H), 5.20 (d, 4H, H-4Gal), 5.16-5.05 (m, 2H, Ar-CH2-), 5.02-4.95 (m, 4H, H-3Gal), 4.48 (d, 4H, H-1Gal), 4.46-4.38 (m, 2H, >CH-), 4.06-3.93 (m, 12H, H-5Gal, H-6Gal, H-6'Gal), 3.90-3.86 (m, 4H, H-2Gal), 3.72-3.17 (m, 56H, -OCH2CH2O-, -OCH2CH2NHC(=O)-, -C(=O)NHCH2CH2CH2N<), 2.33-2.21 (m, 8H, -NHC(=O)CH2CH2CH<,>NC(=O)CH2(CH2)3CH2COOBn), 2.18-2.12 (m, 10H, -NHC(=O)CH2CH2CH2C(=O)NH-), 2.09 (s, 12H, -Ac x 4), 1.99 (s, 12H, -Ac x 4), 1.97-1.92 (m, 2H, -NHC(=O)CH2CH2CH2C(=O)NH-), 1.88 (s, 12H, -Ac x 4), 1.83-1.24 (s, 34H, -Ac x 4, -CH2(CH2)3CH2-, -C(=O)NHCH2CH2CH2N<). MS (ESI): m / z = 2747.1 ([M+H] a H b CH<,-NHC(=O)CH2CH2CH2C(=O)NH-), 2.09 (s, 12H, -Ac x 4), 1.99 (s, 12H, -Ac x 4), 1.97-1.92 (m, 2H, -NHC(=O)CH2CH2CH2C(=O)NH-), 1.88 (s, 12H, -Ac x 4), 1.83-1.24 (s, 34H, -Ac x 4, -CH2(CH2)3CH2-, -C(=O)NHCH2CH2CH2N<). MS (ESI): m / z = 2747.1 ([M+H] a H b CH<),1.88(s,12H,-Ac×4),1.83-1.24(s,34H,-Ac×4,-CH2(CH2)3CH2-,-C(=O)NHCH2CH2CH2N<).MS(ESI):m / z=2747.1([M+H] + ).

[0316] Example 5.3: Synthesis of compound (GalNAc)4-03b

[0317] According to the preparation method in Reference Example 5.2, compound (GalNAc)2-03 was deprotected with TFA / DCM, and then subjected to amidation reaction with compound d3 to obtain compound (GalNAc)4-03b. 1 H NMR (400 MHz, (CD3)2SO) δ: 7.32-7.26 (m, 5H, Ar-H), 5.22 (d, 4H, H-4Gal), 5.13-5.03 (m, 2H, Ar-CH2-), 4.99-4.92 (m, 4H, H-3Gal), 4.47 (d, 4H, H-1Gal), 4.08-3.96 (m, 12H, H-5Gal, H-6Gal, H-6'Gal), 3.88-3.83 (m, 4H, H-2Gal), 3.67-3.46 (m, 8H, -OCH2CH2-), 3.36-3.13 (m, 24H, -C(=O)NHCH2CH2CH2N<), 2.33-2.14 (m, 20H, -CH2C(=O)NH-, -CH2C(=O)N<, -CH2COOBn), 2.08 (s, 12H, -Ac x 4), 1.99 (s, 12H, -Ac x 4), 1.94-1.26 (m, 62H, -Ac x 8, -CH2(CH2)2CH2-, -C(=O)NHCH2CH2CH2N<, >NC(=O)CH2CH2CH2C(=O)NH-, -CH2(CH2)3CH2-). MS (ESI): m / z = 2535.0 ([M+H] + ).

[0318] Example 6: Synthesis of monovalent targeting PEGylated lipids

[0319] Example 6.1: Synthesis of GPL-0102

[0320] GPL-0102 was synthesized by General Preparation Method One, with the following specific method:

[0321] Step 1: Under an argon atmosphere, carboxyl-functionalized PEGylated lipid PEG-L-02 (5.00 g, 2.0 mmol, M n= 2.5 kDa, PDI = 1.01, n ~ 45), compound GalNAc-01 (9.78 g, 20.0 mmol), DMAP (0.49 g, 4.0 mmol) were dissolved in 50 mL of dichloromethane, respectively, and a solution of DCC (6.18 g, 30.0 mmol) in 50 mL of dichloromethane was added dropwise slowly under ice-bath. After stirring at room temperature for 24 h, the precipitate was removed by filtration. The filtrate was dried over anhydrous magnesium sulfate, filtered and concentrated, and purified by column chromatography to give compound GPL-0102-OAc (5.20 g). The molecular weight was determined by MALDI-TOF MS to be 3.0 kDa. The PDI was determined by GPC to be 1.01.

[0322] Step 2: Compound GPL-0102-OAc (2.95 g, 1.0 mmol) was dissolved in ethanol (50 mL) at room temperature, and ammonia water (100 mL) was added and heated at 40 °C for 48 h. After concentration under reduced pressure, monovalent targeting polyethylene glycolylated lipid GPL-0102 (2.29 g) was obtained by column chromatography. 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 1H, H-1Gal), 4.20 (t, 2H, -C(=O)OCH2-), 3.96-3.42 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=O)CH2CH2O-, -O(CH2)2N<, -C(=O)NH(CH2)2NHC(=O)-), 3.18 (t, 2H, >NCH2(CH2) 10 CH3), 2.57 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.46 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.23 (t, 2H, >NC(=O)CH2-), 2.15 (t, 2H, -CH2C(=O)NH-), 1.80 (s, 3H, -Ac), 1.58-1.20 (m, 40H, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3, -CH2(CH2)8CH3), 0.87 (t, 6H, -CH2CH3). The molecular weight was determined by MALDI-TOF MS to be 2.8 kDa. The PDI was determined by GPC to be 1.01.

[0323] Example 6.2: Synthesis of GPL-0202

[0324] Using General Preparation Method One, GalNAc-02 was deprotected after Boc protection and amidated with PEG-L-02, and then deprotected O-acetyl to obtain monovalent targeted PEGylated lipid GPL-0202 (M n = 2.9 kDa, PDI = 1.01, n ~ 45). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 1H, H-1Gal), 4.19 (t, 2H, -C(=O)OCH2-), 3.91-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -OCH2CH2N<-, -CH2OCH2-), 3.26-3.21 (m, 4H, -C(=O)NHCH2-), 3.17 (t, 2H, >NCH2(CH2) 10 CH3), 2.55 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.43 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.22 (t, 2H, >NC(=O)CH2-), 2.14 (t, 2H, -CH2C(=O)NH-), 1.79 (s, 3H, -Ac), 1.59-1.19 (m, 40H, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3, -CH2(CH2)8CH3), 0.86 (t, 6H, -CH2CH3).

[0325] Example 6.3: Synthesis of GPL-0301

[0326] Using General Preparation Method One, GalNAc-03 was amidated with amino-functionalized PEGylated lipid PEG-L-01 (M n = 2.5 kDa, PDI = 1.03, n ~ 45) and then deprotected O-acetyl to obtain monovalent targeted PEGylated lipid GPL-0301 (M n = 2.8 kDa, PDI = 1.03, n ~ 43). 1H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 1H, H-1Gal), 3.92-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -O(CH2)2O-, -OCH2CH2NHC(=O)-, -O(CH2)2N<), 3.26-3.22 (m, 4H, -C(=O)NHCH2-), 3.18 (t, 2H, >NCH2(CH2) 12 CH3), 2.20 (t, 2H, >NC(=O)CH2-), 2.18-2.15 (m, 4H, -NHC(=O)CH2-), 1.79 (s, 3H, -Ac), 1.60-1.21 (m, 48H, -CH2CH2CH2-, 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0327] Example 6.4: Synthesis of GPL-01N01

[0328] The preparation method is as follows:

[0329] Step 1: GalNAc-01 (0.59 g, 1.2 mmol), NHS-PEG-N3 (2.20 g, 1.0 mmol; M n = 2.2 kDa, PDI = 1.02, n ~ 45) and TEA (0.2 mL, 1.5 mmol) were dissolved in dichloromethane (20 mL). The reaction was stirred at room temperature overnight. After the reaction was completed, the reaction liquid was washed with 10% citric acid (10 mL*2), saturated sodium bicarbonate solution (10 mL*2) and saturated sodium chloride solution (10 mL*2) in turn. The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound GP-01-N3 (1.92 g).

[0330] Step 2: N-01 (0.28 g, 0.6 mmol), cuprous bromide (CuBr, 0.14 g, 1.0 mmol), pentamethyldiethylenetriamine (PMDTA, 0.17 g, 1.0 mmol) and GP-01-N3 (1.28 g, 0.5 mmol) were dissolved in anhydrous dichloromethane (10 mL) under argon atmosphere. The reaction was stirred at room temperature for 48 h. After the reaction was completed, the reaction mixture was poured into water (10 mL). The aqueous phase was extracted with dichloromethane (5 mL*2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound GPL-01N01-OAc (1.29 g).

[0331] Step 3: Compound GPL-01N01-OAc (1.21 g, 0.4 mmol) was dissolved in ethanol (10 mL) at room temperature, and ammonia water (20 mL) was added and heated at 40 °C for 48 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and purified by column chromatography to give monovalent PEGylated lipid GPL-01N01 (0.93 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 8.65 & 8.43 (2s, 1H, triazol), 4.49 (t, 2H, -CH2CH2-triazol), 4.44 (d, 1H, H-1Gal), 3.91 (s, 2H, -NHC(=O)CH2O-), 3.84-3.38 (m, PEG; H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6’Gal, -OCH2(CH2)3-, -CH2CH2-triazol, triazol-CH2-N, NCH2-), 3.27-2.96 (m, 4H, -C(=O)NH(CH2)2NHC(=O)-), 2.25 (t, 2H, >NC(=O)CH2-), 2.16 (t, 2H, -CH2C(=O)NH-), 1.79 (s, 3H, -Ac), 1.61-1.21 (m, 50H, -CH2(CH2)2CH2-, 11 CH3, -CH2(CH2) 12 CH3), 0.88 (t, 6H, -CH2CH3). The molecular weight was determined by MALDI-TOF MS to be 2.9 kDa. The PDI was determined by GPC to be 1.02.

[0332] Example 6.5: Synthesis of GPL-03N02

[0333] The preparation method is as follows:

[0334] Step 1 : GalNAc-03 (0.59 g, 1.0 mmol) was dissolved in 10 mL of dichloromethane, EDC HCI (0.21 g, 1.1 mmol) and HOBt H20 (0.17 g, 1.1 mmol) were added successively, followed by the addition of a solution of H2N-PEG-N3 (2.00 g, 1.0 mmol; Mw= 2.0 kDa, PDI = 1.02, n ~ 44) in dichloromethane (10 mL). DIPEA (0.14 g, 1.1 mmol) was added and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (10 mL*2), saturated sodium bicarbonate solution (10 mL*2) and saturated sodium chloride solution (10 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give compound GP-03-N3 (2.10 g). n Step 1 : GalNAc-03 (0.59 g, 1.0 mmol) was dissolved in 10 mL of dichloromethane, EDC HCI (0.21 g, 1.1 mmol) and HOBt H20 (0.17 g, 1.1 mmol) were added successively, followed by the addition of a solution of H2N-PEG-N3 (2.00 g, 1.0 mmol; Mw= 2.0 kDa, PDI = 1.02, n ~ 44) in dichloromethane (10 mL). DIPEA (0.14 g, 1.1 mmol) was added and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with 10% citric acid (10 mL*2), saturated sodium bicarbonate solution (10 mL*2) and saturated sodium chloride solution (10 mL*2). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give compound GP-03-N3 (2.10 g).

