A pegylated lipid and its modified liposome, a pharmaceutical composition containing the liposome, and preparation and application thereof

Abstract

This invention discloses a polyethylene glycol-modified lipid and its preparation method, liposomes containing the polyethylene glycol-modified lipid, pharmaceutical compositions containing the liposomes, their formulations, and applications, belonging to the field of drug delivery. The polyethylene glycol-modified lipid of this invention can be used to modify liposomes, or the polyethylene glycol-modified lipid can be further modified and coupled with a targeting group before being used to modify liposomes to obtain liposomes with a targeting group. Due to the presence of long-chain PEG and the targeting group on the polyethylene glycol-modified lipid, the modified liposomes can not only effectively avoid clearance of liposomes by the reticuloendothelial system in the human body, but also achieve targeting function. Therefore, when liposomes modified with polyethylene glycol-modified lipid deliver active drugs to cells or patients, especially when delivering nucleic acid drugs and anti-tumor drugs, the liposomes can not only achieve long circulation in vivo, improving drug transport efficiency, but also have targeting function, synergistically improving the therapeutic effect of the drug.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery, specifically relating to a pharmaceutical carrier PEGylated lipid, and more particularly to a nitrogen-branched PEGylated lipid and its preparation method, cationic liposomes containing the PEGylated lipid, liposome pharmaceutical compositions containing the PEGylated lipid, their formulations and applications. Background Technology

[0002] Liposomes are microvesicles that encapsulate drugs within a lipid bilayer. Liposome nanoparticles, containing both liposomes and drugs, especially nucleic acid drugs, have a structure similar to a biological membrane. They are biocompatible and non-toxic nanomaterials that can encapsulate both water-soluble and lipid-soluble drugs, offering advantages such as reduced drug dosage, sustained release, targeted drug release, and protection of encapsulated nucleic acids from degradation and clearance in serum. Furthermore, liposome nanoparticles are excellent antigen carriers, capable of encapsulating a range of antigens and adjuvants with different physicochemical properties, protecting protein and peptide antigens from degradation, promoting phagocytosis and presentation of antigens by antigen-presenting cells, and enhancing the body's specific immune response. Therefore, liposome nanoparticles are widely used in drug delivery.

[0003] Cationic liposomes carry a positive charge on their surface, while nucleic acids carry a negative charge. Through electrostatic interactions, they form cationic liposome-nucleic acid drug complexes. However, the overall positive charge on the surface of these complexes makes them prone to non-specific adsorption to serum proteins in plasma, forming large aggregates. These aggregates are easily cleared by the reticuloendothelial system (RES), resulting in short blood circulation time, poor stability, and low transport efficiency. Therefore, surface modification of cationic liposomes is necessary to prepare long-circulating cationic liposomes. Currently, commonly used modifying agents for long-circulating cationic liposomes are polyethylene glycol (PEG)-modified lipid molecules, such as PEG-DMG. PEG forms a hydration layer on the surface of the modified cationic liposome through hydrogen bonds and interactions with water molecules in the solvent, masking the positive charge on the cationic liposome surface, thereby inhibiting protein adsorption and reducing recognition by the phagocytic system. Therefore, PEG-modified lipids are of great significance for preparing long-circulating cationic liposomes and further improving nucleic acid transport efficiency.

[0004] Despite significant progress in the use of PEGylated lipids such as PEG-DMG for drug delivery, there remains a need in the field for new PEGylated lipids suitable for routine therapeutic applications. Summary of the Invention

[0005] This invention provides novel polyethylene glycol-modified lipids and their preparation methods, cationic liposomes containing the polyethylene glycol-modified lipids, and pharmaceutical compositions and formulations containing the cationic liposomes, particularly nucleic acid pharmaceutical compositions and formulations containing the cationic liposomes. The cationic liposome nucleic acid pharmaceutical composition formulations can deliver nucleic acid drugs into cells, improve the transport rate of nucleic acid drugs, and thus improve the therapeutic effect of nucleic acid drugs.

[0006] The above-mentioned objectives of the present invention are achieved through the following technical solutions:

[0007] One embodiment of the present invention provides a polyethylene glycol-modified lipid:

[0008] The structure of a polyethylene glycol-modified lipid is shown in general formula (2):

[0009]

[0010] Or its drug-acceptable salts, tautomers, or stereoisomers.

[0011] Among them, one of L7 and L8 is -O(C=O)O-, -C(=O)-, -O-, -O(CR)O-, -O(C=O)-, -O-, -O(CR)O-, -O(C=O)O-, -C(=O)-, -O-, -O(C=O)O-, -C(=O)-, -O(CR)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR)O-, -O(C=O)-, -O(C=O)-, -O-, -O(CR)O-, -O(C=O)-, - ...-, -O-, -O-, -O-, -O-, -O c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any of C(=O)S-, and the other of L7 and L8 is a linker, -O(C=O), -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR cAny one of C(=O)S-, where R c Each time it appears, it is independently a hydrogen atom or an alkyl group, and s is 2, 3 or 4;

[0012] L3 is any one of the following: linker, -L4-, -Z-L4-, -L4-Z-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, -L4-Z-L5-Z-, -Z-L4-Z-L5-Z-, and -L4-Z-L4-Z-L5-Z-; L4 and L5 are carbon chain linkers, each independently being -(CR... a R b ) t -(CR a R b ) o -(CR a R b ) p - where t, p, and q are each independent integers from 0 to 12, and t, p, and q are not all 0 at the same time; R a and R b Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 Alkyl groups; each occurrence of Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S- and Any one of them, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 alkyl;

[0013] B3 and B4 are each independently a connecting key or C. 1-30 Alkylene;

[0014] R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon groups;

[0015] R represents a hydrogen atom, -R d -OR d -NR d R d -SR d -(C=O)Rd -(C=O)OR d -O(C=O)R d -O(C=O)OR d or Among them, R d Each occurrence is independently represented by C. 1-12 Alkyl group, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R. 01 When j is 0, G1 does not exist; when j is 1, G1 introduces k Fs, where k is an integer from 2 to 8.

[0016] A is -(CR) a R b ) s O- or -O(CR) a R b ) s -, where s is 2, 3 or 4;

[0017] n1 is approximately an integer between 20 and 250;

[0018] The alkyl, alkylene, alkoxy, and aliphatic hydrocarbon groups are each independently substituted or unsubstituted.

[0019] This invention also provides four methods for preparing the above-mentioned polyethylene glycol-modified lipids, as follows:

[0020] Method 1: The aforementioned polyethylene glycol-modified lipids can be prepared through the following steps:

[0021] Step 1: Activate the carboxyl terminus of acid A-1 or A-1' containing a naked carboxyl group using a carboxyl activator to obtain carboxyl terminus-activated ester A-2 or A-2'. Here, B3' and B4' are each independently a linking bond or an alkylene group with one less methylene group than B3 and B4; R1' and R2' are each independently R1 and R2 or an aliphatic hydrocarbon group with one less methylene group than R1 and R2; R7 is a carboxyl activating group. When either B3' or L7 is not a linking bond, R1' is R1; when both B3' and L7 are linking bonds, R1' is an aliphatic hydrocarbon group with one less methylene group than R1; when either B4' or L8 is not a linking bond, R2' is R2; when both B4' and L8 are linking bonds, R2' is an aliphatic hydrocarbon group with one less methylene group than R2.

[0022] Step 2: The carboxyl-terminated activated ester A-2 or A-2' is condensed with a primary amine derivative A-3 or A-3' containing a nitrogen source end group to obtain amide intermediate A-4 or A-4';

[0023] Step 3: Reduce amide intermediate A-4 or A-4' to secondary amine intermediate A-5 or A-5' using a reducing agent;

[0024] Step 4: The secondary amine intermediate is coupled with the bi-functionalized PEG derivative biLPEG molecule A-6 to obtain the polyethylene glycol-modified lipid derivative A-7 or A-7'; wherein biLPEG is monodisperse or polydisperse; wherein the functional groups at both ends of biLPEG can be the same or different; wherein the R' end of biLPEG contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 The group; wherein, F1 contains an active functional group that can react with the secondary amine intermediate A-5 or A-5' to generate a branched central nitrogen atom and a divalent linker L3;

[0025] When R' equals R, the resulting structure A-7 or A-7' corresponds to the structure shown in general formula (2);

[0026] When R' is not equal to R, A-7 or A-7' is modified at the end to obtain A-8 or A-8' corresponding to the structure shown in general formula (2); the end micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0027] Step 1

[0028]

[0029] Step Two

[0030]

[0031] Step 3

[0032]

[0033] Step Four

[0034]

[0035] Method 2: The aforementioned polyethylene glycol-modified lipids can be prepared through the following steps:

[0036] Step 1: Coupling the biLPEG molecule B-1, a bifunctionalized PEG derivative, with a primary amine derivative B-2 or B-2' containing a nitrogen-source end group, yields a polyethylene glycol-modified secondary amine derivative B-3 or B-3'. The biLPEG is monodisperse or polydisperse, and the functional groups at both ends of the biLPEG can be the same or different. The R' end of the biLPEG contains a reactive group R. 01 or contains R 01The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 The group; wherein, F1 contains an active functional group that can react with the amino group of primary amine B-2 or B-2' to generate L3 secondary amine derivatives B-3 or B-3' containing divalent linkers;

[0037] Step 2: The secondary amine derivative B-3 or B-3' reacts with F containing a reactive group. N Compound B-4 or B-4' undergoes an alkylation reaction to yield polyethylene glycol-modified lipid derivatives B-5 or B-5'; The F N The reactive group is capable of reacting with amino or secondary amine groups, preferably -OMs, -OTs, -CHO, -F, -Cl, or -Br;

[0038] When R' equals R, the resulting structure B-5 or B-5' corresponds to the structure shown in general formula (2);

[0039] When R' is not equal to R, B-5 ​​or B-5' is end-micro-modified to obtain B-6 or B-6' corresponding to the structure shown in general formula (2); the end-micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0040] Step 1

[0041]

[0042] Step Two

[0043]

[0044] Method 3: The aforementioned polyethylene glycol-modified lipids can be prepared through the following steps:

[0045] Step 1: React small molecule C-1 with small molecule C-2 to generate a compound containing a divalent linker L7 and a reactive group F at one end. N A small molecular intermediate C-3 with an aliphatic hydrocarbon group R1 at one end; wherein F3 and F4 are each independently reactive groups that can react to form a divalent linker L7; wherein C-2 is a group containing heterofunctional pairs of F3 and F4. N The F N The reactive group is capable of reacting with amino or secondary amine groups, preferably -OMs, -OTs, -CHO, -F, -Cl, or -Br;

[0046] Step 2: Two molecules of the small molecule intermediate C-3 undergo an alkylation reaction with a polyethylene glycol primary amine derivative C-4 containing a nitrogen source end group to obtain a polyethylene glycol-modified lipid C-5, wherein the R' end contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 Groups;

[0047] When R' equals R, the resulting structure C-5 corresponds to the structure shown in general formula (2);

[0048] When R' is not equal to R, C-5 is end-modified to obtain the structure of C-6 corresponding to the general formula (2); the end-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; wherein R1 and R2 are the same, B3 and B4 are the same, and L7 and L8 are the same.

[0049] Step 1

[0050]

[0051] Step Two

[0052]

[0053] Method 4: The aforementioned polyethylene glycol-modified lipids are prepared through the following steps:

[0054] Step 1: React small molecule D-1 with small molecule D-2 to generate a small molecule intermediate D-3 containing a divalent linker L7, a hydroxyl group at one end, and an aliphatic hydrocarbon group R1 at the other end; wherein F3 and F4 are each independently reactive groups that can react to generate the divalent linker L7; wherein D-2 contains heterofunctional groups for F3 and hydroxyl groups;

[0055] Step 2: Oxidize the hydroxyl group of small molecule intermediate D-3 to an aldehyde group to obtain small molecule intermediate D-4 containing an aldehyde group, wherein B3' is an alkylene group with one less methylene group than B3;

[0056] Step 3: Two molecules of the small molecule intermediate D-4 containing aldehyde groups are added to D-5, a polyethylene glycol primary amine derivative containing a nitrogen source end group, to obtain polyethylene glycolated lipid D-6, wherein the R' end contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 Groups;

[0057] When R' equals R, the resulting structure D-6 corresponds to the structure shown in general formula (2);

[0058] When R' is not equal to R, D-6 is end-micro-modified to obtain D-7 corresponding to the structure shown in general formula (2); the end-micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group, wherein R1 and R2 are the same, B3 and B4 are the same, and L7 and L8 are the same.

[0059] Step 1

[0060]

[0061] Step Two

[0062]

[0063] Step 3

[0064]

[0065] The present invention also provides a cationic liposome, wherein the embodiment is as follows:

[0066] A cationic liposome containing polyethylene glycol-modified lipids with the structure shown in formula (2).

[0067] The present invention also provides a cationic liposome pharmaceutical composition, the implementation of which is as follows:

[0068] A liposomal pharmaceutical composition comprising cationic liposomes and a drug, wherein the cationic liposomes comprise polyethylene glycol-modified lipids with the structure shown in formula (2).

[0069] The present invention also provides a liposomal pharmaceutical composition formulation, the implementation of which is as follows:

[0070] A liposomal pharmaceutical composition formulation comprising the aforementioned liposomal pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] The novel polyethylene glycol-modified lipid of this invention has a branched ammonia center that is easily protonated under physiological pH conditions to generate a partially positive charge, which can bind to negatively charged nucleic acids, thereby improving the loading rate of nucleic acid drugs.

[0073] The novel polyethylene glycol-modified lipids of this invention can be used to modify the surface of cationic liposomes to obtain polyethylene glycol-modified cationic liposomes. The presence of long-chain PEG overcomes the disadvantage of conventional cationic liposomes being cleared by phagocytes due to the action of serum proteins in the blood, increases the serum stability of cationic liposomes, prolongs the in vivo circulation time, and further improves the drug transport rate and therapeutic efficiency.

[0074] In this invention, the cationic liposome pharmaceutical composition containing polyethylene glycol-modified lipids has strong gene-complexing ability and high biocompatibility, which helps to improve the therapeutic effect of the drug.

[0075] In this invention, the polyethylene glycol chain ends of the polyethylene glycol-modified lipids may also contain fluorescent groups or targeting groups, further improving the targeted therapeutic or diagnostic effects of the modified cationic liposome drug composition.

[0076] In this invention, the polyethylene glycol chain of the polyethylene glycol-modified lipid can also be degradable. In some therapeutic fields, such as tumor treatment, the polyethylene glycol coating on the surface of liposomes prevents the components of the liposomes from leaving the endosomes, thereby preventing the contents of the liposomes from being delivered into the cytoplasm. The degradable polyethylene glycol can degrade further from the surface of the liposomes in the acidic environment of the endosome vacuoles or tumor tissue, so that the therapeutic agent loaded on the liposomes can be delivered to the target site efficiently. Implementation

[0077] Terminology Explanation

[0078] In this invention, unless otherwise specified, the terms have the following meanings.

[0079] In this invention, when the structure involved has isomers, it can be any one of them unless otherwise specified. For example, for a structure with cis-trans isomers, it can be either the cis or trans structure; for a structure with E / Z isomers, it can be either the E or Z structure; and when it is optically active, it can be either levorotatory or dextrorotatory.

[0080] In this invention, the interpretation of numerical ranges includes both ranges marked with a hyphen (e.g., 0-12) and ranges marked with a wavy line (e.g., 0~12). Unless otherwise specified, integer ranges marked as intervals in this invention can represent groups of all integers within that range, and the range includes two endpoints. For example, the integer range 0-12 represents the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The numerical ranges in this invention include, but are not limited to, ranges of integers, non-integers, percentages, and fractions, all of which, unless otherwise specified, include two endpoints.

[0081] The numerical values ​​used in this invention, when referring to "about" or "around," generally indicate a range of ±10%, which may be increased to ±15% in some cases, but not exceeding ±20%. A preset value is used as the base. For example, if the molar percentage of steroid lipids in the total lipids of a solution containing a solvent is approximately 40%, it can generally be considered that the molar percentage of steroid lipids includes 30%-50%.

[0082] In this invention, unless otherwise specified, the terms "comprising," "including," and "containing," as well as similar expressions, shall be interpreted in an open and inclusive sense as "including but not limited to."

[0083] In this invention, two or more objects are "each independently preferred." When there are multiple levels of preferred conditions, it is not required that they are all selected from the same level of preferred group. One can be a broad-range preferred and the other a narrow-range preferred, or one can be the broadest range and the other any preferred condition, or they can be selected from the same level of preferred. For example, "R1 and R2 are each independently preferred to be straight-chain alkyl; more preferably C..." 1-25 Straight-chain alkyl; more preferably C 1-17 "Straight-chain alkyl" can have R1 as C 1-25 Straight-chain alkyl group, R2 is C 1-17 Straight-chain alkyl groups, or R1 is C 1-17 Straight-chain alkyl group, R2 is C 1-25 Straight-chain alkyl groups, or both R1 and R2 are C1 or C2. 1-25 Straight-chain alkyl groups, or both R1 and R2 are C1 or C2. 1-17 Straight-chain alkyl groups.

[0084] In this invention, the divalent linker, such as alkylene group, alkylene group, arylene group, amide bond, etc., can be selected from either of the two linking ends when it is linked to other groups, unless otherwise specified. For example, when an amide bond is used as the divalent linker between C-CH2CH2- and -CH2-D, it can be C-CH2CH2-C(=O)NH-CH2-D or C-CH2CH2-NHC(=O)-CH2-D.

[0085] In the structural formula of this invention, when the end group of the linking group and the substituents contained in the linking group are easily confused, the following is adopted: To mark the positions where other groups are attached in the linker, such as in the structural formula. In, the method adopted The two positions in the divalent linker that connect to other groups are marked. The two structural formulas mentioned above represent -CH(CH2CH2CH3)2- and -CH2CH2CH(CH3)2-CH2CH2-, respectively.

[0086] In this invention, the range of carbon atoms in a group is indicated by a subscript at the subscript position of C, representing the number of carbon atoms in the group, for example, C. 1-12 Indicates "having 1 to 12 carbon atoms", C 1-30 It indicates "having 1 to 30 carbon atoms". "Substituted C" 1-12 "alkyl" refers to C 1-12 Compounds obtained by substituting hydrogen atoms of alkyl groups. "C" 1-12 "Substituted alkyl" refers to compounds in which the hydrogen atoms of an alkyl group are substituted, resulting in compounds with 1-12 carbon atoms. For example, when a group can be selected from C... 1-12 When alkylene is used, it can be selected from any number of carbon atoms in the range indicated by the subscript, i.e., selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, ... 10 C 11 C 12 Any alkylene group. In this invention, unless otherwise specified, subscripts marked as ranges indicate any integer selected from the range, which includes both endpoints.

[0087] The heteroatoms used in this invention are not particularly limited, and include, but are not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0088] In this invention, the heteroatom used for substitution is referred to as a "substituent atom", and any group used for substitution is referred to as a "substituent".

[0089] In this invention, "substituted" means any of the above-mentioned groups (e.g., aliphatic hydrocarbon group, hydrocarbon group, alkyl group, or alkylene group) in which at least one hydrogen atom is replaced by a bond connected to a non-hydrogen atom, such as, but not limited to: halogen atoms such as F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkyloxy groups (-OR). d , where R d C 1-12 Alkyl group; Carboxyl group (-COOH); Amine group (-NR) c R c Two Rs c Each independently represents H and C. 1-12 Alkyl); C 1-12 Alkyl and cycloalkyl. In some embodiments, the substituent is C. 1-12Alkyl group. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogroup, such as a fluorinated group. In other embodiments, the substituent is an oxogroup. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0090] In this invention, "atomic spacing" or "atomic interval" refers to the number of main chain atoms separated along the main chain, without considering the factors of side groups and side chains. It is usually the shortest atomic spacing and can be used to represent the length of the linker. For example, the atomic spacing between A and B in A-CO-NH-B is 2, the atomic spacing between A and B in Ap-Ph-CH2-B is 5 (p-Ph is para-phenylene), and the atomic spacing between A-CH(CH2CH2CH2CH3)-B is 1. The "main chain atoms" that participate in forming the atomic spacing can only be non-hydrogen atoms. Among them, for divalent linkers containing ring structures, the atomic spacing refers to the shortest number of atoms calculated along the ring atoms. For example, the atomic spacing of para-phenylene, i.e., 1,4-phenylene, is 4, the atomic spacing of meta-phenylene is 3, and the atomic spacing of ortho-phenylene is 2. For example, the atomic spacing of –CH2–, –CH(CH3)–, –C(CH3)2–, –CH(CH2Ph)2–, and –C(CH2OX)– is 1.

[0091] In this invention, a "carbon chain linker" refers to a linker whose main chain atoms are all carbon atoms, while the side chain portion allows heteroatoms or heteroatom-containing groups to replace the hydrogen atoms of the main chain carbons. When the "main chain atom" is a heteroatom, it is also called a "main chain heteroatom," such as AS-CH2-B, AO-CH2-B, etc. (Atomic spacing denoted as 4) is considered to contain heteroatoms in the main chain. Carbon chain linkers can be divided into alkylene groups and carbon chain linkers with heteroatoms in the side groups; the carbon chain linkers with heteroatoms in the side groups include, but are not limited to, oxo (=O), thio (=S), amino (connected to the main chain carbon via a carbon-nitrogen double bond), ether-bonded oxoalkylene groups, thioether-bonded thioalkylene groups, tertiary amino-bonded nitrogen-bonded groups, etc. The main chain of a "carbon chain linker" is entirely composed of carbon atoms, and the side groups of the carbon chain are allowed to contain heteroatoms. That is, it is formed by connecting methylene or substituted methylene groups. The substituted methylene group can be one monovalent substituent, two monovalent substituents, or one divalent substituent (such as divalent oxygen, which together with divalent methylene forms a three-membered ring). The substituted methylene group can be a single hydrogen atom (e.g., -CH(CH3)-), two hydrogen atoms (e.g., -(CH3)C(OCH3)-), or both hydrogen atoms (e.g., carbonyl, thiocarbonyl, -C(=NH)-, -C(=N)-. +H2)-), can also be a cyclic side group (such as H2)-), and can also be a ring-shaped side group The atomic spacing is denoted as 1).

[0092] In this invention, secondary amine bonds and hydrazine bonds refer to "-NH-" with both ends capped by alkylene groups, such as -CH2-NH-CH2-; while -C(=O)-NH- is called an amide bond and is not considered to contain secondary amine bonds.

[0093] In this invention, a compound, a group, or an atom can be simultaneously substituted and hybridized. For example, a nitrophenyl can substitute a hydrogen atom, or -CH2-CH2-CH2- can be replaced with -CH2-S-CH(CH3)-.

[0094] In this invention, a "linking bond" refers to a bond that only serves a connecting function and does not contain any atoms. When a group is defined as a linking bond, it means that the group may not exist.

[0095] In this invention, "each occurrence independently constitutes" not only refers to the fact that different groups can each independently constitute any option in the definition, but also indicates that when the same group appears at different positions, it can also independently constitute any option in the definition. For example, Z can independently constitute -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR each time it appears. c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S- and Any one of them, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 Alkyl group, in the "-NR" group c C(=O)NR c In the "-", there are two R's c Each is independently a hydrogen atom or a carbon atom. 1-12 Alkyl, i.e., two Rs c They can be the same or different.

[0096] In this invention, a "group" containing at least one atom refers to a free radical formed by the loss of one or more atoms from a compound. A group formed after the loss of a portion of a compound is also called a residue. The valence state of a group is not particularly limited, but can be categorized as monovalent, divalent, trivalent, tetravalent, ..., 100valent groups, etc. Groups with a valence of 2 or higher are collectively referred to as linking groups. Linking groups can also contain only one atom, such as oxygen or thio groups.

[0097] In this invention, "hydrocarbon" refers to hydrocarbons composed of carbon atoms and hydrogen atoms.

[0098] In this invention, hydrocarbons are classified into two types according to the type of hydrocarbon group: aliphatic hydrocarbons and aromatic hydrocarbons. Hydrocarbons without a benzene ring or any structure of a benzene ring with substituted hydrocarbon groups are defined as aliphatic hydrocarbons. Hydrocarbons containing at least one benzene ring or a benzene ring with substituted hydrocarbon groups are defined as aromatic hydrocarbons. Aromatic hydrocarbons may contain aliphatic hydrocarbon groups, such as toluene, diphenylmethane, and 2,3-dihydroindene.

[0099] In this invention, hydrocarbons are classified into two types based on their degree of saturation: saturated hydrocarbons and unsaturated hydrocarbons. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also called alkanes. The degree of unsaturation of unsaturated aliphatic hydrocarbons is not particularly limited. Examples include, but are not limited to, alkenes (containing double bonds), alkynes (containing triple bonds), and dienes (containing two conjugated double bonds). When the aliphatic portion of an aromatic hydrocarbon is saturated, it is also called an aromatic alkane, such as toluene.

[0100] In this invention, there are no particular restrictions on the structure of the hydrocarbon, which can take the form of a straight-chain structure without side groups, a branched structure with side groups, a cyclic structure, a dendritic structure, a comb-like structure, a hyperbranched structure, etc. Unless otherwise defined, straight-chain structures without side groups, branched structures with side groups, and cyclic structures are preferred, corresponding to straight-chain hydrocarbons, branched hydrocarbons, and cyclic hydrocarbons, respectively. Hydrocarbons without cyclic structures are collectively referred to as open-chain hydrocarbons, including but not limited to straight-chain structures without side groups and branched structures with side groups. Open-chain hydrocarbons belong to aliphatic hydrocarbons. Therefore, straight-chain hydrocarbons can also be called straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons can also be called branched-chain aliphatic hydrocarbons.

[0101] In this invention, compounds formed by replacing carbon atoms at any position in a hydrocarbon with heteroatoms are collectively referred to as heterohydrocarbons.

[0102] In this invention, aliphatic hydrocarbons refer to hydrocarbons derived from aliphatic hydrocarbons, including aliphatic heterocyclic hydrocarbons and aliphatic open-chain hydrocarbons. Saturated aliphatic hydrocarbons are heteroalkanes.

[0103] In this invention, "hydrocarbon group" refers to the residue formed after a hydrocarbon loses at least one hydrogen atom. Based on the number of hydrogen atoms lost, hydrocarbon groups can be classified as monovalent hydrocarbon groups (losing one hydrogen atom), divalent hydrocarbon groups (losing two hydrogen atoms, also called alkylene groups), trivalent hydrocarbon groups (losing three hydrogen atoms), and so on. When n hydrogen atoms are lost, the valence state of the resulting hydrocarbon group is n. Unless otherwise specified, the hydrocarbon group in this invention specifically refers to a monovalent hydrocarbon group.

[0104] The source of the hydrocarbon group in this invention is not particularly limited. For example, it can originate from aliphatic or aromatic hydrocarbons, saturated or unsaturated hydrocarbons, straight-chain hydrocarbons, branched-chain hydrocarbons, or cyclic hydrocarbons, as well as hydrocarbons or heterocyclic hydrocarbons, etc. From the perspective of saturation, it can originate from alkanes, alkenes, alkynes, dienes, etc.; for cyclic hydrocarbons, it can originate from alicyclic or aromatic hydrocarbons, monocyclic or polycyclic hydrocarbons; for heterocyclic hydrocarbons, it can originate from alicyclic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0105] In this invention, "aliphatic hydrocarbon group" refers to a residue formed after an aliphatic hydrocarbon loses at least one hydrogen atom. Unless otherwise specified, the aliphatic hydrocarbon group in this invention specifically refers to a monovalent aliphatic hydrocarbon group. Aliphatic hydrocarbon groups include saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups.

[0106] In this invention, "alkyl" refers to a hydrocarbon group formed from an alkane. Unless otherwise specified, it refers to a hydrocarbon group formed by losing a hydrogen atom at any position. It can be straight-chain or branched, and can be substituted or unsubstituted. Specifically, for example, propyl refers to either n-propyl or isopropyl, and propylene refers to either 1,3-propylene, 1,2-propylene, or isopropylene.

[0107] In this invention, "unsaturated hydrocarbon group" refers to the hydrocarbon group formed by the loss of hydrogen atoms from an unsaturated hydrocarbon. Hydrocarbon groups formed by the loss of hydrogen atoms from unsaturated carbon atoms in unsaturated hydrocarbons can be classified as alkenyl, alkynyl, dienyl, etc., with examples including propenyl and propynyl. Hydrocarbon groups formed by the loss of hydrogen atoms from saturated carbon atoms in unsaturated hydrocarbons are, depending on the type of unsaturated bond, called alkenyl, alkynyl, dienyl, etc., specifically allyl and propynyl.

[0108] In this invention, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight-chain or branched alkenyl group comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The notation "C" is used to indicate this. 2-15"Alkenyl" refers to a straight-chain or branched alkenyl group consisting of 2-15 carbon atoms and at least one carbon-carbon double bond, whether substituted or unsubstituted. In other words, an alkenyl group can include one, two, three, four, or more carbon-carbon double bonds. Unless otherwise specified, the term alkenyl as used herein refers to both unsubstituted and substituted alkenyl groups.

[0109] In this invention, "alkynyl" or "alkynyl group" means an optionally substituted straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. The notation "C" is used. 2-15 "Alynyl" refers to a straight-chain or branched alkynyl group consisting of 2-15 carbon atoms and at least one carbon-carbon triple bond, whether substituted or unsubstituted. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. Unless otherwise specified, the alkynyl group referred to herein means both unsubstituted and substituted alkynyl groups.

[0110] In this invention, "molecular weight" represents the mass of a compound molecule, and "average molecular weight" represents the mass of a general formula compound component in a macroscopic substance. Unless otherwise specified, "average molecular weight" generally refers to "number-average molecular weight" M. n The number-average molecular weight can refer to the molecular weight of either polydisperse blocks or monodisperse blocks or substances. Unless otherwise specified, the unit of measurement for "molecular weight" and "average molecular weight" is Daltons (Da). The "degree of polymerization" can also characterize the molecular weight of a polyethylene glycol chain, specifically referring to the number of repeating units (ethylene oxide units, EO units) in a compound molecule. Correspondingly, "average degree of polymerization," "number-average degree of polymerization," or "number of EO units" are used to characterize the average or number-mean value of the number of repeating units.

