Use of blank lipid nanoparticles in preparation of in-vivo delivery product

Abstract

The use of blank lipid nanoparticles in the preparation of an in-vivo delivery product, comprising the step of mixing the blank lipid nanoparticles with a biologically active substance in a solvent to obtain a blank lipid nanoparticle-based composition. The blank lipid nanoparticles consist of an ionizable lipid, a phospholipid, a steroid or a derivative thereof, and a polyethylene glycol-lipid conjugate. According to the blank lipid nanoparticles, the dosage of the biologically active substance can be flexibly adjusted according to the requirements of a user, and the blank lipid nanoparticles can be administered via multiple routes such as intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, all of which can achieve an ideal delivery effect.

Description

Application of blank lipid nanoparticles in the preparation of in vivo delivery products

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202410079585.4 filed on January 19, 2024, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application belongs to the field of biomedicine technology, and specifically relates to the application of blank lipid nanoparticles in the preparation of in vivo delivery products. Background Art

[0004] Gene editing therapy has the potential to cure genetic diseases, but it requires the ability to safely and effectively deliver gene editing drugs to the corresponding target organs and tissues in the body. Gene editing drugs can exist in the form of DNA, mRNA, protein or ribonucleoprotein (RNP). The successful delivery of such macromolecular drugs into cells requires breaking through multiple physiological barriers: 1) Before the drug enters the cell, it is necessary to avoid dissociation of the drug from the carrier or drug degradation; 2) targeting specific cells; 3) passing through the cell membrane to enter the cell; 4) releasing the drug in specific organelles. Therefore, how to effectively and safely introduce gene editing drugs into the corresponding target cells in the body is a big problem.

[0005] The existing in vivo introduction technology of gene-editing drugs still has many defects. For example, the most common introduction technology is mediated by viral vectors, which usually uses adeno-associated virus delivery vectors or lentiviral vectors to deliver drugs. Adeno-associated virus delivery vectors have good safety and biocompatibility; they can deliver drugs to tissues such as the eyes, liver, brain, myocardium, and skeletal muscle. Different types of AAV capsid serotypes produced naturally and synthesized in the laboratory can achieve different tissue targeting, but their loading capacity is limited (only 5kb of DNA) and there are potential safety risks. Lentivirus vectors can accommodate DNA drugs up to 10kb, which is enough to load known gene-editing drugs into a single vector, but there is a potential risk of genomic integration. In general, viral vectors face problems such as vector immunogenicity, long-term expression of gene-editing drugs, non-target gene editing, the possibility of genomic integration, manufacturing costs and dose-limiting toxicity, as well as potential unsafety such as integration renaturation and immune response in host cells, which limit their application.

[0006] Cationic polymer-mediated delivery technologies, such as polyethyleneimine, also suffer from low transfection efficiency and high toxicity. While numerous methods exist for delivering gene-editing drugs in vivo, most are expensive, pose safety risks, are toxic, or exhibit low transfection efficiency. Consequently, inefficient nucleic acid delivery in vivo is a bottleneck hindering the further development of gene therapy and gene immunotherapy. Therefore, an in vivo transfection reagent with high transfection efficiency and low toxicity is urgently needed to meet current needs.

[0007] Lipid nanoparticle (LNP) delivery has the following advantages in the field of gene editing drug delivery: 1) LNP delivery of gene editing drugs can achieve transient expression. Compared with the long-term expression of gene editing drugs brought by viral vectors, LNP can minimize the possibility of off-target effects; 2) LNP has much lower immunogenicity than viral vectors, and in some cases it can be repeatedly administered. It has good safety and biocompatibility and can deliver drug doses that meet effective gene editing levels; 3) The large-scale production process of LNP is currently mature, which provides a basis for clinical trials using LNP to deliver gene editing drugs in vivo. Patents CN116832051A and CN116969850A have conducted research in this area. However, in the existing technology, the use of lipid nanoparticles (LNP) to deliver gene editing drugs usually requires a special mixing process, otherwise a good delivery effect cannot be achieved.

[0008] Based on this, the present application provides the use of blank lipid nanoparticles in the preparation of in vivo delivery products. By simply mixing biologically active substances with blank lipid nanoparticles in a solution state, safe and effective delivery of biologically active substances can be achieved. Summary of the Invention

[0009] This application addresses the problems of the prior art and provides the use of blank lipid nanoparticles in the preparation of an in vivo delivery product. The use of the blank lipid nanoparticles in the preparation of an in vivo delivery product comprises the steps of mixing the blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on the blank lipid nanoparticles; the blank lipid nanoparticles are composed of an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol-conjugated lipid. Using these blank lipid nanoparticles, the dosage of the biologically active substance can be flexibly adjusted according to user needs, and administration can be achieved through a variety of routes, all achieving the desired delivery effect.

[0010] To achieve the above-mentioned objectives, in a first aspect, the present application provides the use of blank lipid nanoparticles in the preparation of an in vivo delivery product, comprising the steps of mixing blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on blank lipid nanoparticles; the blank lipid nanoparticles are composed of: 10-60 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-30 mol% of a phospholipid, 15-70 mol% of a steroid or a derivative thereof, and 0-5 mol% of a polyethylene glycol-conjugated lipid.

[0011] In a preferred embodiment, the product is a delivery system or a medicament.

[0012] In a preferred embodiment, the use further comprises the step of introducing the blank lipid nanoparticle-based composition into an animal.

[0013] In a preferred embodiment, the routes of introducing the blank lipid nanoparticle-based composition into an animal include oral administration, intranasal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intraarticular administration, intralesional administration, intratracheal administration, subcutaneous administration or intradermal administration.

[0014] In a preferred embodiment, the animals are mammals and non-mammals.

[0015] In a preferred embodiment, the biologically active substance includes nucleic acid, protein, polypeptide, small molecule active substance; the nucleic acid is DNA and / or RNA.

[0016] In a preferred embodiment, the nucleic acid comprises at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA minicircle, and msDNA.

[0017] In a preferred embodiment, the amount of the nucleic acid is 0.5%-50% of the total amount of the blank lipid nanoparticles and the nucleic acid; and the concentration of the biologically active substance in the composition based on the blank lipid nanoparticles is 50-5000 ng / μl.

[0018] In a preferred embodiment, the first ionizable lipid is selected from a compound comprising the general formula (1) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof;

[0019] Wherein, R1, R2, and R3 are independently H, C5-40 Straight or branched alkyl, C 5-40 Straight-chain or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10-30 Straight-chain or branched alkyl, C 10-30 Straight-chain or branched alkenyl, C 10-30 Straight or branched alkynyl; provided that at most one of R1, R2, and R3 is H;

[0020] M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, a 6-10 membered heteroaryl group containing at least one nitrogen atom, wherein the heterocyclic group and the heteroaryl group are unsubstituted or substituted with one or more -OH, carboxyl, amino, oxo or halogen;

[0021] R4 and R5 are independently H, C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 Straight chain or branched chain alkynyl, the C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, aminoamide, amidino, guanidino or halogen;

[0022] G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS (=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 independently selected from H, hydroxyl, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0023] L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -、-NR 14 -S(=O)2-、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-; wherein m is an integer from 2 to 6, R 7、 R8 are independently H, hydroxy, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 14 、R 15 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0024] L2 is -(CR9R 10 ) n -or-(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR16 C(=O)NR 17 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16 -、NR 16 -S(=O)2-、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-; n is an integer from 1 to 6; k is an integer from 1 to 6; R 9、 R 10、 R 11、 R 12 are independently H, hydroxy, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 16 、R 17 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0025] Where R4 to R 17 The alkyl, cycloalkyl, and alkenyl groups are unsubstituted or substituted by one or more groups selected from hydroxyl, thiol, amino, substituted amino, and halogen;

[0026] The salts do not include quaternary ammonium salts.