[0335] Step 2: N-02 (0.30 g, 0.6 mmol), CuBr (0.14 g, 1.0 mmol), PMDTA (0.17 g, 1.0 mmol) and GP-03-N3 (1.30 g, 0.5 mmol) were dissolved in anhydrous dichloromethane (10 mL) under argon atmosphere. The reaction was stirred at room temperature for 48 hours. After the reaction was completed, the reaction mixture was poured into water (10 mL). The aqueous phase was extracted with dichloromethane (5 mL*2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound GPL-03N02-OAc (1.37 g).

[0336] Step 3: Compound GPL-03N02-OAc (1.24 g, 0.4 mmol) was dissolved in ethanol (10 mL) at room temperature, and ammonia water (20 mL) was added and heated at 40 °C for 48 hours. After concentration under reduced pressure, column chromatography purification gave monovalent targeting polyethylene glycolated lipid GPL-03N02 (1.01 g). 1H NMR (400 MHz, (CD3)2SO) δ: 7.84 (s, 1H, triazol), 4.64-4.40 (m, 5H, H-1Gal, -CH2-triazol-CH2-), 3.86-3.40 (m, PEG; H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -O(CH2)2O-, -OCH2CH2NHC(=O)-, -O(CH2)2N<, -CH2CH2-triazol), 3.24-3.20 (m, 4H, -C(=O)NHCH2-), 3.18 (t, 2H, >NCH2-), 2.19-2.13 (m, 6H, >NC(=O)CH2-, -NHC(=O)CH2-), 1.79 (s, 3H, -Ac), 1.57-1.20 (m, 48H, -CH2CH2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). The molecular weight was determined by MALDI-TOF MS to be 3.0 kDa. The PDI was determined by GPC to be 1.01.

[0337] Example 6.6: Synthesis of GPL-01N09

[0338] Referring to the preparation method of N-04, replacing b24 with b22 gives N-09. Referring to the preparation method of Example 6.4, replacing N-01 with N-09 gives monovalent targeted pegylated lipid GPL-01N09 (M n = 2.9 kDa, PDI = 1.02, n ~ 45). 1H NMR (400 MHz, (CD3)2SO) d: 8.64 & 8.41 (2s, 1 H, triazol), 4.46 (t, 2H, -CH2CH2-triazol), 4.42 (d, 1 H, H-1Gal), 3.87 (s, 2H, -NHC(=0)CH20-), 3.80-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -CH2CH2-triazol, triazol-CH2-N<), 3.32-2.95 (m, 4H, -C(=0)NH(CH2)2NHC(=0)-), 2.69 (t, 4H, >NCH2-), 2.14 (t, 2H, -CH2C(=0)NH-), 1.78 (s, 3H, -Ac), 1.64-1.19 (m, 52H, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0339] Example 6.7: Synthesis of GPL-03N03

[0340] Following the preparation method of Reference Example 6.5, N-02 was replaced by N-03 to give the monovalent targeted pegylated lipid GPL-01N03 (M n = 3.0 kDa, PDI = 1.01, n ~ 44). 1 H NMR (400 MHz, (CD3)2SO) d: 7.81 (s, 1 H, triazol), 4.65-4.38 (m, 5H, H-1Gal, -CH2-triazol-CH2-), 3.83-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -O(CH2)20-, -OCH2CH2NHC(=0)-, -OCH2CH2N<, -CH2CH2-triazol), 3.22-3.17 (m, 4H, -C(=0)NHCH2-), 2.66 (t, 2H, -OCH2CH2N<), 2.59 (t, 4H, >NCH2-), 2.20-2.17 (m, 4H, -NHC(=0)CH2-), 1.79 (s, 3H, -Ac), 1.63-1.21 (m, 50H, -CH2CH2CH2-, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3)

[0341] Example 7: Synthesis of divalent targeting pegylated lipids

[0342] Example 7.1: Synthesis of G2PL-0102

[0343] Using General Preparation Method One, (GalNAc)2-01 was deprotected and amidated with PEG-L-02, followed by removal of O-acetyl group, to obtain divalent targeting pegylated lipid G2PL-0102 (M n = 3.3 kDa, PDI = 1.01, n ~ 45). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.69-4.66 (m, 1H, >CH-), 4.46 (d, 2H, H-1Gal), 4.17 (t, 2H, -C(=O)OCH2-), 3.89-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -OC(=O)NHCH2(CH2)3CH2NHC(=O)-, -O(CH2)2N<), 3.20 (t, 2H, >NCH2(CH2) 10 CH3), 2.58 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.48 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.22 (t, 2H, >NC(=O)CH2-), 2.16 (t, 4H, -CH2C(=O)NH-), 1.78 (s, 6H, -Ac x 2), 1.61-1.23 (m, 50H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 10 CH3, -CH2(CH2)8CH3), 0.87 (t, 6H, -CH2CH3).

[0344] Example 7.2: Synthesis of G2PL-0201

[0345] Using General Preparation Method One, (GalNAc)2-02 was deprotected and amidated with PEG-L-01, followed by removal of O-acetyl group, to obtain divalent targeting pegylated lipid G2PL-0201 (M n = 3.3 kDa, PDI = 1.03, n ~ 43). 1H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 2H, H-1Gal), 4.43-4.36 (m, 1H, >CH-), 3.90-3.41 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -O(CH2)2O-, -O(CH2)2N<), 3.26-3.20 (m, 12H, -C(=O)NH(CH2)2O-), 3.18 (t, 2H, >NCH2(CH2) 12 CH3), 2.34-2.11 (m, 9H, -NHC(=O)CH2-, >NC(=O)CH2-, -NHC(=O)CH2CH a H b CH<), 1.98-1.94 (m, 1H, -NHC(=O)CH2CH a H b CH<), 1.79 (s, 6H, -Ac x 2), 1.59-1.23 (m, 52H, -CH2(CH2)3CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0346] Example 7.3: Synthesis of G2PL-0301

[0347] Using the general preparation method one, (GalNAc)2-03 was deprotected and then amidated with PEG-L-01, followed by deprotection of O-acetyl group, to obtain the bivalent targeting PEGylated lipid G2PL-0301 (M n = 3.2 kDa, PDI = 1.03, n ~ 43). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 2H, H-1Gal), 3.88-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -C(=O)NHCH2CH2CH2N<, -C(=O)NH(CH2)2O-, -OCH2(CH2)3-, -O(CH2)2N<), 3.15 (t, 2H, >NCH2(CH2) 12 CH3), 2.38-2.12 (m, 10H, >NC(=O)CH2-, -CH2C(=O)NH-), 1.83-1.22 (m, 66H, -Ac x 2, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)11 CH3, -CH2(CH2) 12 CH3), 0.88 (t, 6H, -CH2CH3).

[0348] Example 7.4: Synthesis of G2PL-03N02

[0349] Following the preparation method of Reference Example 6.5, the carboxylic acid compound obtained after deprotection of tBu of GalNAc-03 was replaced to give G2PL-03N02-OAc and G2PL-03N02(M n = 3.4 kDa, PDI = 1.01, n ~ 44).

[0350] G2PL-03N02 1 H NMR (400 MHz, (CD3)2SO) δ: 7.85 (s, 1H, triazol), 4.65-4.43 (m, 6H, H-1Gal, -CH2-triazol-CH2-), 3.88-3.37 (m, PEG; H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -C(=O)NHCH2CH2CH2N<, -C(=O)NH(CH2)2O-, -OCH2(CH2)3-, -O(CH2)2N<, -CH2CH2-triazol), 3.20 (t, 2H, >NCH2(CH2) 12 CH3), 2.37-2.15 (m, 10H, >NC(=O)CH2-, -CH2C(=O)NH-), 1.80-1.22 (m, 66H, -Ac x 2, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0351] Example 8: Synthesis of trivalent targeting polyethyleneglycolized lipids

[0352] Example 8.1: Synthesis of G3PL-01b01, G3PL-01b01-0.5k and G3PL-01b01-1k

[0353] Following the general preparation method one, (GalNAc)3-01b was deprotected of tBu and reacted with PEG-L-01 in an amidation reaction to give G3PL-01b01-OAc (M n= 4.3 kDa, PDI = 1.03, n ~ 43), and de-O-acetylation to obtain the trivalent targeting PEGylated lipid G3PL-01b01 (M n = 3.9 kDa, PDI = 1.03, n ~ 43).

[0354] G3PL-01b01-OAc 1 H NMR (400 MHz, (CD3)2SO) δ: 5.20 (d, 3H, H-4Gal), 4.95 (dd, 3H, H-3Gal), 4.47 (d, 3H, H-1Gal), 4.09-3.95 (m, 9H, H-5Gal, H-6Gal, H-6'Gal), 3.90-3.83 (m, 3H, H-2Gal), 3.77-3.41 (m, PEG, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)NH(CH2)2O-, (-OCH2)3C-, -O(CH2)2N<,>NCH2(CH2) 12 CH3), 3.12-2.93 (m, 12H, -C(=O)NHCH2-), 2.35-2.18 (m, 12H, -NHC(=O)(CH2)2C(=O)NH-, -NHC(=O)CH2CH2O-, >NC(=O)CH2-), 2.09 (s, 9H, -OAc x 3), 2.07-2.01 (m, 6H, -(CH2)3CH2C(=O)NH-), 1.99 (s, 9H, -OAc x 3), 1.88 (s, 9H, -OAc x 3), 1.77 (s, 9H, -Ac x 3), 1.57-1.15 (m, 64H, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3), 0.85 (t, 6H, -CH2CH3). 12 CH3), 0.85 (t, 6H, -CH2CH3).

[0355] G3PL-01b01 1 H NMR (400 MHz, (CD3)2SO) δ: 4.20 (d, 3H, H-1Gal), 3.75-3.43 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)NH(CH2)2O-, (-OCH2)3C-, -O(CH2)2N<), 3.20-3.14 (m, 2H, >NCH2(CH2) 12CH3),3.11-2.95(m,12H,-C(=O)NHCH2-),2.35-2.19(m,12H,-NHC(=O)(CH2)2C(=O)NH-,-NHC(=O)CH2CH2O-,>NC(=O)CH2-), 2.12-1.98(m,6H,-(CH2)3CH2C(=O)NH-),1.80(s,9H,-Ac×3),1.55-1.19(m,64H,-CH2CH2CH2-,-CH2(CH2)2CH2-,-CH2(CH2) 11 CH3,-CH2(CH2) 12 CH3), 0.85(t, 6H, -CH2CH3).

[0356] Using the same method described above, but with raw materials of different degrees of polymerization, a material with the same structural formula as G3PL-01b01 was obtained (from...). 1 (Confirmed by H NMR) but with a different molecular weight, the trivalent targeted PEGylated lipid G3PL-01b01-0.5k (M n =2.5kDa, PDI=1.01, n≈11; where the molecular weight of the polyethylene glycol portion is approximately 0.5kDa) and G3PL-01b01-1k(M n =3.0kDa, PDI=1.03, n≈22; where the molecular weight of the polyethylene glycol portion is approximately 1kDa).