[0111] In this invention, for polydisperse cases, the terms "equal," "identical," "equal to," or "approximately equal to" (including other forms of equivalence) for the molecular weight / degree of polymerization of individual compound molecules and the number-average molecular weight / degree of polymerization of compound components in macroscopic substances are not strictly equal in numerical value unless otherwise specified. Rather, they are exponentially close or approximately equal, with the deviation preferably not exceeding ±10%, more preferably not exceeding ±5%, and usually based on a preset value. "Approximately" or "around" generally refers to a numerical range of ±10%, which can be expanded to ±15% in some cases, but not exceeding ±20%. For example, the deviation between 10kDa and 11kDa, and 12kDa is 10% and 20%, respectively. Furthermore, when the molecular weight of a certain PEG component in the general formula is specified to be equal to 5kDa, the corresponding molecular weight or number-average molecular weight is allowed to vary within the range of 5kDa ±10%, that is, 4500 to 5500Da. For monodisperse compounds, the same or equal number of vinyl oxide units in individual compound molecules and general formulas refers to strict numerical equality. For example, if the number of EO units in a PEG component is set to 11, then a value of 12 is not within the set range. However, for macroscopic products obtained by certain preparation methods to obtain compound components containing a set number of EO units, due to limitations in the preparation and purification methods, the macroscopic product may contain impurities of other EO unit arrays besides the target EO unit array. In this case, when the average number of EO units deviates from the preset number of EO units by no more than ±5% (base ≥ 10) or no more than ±0.5% (base ≥ 10), the impurity is considered acceptable. When the number is <10, it is considered that a monodisperse macroscopic product containing the target component has been obtained; in addition, when the content of the component that meets the range of the number of EO units or the average number of EO units reaches a certain percentage (preferably ≥90%, more preferably >95%, more preferably greater than 96%, more preferably greater than 98%, more preferably 99% to 100%), the obtained macroscopic product falls within the protection scope of the present invention; even if the above-mentioned content ratio is not reached, as long as the preparation method of the present invention or a similar method with basically the same preparation idea is used, the product with insufficient content, the component appearing in the form of co-product or by-product, regardless of whether separation and purification are performed, are all within the scope of the present invention.

[0112] In this invention, when the molecular weight of a compound formula of a polydisperse component is described using Da, kDa, number of repeating units, and number of EO units, the value falls within a certain range of the given value for a single compound molecule (including the endpoints, preferably within ±10%). When the molecular weight of a compound formula of a monodisperse component is described using vinyl oxide units, there is no range fluctuation, and it is a discrete point. However, the average number of EO units in the prepared product may fluctuate within a certain range due to the non-uniformity of molecular weight (not exceeding ±10% or ±1, preferably not exceeding ±5% or ±0.5). For example, the molecular weight of mPEG is 5 kDa, meaning that the molecular weight of a single molecule in the general formula is between 4500 and 5500 Da. The average molecular weight of the corresponding component in the prepared product is 5 kDa. That is, the product with an average molecular weight between 4500 and 5500 Da is the target product, and only components with molecular weights within this range contribute to the content of the target component. Similarly, if mPEG is designed to have 22 vinyl oxide units, then the number of EO units in all compound molecules in the general formula should strictly be 22. However, the prepared product may be a mixture of compounds with 20, 21, 22, 23, or 24 EO units. In this case, if the average number of EO units falls within the range of 22 ± 2.2 (preferably within the range of 22 ± 1.1), it is considered to have obtained the target component, and components with molecular weights within this range can be considered as target components for calculating purity. In this invention, a product PDI < 1.005 is considered monodisperse and can be denoted as PDI = 1.

[0113] Regarding the polydispersity index (PDI) in this invention, for different batches of raw materials, if other parameters are the same or considered to be the same, as long as the PDI does not exceed the preset value, it can be regarded as having no significant difference and as the same raw material.

[0114] In this invention, percentages, "about", generally refer to ±0.5%.

[0115] In this invention, the terms "stable existence" and "degradable" of a functional group are relative concepts.

[0116] In this invention, "degradable" (or "can be degraded") refers to the breaking of chemical bonds, specifically the breaking of at least two independent residues. If the structure is altered after a chemical change, but the linker remains a single, intact linker, it still falls under the category of "stable existence." The conditions for degradation are not particularly limited; they can be in vivo physiological conditions, in vitro simulated physiological environments, or other conditions, preferably in vivo physiological conditions and in vitro simulated physiological conditions. The physiological conditions are not particularly limited and include, but are not limited to, sites such as serum, heart, liver, spleen, lungs, kidneys, bones, muscles, fat, brain, lymph nodes, small intestine, and gonads. These can refer to intracellular or extracellular matrix conditions, and can refer to normal physiological tissues or diseased physiological tissues (such as tumors, inflammation, etc.). The in vitro simulated environment is not particularly limited and includes, but is not limited to, physiological saline, buffer solutions, and culture media. The rate of degradation is not particularly limited; for example, it can refer to rapid degradation under enzymatic action or slow hydrolysis under physiological conditions. The in vivo physiological conditions include physiological conditions during treatment, such as ultraviolet irradiation and thermotherapy. The degradation is not limited to conditions such as light, heat, low temperature, enzymes, redox reactions, acidity, alkalinity, physiological conditions, and in vitro simulated environments, with a preference for degradation under these conditions. Degradability refers to degradation occurring under any of the above-mentioned conditions. Light conditions include, but are not limited to, visible light, ultraviolet light, infrared light, near-infrared light, and mid-infrared light. Thermal conditions refer to temperatures above normal physiological temperature, typically above 37°C and usually below 45°C, preferably below 42°C. Low temperature conditions refer to temperatures below human physiological temperature, preferably below 25°C, more preferably ≤10°C, specifically examples include refrigeration temperatures, freezing temperatures, liquid nitrogen therapy temperatures, 2–10°C, 4–8°C, 4°C, 0°C, and -20±5°C. Enzymatic conditions are not particularly limited; all enzymes that can be generated under physiological conditions are included, such as peptidases, proteases, and lyases. Redox conditions are not particularly limited, such as redox transitions between thiol groups and disulfide bonds, and hydrogenation-reduction transitions. The acidic and alkaline conditions mentioned mainly refer to the pH conditions of internal sites such as normal tissues, diseased tissues, and organs or tissues under treatment. For example, the stomach is acidic, and tumor sites are often acidic as well. Here, "degradable" refers to degradation through metabolic processes in the body (such as physiological processes, enzymes, redox reactions, etc.), degradation due to microenvironmental stimulation at specific sites in the body (such as acidity or alkalinity), or degradation under clinical treatment stimuli (such as light, heat, low temperature). It should be noted that bond breaking under extreme conditions in organic chemistry relative to living organisms, such as strong acids, strong bases, and high temperatures (such as above 100°C), is not included in the scope of the degradable conditions of this invention.For example, although ether bonds can break under strong acid conditions such as hydrobromic acid, they are always classified as stable linking groups in this invention.

[0117] In this invention, "stable existence" refers to the ability of a linker to remain as a complete linker (a linker is stably covalently connected to its adjacent groups), which is defined as "stable existence" (be stable or can remain stable). This allows for chemical changes that maintain the integrity of the linker. The chemical changes are not particularly limited and include, but are not limited to, isomerization, oxidation, reduction, ionization, protonation, deprotonation, and substitution reactions. The conditions for stable existence are not particularly limited and include, but are not limited to, light, heat, low temperature, enzymes, redox reactions, neutral, acidic, alkaline, physiological conditions, and in vitro simulated environments. Preferably, it is stable under light, heat, enzymes, redox reactions, acidic, and alkaline conditions. Stable existence here means that, without special stimulation (such as pH conditions at specific sites, light, heat, or low temperatures during treatment), the linker maintains a stable connection during in vivo metabolic circulation and does not experience a decrease in molecular weight due to chain breakage (as long as the overall integrity is maintained).

[0118] In this invention, "stable existence" is not an absolute concept for the same linker. For example, amide bonds are much more stable than ester bonds under acidic or alkaline conditions, and the "stable existence" linkers in this invention include amide bonds. However, peptide bonds, which are amide bonds formed by the dehydration condensation of the α-carboxyl group and the α-amino group of an amino acid molecule, can be broken when exposed to specific enzymes, and are therefore also included in the "degradable" linkers. Similarly, urethane groups, thiourethane groups, etc., can be both stable and degradable linkers. More generally, urethane groups, thiourethane groups, etc., tend to undergo slow degradation, while non-peptide amide bonds are stable during in vivo circulation. Furthermore, common ester bonds can degrade under acidic or alkaline conditions, while ester bonds in special structures can also degrade under ultraviolet light. For example, even if certain chemical bonds can be degraded under the action of specific enzymes, if the circulation pathway does not pass through or basically does not pass through the specific enzyme environment during clinical use (such as in the case of targeted drug administration), the corresponding chemical bonds can still be considered to be stable.

[0119] In this invention, to more clearly define the degradability of compound structures, a reference criterion is provided: the retention of a specific percentage (e.g., 90%) of chemical bonds within a finite time interval. Taking 90% as an example, the pharmacokinetic curve of the functionalized polyethylene glycol-modified product is typically used as a reference, with the percentage of doses meeting clinical evaluation criteria as the benchmark. For instance, for intravenously administered PEGylated drugs, when the blood drug concentration (based on the effective drug component, including the PEGylated drug and the degraded non-PEGylated component) is less than 15% of the initial concentration (or another proportion more consistent with the clinical evaluation of the drug), using the remaining 85% as the base, if the proportion of a linker maintaining chemical bonds exceeds 90%, it is considered a stable group in this invention; conversely, if it is less than 90%, it is considered a degradable group. Hydrolytic stability, enzymatic degradation, etc., reported in published literature are also included in this invention. Taking hydrolytic stability as an example, this includes the hydrolysis rate at which hydrolysis is stable as reported in published literature, preferably referring to a hydrolysis rate under physiological conditions of less than 1-2% per day (generally taken as 2%), in terms of mass or molar mass. The hydrolysis rate of typical chemical bonds can be found in most standard chemistry handbooks.

[0120] In this invention, the "hydroxyl protecting group" includes all groups that can be used as protecting groups for the common hydroxyl group. The hydroxyl protecting group is preferably an alkyl acyl (e.g., acetyl, tert-butyryl), aralkyl acyl (e.g., benzyl), benzyl, triphenylmethyl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal, or ketal. The removal of the acetyl group is generally carried out under alkaline conditions, most commonly by ammonolysis of NH3 / MeOH and methanololysis catalyzed by methanol anion; benzyl is easily removed by palladium-catalyzed hydrogenolysis in neutral solution at room temperature, or by reduction cleavage with metallic sodium in ethanol or liquid ammonia; triphenylmethyl is generally removed by catalytic hydrogenolysis; trimethylsilyl is usually removed using reagents containing fluoride ions (e.g., tetrabutylamine fluoride / anhydrous THF); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (e.g., Zn / CH3OH), and can be removed by fluoride ions (e.g., Bu4N). + F - It can be removed in tetrahydrofuran solution, or it can be removed with aqueous acetic acid at room temperature.

[0121] In this invention, a "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or a deprotection reaction. The carboxyl protecting group is preferably alkyl (e.g., methyl, ethyl, tert-butyl) or aralkyl (e.g., benzyl), more preferably tert-butyl (tBu), methyl (Me), or ethyl (Et). In this invention, a "protected carboxyl group" refers to the group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, or benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under acid or base catalysis, and occasionally by thermal decomposition. For example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0122] In this invention, "amino protecting group" includes all groups that can be used as protecting groups for ordinary amino groups, such as aryl C. 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6 Alkyl sulfonyl, aryl sulfonyl, or silyl groups are preferred. The amino protecting group is preferably Boc tert-butoxycarbonyl, Moz p-methoxybenzyloxycarbonyl, or Fmoc 9-fluorene-methoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably combinations of TFA and H2O, LiOH and MeOH, or LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; TFA is preferred. The deprotecting agent used in the Fmoc protecting group removal reaction is a solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine.

[0123] In this invention, "carboxyl activation" refers to the activation treatment of carboxyl groups with a carboxyl activator. Activated carboxyl groups can promote better condensation reactions, such as inhibiting the generation of racemic impurities in the condensation reaction and accelerating the reaction rate. "Carboxyl activating group" refers to a residue of the carboxyl activator. The carboxyl activator is one or more of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N-dicyclohexylcarbodiimide (DCC), preferably a combination of NHS / EDCI, NHS / DCC, and HONb / DCC, with the most preferred combination being NHS / EDCI.

[0124] In this invention, "cation" refers to a structure that is permanently or non-permanently positively charged in response to certain conditions (e.g., pH). Therefore, cations include both permanent cations and cationizable cations. A permanent cation is a compound, group, or atom that carries a positive charge at any pH or hydrogen ion activity in its environment. Typically, a positive charge arises from the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be called a permanent cation. A cationizable cation is a compound, group, or atom that carries a positive charge at lower pH and no charge at higher pH in its environment. Additionally, in non-aqueous environments where pH cannot be determined, cationizable compounds, groups, or atoms carry a positive charge at high hydrogen ion concentrations and no charge at low hydrogen ion concentrations or activity. It depends on the individual properties of the cationizable or polycationizable compound, particularly the pKa of the corresponding cationizable group or atom, at the pH or hydrogen ion concentration where it is charged or uncharged. In a diluted aqueous environment, the so-called Henderson-Hasselbalch equation, well known to those skilled in the art, can be used to estimate the fraction of positively charged cationizable compounds, groups, or atoms. For example, in some embodiments, if a compound or portion is cationizable, it is preferred that it carries a positive charge at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH equal to or below 9, equal to or below 8, equal to or below 7, and most preferably at physiological pH (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly under physiological saline conditions in vivo. In other embodiments, it is preferred that the cationizable compound or portion is primarily neutral at physiological pH (e.g., about 7.0-7.4) but becomes positively charged at lower pH values. In some embodiments, the preferred range of the pKa of the cationizable compound or portion is about 5 to about 7.

[0125] In this invention, "cationic component / compound" typically refers to a charged molecule that carries a positive charge (cation) at a pH typically of about 1 to 9. In some embodiments, the cationic component / compound is preferably charged at a pH of 9 or less (e.g., 5 to 9), 8 or less (e.g., 5 to 8), or 7 or less (e.g., 5 to 7), and most preferably at a physiological pH (e.g., about 7.3 to 7.4). Thus, cationic peptides, proteins, polysaccharides, lipids, or polymers according to an embodiment of the invention carry a positive charge under physiological conditions, particularly under physiological saline conditions in vivo.

[0126] In this invention, liposome nanoparticles, cationic peptides, proteins, polysaccharides, lipids, or polymers are uncharged, neutrally charged, or electrically neutral under physiological conditions, particularly under physiological salinity conditions in vivo. The cationic peptides or proteins preferably contain a large number of cationic amino acids, such as Arg, His, Lys, or Orn (especially more cationic amino acids than anionic amino acid residues like Asp or Glu) or contain components mainly composed of cationic amino acid residues. The term "cationic" can also refer to "polycationic" components / chemical cationic components / compounds, and can also refer to cationic lipids capable of carrying a positive charge. For example, cationic lipids contain one or more positively charged amine groups, and preferably cationic lipids are ionizable, allowing them to exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles (LNPs) under different pH conditions. This charge state can influence plasma protein uptake, blood clearance and tissue distribution, and the ability to form non-bilayer structures crucial for intracellular nucleic acid delivery.

[0127] In this invention, "polyethylene glycol-modified lipid" refers to a molecule that includes a lipid portion and a polyethylene glycol portion. In addition to those shown in the general formula (2) of this invention, polyethylene glycol-modified lipids also include, but are not limited to, polyethylene glycol-1,2-dimyristoylglycerol (PEG-DMG), polyethylene glycol-distearylphosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), and polyethylene glycol-dialkoxypropyl (PEG-DAA). Specifically, they include polyethylene glycol 500-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearylphosphatidylethanolamine, polyethylene glycol 500-1,2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoylphosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoylglycerol (PEG-DMG).

[0128] In this invention, "neutral lipids" refers to any of a variety of lipid substances that exist in a neutral or zwitterionic form at a selected pH, preferably phospholipids. Such lipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether). 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) Palmitoyl oleoyl phosphatidyl ethanolamine (POPE), distearate-phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidyl ethanolamine (LPE), or combinations thereof. Neutral lipids may be synthetic or of natural origin.

[0129] In this invention, "steroid lipids" are selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, and α-tocopherol, and mixtures thereof.

[0130] In this invention, "amino acid residue" includes amino acids in which a hydrogen atom has been removed from the amino group and / or a hydroxyl group has been removed from the carboxyl group and / or a hydrogen atom has been removed from the thiol group and / or the amino group and / or the carboxyl group and / or the thiol group are protected. Loosely speaking, an amino acid residue may be referred to as an amino acid. The source of the amino acids in this invention is not particularly limited unless specifically specified, and can be of natural origin, non-natural origin, or a mixture of both. The type of amino acid structure in this invention is not particularly limited unless specifically specified, and can refer to L-type, D-type, or a mixture of both. In one embodiment of this invention, the amino acid is a hydrophobic amino acid selected from any one of tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), and tyrosine (Tyr). In another embodiment of the present invention, the amino acid is a hydrophilic amino acid selected from any one of glutamic acid (Glu), aspartic acid (Asp), histidine (His), glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), proline (Pro), glycine (Gly), lysine (Lys), and arginine (Arg), preferably glycine or lysine, and more preferably lysine.

[0131] In this invention, "functional group source" refers to a source that is reactive or potentially reactive, photosensitive or potentially photosensitive, or targeted or potentially targeted. "Potential" means that it can be transformed into a reactive group through chemical processes selected from, but not limited to, functionalization (e.g., grafting, substitution), deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and alteration of the leaving group, and can emit light or exhibit targeting properties under external stimuli such as light, heat, enzymes, specific binding molecules, or the in vivo microenvironment. The luminescence is not particularly limited and includes, but is not limited to, visible light, fluorescence, and phosphorescence.

[0132] The variations in this invention refer to any chemical change process, such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, or alteration of the leaving group, that can transform the structure into the target reactive group.

[0133] In this invention, "the change form of the reactive group" refers to a reactive group that remains active (still a reactive group) after undergoing at least one chemical change process, such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, or change of leaving group, or an inactive form after being protected.

[0134] In this invention, "micro-modification" refers to a chemical modification process that can be completed through a simple chemical reaction. This simple chemical reaction process mainly includes deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and the transformation of leaving groups.

[0135] "Micro-modification" corresponds to "micro-transformation," referring to structural forms that can form the target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and transformation of the leaving group. The transformation of the leaving group includes, for example, the transformation from an ester form to an acyl chloride form.

[0136] The phrase "any suitable linker" or "any suitable reactive group" refers to a structure that conforms to the basic principles of chemical structure and enables the preparation method of this invention to be successfully implemented. A chemical structure described in this way can be considered to have a clear and definite scope.

[0137] When at least two structural types are listed, "any combination" of the listed structural types refers to any combination of any two or more structures among the aforementioned related structural types; and there is no limit to the number of structural units. The number of any structural unit can be zero, one or more. When the number of structural units of the same type is greater than one, they can be structural units with the same or different chemical structures, and the total number of constituent units is at least two. Examples include any combination of alkylene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloynyl, divalent cyclodienyl, aromatic, carbon-carbon double bond, carbon-carbon triple bond, conjugated carbon-carbon double bond, divalent alicyclic linker, divalent aromatic heterocyclic linker, and carbon chain linker with heteroatom-containing side groups, such as -Ph-CH2-Ph-(aromatic-alkylene-aromatic), -CH2-Ph-CH2CH2-(alkylene-aromatic-alkylene, wherein the number of alkylene groups is 2 and they have different chemical structures), or the structure where the benzene ring is replaced by a hexane, diazahexane, or 1-(2-pyridyl)hexahydro-1H-1,4-diazaphene. Another example is cycloalkenyl hydrocarbon group = cycloalkenyl + alkylene group = cycloalkenyl as a substituent of the hydrocarbon group, and cyclodienyl hydrocarbon group = cyclodienyl as a substituent of the hydrocarbon group. The alkylene groups in this invention, also known as divalent alkyl groups, include open-chain alkylene groups and divalent cycloalkyl groups. Open-chain alkylene groups refer to divalent alkyl groups that do not contain a cyclic structure, while divalent cycloalkyl groups refer to divalent alkyl groups that contain a cyclic structure.

[0138] In this invention, "adjuvant or adjuvant component" typically refers to a pharmaceutical agent or composition that can alter (e.g., enhance) the efficacy of other agents (e.g., drugs or vaccines) (e.g., pharmacological or immunological). Generally, in the context of this invention, the term refers to a compound or composition that serves as a carrier or excipient for immunogenic and / or other pharmaceutically active compounds. It should be interpreted broadly and refers to a wide range of substances capable of increasing the immunogenicity of an antigen incorporated into or co-administered with the adjuvant of the invention. In this invention, the adjuvant will preferably enhance the specific immunogenic effect of the active agent of the invention. Typically, "adjuvant" or "adjuvant component" has the same meaning and can be used interchangeably. Adjuvants can be categorized, for example, as immunostimulants, antigen delivery systems, or even combinations thereof.

[0139] In this invention, "N / P ratio" refers to the molar ratio of nitrogen atoms in cationic lipids to phosphate in nucleic acids.

[0140] In this invention, "nucleic acid" refers to DNA or RNA or its modified form, which contains purine or pyrimidine bases present in DNA (adenine "A", cytosine "C", guanine "G", thymine "T") or purine or pyrimidine bases present in RNA (adenine "A", cytosine "C", guanine "G", uracil "U").

[0141] In this invention, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include cap structures, chain-terminating nucleosides, stem-loops, polyadenylated sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide, for example, in vivo translation of mRNA within mammalian cells, can produce the encoded polypeptide. RNA may be selected from the non-restrictive group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), Cas9 mRNA, and mixtures thereof.

[0142] In this invention, antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0143] In this invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate. Therefore, FLuc is often used in mammalian cell culture to measure gene expression and cell activity.

[0144] In this invention, "inhibition of target gene expression" refers to the ability of nucleic acid to silence, reduce, or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium expressing the target gene) is exposed to nucleic acid that inhibits target gene expression. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing the target gene) that has not been exposed to or treated with nucleic acid. The expression of the target gene in the control sample can be specified as a value of 100%. In a particular embodiment, inhibition of target gene expression is achieved when the target gene expression level in the test sample is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the target gene expression level in the control sample or control mammal.

[0145] In this invention, the methods for determining the expression level of target genes include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic determination.

[0146] In this invention, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, in vivo, or in vivo.

[0147] In this invention, "antigen" typically refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and capable of triggering an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or may contain a peptide or protein that can be presented to T cells by the MHC. In the context of this invention, an antigen can be the translation product of a provided nucleic acid molecule (preferably mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins containing at least one epitope are also understood as antigens.

[0148] In this invention, "delivery" refers to providing an entity to a target. For example, delivering a drug and / or a therapeutic agent and / or a preventive agent to a subject, said subject being tissues and / or cells of a human and / or other animal.

[0149] In this invention, a "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium administered co-administered with a therapeutic agent, and which, within the limits of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of this invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers as needed. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Specifically, excipients include, but are not limited to, anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), modifiers, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium hydrogen phosphate, calcium stearate, croscarmellose sodium, croscarmellose polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0150] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered via suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulation, or inhalation administration. For these routes of administration, suitable dosage forms can be used to administer the pharmaceutical compositions of the present invention. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0151] In this invention, the vaccine is a prophylactic 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.

[0152] In this invention, treatment refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom, intended to include delaying the progression of the disease, obstacle, or symptom, alleviating or mitigating symptoms and complications, and / or curing or eliminating the disease, obstacle, or symptom. The patient to be treated is preferably a mammal, especially a human.

[0153] 1.1. The structure of a polyethylene glycol-modified lipid is shown in general formula (2):

[0154]

[0155] Or its drug-acceptable salts, tautomers, or stereoisomers.

[0156] Among them, one of L7 and L8 is -O(C=O)O-, -C(=O)-, -O-, -O(CR)O-, -O(C=O)-, -O-, -O(CR)O-, -O(C=O)O-, -C(=O)-, -O-, -O(C=O)O-, -C(=O)-, -O(CR)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR)O-, -O(C=O)-, -O(C=O)-, -O-, -O(CR)O-, -O(C=O)-, - ...-, -O-, -O-, -O-, -O-, -O c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any of C(=O)S-, and the other of L7 and L8 is a linker, -O(C=O), -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR c R c ) sO-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any one of C(=O)S-, where R c Each time it appears, it is independently a hydrogen atom or an alkyl group, and s is 2, 3 or 4;

[0157] L3 is any one of the following: linker, -L4-, -Z-L4-, -L4-Z-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, -L4-Z-L5-Z-, -Z-L4-Z-L5-Z-, and -L4-Z-L4-Z-L5-Z-; L4 and L5 are carbon chain linkers, each independently being -(CR... a R b ) t -(CR a R b ) o -(CR a R b ) p - where t, p, and q are each independent integers from 0 to 12, and t, p, and q are not all 0 at the same time; R a and R b Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 Alkyl groups; each occurrence of Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S- and Any one of them, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 alkyl;

[0158] B3 and B4 are each independently a connecting key or C.1-30 Alkylene;

[0159] R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon groups;

[0160] R represents a hydrogen atom, -R d -OR d -NR d R d -SR d -(C=O)R d -(C=O)OR d -O(C=O)R d -O(C=O)OR d or Among them, R d Each occurrence is independently represented by C. 1-12 Alkyl group, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R. 01 When j is 0, G1 does not exist; when j is 1, G1 introduces k Fs, where k is an integer from 2 to 8.

[0161] A is -(CR) a R b ) s O- or -O(CR) a R b ) s -, where s is 2, 3 or 4;

[0162] n1 is approximately an integer between 20 and 250;

[0163] The alkyl, alkylene, alkoxy, and aliphatic hydrocarbon groups are each independently substituted or unsubstituted.

[0164] 1.1.1. Divalent connecting bases L3, L4, L5, L7, L8, Z, Z1, Z2

[0165] In this invention, the structures of L3, L4, L5, L7, L8, Z, Z1, and Z2 are not particularly limited, and each can independently include, but is not limited to, a straight chain structure, a branched chain structure, or a ring-shaped structure.

[0166] In this invention, the number of non-hydrogen atoms in L3, L4, L5, L7, L8, Z, Z1, and Z2 is not particularly limited, and each is preferably 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; and even more preferably 1 to 10 non-hydrogen atoms. The non-hydrogen atoms are carbon atoms or heteroatoms. The heteroatoms include, but are not limited to, O, S, N, P, Si, and B. When the number of non-hydrogen atoms is 1, the non-hydrogen atom can be a carbon atom or a heteroatom. When the number of non-hydrogen atoms is greater than 1, the type of non-hydrogen atoms is not particularly limited; it can be one type, or two or more types; when the number of non-hydrogen atoms is greater than 1, it can be any combination of carbon atoms with carbon atoms, carbon atoms with heteroatoms, or heteroatoms with heteroatoms.

[0167] In this invention, two identical or different reactive groups can react to form a divalent linker. The reaction conditions depend on the type of divalent linker generated and can be achieved using existing publicly available techniques. For example, amino groups react with reactive esters, reactive formic acid esters, sulfonates, aldehydes, α,β-unsaturated bonds, carboxylic acid groups, epoxides, isocyanates, and isothiocyanates to obtain amide groups, urethane groups, amino groups, imine groups (which can be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea bonds, thiourea bonds, and other divalent linkers; mercapto groups react with reactive esters, reactive formic acid esters, sulfonates, mercapto groups, maleimides, aldehydes, α,β-unsaturated bonds, carboxylic acid groups, iodoacetamide, and acid anhydrides to obtain thioester groups, thiocarbonates, thioethers, disulfides, thioethers, thiohemiacetals, thioethers, thioesters, thioethers, imides, and other divalent linkers; unsaturated bonds react with mercapto groups to obtain thioether groups; Carboxyl groups or acyl halides react with thiol groups and amino groups to yield thioester groups, amide groups, etc.; hydroxyl groups react with carboxyl groups, isocyanates, epoxides, and chloroformoxy groups to yield ester groups, carbamate groups, ether bonds, carbonate groups, etc.; carbonyl groups or aldehyde groups react with amino groups, hydrazine groups, and acylhydrazine groups to yield imine bonds, hydrazones, acylhydrazones, etc.; reactive groups such as azide, alkynyl, alkenyl, thiol, azide, diene, maleimide, 1,2,4-triazolin-3,5-dione, dithioester, hydroxylamine, acylhydrazine, acrylate, allyloxy, isocyanate, and tetrazolium can undergo click chemistry reactions to generate various divalent linkages containing structures including but not limited to triazole, isoxazole, and thioether bonds.

[0168] There are no particular restrictions on the stability of L3, L4, L5, L7, L8, Z, Z1, and Z2. Any one of the divalent linkers or any one of the divalent linkers formed with an adjacent heteroatom group can be independently a stable linker STAG or a degradable linker DEGG.

[0169] 1.1.1.1 Bivalent connecting bases L7 and L8

[0170] In this invention,

[0171] In this invention, one of L7 and L8 is -O(C=O)O-, -C(=O)-, -O-, or -O(CR). c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any of C(=O)S-, and the other of L7 and L8 is a linker, -O(C=O), -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any one of C(=O)S-, where R c Each time it appears, it is independently a hydrogen atom or an alkyl group, and s is 2, 3 or 4.

[0172] In one specific embodiment of the present invention, preferably one of L7 and L8 is -O(C=O)O-, -C(=O)-, -O-, or -O(CH2). s Any one of the following: O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-, where the other is a linking bond; -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CH2). sAny one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-.

[0173] In one specific embodiment of the present invention, it is more preferable that one of L7 and L8 is -O(C=O)O-, -C(=O)-, -O-, or -O(CH2). s The following are all of the following: O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-, and the other is a linking bond, -O-, -O(C=O)-, and -(C=O)O-.

[0174] In one specific embodiment of the present invention, more preferably, L7 and L8 are selected from any one of the following:

[0175] (1) One of L7 and L8 is -C(=O)-, and the other is a linker;

[0176] (2) One of L7 and L8 is -C(=O)-, and the other is -O(C=O)- or -(C=O)O-;

[0177] (3) Both L7 and L8 are -O-.

[0178] 1.1.1.2 Divalent Connecting Base L3

[0179] In this invention, L3 is any one of the following: linker, -L4-, -Z-L4-, -L4-Z-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, -L4-Z-L5-Z-, -Z-L4-Z-L5-Z-, and -L4-Z-L4-Z-L5-Z-; L4 and L5 are carbon chain linkers, each independently being -(CR a R b ) t -(CR a R b ) o -(CR a R b ) p - where t, p, and q are each independent integers from 0 to 12, and t, p, and q are not all 0 at the same time; R a and R b Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12Alkyl groups; each occurrence of Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S- and Any one of them, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 alkyl.

[0180] In one specific embodiment of the present invention, R c Hydrogen atoms are preferred.