[0027] In a preferred embodiment, R1, R2, and R3 are independently the following groups:

[0028] Where Y does not exist or is C 1-30 Straight chain or branched chain alkyl or cycloalkyl, C 2-20 Straight-chain or branched alkenyl, C 2-20 Straight or branched alkynyl; R1', R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of Y, R1' and R2' is 8-40.

[0029] In a preferred embodiment, R1, R2, and R3 are independently selected from the following groups:

[0030] Wherein, R1' and R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

[0031] In a preferred embodiment, R1, R2, and R3 are independently selected from any one of the following groups:

[0032] In a preferred embodiment, G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

[0033] In a preferred embodiment, L1 is selected from -(CR7R8) m -X1-, wherein X1 is selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-.

[0034] In a preferred embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR 16 C(=O)NR 17 -、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-.

[0035] In a preferred embodiment, M is selected from the following structures:

[0036] wherein m' and n' are independently integers of 0-6, R1" and R2" are independently H, C 1-6 Alkyl, C 2-6 The alkenyl, guanidino, amidino, amide, fatty amine, 3-10 membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and tautomeric forms thereof, which are unsubstituted or optionally substituted with one or more selected from hydroxyl, thiol, amino, substituted amino, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 The aromatic group is substituted with an organic group.

[0037] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1A):

[0038] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1B):

[0039] In a preferred embodiment, the compound of formula (1) is selected from the compound of formula (1C):

[0040] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1D):

[0041] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1E)

[0042] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1F):

[0043] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1G):

[0044] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1H):

[0045] In a preferred embodiment, M is selected from any one of the following groups:

[0046] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (II):

[0047] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1J):

[0048] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1K):

[0049] In a preferred embodiment, Y is absent and the compound of formula (1) is selected from the compounds represented by formula (1L):

[0050] Wherein R1' and R2' are independently selected from H, C1-30 straight chain or branched chain alkyl, C2-30 straight chain or branched chain alkenyl, C2-30 straight chain or branched chain alkynyl, and the total carbon chain length of R1' and R2' is 8-40.

[0051] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1M):

[0052] The compound of formula (1) is selected from:

[0053] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (II) with a compound of formula (III):

[0054] Wherein, Xa and Xb are groups containing a leaving group or a nucleophilic group, and Xa and Xb form L1 through a nucleophilic reaction or a condensation reaction.

[0055] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (IV) with a compound of formula (V):

[0056] wherein Xc and Xd are groups containing a leaving group or a nucleophilic group, and Xc and Xd form L2 through a nucleophilic reaction or a condensation reaction.

[0057] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0058] wherein Xe is a group containing a leaving group or a nucleophilic group, Xf is a compound containing a leaving group or a nucleophilic group, and Xe and Xf form M through a nucleophilic reaction or a condensation reaction.

[0059] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the steps of reacting a compound of formula (VII) with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X) in sequence:

[0060] wherein Xg, Xh, Xi, Xj, Xk, and Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G1 through a nucleophilic reaction or a condensation reaction, Xh and Xk form G2 through a nucleophilic reaction or a condensation reaction, and Xi and Xl form G3 through a nucleophilic reaction or a condensation reaction.

[0061] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (XI) with a compound of formula (XII):

[0062] Steps for reacting the compound of formula (XIII) with the compound of formula (XII):

[0063] wherein Xm is a group containing a nucleophilic group, and Xm and C=CL of the compound of formula (XII) 2a X1-L2 is formed by addition reaction.

[0064] In a preferred embodiment, R1, R2, and R3 are independently of one another the following groups:

[0065] Wherein, Y, R1', and R2' have the same meanings as above;

[0066] It also includes the steps of forming tail chains R1, R2, and R3:

[0067] Wherein X is a leaving group.

[0068] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0069] Wherein N1 is NH or O;

[0070] R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short chain polyethylene oxide; the C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short-chain polyoxyethylene is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C 12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0071] The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, R c " is selected from C1-C6 alkyl groups which are unsubstituted or substituted with amino groups, R c ”' is H,

[0072] Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

[0073] In a preferred embodiment, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines.

[0074] In a preferred embodiment, the condition is that when R a C6-C 24 In the case of alkenyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0075] When R a C6-C 24 When alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines;

[0076] R a For straight chain C6-C 24 When alkyl, A1 is NH or O, R b and R c each independently selected from alkylamines;

[0077] When R a For straight chain C6-C 24 When it is alkyl, A1 is O, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0078] When R a C6-C 24 In the case of short-chain polyoxyethylene, A1 is NH or O, R b and R c each independently selected from alkylamines;

[0079] Or when R a C6-C 24 In the case of alkyl alcohol, A1 is NH or O, R b and R c Each is independently selected from alkylamines.

[0080] In a preferred embodiment, R a Selected from the following compound structures:

[0081] In a preferred embodiment, R b and R c Selected from the following compound structures:

[0082] R d Selected from C1-C6 alkanes or cycloalkanes.

[0083] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0084] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0085] R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form ionizable lipid compound③:

[0086] Among them, R a -NH2 is R a -NH2 or

[0087] R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0088] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0089] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤;

[0090] 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③;

[0091] Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a -NH2 is selected from R a -NH2 or

[0092] R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -Rc '-A1'-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0093] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0094] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form compound⑦;

[0095] 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨;

[0096] 3) Compound ⑨ reacts with nucleophile ⑩ to form an ionizable lipid compound

[0097] Among them, R a -NH2 is R a -NH2, Z3 is a leaving group, A3 reacts with Z3 to obtain A1.

[0098] In a preferred embodiment, in Formula 2a-Formula 2c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to generate the ionizable lipid compound with two forks containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0099] In a preferred embodiment, Formula 2a-Formula 2b further comprises the step of using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a-Formula 2b as a starting material, and performing an iterative reaction according to Step 1 in Formula 2a or Steps 1-2 in Formula 2b;

[0100] The compound of formula (2) with a terminal amino group is

[0101] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0102] In a preferred embodiment, the reaction formula 2a-2c further comprises using the compound of formula (2) with a terminal amino group obtained by the reaction formula 2a-2c as a raw material, and The steps of the reaction;

[0103] The compound of formula (2) with a terminal amino group is

[0104] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0105] The leaving group mentioned above refers to the leaving part in the nucleophilic reaction or condensation reaction, including but not limited to: H,

[0106] OH, H2O, halogen (such as F, Cl, Br and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy (RO-), amino (-NHR, wherein R is an alkyl or aryl group with H removed), phenoxy, tertiary carbon cation (such as tert-butyl cation), carbon cation stabilized by an unsaturated system or a heteroatom, or the various protecting groups mentioned above.

[0107] The nucleophilic groups mentioned above refer to molecules or ions that can provide electron pairs to form new chemical bonds in chemical reactions. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, nucleophilic halogens (such as Cl - Br - or I - ), hydrogen ions (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxy anion (such as alcohol with hydroxyl hydrogen removed), amine group (including primary amine, secondary amine and tertiary amine) or amine anion, carbon anion (such as carbon anion in organometallic reagents such as Grignard reagent, organic lithium reagent, Gilman reagent), sulfhydryl or sulfhydryl anion, sulfide, enol or enol anion, alkenyl ether, enamine, carboxylic acid or carboxylic acid anion, alkyl or aryl phosphine (such as triphenylphosphine), aromatic heterocycle with lone pair electrons (such as pyridine), etc.