[0357] Example 8.2: Synthesis of G3PL-02b01~G3PL-07b01 and G3PL-01b10~G3PL-01b12

[0358] Using general preparation method one, any one of (GalNAc)3-02b to (GalNAc)3-07b, after deprotection of Bn, is subjected to an amidation reaction with PEG-L-01, followed by removal of the O-acetyl group, to obtain trivalent targeted polyethylene glycol-modified lipids G3PL-02b01 to G3PL-07b01 (n≈43):

[0359] 1 H NMR(400MHz,(CD3)2SO)δ:4.46-4.38(m,4H,>CH-,H-1Gal),3.88-3.39(m,PEG,H-4Gal,H-3Gal,H-2Gal,H-5Gal,H-6G al,H-6'Gal,-OCH2(CH2)3-,-C(=O)NHCH2CH2O-,-O(CH2)2N<),3.27-3.13(m,14H,-C(=O)NHCH2-,-CH2N<,>NCH2(CH2)12 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) n CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)

[0360] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.60-4.53 (m, 1H, >CHC(=0)0-), 4.47 (d, 3H, H-1Gal), 4.34 (t, 2H, -C(=0)OCH2-), 3.92-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2CH20-, -C(=0)NH(CH2)2N<, -0(CH2)2N<), 3.28-3.24 (m, 2H, -C(=0)NHCH2CH20-), 3.20 (t, 2H, >NCH2(CH2) 12 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) n CH3), 2.22 (t, 2H, >NC(=0)CH2-), 2.19-2.13 (m, 10H, -NHC(=0)CH2-), 1.85-1.23 (m, 79H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)

[0361] 1H NMR (400 MHz, (CD3)2SO) δ: 4.60-4.42 (m, 5H, >CHC(=0)NH-, >CHC(=0)0-, H-1Gal), 4.10 (t, 2H, >CHC(=0)OCH2-), 3.90-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2CH20-, -0(CH2)2N<), 3.28-3.21 (m, 8H, -C(=0)NHCH2CH20-, >NCH2(CH2) 12 CH3, -C(=0)NHCH2(CH2)3-), 2.20-2.10 (m, 10H, -CH2C(=0)NH-, >NC(=0)CH2-), 1.84-1.22 (m, 81H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3, -CH2CH<), 0.86 (t, 6H, -CH2CH3). M n = 3.6 kDa, PDI = 1.03.

[0362] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.25 (dd, 1H, >CHCH a H b OC(=0)-), 4.08-3.42 (m, PEG, H-4Gal, H-3Gal, H-5Gal, H-6Gal, H-6'Gal, H-2Gal, H-1, H-2, H-3, H-4, H-5, -0(CH2)20-, -0(CH2)2NHC(=0)-, -NHC(=0)CH2CH20-, >CHCH a H b OC(=0)-, -0(CH2)2N<, >NCH2(CH2) 12 CH3), 3.14-3.07 (m, 1H, H-1'), 2.47-2.31 (m, 8H, -NHC(=0)CH2CH20-, -OC(=0)CH2-), 2.21-2.16 (m, 4H, -(CH2)2CH2C(=0)NH-, >NC(=0)CH2-), 1.86-1.20 (m, 57H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2) 11CH3), 0.87 (t, 6H, -CH2CH3). M 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.9 kDa, PDI = 1.04.

[0363] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.42-4.31 (m, 2H, >CHNHC(=0)-), 3.89-3.39 (m, PEG, H-4Gal, H-3Gal, H-5Gal, H-6Gal, H-6'Gal, H-2Gal, -0(CH2)20-, >NCH2(CH2) 12 CH3), 2.27-2.00 (m, 14H, -NHC(=0)CH2-, -CH2CH<,>NC(=0)CH2-), 1.79-1.18 (m, 57H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2) 11 CH3), 0.87 (t, 6H, -CH2CH3). M 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.8 kDa, PDI = 1.02.

[0364] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 3.90-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -0(CH2)20-, -C(=0)NH(CH2)20-, -NHC(=0)CH2CH20-, (-OCH2)3C-, -0(CH2)2N<), 3.18 (t, 2H, >NCH2(CH2) 12 CH3), 2.47-2.40 (m, 6H, -NHC(=0)CH2CH20-), 2.26-2.14 (m, 6H, -NHC(=0)CH2(CH2)2CH2C(=0)NH-, >NC(=0)CH2-), 1.80 (s, 9H, -Ac x 3), 1.68-1.22 (m, 50H, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3), 0.87 (t, 6H, -CH2CH3). M 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.8 kDa, PDI = 1.03.

[0365] Using general preparation method one, (GalNAc)3-01b was deprotected and amidated with any one of PEG-L-10~PEG-L-12, followed by deprotection of O-acetyl group, to obtain trivalent targeting PEGylated lipid G3PL-01b10~G3PL-01b12:

[0366] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.17 (s, 2H, -OCH2C(=O)N<), 3.87-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)NHCH2CH2O-, (-OCH2)3C-, -CH2OCH3), 3.24 (s, 6H, -OCH3), 3.21-3.14 (m, 18H, -C(=O)NHCH2-, >NCH2-), 2.66-2.48 (m, 4H, -NHC(=O)(CH2)2C(=O)NH-), 2.25 (t, 6H, -NHC(=O)CH2CH2O-), 2.18-2.12 (m, 6H, -(CH2)3CH2C(=O)NH-), 1.84-1.24 (m, 71H, -Acx3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH2-).M n = 4.0 kDa, PDI = 1.03, n ~ 45.

[0367] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 3.91-3.41 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)NHCH2CH2O-, (-OCH2)3C-), 3.27 (s, 2H, -NHC(=O)CH2N<), 3.26-3.21 (m, 16H, -C(=O)NHCH2-), 2.69-2.50 (m, 4H, -NHC(=O)(CH2)2C(=O)NH-), 2.40 (t, 4H, >NCH2(CH2) 12CH3),2.29(t,6H,-NHC(=O)CH2CH2O-),2.19-2.13(m,6H,-(CH2)3CH2C(=O)NH-),1.84-1.25(m,75H,-Ac×3,-CH2CH2CH2-,-CH2(CH2)2CH2-,-CH2(CH2) 12 CH3), 0.88(t, 6H, -CH2CH3). M n =4.0kDa, PDI=1.02, n≈45.

[0368] 1 H NMR(400MHz,(CD3)2SO)δ:4.48(d,3H,H-1Gal),3.92-3.40(m,PEG,H-4Gal,H-3Ga l,H-2Gal,H-5Gal,H-6Gal,H-6'Gal,-OCH2(CH2)3-,-NHC(=O)CH2CH2O-,-C(=O)NH CH2CH2O-,(-OCH2)3C-),3.28-3.22(m,16H,-C(=O)NHCH2-),2.76(t,2H,-NHC(=O) CH2CH2N<),2.68-2.48(m,4H,-NHC(=O)(CH2)2C(=O)NH-),2.39(t,4H,>NCH2(CH2) 12 CH3),2.36-2.29(m,8H,-NHC(=O)CH2CH2N<,-NHC(=O)CH2CH2O-),2.18-2.13(m,6H,-(CH 2)3CH2C(=O)NH-),1.81-1.24(m,75H,-Ac×3,-CH2CH2CH2-,-CH2(CH2)2CH2-,-CH2(CH2) 12 CH3), 0.88(t, 6H, -CH2CH3). M n =3.9kDa, PDI=1.04, n≈44.

[0369] Example 8.3: Synthesis of G3PL-01c02

[0370] Using the general preparation method one, (GalNAc)3-01c was deprotected from TBS and then esterified with PEG-L-02, followed by the removal of the O-acetyl group to obtain the trivalent targeted polyethylene glycol-modified lipid G3PL-01c02(M n =3.9kDa, PDI=1.01, n≈45). 1H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.29-4.21 (m, 4H, -C(=O)OCH2-), 3.91-3.42 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, (-OCH2)3C-, -C(=O)OCH2CH2O-, -O(CH2)2N<), 3.27-3.22 (m, 12H, -C(=O)NHCH2CH2CH2NHC(=O)-), 3.19 (t, 2H, >NCH2(CH2) 10 CH3), 2.67-2.61 (m, 4H, -OC(=O)(CH2)2C(=O)O-), 2.43 (t, 2H, -NHC(=O)CH2CH2OC(=O)-), 2.25 (t, 6H, -NHC(=O)CH2CH2O-), 2.23 (t, 2H, >NC(=O)CH2-), 2.16 (t, 6H, -(CH2)3CH2C(=O)NH-), 1.79-1.56 (m, 27H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-), 1.55-1.22 (m, 36H, -CH2(CH2)8CH3, -CH2(CH2) 10 CH3), 0.86 (t, 6H, -CH2CH3).

[0371] Example 8.4: Synthesis of G3PL-02c02~G3PL-07c02

[0372] Using the general preparation method one, any one of (GalNAc)3-02c~(GalNAc)3-07c was deprotected from TBS and esterified with PEG-L-02, and then deprotected from O-acetyl to obtain trivalent targeting polyethyleneglycolated lipid G3PL-02c02~G3PL-07c02 (n ~ 45):

[0373] 1H NMR (400 MHz, (CD3)2SO) δ: 4.50-4.38 (m, 4H, H-1Gal, >CH-), 4.29-4.20 (m, 4H, -C(=0)OCH2-), 3.92-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)OCH2CH20-, -0(CH2)2N<, -C(=0)NHCH2-, -CH2N<), 2.65-2.60 (m, 4H, -OC(=0)(CH2)2C(=0)0-), 2.42 (t, 2H, -NHC(=0)CH2CH2OC(=0)-), 2.24-2.20 (m, 8H, >NC(=0)CH2-, -CH2C(=0)NHCH2-), 1.77-1.20 (m, 63H, -Ac x 3, -(CH2)3CH2CH<, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.7 kDa, PDI = 1.02.

[0374] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 3H, H-1Gal), 4.60-4.51 (m, 1H, >CHC(=0)0-), 4.24 (t, 2H, -C(=0)OCH2CH20-), 4.26-4.18 (m, 4H, -C(=0)OCH2CH2CH2OC(=0)-), 3.87-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)OCH2CH20-, -0(CH2)2N<, -C(=0)NH(CH2)2N<), 3.18 (t, 2H, >NCH2(CH2) 10CH3), 2.66-2.59 (m, 4H, -OC(=0)(CH2)2C(=0)0-), 2.33-1.90 (m, 14H, >NC(=0)(CH2)2CH<, -CH2C(=0)NH-, >NC(=0)CH2(CH2)8CH3, -C(=0)OCH2CH2CH2OC(=0)-), 1.80-1.23 (m, 57H, -Ac x 3, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.6 kDa, PDI = 1.01.

[0375] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.57-4.38 (m, 9H, H-1Gal, >CHC(=0)NH-, >CHC(=0)0-, -C(=0)0(CH2)2OC(=0)-), 4.25 (t, 2H, -C(=0)OCH2CH20-), 3.89-3.43 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)OCH2CH20-, -0(CH2)2N<), 3.27-3.20 (m, 4H, -C(=0)NHCH2-), 3.16 (t, 2H, >NCH2(CH2) 10 CH3), 2.66-2.60 (m, 4H, -OC(=0)(CH2)2C(=0)0-), 2.26 (t, 2H, >NC(=0)CH2-), 2.18-2.12 (m, 6H, -CH2C(=0)NH-), 1.84-1.25 (m, 69H, -Ac x 3, >CHCH2-, -CH2(CH2)2CH2-, -CH2(CH2)8CH3, -CH2(CH2) 10 CH3), 0.88 (t, 6H, -CH2CH3). M n = 3.7 kDa, PDI = 1.02.

[0376] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 3H, H-1Gal), 4.29 (t, 2H, -OC(=0)CH2CH2OC(=0)-), 4.27-4.23 (m, 3H, >CHCH a H bOC(=O)-,-C(=O)OCH2CH2O-),4.04-3.38(m,PEG,H-4Gal,H-3Gal,H-2Gal,H-5Gal,H-6Gal,H-6'Gal,H-1,H- 2,H-3,H-4,H-5,-NHC(=O)CH2CH2O-,-O(CH2)2O-,-O(CH2)2NHC(=O)-,-C(=O)OCH2CH2O-,-O(CH2)2N<,>CHCH a H b OC(=O)-),3.18-3.10(m,3H,H-1',>NCH2(CH2) 10 CH3),2.67(t,2H,-OC(=O)CH2CH2OC(=O)-),2.66-2.61(m,4H,-OC(=O)(CH2)2C(=O)O-),2.46-2.41(m,6H,-NHC(=O)CH2CH2O-),2.25(t,2H,>NC(=O)CH2-),1.79(s,9H,-Ac×3),1.58-1.23(m,36H,-CH2(CH2)8CH3,-CH2(CH2) 10 CH3),0.87(t,6H,-CH2CH3).M n =3.9kDa,PDI=1.01.