[0181] In one specific embodiment of the present invention, L3 contains a degradable group, which refers to a group that can degrade under any conditions such as light, heat, low temperature, enzymes, redox reactions, acidity, alkalinity, physiological conditions, or in vitro simulated environments; preferably, L4 and L5 in L3 are each independently -(CH2). t -, L3 is -(CH2) t -、-(CH2) t Z-, -Z(CH2) t -、-(CH2) t Z(CH2) t -、-Z(CH2) t Z-, -(CH2) t Z(CH2) t Z-, -Z(CH2) t Z(CH2) t - and -Z(CH2) t Z(CH2) t Z- can be any one of the following, where t is an integer from 1 to 12, and each occurrence of Z is independently one of -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-; preferably L3 is -(CH2). t -、-(CH2) tO- -(CH2) t C(=O)-、-(CH2) t C(=O)O-、-(CH2) t OC(=O)-、-(CH2) t C(=O)NH-、-(CH2) t NHC(=O)-、-(CH2) t OC(=O)O-、-(CH2) t NHC(=O)O-、-(CH2) t OC(=O)NH-、-(CH2) t NHC(=O)NH-、-O(CH2) t -C(=O)(CH2) t -C(=O)O(CH2) t -、-OC(=O)(CH2) t -、-C(=O)NH(CH2) t - -NHC(=O)(CH2) t -、-OC(=O)O(CH2) t - -NHC(=O)O(CH2) t -、-OC(=O)NH(CH2) t - -NHC(=O)NH(CH2) t -、-(CH2) t O(CH2) t -、-(CH2) t C(=O)(CH2) t -、-(CH2) t C(=O)O(CH2) t -、-(CH2) t OC(=O)(CH2) t -、-(CH2) t C(=O)NH(CH2) t -、-(CH2) t NHC(=O)(CH2) t -、-(CH2) t OC(=O)O(CH2) t -、-(CH2) t NHC(=O)O(CH2) t -、-(CH2) t OC(=O)NH(CH2) t -、-(CH2) t NHC(=O)NH(CH2) t -O(CH2) tO- -C(=O)(CH2) t C(=O)-、-C(=O)O(CH2) t C(=O)O-、-OC(=O)(CH2) t OC(=O)-、-C(=O)O(CH2) t OC(=O)-、-OC(=O)(CH2) t C(=O)O-、-OC(=O)O(CH2) t OC(=O)O-、-C(=O)NH(CH2) t C(=O)NH-、-NHC(=O)(CH2) t NHC(=O)-、-NHC(=O)(CH2) t C(=O)NH-、-C(=O)NH(CH2) t NHC(=O)-、-NHC(=O)O(CH2) t NHC(=O)O- 、 -OC(=O)NH(CH2) t OC(=O)NH- 、-NHC(=O)O(CH2) t OC(=O)NH- 、 -OC(=O)NH(CH2) t NHC(=O)O- 、-NHC(=O)NH(CH2) t NHC(=O)NH-、-C(=O)(CH2) t O- -C(=O)(CH2) t C(=O)O-、-C(=O)(CH2) t OC(=O)-、-C(=O)(CH2) t OC(=O)O-、-C(=O)(CH2) t NHC(=O)O-、-C(=O)(CH2) t OC(=O)NH-、-C(=O)(CH2) t NHC(=O)NH-、-C(=O)(CH2) t C(=O)O(CH2) t -C(=O)(CH2) t OC(=O)(CH2) t - 、 -C(=O)(CH2) t OC(=O)O(CH2) t - -C(=O)(CH2) t NHC(=O)O(CH2) t - 、 -C(=O)(CH2) tOC(=O)NH(CH2) t - 、-C(=O)(CH2) t NHC(=O)NH(CH2) t - and -C(=O)(CH2) t C(=O)(CH2) t Any one of NHC(=O)O-, where t is an integer from 2 to 12; the most preferred are -C(=O)O- and -(CH2). t O-, -(CH2) t C(=O)O-、-(CH2) t OC (=O)-、-(CH2) t O-, -C(=O)(CH2) t O-, -C(=O)(CH2) t OC(=O)-、-C(=O)(CH2) t C(=O)O-、-C(=O)(CH2) t C(=O)NH-、-C(=O)(CH2) t OC(=O)NH-、-C(=O)(CH2) t NHC(=O)O-、-C(=O)(CH2) t OC(=O)NH(CH2) t - and -C(=O)(CH2) t C(=O)(CH2) t Any one of NHC(=O)O-.

[0182] 1.1.2. Explanation of Stabilizable and Degradable Groups

[0183] The stable linker STAG or the degradable linker DEGG in this invention can exist in any of the divalent linkers L3, L4, L5, L7, L8, Z, Z1, and Z2, or in any divalent linker composed of a divalent linker and an adjacent heteroatom group.

[0184] 1.1.2.1 The STAG divalent linker that can exist stably in this invention

[0185] The divalent linker STAG can exist stably under any of the following conditions: light, heat, low temperature, enzymes, redox, acidic, alkaline, physiological conditions, and in vitro simulated environment. It is preferred that it can exist stably under any of the following conditions: light, heat, enzymes, redox, acidic, and alkaline.

[0186] There are no particular restrictions on the type of stable divalent linker STAG, including but not limited to alkylene, divalent heteroalkyl, double bond, triple bond, divalent dienyl, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloolefinyl, divalent cycloalkynyl, aromatic ring, aliphatic heterocycle, heterobenzene ring, arylheterocyclic ring, heterofused heterocycle, substituted alkylene, substituted heteroalkyl, substituted divalent heteroalkyl, substituted double bond, substituted triple bond, substituted diene, substituted divalent cycloalkyl, substituted divalent cycloalkenyl, substituted divalent cycloolefinyl, substituted divalent cycloalkynyl, substituted aromatic ring, substituted alkylene, substituted heteroalkyl, substituted divalent cycloalkynyl, substituted alkylene ... Rings, substituted alicyclic rings, substituted heterobenzene rings, substituted aryl heterocyclic rings, substituted heterofused heterocyclic rings, ether bonds, thioether bonds, urea bonds, thiourea bonds, carbamate groups, thiocarbamate groups, -P(=O)-, divalent silicon groups without active hydrogen, divalent linking groups containing boron atoms, secondary amino groups, tertiary amino groups, carbonyl groups, thiocarbonyl groups, amide groups, thioamide groups, sulfonamide groups, enamine groups, triazoles, 4,5-dihydroisoxazoles, any one of the divalent linking groups in the skeleton of amino acids and their derivatives, and stable divalent linking groups composed of any two or more groups.

[0187] Specifically, the stable bivalent linker STAG includes, but is not limited to, the structures described and listed in documents CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, the corresponding sections

[0627] to

[0704] are shown. There are no particular restrictions on the way two or more stable bivalent linkers can be combined to form a STAG. This includes, but is not limited to, section

[704] of CN104530417A.

[0188] 1.1.2.2 The biodegradable divalent linker DEGG in this invention

[0189] The degradable divalent linker DEGG is not particularly limited by the conditions under which it can be degraded. It can be degraded under any of the following conditions, including but not limited to light, heat, low temperature, enzymes, redox, acidity, alkalinity, physiological conditions, and in vitro simulated environment. It is preferred that it can be degraded under any of the following conditions: light, heat, enzymes, redox, acidity, and alkalinity.

[0190] A divalent linker formed by combining any degradable divalent linker DEGG with any stable divalent linker STAG is still a degradable linker. For degradable divalent linkers containing aromatic rings, they can also be formed by combining aromatic rings with degradable divalent linkers.

[0191] There are no particular restrictions on the type of degradable divalent linker DEGG, including but not limited to those containing disulfide bonds, vinyl ether bonds, ester groups, thioester groups, thioester groups, dithioester groups, carbonate groups, thiocarbonate groups, dithiocarbonate groups, trithiocarbonate groups, carbamate groups, thiocarbamate groups, dithiocarbamate groups, acetals, cycloacetals, thioacetals, azaacetals, azacyclic acetals, nitrothiacetals, dithioacetals, hemiacetals, thiohemiacetals, azahemiacetals, ketals, thioketals, azaketals, azacyclic ketals, nitrothiacetals, imine bonds, hydrazone bonds, acylhydrazone bonds, oxime bonds, thiooxime ether groups, hemicarbazone bonds, thiohemicarbazone bonds, hydrazyl groups, acylhydrazyl groups, thiocarbonylhydrazyl groups, azocarbonylhydrazyl groups, thioazocarbonylhydrazyl groups, and hydrazocarboxylic acid. Ester group, hydrazine thiocarbamate group, carbazine, thiocarbazine, azo group, isourea group, isothiourea group, urea carbamate group, thiourea carbamate group, guanidine group, amidine group, aminoguanidine group, aminoamidinyl group, imine group, imine thioester group, sulfonate group, sulfinate group, sulfonylhydrazine group, sulfonylurea group, maleimide, orthoester group, phosphate group, phosphite group, hypophosphite group, phosphosilicate group, silane ester group, carbamide, thioamide, sulfonamide group, polyamide, phosphoramide, phosphorous acid, pyrophosphoramide, cyclophosphoramide, isocyclophosphoramide, thiophosphoramide, aconityl group, polypeptide fragment, nucleotide and its derivative backbone, deoxynucleotide and its derivative backbone, any one of the following divalent linkers, or a combination of any two or more divalent linkers.

[0192] The urethane groups, thiourethane groups, carbamates, and phosphoramides mentioned here can serve as either stable linkers or biodegradable linkers, depending on the environmental conditions in which they are used.

[0193] Specifically, the degradable divalent linker DEGG includes, but is not limited to, the structures described and listed in documents CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, the corresponding paragraphs are

[705] to

[0725] .

[0194] 1.1.3. Alkylene B3, B4

[0195] In this invention, B3 and B4 are each independently a connecting key or a C. 1-30 Alkyl groups, more preferably each independently formed by a linking bond or a C-bond. 1-20 Alkylene.

[0196] In one specific embodiment of the present invention, B3 and B4 are both connecting keys.

[0197] In one specific embodiment of the present invention, one of B3 and B4 is a connecting key, and the other is a C. 1-20 Alkylene.

[0198] In one specific embodiment of the present invention, B3 and B4 are preferably each independently C. 1-20 Alkylene; specifically, each independently being any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecanylene, octadecylene, nonadecanylene, and eicosylene.

[0199] 1.1.4. Aliphatic hydrocarbon groups R1, R2

[0200] In this invention, R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon group.

[0201] In one specific embodiment of the present invention, R1 and R2 are preferably each C 1-20 Aliphatic hydrocarbon group.

[0202] In one specific embodiment of the present invention, R1 and R2 are each independently a straight-chain alkyl group, a branched alkyl group, a straight-chain alkenyl group, a branched alkenyl group, a straight-chain alkynyl group, or a branched alkynyl group; preferably a straight-chain alkyl group; more preferably, each independently a C1 group. 1-25 Straight-chain aliphatic hydrocarbon groups; more preferably, each is independently C 1-20Straight-chain aliphatic hydrocarbon groups; most preferably, each independently, is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, (Z)-tridecane-8-enyl, (Z)-tetradecane-9-enyl, (Z)-pentadecanane-8-enyl, (Z)-hexadecane-9-enyl, (Z)-heptadecane-5-enyl, (Z)-heptadecane-8-enyl, (E)-heptadecane-8-enyl, (Z)-heptadecane-10-enyl, (8Z,11Z)-heptadecane-8,11-dienyl, (Z)-octadecane-6-enyl, (Z)-octadecane-9-enyl, (E)-octadecane-8-enyl, (Z)-octadecane-9-enyl, (E)-octadecane-8-enyl, (Z)-octadecane-8-enyl, (Z)-octadecane-9-enyl, (Z)-octadecane-8 ... The following is a list of compounds: alkan-9-enyl, (Z)-octadecane-11-enyl, (9Z,12Z)-octadecane-9,12-dienyl, (9Z,12Z,15Z)-octadecane-9,12,15-trienyl, (8Z,11Z,14Z)-octadecane-8,11,14-trienyl, (Z)-eicosane-11-enyl, (11Z,14Z)-eicosane-11,14-dienyl, (Z)-nonadecane-10-enyl, (10Z,13Z)-nonadecane-10,13-dienyl, 2,6,10-trimethylundecane-1,5,9-trienyl, 3,7,11-trimethyldodecane-2,6,10-trienyl, or 3,7,11,15-tetramethylhexadecane-2-enyltridecyl.

[0203] In one specific embodiment of the present invention, it is preferred that both R1 and R2 mentioned above are methyl groups.

[0204] In one specific embodiment of the present invention, R1 and R2 are each independently a branched alkyl group, a branched alkenyl group, or a branched alkynyl group, and are each independently represented as follows: Among them, R e R f Each independently is C1-C 15 Alkyl, C2-C 15 alkenyl and C2-C 15The alkynyl group is selected from any one of the following groups; more preferably, each is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propynyl, allyl, butenyl, allenyl, pentenyl, allenyl, hexenyl, heptenyl, allenylheptyl, octenyl, allenyl, nonenyl, decenyl, ethynyl, propynyl, propynyl, butynyl, ethynylbutyryl, pentynyl, ethynyl, hexynyl, heptynyl, ethynylheptyl, octyryl, ethynyloctyl, nonynyl, ethynylnonyl, decynyl, and ethynyldecyl; more preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Further preferably, R1 and R2 are each independently selected from any one of the following structures:

[0205]

[0206]

[0207] Where t is an integer between 0 and 12.

[0208] 1.1.5.R

[0209] In this invention, R is a hydrogen atom, -R d -OR d -NR d R d -SR d -(C=O)R d -(C=O)OR d -O(C=O)R d -O(C=O)OR d or Among them, R d Each occurrence is independently represented by C. 1-12 Alkyl group, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R. 01 When j is 0, G1 does not exist; when j is 1, G1 introduces k Fs, where k is an integer from 2 to 8.

[0210] 1.1.5.1.R d

[0211] In this invention, R d Each occurrence is independently represented by C. 1-12 alkyl.

[0212] In one specific embodiment of the present invention, R d C is preferred 1-8 Alkyl, specifically, R dPreferably, it is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

[0213] 1.1.5.2.G1

[0214] In this invention, j is 0 or 1. When j is 0, G1 does not exist. When j is 1, G1 exists and is a terminal branched group with a valence of k+1, which leads to k F groups containing functional groups. Here, k is an integer from 2 to 8, preferably k is 2, 3 or 4.

[0215] In one specific embodiment of the present invention, when G1 is preferably a trivalent or higher branched group, it is more preferably a trivalent or tetravalent branched group; more preferably, G1 is a trivalent branched group; and even more preferably, G1 is a trivalent branched group of glycerol or an amino acid residue.

[0216] In this invention, when performing terminal difunctionalization, G1 is preferably derived from alcohols, thiols, primary amines, secondary amines, sulfonates, or halogens containing two exposed hydroxyl groups or two protected hydroxyl groups (e.g., triethanolamine p-toluenesulfonate, glyceryl monothioacetate, 3,4-dihydroxy-2'-chloroacetophenone, and hydroxyl-protected forms), alcohols, thiols, primary amines, secondary amines, sulfonates, or halogens containing two mercapto groups or two protected mercapto groups (e.g., dimercaprol and its mercapto-protected forms), alcohols, thiols, primary amines, secondary amines, sulfonates, or halogens containing two primary amino groups, two secondary amino groups, two protected primary amino groups, or two protected secondary amino groups, etc. Examples of alcohols containing two primary amines include 1,3-diamino-2-propanol, aldehydes containing one epoxy group, and alcohols containing one epoxy group (e.g.,...). The group consists of sulfonates containing one epoxy group, halogenated compounds containing one epoxy group, and compounds containing one epoxy group and one other reactive group. It also includes combinations of primary amines reacting with two molecules of acrylate via Michael addition. Alternatively, ring-opening can be achieved by reducing the disulfide bond after thioctic acid capping, yielding two terminal thiol groups.

[0217] In this invention, when performing terminal trifunctionalization, G1 is preferably derived from a tetrafunctionalized small molecule htetraSM containing three hydroxyl groups and another reactive group, including but not limited to: N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, tris(hydroxymethyl)aminopropanesulfonic acid, methyl-6-O-p-toluenesulfonyl-α-D-glucoside, 2-(bromomethyl)-2-(hydroxymethyl)-1,3-propanediol, tris(hydroxymethyl)aminomethane, 2-amino-1,3,4-octadecanetriol, 3-aminopropylsilanetriol, 4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, 4-[(2-isopropylamino-1-hydroxy)ethyl]-1,2-benzenediol, 3,4-dihydroxy-α-((methylamino)methyl)benzyl alcohol, 2,5-dehydrated-1-dihydroxymethyl-2- ... Nitrogen-1-deoxy-D-glucanol, 2,3,4-trihydroxybutanal (L-erythrose, D-erythrose, L-(+)-threose, D-(+)-threose), 2,3,4-trihydroxybenzaldehyde, 3,4,5-trihydroxybenzaldehyde, tris(hydroxymethyl)methylglycine, 2,3,4-trihydroxybutyric acid (including but not limited to erythric acid and threonic acid), 2,4,6-trihydroxybenzoic acid, shikimic acid, 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, ajangalamic acid, 1,4,7-tritert-butoxycarbonyl-1,4,7,10-tetraazacyclododecane, tritert-butoxycarbonylspermine, 1,4,7-tritert-butoxycarbonyl-1,4,7,10-tetraazacyclododecane, and the hydroxyl groups of any of the above are protected forms. It can also be selected from groups composed of citric acid, muscarinic acid, N-hydroxyethylethylenediaminetriacetic acid, pentaerythritol triacrylate, aminomethanetripropionic acid, and tri-tert-butyl aminomethanetripropionic acid. It also includes terminal branching reactions based on alkenyl, trichlorosilane, and allyl magnesium chloride, referring to the literature "Macromolecules, Vol. 33, No. 12, 2000," to form tetravalent silyl branching centers. It also includes terminal branching reactions based on alkenyl, trichlorosilane, and allyl alcohol to form tetravalent siloxane branching centers. It can also include trifunctionalized small molecules such as 1,4,7-tris(tert-butoxycarbonylmethyl)-1,4,7,10-azacyclotetradecane (NOTA), in which case these trifunctionalized small molecules need to be in excess during the reaction.

[0218] 1.1.5.3. F containing functional groups

[0219] In one specific embodiment of the present invention, the structure of F is preferably -(Z2). q -(Z1) q1 -R 01 Where q and q1 are each independently 0 or 1; Z1 and Z2 are each independently a divalent linker; R 01 It is a functional group that can react with biologically related substances.

[0220] In one specific embodiment of the present invention, more preferably, Z1 and Z2 are each independently any one of -L4-, -L4-Z-, -Z-L4-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5- and -L4-Z-L5-Z-, and t is an integer from 1 to 12.

[0221] In a more specific embodiment of the present invention, R 01 Preferably selected from: reactive groups, variations of reactive groups, therapeutically targeted functional groups, and fluorescent functional groups; wherein, the variations include any one of the following: a precursor of the reactive group, an active form using it as a precursor, a substituted active form, a protected form, and a deprotected form; wherein, the precursor of the reactive group refers to a structure that can be transformed into the structure of the reactive group through at least one of the following processes: oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation; wherein, the variation of the reactive group refers to a form in which a reactive group remains active after undergoing at least one of the following processes: oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, and deprotection, or an inactive form after being protected; the R 01 More preferably, the group consisting of any one functional group from the following categories A to H and their variations, or the group consisting of functional groups from categories I to J:

[0222] Class A: Active ester groups and similar structures of active ester groups; wherein, active ester groups include: succinimide active ester group, p-nitrobenzene active ester group, o-nitrobenzene active ester group, benzotriazole active ester group, 1,3,5-trichlorobenzene active ester group, 1,3,5-trifluorobenzene active ester group, pentafluorobenzene active ester group, and imidazole active ester group; wherein, similar structures of active ester groups include: 2-thionone-3-thiazolyl carboxylate group, 2-thiooxothiazolyl-3-carboxylate group, 2-thionone pyrrolidine-N-carboxylate group, 2-thionone pyrrolidine-N-carboxylate group, 2-thionone benzothiazolyl-N-carboxylate group, and 1-oxo-3-thiooxoisoindoline-N-carboxylate group;

[0223] Class B: sulfonate group, sulfinate group, sulfone group, sulfoxide group, 1,3-disulfonyl-2-propylcarbonylphenyl, sulfonemethylacryloyl group;

[0224] Class C: hydroxylamine, mercapto, primary amino, secondary amino, halogen atom, haloacetamide, tetramethylpiperidinoxy, dioxapiperidinoxy, ammonium salt, hydrazine, disulfide compound, ester, thioester, thioester, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, xanthate, perthiocarbonate, tetrathiodiester, O-carbonylhydroxylamine, amide, imide, acylhydrazine, sulfonylhydrazine, hydrazone, imine, enamine, acetylenyl, carbamate, monothiocarbamate, dithiocarbamate, protected amino group;

[0225] Class D: Carboxyl, sulfonic acid, hyposulfonic acid, isohydroxamic acid, thioisohydroxamic acid, xanthic acid, acyl halide, sulfonyl chloride, aldehyde, glyoxal, acetal, hemiacetal, hydrated aldehyde, ketone, ketal, hemiketal, hemiketal alcohol, ketal alcohol, hydrated ketone, orthoic acid, orthoester, cyanate, thiocyanate, isocyanate, isothiocyanate, ester, oxycarbonyl acyl halide, oxazoline, isoxazoline, thioaldehyde, thioketone, thioacetal, thioketone hydrate, ketethiothyl alcohol, thioester, sulfur Ester group, dithioester group, thiohemiacetal group, monothiohydrate group, dithiohydrate group, thiol hydrate group, thiocarbonyl monothiocarboxylic acid group, thiohydroxy monothiocarboxylic acid group, dithiocarboxylic acid group, urea group, thiourea group, guanidinyl group and its protonated form, amidinyl group and its protonated form, acid anhydride group, squaric acid group, squaric acid ester group, hemisquaric acid group, hemisquaric acid ester group, N-carbamoyl-3-imidazolyl group, N-carbamoyl-3-methyliodimidazolium group, imine group, imine ester group, nitroketone group, oxime group, pseudourea group;

[0226] Class E: maleimide group, acrylate group, N-acrylamide group, methacrylate group, N-methacrylamide group, protected maleimide group, maleamic acid group, 1,2,4-triazolin-3,5-dione group, linear azo compound group, cyclic azo compound group, cyclic olefin group; wherein, the cyclic olefin group includes cyclooctene group, norbornenyl group, 7-oxa-bicyclo[2.2.1]hept-5-en-2-yl group, bicycloheptadienyl group, 7-oxa-bicycloheptadienyl group;

[0227] Class F: epoxy, vinyl, propenyl, alkenyl, alkynyl, alkynyl hydrocarbon;

[0228] Class G,

[0229] Ga-like groups: cycloalkyne group, cycloalkyne heterohydrazine group, linear conjugated diene group, cyclic conjugated diene group, hybrid cyclic conjugated diene group, 1,2,4,5-tetraazine group;

[0230] Gb-like groups: azide, nitrile, cyano, isocyanate, aldoxime, diazo, diazoonium ion, azo group, nitrile imine, N-oxyaldiimine, tetrazolium, 4-acetyl-2-methoxy-5-nitrophenoxy and their diazotized forms; other functionalized groups that can undergo 1,3-dipolar cycloaddition reactions;

[0231] H-type: hydroxyl group, protected hydroxyl group, siloxy group, protected dihydroxyl group, trihydroxysilyl group, protected trihydroxysilyl group; wherein, hydroxyl group includes alcohol hydroxyl group, phenolic hydroxyl group, enol hydroxyl group, hemiacetal hydroxyl group;

[0232] Class I: Targeting groups and their pharmaceutically acceptable salts;

[0233] Class J: Fluorescent groups, including residues of any one of fluorescein, rhodamine, anthracene, pyrene, coumarin, fluorescein 3G, carbazole, imidazole, indole, alizarin violet, and any functional derivative thereof.

[0234] Furthermore, R 01 The functional groups are preferably selected from any one of the following categories A to J, variations of categories A to H, and functional derivatives of categories I to J; the variations are selected from any one of the following: precursors of reactive groups, active forms using these as precursors, substituted active forms, protected forms, and deprotected forms.

[0235] Class A:

[0236]

[0237] Or class B:

[0238]

[0239] Or class C:

[0240]

[0241] Or class D:

[0242]

[0243] Or class E:

[0244]

[0245] Or class F:

[0246]

[0247] Or class G: class Ga:

[0248]

[0249] Or class Gb:

[0250]

[0251] Or class H:

[0252]

[0253] Or class I:

[0254]

[0255] Or class J:

[0256]

[0257] Wherein, M5 is a cyclic atom selected from carbon, nitrogen, phosphorus, and silicon atoms; the ring structure containing M5 is a 3- to 50-membered ring, preferably a 3- to 32-membered ring, more preferably a 3- to 18-membered ring, and even more preferably a 5- to 18-membered ring; the ring structure is preferably any one of the following groups, any substituted form of any one, or any hybridized form of any one: cyclohexane, furanose ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidinyl ether, cyclohexene, tetrahydropyran, piperidine, 1, 4-Dioxane, pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,4,7-triazacyclononane, cyclic tripeptide, indene, dihydroindene, indole, isoindole, purine, naphthalene, dihydroanthracene, oxanthracene, thioxanthraphenanthrene, dihydrophenanthrene, 10,11-dihydro-5H-dibenzo[a,d]cycloheptane, dibenzocycloheptene, 5-dibenzocycloheptene, quinoline, isoquinoline, fluorene, carbazole, iminodibenzyl, naphthylene, dibenzocyclooctylene, azadibenzocyclooctylene;

[0258] Wherein, Y1 is a leaving group that connects to a sulfonyl group, a sulfinyl group, an oxysulfonyl group, or an oxysulfinyl group, and is selected from any one of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, vinyl, phenyl, benzyl, p-methylphenyl, 4-(trifluoromethoxy)phenyl, trifluoromethyl, and 2,2,2-trifluoroethyl;

[0259] Wherein, W is F, Cl, Br or I;

[0260] Wherein, W2 is F, Cl, Br or I;

[0261] Wherein, W3 is a leaving group selected from F, Cl, Br, I, and PhS;

[0262] in, These are cyclic structures containing nitrogen atoms, nitronium ions, double bonds, azo bonds, triple bonds, disulfide bonds, acid anhydrides, imides, and dienes on their ring skeletons, wherein the cyclic structures are selected from carbocyclic rings, heterocyclic rings, benzo[a]heterocyclic rings, substituted carbocyclic rings, substituted heterocyclic rings, or substituted benzo[a]heterocyclic rings.

[0263] Where M is a carbon atom, nitrogen atom, phosphorus atom, or silicon atom on the ring;

[0264] Wherein, M8 is a carbon atom, nitrogen atom, phosphorus atom or silicon atom located on the ring; the number of cyclic atoms in the ring containing M8 is 4 to 50; preferably 4 to 32; more preferably 5 to 32;

[0265] Among them, M 22 It is a carbon atom, nitrogen atom, phosphorus atom, or silicon atom located on an alicyclic or heterocyclic ring; M 22 The ring contains 4, 5, 6, 7, or 8 ring atoms;

[0266] Among them, R 22 It is an end group or divalent linker that connects oxygen or sulfur atoms, selected from hydrogen atoms, R 21 or R 33 Any one of the atoms or groups;

[0267] Among them, R 21 It is a divalent linker and participates in ring formation; R 21 Selected from C 1-20 Hydroxyl group, divalent C 1-20 heteroalkyl groups, substituted C 1-20 Hydroxyl group, substituted divalent C 1-20 R is a divalent linker formed by any one or any combination of two or three of the heteroalkyl groups; 21 Preferred compounds include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, 1,2-phenylene, benzylene, and C. 1-20 oxaalkylene, C 1-20 Thionide, C 1-20 Any one of the aziridine and azirane groups, any substituted form of the group, any combination of two or more identical or different groups or their substituted forms;

[0268] Among them, R 33 The terminal group is the one that connects to an oxygen or sulfide group, selected from C 1-20 hydrocarbon group, C 1-20 heterohydrocarbon group, C 1-20 Substituted hydrocarbon group, C 1-20 Any one of the substituted heteroalkyl groups; preferably any one or a substituted form of methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, benzyl, allyl;

[0269] Where R4 is -(R4)C=N + =N — or -(R4)C - -N + The hydrogen atom, substituted atom, or substituent on C in the ≡N structure; preferably any one of the following atoms or groups: hydrogen atom, methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, allyl, propenyl, vinyl, phenyl, methylphenyl, butylphenyl, and benzyl.

[0270] Among them, R8, R9, R 10 R 11 R 12 Each of the following is an independent hydrogen atom, substituent atom, or substituent on a double bond (-C=C-), and in the same molecule, R8, R9, R... 10 R 11 R 12 They can be the same or different; R8, R9, R 10 R 11 R 12 Each atom is independently selected from: hydrogen atom, fluorine atom, and methyl group; among E3, R8 is preferably methyl group;

[0271] Among them, R 24 As the end group attached to the disulfide bond, it is selected from: C 1-20 Alkyl, aryl, and hybrid phenyl groups;

[0272] Among them, R 27 The substituent attached to the azo group is selected from: phenyl, substituted phenyl, or hybridized phenyl;

[0273] Among them, R 30 It is a hydrocarbon group, selected from: C 1-20 Alkyl, benzyl, and benzene ring hydrogen atoms are C 1-20 Benzyl groups substituted with hydrocarbon groups;

[0274] Among them, M 19 M 20 M 21 Each atom can be an oxygen atom or a sulfur atom, and within the same molecule, they can be the same or different from each other;

[0275] Wherein, X6 is the terminal group attached to the oxygen atom in the ester group, selected from a hydroxyl protecting group or the group LG4; LG4 is selected from C 1-20 Alkyl, aryl, aralkyl, C 1-20 heteroalkyl, heteroaryl, heteroarylalkyl, C 1-20 Alkyl carbonyl, aryl carbonyl, aralkyl carbonyl, C 1-20 heteroalkyl carbonyl, heteroaryl carbonyl, heteroarylalkyl carbonyl, C 1-20Alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, C 1-20 Alkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbonyl, C 1-20 Alkylaminocarbonyl, arylaminocarbonyl, aralkylaminocarbonyl, C 1-20 Heteroalkyloxycarbonyl, heteroaryloxycarbonyl, heteroarylalkyloxycarbonyl, C 1-20 heteroalkylthiocarbonyl, heteroarylthiocarbonyl, heteroarylalkylthiocarbonyl, C 1-20 Heteroalkylaminocarbonyl, heteroarylaminocarbonyl, heteroarylalkylaminocarbonyl, C 1-20 Alkyl thiocarbonyl, aryl thiocarbonyl, aralkyl thiocarbonyl, C 1-20 Heteroalkyl thiocarbonyl, heteroaryl thiocarbonyl, heteroarylalkyl thiocarbonyl, C 1-20 Alkoxythiocarbonyl, aryloxythiocarbonyl, aralkyloxythiocarbonyl, C 1-20 Alkylthiocarbonyl, arylthiocarbonyl, aralkylthiocarbonyl, C 1-20 Alkylaminothiocarbonyl, arylaminothiocarbonyl, aralkylaminothiocarbonyl, C 1-20 Heteroalkyloxythiocarbonyl, heteroaryloxythiocarbonyl, heteroarylalkyloxythiocarbonyl, C 1-20 heteroalkylthiocarbonyl, heteroarylthiocarbonyl, heteroarylalkylthiocarbonyl, C 1-20 The substituent is any one of the following groups: heteroalkylaminothiocarbonyl, heteroarylaminothiocarbonyl, and heteroarylalkylaminothiocarbonyl; wherein the substituent atom or substituent is a fluorine atom, an alkoxy group, or a nitro group.