[0108] In a preferred embodiment, the raw materials used in the reaction process also contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0109] In a preferred embodiment, the blank liposome-based in vivo delivery method further comprises 0-60 mol% of other ionizable lipids, wherein the other ionizable lipids are selected from at least one of the following compounds:

[0110] In a preferred embodiment, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl at least one of 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0111] In a preferred embodiment, the steroid or its derivative comprises at least one of cholesterol, cholesterol stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, salinarate, tomatine, ursolic acid, and α-tocopherol.

[0112] In a preferred embodiment, the polyethylene glycol-conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0113] In a preferred embodiment, the polyethylene glycol conjugated lipid includes at least one of PEG-distearyloxypropyl (PEG-DSA), PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate (PEG-DMA), 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroylphosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoylphosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0114] In a preferred embodiment, in the blank lipid nanoparticle-based composition, the biologically active substance is encapsulated inside the blank lipid nanoparticle and / or adsorbed on the surface of the blank lipid nanoparticle to form a complex.

[0115] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffered saline solution.

[0116] In a preferred embodiment, the pH of the buffered salt solution is 1-9, the buffer salt concentration is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration of <50%.

[0117] In a preferred embodiment, the alcohol is ethanol.

[0118] In a preferred embodiment, the buffered saline solution is selected from at least one of a citrate solution, an acetate solution, a tartrate solution, a phosphate solution, a carbonate solution, a Tris-HCl solution, and a sodium chloride solution.

[0119] In a preferred embodiment, at least one of sugar, glycerol, DMSO, salt, antibiotics, and surfactants is further added to the blank lipid nanoparticle-based composition.

[0120] In a second aspect, the present application provides an in vitro gene delivery method, comprising the steps of mixing blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on blank lipid nanoparticles; the blank lipid nanoparticles are composed of: 10-60 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-30 mol% of a phospholipid, 15-70 mol% of cholesterol, and 0-10 mol% of a polyethylene glycol-conjugated lipid.

[0121] Compared with the prior art, this application has the following beneficial effects:

[0122] 1. The use of the blank lipid nanoparticles in the preparation of an in vivo delivery product comprises the steps of mixing the blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on the blank lipid nanoparticles; the blank lipid nanoparticles comprise: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-conjugated lipids.

[0123] 2. The blank lipid nanoparticles used in this application can flexibly adjust the dosage of biologically active substances according to user needs, and can be administered through multiple routes to achieve ideal delivery effects.

[0124] 3. The preparation process of this application is simple and can be completed without the help of equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 is the whole-body in vivo imaging of blank lipid nanoparticle-luciferase mRNA complex in Example 5 3h / 6h after intravenous administration to mice;

[0126] FIG2 is an in vivo imaging of the abdomen and lateral decubitus of mice 3 / 6 hours after intramuscular injection of blank lipid nanoparticle-luciferase mRNA complexes according to Example 10;

[0127] Figure 3 shows the in vivo imaging of the blank lipid nanoparticle-luciferase mRNA complex of Example 16 administered intraperitoneally and subcutaneously to mice for 3h / 6h. DETAILED DESCRIPTION

[0128] The synthetic process of the present application can tolerate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, but in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present application can use commercially available starting materials, compounds known in the literature, or intermediates that are easy to prepare, by using standard synthetic methods and procedures known to those skilled in the art or obvious to the skilled person based on the teachings of this article, prepared in a variety of ways. Standard synthetic methods and procedures for preparing organic molecules and functional group transformations and operations can be obtained from relevant scientific literature or from standard textbooks in this area. The following description of the synthetic method is designed to illustrate, but not limit, the general procedures for preparing the compounds of the present application.

[0129] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables in each general synthetic route (e.g., R1, R2, and R3, etc.) are as defined herein. One of ordinary skill in the art will note that in the reaction procedures and synthetic schemes described herein, the order of certain steps may be varied, such as the introduction and removal of protecting groups.

[0130] In the reaction schemes described herein, a variety of stereoisomers can be produced. When a specific stereoisomer is not indicated, this is understood to include all possible stereoisomers produced by the reaction. One of ordinary skill in the art will recognize that the reaction can be optimized to preferentially obtain a single isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0131] (1) Synthesis of the first ionizable lipid in Example 1

[0132] The general synthetic route for the first ionizable lipid is as shown in general synthetic route 1-5.

[0133] General synthetic route 1

[0134] Wherein, M' is M or M containing a protecting group.

[0135] As described in the general synthetic route 1 above, Boc-aminotris(hydroxymethyl)methane reacts with an acid (Compound 2) to form Compound 3. Step 1 can be carried out in an organic solvent (e.g., dichloromethane (DCM)) in the presence of, for example, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 4-dimethylaminopyridine (DMAP). Step 1 can be carried out at room temperature for 24 hours.

[0136] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0137] Next, compound 4 and compound 5 undergo condensation reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) using EDCl and DMAP or dicyclohexylcarbodiimide (DCC) as a catalyst.

[0138] If the M' group of compound 6 contains the aforementioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the selected protecting group removal reaction conditions.

[0139] General synthetic route 2

[0140] Wherein M' is M or M containing a protecting group; A is O, NH or S.

[0141] As described in the above general synthetic route 2, compound 1 and compound 2 undergo condensation reaction to give compound 3. Step 1 can be carried out in an organic solvent (eg, DCM) in the presence of EDCl and DMAP.

[0142] Next, the tert-butyloxy protecting group of compound 3 is removed to obtain compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) in the presence of an acid (eg, trifluoroacetic acid) and a positive ion scavenger (eg, triisopropylsilane (TiPS)).

[0143] Next, compound 4 undergoes a condensation reaction with a compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0144] If the M' group of compound 6 contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0145] General synthetic route 3

[0146] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0147] As described in the general synthetic route 3 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0148] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0149] Next, compound 4 is reacted with a halogen-substituted aldehyde (compound 5) through condensation and reduction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0150] Next, compound 6 reacts with an amine (compound 7) to produce compound 8. Step 4 can be carried out in an organic solvent (such as DMF) in the presence of a base (such as a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0151] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0152] General synthetic route 4

[0153] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0154] As described in the general synthetic route 4 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0155] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0156] Next, compound 4 undergoes a condensation reaction with a halogen-substituted compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF using EDCl and DMAP or DCC as catalyst.

[0157] Next, compound 6 reacts with an amine (compound 7) to form compound 8. Step 4 can be carried out in an organic solvent (e.g., DMF) in the presence of a base (e.g., a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0158] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0159] General synthetic route 5

[0160] Among them, M pro is M or M containing a protecting group.

[0161] As described in the general synthetic route 5 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0162] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0163] Next, compound 4 and compound 5 are subjected to condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0164] If the M of compound 6 pro If the group contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0165] Furthermore, it should be understood that any particular embodiment of the present application that is within the prior art may be explicitly excluded from any one or more claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if such exclusion is not explicitly stated herein.

[0166] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the present application, the present application shall prevail.

[0167] (1) Synthesis of a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L) or (1M)

[0168] A. General considerations

[0169] It is worth noting that the raw materials used in this application are all common commercially available products, and their sources are not specifically limited.

[0170] The process routes described below can be used to synthesize compounds 1001-3422 of this application.