[0377] 1 H NMR(400MHz,(CD3)2SO)δ:4.48(d,3H,H-1Gal),4.46-4.35(m,2H,>CHNHC(=O)-),4.24(t,2H,-C(=O)OCH2CH2O-),4.06(t,2H,-(CH2)4CH2OC(=O)-),3.91-3.40(m,PEG,H-4Gal,H-3Gal,H-2Gal,H-5Gal,H-6Gal,H-6'Gal,-O(CH2)2O-,-O(CH2)2NHC(=O)-,-C(=O)OCH2CH2O-,-O(CH2)2N<),3.14(t,2H,>NCH2(CH2) 10CH3),2.67-2.63(m,4H,-OC(=O)(CH2)2C(=O)O-),2.39-1.97(m,12H,-NHC(=O)CH2-,-CH2CH<,>N C(=O)CH2-),1.79(s,9H,-Ac×3),1.65-1.26(m,42H,-CH2(CH2)3CH2-,-CH2(CH2)8CH3,-CH2(CH2) 10 CH3), 0.87(t, 6H, -CH2CH3). M n =3.8kDa, PDI=1.01.

[0378] 1 H NMR(400MHz,(CD3)2SO)δ:4.45(d,3H,H-1Gal),4.27-4.20(m,4H,-C(=O)OCH2-),3.89-3.42(m,PEG,H-4Gal,H-3Gal,H-2Gal,H-5Gal,H-6 Gal,H-6'Gal,-O(CH2)2O-,-O(CH2)2NHC(=O)-,-NHC(=O)CH2CH2O-,(-OCH2)3C-,-C(=O)OCH2CH2O-,-O(CH2)2N<),3.18(t,2H,>NCH2(CH2) 10 CH3),2.66-2.59(m,4H,-OC(=O)(CH2)2C(=O)O-),2.46-2.40(m,8H,-NHC(=O)CH2CH2OC(=O)-,-NHC(=O)C H2CH2O-),2.22(t,2H,>NC(=O)CH2-),1.79(s,9H,-Ac×3),1.55-1.21(m,36H,-CH2(CH2)8CH3,-CH2(CH2) 10 CH3), 0.86(t, 6H, -CH2CH3). M n =3.9kDa, PDI=1.01.

[0379] Example 8.5: Synthesis of G3PL-01a02

[0380] Using general preparation method one, (GalNAc)3-01a was deprotected from Cbz and then subjected to amidation reaction with PEG-L-02, followed by removal of the O-acetyl group to obtain trivalent targeted PEGylated lipid G3PL-01a02(M n =3.9kDa, PDI=1.01, n≈45). 1H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.25 (t, 2H, -C(=0)OCH2-), 3.87-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, (-OCH2)3C-, -C(=0)OCH2CH20-, -0(CH2)2N<), 3.27-3.22 (m, 12H, -C(=0)NHCH2CH2CH2NHC(=0)-), 3.17 (t, 2H, >NCH2(CH2) 10 CH3), 2.66 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.48 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.28 (t, 6H, -NHC(=0)CH2CH20-), 2.23 (t, 2H, >NC(=0)CH2-), 2.18-2.12 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.85-1.24 (m, 63H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3).

[0381] Example 8.6: Synthesis of G3PL-02a02, G3PL-06a02 and G3PL-07a02

[0382] Using the general preparation method one, any one of (GalNAc)3-02a, (GalNAc)3-06a and (GalNAc)3-07a after removal of Cbz or Boc protection was subjected to amidation reaction with PEG-L-02, followed by removal of O-acetyl, to obtain trivalent targeting polyethyleneglycolated lipid G3PL-02a02, G3PL-06a02 and G3PL-07a02 (n ~ 45):

[0383] 1H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.46-4.40 (m, 1H, >CH-), 4.23 (t, 2H, -C(=0)OCH2-), 3.88-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)OCH2CH20-, -0(CH2)2N<, -C(=0)NHCH2(CH2)3-, -C(=0)NHCH2CH2CH2N<), 3.17 (t, 2H, >NCH2(CH2) 10 CH3), 2.67 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.50 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.25-2.22 (m, 8H, -(CH2)3CH2C(=0)NH-, >NC(=0)CH2-), 1.81-1.22 (m, 67H, -Ac x 3, -(CH2)3CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.6 kDa, PDI = 1.01.

[0384] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 3H, H-1Gal), 4.44-4.32 (m, 2H, >CHNHC(=0)-), 4.25 (t, 2H, -C(=0)OCH2-), 3.90-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -0(CH2)20-, -0(CH2)2NHC(=0)-, -C(=0)OCH2CH20-, -0(CH2)2N<), 3.17 (t, 2H, >NCH2(CH2) 10 CH3), 2.65 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.46 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.36-1.96 (m, 10H, -NHC(=0)(CH2)2CH<, >NC(=0)CH2-), 1.79 (s, 9H, -Ac x 3), 1.57-1.22 (m, 36H, -CH2(CH2)8CH3, -CH2(CH2)10 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.7 kDa, PDI = 1.02.

[0385] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 3H, H-1Gal), 4.25 (t, 2H, -C(=0)OCH2-), 3.89-3.43 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -0(CH2)20-, -0(CH2)2NHC(=0)-, -C(=0)OCH2CH20-, -0(CH2)2N<, -NHC(=0)CH2CH20-, (-OCH2)3C-), 3.18 (t, 2H, >NCH2(CH2) 10 CH3), 2.68 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.49 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.45-2.38 (m, 6H, -NHC(=0)CH2CH20-), 2.24 (t, 2H, >NC(=0)CH2-), 1.79 (s, 9H, -Ac x 3), 1.56-1.20 (m, 36H, -CH2(CH2)8CH3, -CH2(CH2) 10 CH3), 0.86 (t, 6H, -CH2CH3). M n = 3.8 kDa, PDI = 1.01.

[0386] Example 8.7: Synthesis of G3PL-03a01 and G3PL-04a01

[0387] Using the general preparation method one, (GalNAc)3-03a or (GalNAc)3-04a was deprotected from tBu or Bn protection and then reacted with PEG-L-01 to form an amide, and then deprotected from O-acetyl to obtain trivalent targeting polyethylene glycolated lipids G3PL-03a01 and G3PL-04a01:

[0388] 1H NMR (400 MHz, (CD3)2SO) δ: 4.47 (d, 3H, H-1Gal), 4.42-4.35 (m, 1H, >CHNHC(=0)-), 3.93-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2CH20-, -C(=0)NH(CH2)2N<, -0(CH2)2N<), 3.27-3.23 (m, 2H, -C(=0)NHCH2CH20-), 3.19 (t, 2H, >NCH2(CH2) 12 CH3), 2.36-1.95 (m, 12H, >NC(=0)(CH2)2CH<, -CH2C(=0)NH-, >NC(=0)CH2-), 1.79 (s, 9H, -Ac x 3), 1.57-1.18 (m, 58H, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.86 (t, 6H, -CH2CH3). M n = 3.5 kDa, PDI = 1.04, n ~ 43.

[0389] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.43-4.35 (m, 2H, >CHNHC(=0)-), 3.89-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2CH20-, -0(CH2)2N<), 3.26-3.17 (m, 8H, >NCH2(CH2) 12 CH3, -C(=0)NHCH2-), 2.23 (t, 2H, >NC(=0)CH2-), 2.17-2.11 (m, 6H, -CH2C(=0)NH-), 1.83-1.20 (m, 79H, -Ac x 3, -CH2(CH2)2CH2-, -CH2CH<, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.5 kDa, PDI = 1.03, n ~ 43.

[0390] Example 8.8: Synthesis of G3PL-05a02

[0391] Using General Preparation Method One, (GalNAc)3-05a was deprotected from TIPS protection and esterified with PEG-L-02, followed by deprotection of O-acetyl groups, to give the trivalent targeted PEGylated lipid G3PL-05a02 (M n = 3.8 kDa, PDI = 1.01, n ~ 45). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.27-4.23 (m, 3H, >CHCH a H b OC(=0)-, -C(=0)OCH2CH20-), 3.99-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, H-1, H-2, H-3, H-4, H-5, -NHC(=0)CH2CH20-, a H b OC(=0)-), 3.16 (t, 2H, >NCH2(CH2) 10 CH3), 3.12-3.07 (m, 1H, H-1'), 2.67-2.62 (m, 4H, -OC(=0)(CH2)2C(=0)0-), 2.49-2.43 (m, 6H, -NHC(=0)CH2CH20-), 2.22 (t, 2H, >NC(=0)CH2-), 1.79 (s, 9H, -Ac x 3), 1.57-1.19 (m, 36H, -CH2(CH2)8CH3, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3).

[0392] Example 8.9: Synthesis of G3PL-01b03

[0393] Using General Preparation Method One, (GalNAc)3-01b was deprotected from tBu protection and esterified with PEG-L-03 (M n = 2.5 kDa, PDI = 1.01, n ~ 45) followed by deprotection of O-acetyl groups, to give the trivalent targeted PEGylated lipid G3PL-01b03 (M n = 4.0 kDa, PDI = 1.01, n ~ 45). 1H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.22 (t, 2H, -C(=O)OCH2-), 3.91-3.42 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)OCH2CH2O-, (-OCH2)3C-, -O(CH2)2N<), 3.27-3.21 (m, 12H, -C(=O)NHCH2-), 3.18 (t, 2H, >NCH2(CH2) 12 CH3), 2.64 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.48 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.26 (t, 6H, -NHC(=O)CH2CH2O-), 2.22 (t, 2H, >NC(=O)CH2-), 2.19-2.13 (m, 6H, -(CH2)3CH2C(=O)NH-), 1.82-1.22 (m, 73H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0394] Example 8.10: Synthesis of G3PL-01b04~G3PL-01b09

[0395] Using the general preparation method one, after removing tBu protection of (GalNAc)3-01b, esterification reaction was carried out with any one of PEG-L-04~PEG-L-09, and then O-acetyl was removed, to obtain trivalent targeting polyethylene glycolated lipid G3PL-01b04~G3PL-01b09:

[0396] 1H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.24 (t, 2H, -C(=0)OCH2-), 3.90-3.43 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)OCH2CH20-, (-OCH2)3C-, -OCH2CH2N<, -(CH2)6CH2OCH2(CH2)5-), 3.27-3.22 (m, 12H, -C(=0)NHCH2-), 2.67-2.64 (m, 4H, -NHC(=0)CH2CH2C(=0)0-, -OCH2CH2N<), 2.51 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.46 (t, 4H, >NCH2(CH2)50-, >NCH2(CH2) 12 CH3), 2.27 (t, 6H, -NHC(=0)CH2CH20-), 2.18-2.11 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.82-1.20 (m, 71H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)5CH2-, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 4.0 kDa, PDI = 1.04, n ~ 45.

[0397] 1H NMR (400 MHz, (CD3)2SO) δ: 4.48 (d, 3H, H-1Gal), 4.06 (t, 2H, -C(=0)0CH2(CH2)7CH3), 3.88-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)0CH2CH20-, (-OCH2)3C-, -0(CH2)2N<), 3.26-3.20 (m, 14H, -C(=0)NHCH2-), 3.16 (t, 2H, >NCH2(CH2)8CH3), 2.68-2.50 (m, 4H, -NHC(=0)(CH2)2C(=0)0-), 2.33 (t, 2H, -(CH2)4CH2C(=0)0-), 2.27 (t, 6H, -NHC(=0)CH2CH20-), 2.23 (t, 2H, >NC(=0)CH2-), 2.17-2.11 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.82-1.21 (m, 63H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)7CH3, -CH2(CH2)8CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.9 kDa, PDI = 1.02, n ~ 43.