[0276] Among them, X 11 The terminal group is selected from C10 to connect a carbonyl or thiocarbonyl group. 1-20 alkyl;

[0277] Among them, X 12 The end group is selected from C to connect a carbonate group or a thiocarbonate group. 1-20 hydrocarbon group;

[0278] Among them, X 13 The terminal group connecting the sulfur group is selected from: mercapto protecting group, group LG2;

[0279] LG2 is selected from methyl, ethyl, n-propyl, isopropyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, allyl, triphenylmethyl, phenyl, benzyl, methylbenzyl, nitrobenzyl, tert-butylthio, benzylthio, 2-pyridylthio, acetyl, benzoyl, methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, phenoxycarbonyl, benzyloxycarbonyl, methylthiocarbonyl, ethylthiocarbonyl, tert-butyl... Butylthiocarbonyl, phenylthiocarbonyl, benzylthiocarbonyl, 2-pyridylcarbonyl, methylaminocarbonyl, ethylaminocarbonyl, tert-butylaminocarbonyl, benzylaminocarbonyl, ethylthiocarbonyl, phenylmethylthiocarbonyl, methoxythiocarbonyl, ethoxythiocarbonyl, tert-butyloxythiocarbonyl, phenoxythiocarbonyl, benzyloxythiocarbonyl, methylthiocarbonyl, ethylthiocarbonyl, tert-butylthiocarbonyl, phenylthiocarbonyl, benzylthiocarbonyl, methylaminothiocarbonyl, ethylaminothiocarbonyl, tert-butylaminothiocarbonyl, benzylaminothiocarbonyl, C 1-10 The substituted form of any one of the following groups: halogenated hydrocarbon group, trifluoroacetyl group, and nitrophenyl group; wherein the substituted atom or substituent is a fluorine atom, an alkoxy group, or a nitro group;

[0280] Wherein, Q is an atom or substituent that contributes to the induction and conjugation effect of unsaturated bond electrons; when Q is on a ring, there are one or more; when there are multiple, they are of the same structure or a combination of two or more different structures; when it is a substituent, Q has a straight chain structure, a branched chain structure with side groups, or a ring structure.

[0281] Wherein, Q3 is a H atom or a group that contributes to the induction or conjugation effect of unsaturated bond electrons, selected from any one atom or group, or a substituted form of any one of the following: hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom, methyl, ethyl, n-propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, vinyl, propenyl, allyl, propynyl, propynyl, cyclopropyl, cyclopropenyl, phenyl, benzyl, butylphenyl, p-methylphenyl, p-nitrophenyl, o-nitrophenyl, p-methoxyphenyl, azaphenyl, methoxy, ethoxy, phenoxy, benzyloxy, methylthio, ethylthio, phenylthio, benzylthio, trifluoromethyl, 2,2,2-trifluoroethyl.

[0282] Wherein, Q5 is a H atom, methyl, ethyl, or propyl; when Q5 is located on a ring, there is one or more; when there is more than one, it is the same structure, or a combination of two or more different structures;

[0283] In this molecule, Q6 is a hydrogen atom or a methyl group; Q7 is a hydrogen atom, a methyl group, a phenyl group, or a substituted phenyl group; Q6 and Q7 can be the same or different in the same molecule.

[0284] Wherein, Q8 is a substituent atom or substituent on the imidazole group, selected from any one of H atom, methyl, ethyl, propyl, butyl, and phenyl; when there is one or more Q8, it is the same structure, or a combination of two or more different structures;

[0285] Among them, Q 11 The substituent on the nitrogen atom of tetrazolium is selected from any one of phenyl, substituted phenyl, and aziridine;

[0286] Wherein, PG2 is a thiol protecting group, and the protected thiol group is represented as SPG2, preferably any one of thioether, dithioether, silyl thioether, and thioester;

[0287] Among them, PG3 is an alkynyl protecting group, preferably silicon-based;

[0288] Wherein, PG4 is a hydroxyl protecting group, and the protected hydroxyl group is represented as OPG4, preferably any one of ether, silicone ether, ester, carbonate, or sulfonate;

[0289] Wherein, PG5 is an amino protecting group, and the protected amino group is represented as NPG5, preferably any one of urethane, amide, imide, N-alkylamine, N-arylamine, imine, enamine, imidazole, pyrrole, and indole;

[0290] Wherein, PG6 is a dihydroxy protecting group, and PG6 forms a five-membered or six-membered ring acetal structure with two oxygen atoms; PG6 is methylene or a substituted methylene group; wherein, the substituent of PG6 is a hydrocarbon substituent or a substituent containing heteroatoms, selected from any one of: methylene, 1-methylmethylene, 1,1-dimethylmethylene, 1,1-cyclopentylene, 1,1-cyclohexylene, 1-phenylmethylene, 3,4-dimethylphenylmethylene;

[0291] PG8 is a protecting group for orthocarbonic acid or orthosilicic acid.

[0292] 1.1.5.3. Specific Examples of R

[0293] In one specific embodiment of the present invention, R preferably contains any one of the following: hydrogen atom, alkyl group, alkoxy group, alcohol hydroxyl group, protected alcohol hydroxyl group, thiol hydroxyl group, protected thiol hydroxyl group, carboxyl group, protected carboxyl group, amino group, protected amino group, aldehyde group, protected aldehyde group, ester group, carbonate group, carbamate group, succinimide group, maleimide group, protected maleimide group, dimethylamino group, alkenyl group, acrylate group, azide group, alkynyl group, folic acid group, rhodamine group, biotinyl group, monosaccharide group, and polysaccharide group; more preferably, R contains H, -CH3, -CH2CH3, or -(CH2). t OH, -(CH2) t SH, -OCH3, -OCH2CH3, -(CH2) t NH2、-(CH2) t C(=O)OH, -C(=O)(CH2) t C(=O)OH, -C(=O)CH3, -(CH2) t N3, -C(=O)CH2CH3, -C(=O)OCH3, -OC(=O)OCH3, -C(=O)OCH2CH3, -OC(=O)OCH2CH3, -(CH2) t N(CH3)2、-(CH2) t N(CH2CH3)2、-(CH2) t CHO Any one of the following, where t is an integer from 0 to 12.

[0294] 1.1.6.A

[0295] In this invention, A is -(CR) a R b ) s O- or -O(CR) a R b ) s - where s is 2, 3, or 4, R a and R b Each is independently a hydrogen atom or a carbon atom. 1-12 alkyl.

[0296] In one embodiment of the present invention, R a R b Both are hydrogen atoms, and s is 2, specifically corresponding to A as -CH2CH2O- or -OCH2CH2-.

[0297] In this invention, the number average molecular weight of the polyethylene glycol chain of the polyethylene glycol-modified lipid is 900, 1000, 1500, 2000, 2500, 3000, 3350, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 11000.

[0298] In one embodiment of the present invention, the polyethylene glycol chain of the polyethylene glycol-modified lipid is polydisperse or monodisperse.

[0299] In one embodiment of the present invention, the polyethylene glycol chain is preferably polydisperse and the number-average degree of polymerization n1 is preferably an integer of about 20-100, more preferably an integer of about 20-60, more preferably an integer of about 40-60; more preferably any one of 44, 45, 46, 48, 50, 52, 54, 56, 58, and 60.

[0300] In one embodiment of the present invention, the polyethylene glycol chain is preferably monodisperse and the number of its EO units is preferably 20-70; more preferably 20-60; and even more preferably any one of 20, 22, 24, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 56, 58, and 60.

[0301] 1.1.7. Examples of Specific Structural Formulas In a specific embodiment of the present invention, the structure of the polyethylene glycol-modified lipid is preferably any one of the following structural formulas:

[0302]

[0303]

[0304]

[0305] 1.1.8. Examples of specific structures of polyethylene glycol-modified lipids

[0306] Some specific embodiments of the present invention ultimately yield polyethylene glycol-modified lipids with the structures shown below, including but not limited to any of the following structures:

[0307]

[0308]

[0309]

[0310]

[0311] Where n1 is an integer between 20 and 250.

[0312] 2. Preparation of polyethylene glycol-modified lipids

[0313] In this invention, the preparation of any of the aforementioned polyethylene glycol-modified lipids can be carried out by methods including but not limited to the following:

[0314] 2.1. Method 1:

[0315] Step 1: Activate the carboxyl terminus of acid A-1 or A-1' containing a naked carboxyl group using a carboxyl activator to obtain carboxyl terminus-activated ester A-2 or A-2'. Here, B3' and B4' are each independently a linking bond or an alkylene group with one less methylene group than B3 and B4; R1' and R2' are each independently R1 and R2 or an aliphatic hydrocarbon group with one less methylene group than R1 and R2; R7 is a carboxyl activating group. When either B3' or L7 is not a linking bond, R1' is R1; when both B3' and L7 are linking bonds, R1' is an aliphatic hydrocarbon group with one less methylene group than R1; when either B4' or L8 is not a linking bond, R2' is R2; when both B4' and L8 are linking bonds, R2' is an aliphatic hydrocarbon group with one less methylene group than R2.

[0316] Step 2: The carboxyl-terminated activated ester A-2 or A-2' is condensed with a primary amine derivative A-3 or A-3' containing a nitrogen source end group to obtain amide intermediate A-4 or A-4';

[0317] Step 3: Reduce amide intermediate A-4 or A-4' to secondary amine intermediate A-5 or A-5' using a reducing agent;

[0318] Step 4: The secondary amine intermediate is coupled with the bi-functionalized PEG derivative biLPEG molecule A-6 to obtain the polyethylene glycol-modified lipid derivative A-7 or A-7'; wherein biLPEG is monodisperse or polydisperse; wherein the functional groups at both ends of biLPEG can be the same or different; wherein the R' end of biLPEG contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 The group; wherein, F1 contains an active functional group that can react with the secondary amine intermediate A-5 or A-5' to generate a branched central nitrogen atom and a divalent linker L3;

[0319] When R' equals R, the resulting structure A-7 or A-7' corresponds to the structure shown in general formula (2);

[0320] When R' is not equal to R, A-7 or A-7' is modified at the end to obtain A-8 or A-8' corresponding to the structure shown in general formula (2); the end micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0321] Step 1

[0322]

[0323] Step Two

[0324]

[0325] Step 3

[0326]

[0327] Step Four

[0328]

[0329] L3, L7, L8, B3, B4, R, R1, R2 and n1 are consistent with those described in general formula (2), and will not be repeated here.

[0330] In one specific embodiment of the present invention, in method 1, A-1 is preferably R1'-COOH or R2'-COOH, A-3 is preferably R1-NH2 or R2-NH2, the carboxyl activator is preferably any one of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N-dicyclohexylcarbodiimide (DCC), and the polyethylene glycol-modified lipid is preferably prepared using the following route:

[0331]

[0332] 2.2. Method 2:

[0333] Step 1: Coupling the biLPEG molecule B-1, a bifunctionalized PEG derivative, with a primary amine derivative B-2 or B-2' containing a nitrogen-source end group, yields a polyethylene glycol-modified secondary amine derivative B-3 or B-3'. The biLPEG is monodisperse or polydisperse, and the functional groups at both ends of the biLPEG can be the same or different. The R' end of the biLPEG contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R.01 The group; wherein, F1 contains an active functional group that can react with the amino group of primary amine B-2 or B-2' to generate L3 secondary amine derivatives B-3 or B-3' containing divalent linkers;

[0334] Step 2: The secondary amine derivative B-3 or B-3' reacts with F containing a reactive group. N Compound B-4 or B-4' undergoes an alkylation reaction to yield polyethylene glycol-modified lipid derivatives B-5 or B-5'; The F N The reactive group is capable of reacting with amino or secondary amine groups, preferably -OMs, -OTs, -CHO, -F, -Cl, or -Br;

[0335] When R' equals R, the resulting structure B-5 or B-5' corresponds to the structure shown in general formula (2);

[0336] When R' is not equal to R, B-5 ​​or B-5' is end-micro-modified to obtain B-6 or B-6' corresponding to the structure shown in general formula (2); the end-micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0337] Step 1

[0338]

[0339] Step Two

[0340]

[0341] L3, L7, L8, B3, B4, R, R1, R2 and n1 are consistent with those described in general formula (2), and will not be repeated here.

[0342] In one specific embodiment of the present invention, B-3 or B-3' in the aforementioned method 2 is R1-NH2 or R2-NH2, B-4 or B-4' is R2-L8-B4-Br or R1-L7-B3-Br, and F1 contains an -OMS group.

[0343] 2.3. Method 3:

[0344] Step 1: React small molecule C-1 with small molecule C-2 to generate a compound containing a divalent linker L7 and a reactive group F at one end. N A small molecular intermediate C-3 with an aliphatic hydrocarbon group R1 at one end; wherein F3 and F4 are each independently reactive groups that can react to form a divalent linker L7; wherein C-2 is a group containing heterofunctional pairs of F3 and F4. N The F NThe reactive group is capable of reacting with amino or secondary amine groups, preferably -OMs, -OTs, -CHO, -F, -Cl, or -Br;

[0345] Step 2: Two molecules of the small molecule intermediate C-3 undergo an alkylation reaction with a polyethylene glycol primary amine derivative C-4 containing a nitrogen source end group to obtain a polyethylene glycol-modified lipid C-5, wherein the R' end contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 Groups;

[0346] When R' equals R, the resulting structure C-5 corresponds to the structure shown in general formula (2);

[0347] When R' is not equal to R, C-5 is end-modified to obtain the structure of C-6 corresponding to the general formula (2); the end-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; wherein R1 and R2 are the same, B3 and B4 are the same, and L7 and L8 are the same.

[0348] L3, L7, L8, B3, B4, R, R1, R2 and n1 are consistent with those described in general formula (2), and will not be repeated here.

[0349] Step 1

[0350]

[0351] Step Two

[0352]

[0353] 2.4. Method 4:

[0354] Step 1: React small molecule D-1 with small molecule D-2 to generate a small molecule intermediate D-3 containing a divalent linker L7, a hydroxyl group at one end, and an aliphatic hydrocarbon group R1 at the other end; wherein F3 and F4 are each independently reactive groups that can react to generate the divalent linker L7; wherein D-2 contains heterofunctional groups for F3 and hydroxyl groups;

[0355] Step 2: Oxidize the hydroxyl group of small molecule intermediate D-3 to an aldehyde group to obtain small molecule intermediate D-4 containing an aldehyde group, wherein B3' is an alkylene group with one less methylene group than B3;

[0356] Step 3: Two molecules of the small molecule intermediate D-4 containing aldehyde groups are added to D-5, a polyethylene glycol primary amine derivative containing a nitrogen source end group, to obtain polyethylene glycolated lipid D-6, wherein the R' end contains a reactive group R. 01 or contains R 01 The micro-change forms; the micro-change forms refer to those that, through any one of the following chemical processes—deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or alteration of the leaving group—can be transformed into R. 01 Groups;

[0357] When R' equals R, the resulting structure D-6 corresponds to the structure shown in general formula (2);

[0358] When R' is not equal to R, D-6 is end-micro-modified to obtain D-7 corresponding to the structure shown in general formula (2); the end-micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group, wherein R1 and R2 are the same, B3 and B4 are the same, and L7 and L8 are the same.

[0359] Step 1

[0360]

[0361] Step Two

[0362]

[0363] Step 3

[0364]

[0365] L3, L7, L8, B3, B4, R, R1, R2 and n1 are consistent with those described in general formula (2), and will not be repeated here.

[0366] 2.5. Description of relevant raw materials and / or steps in the preparation process

[0367] 2.5.1. Carboxyl activators, condensing agents, oxidizing agents, reducing agents

[0368] In this invention, "carboxyl activation" refers to the activation treatment of carboxyl groups with a carboxyl activator. Activated carboxyl groups can promote better condensation reactions, such as inhibiting the generation of racemic impurities in the condensation reaction and accelerating the reaction rate. "Carboxyl activating group" refers to a residue of the carboxyl activator. The carboxyl activator is one or more of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N-dicyclohexylcarbodiimide (DCC), preferably a combination of NHS / EDCI, NHS / DCC, and HONb / DCC, with the most preferred combination being NHS / EDCI.

[0369] In this invention, the condensing agent used in the reaction is not limited, but N,N'-dicyclohexylcarbonyldiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) are preferred, with DCC being the most preferred. The amount of condensing agent used is generally 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 to 10 times. A suitable catalyst (such as 4-dimethylaminopyridine) can be added to this reaction.

[0370] In this invention, the oxidizing agent used in the reaction is not particularly limited, as long as it is a compound or combination of compounds that can increase the oxidation state of the substrate. Preferred oxidizing agents include phenyl iodide di(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluoride, sodium hypochlorite, cobalt acetate, cobalt acetate, manganese acetate, palladium acetate, copper acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzoylbenzene, 2-iodobenzoic acid, dimethyldioxane, dimethyl sulfoxide-oxalyl chloride, dimethyl sulfoxide-acetic anhydride, DDQ, ruthenium dichlorotris(triphenylphosphine)diruthenium, manganese dioxide, diacetoxyiodobenzoylbenzene, periodic acid, sodium periodate, sodium periodate-osmium tetroxide, potassium permanganate, sodium perborate, peroxybenzoic acid, and benzoyl peroxide. The oxidant comprises one or a combination of nickel peroxide, hydrogen peroxide, cumene hydrogen peroxide, tert-butanol peroxide, peracetic acid, m-chloroperoxybenzoic acid, N-chlorosuccinimide, pyridine chlorochromate, palladium chloride-copper chloride, urea hydrogen peroxide complex, triphenylmethyltetrafluoroborate, tributyltin oxide, cobalt trifluoride, vanadium trifluorooxyfluoride, chromium trioxide, manganese triacetate, TEMPO, cerium ammonium nitrate, bromine, N-pyridine oxide, silver oxide, O-ethylperoxycarbonate, manganese acetylacetone, vanadium acetylacetone, aluminum isopropoxide, potassium persulfate, and dichloroiodobenzene, more preferably one or a combination of oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, and potassium persulfate, wherein the amount of oxidant is 1 to 50 times, preferably 1 to 20 times, and more preferably 5 to 10 times, the molar equivalent of hydroxyl groups in the intermediate compound.

[0371] In this invention, the reducing agent used in the reaction is not particularly limited, as long as it can reduce the Schiff base formed by ammonia and aldehyde or ketone to an amino group; preferably, it is one or a combination of sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopineborneolborane, lithium borohydride, zinc borohydride, borane-pyridine, borane-methyl sulfide, borane-tetrahydrofuran, etc.; more preferably, it is sodium cyanoborohydride, and the equivalent of the reducing agent is 1 to 50 times the molar equivalent of the amino group to be modified, preferably 1 to 20 times, more preferably 5 to 10 times.

[0372] In this invention, the reaction temperature is 0 to 200°C, preferably 0 to 100°C, more preferably 0 to 25°C, and the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours. The obtained product can be purified by methods such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction.

[0373] In this invention, the solvent for the reaction can be a solvent-free solvent or an aprotic solvent. The aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or dimethylformamide.

[0374] In this invention, the base used in the reaction is generally an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or diisopropylethylamine), preferably triethylamine or pyridine. The amount of base used is 1 to 50 times the molar equivalent of the carboxylic acid, preferably 1 to 10 times, and more preferably 2 to 3 times.

[0375] 2.5.2. The "protection" and "deprotection" of relevant functional groups involved in the reaction process.

[0376] In this invention, the reaction process also involves the "protection" and "deprotection" of relevant functional groups. To prevent the functional group from affecting the reaction, it is typically protected. Furthermore, when there are two or more functional groups, only the target functional group is selectively reacted, thus protecting the other functional groups. The protecting group not only stably protects the target functional group but also needs to be easily removed as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting groups bonded to the specified functional group under appropriate conditions.

[0377] In this invention, a "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or a deprotection reaction. The carboxyl protecting group is preferably alkyl (e.g., methyl, ethyl, tert-butyl) or aralkyl (e.g., benzyl), more preferably tert-butyl (tBu), methyl (Me), or ethyl (Et). In this invention, a "protected carboxyl group" refers to the group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, or benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under acid or base catalysis, and occasionally by thermal decomposition. For example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0378] In this invention, "amino protecting group" includes all groups that can be used as protecting groups for ordinary amino groups, such as aryl C. 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6Alkyl sulfonyl, aryl sulfonyl, or silyl groups are preferred. The amino protecting group is preferably Boc tert-butoxycarbonyl, Moz p-methoxybenzyloxycarbonyl, or Fmoc 9-fluorene-methoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably combinations of TFA and H2O, LiOH and MeOH, or LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; TFA is preferred. The deprotecting agent used in the Fmoc protecting group removal reaction is a solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine.

[0379] In this invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, an alcohol hydroxyl group, a phenolic hydroxyl group, etc. The amino group protected by the amino protecting group is not particularly limited, and can be, for example, derived from primary amines, secondary amines, hydrazines, amides, etc. The amino group in this invention is not particularly limited, and includes, but is not limited to, primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium ions.

[0380] In this invention, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. There are no particular limitations on the type of hydroxyl protecting group; taking benzyl, silyl ether, acetal, and tert-butyl groups for protecting the terminal hydroxyl group as examples, the corresponding deprotection methods include:

[0381] A: Deprotection of benzyl groups

[0382] Benzyl deprotection can be achieved through hydrogenation using a hydrogen reducing agent and a hydrogen donor. For this reaction to proceed smoothly, the water content in the system should be less than 1%. When the water content exceeds 1%, polyethylene glycol chains will break, producing low-molecular-weight polyethylene glycol with hydroxyl groups. This low-molecular-weight polyethylene glycol can participate in subsequent polymerization reactions or functional group modifications, introducing impurities into the target product. Furthermore, it can react with biologically related substances, altering the properties of the formulation.

[0383] The hydrogenation reduction catalyst is not limited, but palladium and nickel are preferred. The support is not limited, but alumina or carbon is preferred, and carbon is more preferred. The amount of palladium used is 1 to 100 wt% of the protected hydroxyl compound, preferably 1 to 20 wt%. When the amount of palladium is less than 1 wt%, the deprotection rate and conversion decrease, and the unprotected portion cannot undergo subsequent polymerization or functionalization, resulting in a low functional group content in the final product. However, when the amount of palladium is greater than 100 wt%, it easily leads to the cleavage of polyethylene glycol chains.

[0384] There are no particular restrictions on the reaction solvent, as long as both the raw materials and products can be used as solvents. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is even more preferred. There are no particular restrictions on the hydrogen donor, but hydrogen, cyclohexene, 2-propanol, and ammonium formate are preferred. The reaction temperature is preferably 25 to 40°C. When the temperature is above 40°C, polyethylene glycol chain scission is more likely to occur. There are no particular restrictions on the reaction time, but the reaction time is negatively correlated with the amount of catalyst used. It is preferably 1 to 5 hours. When the reaction time is less than 1 hour, the conversion rate is low; when the reaction time is greater than 5 hours, polyethylene glycol chain scission is more likely to occur.

[0385] B: Deprotection of acetals and ketals

[0386] The preferred acetals or ketals for this type of hydroxyl protection are ethyl vinyl ethers, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, etc. Deprotection of these acetals and ketals is achieved under acidic conditions, with the solution pH preferably between 0 and 4. When the pH is greater than 4, the acidity is too weak to completely remove the protecting group; when the pH is less than 0, the acidity is too strong, and polyethylene glycol chain scission easily occurs. There are no particular limitations on the acid, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. There are no particular limitations on the reaction solvent, as long as it can dissolve the reactants and products; water is preferred. The preferred reaction temperature is between 0 and 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed; when the temperature is above 30°C, under acidic conditions, polyethylene glycol chain scission easily occurs.

[0387] C: Deprotection of silyl ether

[0388] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, and tert-butyldiphenylsilyl ether. Deprotection of these silyl ethers is achieved using fluoride-containing compounds, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, and potassium fluoride, more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of fluoride-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the amount of fluoride is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will occur; if the amount of deprotecting reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, excess reagent or compound will cause purification problems, may be introduced into subsequent steps, and thus cause side reactions. There are no particular restrictions on the reaction solvent, as long as it can dissolve the reactants and products. Aprotic solvents are preferred, more preferably tetrahydrofuran and dichloromethane. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed.

[0389] D: Deprotection of tert-butyl

[0390] The deprotection of tert-butyl groups is carried out under acidic conditions, preferably with a solution pH of 0 to 4. When the pH is greater than 4, the acidity is too weak to completely remove the protecting group; when the pH is less than 0, the acidity is too strong, and polyethylene glycol chain scission easily occurs. There are no particular limitations on the acid, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. There are no particular limitations on the reaction solvent, as long as it can dissolve the reactants and products; water is preferred. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed; when the temperature is above 30°C, under acidic conditions, polyethylene glycol chain scission easily occurs.

[0391] In the linear functionalization method described below for the terminal hydroxyl groups of polyethylene glycol, q = 0, q1 = 1, and Z1 is 1,2-methylene. When q is not 0, such as with PEG and R... 01 When there are linking groups such as amino acids or succinyl groups between them, existing technologies in this field that can generate Z2 or Z1 (including but not limited to alkylation, condensation, click reaction, etc.) can be used, and the preparation can be carried out with reference to the linear functionalization described below.

[0392] 2.5.3. Alkylation reaction

[0393] The alkylation reaction of the present invention is preferably based on the alkylation of hydroxyl, mercapto, or amino groups, corresponding sequentially to the formation of ether bonds, thioether bonds, secondary amino groups, or tertiary amino groups. Examples are as follows:

[0394] 2.5.3.1. Alkylation of substrate alcohols with sulfonates and halogenated derivatives

[0395] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of the substrate alcohol with a sulfonate derivative and a halide. The molar equivalents of the sulfonate and halide are 1 to 50 times that of the substrate alcohol, preferably 1 to 5 times. When the molar equivalents of the sulfonate and halide are less than one molar equivalent of the substrate alcohol, the substitution reaction is incomplete, making purification difficult. Conversely, when the molar equivalents of the sulfonate and halide are greater than 50 times that of the substrate alcohol, excess reagents complicate purification, potentially contaminating subsequent steps and increasing the likelihood of side reactions, thus increasing the difficulty of purification.

[0396] The resulting product is a mixture of an ether intermediate and excess sulfonate and halogenated derivatives, which can be purified by anion exchange resin, permeation, ultrafiltration, etc. The anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin. Preferably, it is an ion exchange resin with tertiary amines or quaternary ammonium salts as the backbone, such as dextran, agarose, polypropylene ester, polystyrene, or polystyrene. The solvents for permeation and ultrafiltration are not limited; generally, water or organic solvents are acceptable. The organic solvent is not particularly limited, as long as the product can dissolve in it. Preferred solvents include dichloromethane and trichloromethane.

[0397] The reaction solvent is not limited, but aprotic solvents are preferred, such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0398] The base includes organic bases (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or diisopropylethylamine) or inorganic bases (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate, or potassium hydroxide), preferably organic bases, and more preferably triethylamine or pyridine. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonate or halide, preferably 1 to 10 times, and more preferably 3 to 5 times.

[0399] 2.5.3.2. Alkylation of substrate amines with sulfonates and halogenated derivatives

[0400] A. Alkylation of the substrate amine with sulfonates and halogens

[0401] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of the substrate amine with a sulfonate derivative and a halide. The molar equivalent of the sulfonate and halide is 1 to 50 times that of the substrate amine, preferably 1 to 5 times. When the molar equivalent of the sulfonate and halide is less than one molar equivalent of the substrate amine, the substitution reaction is incomplete, making purification difficult. Conversely, when the molar equivalent of the sulfonate and halide is greater than 50 times that of the substrate amine, excess reagents complicate purification, potentially contaminating subsequent steps and increasing the likelihood of side reactions, thus further complicating purification.

[0402] The resulting product is a mixture of amine intermediates and excess sulfonates and halogenated derivatives, which can be purified by anion exchange resins, permeation, ultrafiltration, etc. The anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin. Preferably, it is an ion exchange resin with tertiary amines or quaternary ammonium salts as the backbone, such as dextran, agarose, polypropylene ester, polystyrene, or polystyrene. The solvents for permeation and ultrafiltration are not limited; generally, water or organic solvents are acceptable. The organic solvent is not particularly limited, as long as the product can dissolve in it. Preferred solvents include dichloromethane and trichloromethane.

[0403] The reaction solvent is not limited, but aprotic solvents are preferred, such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0404] The base includes organic bases (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or diisopropylethylamine) or inorganic bases (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate, or potassium hydroxide), preferably organic bases, and more preferably triethylamine or pyridine. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonate or halide, preferably 1 to 10 times, and more preferably 3 to 5 times.

[0405] 2.5.3.3. Alkylation reaction of substrate amines with aldehyde derivatives

[0406] The reaction of a substrate amine with an aldehyde derivative yields an imine intermediate, which is then further purified by a reducing agent to obtain the intermediate. The molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, excess reagent complicates purification, potentially contaminating subsequent steps and increasing purification difficulty. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete, further complicating purification. The product after the reaction can be purified to obtain the intermediate through cation exchange resin, permeation, ultrafiltration, etc. The cation exchange resin is not particularly limited, as long as it can exchange with quaternary ammonium cations to achieve separation. The solvents for permeation and ultrafiltration are not limited; generally, water or organic solvents are acceptable. The organic solvent is not particularly limited, as long as the product can dissolve in it; dichloromethane and trichloromethane are preferred.

[0407] The reaction solvent is not limited, but organic solvents are preferred, such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide; water and methanol are more preferred.

[0408] There are no particular limitations on the reducing agent, as long as it can reduce imine to amine. Sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, etc. are preferred, with sodium cyanoborohydride being more preferred. Generally, the amount of reducing agent used is 0.5 to 50 times the amount of the aldehyde derivative, more preferably 1 to 10 times.

[0409] 2.5.4. Linear dual-terminal functionalized PEG derivative biLPEG

[0410] The biLPEG has a functional group at one end that can react with an amino group (primary or secondary amino group), and forms a divalent linker L3 through coupling.