[0171] This article uses the following abbreviations:

[0172] THF: Tetrahydrofuran

[0173] MeCN: acetonitrile

[0174] MeOH: methanol

[0175] PE: Petroleum ether

[0176] EA: ethyl acetate

[0177] DMF: N,N-dimethylformamide

[0178] EDCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0179] LAH: lithium aluminum hydride

[0180] DCM: dichloromethane

[0181] DMAP: 4-dimethylaminopyridine

[0182] LDA: lithium diisopropylamide

[0183] rt: room temperature

[0184] DCE: 1,2-dichloroethane

[0185] n-BuLi: n-butyllithium

[0186] i-Pr2EtN:N,N-diisopropylethylamine

[0187] B. Intermediate Synthesis

[0188] Intermediate A:

[0189] Intermediate A is obtained by the following synthetic process:

[0190] Tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl carbonic anhydride (Boc2O) (99.1 g) were dissolved in a methanol (300 mL) / H2O (30 mL) mixed solvent and reacted at room temperature for 72 h. The mixture was purified by silica gel column chromatography (DCM:MeOH = 20:1-10:1) to obtain a white solid compound intermediate A (N-Boc-aminotrihydroxymethylmethane) (75.0 g). LCMS (ESI) calculation for C9H 19 NO 5， [M+H] + m / z 222.13, found 222.25.

[0191] Intermediate B:

[0192] Intermediate B is obtained by the following synthetic process:

[0193] To a single-necked flask, diethyl 2-ethyl-1,3-propanedioate (29.0 g), THF (90 mL), and DMF (30 mL) were added, and NaH (3.7 g) was added under ice bath. The mixture was stirred at room temperature for 30 min under nitrogen protection; pentadecane bromide (15.0 g) was added, and the mixture was reacted at 80° C. for 2 h under nitrogen protection; THF was concentrated under reduced pressure to remove THF, and the reaction solution was added dropwise to ice water. Ethyl acetate was added and stirred, and the mixture was filtered through diatomaceous earth. The organic phase was collected and then separated. The organic phase was washed with saturated brine, separated, and purified by silica gel column chromatography (PE:EA=100:1-50:1) to obtain the compound diethyl 2-ethyl-2-pentadecylamealate (16.5 g). To diethyl 2-ethyl-2-pentadecylmaleate (8.0 g) was added EtOH (50 mL), H2O (50 mL), and KOH (11.3 g), and the mixture was reacted at 90°C for 12 h; the EtOH was removed by concentration under reduced pressure, and the reaction system was adjusted to pH 4-5 by adding dilute hydrochloric acid. Water and ethyl acetate were added, stirred, and separated, and the organic phase was washed with saturated brine, separated, and purified by column chromatography (PE:EA=5:1-1:1) to obtain 2-ethyl-2-pentadecylmaleic acid (5.2 g). 2-ethyl-2-pentadecylmaleic acid (10.0 g) was reacted at 170°C for 6 h under open conditions, cooled to room temperature, and water and ethyl acetate were added, stirred, separated, and the organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (DCM:MeOH=20:1-10:1) to obtain intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1HNMR (400MHz, CDCl3) δ2.31 (tt, J = 8.6, 5.3Hz, 1H), 1.71-1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4Hz, 3H), 0.90 (t, J = 6.7Hz, 3H).

[0194] Intermediate C:

[0195] Intermediate C is obtained by the following synthetic process:

[0196] To a three-necked flask, add decanoic acid (50 g) and THF (500 mL). After cooling the system to 0°C, slowly add NaH (23.22 g). After stirring at 0°C under nitrogen for 1 hour, slowly add LDA (62.19 g) dropwise. Stirring at 0°C under nitrogen for another 1 hour is then continued. Finally, add iodonane (88.52 g) dropwise, warm to room temperature, stir overnight, then dilute with 1 L of DCM and wash with saturated NH4Cl solution and water, respectively. The organic layer is dried over anhydrous Na2SO4, filtered, and concentrated. Purification by silica gel chromatography (PE:EA = 50:1) yields intermediate C (2-octylundecanoic acid) (11 g). 1H NMR (400MHz, Chloroform-d) δ2.37 (tt, J = 8.7, 5.1 Hz, 1H), 1.71-1.58 (m, 2H), 1.48 (dt, J = 13.3, 6.7 Hz, 2H), 1.29 (d, J = 9.4 Hz, 25H), 0.90 (t, J = 6.8 Hz, 6H).

[0197] Intermediate D:

[0198] Intermediate D is obtained by the following synthetic route:

[0199] Tridecanoic acid (79.5 g) and THF (800 mL) were added to a three-necked flask. The temperature was lowered to 0°C, and NaH (22.25 g) was slowly added. The reaction mixture was stirred at 0°C under nitrogen for 1 hour. LDA (317.87 g) was then slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 0°C under nitrogen for another 1 hour. After adding n-hexyl iodide (94.3 g) dropwise, the mixture was warmed to room temperature and stirred overnight. The reaction mixture was then diluted with 1 L of DCM and washed with saturated NH4Cl solution and then water. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (PE:EA = 50:1) to yield intermediate D (2-hexyltridecanoic acid) (26 g). 1H NMR(400MHz,Chloroform-d)δ2.36(tt,J=8.7,5.4Hz,1H),1.63(ddd,J=14.3,8.7,5.5 Hz, 2H), 1.49 (dq, J = 13.5, 6.6 Hz, 2H), 1.29 (d, J = 10.1 Hz, 26H), 0.90 (t, J = 6.6 Hz, 6H).

[0200] Intermediate E:

[0201] Intermediate E is obtained by the following synthetic process:

[0202] Tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1 1) were dissolved in EtOH (150 mL) and reacted at 45°C under nitrogen for 30 h. The mixture was then concentrated under reduced pressure to remove the EtOH. Three 100 mL portions of (PE:EA=20:1) were added for slurrying, and the mixture was filtered to obtain Intermediate E (11.5 g) as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.60 (s, 6H), 2.84 (t, J=5.9 Hz, 2H), 2.47 (t, J=5.8 Hz, 2H), 1.48 (s, 9H).

[0203] Intermediate F:

[0204] Intermediate F is obtained by the following synthetic route:

[0205] To a single-necked flask, tert-butyl (2-aminoethyl)carbamate (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) were added. After reacting at 80°C overnight, water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (5.5 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2, stirred at room temperature for 3 h, and then concentrated under reduced pressure to obtain compound 3, intermediate F (6 g). LCMS (ESI) calculated for C 20 H 28 N2O2,[M+H] + m / z 329.22, found 329.24.

[0206] Intermediate G:

[0207] Intermediate G is obtained by the following synthetic process:

[0208] Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropane bromide (9.45 g), and K2CO3 (12.94 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (6.9 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.9 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate G (7.5 g). LCMS (ESI) calculated for C 22 H 32 N2O2,[M+H] + m / z 357.25, found 357.51.

[0209] Intermediate H:

[0210] Intermediate H is obtained by the following synthetic process:

[0211] Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), and K2CO3 (6.47 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 20:1-10:1) to obtain compound 2 (4.1 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (4.1 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate H (4.9 g). LCMS (ESI) calculated for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56.