[0398] 1H NMR (400 MHz, (CD3)2SO) δ: 4.45 (d, 3H, H-1Gal), 4.07 (t, 2H, -C(=0)OCH2CH<), 3.89-3.41 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)OCH2CH20-, (-OCH2)3C-, -0(CH2)2N<), 3.26-3.20 (m, 14H, -C(=0)NHCH2-), 3.15 (t, 2H, >NCH2(CH2)8CH3), 2.66-2.48 (m, 4H, -NHC(=0)(CH2)2C(=0)0-), 2.30-2.25 (m, 7H, -NHC(=0)CH2CH20-, -OC(=0)CH<), 2.22 (t, 2H, >NC(=0)CH2-), 2.16-2.11 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.79-1.18 (m, 75H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)4CH2-, -CH2(CH2)5CH3, -CH2(CH2)7CH3, -CH2(CH2)8CH3), 0.86 (t, 9H, -CH2CH3). M n = 4.0 kDa, PDI = 1.05, n ~ 45.

[0399] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.17-4.13 (m, 2H, -C(=0)CH2CH<), 3.93-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -OCH2)3C-, -0(CH2)2N<, >CHCH2OH), 3.27-3.22 (m, 12H, -C(=0)NHCH2-), 3.17 (t, 2H, >NCH2(CH2) 12CH3),2.68(t,2H,-NHC(=O)CH2CH2C(=O)O-),2.50(t,2H,-NHC(=O)CH2CH2C(=O)O-),2.27(t,6H,-NHC(=O)CH2CH2O-),2.24(t,2H,>NC(=O)CH2-),2.17-2.11(m,6H,-(CH2)3CH2C(=O)NH-),1.84-1.24(m,73H,-Ac×3,-CH2CH2CH2-,-CH2(CH2)2CH2-,-CH2(CH2) 11 CH3,-CH2(CH2) 12 CH3),0.88(t,6H,-CH2CH3).M n =4.0kDa,PDI=1.03,n≈45.

[0400] 1 H NMR(400MHz,(CD3)2SO)δ:4.49(d,3H,H-1Gal),4.25-4.20(m,4H,-C(=O)OCH2-),3.92-3.42(m,PEG,H-4Gal,H-3Gal,H-2Gal,H-5Gal,H-6Gal,H-6'Gal,-OCH2(CH2)3-,-NHC(=O) CH2CH2O-,-C(=O)OCH2CH2O-,(-OCH2)3C-),3.31(s,2H,-OC(=O)CH2N<),3.28-3.23(m,12H,-C(=O)NHCH2-),2.68(t,2H,-NHC(=O)CH2CH2C(=O)O-),2.60-2.44(m,4H,>NCH2(CH2) 12 CH3),2.51(t,2H,-NHC(=O)CH2CH2C(=O)O-),2.29(t,6H,-NHC(=O)CH2CH2O-),2.18-2.13(m,6H,-(CH2)3CH2C(=O)NH-),1.85-1.23(m,75H,-Ac×3,-CH2CH2CH2-,-CH2(CH2)2CH2-,-CH2(CH2) 12 CH3),0.89(t,6H,-CH2CH3).M n =3.9kDa,PDI=1.02,n≈43.

[0401] 1H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.23-4.18 (m, 4H, -C(=0)OCH2-), 3.90-3.44 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)OCH2CH20-, (-OCH2)3C-), 3.28-3.21 (m, 12H, -C(=0)NHCH2-), 2.70-2.66 (m, 4H, -NHC(=0)CH2CH2C(=0)0-, -OC(=0)CH2CH2N<), 2.48 (t, 2H, -NHC(=0)CH2CH2C(=0)0-), 2.43-2.37 (m, 6H, >NCH2(CH2) 12 CH3, -OC(=0)CH2CH2N<), 2.28 (t, 6H, -NHC(=0)CH2CH20-), 2.20-2.14 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.82-1.20 (m, 75H, -Ac x 3, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3). M n = 3.9 kDa, PDI = 1.02, n ~ 43.

[0402] Example 8.11: Synthesis of G3PL-03e02

[0403] Using the general preparation method one, (GalNAc)3-03e was deprotected and amidated with PEG-L-02, followed by removal of O-acetyl group, to obtain the trivalent targeting polyethylene glycolized lipid G3PL-03e02 (M n = 3.6 kDa, PDI = 1.01, n ~ 45). 1H NMR (400 MHz, (CD3)2SO) δ: 4.44 (d, 3H, H-1Gal), 4.40-4.33 (m, 1H, >CHNHC(=0)-), 4.18 (t, 2H, -C(=0)OCH2-), 3.87-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NH(CH2)2N<, -C(=0)OCH2CH2O-, -0(CH2)2N<, -C(=0)NH(CH2)2NHC(=0)-), 3.15 (t, 2H, >NCH2(CH2) 12 CH3), 2.54 (t, 2H, -NHC(=0)CH2CH2C(=0)O-), 2.44 (t, 2H, -NHC(=0)CH2CH2C(=0)O-), 2.33-1.92 (m, 12H, >NC(=0)(CH2)2CH<, -(CH2)3CH2C(=0)NH-, >NC(=0)CH2(CH2)8CH3), 1.78 (s, 9H, -Ac x 3), 1.55-1.16 (m, 48H, -CH2(CH2)2CH2-, -CH2(CH2)8CH3, -CH2(CH2) 10 CH3), 0.86 (t, 6H, -CH2CH3).

[0404] Example 8.12: Synthesis of G3PL-03d03

[0405] G3PL-03d03 was synthesized using the general preparation method two, with the following specifics:

[0406] Step 1: PEGylated lipid PEG-L-03 (2.50 g, 1.0 mmol, M n= 2.5 kDa, PDI = 1.01, n ~ 45), DSC (3.07 g, 12.0 mmol) and TEA (4.2 mL, 30.0 mmol) were dissolved in 30 mL of anhydrous dichloromethane and stirred at room temperature overnight. A solution of compound (GalNAc)3-03d-NH2(18.77 g, 12.0 mmol; obtained by removing Boc protection from (GalNAc)3-03d) in dichloromethane (200 mL) was added and the reaction was continued to be stirred at room temperature for 24 hours. After the reaction was completed, it was washed with saturated sodium chloride solution. The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain compound G3PL-03d03-OAc (3.55 g). The molecular weight was determined to be 4.1 kDa by MALDI-TOF MS. The PDI was determined to be 1.01 by GPC.

[0407] Step 2: Compound G3PL-03d03-OAc (4.08 g, 1.0 mmol) was dissolved in ethanol (50 mL) at room temperature, and ammonia water (100 mL) was added and heated at 40°C for 48 hours. After concentration under reduced pressure, column chromatography purification gave trivalent target PEGylated lipid G3PL-03d03 (2.76 g). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.62-4.56 (m, 1H, >CHC(=O)O-), 4.48 (d, 3H, H-1Gal), 4.10-3.95 (m, 4H, -NHC(=O)OCH2CH2O-, -C(=O)OCH2CH2NHC(=O)O-), 3.94-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)OCH2CH2O-, -C(=O)NH(CH2)2N<, -O(CH2)2N<, -C(=O)OCH2CH2NHC(=O)O-), 3.21 (t, 2H, >NCH2(CH2) 12 CH3), 2.41-1.97 (m, 12H, >NC(=O)(CH2)2CH<, -(CH2)3CH2C(=O)NH-, >NC(=O)CH2(CH2) 11 CH3), 1.80 (s, 9H, -Ac x 3), 1.58-1.22 (m, 58H, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH2(CH2) 12CH3), 0.87 (t, 6H, -CH2CH3). MALDI-TOF MS determined the molecular weight to be 3.7 kDa. GPC determined the PDI to be 1.01.

[0408] Example 8.13: Synthesis of G3PL-05d03

[0409] Using General Preparation Method Two, (GalNAc)3-05d was deprotected and subjected to a carbamating reaction with PEG-L-03, followed by removal of the O-acetyl group, to obtain the trivalent targeting polyethyleneglycolated lipid G3PL-05d03 (M n = 4.0 kDa, PDI = 1.01, n ~ 45). 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 3H, H-1Gal), 4.26 (dd, 1H, >CHCH a H b OC(=O)NH-), 4.19 (dd, 1H, >CHCH a H b OC(=O)NH-), 4.09 (t, 2H, -NHC(=O)OCH2CH2O-), 4.05-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, H-1, H-2, H-3, H-4, H-5, -NHC(=O)CH2CH2O-, -O(CH2)2O-, -O(CH2)2NHC(=O)-, -NHC(=O)OCH2CH2O-, -O(CH2)2N<), 3.26-3.05 (m, 7H, H-1', -OC(=O)NHCH2-, >NCH2(CH2) 12 CH3), 2.45-2.40 (m, 6H, -NHC(=O)CH2CH2O-), 2.23 (t, 2H, >NC(=O)CH2-), 1.79 (s, 9H, -Ac x 3), 1.70-1.24 (m, 48H, -CH2CH2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.87 (t, 6H, -CH2CH3).

[0410] Example 8.14: Synthesis of G3PL-01bN02, G3PL-01bN04, G3PL-02bN03 and G3PL-07bN04

[0411] Reference is made to the preparation method of G2PL-03N02 in Example 7.4, replacing (GalNAc)2-03 with (GalNAc)3-01b, (GalNAc)3-02b or (GalNAc)3-07b, and optionally replacing N-02 with N-03 or N-04, to obtain trivalent targeting pegylated lipids G3PL-01bN02 (M n = 4.0 kDa, PDI = 1.02, n ~ 44), G3PL-01bN04 (M n = 4.0 kDa, PDI = 1.02, n ~ 44), G3PL-02bN03 (M n = 3.8 kDa, PDI = 1.01, n ~ 44) and G3PL-07bN04 (M n = 4.0 kDa, PDI = 1.01, n ~ 44):

[0412] G3PL-01bN02 1 H NMR (400 MHz, (CD3)2SO) d: 7.82 (s, 1H, triazol), 4.62-4.40 (m, 4H, -CH2-triazol-CH2-), 4.26 (d, 3H, H-1Gal), 3.77-3.39 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=O)CH2CH2O-, -C(=O)NH(CH2)2O-, (-OCH2)3C-, -O(CH2)2N<, -CH2CH2-triazol), 3.19-2.90 (m, 14H, >NCH2(CH2) 12 CH3, -C(=O)NHCH2-), 2.38-2.18 (m, 12H, -NHC(=O)(CH2)2C(=O)NH-, -NHC(=O)CH2CH2O-, >NC(=O)CH2-), 2.16-2.00 (m, 6H, -(CH2)3CH2C(=O)NH-), 1.82 (s, 9H, -Ac x 3), 1.56-1.18 (m, 64H, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3, -CH2(CH2)

[0413] G3PL-01bN04 1H NMR (400 MHz, (CD3)2SO) δ: 7.85 (s, 1H, triazol), 4.63-4.47 (m, 2H, -CH2CH2-triazol), 4.29 (d, 2H, triazol-CH2NHC(=0)-), 4.25 (d, 3H, H-1Gal), 3.75-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -NHC(=0)CH2CH20-, -C(=0)NH(CH2)20-, (-OCH2)3C-, -CH2CH2-triazol), 3.25-3.06 (m, 12H, -C(=0)NHCH2-), 3.03 (s, 2H, -NHC(=0)CH2N<), 2.47 (t, 4H, >NCH2-), 2.34-2.20 (m, 10H, -NHC(=0)(CH2)2C(=0)NH-, -NHC(=0)CH2CH20-), 2.15-2.06 (m, 6H, -(CH2)3CH2C(=0)NH-), 1.78 (s, 9H, -Ac x 3), 1.65-1.20 (m, 66H, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 0.85 (t, 6H, -CH2CH3).

[0414] G3PL-02bN03 1 H NMR (400 MHz, (CD3)2SO) δ: 7.79 (s, 1H, triazol), 4.56-4.34 (m, 8H, -CH2-triazol-CH2-, 4H, >CH-, H-1Gal), 3.85-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2CH20-, -CH2CH2-triazol, -OCH2CH2N<), 3.24-3.14 (m, 12H, -C(=0)NHCH2-, -CH2N<), 2.66 (t, 2H, -OCH2CH2N<), 2.56 (t, 4H, >NCH2-), 2.17-2.09 (m, 10H, -NHC(=0)CH2-), 1.78-1.19 (m, 81H, -Ac x 3, -CH2CH<, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2) 12CH3), 0.86 (t, 6H, -CH2CH3).