[0411] The other end of the biLPEG may be the same as or different from the target structure R, and its functional group may be the same as or different from the target functional group. The functional group carried at this end is selected from, but is not limited to, any one of the functional groups in AJ above, including precursors of any reactive group, variations of the precursor, substituted forms, protected forms, and deprotected forms.

[0412] The functional groups at both ends of the biLPEG can be the same or different, preferably a linear heterofunctionalized PEG derivative (biheteroLPEG) with two different functional groups at both ends. The heterofunctional group pairs that can coexist in this invention include, but are not limited to: hydroxyl groups with protected hydroxyl groups, hydroxyl groups or protected hydroxyl groups with non-hydroxyl reactive groups of type A to H (such as amino, protected amino, amine salts, aldehydes, active esters, maleimides, carboxyl groups, protected carboxyl groups, alkynyl groups, protected alkynyl groups, azides, alkenyl groups, acrylate groups, methacrylate groups, epoxy groups, isocyanate groups, etc.), hydroxyl groups or protected hydroxyl groups with functional groups of type I to J or their derivatives (such as targeting groups, photosensitizing groups, etc.), active ester groups with maleimides, active ester groups with aldehydes, active ester groups with azides, active ester groups with alkynyl groups, or... Protected alkynyl groups, active ester groups and acrylate groups, active ester groups and methacrylate groups, active ester groups and acrylate groups, maleimide groups and azide groups, maleimide groups and alkynyl groups or protected alkynyl groups, maleimide groups and acrylate groups, maleimide groups and methacrylate groups, maleimide groups and acrylate groups, maleimide groups and carboxyl groups, maleimide groups and amino groups or protected amino or amine salts, maleimide groups and isocyanate groups, maleimide groups and protected thiol groups, aldehyde groups and azide groups, aldehyde groups and acrylate groups, aldehyde groups and methacrylate groups, aldehyde groups and acrylate groups, aldehyde groups and epoxy groups, aldehyde groups and carboxyl groups, aldehyde groups and alkynyl groups or protected alkynyl groups, Azido group with a mercapto group or a protected mercapto group; azido group with an amino group or a protected amino or amine salt; azido group with an acrylate group; azido group with a methacrylate group; azido group with an acrylate group; azido group with a carboxyl group; acrylate group with an amino group or a protected amino or amine salt; acrylate group with an isocyanate group; acrylate group with an epoxy group; acrylate group with a methacrylate group; acrylate group with a carboxyl group; methacrylate group with a carboxyl group; methacrylate group with an amino group or a protected amino or amine salt; methacrylate group with an isocyanate group; methacrylate group with an epoxy group; alkynyl group or a protected alkynyl group with an amino group or a protected amino or amine salt; alkynyl group or a protected alkynyl group with isocyanate. Ester group, alkynyl group or protected alkynyl group with acrylate group, alkynyl group or protected alkynyl group with methacrylate group, alkynyl group or protected alkynyl group with acrylate group, alkynyl group or protected alkynyl group with epoxy group, alkynyl group or protected alkynyl group with carboxyl group, protected alkynyl group with azide group, acrylate group with isocyanate group, acrylate group with acrylate group, acrylate group with epoxy group, acrylate group with carboxyl group, carboxyl group with mercapto group or protected mercapto group, carboxyl group with amino group or protected amino or amine salt, carboxyl group with isocyanate group, carboxyl group with epoxy group, amino group or protected amino or amine salt with mercapto group or protected mercapto group, targeting group with non-hydroxyl reactive group, photosensitive group with non-hydroxyl reactive group, etc.The active esters include, but are not limited to, any of the succinimide active esters (such as succinimide carbonate group), p-nitrobenzene active esters, o-nitrobenzene active esters, benzotriazole active esters, 1,3,5-trichlorobenzene active esters, 1,3,5-trifluorobenzene active esters, pentafluorobenzene active esters, imidazole active esters, 2-thiooxothiazolidin-3-carboxylic acid esters, 2-thiononepyrrolidine-1-carboxylic acid esters, etc.; the amino groups include primary and secondary amino groups. The amine salt is preferably the hydrochloride salt form of the amino group, such as NH₂HCl.

[0413] The biLPEG can be polydisperse or monodisperse.

[0414] When biLPEG is polydisperse, its polydispersity index (PDI) is not particularly limited, but it is preferred to have a PDI of less than 1.15, more preferably less than 1.10, more preferably less than 1.08, and even more preferably less than 1.05. The lower the PDI, the more uniform the molecular weight, and the narrower the molecular weight distribution of the resulting product. When used to modify molecules such as drugs, the quality of the modified product is higher, and it can better meet the actual needs.

[0415] When biLPEG is monodisperse, PDI = 1, resulting in polyethylene glycol-modified lipids with defined molecular structures and molecular weights. Products prepared using monodisperse raw materials have relatively uniform molecular weights, but due to limitations in the preparation methods, the molecular weight is often restricted, and the steps are lengthy. The advantage of using polydisperse raw materials is the simplicity of the steps and the wide range of molecular weight adjustment.

[0416] The preparation method for monodisperse polyethylene glycol chains can adopt well-known technologies in this field, including but not limited to the literature "J. Org. Chem. 2006, 71, 9884-9886" and its cited literature, literature "Angew. Chem. 2009, 121, 1274-1278" and its cited literature, literature "Expert Rev. Mol. Diagn. 2013, 13(4), 315-319" and its cited literature, literature "Angew. Chem. Int. Ed. 2014, 53, 6411-6413" and its cited literature, literature "Bioorganic & Medicinal Chemistry Letters, 2015, 25: 38-42" and its cited literature, literature "Angew. Chem. Int. Ed., 2015, 54: 3763-3767" and its application literature, etc.

[0417] 2.5.5. Linear functionalization of polyethylene glycol chain ends

[0418] There are no particular limitations on the methods for linear functionalization of polyethylene glycol (PEG) chains, depending on the type of the final functional group or its protected form. These methods can be based on linear functionalization of PEG chain-terminal hydroxyl groups, conversion of reactive groups to the target functional group or its protected form, or a combination of both.

[0419] A method for preparing linear functionalized hydroxyl groups at the ends of polyethylene glycol chains involves starting from the terminal hydroxyl groups of the polyethylene glycol chain and obtaining functional groups of type A to J or their protected forms through functionalization. Specific preparation methods are described in paragraphs

[0960] to

[1205] of document CN104530417A. The general reaction formula is as follows:

[0420]

[0421] The structure of -PEG-OH is -(CH2CH2O). n CH2CH2OH, n is n1-1; q, Z2, q1, Z1, R 01 The definition is consistent with the one above.

[0422] The transformation of a reactive group into a target functional group or its protected form can be achieved through any of the following methods:

[0423] Method 1: Direct modification, based on the direct modification of reactive groups, to obtain the target functional group or its protected form. Examples include the transformation of carboxyl groups into acyl halides, acyl hydrazides, esters, thioesters, and dithioesters; and the transformation of hydroxyl, mercapto, alkynyl, amino, and carboxyl groups into their corresponding protected structures. Another example is the modification of hydroxyl and amino groups by acid anhydrides.

[0424] Method 2: Coupling reaction between two reactive groups. Using a heterofunctionalizing agent containing one reactive group and a target functional group or its protected form as raw materials, the target functional group or its protected form is introduced through a reaction between one of the reactive groups and the reactive group at the end of the polyethylene glycol chain. There are no particular restrictions on the reaction mode or method between the two reactive groups; the reaction conditions are related to the type of divalent linker generated and existing publicly available techniques can be used. Examples include alkylation, alkenyl addition reactions, alkynyl addition reactions, Schiff base reactions combined with reduction reactions, and condensation reactions. Alkylation reactions are preferably based on thiol or amino alkylation, corresponding sequentially to the formation of thioether bonds, secondary amino groups, or tertiary amino groups. Condensation reactions include, but are not limited to, condensation reactions that generate ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, and urethane groups. For example, using isofunctionalizing reagents containing groups such as azide, alkynyl, alkenyl, trithioester, mercapto, dienyl, furanyl, 12,4,5-tetraazine, and cyanate, along with the target functional group or its protected form, as raw materials, the target functional group or its protected form is introduced through a click reaction. The reaction between two reactive groups is accompanied by the formation of a new bond. Typical examples of the newly formed divalent linker include amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, and triazole groups.

[0425] Method 3: Obtain the target functional group or its protected form through a combination of direct modification and coupling reaction.

[0426] In this invention, the raw materials used in each preparation method can be purchased or synthesized by the user.

[0427] The intermediates and final products prepared in this invention can be purified by methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction. The structure and molecular weight of the final products can be characterized using methods including but not limited to NMR, electrophoresis, UV-Vis spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, and circular dichroism.

[0428] 3. Cationic liposomes

[0429] 3.1. Cationic liposomes

[0430] In this invention, a cationic liposome contains any of the structures described above, such as the polyethylene glycol-modified lipid represented by general formula (2).

[0431] In one specific embodiment of the present invention, the cationic liposome preferably contains, in addition to any of the polyethylene glycol-modified lipids with the structure shown in general formula (2) described above, one or more of neutral lipids, steroid lipids and cationic lipids; more preferably, it contains all three types of lipids: neutral lipids, steroid lipids and cationic lipids.

[0432] In one specific embodiment of the present invention, the neutral lipids in the cationic liposomes preferably include, but are not limited to, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether). 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) Palmitoyl oleoyl phosphatidyl ethanolamine (POPE), distearate-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidyl ethanolamine (LPE), or any one thereof, or a combination thereof.

[0433] In one specific embodiment of the present invention, the polyethylene glycol-modified lipids in the cationic liposomes are preferably polyethylene glycol 500-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-disstearoylphosphatidylethanolamine, or polyethylene glycol 500-1,2-oleoylphosphatidyl The polyethylene glycol 2000-1,2-oleoylphosphatidylethanolamine or polyethylene glycol 2000-2,3-dimyristoylglycerol is preferred, and more preferably, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-distearatephosphatidylethanolamine or polyethylene glycol 2000-1,2-acylphosphatidylethanolamine is preferred.

[0434] In one specific embodiment of the present invention, the steroid lipids in the cationic liposomes are preferably any one or a mixture thereof, including cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, and α-tocopherol.

[0435] In one specific embodiment of the present invention, the structure of the cationic lipid in the cationic liposome is preferably as shown in general formula (1):

[0436]

[0437] Where N is the nitrogen branching center;

[0438] L1 and L2 are divalent linker groups, each independently selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -S-, -C(=O)S-, -SC(=O)-, and -NR-. c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any one of C(=O)S-, wherein R c It is a hydrogen atom or a carbon atom. 1-12 alkyl;

[0439] L3 is a linker bond or -L4-Z-L5-; L4 and L5 are carbon chain linkers, each independently being -(CR) a R b ) t -or-(CR) a Rb ) t -(CR a R b ) p -(CR a R b ) q - where t, p, and q are each independent integers from 0 to 12, and R a and R b Each is independently a hydrogen atom or a carbon atom. 1-12 Alkyl group; wherein Z is a linking bond or selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any one of C(=O)S-;

[0440] B1 and B2 are each independently C 1-12 Alkylene;

[0441] R1 and R2 are each independently C 2-30 Aliphatic hydrocarbon groups;

[0442] R3 represents a hydrogen atom, alkyl group, alkoxy group, or -(C=O)R. d -(C=O)OR d -O(C=O)R d -O(C=O)OR d or Among them, R d C 1-12 Alkyl group, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, F contains a functional group, when j is 0, G1 does not exist, when j is 1, G1 leads to k F, where k is an integer from 2 to 8;

[0443] A is selected from -(CR) a R b ) s O-、-O(CR a R b ) s -、-(CR a R b ) s S-、-S(CR a Rb ) s -、-(CR a R b ) s O(CR a R b ) s S-、-(CR a R b ) s S(CR a R b ) s O-、-(CR a R b ) s NR c (CR a R b ) s S-、-(CR a R b ) s S(CR a R b ) s NR c -、-(CR a R b ) s NR c (CR a R b ) s O- and -(CR a R b ) s O(CR a R b ) s NR c - any one of the following, where s is 2, 3, or 4, R a and R b Each is independently a hydrogen atom or a carbon atom. 1-12 alkyl;

[0444] When A is -(CR) a R b ) s O- or -O(CR) a R b ) s When -, n is an integer between 2 and 6; when A is not -(CR a R b ) s O- or -O(CR) a R b ) s When n is an integer from 1 to 6;

[0445] The alkyl, alkylene, alkoxy, and aliphatic hydrocarbon groups are each independently substituted or unsubstituted.

[0446] In one specific embodiment of the present invention, the structure of the cationic lipid in the cationic liposome is shown in general formula (1), preferably any one of the following structures:

[0447]

[0448]

[0449]

[0450] In one specific embodiment of the present invention, the cationic lipid in the cationic liposomes, in addition to the cationic lipid shown in the aforementioned general formula (1), may also be N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 3-(bis(dodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine) (KL10), N1-[2-(bis(dodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine), etc. The following are included in the list of ethyl[2-ethyl]-N1,N4,N4-tri-dodecyl-1,4-piperazine diethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (DLin-K-DMA), 4-(dimethylamino)butyric acid 37-carbon-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (DLin-KC2-DMA), and mixtures thereof.

[0451] In one specific embodiment of the present invention, preferably any of the aforementioned cationic liposomes contains 20-80% cationic lipids of formula (1), 5-15% neutral lipids, 25-55% steroid lipids and 0.5-10% polyethylene glycol-modified lipids, wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

[0452] In one specific embodiment of the present invention, preferably, in any of the aforementioned cationic liposomes, the molar percentage of cationic lipids in the total lipids in the solution containing the solvent is 30-65%; more preferably, it is any one of about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%.

[0453] In one specific embodiment of the present invention, preferably, in any of the aforementioned cationic liposomes, the molar percentage of neutral lipids in the total lipids in the solution containing the solvent is 7.5-13%; more preferably, it is about 8%, 9%, 10%, 11%, or 12%.

[0454] In one specific embodiment of the present invention, preferably, in any of the aforementioned cationic liposomes, the steroid lipids account for 35-50% of the total lipids in the solution containing the solvent, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

[0455] In one specific embodiment of the present invention, preferably, in any of the aforementioned cationic liposomes, the polyethylene glycol-modified lipid accounts for 0.5-5% of the total lipid molar percentage in the solution containing the solvent; more preferably, 1-3%; and more preferably, about 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%.

[0456] 3.2. Preparation of cationic liposomes

[0457] In this invention, cationic liposomes can be prepared by the following methods, including but not limited to thin film dispersion, ultrasonic dispersion, reverse phase evaporation, freeze drying, freeze-thaw, double emulsion and / or injection, preferably thin film dispersion, ultrasonic dispersion and / or reverse phase evaporation.

[0458] In the method for preparing cationic liposomes of the present invention, the thin-film dispersion method may include the following steps:

[0459] (1) Weigh cationic lipids, steroid lipids, neutral lipids and polyethylene glycol-modified lipids, dissolve them thoroughly in an organic solvent, shake well, remove the organic solvent by rotary evaporation under reduced pressure to form an oil film, and then dry it with a vacuum pump to remove the organic solvent.

[0460] (2) Add phosphate buffer solution containing cryoprotectant, sonicate in water bath to form a semi-transparent emulsion;

[0461] (3) The emulsion is added to a high-pressure homogenizer for overpressure, and then the overpressured emulsion is added to a liposome extruder for membrane processing to form cationic liposomes; and

[0462] (4) Optionally, the cationic liposomes are dried in a freeze dryer to form cationic liposome powder;

[0463] Preferably, the organic solvent is dichloromethane, chloroform, and / or methanol, more preferably chloroform and methanol; preferably, the rotary evaporation speed is 30-300 rpm, more preferably 50-200 rpm, and most preferably 100-170 rpm; preferably, the rotary evaporation temperature is 10-200°C, more preferably 20-100°C, and most preferably 40-80°C.

[0464] Preferably, the vacuum pump drying time is 1 to 72 hours, more preferably 5 to 48 hours, and most preferably 15 to 36 hours;

[0465] Preferably, the cryoprotectant is dissolved in phosphate buffer at a concentration of 0.1-80%, more preferably 1-50%, and even more preferably 5-20%.

[0466] Preferably, the frequency of the water bath ultrasound is 10-300, more preferably 30-200, and most preferably 60-150.

[0467] Preferably, the water bath ultrasound time is 0.1 to 5 hours, more preferably 0.2 to 2 hours, and most preferably 0.25 to 1 hour;

[0468] Preferably, the pressure of the high-pressure homogenizer is 50-240 MPa, more preferably 80-200 MPa, and most preferably 100-150 MPa;

[0469] Preferably, the number of overpressure cycles of the high-pressure homogenizer is any integer between 1 and 50, more preferably any integer between 3 and 20, and most preferably any integer between 5 and 10.

[0470] Preferably, the pressure of the liposome extruder is 50-300 MPa, more preferably 80-250 MPa, and most preferably 120-200 MPa;

[0471] Preferably, the number of times the liposome extruder passes through the membrane is any integer between 1 and 50, more preferably any integer between 3 and 30, and most preferably any integer between 5 and 20.

[0472] Preferably, the drying time of the freeze dryer is 1 to 120 hours, more preferably 5 to 72 hours, and most preferably 10 to 36 hours.

[0473] In the preparation method of cationic liposomes of the present invention, the ratio of cationic liposomes to phosphate buffer solution containing cryoprotectant can be 1 mg: (0.1-100) mL, preferably 1 mg: (0.3-50) mL, and more preferably 1 mg: (0.5-5) mL.

[0474] 4. Cationic liposome pharmaceutical compositions

[0475] In one embodiment of the present invention, a cationic liposome pharmaceutical composition comprises any of the cationic liposomes and the drug described above, wherein the cationic liposomes comprise any of the polyethylene glycol-modified lipids with the structure of general formula (2) described above.

[0476] In one specific embodiment of the present invention, the cationic liposome pharmaceutical composition preferably contains nucleic acid drugs and antitumor agents. The nucleic acid drugs are selected from any one of DNA, antisense nucleic acids, plasmids, mRNA (messenger RNA), interfering nucleic acids, aptamers, miRNA inhibitors (antagomir), microRNAs (miRNAs), ribozymes, and small interfering RNAs (siRNAs); preferably any one of RNA, miRNAs, and siRNAs.

[0477] In one specific embodiment of the present invention, the cationic liposome pharmaceutical composition is preferably used as a drug and is selected from any of the following drugs: drugs for treating cancer, malignant tumors, anti-infective agents, antiviral agents, antifungal agents, vaccines; esophageal cancer, gastric cancer, colorectal cancer, nasopharyngeal carcinoma, brain tumors, cervical cancer, leukemia, bone cancer, AIDS, and viral infections.

[0478] In this invention, the drugs are further preferably including, but not limited to, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, lactamase, mefenamic acid, carmustine, romustine, busulfan, dibromomannitol, mitomycin C, cisdichlorodiamine cycloplatin(II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, debucaine, chlorpromazine, propranolol, dimolol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, and phenytoin. The following are prohibited: diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butonazole, clotrimazole, itraconazole, nystatin, neftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma eye drops, vitamins, sedatives and imaging agents, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, dihydroxyanthraquinone, safflowerin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, and serotonin.

[0479] In one specific embodiment of the present invention, the N / P ratio of the cationic liposome to the nucleic acid is preferably (0.1-100):1, more preferably (0.2-30):1, and most preferably (0.5-20):1;

[0480] In one specific embodiment of the present invention, the working solution of the nucleic acid drug formulation is preferably deionized water, ultrapure water, phosphate buffer, or physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline; preferably, the ratio of cationic liposomes to working solution is (0.05-20) g: 100 mL, more preferably (0.1-10) g: 100 mL, and most preferably (0.2-5) g: 100 mL.

[0481] 5. A cationic liposome pharmaceutical composition formulation

[0482] In this invention, a cationic liposome pharmaceutical composition formulation contains the aforementioned cationic liposome pharmaceutical composition and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.

[0483] In this invention, the preparation of a cationic liposome nucleic acid drug composition formulation includes the following steps:

[0484] (1) Equilibrate the cationic liposomes in the diluent or excipient;

[0485] (2) Nucleic acid drugs are compounded by adding them to a mixture of cationic liposomes and diluents or excipients after equilibration;

[0486] Preferably, the balancing time is 0.1 to 12 hours, more preferably 0.2 to 6 hours, and even more preferably 0.5 to 3 hours; preferably, the compounding time is 0.1 to 12 hours, more preferably 0.2 to 5 hours, and even more preferably 0.5 to 2 hours.

[0487] The following describes in further detail the preparation methods of PEGylated lipids, cationic liposomes, and cationic liposome pharmaceutical compositions, as well as the bioactivity testing of the cationic liposome pharmaceutical compositions, using specific embodiments. These specific embodiments are for further detailed explanation of the invention and are not intended to limit the scope of protection of the invention. In the embodiments for preparing PEGylated lipids, the final product was characterized by NMR spectroscopy or its molecular weight was confirmed by MALDI-TOF.

[0488] Example 1: Synthesis of polyethylene glycol-modified lipids E1-1 and E1-3

[0489] Example 1.1: Synthesis of methoxylated polyethylene glycol lipid E1-1

[0490]

[0491] In general formula (2), E1-1, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b It consists of hydrogen atoms, s = 2, n1 ≈ 45, R = methoxy group, and the total molecular weight is approximately 2460.

[0492] The preparation process is as follows:

[0493] Step a: Compound S1-1 (20.00 g, 10.0 mmol, Mw approximately 2000, n1 ≈ 45, PDI = 1.03) and toluene (200 mL) were azeotropically dehydrated at 140 °C. After distilling off 60 mL of solvent, the reaction mixture was cooled to room temperature. Triethylamine (TEA, 2.02 g, 20.0 mmol) and methanesulfonyl chloride (MsCl, 2.05 g, 18.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S1-2 (18.00 g).

[0494] Step b: Compound S1-2 (18.00 g, 9.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (K2CO3, 12.42 g, 90.0 mmol), compound S1-3 (9.58 g, 45.0 mmol), and tetrabutylammonium bromide (TBABr, 0.29 g, 0.9 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S1-4. The crude compound S1-4 was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S1-4 (12.00 g).

[0495] Step c: Compound S1-4 (11.26 g, 5.0 mmol), compound S1-5 (1.95 g, 6.0 mmol), and TEA (0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E1-1 (10.1 g). 1¹H NMR (500MHz, CDCl₃) δ: 3.84–3.45 (m, 182H), 3.37 (s, 3H), 3.35 (t, 2H), 3.18 (t, 2H), 2.27 (t, 2H), 1.56–1.40 (m, 4H), 1.36–1.18 (m, 42H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E1-1 to be 2461 Da, with a PDI of 1.03.

[0496]

[0497] Example 1.2: Synthesis of hydroxylated polyethylene glycol lipid E1-3

[0498]

[0499] In general formula (2), E1-3, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is a hydroxyl group, and the total molecular weight is approximately 2450.

[0500] The preparation process is as follows:

[0501] Step a: Compound S1-6 (19.95 g, 9.5 mmol, mPEG-OH, Mw approximately 2100, n1 ≈ 45, PDI = 1.03) and toluene (200 mL) were azeotropically dehydrated at 140 °C. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (2.02 g, 20.0 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S1-7 (18.93 g).

[0502] Step b: Compound S1-7 (18.00 g, 8.5 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (12.42 g, 90.0 mmol), compound S1-3 (9.58 g, 45.0 mmol), and tetrabutylammonium bromide (0.29 g, 0.9 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S1-8. The crude compound S1-8 was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S1-8 (12.93 g).

[0503] Step c: Compound S1-8 (10.00 g, 4.3 mmol), compound S1-5 (2.05 g, 6.3 mmol), and TEA (0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E1-2 (8.88 g).

[0504] Step d: The above-mentioned substituted product (E1-2, 8.00 g, 3.1 mmol) was dissolved in 50 mL of THF solution and placed in a nitrogen-protected flask. Tetrabutylammonium fluoride solution (TBAF, 50 mL, 1 N in THF) was added. The reaction was allowed to proceed overnight to remove TBS protection. The product was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography, and the target eluent was collected and concentrated to obtain product E1-3 (6.60 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.85–3.44 (m, 182H), 3.35 (t, 2H), 3.17 (t, 2H), 2.28 (t, 2H), 1.56–1.43 (m, 4H), 1.36–1.20 (m, 42H), 0.88 (t, 6H). MALDI-TOF analysis determined the molecular weight of E1-3 to be 2447 Da, with a PDI of 1.03.

[0505]

[0506] Example 2: Synthesis of methoxylated polyethylene glycol lipid E2-1

[0507]

[0508] In general formula (2), E2-1, R1 is tetradecyl, R2 is decyl, B3 is a linking bond, B4 is a linking bond, L7 is a linking bond, L8 is a carbonyl group, L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈60, R is methoxy group, and the total molecular weight is approximately 3070.

[0509] The preparation process is as follows:

[0510] Step a: Compound S2-1 (27.00 g, 10.0 mmol, mPEG-OH, Mw approximately 2700, n1 ≈ 60, PDI = 1.03) and toluene (200 mL) were azeotropically dehydrated at 140 °C. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (2.02 g, 20.0 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S2-2 (24.75 g).

[0511] Step b: Compound S2-2 (24.75 g, 9.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (12.42 g, 90.0 mmol), compound S1-3 (9.58 g, 45.0 mmol), and tetrabutylammonium bromide (0.29 g, 0.9 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S2-3. The crude compound S2-3 (17.55 g) was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S2-3.

[0512] Step c: The above-mentioned substituted product S2-3 (14.55 g, 5.0 mmol), compound S2-4 (1.22 g, 6.3 mmol), and TEA (0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After the reaction solution was concentrated, it was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the mixture was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E2-1 (13.13 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.85–3.44 (m, 242H), 3.39 (s, 3H), 3.34 (t, 2H), 3.18 (t, 2H), 2.25 (t, 2H), 1.56–1.42 (m, 4H), 1.36–1.19 (m, 36H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E₂-1 to be 3079 Da, with a PDI of 1.03.

[0513]

[0514] Example 3: Synthesis of methoxylated polyethylene glycol lipid E3-1

[0515]

[0516] In general formula (2), E3-1, R1 is tetradecyl, R2 is heptyl, B3 is a linking bond, B4 is pentylene, L7 is a linking bond, L8 is an amide bond (-NHC(=O)-), L3 is carbonyl, and A is -OCH2CH2-. Wherein, R... a and R b It consists of hydrogen atoms, s = 2, n1 ≈ 45, R = methoxy group, and the total molecular weight is approximately 2500.

[0517] The preparation process is as follows:

[0518] Step a: Compound S3-2 (10.00 g, 34.4 mmol) was dissolved in 30 mL of isopropanol, and then compound S3-1 (12.40 g, 33.8 mmol), potassium carbonate (4.75 g, 34.4 mmol), and potassium iodide (0.16 g, 1.0 mmol) were added sequentially. The mixture was heated under reflux for 36 h. TLC showed that a small amount of the starting material remained. The isopropanol was concentrated, quenched with 200 mL of purified water, and then extracted with dichloromethane (100 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain product S3-3 (10.83 g).

[0519] Step b: Compound S3-3 (2.13 g, 5.0 mmol), compound S3-4 (8.80 g, 4.0 mmol), and TEA (0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E3-1 (9.25 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.07(t, 2H), 3.83–3.45(m, 182H), 3.36(s, 3H), 3.26(t, 2H), 3.25–3.20(q, 2H), 3.14(t, 2H), 2.17(t, 2H), 1.66–1.46(m, 8H), 1.34–1.25(m, 32H), 0.87(t, 6H). MALDI-TOF analysis determined the molecular weight of E₃-1 to be 2508 Da, with a PDI of 1.02.

[0520]

[0521] Example 4: Synthesis of hydroxylated polyethylene glycol lipid E4-2

[0522]

[0523] In general formula (2), E4-2, R1 is tetradecyl, R2 is heptyl, B3 is a linking bond, B4 is heptyl, L7 is a linking bond, L8 is an ether bond (-O-), L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is a hydroxyl group, and the total molecular weight is approximately 2450.

[0524] The preparation process is as follows:

[0525] Step a: Compound S4-1 (10.00 g, 34.2 mmol) was dissolved in 100 mL of isopropanol. Then, compound S3-1 (12.40 g, 33.8 mmol), potassium carbonate (4.75 g, 34.4 mmol), and potassium iodide (0.16 g, 1.0 mmol) were added sequentially, and the mixture was heated under reflux for 36 h. TLC showed a small amount of reactant remaining. The isopropanol was concentrated, quenched with 100 mL of purified water, and then extracted with dichloromethane (100 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain product S4-2 (10.53 g).

[0526] Step b: In a 500 mL round-bottom flask, naphthalene (9.60 g, 75.0 mmol) was added and dissolved in 120 mL of THF. Sodium (2.59 g, 112.5 mmol) was then added, and the mixture was heated to 70 °C with stirring for 18 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonamide compound S4-2 (8.70 g, 15.0 mmol) dissolved in 20 mL of THF was slowly added dropwise. After the addition was complete, the mixture was kept at 1.5 h, then heated to 50 °C with stirring for 16 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was slowly added dropwise to quench the reaction, followed by water quenching. The mixture was then extracted with PE, dried, concentrated, dissolved in methanol at 60 °C, crystallized under ice bath conditions, and filtered to obtain amine compound S4-3 (4.65 g).

[0527] Step c: Compound S1-7 (4.40 g, 2.0 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.02) was dissolved in 80 mL of water by stirring at room temperature. Potassium carbonate (3.04 g, 22.0 mmol), compound S4-3 (4.26 g, 10.0 mmol), and tetrabutylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E4-1. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E4-1 (3.50 g).

[0528] Step d: Dissolve the above-mentioned substituted product E4-1 (2.50 g, 1.0 mmol, Mw approximately 2500, n1 ≈ 45, PDI = 1.02) in 50 mL of THF solution, place in a nitrogen-protected flask, and add TBAF (50 mL, 1 N in THF). React overnight to remove TBS protection. Dry the product over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify the crude product by column chromatography, collect the target eluent, and concentrate to obtain product E4-2 (2.14 g). In the 1H NMR spectrum, the characteristic peak of TBS disappears. 1 ¹H NMR (500MHz, CDCl₃) δ: 3.86–3.46 (m, 186H), 2.65 (t, 2H), 2.49 (m, 4H), 1.65–1.42 (m, 8H), 1.36–1.18 (m, 36H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E₄⁻ to be 2449 Da, with a PDI of 1.02.