[0212] Intermediate I:

[0213] Intermediate I is obtained by the following synthetic route:

[0214] Compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), and K2CO3 (15.46 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=20:1-10:1) to obtain compound 2 (6.8 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.8 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calculated for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0215] Intermediate J:

[0216] Intermediate J is obtained using the following synthetic route:

[0217] To a single-necked flask, butyric acid (5.0 g) and THF (100 mL) were added. NaH (2.73 g) was added at 0°C, and LDA (56.8 mL) was slowly added dropwise. The mixture was reacted at room temperature for 30 min. 1-Bromotridecane was added and the reaction was continued at room temperature overnight. Ice water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 10:1-5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calculated for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0218] Intermediate K:

[0219] Intermediate K is obtained by the following synthetic process:

[0220] To a three-necked flask, reactant 1 (24 g), imidazole (19.00 g), and DCM (200 mL) were added. The temperature was lowered to 0°C, and then TBDMSCI (38.57 g) was slowly added. After stirring at 25°C for 4 h, the mixture was diluted with 300 mL of DCM. The organic phase was washed twice with 1 L of water and dried over anhydrous Na2SO4. After filtration and concentration, silica gel was added and column chromatography with 1:20 DCM:MeOH was performed. The sample was collected and concentrated to yield intermediate K (28.00 g). 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 3.58 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.67-1.40 (m, 4H), 0.84 (s, 9H).

[0221] Intermediate L:

[0222] Intermediate L is obtained by the following synthetic process:

[0223] Compound DMSO (3.58 g) and anhydrous DCM (30 mL) were added to a three-necked flask. The system was cooled to -78°C and oxalyl chloride (2.91 g) was slowly added. The reaction solution was stirred at -78°C under N2 protection for 10 minutes, and compound 1 (3 g) was slowly added dropwise. Stirring was continued at -78°C under N2 protection for 1 hour. Finally, TEA (9.28 g) was added dropwise. Stirring was continued at -78°C under N2 protection for 0.5 hour, and the mixture was diluted with 100 mL DCM and washed with saturated NH4Cl solution and water, respectively. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain intermediate L (3.0 g). LCMS (ESI) calculated for C 11 H 14 O3, [M+H] + m / z 195.09, found 195.23.

[0224] Intermediate M:

[0225] Intermediate M is obtained by the following synthetic process:

[0226] To a sealed tube, intermediate K (2.0 g), compound 1 (1.79 g), and ACN (20 mL) were added. The reactants, KCO (3.81 g) and KI (1.52 g), were then added. The mixture was stirred overnight at 70°C under N2 protection, then diluted with 100 mL of EA and washed with saturated NH4Cl solution and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The mixture was then added to a silica gel column and filtered with 15:1 DCM:MeOH. The sample was collected and concentrated to yield compound 2 (2.0 g). Reactant 2 (1.80 g) and solvent DCM (20 mL) were added to a three-necked flask. The system was purged with nitrogen and the temperature was lowered to -78°C. DIBAL-H (1.00 g) was then added dropwise. After stirring at -78°C for 4 h, methanol and sodium carbonate solution were added dropwise to the reaction solution to quench the mixture. The mixture was then extracted with DCM (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate M (1.36 g). LCMS (ESI) calculated for C 15 H 33 NO2Si,[M+H] + m / z 288.52, found 288.23.

[0227] C. Compound 1003 was synthesized according to general synthetic route 1

[0228] Structural formula:

[0229] Chemical formula: C 63 H 122 N2O7

[0230] Molecular weight: 1019.68

[0231] Step 1: Synthesis of compound 3 in general synthetic route 1

[0232] Compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) were added to compound 2 (intermediate B) (4.3 g). The mixture was stirred at room temperature for 12 h under nitrogen protection, and then water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated. The organic phase was purified by silica gel column chromatography (PE:EA=40:1-30:1) to give compound 3 (2.6 g).

[0233] Step 2: Synthesis of compound 4 in general synthetic route 1

[0234] DCM (15 mL) and TFA (5 mL) were added to compound 3 (2.6 g), and the mixture was stirred at room temperature for 3 h, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to give compound 4 (3.2 g).

[0235] Step 3: Synthesis of compound 6 in general synthetic route 1

[0236] To compound 4 (3.2 g) were added DCM (20 mL), Boc-glycine (compound 5, 578 mg), and DCC (6.8 g). The mixture was stirred at room temperature under nitrogen for 12 h, and then water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA=5:1-2:1) to give compound 6 (2.2 g).

[0237] Step 4: Compound 1003

[0238] To compound 6 (2.2 g) were added DCM (15 mL) and TFA (5 mL), and the mixture was stirred at room temperature for 12 h and concentrated. The mixture was purified by column chromatography (DCM:MeOH=30:1-20:1) to give compound 1003 (1.3 g). 1 H NMR (400MHz, CDCl3) δ4.49 (d, J=2.6Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J=8.6, 5. 5Hz, 3H), 1.62 (q, J = 7.1Hz, 12H), 1.27 (d, J = 3.4Hz, 78H), 0.94-0.86 (m, 18H).

[0239] D. Compound 1002 was synthesized according to general synthetic route 1

[0240] Structural formula:

[0241] Chemical formula: C 60 H 116 N2O7

[0242] Molecular weight: 977.60

[0243] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that intermediate C was used as compound 2 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5Hz, 3H), 1 .64(d,J=6.9Hz,6H),1.50-1.42(m,6H),1.27(s,72H),0.90(t,J=6.7Hz,18H).

[0244] E. Compound 1004 was synthesized according to general synthetic route 1

[0245] Structural formula:

[0246] Chemical formula: C 54 H 104 N2O7

[0247] Molecular weight: 893.43

[0248] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that palmitic acid was used as compound 2 in the general synthetic route 1. 1 HNMR (400MHz, CDCl3) δ7.55(s,1H),4.45(s,6H),3.74(s,2H),2.35(t,J=7.6Hz,6H),1.60(p,J=6.9Hz,6H),1.27(s,72H),0.90(t,J=6.7Hz,9H).

[0249] F. Compound 1001 was synthesized according to general synthetic route 1

[0250] Structural formula:

[0251] Chemical formula: C 59 H 108 N2O7

[0252] Molecular weight: 957.52

[0253] The product was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that (9Z)-9-hexadecenoic acid was used as compound 2 in the general synthetic route 1, and 5-(N,N-dimethylamino)pentanoic acid was used as compound 5 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ5.42-5.30(m,6H),4.51-4.38(m,6H),3.19(dt,J=11.3,5 .6Hz,2H),2.97(t,J=5.9Hz,6H),2.46(t,J=6.6Hz,1H),2.35(td,J=7.6,3.1Hz, 5H),2.20(t,J=7.6Hz,2H),2.03(d,J=6.2Hz,12H),1.89-1.81(m,3H),1.74(q, J=7.8Hz,2H),1.67-1.51(m,6H),1.31(d,J=7.8Hz,48H),0.90(t,J=6.7Hz,9H).

[0254] G. Compound 1014 was synthesized according to general synthetic route 2

[0255] Structural formula:

[0256] Chemical formula: C 70 H 137 N3O9

[0257] Molecular weight: 1164.88

[0258] Step 1: Synthesis of Intermediate 3 in General Synthesis Route 2

[0259] Intermediate 3 was synthesized according to the method shown in step 1 of general synthetic route 1, except that intermediate E was used as compound 1 in general synthetic route 1.

[0260] Step 2: Synthesis of compound 4 of general synthetic route 2

[0261] DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added to compound 3 (4.8 g). After reacting at room temperature for 12 h, water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH=20:1-10:1) to give compound 4 (3.3 g).

[0262] Step 3: Synthesis of compound 6 of general synthetic route 2

[0263] Compound 4 (4.0 g), DCM (80 mL), EDCI (1.1 g), 1-hydroxybenzotriazole (HOBt) (0.78 g), and DIEA (2.5 g) were added to compound 5 (1.9 g). After stirring at room temperature for 12 h, water and dichloromethane were added, stirred and separated, and the organic phase was washed with saturated brine. The organic phase was separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH=30:1-20:1) to give compound 6 (4.0 g).