[0415] G3PL-07bN04 1 H NMR (400 MHz, (CD3)2SO) δ: 7.80 (s, 1H, triazol), 4.60-4.42 (m, 5H, -CH2CH2-triazol, H-1Gal), 4.25 (d, 2H, triazol-CH2NHC(=0)-), 3.86-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -0(CH2)2-, -C(=0)NH(CH2)20-, -NHC(=0)CH2CH20-, (-OCH2)3C-, -CH2CH2-triazol), 3.01 (s, 2H, -NHC(=0)CH2N<), 2.49-2.41 (m, 10H, -NHC(=0)CH2CH20-, >NCH2-), 2.22-2.15 (m, 4H, -NHC(=0)CH2-), 1.79 (s, 9H, -Ac x 3), 1.63-1.20 (m, 52H, -CH2(CH2)2CH2-, -CH2(CH2) 12 CH3), 0.86 (t, 6H, -CH2CH3).

[0416] Example 9: Synthesis of tetravalent targeting PEGylated lipids

[0417] Example 9.1: Synthesis of G4PL-01a02

[0418] Using the general preparation method one, (GalNAc)4-01a was deprotected and reacted with PEG-L-02 to give amide, then deprotected O-acetyl group to obtain tetravalent targeting PEGylated lipid G4PL-01a02 (M n = 4.2 kDa, PDI = 1.01, n ~ 45). 1H NMR (400 MHz, (CD3)2SO) δ: 4.66-4.62 (m, 2H, >CHOC(=O)NH-), 4.46 (d, 4H, H-1Gal), 4.44-4.35 (m, 1H, >CHNHC(=O)-), 4.19 (t, 2H, -C(=O)OCH2-), 3.88-3.41 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=O)NHCH2-, -OC(=O)NHCH2-, -C(=O)OCH2CH2O-, -O(CH2)2N<), 3.19 (t, 2H, >NCH2(CH2) 10 CH3), 2.55 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.48 (t, 2H, -NHC(=O)CH2CH2C(=O)O-), 2.35-2.15 (m, 13H, -CH2NHC(=O)CH2-, >NC(=O)CH2-, -NHC(=O)CH2CH a H b CH<), 1.97-1.94 (m, 1H, -NHC(=O)CH2CH a H b CH<), 1.79 (s, 12H, -Ac x 4), 1.59-1.25 (m, 64H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 10 CH3), 0.87 (t, 6H, -CH2CH3).

[0419] Example 9.2: Synthesis of G4PL-01b01~G4PL-03b01

[0420] Using the general preparation method one, any one of (GalNAc)4-01b~(GalNAc)4-03b after removing Bn protection was subjected to amidation reaction with PEG-L-01, and then removing O-acetyl, to obtain the tetravalent targeting polyethylene glycolated lipid G4PL-01b01~G4PL-03b01 (n ~ 43).

[0421] 1H NMR (400 MHz, (CD3)2SO) δ: 4.65-4.60 (m, 2H, >CHOC(=0)NH-), 4.46 (d, 4H, H-1Gal), 4.43-4.33 (m, 1H, >CHNHC(=0)-), 3.89-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2-, -OC(=0)NHCH2-, -0(CH2)2N<, -C(=0)NH(CH2)20-), 3.17 (t, 2H, >NCH2(CH2) 10 CH3), 2.36-2.12 (m, 17H, -NHC(=0)CH2-, >NC(=0)CH2-, -NHC(=0)CH2CH a H b CH<), 1.98-1.94 (m, 1H, -NHC(=0)CH2CH a H b CH<), 1.78 (s, 12H, -Ac x 4), 1.58-1.20 (m, 80H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.86 (t, 6H, -CH2CH3). M n = 4.3 kDa, PDI = 1.03.

[0422] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.46 (d, 4H, H-1Gal), 4.46-4.38 (m, 2H, >CHNHC(=0)-), 3.89-3.38 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -0(CH2)20-, -0(CH2)2N<, -C(=0)NH(CH2)20-, -C(=0)NHCH2-, -CH2N<), 2.32-2.13 (m, 20H, -NHC(=0)CH2-, >NC(=0)CH2-, -NHC(=0)CH2CH a H b CH<,), 1.97-1.93 (m, 2H, -NHC(=0)CH2CH a H bCH3, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.86 (t, 6H, -CH2CH3). M n = 4.5 kDa, PDI = 1.03.

[0423] 1 H NMR (400 MHz, (CD3)2SO) δ: 4.49 (d, 4H, H-1Gal), 3.92-3.43 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -O(CH2)2N<-, -C(=O)NH(CH2)2O-, -C(=O)NHCH2-, -CH2N<), 2.35-2.14 (m, 22H, -NHC(=O)CH2-, >NC(=O)CH2-), 1.87-1.25 (m, 96H, -Ac x 4, -CH2CH2CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.88 (t, 6H, -CH2CH3). M n = 4.3 kDa, PDI = 1.05.

[0424] Example 9.3: Synthesis of G4PL-01c01

[0425] Using General Preparation Method Two, (GalNAc)4-01c was deprotected from TBS protection, then reacted with PEG-L-01 for carbamate formation, and then deprotected from O-acetyl to obtain the tetravalent targeting PEGylated lipid G4PL-01c01 (M n = 4.4 kDa, PDI = 1.03, n ~ 43). 1H NMR (400 MHz, (CD3)2SO) δ: 4.67-4.62 (m, 2H, >CHOC(=0)NH-), 4.48 (d, 4H, H-1Gal), 4.45-4.34 (m, 1H, >CHNHC(=0)-), 3.99 (t, 2H, -CH2OC(=0)NH-), 3.89-3.37 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2-, -OC(=0)NHCH2-, >NCH2-, -OC(=0)NHCH2CH2O-, -OCH2CH2N<), 2.36-1.94 (m, 16H, >NC(=0)CH2-, -CH2C(=0)NH-, -NHC(=0)CH2CH2CH<), 1.80 (s, 12H, -Ac x 4), 1.60-1.20 (m, 90H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 11 CH3, -CH2(CH2) 12 CH3), 0.88 (t, 6H, -CH2CH3).

[0426] Example 9.4: Synthesis of G4PL-01bN01

[0427] Following the procedure for the preparation of G2PL-03N02 in Reference Example 7.4, (GalNAc)2-03 was replaced by (GalNAc)4-01b and N-02 was replaced by N-01 to give the tetra-valent targeting PEGylated lipid G4PL-01bN01 (M n = 4.8 kDa, PDI = 1.02, n ~ 44):

[0428] G4PL-01bN01 1H NMR (400 MHz, (CD3)2SO) δ: 8.57 & 8.36 (2s, 1H, triazol), 4.64-4.58 (m, 2H, >CHOC(=0)NH-), 4.50 (t, 2H, -CH2CH2-triazol), 4.46 (d, 4H, H-1Gal), 4.43-4.33 (m, 1H, >CH-), 3.79-3.40 (m, PEG, H-4Gal, H-3Gal, H-2Gal, H-5Gal, H-6Gal, H-6'Gal, -OCH2(CH2)3-, -C(=0)NHCH2-, -OC(=0)NHCH2-, -CH2CH2-triazol-CH2-, -C(=0)NH(CH2)20-), 3.15 (t, 2H, >NCH2-), 2.33-2.14 (m, 17H, -NHC(=0)CH2-, >NC(=0)CH2-, -NHC(=0)CH2CH a H b CH2CH2-), 2.00-1.95 (m, 1H, -NHC(=0)CH2CH a H b CH2CH2-), 1.79 (s, 12H, -Ac x 4), 1.60-1.21 (m, 80H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 11 CH2CH2-), 1.79 (s, 12H, -Ac x 4), 1.60-1.21 (m, 80H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2) 12 CH2CH2-), 1.79 (s, 12H, -Ac x 4), 1.60-1.21 (m, 80H, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)

[0429] Example 10: Preparation of a lipid pharmaceutical composition

[0430] Lipid drug composition nanoparticles (LNP / Fluc-mRNA) containing Fluc-mRNA were prepared in this example, which contained DSPC as phospholipid, cholesterol as sterol lipid, and DHA-1 as cationic lipid. The difference was the pegylated lipid. DHA-1 was synthesized according to the method disclosed in CN113402405A. Fluc-mRNA can express luciferase protein, which can catalyze the oxidation reaction of luciferin substrate to produce bioluminescence. The non-targeted pegylated lipid used was PEG2k-DMG, PEG2k-DTA, PEG1k-DTA or PEG500-DTA. The targeted pegylated lipid used was G3PL-D1, G3PL-D2, GPL-D3 or the monovalent, divalent, trivalent or tetravalent targeted pegylated lipid in the previous examples. Among them, G3PL-D1 and G3PL-D2 were synthesized according to the method in Nature Communications, 2023, 14, 2776; GPL-D3 was synthesized according to the method in WO2008042973A2.

[0431] The preparation method of LNP / Fluc-mRNA is as follows:

[0432] Step (1): A certain amount of DHA-1, DSPC, cholesterol, non-targeted pegylated lipid and targeted pegylated lipid stock solution was dissolved in ethanol according to the molar ratio given in Table 1 to obtain an ethanol phase solution.

[0433] Step (2): Fluc-mRNA was added to 10-50 mM citrate buffer (pH=4) to obtain an aqueous phase solution.

[0434] Step (3): The ethanol phase solution and the aqueous phase solution were mixed (1:3 v / v) to prepare LNP / Fluc-mRNA, and then washed by multiple DPBS ultrafiltration to remove ethanol and free molecules, and finally passed through a 0.2 μm sterile filter for standby.

[0435] Table 1 Lipid formulation in lipid drug composition

[0436] Example 11: Biological activity test of lipid drug composition

[0437] Example 11.1: Nanoparticle size, nucleic acid complexing ability and encapsulation efficiency determination

[0438] Particle size determination: According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), the particle size of LNP preparations encapsulating nucleic acid drugs can exert better efficacy when the particle size is in the range of 60-150 nm. In this embodiment, the particle size of LNP / Fluc-mRNA was determined by dynamic light scattering (DLS). The LNP / Fluc-mRNA size uniformity determined was high, and the PDI was less than 0.3. The experimental results showed that the LNP / Fluc-mRNA (LNP01-LNP55, including LNP07-d1-LNP07-d10) prepared by using the targeted pegylated lipid of the present application had a particle size of 64 nm-117 nm (as shown in Table 2), which was in the particle size range capable of achieving better efficacy.

[0439] Nucleic acid complexing ability determination: An appropriate amount of agarose was dissolved in TAE buffer, heated in a microwave oven to completely dissolve the agarose particles, cooled, and then nucleic acid dye GelGreen was added to the cooled agarose gel. The gel was placed in a gel tank and naturally air-dried. The lipid drug composition nanoparticles were mixed with the loading buffer and then added to the agarose gel well. The electrophoresis voltage was set to 90 V, and the electrophoresis experiment was performed at room temperature for 10 min. No obvious free Fluc-mRNA was found in the electrophoresis results of LNP-D0-LNP-D3 and LNP01-LNP55, indicating that the targeted ligand modified pegylated lipid of the present application did not affect the nucleic acid complexing ability of the lipid nanoparticles.

[0440] Encapsulation efficiency determination: The LNP / Fluc-mRNA was ultracentrifuged using an ultracentrifuge (4°C, 60000 rpm, 1 h), and the concentration of unencapsulated Fluc-mRNA in the supernatant was detected using a nucleic acid quantifier. The encapsulation efficiency of LNP for Fluc-mRNA was calculated. The encapsulation efficiency of LNP-D0-LNP-D3 and LNP01-LNP55 was all above 84% (as shown in Table 2), indicating that the lipid composition of the present application had a high encapsulation efficiency for nucleic acid drugs.