[0529]

[0530] Example 5: Synthesis of methoxylated polyethylene glycol lipid E5-1

[0531]

[0532] In general formula (2), E5-1, R1 is tetradecyl, R2 is butyl, B3 is a linking bond, B4 is heptyl, L7 is a linking bond, L8 is -OCH2CH2O-, L3 is -CH2CH2-, and A is -OCH2CH2-, where R a and R b It consists of hydrogen atoms, s = 2, n1 ≈ 45, R = methoxy group, and the total molecular weight is approximately 2460.

[0533] The preparation process is as follows:

[0534] Step a: Compound S5-1 (10.00 g, 34.0 mmol) was dissolved in 30 mL of isopropanol. Then, compound S3-1 (12.40 g, 33.8 mmol), potassium carbonate (4.75 g, 34.4 mmol), and potassium iodide (0.16 g, 1.0 mmol) were added sequentially, and the mixture was heated under reflux for 36 h. TLC showed a small amount of reactant remaining. The isopropanol was concentrated, quenched with 100 mL of purified water, and then extracted with dichloromethane (100 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain product S5-2 (10.88 g).

[0535] Step b: In a 500 mL round-bottom flask, naphthalene (9.60 g, 75.0 mmol) was added and dissolved in 120 mL of THF. Sodium (2.59 g, 112.5 mmol) was then added, and the mixture was heated to 70 °C with stirring for 18 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonamide compound S5-2 (8.73 g, 15.0 mmol) dissolved in 20 mL of THF was slowly added dropwise. After the addition was complete, the mixture was kept at 1.5 h, then heated to 50 °C with stirring for 16 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was slowly added dropwise to quench the reaction, followed by water quenching. The mixture was then extracted with PE, dried, concentrated, dissolved in methanol at 60 °C, crystallized under ice bath conditions, and filtered to obtain amine compound S5-3 (4.75 g).

[0536] Step c: Compound S1-2 (4.20 g, 2.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.02) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (3.04 g, 22.0 mmol), compound S5-3 (4.28 g, 10.0 mmol), and tetrabutylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E5-1. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E5-1 (3.56 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.85–3.45 (m, 190H), 3.38 (s, 3H), 2.65 (t, 2H), 2.49 (m, 4H), 1.66–1.43 (m, 8H), 1.36–1.19 (m, 30H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E5-1 to be 2467 Da, with a PDI of 1.02.

[0537]

[0538] Example 6: Synthesis of methoxylated polyethylene glycol lipid E6-1

[0539]

[0540] In general formula (2), E6-1, R1 and R2 are methyl groups, B3 and B4 are tridecylene groups, L7 and L8 are ether bonds (-O-), L3 is -CH2CH2O-, and A is -CH2CH2O-. Among these, R... a and R bThe atoms are hydrogen atoms, s is 2, n1≈40, R is methyl, and the total molecular weight is approximately 2260.

[0541] The preparation process is as follows:

[0542] Step a: In a 500 mL round-bottom flask, compound 13-bromotridecyl-1-ol (S6-1, 20.00 g, 71.9 mmol) was dissolved in THF (100 mL), and Na2CO3 (15.20 g, 143.8 mmol) was added. Under ice bath conditions, magnesium sulfate (13.60 g, 107.8 mmol) was slowly added dropwise, and the reaction was stirred at room temperature for 24 h. After the starting material was consumed by TLC, the reaction solution was quenched with saturated ammonium chloride solution, then extracted with EtOAc (200 mL * 2), dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the methyl ether compound S6-2 (19.66 g).

[0543] Step b: The above compound S6-2 (19.66 g, 67.3 mmol) was dissolved in 60 mL of isopropanol, and then p-toluenesulfonamide (TsNH2, 3.82 g, 22.3 mmol), potassium carbonate (9.24 g, 67.3 mmol), and potassium iodide (0.36 g, 2.2 mmol) were added sequentially. The mixture was heated under reflux for 36 h. TLC showed that a small amount of the starting material remained. The isopropanol was concentrated, quenched with 200 mL of purified water, and then extracted with dichloromethane (200 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain product S6-3 (9.30 g).

[0544] Step c: In a 500 mL round-bottom flask, naphthalene (10.00 g, 78.2 mmol) was added and dissolved in 120 mL of THF. Sodium (2.70 g, 117.2 mmol) was then added, and the mixture was heated to 70 °C and stirred for 18 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonamide compound S6-3 (9.30 g, 15.4 mmol) dissolved in 20 mL of THF was slowly added dropwise. After the addition was complete, the mixture was kept in this state for 1.5 h, then heated to 50 °C and stirred for 16 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was slowly added dropwise to quench the reaction, followed by water. The mixture was then extracted with PE, dried, and concentrated to obtain a pale yellow solid. The solid was dissolved in methanol at 60 °C, crystallized under ice bath conditions, and filtered to obtain amine compound S6-4 (5.00 g).

[0545] Step d: Compound S6-5 (4.37 g, 2.3 mmol, Mw approximately 1900, n1 ≈ 40, PDI = 1.02) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (3.17 g, 23.01 mmol), amine compound S6-4 (5.00 g, 11.3 mmol), and tetrabutylammonium bromide (0.74 g, 0.23 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E6-1. The crude compound E6-1 was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E6-1 (3.95 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.45 (m, 166H), 3.38 (s, 3H), 3.32 (s, 6H), 2.65 (t, 2H), 2.49 (m, 4H), 1.66–1.42 (m, 8H), 1.35–1.19 (m, 36H). MALDI-TOF analysis determined the molecular weight of E6-1 to be 2259 Da, with a PDI of 1.02.

[0546]

[0547] Example 7: Synthesis of methoxylated polyethylene glycol lipid E7-1

[0548]

[0549] In general formula (2), E7-1, R1 and R2 are heptyl groups, B3 and B4 are heptyl derivatives, L7 and L8 are ether bonds (-O-), L3 is -CH2CH2-, and A is -OCH2CH2-. Among these, R... a and R b It consists of hydrogen atoms, s = 2, n1 = 45, R is a methoxy group, and the total molecular weight is approximately 2482.

[0550] The preparation process is as follows:

[0551] Step a: In a 500 mL round-bottom flask, the above compound S4-1 (10.00 g, 34.2 mmol) was dissolved in 100 mL of isopropanol. p-Toluenesulfonamide (TsNH2, 1.95 g, 11.4 mmol), potassium carbonate (4.72 g, 34.2 mmol), and potassium iodide (0.18 g, 1.1 mmol) were added, and the mixture was heated under reflux for 36 h. TLC showed a small amount of reactant remaining. The isopropanol was concentrated, quenched with 100 mL of purified water, and then extracted with dichloromethane (100 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain compound S7-1 (5.01 g).

[0552] Step b: Naphthalene (5.38 g, 42.0 mmol) was added to a 500 mL round-bottom flask and dissolved in 120 mL THF. Sodium (1.45 g, 63.0 mmol) was then added, and the mixture was heated to 70 °C for 18 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonamide compound S7-1 (5.00 g, 8.4 mmol) dissolved in 20 mL THF was slowly added dropwise. After the addition was complete, the mixture was kept in this state for 1.5 h, then heated to 50 °C for 16 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was slowly added dropwise to quench the reaction, followed by water. The mixture was then extracted with PE, dried, and concentrated to obtain a pale yellow solid. The solid was dissolved in methanol at 60 °C, crystallized under ice bath conditions, and filtered to obtain compound S7-2 (2.78 g).

[0553] Step c: Compound S7-3 (2.78 g, 1.3 mmol, Mw = 2137, n1 = 45) was added to 50 mL of water and dissolved by stirring at room temperature. Potassium carbonate (1.79 g, 13.0 mmol), compound S7-2 (2.78 g, 6.3 mmol), and tetrabutylammonium bromide (0.03 g, 0.1 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (50 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (50 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E7-1. The crude compound E7-1 was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E7-1 (2.43 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.43 (m, 190H), 3.37 (s, 3H), 2.65 (t, 2H), 2.49 (m, 4H), 1.61–1.47 (m, 12H), 1.34–1.19 (m, 28H), 0.88 (t, 6H). MALDI-TOF analysis determined the molecular weight of E7-1 to be 2481 Da, with a PDI of 1.

[0554]

[0555] Example 8: Synthesis of methoxylated polyethylene glycol lipids E8-1 and E8-2

[0556] Example 8.1. Synthesis of methoxylated polyethylene glycol lipid E8-1

[0557]

[0558] In general formula (2), E8-1, R1 and R2 are heptyl groups, B3 and B4 are pentylenes, L7 and L8 are amide bonds (-NHC(=O)-), L3 is -CH2C(=O)-, and A is -OCH2CH2-. Among these, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈60, R is methoxy group, and the total molecular weight is approximately 3150.

[0559] The preparation process is as follows:

[0560] Step a: Compound S8-1 (20.00 g, 103.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 20.08 g, 104.6 mmol), and N-hydroxysuccinimide (NHS, 11.57 g, 101.5 mmol) were dissolved in dichloromethane (400 mL), and the reaction was carried out overnight at room temperature with stirring. After the reaction was completed, the reaction solution was backwashed twice with 0.1 mol / L HCl (200 mL * 2), and once with saturated sodium chloride (200 mL). The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the target compound S8-2 (29.31 g).

[0561] Step b: Compound S8-2 (29.00 g, 99.7 mmol) was dissolved in dichloromethane (100 mL), and compound S8-3 (11.67 g, 101.5 mmol) and TEA (14.15 g, 140.1 mmol) were added. The mixture was reacted overnight at room temperature, and a large amount of white solid precipitated. The solid was filtered, slurried with methanol (60 mL), filtered, washed twice with methanol, and the solid was collected and dried to obtain the target compound S8-4 (24.55 g).

[0562] Step c: The above compound S8-4 (20.00 g, 68.7 mmol) was dissolved in 60 mL of isopropanol, and then p-toluenesulfonamide (TsNH2, 3.93 g, 22.9 mmol), potassium carbonate (9.48 g, 68.7 mmol), and potassium iodide (0.36 g, 2.2 mmol) were added sequentially. The mixture was heated under reflux for 36 h. TLC showed that a small amount of the starting material remained. The isopropanol was concentrated, quenched with 200 mL of purified water, and then extracted with dichloromethane (200 mL * 2). The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography and concentrated to obtain product S8-5 (9.68 g).

[0563] Step d: Naphthalene (9.60 g, 75.0 mmol) was added to a 500 mL round-bottom flask and dissolved in 120 mL of THF. Sodium (2.59 g, 112.5 mmol) was then added, and the mixture was heated to 70 °C with stirring for 18 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonamide compound S8-5 (8.91 g, 15.0 mmol) dissolved in 20 mL of THF was slowly added dropwise. After the addition was complete, the mixture was kept at 1.5 h, then heated to 50 °C with stirring for 16 h. After the reaction was complete, the mixture was cooled to 0 °C, and methanol was slowly added dropwise to quench the reaction, followed by water. The mixture was then extracted with PE, dried, and concentrated to obtain a pale yellow solid. The solid was dissolved in methanol at 60 °C, crystallized under ice bath conditions, and filtered to obtain amine compound S8-6 (4.79 g).

[0564] Step e: Compound S8-7 (5.60 g, 2.0 mmol, Mw approximately 2800, n1 ≈ 60, PDI = 1.02) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (2.76 g, 20.0 mmol), secondary amine compound S8-6 (4.40 g, 10.0 mmol), and tetrabutylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E8-1. Purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E8-1 (5.12 g). 1 H NMR(500MHz, CDCl3)δ:4.17(s,2H),3.85-3.45(m,238H),3.37(s,3H),3.26(t,2H),3.25-3.20(q, 4H),3.14(t,2H),3.06(t,2H),2.17(t,4H),1.68-1.41(m,12H),1.33-1.25(m,20H),0.88(t,6H).

[0565] MALDI-TOF testing determined the molecular weight of E8-1 to be 3153 Da, with a PDI of 1.02.

[0566]

[0567] Example 8.2. Synthesis of methoxylated polyethylene glycol lipid E8-2

[0568]

[0569] In general formula (2), E8-2, R1 and R2 are heptyl groups, B3 and B4 are pentylenes, L7 and L8 are amide bonds (-NHC(=O)-), L3 is -CH2CH2C(=O)-, and A is -OCH2CH2-. Among these, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈150, R is methoxy group, and the total molecular weight is approximately 7130.

[0570] The preparation process is as follows:

[0571] Compound S8-8 (13.60 g, 2.0 mmol, Mw approximately 6800, n1 ≈ 150, PDI = 1.02) was dissolved in 80 mL of water by stirring at room temperature. Potassium carbonate (2.76 g, 20.0 mmol), secondary amine compound S8-6 (4.40 g, 10.0 mmol), and tetrabutylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E8-2. This crude compound was purified by column chromatography, concentrated, and dried by pumping to obtain the target compound E8-2 (11.12 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.07(t, 2H), 3.83–3.46(m, 598H), 3.36(s, 3H), 3.26(t, 2H), 3.25–3.21(q, 4H), 3.14(t, 2H), 3.06(t, 2H), 2.38(t, 2H), 2.16(t, 4H), 1.66–1.41(m, 12H), 1.32–1.26(m, 20H), 0.87(t, 6H). MALDI-TOF analysis determined the molecular weight of E₈-2 to be 7129 Da, with a PDI of 1.02.

[0572]

[0573] Example 9: Synthesis of polyethylene glycol ethylamined lipid E9-2

[0574]

[0575] In general formula (2), E9-1, R1 is undecyl, R2 is tetradecyl, B3 is propylene, B4 is a linking bond, L7 is an ether bond, L8 is a linking bond, L3 is -C(=O)CH2-, and A is -OCH2CH2-, where R a and R b It consists of hydrogen atoms, s = 2, n1 ≈ 45, R = -CH2CH2NH2, and the total molecular weight is approximately 2500.

[0576] The preparation process is as follows:

[0577] Step a: In a clean, dry 500 mL round-bottom flask, 80 mL of water was added to compound S9-1 (12.00 g, 41.1 mmol), and the mixture was stirred and dissolved at room temperature. Potassium carbonate (56.72 g, 411.0 mmol), compound S9-2 (47.06 g, 205.5 mmol), and tetrabutylammonium bromide (1.32 g, 4.1 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S9-3. The crude compound S9-3 (10.37 g) was purified by column chromatography, concentrated, and dried under oil pressure to obtain the target compound S9-3.

[0578] Step b: Compound S9-4 (7.92 g, 3.6 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.03), compound S9-3 (2.29 g, 5.4 mmol), EDCI (1.38 g, 7.2 mmol), HOBt (0.73 g, 5.4 mmol), and TEA (0.73 g, 7.2 mmol) were successively dissolved in dichloromethane (100 mL), and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (100 mL * 2), and then backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound E9-1 (7.75 g).

[0579] Step c: Removal of the Boc protecting group. In a clean, dry 500 mL round-bottom flask, prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution. Slowly add a dichloromethane solution of E9-1 (7.00 g, 2.7 mmol) dropwise under ice bath conditions, and react at room temperature for 2 hours. Concentrate the reaction solution, add purified water, extract with dichloromethane, dry the extract with anhydrous magnesium sulfate, filter, concentrate the filtrate, and recrystallize to obtain E9-2 (6.16 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.17 (s, 2H), 3.83–3.43 (m, 186H), 3.26 (t, 2H), 3.17 (t, 2H), 2.83 (t, 2H), 1.66–1.46 (m, 6H), 1.36–1.21 (m, 38H), 0.89 (t, 6H). MALDI-TOF analysis determined the molecular weight of E9-2 to be 2506 Da, with a PDI of 1.03.

[0580]

[0581] Example 10: Synthesis of methoxylated polyethylene glycol lipid E10-1

[0582]

[0583] In the corresponding general formula (2), E10-1, R1 is nonyl, R2 is decyl, L7 is -OC(=O)-, L8 is -C(=O)-, B3 is pentylene, B4 is a linking bond, L3 is -CH2CH2O, and A is -CH2CH2O-, where R a and R b The atoms are hydrogen atoms, s is 2, n1≈100, R is methyl, and the total molecular weight is approximately 4880.

[0584] The preparation process is as follows:

[0585] Step a: Under an argon atmosphere, dicyclohexylcarbodiimide (DCC, 4.55 g, 22.0 mmol) was added to a round-bottom flask containing S10-2 (1.45 g, 10.0 mmol), S10-1 (3.88 g, 20.0 mmol), and 4-(dimethylamino)pyridine (DMAP, 0.30 g, 2.5 mmol) dissolved in dichloromethane (20 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue was purified by silica gel column chromatography to obtain a colorless oily substance S10-3 (2.65 g).

[0586] Step b: Under nitrogen protection, acetonitrile was added, and brominated compound S10-3 (2.41 g, 7.5 mmol), methoxy polyethylene glycolamine S10-4 (27.00 g, 6.0 mmol, Mw approximately 4500, n1 ≈ 100, PDI = 1.03), and N,N-diisopropylethylamine (DIPEA, 1.04 mL, 6.0 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for approximately 20 h. After the reaction was complete, the reaction solution was concentrated and dissolved in dichloromethane. Extraction was performed sequentially with 0.6 M hydrochloric acid (10% sodium chloride) and saturated sodium bicarbonate solution. After ensuring no loss of aqueous phase, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain S10-5 (22.25 g).

[0587] Step c: At room temperature, a solution of undecanoyl chloride (S10-6, 0.84 g, 4.1 mmol) in benzene (50 mL) was slowly added dropwise via a constant pressure burette to a solution of compound S10-5 (16.10 g, 3.4 mmol) in benzene (100 mL) along with triethylamine (17.0 mmol, 2.3 mL, 5 equivalents) and DMAP (66.00 mg). After the reaction was complete, the mixture was diluted with a mixture of hexane and ethyl acetate (approximately 5%), washed with water, washed with brine, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain product E10-1 (12.98 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.07(t, 2H), 3.84–3.45(m, 402H), 3.39(s, 3H), 3.34(t, 2H), 3.18(t, 2H), 2.36(t, 2H), 2.25(t, 2H), 1.71–1.19(m, 36H), 0.86(t, 6H). MALDI-TOF analysis determined the molecular weight of E10⁻¹ to be 4886 Da, with a PDI of 1.03.

[0588]

[0589] Example 11: Synthesis of ethoxylated polyethylene glycol lipid E11-1

[0590]

[0591] In general formula (2), E11-1, R1 is pentadecyl, R2 is nonyl, B3 is a linking bond, B4 is propylene, L7 is a linking bond, L8 is an amide bond (-C(=O)NH-), L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈20, R is ethoxy, and the total molecular weight is approximately 1390.

[0592] The preparation process is as follows:

[0593] Step a: Compound S11-1 (5.53 g, 18.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (24.84 g, 180.0 mmol), compound S11-2 (20.52 g, 90.0 mmol), and tetrabutylammonium bromide (0.58 g, 1.8 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S11-3. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S11-3 (5.27 g).

[0594] Step b: S11-4 (2.00 g, 2.0 mmol, Mw approximately 1000, n1 ≈ 20, PDI = 1.03) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (2.76 g, 20.0 mmol), secondary amine compound S11-3 (4.39 g, 10.0 mmol), and tetrabutylammonium bromide (0.06 g, 0.2 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E11-1. The crude compound E11-1 was purified by column chromatography, concentrated, and dried by oil pump to obtain the target compound ethoxylated polyethylene glycol lipid E11-1 (1.98 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.47 (m, 84H), 3.25–3.20 (q, 2H), 2.62 (t, 2H), 2.49 (m, 4H), 2.19 (t, 2H), 1.74–1.46 (m, 6H), 1.33–1.25 (m, 36H), 1.12 (t, 3H), 0.86 (t, 6H). MALDI-TOF analysis determined the molecular weight of E11-1 to be 1390 Da, with a PDI of 1.03.

[0595]

[0596] Example 12: Synthesis of methoxylated polyethylene glycol lipid E12-1

[0597]

[0598] In general formula (2), E12-1, R1 is undecyl, R2 is tetradecyl, B3 is propylene, B4 is a linking bond, L7 is an ether bond, L8 is a linking bond, L3 is -C(=O)CH2CH2C(=O)-, and A is -OCH2CH2-, where R a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is methoxy group, and the total molecular weight is approximately 2520.

[0599] The preparation process is as follows:

[0600] Compound S12-1 (10.50 g, 5.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.02), compound S9-3 (3.19 g, 7.5 mmol) from Example 9, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.92 g, 10.0 mmol), 1-hydroxybenzotriazole (HOBt, 1.01 g, 7.5 mmol), and TEA (1.01 g, 10.0 mmol) were dissolved sequentially in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After the reaction was complete, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (100 mL * 2), and then backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The target compound, methoxylated polyethylene glycol lipid E12-1 (12.15 g), was obtained by column chromatography purification, concentration, and oil pump drying. 1 ¹H NMR (500MHz, CDCl₃) δ: 4.17(t, 2H), 3.84–3.45(m, 182H), 3.39(s, 3H), 3.20(t, 2H), 3.14(t, 2H), 2.62(t, 2H), 2.36(t, 2H), 1.62–1.46(m, 6H), 1.24–1.19(m, 38H), 0.87(t, 6H). MALDI-TOF analysis determined the molecular weight of E12-1 to be 2519 Da, with a PDI of 1.02.

[0601]

[0602] Example 13: Synthesis of methoxylated polyethylene glycol lipid E13-2

[0603]

[0604] In general formula (2), E13-2, R1 is dodecyl, R2 is decyl, B3 and B4 are connecting bonds, L7 is a connecting bond, L8 is a carbonyl group, L3 is -CH2CH2-, and A is -OCH2CH2-, where R aand R b The atoms are hydrogen atoms, s is 2, n1≈45, R is -OC(=O)CH2CH2COOH, and the total molecular weight is approximately 2480.

[0605] The preparation process is as follows:

[0606] Step a: Compound S13-1 (10.50 g, 5.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.03) and toluene (200 mL) were azeotropically heated at 140 °C to remove water. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (1.01 g, 10.0 mmol) and MsCl (1.04 g, 9.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S13-2 (10.01 g).

[0607] Step b: Compound S13-2 (8.80 g, 4.0 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.03) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S13-3 (3.70 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S13-4. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S13-4 (6.38 g).

[0608] Step c: Compound S13-4 (5.75 g, 2.5 mmol, Mw approximately 2300, n1 ≈ 45, PDI = 1.03), compound S13-5 (0.79 g, 2.8 mmol), and TEA (0.38 g, 3.8 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E13-1 (5.16 g).

[0609] Step d: Removal of the tBu protecting group. In a clean, dry 500 mL round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared. Under ice bath conditions, a dichloromethane solution of E13-1 (5.00 g, 2.0 mmol, Mw approximately 2500, n1 ≈ 45, PDI = 1.03) was slowly added dropwise. The reaction was carried out at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated, purified water was added, and the mixture was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain the final product, methoxylated polyethylene glycol lipid E13-2 (4.58 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.21(t, 2H), 3.84–3.45(m, 180H), 3.35(t, 2H), 3.18(t, 2H), 2.38(t, 2H), 2.30(t, 2H), 2.25(t, 2H), 1.56–1.45(m, 4H), 1.36–1.20(m, 32H), 0.86(t, 6H). MALDI-TOF analysis determined the molecular weight of E13-2 to be 2477 Da, with a PDI of 1.03.

[0610]

[0611] Example 14: Synthesis of polyethylene glycol succinimide-modified lipid E14-1

[0612]

[0613] In general formula (2), E14-1, R1 is tetradecyl, R2 is heptyl, B3 is a linking bond, B4 is heptylene, L7 is a linking bond, L8 is an ether bond (-O-), L3 is -CH2CH2-, and A is -OCH2CH2-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2570.

[0614] The preparation process is as follows:

[0615] S14-1 (8.80 g, 4.0 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.03) was dissolved in 80 mL of water by stirring at room temperature. Potassium carbonate (5.52 g, 50.0 mmol), secondary amine compound S4-2 (8.52 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E14-1. This crude compound was purified by column chromatography, concentrated, and dried by pumping to obtain the target compound, polyethylene glycol succinimide lipid E14-1 (8.35 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.87–3.45 (m, 184H), 2.86 (m, 4H), 2.62 (t, 2H), 2.45 (m, 4H), 2.28 (t, 2H), 1.61–1.46 (m, 8H), 1.35–1.19 (m, 36H), 0.88 (t, 6H). MALDI-TOF analysis determined the molecular weight of E14-1 to be 2574 Da, with a PDI of 1.03.

[0616]

[0617] Example 15: Synthesis of polyethylene glycol maleimide-modified lipid E15-2

[0618]

[0619] In general formula (2), E15-2, R1 is tridecyl, R2 is tetradecyl, B3 and B4 are connecting bonds, L7 is carbonyl, L8 is connecting bond, L3 is -CH2CH2-, and A is -OCH2CH2-, where R a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2600.

[0620] Synthesis Method 1:

[0621] The preparation process is as follows:

[0622] Step a: Compound S15-1 (11.30 g, 5.0 mmol, Mw approximately 2260, n1 ≈ 45, PDI = 1.02) and toluene (200 mL) were azeotropically heated at 140 °C to remove water. After distilling off 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (1.01 g, 10.0 mmol) and methanesulfonyl chloride (MsCl, 1.03 g, 9.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S15-2 (10.48 g).

[0623] Step b: Compound S15-2 (9.36 g, 4.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S1-3 (4.26 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S15-2. This crude compound was purified by column chromatography, concentrated, and dried by pumping to obtain the target compound S15-3 (6.45 g).

[0624] Step c: Compound S15-3 (6.14 g, 2.5 mmol), compound S1-5 (0.89 g, 2.8 mmol), and TEA (0.38 g, 3.8 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by oil pump to obtain the target compound E15-1 (5.23 g). The furan protecting group of E15-1 was removed to obtain the final product, polyethylene glycol maleimide lipid E15-2 (4.68 g). 1¹H NMR (500MHz, CDCl₃) δ: 6.49 (s, 2H), 4.25–4.10 (m, 4H), 3.85–3.46 (m, 180H), 3.35 (t, 2H), 3.17 (t, 2H), 2.73 (t, 2H), 2.24 (t, 2H), 1.58–1.48 (m, 4H), 1.36–1.17 (m, 42H), 0.88 (t, 6H). MALDI-TOF analysis determined the molecular weight of E15-2 to be 2598 Da, with a PDI of 1.02.

[0625]

[0626] Synthesis Method 2:

[0627] The preparation process is as follows:

[0628] Under an argon atmosphere, DCC (2.28 g, 11.0 mmol) was added to a round-bottom flask containing E1-3 (12.24 g, 5.0 mmol), S15-4 (1.69 g, 10.0 mmol), and DMAP (0.15 g, 1.25 mmol) dissolved in dichloromethane (20 mL), and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The mixture was backwashed twice with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography to give the final product, polyethylene glycol maleimide lipid E15-2 (11.00 g, 84.7%). 1 ¹H NMR (500MHz, CDCl₃) δ: 6.49 (s, 2H), 4.25–4.10 (m, 4H), 3.85–3.46 (m, 180H), 3.35 (t, 2H), 3.17 (t, 2H), 2.73 (t, 2H), 2.24 (t, 2H), 1.58–1.48 (m, 4H), 1.36–1.17 (m, 42H), 0.88 (t, 6H). MALDI-TOF analysis determined the molecular weight of E15-2 to be 2598 Da, with a PDI of 1.02.

[0629]

[0630] Example 16: Synthesis of polyethylene glycol azide lipid E16-1

[0631]

[0632] In general formula (2), E16-1, R1 is undecyl, R2 is tetradecyl, B3 is propylene, B4 is a linking bond, L7 is an ether bond, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b It consists of hydrogen atoms, s = 2, n1 ≈ 45, R = -CH2CH2N3, and the total molecular weight is approximately 2520.

[0633] The preparation process is as follows:

[0634] S16-1 (8.80 g, 4.0 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.03) was dissolved in 80 mL of water by stirring at room temperature. Potassium carbonate (5.52 g, 50.0 mmol), secondary amine compound S9-3 (8.52 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E16-1. This crude compound was purified by column chromatography, concentrated, and dried by a pump to obtain the target compound, polyethylene glycol azidolipid E16-1 (8.03 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.85–3.42 (m, 186H), 3.99 (t, 2H), 2.66 (t, 2H), 2.48 (m, 4H), 1.58–1.48 (m, 8H), 1.38–1.19 (m, 38H), 0.85 (t, 6H). MALDI-TOF analysis determined the molecular weight of E16-1 to be 2518 Da, with a PDI of 1.03.

[0635]

[0636] Example 17: Synthesis of polyethylene glycol glycerol-modified lipid E17-2

[0637]

[0638] In general formula (2), E17-2, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈250, R is The total molecular weight is approximately 11,540.

[0639] The preparation process is as follows:

[0640] Step a: Compound S17-1 (56.50 g, 5.0 mmol, Mw approximately 11300, n1 ≈ 250, PDI = 1.02) and toluene (200 mL) were azeotropically heated at 140 °C to remove water. After distilling off 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (1.01 g, 10.0 mmol) and methanesulfonyl chloride (MsCl, 1.03 g, 9.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S17-2 (51.63 g).

[0641] Step b: Compound S17-2 (45.60 g, 4.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S1-3 (4.26 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S17-3. The crude compound S17-3 (31.16 g) was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S17-3.

[0642] Step c: Compound S17-3 (28.90 g, 2.5 mmol), compound S1-5 (0.89 g, 2.8 mmol), and TEA (0.38 g, 3.8 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by oil pump to obtain compound E17-1 (23.83 g).

[0643] Step d: Dissolve the above-mentioned substituted product E17-1 (23.54 g, 2.0 mmol) in 50 mL of THF solution, place in a nitrogen-protected flask, and add TBAF (50 mL, 1 N in THF). React overnight to remove TBS protection. Dry the product over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify the crude product by column chromatography, collect the target eluent, and concentrate to obtain the product polyethylene glycol glycerolized lipid E17-2 (20.20 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.45 (m, 100⁶H), 3.35 (t, 2H), 3.30 (m, 1H), 3.18 (t, 2H), 2.28 (t, 2H), 1.56–1.46 (m, 4H), 1.39–1.19 (m, 42H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E17-2 to be 11542 Da, with a PDI of 1.02.

[0644]

[0645] Example 18: Synthesis of polyethylene glycol lysine-modified lipid E18-2

[0646]

[0647] In general formula (2), E18-2, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2580.

[0648] The preparation process is as follows:

[0649] Step a: Compound S18-1 (13.00 g, 5.0 mmol, Mw approximately 2600, n1 ≈ 45, PDI = 1.02, prepared by coupling lysine protected by bis-Fmoc with polyethylene glycol) was azeotropically dehydrated with toluene (200 mL) at 140 °C. After evaporating 60 mL of solvent, the reaction was allowed to cool to room temperature. TEA (1.01 g, 10.0 mmol) and MsCl (1.03 g, 9.0 mmol) were added, and the reaction was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath, and filtered to obtain compound S18-2 (12.18 g).