[0264] Step 4: Compound 1014

[0265] To compound 6 (4.0 g) were added MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)). The mixture was stirred at room temperature overnight under hydrogen atmosphere, filtered through celite, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1-15:1) to give compound 1014 (2 g). 1 H(400MHz, CDCl3)δ7.38(t,J=5.8Hz,1H),4.14-4.05(m,6H),3.58(t,J=4.9Hz,4H),3.31(q,J=5.7Hz,2H),2.89(t,J=6.3Hz, 2H), 2.64 (q, J=5.2Hz, 6H), 2.29 (dq, J=10.8, 4.4Hz, 6H), 1.66-1.37 (m, 12H), 1.24 (d, J=4.3Hz, 78H), 0.87 (t, J=7.1Hz, 18H).

[0266] H. Compound 1015 was synthesized according to general synthetic route 2

[0267] Structural formula:

[0268] Chemical formula: C 72 H 141 N3O9

[0269] Molecular weight: 1192.93

[0270] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate G was used as compound 5 in general synthetic route 2. 1H(400MHz, CDCl3)δ7.58(t,J=5.7Hz,1H),4.15-4.01(m,6H),3.70(t,J=5.4Hz,4H),3.39(q,J=5.7Hz,2H),2.86(t,J=6.2Hz,2H),2 .57(dt,J=20.5,5.8Hz,6H),2.36-2.22(m,5H),1.76-1.67(m,4H),1.62-1.35(m,13H),1.23(d,J=4.6Hz,78H),0.92-0.80(m,18H).

[0271] I. Compound 1020, synthesized according to general synthetic route 2

[0272] Structural formula:

[0273] Chemical formula: C 74 H 145 N3O9

[0274] Molecular weight: 1220.99

[0275] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate H was used as compound 5 in the general synthetic route 2.

[0276] J. Compound 1025 was synthesized according to general synthetic route 2

[0277] Structural formula:

[0278] Chemical formula: C 69 H 135 N3O8

[0279] Molecular weight: 1134.85

[0280] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate I was used as compound 5 in the general synthetic route 2. 1 H(400MHz, CDCl3)δ6.98(t,J=5.7Hz,1H),4.10(m,J=3.8Hz,6H),3.63(t,J=5.2Hz,2H),3.35(q,J=5.8Hz,2H),2.89(t,J=6.1Hz,2 H), 2.57 (q, J = 5.3Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1Hz, 5H), 1.65-1.41 (m, 13H), 1.24 (d, J = 4.4Hz, 78H), 0.87 (t, J = 7.1Hz, 18H).

[0281] K. Compound 1005, synthesized according to general synthetic route 2

[0282] Structural formula:

[0283] Chemical formula: C 68 H 133 N3O7

[0284] Molecular weight: 1104.83

[0285] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-dimethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 HNMR (400MHz, CDCl3) δ4.19-4.07(m,6H),3.40(d,J=5.7Hz,2H),2.95-2.87(m,2H),2.82(s,4H),2.64-2.55(m,2H),2.3 8(s,6H),2.33-2.29(m,3H),1.63-1.55(m,6H),1.49-1.44(m,2H),1.26(d,J=5.4Hz,78H),0.89(td,J=7.0,2.8Hz,18H).

[0286] L. Compound 1006 was synthesized according to general synthetic route 2

[0287] Structural formula:

[0288] Chemical formula: C 53 H 103 N3O7

[0289] Molecular weight: 894.42

[0290] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that tetradecanoic acid was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ7.99(t,J=5.8Hz,1H),4.13(s,6H),3.60(q,J=5.3Hz,2H),3.38-3.34(m,2H),3.00(s,6H),2.94(t, J=5.8Hz,2H),2.45(t,J=5.8Hz,2H),2.35(t,J=7.6Hz,6H),1.66-1.56(m,6H),1.33-1.23(m,60H),0.90(t,J=6.8Hz,9H).

[0291] M. Compound 1007, synthesized according to general synthetic route 2

[0292] Structural formula:

[0293] Chemical formula: C 68 H 133 N3O7

[0294] Molecular weight: 1104.83

[0295] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate D was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H NMR (400MHz, Chloroform-d) δ7.13(t,J=5.0Hz,1H),4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=6.0Hz,2H),2.47-2.28(m,7H ), 2.24 (s, 6H), 1.60 (dq, J = 14.7, 7.2Hz, 6H), 1.47 (dd, J = 14.2, 6.7Hz, 6H), 1.27 (d, J = 3.4Hz, 78H), 0.89 (td, J = 6.7, 2.5Hz, 18H).

[0296] N. Compound 1008, synthesized according to general synthetic route 2

[0297] Structural formula:

[0298] Chemical formula: C 68 H 133 N3O7

[0299] Molecular weight: 1104.01

[0300] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1H NMR(400MHz,Chloroform-d)δ4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=5.9Hz,2H),2.51-2.26(m,7H),2.24 (s, 6H), 1.59 (ddt, J = 14.8, 10.9, 6.4Hz, 6H), 1.47 (tq, J = 11.0, 5.4Hz, 6H), 1.26 (s, 77H), 0.89 (t, J = 6.7Hz, 18H).

[0301] O. Compound 1009, synthesized according to general synthetic route 2

[0302] Structural formula:

[0303] Chemical formula: C 62 H 121 N3O7

[0304] Molecular weight: 1020.66

[0305] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate J was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H(400MHz, CDCl3)δ7.04(t,J=5.0Hz,1H),4.12(m,J=3.3Hz,6H),3.30(q,J=5.6Hz,2H),2.91(t,J=6.0Hz,2H),2.39 (t,J=6.0Hz,2H),2.33-2.25(m,5H),2.22(s,6H),1.76-1.54(m,12H),1.24(m,J=3.7Hz,66H),0.90-0.84(m,18H).

[0306] P. Compound 1011 was synthesized according to general synthetic route 2

[0307] Structural formula:

[0308] Chemical formula: C 70 H 137 N3O7

[0309] Molecular weight: 1132.88

[0310] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.19-4.08(m,6H),3.31(q,J=5.8Hz,2H),2.92(t,2H),2.57(t,J=6.9Hz,6H),2.37-2.26( m,5H),1.62(ddd,J=18.3,9.0,4.3Hz,12H),1.27(d,J=4.7Hz,78H),1.04(t,J=7.1Hz,6H),0.94-0.86(m,18H).

[0311] Q. Compound 1012, synthesized according to general synthetic route 2

[0312] Structural formula:

[0313] Chemical formula: C 72 H 141 N3O7

[0314] Molecular weight: 1160.93

[0315] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ6.59 (s, 1H), 4.18-4.07 (m, 6H), 3.29 (q, J = 5.7Hz, 2H), 2.91 (t, J = 6.2Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5Hz, 4H), 2.35-2.27 (m, 5H), 1.63-1.42 (m, 16H), 1.27 (m, J = 4.5Hz, 78H), 0.93-0.85 (m, 24H).

[0316] R. Compound 1013, synthesized according to general synthetic route 2

[0317] Structural formula:

[0318] Chemical formula: C 74 H 145 N3O7

[0319] Molecular weight: 1188.99

[0320] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1HNMR (400MHz, CDCl3) δ4.10(d,J=4.1Hz,6H),3.26(q,J=5.8Hz,2H),2.89(t,J=6.2Hz,2H),2.52(d,J=6.6Hz,2H) ,2.41(t,J=7.4Hz,4H),2.33-2.25(m,5H),1.60(d,J=11.8Hz,16H),1.24(d,J=4.5Hz,82H),0.96-0.79(m,24H).