[0441] Table 2 Particle size and encapsulation efficiency of LNP / Fluc-mRNA

[0442] Example 11.2: Serum stability evaluation

[0443] LNP01-LNP55 were added into the culture medium containing 10% fetal bovine serum (FBS) and stirred at 37°C, and the particle size change of LNP / Fluc-mRNA was determined by sampling at regular time intervals. The experimental results show that the particle size change of LNP01-LNP55 (including LNP07-d1-LNP07-d10) is in the range of 0-6% within 7 days, indicating that the LNP / Fluc-mRNA of the application has good serum stability.

[0444] Example 11.3: Evaluation of cytotoxicity

[0445] The commercial transfection reagent Lipofectamine 2000 (L2K) was used to encapsulate Fluc-mRNA to prepare the lipid drug composition L2K / Fluc-mRNA as a positive control group. HeLa was inoculated in a 96-well plate at a ratio of 6000 cells / well and a volume of 100 μL, and was divided into a blank control group, a positive control group and an experimental group, and was incubated at 37°C and 5% CO2. After 24 hours of cell incubation, 10 μL of PBS solution was added to the blank control group, 3.3 μg / mL of L2K / Fluc-mRNA (10 μL) was added to the positive control group, and 3.3 μg / mL of LNP01-LNP55 (10 μL) was added to the experimental group, and the incubation was continued at 37°C and 5% CO2. After 24 hours of incubation, the 96-well plate was taken out under light shielding conditions, and the culture solution was aspirated, and 120 μL of CCK-8 solution diluted was added to each well. After incubation at 37°C and 5% CO2 for 4 hours, the absorbance value of each well at 450 nm wavelength was determined by an enzyme-labeled instrument.

[0446] Each group was tested three times, and the average value was taken. The results show that the cell survival rate of the positive control group is 93%, and the cell survival rate of all experimental groups is greater than 97%, indicating that the lipid nanoparticles of the application have no obvious cytotoxicity.

[0447] Example 11.4: Evaluation of in vitro targeting effect

[0448] The efficiency of the LNP-D0, LNP-D1, LNP-D2, LNP-D3, LNP01-LNP55 in transfecting nucleic acid drugs was evaluated by Luciferase bioluminescence. Mouse primary hepatocytes were seeded in a black clear bottom 96-well plate at a density of 150000 cells per well. After incubation in a cell incubator for 24 hours, the aforementioned lipid drug compositions were used for transfection at a dose of 0.2 ug Fluc-mRNA per well. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-luciferin sodium (1.5 mg / mL) substrate. After 5 minutes of incubation, the bioluminescence was detected using a microplate reader, and the stronger the fluorescence, the more Fluc-mRNA entered the cytoplasm and translated into the corresponding fluorescent protein. The relative fluorescence value of the LNP-D0 group without a targeting group was set to 1. The results showed (as shown in Table 3) that the in vitro transfection effect of the control groups (LNP-D1, LNP-D2, LNP-D3) and the experimental groups (LNP01-LNP55) containing the targeting polyethylene glycolized lipid was better than that of the control group (LNP-D0) without the targeting polyethylene glycolized lipid. Among them, the relative fluorescence values (1.5-2.2) of the LNP01-LNP03 and LNP46-LNP49 groups were greater than 1, indicating that the monovalent targeting polyethylene glycolized lipid contained therein could promote cell uptake to a certain extent. In contrast, the relative fluorescence value of the control group LNP-D3, which also contained a monovalent targeting polyethylene glycolized lipid, was only 1.2. The LNP04-LNP06 group and the LNP50 group containing divalent targeting polyethylene glycolized lipids showed relatively better transfection effect, with a relative fluorescence value of 2.2-2.7. The relative fluorescence values of the LNP07-LNP40 group and the LNP51-LNP54 group containing trivalent targeting polyethylene glycolized lipids were significantly improved (4.7-6.6) and higher than those of the LNP-D1 group (3.9) and the LNP-D2 group (3.4), which may be because the trivalent targeting polyethylene glycolized lipids of the present application can more effectively promote ASGPR-mediated endocytosis, thereby significantly improving the liver-targeted drug delivery efficiency of the nanoparticles. The LNP41-LNP45 group and the LNP55 group containing tetravalent targeting polyethylene glycolized lipids also showed good in vitro transfection effect.

[0449] Table 3 Cell transfection test results

[0450] Example 11.5: Evaluation of in vivo targeting effect

[0451] LNP-D0, LNP-D1, LNP-D2, LNP-D3, LNP01, LNP07 and LNP08 were delivered to 6-8 weeks old female BALB / c mice by tail vein injection at a dose of 10 ug / mouse, two mice per group. Small animal live fluorescence imaging was performed at 6h, 12h and 24h post-dose. After the last time point imaging, the mice were euthanized and the major organs were imaged. The fluorescence intensity of mice in LNP-D1, LNP-D2, LNP-D3, LNP01, LNP07 and LNP08 groups were higher than that in LNP-D0 group at each time point. The fluorescence intensity of LNP01 group was higher than that of LNP-D3 group, and the fluorescence intensity of LNP07 and LNP08 groups was significantly higher than that of LNP-D1 and LNP-D2 groups. In particular, LNP07 group showed high liver selectivity (as shown in Figure 6). These results indicate that the targeting PEGylated lipids of the present application can be used for liver-targeted delivery of lipid drug composition nanoparticles, and the special structure (including the nitrogen branched lipid moiety) can significantly improve the efficiency of targeted drug delivery.

Claims

1. A target ligand-modified polyethylene glycolized lipid, characterized in that, The structure is shown in general formula (1): or a salt, tautomer, stereoisomer, deuterated form, or solvate thereof; wherein, each T is independently a residue of a monosaccharide or a derivative thereof; preferably, each T is independently a residue of any one of galactose, galactosamine, glucose, glucosamine, mannose, mannosamine, fucose, and fucosamine, or a derivative thereof; y is 1 and X is a bond; or, y is an integer from 2 to 4, X is a (y+1)-valent linking group, and X is selected from any of the foregoing, wherein any of X is connected to M; each G3is independently >CH-, >N-, or a trivalent cyclic group; G4is a tetravalent carbon atom or a tetravalent cyclic group; each L X is independently -B X -, -B X -Z X -B X - or -B X -Z X -B X -Z X -B X -, wherein each Bx is independently a bond or an optionally substituted C 1-6 alkylene, and any B X that is a bond is not simultaneously connected to two Z X ; each Z X is independently a heteroatom-containing divalent linking group; when y is an integer from 2 to 4, any two T are the same or different from each other, and any two L0 are the same or different from each other; N core a trivalent linker with >N- as branching core; each R is independently -B R -(Z R -B R ) r -E R wherein r is an integer from 0 to 2; each B R is independently a bond or an optionally substituted C 1-29 alkylene, and any B R that is a bond is not simultaneously connected to two Z R ; each Z R is independently a heteroatom-containing divalent linker; E R is an optionally substituted C 1-30 alkyl; two R's are the same or different; the sum of all B R in each R and the carbon chain length of E R is independently an integer from 5 to 30; P0is -(OCH2CH2) n and its oxygen end is connected to M, wherein n is an integer from 1 to 250; each L0is independently -B L -(Z L -B L ) j - wherein j is an integer from 1 to 6; each B L is independently a bond or an optionally substituted C 1-12 alkylene, and any B L that is a bond is not simultaneously connected to two Z L ; each Z L is independently a heteroatom-containing divalent linking group; the sum of the carbon chain lengths of all B L in each L0is independently an integer from 2 to 24; M is -B M -(Z M -B M ) k - wherein k is an integer from 1 to 3; each B M is independently a bond or an optionally substituted C 1-12 alkylene, and at most one B M is a bond to two Z M groups; each Z M is independently a heteroatom-containing divalent linking group; the sum of the carbon chain lengths of all B M in M is an integer from 2 to 24; Z X , Z R , Z L and Z M each occurrence is independently selected from the group consisting of -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and any one of; wherein each Y is independently O, S, or NR c ; each R c is independently a hydrogen atom or C 1-12 alkyl; the pegylated lipid is monodisperse or polydisperse.

2. The targeted ligand-modified polyethylene glycolized lipid according to claim 1, wherein, The monosaccharide derivative is preferably in the form of an N-substitution, an O-substitution, or a combination thereof, and each substituent for the N-substitution or O-substitution is independently selected from R T , -C(=O)R T , -C(=O)NH2, -C(=O)N(R T )2, -S(=O)2OH, -S(=O)2R T , -P(=O)(OH)2, and -P(=O)(R T )2; wherein each R T is independently an optionally substituted C 1-3 alkyl, phenyl, or benzyl; preferably, the number of substituents for each R T is independently 0 to 5; preferably, each substituent in each R T is independently selected from any one of -OH, -F, -Cl, -Br, -I, and -N3.

3. The targeted ligand-modified PEGylated lipid of claim 1, wherein, each T is independently a residue of any one of galactose, galactosamine, and a derivative thereof; the galactose derivative is preferably an O-substituted form; the galactosamine derivative is preferably an N-substituted and / or O-substituted form; Preferably, each T is independently wherein R a selected from any of the compounds of the formula (I), preferably R b is -H or -CH3, preferably -H; each R d independently is -H, -C(=O)CH3, More preferably, each T is independently any one of the following: Most preferably, each of the y T is 4. The targeted ligand-modified PEGylated lipid of claim 1, wherein, Each Z R It is independently selected from any one of -O-, -SS-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, and -NHC(=O)NH-; More preferably, each Z is independently -O-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, or -C(=O)NH-. R is independently -O-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, or -C(=O)NH-.

5. The targeted ligand-modified pegylated lipid of claim 1, wherein, two R are each independently selected from -E R , -B R -Z R -E R , -B R -Z R -B R -Z R -E R , wherein each B R is independently optionally substituted C 1-29 alkylene, E R is optionally substituted C 1-30 alkyl or optionally substituted C 2-30 alkenyl; preferably, each R is independently selected from any one of the following structures: wherein each t R is independently an integer from 0 to 10; More preferably, each of the two R is independently selected from any one of the following structures:

6. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, N core The structure of the compound of formula (I) is as follows: wherein, each B is independently a bond or optionally substituted C N is independently a bond or optionally substituted C 1-12 alkylene, preferably a bond or unsubstituted C 1-12 alkylene, more preferably a bond, methylene, ethylene, propylene, butylene, pentylene or hexylene; each Z is independently -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and N independently a heteroatom-containing divalent linker, preferably -Y-, -C(=Y)-, -YC(=Y)-, -C(=Y)Y-, -YC(=Y)Y-, -S-S-, and each Y is independently O, S, or NR c each R c is independently a hydrogen atom or a C 1-12 alkyl group; preferably, each Z N is independently -O-, -S-, -S-S-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and any one of the following: m1, m2, and m3 are each independently 0, 1, 2, or 3; preferably, m1 and m2 are each independently 0 or 1; m3 is preferably 1, 2, or 3. N core one end is connected with P0, and the other two ends are each connected with one R; Preferably, said N core The structure of any of the following structures: wherein each tn is independently an integer from 1 to 4, preferably 1 or 2; more preferably, N core selected from any of the following structures:

7. The targeted ligand-modified pegylated lipid of claim 1, wherein, each Z is independently selected from -0-, -S-, -S-S-, -C(=0)-, -OC(=0)-, -C(=0)0-, -OC(=0)0-, -NHC(=0)-, -C(=0)NH-, -NHC(=0)0-, -OC(=0)NH-, -NHC(=0)NH-, and M each Z is independently selected from -0-, -S-, -S-S-, -C(=0)-, -OC(=0)-, -C(=0)0-, -OC(=0)0-, -NHC(=0)-, -C(=0)NH-, -NHC(=0)0-, -OC(=0)NH-, -NHC(=0)NH-, and any one of.