[0650] Step b: Compound S18-2 (10.60 g, 4.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S1-3 (4.26 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S18-3. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S18-3 (7.80 g).

[0651] Step c: Compound S18-3 (7.02 g, 2.5 mmol), compound S1-5 (0.89 g, 2.8 mmol), and TEA (0.38 g, 3.8 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by oil pump to obtain compound E18-1 (6.24 g).

[0652] Step d: Removal of the Fmoc protecting group. The concentrated product E18-1 (6.04 g, 2.0 mmol) was treated with 20% piperidine / DMF solution, the solvent was removed by rotary evaporation, dissolved in dichloromethane, precipitated with anhydrous diethyl ether, filtered, and recrystallized from isopropanol to obtain product E18-2 (3.50 g) with two amino groups exposed lysine. The main data of the 1H NMR spectrum of E18-2 are as follows: 4.36 (t, 1H). In the 1H NMR spectrum, the characteristic peak of Fmoc disappeared. 1 ¹H NMR (500MHz, CDCl₃) δ: 4.36(t, 1H), 4.20(t, 2H), 3.83–3.45(m, 180H), 3.35(t, 2H), 3.16(t, 2H), 2.38(t, 2H), 2.27(t, 2H), 2.00–1.76(t, 2H), 1.56–1.46(m, 6H), 1.36–1.19(m, 44H), 0.86(t, 6H). MALDI-TOF analysis determined the molecular weight of E18-2 to be 2575 Da, with a PDI of 1.02.

[0653]

[0654] Example 19: Synthesis of polyethylene glycol tripolylysine-modified lipid E19-2

[0655]

[0656] In general formula (2), E19-2, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2830.

[0657] The preparation process is as follows:

[0658] Step a: Compound S19-1 (14.00 g, 5.0 mmol, Mw approximately 2800, n1 ≈ 45, PDI = 1.03, prepared by polymerization of lysine under double Boc protection followed by coupling with polyethylene glycol) was azeotropically heated with toluene (200 mL) at 140 °C to remove water. After 60 mL of solvent was distilled off, the reaction mixture was cooled to room temperature. TEA (1.01 g, 10.0 mmol) and MsCl (1.03 g, 9.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath, and filtered to obtain compound S19-2 (12.92 g).

[0659] Step b: Compound S19-2 (11.38 g, 4.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S1-3 (4.26 g, 20.0 mmol), and tetrabutylammonium bromide (0.13 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S19-3. This crude compound was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S19-3 (8.66 g).

[0660] Step c: Compound S19-3 (7.52 g, 2.5 mmol), compound S1-5 (0.89 g, 2.8 mmol), and TEA (0.38 g, 3.8 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by oil pump to obtain compound E19-1 (6.85 g).

[0661] Step d: Removal of the Boc protecting group. In a clean, dry 500 mL round-bottom flask, a trifluoroacetic acid / dichloromethane (1:2, v / v) solution was prepared. Under ice bath conditions, a dichloromethane solution containing E19-1 (6.43 g, 2.0 mmol) was slowly added dropwise, and the reaction was carried out at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated, purified water was added, and the mixture was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain polyethylene glycol tripolylysine-modified lipid E19-2 (5.25 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.67(t, 1H), 4.36(t, 2H), 4.20(t, 2H), 3.83–3.45(m, 180H), 3.35(t, 2H), 3.25(t, 2H), 3.18(t, 2H), 2.36(t, 4H), 2.25(t, 2H), 2.00–1.76(t, 6H), 1.56–1.46(m, 10H), 1.36–1.19(m, 48H), 0.86(t, 6H). MALDI-TOF analysis determined the molecular weight of E19-2 to be 2831 Da, with a PDI of 1.03.

[0662]

[0663] Example 20: Synthesis of methoxylated polyethylene glycol lipid E20-1

[0664]

[0665] In the corresponding general formula (2), in E20-1, R1 is R2 is pentadecyl, B3 is hexanediol, B4 is a linking bond, L7 is an amide bond (-C(=O)NH-), L8 is a linking bond, L3 is -CH2CH2-, and A is -OCH2CH2-. Among these, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈40, R is methoxy group, and the total molecular weight is approximately 2380.

[0666] Synthesis Method 1:

[0667] The preparation process is as follows:

[0668] Step a: Compound S20-1 (5.12 g, 20.0 mmol), EDCI (4.61 g, 24.0 mmol), and NHS (2.51 g, 22.0 mmol) were dissolved in dichloromethane (100 mL), and the reaction was carried out overnight at room temperature with stirring. After the reaction was completed, the reaction solution was backwashed twice with 0.1 mol / L HCl (50 mL * 2), and once with saturated sodium chloride (50 mL). The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the target compound S20-2 (6.46 g).

[0669] Step b: Compound S20-2 (6.35 g, 18.0 mmol) was dissolved in dichloromethane (100 mL), and compound S20-3 (4.57 g, 19.8 mmol) and TEA (2.73 g, 27.0 mmol) were added. The mixture was reacted overnight at room temperature, precipitating a large amount of white solid. The solid was filtered, slurried with methanol (30 mL), filtered, washed twice with methanol, and the solid was collected and dried to obtain the substituted product (4.51 g). The substituted product (4.37 g, 15.0 mmol) was dissolved in 50 mL of THF solution and placed in a nitrogen-protected flask. TBAF (50 mL, 1 N in THF) was added. The reaction was allowed to proceed overnight to remove TBS protection. The product was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography, and the target eluent was collected and concentrated to obtain product S20-4 (4.50 g).

[0670] Step c: Compound S20-4 (4.26 g, 12.0 mmol) and toluene (200 mL) were azeotropically heated at 140 °C to remove water. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (2.42 g, 24.0 mmol) and MsCl (2.46 g, 21.6 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath, and filtered to obtain compound S20-5 (4.70 g).

[0671] Step d: Compound S20-5 (4.33 g, 10.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (13.80 g, 100.0 mmol), compound S20-6 (11.35 g, 50.0 mmol), and tetrabutylammonium bromide (0.32 g, 1.0 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S20-7. The crude compound S20-7 (3.98 g) was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S20-7.

[0672] Step e: S20-8 (1.90 g, 1.0 mmol, Mw approximately 1900, n1 ≈ 40, PDI = 1.03) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (1.38 g, 10.0 mmol), secondary amine compound S20-7 (2.83 g, 5.0 mmol), and tetrabutylammonium bromide (0.03 g, 0.1 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E20-1. The crude compound E20-1 was purified by column chromatography, concentrated, and dried by oil pump to obtain the target compound methoxylated polyethylene glycol lipid E20-1 (1.75 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.46 (m, 162H), 3.37 (s, 3H), 3.25–3.20 (q, 2H), 2.64 (t, 2H), 2.48 (m, 4H), 2.27–2.25 (m, 1H), 1.56–1.46 (m, 10H), 1.32–1.25 (m, 48H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E₂O₁ to be 2382 Da, with a PDI of 1.03.

[0673]

[0674] Synthesis Method 2:

[0675] The preparation process is as follows:

[0676] Step a: Compound S20-2 (6.35 g, 18.0 mmol) was dissolved in dichloromethane (100 mL), and compound S20-9 (4.57 g, 19.8 mmol) and TEA (2.73 g, 27.0 mmol) were added. The mixture was reacted overnight at room temperature, and a large amount of white solid precipitated. The solid was filtered, slurried with methanol (30 mL), filtered, washed twice with methanol, and the solid was collected and dried to give compound 20-10 (5.45 g).

[0677] Step b: Compound S20-10 (3.54 g, 10.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (13.80 g, 100.0 mmol), compound S20-11 (15.3 g, 50.0 mmol), and tetrabutylammonium bromide (0.32 g, 1.0 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S20-7. The crude compound S20-7 (4.07 g) was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S20-7.

[0678] Step c: S20-8 (1.90 g, 1.0 mmol, Mw approximately 1900, n1 ≈ 40, PDI = 1.03) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (1.38 g, 10.0 mmol), secondary amine compound S20-7 (2.83 g, 5.0 mmol), and tetrabutylammonium bromide (0.03 g, 0.1 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound E20-1. The crude compound E20-1 was purified by column chromatography, concentrated, and dried by oil pump to obtain the target compound methoxylated polyethylene glycol lipid E20-1 (1.82 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 3.83–3.46 (m, 162H), 3.37 (s, 3H), 3.26 (t, 2H), 3.25–3.20 (q, 2H), 3.14 (t, 2H), 2.27–2.25 (m, 3H), 1.56–1.46 (m, 8H), 1.32–1.25 (m, 50H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E₂O₁ to be 2382 Da, with a PDI of 1.03.

[0679]

[0680] Example 21: Synthesis of methoxylated polyethylene glycol lipid E21-1

[0681]

[0682] In the corresponding general formula (2), E21-1, R1 is R2 is decyl, B3 is hexylene, B4 is a linking bond, L7 is a carboxyl group (-C(=O)O-), L8 is a carbonyl group, L3 is -CH2CH2-, and A is -OCH2CH2-. Among these, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is methoxy group, and the total molecular weight is approximately 2560.

[0683] The preparation process is as follows:

[0684] Step a: 2-Hexyldecanoic acid (S21-1, 10.00 g, 39.1 mmol) was dissolved in anhydrous dichloromethane (DCM, 100 mL) in a nitrogen-protected flask. After the mixture cooled to 0–10 °C, 1,6-hexanediol (S21-2, 9.23 g, 78.1 mmol) and DMAP (5.72 g, 46.9 mmol) were carefully added to the solution, followed by the addition of EDCI (81.60 g, 430.0 mmol) in portions. The reaction was then brought back to room temperature to continue. After 16 h of reaction, TLC showed that S21-1 was completely consumed. The reaction solution was washed twice with 500 mL of a 0.4 N HCl / 10% NaCl mixed solution, and then once with saturated brine. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated and purified by silica gel column chromatography. The target eluent was collected and concentrated to obtain product S21-3 (8.12 g).

[0685] Step b: The condensation product (S21-3, 5.00 g, 14.1 mmol) was dissolved in 500 mL of DCM solution. After the solution cooled to 0 °C, 2,2,6,6-tetramethylpiperidine oxide (Tempo, 1.10 mg) and KBr solution (2.01 g, 16.9 mmol) were added to the solution and dissolved in 50 mL of purified water. NaClO solution (18.3 mmol) was slowly added dropwise. After the addition was complete, the raw material was monitored by TLC until it was completely consumed. The reaction was quenched with sodium sulfite solution, and the mixture was brought to room temperature and extracted twice with 50 mL of DCM. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography, and the target eluent was collected and concentrated to obtain the oxidation product S21-4 (2.40 g).

[0686] Step c: Dissolve the above-mentioned oxidation product S21-4 (2.00 g, 5.7 mmol) in 50 mL of THF and 50 mL of methanol solution. After the solution is cooled to 0 °C, add methoxy polyethylene glycol ethylamine (S21-5, 12.92 g, 6.3 mmol, Mw approximately 2050, n1 ≈ 45, PDI = 1.02) and glacial acetic acid (0.38 g, 6.3 mmol) to the solution. Slowly add NaBH(OAc)3 (5.38 g, 19.4 mmol) in portions. After the addition is complete, continue the reaction for 2 h, and monitor the consumption of the raw materials by TLC. Quench with saturated sodium bicarbonate solution, return to room temperature, concentrate and remove THF and methanol, then extract the concentrate twice with 200 mL of DCM. Combine the organic phases, dry with anhydrous magnesium sulfate, filter and concentrate to obtain the crude product. The crude product was separated and purified by silica gel column chromatography. The target eluent was collected and concentrated to obtain the final product S21-6 (8.41 g).

[0687] Step d: Compound S21-6 (8.00 g, 3.3 mmol), compound S21-7 (0.82 g, 4.0 mmol), and TEA (0.51 g, 5.0 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E21-1 (7.02 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.09 (m, 2H), 3.83–3.43 (m, 182H), 3.37 (s, 3H), 3.35 (t, 2H), 3.15 (t, 2H), 2.26–2.25 (m, 3H), 1.56–1.46 (m, 10H), 1.32–1.25 (m, 38H), 0.88 (t, 9H). MALDI-TOF analysis determined the molecular weight of E₂₁-1 to be 2561 Da, with a PDI of 1.02.

[0688]

[0689] Example 22: Synthesis of polyethylene glycol-folic acidified lipid E22-1

[0690]

[0691] In general formula (2), E22-1, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2910.

[0692] Step a: Compound S22-1 (13.60 g, 6.8 mmol, Mw approximately 2000, n1 ≈ 45, PDI = 1.03) was dissolved in dichloromethane (300 mL), and TEA (1.01 g, 10.1 mmol) and di-tert-butyl dicarbonate (Boc2O, 2.02 g, 10.1 mmol) were added at room temperature. The reaction was carried out overnight at room temperature. After the reaction was completed, the reaction solution was concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S22-2 (12.50 g).

[0693] Step b: Compound S22-2 (10.70 g, 5.0 mmol) and toluene (200 mL) were azeotropically heated at 140 °C to remove water. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (1.01 g, 10.0 mmol) and MsCl (1.02 g, 9.0 mmol) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice more with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath conditions, and filtered to obtain compound S22-3 (9.00 g).

[0694] Step c: Compound S22-3 (8.88 g, 4.0 mmol) was added to 80 mL of water and dissolved by stirring at room temperature. Potassium carbonate (5.52 g, 40.0 mmol), compound S1-3 (4.26 g, 20.0 mmol), and tetrabutylammonium bromide (0.12 g, 0.4 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, and the mixture was backwashed once with saturated sodium chloride aqueous solution (100 mL). The organic phase was retained, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S22-4. The crude compound S22-4 was purified by column chromatography, concentrated, and dried by pumping dry to obtain the target compound S22-4 (7.27 g).

[0695] Step d: Compound S22-4 (7.14 g, 3.0 mmol), compound S1-5 (1.46 g, 4.5 mmol), and TEA (0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound S22-5 (6.41 g).

[0696] Step e: Removal of the Boc protecting group. In a clean, dry 500 mL round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). S22-5 (6.00 g, 2.3 mmol) in dichloromethane is slowly added dropwise under ice bath conditions, and the reaction is carried out at room temperature for 2 hours. After the reaction is complete, the reaction solution is concentrated, purified water is added, and the mixture is extracted with dichloromethane. The extract is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to obtain S22-6 (5.35 g).

[0697] Step f: In a clean, dry 1000 mL round-bottom flask, add folic acid dissolved in DMSO (S22-7, 1.49 g, 3.4 mmol), polyethylene glycol ethylamine lipids (S22-6, 5.00 g, 2.0 mmol), pyridine (20 mL), and DCC (1.85 g, 9.0 mmol). Stir the mixture at room temperature for 4 h. Monitor the reaction using TLC until the production of the final product is detected and the starting material, polyethylene glycol ethylamine lipids, disappears from the reaction mixture. Then, concentrate the solution under reduced pressure using a rotary evaporator to remove pyridine. Add 300 mL of water to the solution, centrifuge to remove trace amounts of insoluble matter, and dialyze the supernatant obtained from centrifugation in saline and water. Monitor the dialysate using TLC until it contains only the final product (only one spot is detected on the TLC plate). Freeze-dry the aqueous solution of the dialysate to obtain the final product, polyethylene glycol ethylamine lipid folic acid derivative (E22-1, 5.35 g). 1¹H NMR (500MHz, CDCl₃) δ: 8.71 (s, 1H), 7.66–7.64 (d, 2H), 6.67 (d, 2H), 4.49–4.48 (d, 2H), 4.32–4.20 (m, 1H), 3.83–3.45 (m, 184H), 3.35 (t, 2H), 3.25–3.20 (q, 2H), 3.19 (t, 2H), 2.34 (t, 2H), 2.28 (t, 2H), 2.08–1.94 (m, 2H), 1.56–1.46 (m, 4H), 1.36–1.19 (m, 42H), 0.86 (t, 6H). MALDI-TOF analysis determined the molecular weight of E₂₂-1 to be 2913 Da, with a PDI of 1.03.

[0698]

[0699] Example 23: Synthesis of polyethylene glycol biotinylated lipid E23-1

[0700]

[0701] In general formula (2), E23-1, R1 is tridecyl, R2 is tetradecyl, B3 is a linking bond, B4 is a linking bond, L7 is a carbonyl group, L8 is a linking bond, L3 is -CH2CH2O-, and A is -CH2CH2O-. Wherein, R... a and R b For hydrogen atoms, s is 2, n1≈45, and R is... The total molecular weight is approximately 2720.

[0702] The preparation process is as follows:

[0703] In a clean, dry 1000 mL round-bottom flask, biotin succinimide ester dissolved in DMF (S23-1, 1.87 g, 5.5 mmol) and polyethylene glycol ethylamined lipid dissolved in DCM (S22-6, 12.50 g, 5.0 mmol, Mw approximately 2500, n1 ≈ 45, PDI = 1.03) were added and stirred until homogeneous. Then, TEA (1.52 g, 15.0 mmol) was added, and the reaction was carried out at room temperature. TLC monitoring was used until the reaction was complete. After the reaction, the mixture was filtered, concentrated, purified by column chromatography, and the eluent was collected, concentrated, and lyophilized to obtain the polyethylene glycol-modified lipid biotin derivative (E23-1, 9.82 g). 1¹H NMR (CDCl₃) δ: 4.36–4.25 (m, 2H), 3.83–3.45 (m, 184H), 3.35 (t, 2H), 3.20 (m, 1H), 3.18 (t, 2H), 2.81 (t, 2H), 2.26 (t, 2H), 2.22–2.18 (m, 2H), 1.70–1.40 (m, 8H), 1.36–1.21 (m, 42H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E₂₃⁻ to be 2716 Da, with a PDI of 1.03.

[0704]

[0705] Example 24: Synthesis of methoxylated polyethylene glycol lipid E24-1

[0706]

[0707] In general formula (2), E24-1, R1 and R2 are methyl groups, B3 and B4 are tridecylene groups, L7 and L8 are ether bonds (-O-), L3 is -(C=O)CH2CH2(C=O)NH-, and A is -CH2CH2O-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is methyl, and the total molecular weight is approximately 2530.

[0708] The preparation process is as follows:

[0709] The methoxy-polyethylene glycol succinic acid derivative S24-1 (10.50 g, 5.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.02), compound S6-4 (3.32 g, 7.5 mmol), EDCI (1.92 g, 10.0 mmol), HOBt (1.01 g, 7.5 mmol), and TEA (1.01 g, 10.0 mmol) containing amide bonds were successively dissolved in dichloromethane (100 mL), and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (100 mL * 2), and then backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The target compound, methoxylated polyethylene glycol lipid E24-1 (10.55 g), was obtained by column chromatography purification, concentration, and oil pump drying. 1¹H NMR (500MHz, CDCl₃) δ: 3.83–3.45 (m, 184H), 3.38 (s, 3H), 3.32 (s, 6H), 3.26–3.17 (m, 4H), 2.43–2.36 (m, 4H), 1.66–1.42 (m, 8H), 1.35–1.19 (m, 36H). MALDI-TOF analysis determined the molecular weight of E24-1 to be 2536 Da, with a PDI of 1.02.

[0710]

[0711] Example 25: Synthesis of methoxylated polyethylene glycol lipid E25-1

[0712]

[0713] In general formula (2), E25-1, R1 and R2 are methyl groups, B3 and B4 are tridecylene groups, L7 and L8 are ether bonds (-O-), L3 is -(C=O)CH2CH2CH2NH(C=O)O-, and A is -CH2CH2O-. Wherein, R... a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is methyl, and the total molecular weight is approximately 2560.

[0714] The preparation process is as follows:

[0715] Step a: S25-1 (1.91 g, 12.0 mmol) and compound S25-2 (16.80 g, 8.0 mmol, Mw approx. 2100, n1 ≈ 45, PDI = 1.02) were dissolved in DCM and cooled in an ice bath. Pyridine (20 mL) was added to the mixture and stirred overnight. After the reaction was complete, the solvent was removed and the residue was dried under high vacuum. The residue was dissolved in dichloromethane and washed with bicarbonate and water. The crude product was purified by column chromatography to give compound S25-3 (15.84 g).

[0716] Step b: The above compound S25-3 (15.40 g, 7.0 mmol, Mw approximately 2200, n1 ≈ 45, PDI = 1.02) was dissolved in dichloromethane (80 mL), and then trifluoroacetic acid (80 mL) was added. The reaction was carried out at room temperature for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain compound S25-4 (10.85 g).

[0717] Step c: S25-1 (10.50 g, 5.0 mmol, Mw approx. 2100, n1 ≈ 45, PDI = 1.02), compound S6-4 (3.32 g, 7.5 mmol), EDCI (1.92 g, 10.0 mmol), HOBt (1.01 g, 7.5 mmol), and TEA (1.01 g, 10.0 mmol) were dissolved sequentially in dichloromethane (100 mL), and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (100 mL * 2), and then backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried by pumping to obtain the target compound, methoxylated polyethylene glycol lipid E25-1 (10.30 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.04–3.45 (m, 184H), 3.38 (s, 3H), 3.32 (s, 6H), 3.26–3.17 (m, 4H), 3.23 (m, 2H), 2.34 (m, 2H), 1.84 (m, 2H), 1.66–1.42 (m, 8H), 1.35–1.19 (m, 36H). MALDI-TOF analysis determined the molecular weight of E25-1 to be 2566 Da, with a PDI of 1.02.

[0718]

[0719] Example 26: Synthesis of methoxylated polyethylene glycol lipid E26-1

[0720]

[0721] In general formula (2), E26-1, R1 is nonyl, R2 is tridecyl, B3 is pentylenetene, B4 is a linking bond, L7 is -OC(=O)-, L8 is -C(=O)-, L3 is -CH2CH2(C=O)O-, and A is -CH2CH2O-, where R a and R b The atoms are hydrogen atoms, s is 2, n1≈45, R is methyl, and the total molecular weight is approximately 2530.

[0722] Synthesis Method 1:

[0723] The preparation process is as follows:

[0724] Step a: Under nitrogen protection, acetonitrile was added, and brominated derivative S10-3 (2.41 g, 7.5 mmol), methoxy polyethylene glycol alanine ester derivative S26-1 (12.60 g, 6.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.03, prepared by reacting methoxy polyethylene glycol with β-alanine containing a Boc-protected amino group), and DIPEA (1.04 mL, 6.0 mmol) were added sequentially with slow stirring. The reaction was stirred at room temperature for approximately 20 h. After the reaction was completed, the reaction solution was concentrated and dissolved in dichloromethane. Extraction was performed sequentially with 0.6 M hydrochloric acid (10% sodium chloride) and saturated sodium bicarbonate solution. After ensuring no loss of aqueous phase, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain S26-2 (10.63 g).

[0725] Step b: S26-2 (9.20 g, 4.0 mmol, Mw approx. 2300, n1 ≈ 45, PDI = 1.03), compound S1-5 (1.56 g, 4.8 mmol), and TEA (0.61 g, 6.0 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E26-1 (8.16 g). 1 ¹H NMR (500MHz, CDCl₃) δ: 4.15 (m, 2H), 4.07 (t, 2H), 3.83–3.45 (m, 178H), 3.39 (s, 3H), 3.45–3.18 (m, 4H), 2.36 (t, 2H), 2.25 (t, 2H), 2.61 (m, 2H), 1.71–1.19 (m, 42H), 0.86 (t, 6H). MALDI-TOF analysis determined the molecular weight of E26-1 to be 2535 Da, with a PDI of 1.03.

[0726]

[0727] Synthesis Method 2:

[0728] The preparation process is as follows:

[0729] Step a: The methoxy polyethylene glycol 3-hydroxypropionate derivative S26-3 (21.00 g, 10.0 mmol, Mw approximately 2100, n1 ≈ 45, PDI = 1.03, obtained by reacting methoxy polyethylene glycol with 3-hydroxypropionic acid containing TBS-protected hydroxyl groups and then deprotecting) and toluene (200 mL) were azeotropically dehydrated at 140 °C. After 60 mL of solvent was distilled off, the reaction was cooled to room temperature. TEA (2.02 g, 20.0 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), and the aqueous phase was retained. The aqueous phase was then extracted twice with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, recrystallized under ice bath, and filtered to obtain compound S26-4 (19.62 g).

[0730] Step b: S26-4 (17.60 g, 8.0 mmol, Mw approx. 2200, n1 ≈ 45, PDI = 1.03) was added to 80 mL of water and stirred to dissolve at room temperature. Potassium carbonate (11.04 g, 80.0 mmol), compound S26-5 (10.28 g, 40.0 mmol, obtained by reacting nonanol with 6-aminohexanoic acid containing a Boc-protected amino group and then deprotecting, see step a of Example 10), and tetrabutylammonium bromide (0.26 g, 0.8 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, backwashed once with saturated sodium chloride aqueous solution (100 mL), the organic phase was retained, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by column chromatography to obtain compound S26-2 (5.72 g).

[0731] Step c: S26-2 (4.60 g, 2.0 mmol, Mw approx. 2300, n1 ≈ 45, PDI = 1.03), compound S1-5 (0.78 g, 2.4 mmol), and TEA (0.30 g, 3.0 mmol) were dissolved in dichloromethane (100 mL) and reacted overnight at room temperature with stirring. After concentration, the reaction solution was dissolved in 100 mL of water and extracted twice with EtOAc (100 mL * 2). The aqueous phase was retained, sodium chloride was added, and the solution was extracted twice with dichloromethane (100 mL * 2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography, concentrated, and dried with a pump to obtain the target compound E26-1 (4.03 g). 1¹H NMR (500MHz, CDCl₃) δ: 4.15 (m, 2H), 4.07 (t, 2H), 3.83–3.45 (m, 178H), 3.39 (s, 3H), 3.45–3.18 (m, 4H), 2.36 (t, 2H), 2.25 (t, 2H), 2.61 (m, 2H), 1.71–1.19 (m, 42H), 0.86 (t, 6H). MALDI-TOF analysis determined the molecular weight of E26-1 to be 2535 Da, with a PDI of 1.03.

[0732]

[0733] Example 27: Preparation of cationic lipid P-9

[0734]

[0735] In the corresponding general formula (1), P-9, L1 and L2 are the same and both are -(C=O)O-, L3 is a connecting bond, and B1 and B2 are both... R1 and R2 are both R3 is a hydrogen atom, and A is -(CR) a R b ) s O-, R a and R b All are hydrogen atoms, with 2 s and 2 n.

[0736] Step a: 2-Hexyldecanoic acid (S27-1, 100.00 g, 390.6 mmol) was dissolved in anhydrous dichloromethane (DCM, 1 L) in a nitrogen-protected flask. After the mixture cooled to 0–10 °C, 1,6-hexanediol (S27-2, 92.30 g, 781.3 mmol) and DMAP (57.20 g, 468.7 mmol) were carefully added to the solution, followed by the addition of EDCI (81.60 g, 430.0 mmol) in portions. The reaction was then brought back to room temperature to continue. After 16 h of reaction, TLC showed that S27-1 was completely consumed. The reaction solution was washed twice with 500 mL of a 0.4 N HCl / 10% NaCl mixed solution, and then once with saturated brine. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated to obtain the crude product. The crude product was separated and purified by silica gel column chromatography. The target eluent was collected and concentrated to obtain product S27-3 (79.00 g).

[0737] Step b: The condensation product (S27-3, 50.0 g, 140.5 mmol) was dissolved in 500 mL of DCM solution. After the solution cooled to 0 °C, 2,2,6,6-tetramethylpiperidine oxide (Tempo, 11.00 mg) and KBr solution (20.10 g, 168.6 mmol) were added to the solution and dissolved in 50 mL of purified water. NaClO solution (182.6 mmol) was slowly added dropwise. After the addition was complete, the raw material was monitored by TLC until it was completely consumed. The reaction was quenched with sodium sulfite solution, and the mixture was brought to room temperature and extracted twice with 500 mL of DCM. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography, and the target eluent was collected and concentrated to obtain the oxidation product S27-4 (23.00 g).

[0738] Step c: The above oxidation product (S27-4, 20.00 g, 56.5 mmol) was dissolved in 200 mL of THF and 20 mL of methanol solution. After the solution was cooled to 0 °C, diethylene glycolamine (S27-5, 2.80 g, 26.9 mmol) and glacial acetic acid (1.61 g, 26.9 mmol) were added to the solution. NaHB(OAc)3 (17.50 g, 82.5 mmol) was slowly added in portions. After the addition was complete, the reaction was continued for 2 h, and the raw material was monitored by TLC until it was completely consumed. The reaction was quenched with saturated sodium bicarbonate solution, and the mixture was brought back to room temperature. THF and methanol were concentrated to remove the residues. The concentrate was then extracted twice with 200 mL of DCM. The organic phase was dried over anhydrous MgSO4, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography. The target eluent was collected and concentrated to obtain product P-9 (12.00 g). The main data of the 1H NMR spectrum of P-9 are as follows: 1 H NMR (400MHz, CDCl3) δ4.07 (t, J = 5.9Hz, 4H), 3.70 (s, 2H), 3.63 (m, 4H), 2.65 ( m,2H),2.49(m,4H),2.32(m,2H),1.70-1.22(m,64H),0.88(d,J=6.3Hz,12H).

[0739]

[0740] Example 28: Preparation of cationic liposomes

[0741] In this embodiment, multiple groups of cationic liposomes were prepared for comparison. The neutral lipids in each group of cationic liposomes were all DSPC, and the sterol lipids were all cholesterol. The difference lay in the two components: cationic lipids and polyethylene glycol-modified lipids. Specifically, the control group (L-0) contained DLin-MC3-DMA (MC3) as cationic lipids and polyethylene glycol-modified lipids PEG2k-DMG (DMG); the experimental groups (L-1 to L-28 and L-31) contained MC3 as cationic lipids and polyethylene glycol-modified lipids prepared in the examples of this application; the experimental groups (L-29 to L-30) contained P-9 as cationic lipids prepared in the examples of this application and polyethylene glycol-modified lipids E1-1 or E22-1 prepared in this application; as shown in Table 1.

[0742] The preparation method of cationic liposomes is as follows:

[0743] Step a: According to the molar ratio of each lipid as cationic lipid:DSPC:cholesterol:polyglycolic lipid of 50:10:40:1.5, weigh the cationic lipid CL (15.0 μmol), distearate phosphatidylcholine (DSPC, 3.0 μmol), cholesterol (12.0 μmol), and polyethylene glycol-modified lipid PL (0.45 μmol) from groups L-0 to L-30 in Table 1 into a 100 mL round-bottom flask, add 30 mL of chloroform to fully dissolve the solids, and shake well;

[0744] Step b: Use a rotary evaporator at a speed of 140 rpm and a temperature of 55°C to remove the solvent chloroform by reduced pressure to form a thin oil film. Then use a vacuum pump to dry for 12 hours to ensure that all chloroform is removed.