[0321] S. Compound 1029, synthesized according to general synthetic route 2

[0322] Structural formula:

[0323] Chemical formula: C 68 H 132 N2O8

[0324] Molecular weight: 1105.81

[0325] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethyl-2-hydroxyethylamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.16(t,J=5.8Hz,2H),4.13-4.05(m,6H),2.86(t,J=6.3Hz,2H),2.55(t,J=5.8Hz,2H) ,2.44(t,J=6.3Hz,2H),2.35-2.20(m,9H),1.60-1.39(m,11H),1.24(d,J=4.5Hz,80H),1.02-0.79(m,18H).

[0326] T. Compound 1111, synthesized according to general synthetic route 2

[0327] Structural formula:

[0328] Chemical formula: C 70 H 136 N4O7

[0329] Molecular weight: 1145.88

[0330] The product was synthesized according to general synthetic route 2, which was similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate was used as compound 5 in general synthetic route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97-2.80 (m, 5H), 2.70 (s, 5H), 2.46-2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54-1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0331] U. Compound 1118, synthesized according to general synthetic route 2

[0332] Structural formula:

[0333] Chemical formula: C 71 H 139 N3O8

[0334] Molecular weight: 1162.91

[0335] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and intermediate K was used as compound 5 in general synthetic route 2. 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.61(s,2H),3.42(d,J=33.5Hz,2H),2.91(t,J=6.2Hz,3H ), 2.36 (dd, J = 9.2, 3.6Hz, 6H), 1.47 (dd, J = 13.9, 6.2Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7Hz, 18H).

[0336] V. Compound 1010, synthesized according to general synthetic route 3

[0337] Structural formula:

[0338] Chemical formula: C 67 H 133 N3O6

[0339] Molecular weight: 1076.82

[0340] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 3

[0341] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0342] Step 3: Synthesis of compound 6 of general synthetic route 3

[0343] Compound 4 (600 mg), chloroacetaldehyde (compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) were added to DCE (60 mL). After reacting at room temperature overnight, water and dichloromethane were added, stirred, and the liquids were separated. The organic phase was washed with saturated brine, separated, and concentrated. Compound 6 (270 mg) was obtained by silica gel column chromatography (DCM:MeOH=30:1-15:1).

[0344] Step 4: Synthesis of Compound 1010

[0345] MeCN (20 mL), N,N-dimethylethylenediamine (compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) were added to compound 6 (270 mg). The mixture was reacted at 70°C under nitrogen protection overnight, and then silica gel column chromatography was used to obtain compound 1010 (16 mg). 1 H(400MHz, CDCl3)δ4.10(m,J=2.9Hz,6H),2.76-2.64(m,6H),2.40(t,J=6.2Hz,2H),2.29(ddd,J=8.5,5.6 ,2.9Hz,3H),2.22(s,6H),1.97(s,6H),1.50-1.41(m,6H),1.24(d,J=4.2Hz,78H),0.87(t,J=7.1Hz,18H).

[0346] W. Compound 1059, synthesized according to general synthetic route 3

[0347] Structural formula:

[0348] Chemical formula: C 67 H 132 N2O7

[0349] Molecular weight: 1077.80

[0350] The synthesis was carried out according to the general synthetic route 3, which is similar to the synthesis process of compound 1010, except that intermediate C and intermediate L were used as compound 2 and compound 5 in the general synthetic route 3, respectively. In addition, compound 6 in the general synthetic route 3 was subjected to the following process steps for functional group transformation:

[0351] To intermediate 6a (1.5 g) was added MeOH (5 mL) and THF (5 mL), and the reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g) were added. The mixture was reacted under H2 at 25°C overnight, filtered through celite, and the filtrate was concentrated and chromatographed on a silica gel column (PE:EA 30:1-10:1) to afford intermediate 6b (520 mg). To intermediate 6b (470 mg) was added DCM (5 mL), and SOCl2 (1064.26 mg, 8.946 mmol) was added dropwise at 0°C. The mixture was allowed to react at room temperature for 3 hours, and then the reaction mixture was extracted with water and DCM. The organic phase was concentrated and chromatographed on a silica gel column (PE:EA 20:1) to afford intermediate 6c (181 mg). To intermediate 6c (170 mg) were added dimethylamine (143.39 mg) and DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg). The reaction solution was stirred at 70°C, filtered, concentrated, and purified by silica gel column chromatography (DCM:MeOH 20:1) to give compound 1059 (91 mg, 53.22% yield). 1 H NMR(400MH z,Chloroform-d)δ4.11(s,6H),3.51(dt,J=18.3,5.6Hz,4H),2.81(dd,J=10.3,5.1Hz,2H),2.50(t,J=6.0Hz,2 H),2.42-2.32(m,3H),2.28(s,6H),2.08-1.98(m,1H),1.53-1.38(m,7H),1.27(s,77H),0.90(t,J=6.7Hz,18H).

[0352] X. Compound 1112, synthesized according to general synthetic route 3

[0353] Structural formula:

[0354] Chemical formula: C 69 H 136 N2O6

[0355] Molecular weight: 1089.85

[0356] The compound was synthesized according to the general synthetic route 3, which is similar to the synthetic process of compound 1010, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in the general synthetic route 3, respectively. 1HNMR(400MHz,Chloroform-d)δ4.11(s,6H),3.11(s,1H),2.57(t,J=6.8Hz,2H),2.44-2.19 (m,12H),2.04(q,J=6.5Hz,2H),1.53-1.38(m,13H),1.27(s,92H),0.90(t,J=6.7Hz,20H).

[0357] Y. Compound 1113, synthesized according to general synthetic route 4

[0358] Structural formula:

[0359] Chemical formula: C 71 H 138 N2O8

[0360] Molecular weight: 1147.89

[0361] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 4

[0362] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0363] Step 3: Synthesis of compound 6 of general synthetic route 4

[0364] DIEA (0.30 g) and DCM (20 mL) were added to compound 4. Under nitrogen, the temperature was cooled to 0°C, and 5-bromovaleryl chloride (compound 5, 0.27 g) was added. The mixture was stirred at 25°C for 2 h. After dilution with 100 mL of DCM, the organic phase was washed twice with 100 mL of water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (PE:EA 20:1) to obtain compound 6 (800 mg).

[0365] Step 4: Synthesis of compound 8 of general synthetic route 4

[0366] Compound 6 (750.00 mg), intermediate K (compound 7, 217.31 mg), and DMF (20 mL) were added to a three-necked flask. KCO (184.16 mg) and KI (110.60 mg) were then added and stirred at 25°C for 16 h. The temperature was then raised to 100°C and stirred for 3 h. The reaction solution was diluted with 200 mL of EA, and the organic phase was washed twice with 300 mL of saturated brine. The organic phase was dried over anhydrous NaSO, filtered, and concentrated. Silica gel was added and the mixture was filtered through a column using 20:1 DCM:MeOH. The sample was collected and concentrated to yield compound 8 (700.00 mg).

[0367] Step 5: Synthesis of Compound 1113

[0368] Compound 8 (700.00 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (5.55 mL). The reaction mixture was stirred at 25°C for 2 h, diluted with 50 mL of EA, and the organic phase was washed with a saturated NaHCO solution and water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1113 (201.1 mg, 31.59% yield). 1 H NMR (400MHz, CDCl3) δ4.50-4.39(m,6H),3.59(t,J=4.6Hz,2H),2.49(s,3H),2.35-2.26(m,6H),2.14 (t,J=7.5Hz,2H),1.75-1.69(m,4H),1.66-1.43(m,17H),1.27(d,J=3.7Hz,78H),0.94-0.87(m,18H).