8. The targeted ligand-modified pegylated lipid of claim 1, wherein, each B is independently M is independently a bond or optionally substituted C 1-12 alkylene; any B M is substituted C 1-12 when alkylene, preferably the number of substituents thereof is 1 and is -OH, -CH2OH or 9. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, said M is -B M -Z M -B M - and is selected from -B M -NHC(=O)-B M -, -B M -C(=O)NH-B M -, -B M -OC(=O)-B M -, -B M -C(=O)O-B M -, -B M -OC(=O)O-B M -, -B M -NHC(=O)O-B M - and -B M -OC(=O)NH-B M -; or, said M is -B M -(Z M -B M )2- and is selected from -B M -NHC(=O)-B M -C(=O)NH-B M -, M -NHC(=O)-B M -C(=O)O-B M -, M -OC(=O)-B M -C(=O)NH-B M -, M -OC(=O)-B M -C(=O)O-B M -, M -C(=O)NH-B M -C(=O)NH-B M -, M -C(=O)NH-B M -C(=O)O-B M -, M -C(=O)O-B M -C(=O)NH-B M -, M -C(=O)O-B M -C(=O)O-B M -, M -NHC(=O)-B M -C(=O)-B M -, M -OC(=O)-B M -C(=O)-B M -, M -C(=O)NH-B M -C(=O)-B M -, M -C(=O)O-B M -C(=O)-B M -, M -C(=O)-B M -NHC(=O)-B M -, M -C(=O)-B M -OC(=O)-B M -, M -C(=O)-B M -C(=O)NH-B M -, M -C(=O)-B M -C(=O)OB M -、-B M -C(=O)-B M -C(=O)-B M -、-B M -C(=O)NH-B M -NHC(=O)OB M -、-B M -C(=O)OB M -NHC(=O)OB M -、-B M -C(=O)NH-B M -NHC(=O)NH-B M -、-B M -C(=O)OB M -NHC(=O)NH-B M -、-B M -NHC(=O)-B M -OC(=O)NH-B M -、-B M -OC(=O)NH-B M -NHC(=O)OB M -、-B M -OC(=O)NH-B M -OC(=O)NH-B M -、-B M -NHC(=O)OB M -NHC(=O)OB M -、-B M -NHC(=O)OB M -OC(=O)NH-B M -、-B M -NHC(=O)NH-B M -NHC(=O)OB M -、-B M -OC(=O)NH-B M -NHC(=O)NH-B M -and-B M -NHC(=O)NH-B M -NHC(=O)NH-B M - any one of the following or its alternative forms, and its left end is connected to X; or, said M is -B M -(Z M -B M )3- and is selected from -B M -NHC(=O)-B M -NHC(=O)-B M -C(=O)-B M -, M -NHC(=O)-B M -OC(=O)-B M -C(=O)-B M -, M -OC(=O)-B M -NHC(=O)-B M -C(=O)-B M -, M -OC(=O)-B M -OC(=O)-B M -C(=O)-B M -, M -C(=O)NH-B M -NHC(=O)-B M -C(=O)-B M -, M -C(=O)NH-B M -OC(=O)-B M -C(=O)-B M -, M -C(=O)O-B M -NHC(=O)-B M -C(=O)-B M -, M -C(=O)O-B M -OC(=O)-B M -C(=O)-B M -, M -OC(=O)NH-B M -NHC(=O)-B M -C(=O)-B M -, M -C(=O)O-B M -S-S-B M -OC(=O)-B M -, M -C(=O)O-B M -S-S-B M -OC(=O)NH-B M -, M -NHC(=O)O-B M -S-S-B M -OC(=O)-B M - and -B M -NHC(=O)O-B M -S-S-B M -OC(=O)NH-B M - any one of -S-, -S(O)-, -S(O2)-, -S(O2)O-, -S(O2)NH-, -S(O2)NRi2-, -O-, -OCH2-, -CH2O-, -NRi2CH2-, -CH2NRi2-, -OC(O)-, -C(O)O-, -OC(O)O-, -C(O)NRi2-, -NRi2C(O)-, -NRi2C(O)O-, -NRi2C in any of the foregoing cases, the substituted form of M is preferably a form in which one hydrogen atom on the carbon chain of M is replaced by one -OH or -CH2OH.

10. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, each Z is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, and -OC(=O)NH-. X each Z is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, and -OC(=O)NH-.

11. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, X is selected from any one of the following: (i) X is Preferably any one of the following, wherein the * end of X is connected to M; (ii) X is Preferably any of the foregoing, wherein any of X is connected to M at either end; tx is an integer from 0 to 6; the number of Rx is 0 to 2, each R X independently is a hydroxyl or C 1-3 alkoxy; more preferably, X is selected from any one of the following, wherein the * end of X is connected to M; (iii) X is Preferably any one of the following, wherein the * end of X is connected to M.

12. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, each Z is independently selected from -O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and L each Z is independently selected from -O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and each Z is independently selected from -O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)O-, -OC(=O) 13. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, said L0 is selected from the group consisting of -(CH2) ta -C(=O)NH-(CH2) ta - -(CH2) ta -O-(CH2) tb -O-(CH2) tb -NHC(=O)-(CH2) ta - -(CH2) ta -C(=O)NH-(CH2) tb -NHC(=O)-(CH2) tb -O-(CH2) ta - -(CH2) ta -O-(CH2) tb -O-(CH2) tb -NHC(=O)-(CH2) tb -O-(CH2) ta - and -(CH2) ta -O-(CH2) tb -O-(CH2) tb -O-(CH2) tb -C(=O)NH-(CH2) ta -; and either end of L0 is attached to X; wherein each ta is independently an integer from 0 to 12, and each tb is independently an integer from 1 to 12.

14. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, the spacer length between any one T and M is 5 to 30 atoms; when y is 1, 2, or 3, the spacer length is preferably 7 to 17 atoms; when y is 4, the spacer length is preferably 19 to 26 atoms; Preferably, (T - L0) y - X - is selected from any one of the following structures:

15. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, the pegylated lipid is monodisperse, and n is an integer from 1 to 100, preferably an integer from 3 to 60, more preferably an integer from 10 to 47; alternatively, the pegylated lipid is polydisperse, and n is an integer from 10 to 250, preferably an integer from 20 to 60.

16. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, the number average molecular weight of the pegylated lipid is selected from 2000 Da to 5000 Da.

17. The targeted ligand-modified polyethylene glycolized lipid of claim 1, wherein, The pegylated lipid is selected from any one of the following structures:

18. A lipid composition, characterized in that, the lipid composition further comprises one or more non-targeting pegylated lipids and / or one or more cationic lipids and / or one or more phospholipids and / or one or more sterol lipids and / or one or more anionic lipids.

19. [Amended according to Rule 91 on 16.10.2025] The lipid composition according to claim 18, characterized in that, the lipid composition further comprises one or more non-targeting pegylated lipids and / or one or more cationic lipids and / or one or more phospholipids and / or one or more sterol lipids and / or one or more anionic lipids. the non-targeted pegylated lipid is preferably selected from the group consisting of polyethylene glycol-dipalmitoyl phosphatidyl choline, polyethylene glycol-dimyristoyl glyceride, polyethylene glycol-distearyl phosphatidyl ethanolamine, polyethylene glycol-dioleoyl phosphatidyl ethanolamine, polyethylene glycol-cholesterol, polyethylene glycol-diacylglycerol and polyethylene glycol-dialkyloxypropyl, more preferably from the group consisting of polyethylene glycol 500-dipalmitoyl phosphatidyl choline, polyethylene glycol 2000-dipalmitoyl phosphatidyl choline, polyethylene glycol 500-distearyl phosphatidyl ethanolamine, polyethylene glycol 2000-distearyl phosphatidyl ethanolamine, polyethylene glycol 500-dioleoyl phosphatidyl ethanolamine, polyethylene glycol 2000-dioleoyl phosphatidyl ethanolamine, polyethylene glycol 500-dimyristoyl glyceride and polyethylene glycol 2000-dimyristoyl glyceride; Alternatively, the non-targeted pegylated lipid is preferably selected from the following structures: wherein n1 is an integer from 10 to 250; the phospholipid is preferably selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearyl-sn-glycero-3-phosphocholine, 1,2-didecanyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemi succinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoyl phosphatidylserine, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidyl ethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidyl choline, lysophosphatidyl choline and lysophosphatidyl ethanolamine; The sterol lipid is preferably selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, elaidosterol, stigmasterol, brassicasterol, tomatidine and ursolic acid; The cationic lipid is preferably selected from 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate), 1,2-dioleyloxy-3-trimethylammonium propane chloride, 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazoline chloride, 1,2-dioleoyl-3-dimethylamino-propane, 2,3-bis(tetradecanoyloxy)propyltrimethylammonium chloride, didecyldimethylammonium chloride, didecyldimethylammonium bromide, N,N-dioleyl-N,N-dimethylammonium chloride, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), 4-(N,N-dimethylamino)butanoic acid (dilinoleyl) methyl ester, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, ((4-hydroxybutyl)azanediyl)bis(hexan-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoic acid (heptadecan-9-yl) ester, The anionic lipid is preferably selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt, ammonium bis(mono-oleoylglycero)phosphate and cardiolipin.

20. The lipid composition of claim 19, wherein, The lipid composition consists of one cationic lipid, one phospholipid, one sterol lipid and one targeting PEGylated lipid, optionally further comprising one non-targeting PEGylated lipid; wherein, The molar percentage of the cationic lipid in the total lipid is preferably 30% to 65%, more preferably 35% to 55%; The molar percentage of the phospholipid in the total lipid is preferably 2% to 15%, more preferably 5% to 12%; The molar percentage of the sterol lipid in the total lipid is preferably 25% to 50%, more preferably 38% to 50%; The molar percentage of the total PEGylated lipid in the total lipid is preferably 0.5% to 10%, more preferably 1% to 3%, more preferably 1.2% to 2.1%; the total PEGylated lipid consists of the non-targeting PEGylated lipid and the targeting PEGylated lipid; wherein the molar percentage of the targeting PEGylated lipid in the total lipid is preferably 0.001% to 10%, more preferably 0.005% to 1.5%, more preferably 0.15% to 0.65%, more preferably 0.5%.

21. A lipid pharmaceutical composition, characterized by, The lipid composition of any one of claims 18-20, further comprising one or more than one pharmaceutically active ingredient; the pharmaceutically active ingredient is preferably any one of nucleic acid, small molecule, polypeptide and protein, more preferably nucleic acid, more preferably any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme; wherein the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably, the pharmaceutically active ingredient in the lipid composition is selected from any one of DNA, mRNA, miRNA and siRNA.

22. Use of a lipid pharmaceutical composition for the manufacture of a therapeutic agent, characterized in that, The lipid pharmaceutical composition is the lipid pharmaceutical composition of claim 21.

23. Use of a lipid pharmaceutical composition according to claim 22 for the manufacture of a medicament, characterized in that, The therapeutic agent is selected from any one of antitumor agent, antibiotic agent, antiviral agent, antifungal agent, antiparasitic agent and vaccine.

24. Use of a lipid pharmaceutical composition according to claim 22 for the manufacture of a medicament, characterized in that, The therapeutic agent is selected from therapeutic agents for treating liver diseases; preferably, the liver disease is selected from any one of liver fibrosis, liver cirrhosis and liver cancer.

25. A lipid pharmaceutical composition formulation, characterized by, The lipid pharmaceutical composition of claim 21, further comprising a working solution; the working solution is preferably pharmaceutically acceptable diluent or excipient, more preferably any one of deionized water, ultrapure water, phosphate buffer and physiological saline, more preferably phosphate buffer or physiological saline, most preferably physiological saline.

26. The lipidic pharmaceutical composition formulation of claim 25, wherein, The lipid pharmaceutical composition in the formulation forms drug-loaded lipid nanoparticles.

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