[0745] Step c: Add 30 mL of phosphate buffer (PBS, pH = 7.4) containing 10% lactose to the flask, and use an ultrasonic cleaner to sonicate for 30 min at a frequency of 90% to form a translucent emulsion;

[0746] Step d: Add the emulsion to a high-pressure homogenizer and over-press it 5 times at a pressure of 100 MPa; then add the emulsion to a liposome extruder and over-press it 10 times at a pressure of 150 MPa to prepare cationic liposome powders from L-0 to L-30.

[0747] Using the above steps ad, the molar ratio of each lipid was cationic lipid:DSPC:cholesterol:polyethylene glycol-modified lipid of 48:9:42:1.5. Cationic lipid CL (16.0 μmol), distearate phosphatidylcholine (DSPC, 3.0 μmol), cholesterol (14.0 μmol), and polyethylene glycol-modified lipid PL (0.5 μmol) from the L-31 group in Table 1 were weighed out. Under the same conditions, L-31 cationic liposome powder was prepared.

[0748] Example 29: Preparation of cationic liposome nucleic acid drug composition formulation

[0749] Step a: Take 0.03 mL of physiological saline as the working solution for the nucleic acid drug composition formulation;

[0750] Step b: Weigh 0.1 mg of the cationic liposomes (L-0 to L-31) prepared in Example 28, dissolve them in physiological saline, and equilibrate for 30 min. Weigh 1.00 μg of siRNA and dissolve it in 10 μL of physiological saline according to a N / P ratio of 10 / 1. Combine this with the physiological saline containing the cationic liposomes and equilibrate for 30 min to prepare a control group (L-0 / siRNA) and the cationic liposome nucleic acid drug composition formulation described in this invention (referred to as LNP / siRNA formulation, L-1 / siRNA to L-31 / siRNA).

[0751] Example 30: Biological activity test of cationic liposome nucleic acid drug composition formulation

[0752] (1) Research on gene compounding ability

[0753] The gene recombination capacity of LNP / siRNA in each group was investigated using gel permeation electrophoresis. 0.8 g of agarose was dissolved in 40 mL of TAE solution and heated in a microwave oven until the agarose particles were completely dissolved. After cooling, 5 μL of the nucleic acid dye GelGreen was added to the cooled agarose gel. The gel was then placed in a gel bath and allowed to air dry. A mixture of LNP / siRNA and 2 μL of Loading Buffer was added to the wells of the agarose gel, and electrophoresis was performed at 90 V for 10 min at room temperature. The results showed that the experimental groups (L-1 / siRNA to L-31 / siRNA) and the control group (L-0 / siRNA) contained virtually no free siRNA, indicating that the polyethylene glycol-modified lipid-modified cationic liposome nucleic acid drug composition of this invention has a strong mRNA recombination capacity.

[0754] Table 1 - Summary of the formulations of various liposomes and the particle size and encapsulation efficiency of the LNP / siRNA prepared from them.

[0755]

[0756] Encapsulation efficiency determination: LNP / siRNA was ultracentrifuged (4℃, 60000rpm, 1h) using an ultracentrifuge. The concentration of unencapsulated siRNA in the supernatant was detected using a nucleic acid quantification instrument. The encapsulation efficiency of siRNA by liposomes was calculated. The results are summarized in Table 1, showing that the cationic liposomes (L-1 to L-31) of the present invention have a high nucleic acid encapsulation efficiency for nucleic acid drugs. DMG is a polyethylene glycol-modified lipid that has been successfully applied to mRNA vaccines. In contrast, when paired with the same cationic lipid MC3, the encapsulation rates of siRNA by L-1 (polyethylene glycol-modified lipid E1-1), L-3 (polyethylene glycol-modified lipid E2-1), L-7 (polyethylene glycol-modified lipid E6-1), L-12 (polyethylene glycol-modified lipid E10-1), L-16 (polyethylene glycol-modified lipid E14-1), L-17 (polyethylene glycol-modified lipid E15-2), and L-26 (polyethylene glycol-modified lipid E24-1) of this application are all greater than those by DMG. The cationic lipids used in L-30 to L-31 are cationic lipids prepared in this application, which also show high encapsulation rates. The cationic liposomes prepared by L-29 using different lipid ratios also show high nucleic acid encapsulation rates.

[0757] Particle size determination: In this embodiment, the particle size of LNP / siRNA was determined by dynamic light scattering (DLS). The measured LNP / siRNA showed high size uniformity, with a PDI of less than 0.3 for all samples. Using the polyethylene glycol-modified lipids of this invention, when n1 is approximately in the range of 40-100, the particle size of the prepared LNP / siRNA is in the range of 90-130 nm.

[0758] (2) Study on serum stability

[0759] In this experiment, the serum stability of the PEGylated lipid-modified cationic liposome nucleic acid drug composition was tested using the liposome particle size change method. In this example, 0.5 mL of LNP / siRNA formulations L-0 to L-31 (N / P = 10 / 1) were added to 0.5 mL of culture medium containing 10% fetal bovine serum (FBS). The mixture was stirred at 37°C, and samples were taken periodically to measure the particle size change of the cationic liposome nucleic acid drug composition. The serum stability of the cationic liposome nucleic acid drug composition was analyzed by measuring the particle size change. The serum stability of the cationic liposome nucleic acid drug composition was also measured using the same method with 0.5 mL of PBS added instead of the culture medium. The experimental results showed that, within 48 hours, compared to L-0, the particle size changes of the L-1 to L-31 cationic liposome nucleic acid drug compositions were smaller in both PBS solution and serum. This indicates that the PEGylated lipid-modified cationic liposomes of the present invention are relatively stable and have good serum stability, meeting the requirements for long-circulating cationic liposomes.

[0760] (3) Cytotoxicity (biocompatibility) studies

[0761] The cytotoxicity of the cationic liposome nucleic acid drug composition of the present invention was tested using the MTT staining method. The cationic liposome nucleic acid drug composition was dissolved in culture medium to prepare the required concentration. If necessary, an appropriate amount of solubilizer could be added. HeLa cells were used as a cell model, with a seeding density of 1×10⁶ cells / year. 4 100 μL of cell suspension per well was seeded into 96-well plates. After seeding, the cells were incubated at 37°C and 4% CO2 for 24 h. Then, the old culture medium was discarded, and 100 μL of culture medium containing 3.3 μg / mL LNP / siRNA (a cationic liposome nucleic acid drug composition, prepared in Example 41) was added to each well. For the blank control group, 100 μL of fresh culture medium was added. Each concentration was repeated in 6 replicates. After co-incubating the cationic liposome nucleic acid drug composition with HeLa cells for 24 h, 20 μL of 5 mg / mL MTT in PBS buffer was added to each well. After incubating MTT with cancer cells for 4 h, the mixture of culture medium and MTT buffer was discarded, and 150 μL of DMSO per well was added to dissolve the purple formazan crystals from the live cells. After thorough shaking, the absorbance was measured using a microplate reader. Calculations based on the measured absorbance values ​​showed that, compared with the blank control group, the cell viability of the cationic liposome nucleic acid drug composition prepared in this invention was greater than 93%, indicating that the PEGylated lipid-modified cationic liposome nucleic acid drug composition of this invention has good biocompatibility.

[0762] (4) Research on nucleic acid transport efficiency

[0763] To investigate the nucleic acid transport efficiency of the various cationic liposome nucleic acid drug formulations prepared by polyethylene glycol-modified lipids according to the present invention, this experiment used Luc-Hela cells stably expressing luciferase as a model to investigate the nucleic acid transport efficiency of the various cationic liposome nucleic acid drug formulations (described in Example 29) with an N / P ratio of 10 / 1. In 96-well plates, when the cell density reached 80%, the various cationic liposome nucleic acid drug formulations were added to the culture medium and incubated for 24 h at 37°C and 4% CO2. The old culture medium was then discarded, and cultured again for 24 h with medium containing 3.3 μg / mL LNP / siRNA. Cells were treated with a dissolution buffer, and fluorescence intensity was measured using a luciferase assay kit on a substrate chemiluminescence detector. A blank / siRNA group was also included, where the same dose of naked siRNA was added to the cultured cells. Since siRNA transcribed into cells can inhibit the expression of the luciferase gene Fluc mRNA, untreated cells were used as a negative control group. The expression of the target gene (i.e., fluorescence intensity) in the blank cell group was set as 100%. The results showed that the fluorescence intensity of the blank / siRNA group was approximately 78%, the fluorescence intensity of the L-0 / siRNA group was approximately 43%, the fluorescence intensity of the L-1 / siRNA group was approximately 29%, the fluorescence intensity of the L-7 / siRNA group was approximately 31%, the fluorescence intensity of the L-26 / siRNA group was approximately 39%, and the fluorescence intensity of the L-27 / siRNA group was approximately 36%. Compared with the blank cell group and the siRNA group, both the control group and the experimental group showed better luciferase gene inhibition, indicating that cationic liposomes can improve the transport efficiency of nucleic acid drugs. Except for the L-10 / siRNA, L-12 / siRNA and L-19 / siRNA groups, the other experimental groups showed better luciferase gene inhibition (fluorescence intensity less than 43%) than the control group L-0 / siRNA, indicating that the cationic liposomes prepared by the polyethylene glycol-modified lipids of the present invention can improve the nucleic acid transport efficiency of liposomes.

[0764] (5) Study on tumor cell uptake rate

[0765] To investigate whether PEGylated cationic liposomes with folic acid targeting groups can enhance the uptake of cationic liposomes by tumor cells, this invention used nasopharyngeal carcinoma cells (KB) with high expression of folic acid receptors on their cell surfaces and hepatocellular carcinoma cells (HepG2) with low expression of folic acid receptors on their cell surfaces as models to evaluate the cell uptake of liposomes. Cells were seeded in 6-well plates, and when the cell density was approximately 80%, the supernatant was discarded. 0.2 mL and 1.8 mL of culture medium were added to each well for L-29 / siRNA (without folic acid targeting groups) and L-30 / siRNA (with folic acid targeting groups), respectively, with three replicates per sampling point. After 1, 3, and 6 hours of culture, the supernatant was collected, cells were digested with trypsin and washed with PBS, and transferred to flow cytometry tubes. Cell counts were collected at 1×10⁶ cells / well using a flow cytometer. 4 Cells were counted, and the percentage of positive cells (i.e., cells with fluorescence intensity greater than 10 units) was calculated. Uptake rate is expressed as the percentage of positive cells. Experimental results showed that in hepatocellular carcinoma cells with low folate receptor expression, the difference in uptake rate of L-29 / siRNA without a folate target group and L-30 / siRNA with a folate target group was small. Compared with L-29 / siRNA without a folate target group, HepG2 cells showed approximately a 4% increase in uptake of L-30 / siRNA with a folate target group. However, in nasopharyngeal carcinoma cells with high folate receptor expression, KB cells showed a significantly higher uptake rate of L-30 / siRNA with a folate target group compared to L-29 / siRNA without a folate target group, increasing by approximately 16%. This indicates that folate targeting and the high transport rate of nucleic acid drugs by cationic liposomes can synergistically enhance the efficacy of gene therapy for tumors.

[0766] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

[0767] For those skilled in the art, the invention can be practiced in a wide range of ways with...

Claims

1. A polyethylene glycol-modified lipid, the structure of which is shown in general formula (2): Or its drug-acceptable salts, tautomers, or stereoisomers. in, L7 is -OC(=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any of C(=O)S-, L8 is -OC(=O)O-, -O-, -O(CR) c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -and-NR c Any one of C(=O)S-, where R c Each time it appears, it is independently a hydrogen atom or an alkyl group, and s is 2, 3 or 4; L3 is any one of the following: linker, -L4-, -Z-L4-, -L4-Z-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, -L4-Z-L5-Z-, -Z-L4-Z-L5-Z-, and -L4-Z-L4-Z-L5-Z-; L4 and L5 are carbon chain linkers, each independently being -(CR... a R b ) t -(CR a R b ) o -(CR a R b ) p - where t, o, and p are each an independent integer from 0 to 12, and t, o, and p are not all 0 at the same time; R a and R b Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 Alkyl groups; each occurrence of Z is independently -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-、-C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-、-SC(=O)NR c -、-NR c C(=O)S- and Any one of them, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 alkyl; B3 and B4 are each independently a connecting key or C. 1-30 Alkylene; R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon groups; R represents a hydrogen atom, -R d -OR d -NR d R d -SR d -C(=O)R d -C(=O)OR d -OC(=O)R d -OC(=O)OR d or Among them, R d Each occurrence is independently represented by C. 1-12 Alkyl group, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R. 01 When j is 0, G1 does not exist; when k is 1, G1 is a trivalent branched group of glycerol or amino acid residues; when k is 2, the structure of F is -(Z2). q -(Z1) q1 -R 01 q and q1 are each independently 0 or 1; Z1 and Z2 are each independently a binary linker, each independently being any one of -L4-, -L4-Z-, -Z-L4-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, and -L4-Z-L5-Z-, where t in L4 and L5 is an integer from 1 to 12; R 01 The functional group is capable of reacting with biologically related substances and is selected from any one of the following: alcohol hydroxyl, protected alcohol hydroxyl, thiol hydroxyl, protected thiol hydroxyl, carboxyl, protected carboxyl, amino, protected amino, aldehyde, protected aldehyde, succinimide, maleimide, protected maleimide, alkenyl, acrylate, azide, alkynyl, folic acid, rhodamine, biotinyl, monosaccharide, and polysaccharide. A is -(CR) a R b ) s O- or -O(CR) a R b ) s -, where s is 2, 3 or 4; n1 is an integer between 20 and 250; The alkyl, alkylene, alkoxy, and aliphatic hydrocarbon groups are each independently substituted or unsubstituted.

2. The polyethylene glycol-modified lipid according to claim 1, characterized in that, B3 and B4 are each independently a connecting key or a C. 1-20 Alkylene.

3. The polyethylene glycol-modified lipid according to claim 2, characterized in that, Both B3 and B4 are connection keys.

4. The polyethylene glycol-modified lipid according to claim 2, characterized in that, Of B3 and B4, one is a connector key and the other is a C. 1-20 Alkylene.

5. The polyethylene glycol-modified lipid according to claim 2, characterized in that, B3 and B4 are each independently one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecanylene, octadecylene, nonadecanylene, and eicosylene.

6. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The L7 is -OC(=O)O-, -O-, or -O(CH2). s Any one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-, and L8 is -OC(=O)O-, -O-, or -O(CH2). s Any one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH- and -NHC(=O)S-.

7. The polyethylene glycol-modified lipid according to claim 6, characterized in that, The L7 is -OC(=O)O-, -O-, or -O(CH2). s Any one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH- and -NHC(=O)S-, and L8 is -O-.

8. The polyethylene glycol-modified lipid according to claim 6, characterized in that, Both L7 and L8 are -O-.

9. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The L3 contains a degradable group, which refers to a group that can be degraded under any of the following conditions: light, heat, low temperature, enzyme, redox, acidity, alkalinity, physiological conditions, or in vitro simulated environment. The low temperature condition refers to a temperature below 25°C.

10. The polyethylene glycol-modified lipid according to claim 9, characterized in that, L4 and L5 in L3 are each independently -(CH2). t -, L3 is -(CH2) t -、-(CH2) t Z-, -Z(CH2) t -、-(CH2) t Z(CH2) t -、-Z(CH2) t Z-, -(CH2) t Z(CH2) t Z-, -Z(CH2) t Z(CH2) t - and -Z(CH2) t Z(CH2) t Z- is any one of the following, where t is an integer from 1 to 12, and each occurrence of Z is independently any one of -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH- and -NHC(=O)S-.

11. The polyethylene glycol-modified lipid according to claim 10, characterized in that, L3 is -(CH2) t -, -(CH2) t O-, -(CH2) t C(=O)-, -(CH2) t C(=O)O-, -(CH2) t OC(=O)-, -(CH2) t C(=O)NH-, -(CH2) t NHC(=O)-, -(CH2) t OC(=O)O-, -(CH2) t NHC(=O)O-, -(CH2) t OC(=O)NH-, -(CH2) t NHC(=O)NH-, -O(CH2) t -, -C(=O)(CH2) t -, -C(=O)O(CH2) t -, -OC(=O)(CH2) t -, -C(=O)NH(CH2) t -, -NHC(=O)(CH2) t -, -OC(=O)O(CH2) t -, -NHC(=O)O(CH2) t -, -OC(=O)NH(CH2) t -, -NHC(=O)NH(CH2) t -, -(CH2) t O(CH2) t -, -(CH2) t C(=O)(CH2) t -, -(CH2) t C(=O)O(CH2) t -, -(CH2) t OC(=O)(CH2) t -, -(CH2) t C(=O)NH(CH2) t -, -(CH2) t NHC(=O)(CH2) t -, -(CH2) t OC(=O)O(CH2) t -, -(CH2) t NHC(=O)O(CH2) t -, -(CH2) t OC(=O)NH(CH2) t -, -(CH2) t NHC(=O)NH(CH2) t -O(CH2) t O- -C(=O)(CH2) t C(=O)-, -C(=O)O(CH2) t C(=O)O-, -OC(=O)(CH2) t OC(=O)-、-C(=O)O(CH2) t OC(=O)-、-OC(=O)(CH2) t C(=O)O-、-OC(=O)O(CH2) t OC(=O)O-、-C(=O)NH(CH2) t C(=O)NH-、-NHC(=O)(CH2) t NHC(=O)-, -NHC(=O)(CH2) t C(=O)NH-, -C(=O)NH(CH2) t NHC(=O)-, -NHC(=O)O(CH2) t NHC(=O)O-、-OC(=O)NH(CH2) t OC(=O)NH-、-NHC(=O)O(CH2) t OC(=O)NH-、-OC(=O)NH(CH2) t NHC(=O)O-, -NHC(=O)NH(CH2) t NHC(=O)NH-、-C(=O)(CH2) t O- -C(=O)(CH2) t C(=O)O-, -C(=O)(CH2) t OC(=O)-、-C(=O)(CH2) t OC(=O)O-、-C(=O)(CH2) t NHC(=O)O-、-C(=O)(CH2) t OC(=O)NH-、-C(=O)(CH2) t NHC(=O)NH-、-C(=O)(CH2) t C(=O)O(CH2) t -C(=O)(CH2) t OC(=O)(CH2) t -C(=O)(CH2) t OC(=O)O(CH2) t -C(=O)(CH2) t NHC(=O)O(CH2) t -、-C(=O)(CH2) t OC(=O)NH(CH2) t -、-C(=O)(CH2) t NHC(=O)NH(CH2) t - and -C(=O)(CH2) t C(=O)(CH2) t Any one of NHC(=O)O-, where t is an integer from 2 to 12.

12. The polyethylene glycol-modified lipid according to claim 10, characterized in that, The L3 is -C(=O)O- or -(CH2). t O-, -(CH2) t C(=O)O-、-(CH2) t OC(=O)-、-(CH2) t O-, -C(=O)(CH2) t O-, -C(=O)(CH2) t OC(=O)-、-C(=O)(CH2) t C(=O)O-、-C(=O)(CH2) t C(=O)NH-、-C(=O)(CH2) t OC(=O)NH-、-C(=O)(CH2) t NHC(=O)O-、-C(=O)(CH2) t OC(=O)NH(CH2) t - and -C(=O)(CH2) t C(=O)(CH2) t Any one of NHC(=O)O-.

13. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The number-average molecular weight of the polyethylene glycol chains is 900, 1000, 1500, 2000, 2500, 3000, 3350, 3500, 4000, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 11000.

14. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The polyethylene glycol chains are polydisperse.

15. The polyethylene glycol-modified lipid according to claim 14, characterized in that, The number-average degree of polymerization n1 of the polyethylene glycol chain is an integer between 20 and 100.

16. The polyethylene glycol-modified lipid according to claim 14, characterized in that, The number-average degree of polymerization n1 of the polyethylene glycol chain is an integer between 20 and 60.

17. The polyethylene glycol-modified lipid according to claim 14, characterized in that, The number-average degree of polymerization n1 of the polyethylene glycol chain is an integer between 40 and 60.

18. The polyethylene glycol-modified lipid according to claim 14, characterized in that, The number-average degree of polymerization n1 of the polyethylene glycol chain is any one of 44, 45, 46, 48, 50, 52, 54, 56, 58, and 60.

19. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The polyethylene glycol chains are monodisperse.

20. The polyethylene glycol-modified lipid according to claim 19, characterized in that, The number of EO units in the polyethylene glycol chain is an integer from 20 to 70.

21. The polyethylene glycol-modified lipid according to claim 19, characterized in that, The number of EO units in the polyethylene glycol chain is an integer ranging from 20 to 60.

22. The polyethylene glycol-modified lipid according to claim 19, characterized in that, The number of EO units in the polyethylene glycol chain is any one of 20, 22, 24, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 56, 58, and 60.

23. The polyethylene glycol-modified lipid according to claim 1, characterized in that, R1 and R2 are each independently C 5-30 Aliphatic hydrocarbon group.

24. The polyethylene glycol-modified lipid according to claim 23, characterized in that, R1 and R2 are each independently C 10-30 Aliphatic hydrocarbon group.

25. The polyethylene glycol-modified lipid according to claim 23, characterized in that, R1 and R2 are each independently C 10-20 Aliphatic hydrocarbon group.

26. The polyethylene glycol-modified lipid according to claim 1, characterized in that, R1 and R2 are each independently C 1-30 Straight-chain alkyl, C 1-30 Branched alkyl groups, C 1-30 Straight-chain alkenyl, C 1-30 Branched alkenyl groups, C 1-30 Straight-chain alkynyl or C 1-30 Branched alkynyl group.

27. The polyethylene glycol-modified lipid according to claim 26, characterized in that, R1 and R2 are each independently C 1-30 Straight-chain aliphatic hydrocarbon group.

28. The polyethylene glycol-modified lipid according to claim 26, characterized in that, R1 and R2 are each independently C 1-25 Straight-chain aliphatic hydrocarbon group.

29. The polyethylene glycol-modified lipid according to claim 26, characterized in that, R1 and R2 are each independently C 1-20 Straight-chain aliphatic hydrocarbon group.

30. The polyethylene glycol-modified lipid according to claim 26, characterized in that, R1 and R2 are each independently methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, (Z)-tridecane-8-enyl, (Z)-tetradecane-9-enyl, (Z)-pentadecanane-8-enyl, (Z)-hexadecane-9-enyl, (Z)-heptadecane-5-enyl, (Z)-heptadecane-8-enyl, (E)-heptadecane-8-enyl, (Z)-heptadecane-10-enyl, (8Z,11Z)-heptadecane-8,11-dienyl, (Z)-octadecane-6-enyl, (Z)-octadecane- The following is a list of 9-alkenyl, (E)-octadecane-9-alkenyl, (Z)-octadecane-11-alkenyl, (9Z,12Z)-octadecane-9,12-dienyl, (9Z,12Z,15Z)-octadecane-9,12,15-trienyl, (8Z,11Z,14Z)-octadecane-8,11,14-trienyl, (Z)-eicosane-11-alkenyl, (11Z,14Z)-eicosane-11,14-dienyl, (Z)-nonadecane-10-alkenyl, (10Z,13Z)-nonadecane-10,13-dienyl, 2,6,10-trimethylundecane-1,5,9-trienyl, or 3,7,11-trimethyldodecane-2,6,10-trienyl.

31. The polyethylene glycol-modified lipid according to claim 26, characterized in that, R1 and R2 are each independently a branched alkyl, branched alkenyl, or branched alkynyl group, and are each independently represented as follows: , where R e R f Each independently is C1-C 15 Alkyl, C2-C 15 alkenyl and C2-C 15 Any of the alkynyl groups, where t in R1 and R2 is an integer from 0 to 12.

32. The polyethylene glycol-modified lipid according to claim 31, characterized in that, The R e R f Each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, allenyl, pentenyl, allenyl, hexenyl, allenyl, heptyl, octenyl, allenyl, nonenyl, decenyl, ethynyl, propynyl, propynyl, butynyl, ethynyl, pentynyl, ethynyl, hexynyl, heptynyl, ethynyl-heptyl, octyynyl, ethynyl-octyl, nonynyl, ethynyl-nonyl, decynyl, and ethynyl-decyl.

33. The polyethylene glycol-modified lipid according to claim 31, characterized in that, The R e R f Each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

34. The polyethylene glycol-modified lipid according to claim 31, characterized in that, R1 and R2 are each independently selected from any of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , where t is an integer from 0 to 12.

35. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The R contains any one of the following: hydrogen atom, alkyl group, alkoxy group, alcohol hydroxyl group, protected alcohol hydroxyl group, thiol hydroxyl group, protected thiol hydroxyl group, carboxyl group, protected carboxyl group, amino group, protected amino group, aldehyde group, protected aldehyde group, ester group, carbonate group, carbamate group, succinimide group, maleimide group, protected maleimide group, dimethylamino group, alkenyl group, acrylate group, azide group, alkynyl group, folic acid group, rhodamine group, biotin group, monosaccharide group, and polysaccharide group.

36. The polyethylene glycol-modified lipid according to claim 35, characterized in that, The R contains H, -CH3, -CH2CH3, or -(CH2). t OH, -(CH2) t SH, -OCH3, -OCH2CH3, -(CH2) t NH2、-(CH2) t C(=O)OH, -C(=O)(CH2) t C(=O)OH, -C(=O)CH3, -(CH2) t N3, -C(=O)CH2CH3, -C(=O)OCH3, -OC(=O)OCH3, -C(=O)OCH2CH3, -OC(=O)OCH2CH3, -(CH2) t N(CH3)2、-(CH2) t N(CH2CH3)2、-(CH2) t CHO , , , and Any one of them.

37. The polyethylene glycol-modified lipid according to claim 1, characterized in that, Its structure can be any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 38. The polyethylene glycol-modified lipid according to claim 1, characterized in that, The structure of the polyethylene glycol-modified lipid is selected from any of the following structures: , , , , , , , , , , , , , , , , , , , and .

39. A cationic liposome, characterized in that, Contains polyethylene glycol-modified lipids as described in any one of claims 1-38.

40. The cationic liposome according to claim 39, characterized in that, It also contains one or more of the following: neutral lipids, steroid lipids, and cationic lipids.

41. The cationic liposomes according to claim 40, characterized in that, The neutral lipids mentioned are phospholipids.

42. The cationic liposomes according to claim 40, characterized in that, The neutral lipids are selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, and 1,2-di-O-decanoyl-sn-glycerol-3-phosphate choline. Octadectenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diphydanyl-sn 1,2-Distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt, dioleoylphosphatidyl Serine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and any one of these and combinations thereof.

43. The cationic liposomes according to claim 40, characterized in that, The steroid lipids are selected from any one of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof.

44. The cationic liposomes according to claim 40, characterized in that, The structure of the cationic lipid is selected from any of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

45. The cationic liposomes according to claim 40, characterized in that, The cationic lipid is selected from N,N-dioleoyl-N,N-dimethylammonium chloride, N,N-distearate-N,N-dimethylammonium bromide, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, N,N-dimethyl-2,3-dioleoyloxypropylamine, 3-(bisdodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine, and N1-[2-(bisdodecylamino)ethyl]-N1,N4 The following are all of the following: N4-tri-dodecyl-1,4-piperazine diethylamine, 14,25-di-tetrazyl-15,18,21,24-tetraaza-octacosane, 1,2-dilinoleyloxy-N,N-dimethylaminopropane, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane, 4-(dimethylamino)butyric acid 37-carbon-6,9,28,31-tetraen-19-yl ester, and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane, and mixtures thereof.

46. ​​The cationic liposomes according to any one of claims 40-45, characterized in that, It contains 20-65% cationic lipids, 5-15% neutral lipids, 25-55% steroidal lipids and 0.5-10% polyethylene glycol-modified lipids as shown in formula (2), wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

47. The cationic liposome according to any one of claims 40-45, characterized in that, The cationic lipids comprise 30-65% of the total lipids in the solvent-containing solution.

48. The cationic liposomes according to claim 47, characterized in that, The cationic lipid accounts for any one of the following molar percentages of the total lipids in the solvent-containing solution: 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%.

49. The cationic liposomes according to any one of claims 40-45, characterized in that, The neutral lipids comprise 7.5-13% of the total lipids in the solvent-containing solution.

50. The cationic liposome according to claim 49, characterized in that, The neutral lipids constitute any one of the following molar percentages of the total lipids in the solvent-containing solution: 8%, 9%, 10%, 11%, and 12%.

51. The cationic liposome according to any one of claims 40-45, characterized in that, The steroid lipids comprise 35-50% of the total lipids in the solvent-containing solution.

52. The cationic liposomes according to claim 51, characterized in that, The steroid lipids constitute any one of the following molar percentages of the total lipids in the solvent-containing solution: 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

53. The cationic liposomes according to any one of claims 40-45, characterized in that, The polyethylene glycol-modified lipids comprise 0.5-5% of the total lipids in the solvent-containing solution.

54. The cationic liposomes according to claim 53, characterized in that, The polyethylene glycol-modified lipids comprise 1-3% of the total lipids in the solvent-containing solution.

55. The cationic liposomes according to claim 53, characterized in that, The polyethylene glycol-modified lipids constitute any one of the following molar percentages of the total lipids in the solvent-containing solution: 1.5%, 1.6%, 1.7%, 1.8%, and 1.9%.

56. A cationic liposome pharmaceutical composition, characterized in that, The cationic liposomes and the drug contained in any one of claims 40-45.

57. The cationic liposome pharmaceutical composition according to claim 56, characterized in that, The drug is a nucleic acid drug or an antitumor agent, wherein the nucleic acid drug is selected from any one of DNA, antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA.

58. The cationic liposome pharmaceutical composition according to claim 57, characterized in that, The drug is any one of DNA, mRNA, miRNA, and siRNA.

59. The cationic liposome pharmaceutical composition according to claim 56, characterized in that, The pharmaceutical composition is used to prepare a drug and is selected from any of the following drugs: drugs for treating cancer and malignant tumors, anti-infective agents, antiviral agents, antifungal agents, and vaccines.

60. A cationic liposome pharmaceutical composition formulation, characterized in that, The liposomal pharmaceutical composition comprising any one of claims 56-59 and a pharmaceutically acceptable diluent or excipient.

61. The cationic liposome pharmaceutical composition formulation according to claim 60, characterized in that, The diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer, and physiological saline.

62. The cationic liposome pharmaceutical composition formulation according to claim 61, characterized in that, The diluent or excipient is phosphate buffer or physiological saline.

63. The cationic liposome pharmaceutical composition formulation according to claim 61, characterized in that, The diluent or excipient is physiological saline.

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