[0369] Z. Compound 1115, synthesized according to general synthetic route 4

[0370] Structural formula:

[0371] Chemical formula: C 67 H 131 N3O7

[0372] Molecular weight: 1090.80

[0373] The compound was synthesized according to general synthetic route 4, which is similar to the synthetic process of compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine were used to synthesize compound 5 and compound 7 in general synthetic route 4, respectively. 1H NMR(400MHz, CDCl3)δ7.42(s,1H),4.48(tt,J=11.9,5.7Hz,6H),3.27(s,2H),3.13(s,2H),3.01(s,2H),2.83(s,6 H), 2.38-2.25 (m, 3H), 1.55 (dtt, J = 36.3, 14.1, 7.3Hz, 12H), 1.27 (d, J = 3.3Hz, 78H), 0.90 (td, J = 7.2, 3.4Hz, 18H).

[0374] AA. Compound 1114, synthesized according to general synthetic route 5

[0375] Structural formula:

[0376] Chemical formula: C 70 H 138 N2O7

[0377] Molecular weight: 1119.88

[0378] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 5

[0379] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0380] Step 3: Synthesis of compound 6 of general synthetic route 5

[0381] Compound 4 (500 mg), intermediate M (compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) were added to a three-necked flask. The reaction solution was stirred under N2 at 25°C for 16 h, then 100 mL of water was added to the reaction solution. The mixture was extracted twice with EA (100 mL). The organic phases were combined and concentrated. Silica gel was added and the mixture was passed through a column with 1:20 DCM:MeOH. The sample was collected and concentrated to give compound 6 (500 mg).

[0382] Step 4: Synthesis of Compound 1114

[0383] Compound 6 (450 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (3.65 mL). After stirring at 25°C for 2 h, the mixture was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO solution and water. The organic layer was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1114 (107 mg, 24.84% yield). 1 H NMR (400MHz, CDCl3) δ4.16-4.07(m,6H),3.60(t,J=4.8Hz,2H),2.62(t,J=7.0Hz,2H),2.49(s,3H),2.31(tt, J=8.4,5.5Hz,6H),1.70(d,J=4.6Hz,3H),1.64-1.39(m,18H),1.27(d,J=4.0Hz,78H),0.90(t,J=7.1Hz,18H).

[0384] Z. Compound 1120, synthesized according to general synthetic route 5

[0385] Structural formula:

[0386] Chemical formula: C 72 H 142 N2O7

[0387] Molecular weight: 1147.90

[0388] The product was synthesized according to general synthetic route 5, which was similar to the synthetic process of compound 1114, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in general synthetic route 5, respectively. 1H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.63 (s, 2H), 2.57 (t, J = 6.8 Hz, 4H), 2.37 (ddd, J = 8.5, 7.1, 4.3 Hz, 5H), 1.67-1.41 (m, 24H), 1.27 (s, 79H), 0.90 (t, J = 6.7 Hz, 18H).

[0389] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0390] 1. Preparation of Compound 6001

[0391] Structural formula:

[0392] Molecular weight: 499.43

[0393] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h and then passed through a reverse column to obtain compound 6002.

[0394] 0.7 g of compound 6002, 0.4 g of palladium on carbon and 28 mL of tetrahydrofuran were added to a reaction flask and stirred. The gas was replaced with hydrogen and the pressure was maintained with a hydrogen balloon. After 16 h, the mixture was filtered and passed through a column (methanol-DCM system) in the forward direction to obtain 200 mg of compound 6001. 1 H NMR (400MHz, CDCl3) δ7.54(t,J=5.5Hz,2H),3.79-3.68(m,4H),3.42(dd,J=10.0,5.4Hz,4H),2.89(t,J =6.0Hz, 4H), 2.55 (dt, J = 11.8, 6.8Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2Hz, 32H), 0.91 (t, J = 6.8Hz, 3H).

[0395] 2. Preparation of Compound 6002

[0396] Structural formula:

[0397] Molecular weight: 497.42

[0398] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h, and the organic solvent was concentrated. The mixture was passed through a reverse phase column and freeze-dried to obtain 400 mg of compound 6002. 1 H NMR(400MHz, CDCl3) δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79-3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2. 82-2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10-1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0399] 3. Preparation of Compound 6003

[0400] Structural formula:

[0401] Molecular weight: 445.34

[0402] 4.00 g of tetradecylamine, 6.53 g of hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70°C. After 29 h, the organic solvent was concentrated, passed through a reverse phase column and freeze-dried to obtain 1.5 g of compound 6003. 1 H NMR(400MHz, CDCl3)δ4.29(t,J=7.5Hz,4H),3.89-3.75(m,4H),2.81(t,J=6.2Hz,4H), 2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0403] 4. Preparation of Compound 6004

[0404] Structural formula:

[0405] Molecular weight: 553.48

[0406] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate, and 5.6 mL of n-butanol were added to a reaction flask, stirred, and heated to 100°C. After 4 h, 1 mL of n-butyl acrylate was added, and the reaction was continued for 1 h. The mixture was passed through a normal phase column (petroleum ether-ethyl acetate system), and 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added, stirred and hydrolyzed for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to obtain a mixed solution, and the pH was adjusted to neutral. The solution was spin-dried, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was spin-dried. 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT, and 2.3 g of EDCI were added, and the reaction was allowed to react at room temperature for 18 h. After concentrating the organic solvent, the solution was passed through a reverse phase column and freeze-dried to obtain 240 mg of compound 6004. 1 HNMR (400MHz, CDCl3) δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17( s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0407] 5. Preparation of Compound 6005

[0408] Structural formula:

[0409] Molecular weight: 597.54

[0410] 25.00 g of 11-henicosone, 62.05 g of ammonium acetate, and 500 mL of methanol were added to a reaction flask and stirred. 6.55 g of sodium cyanoborohydride was added and reacted for 16 h. 250 mL of water and 250 mL of DCM were added, and the layers were separated. The aqueous phase was extracted with 50 mL of DCM, and the combined organic phases were passed through a normal phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0411] 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex were stirred in a reaction flask and heated to 100° C. 10 mL of n-butyl acrylate was added. After 16 h, the mixture was passed through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0412] A solution prepared by mixing 31.00 g of compound 6005-B with 6.55 g of sodium hydroxide, 310 mL of methanol, and 31 mL of water was added to a reaction flask and stirred. After 20 minutes, 150 mL of THF was added, and the temperature was raised to 50°C with stirring. After 30 minutes, 16.13 g of concentrated hydrochloric acid and 160 mL of methanol were added and stirred. After 30 minutes, the solvent was dried by spin-drying, and 300 mL of DCM was added to dissolve the mixture. The mixture was dried over anhydrous magnesium sulfate, filtered, and dried to obtain 27.00 g of compound 6005-C as an oil.

[0413] 27.00 g of compound 6005-C, 25.00 g of 4-amino-1-butanol, 41.85 g of EDCI, 14.74 g of HOBT, and 310 mL of DCM were added to a reaction flask and stirred. After 18 h, the organic solvent was concentrated and filtered through a column (methanol-DCM system) to remove excess 4-amino-1-butanol, yielding 6.80 g of compound 6005. 1 H NMR (400MHz, CDCl3) δ3.70(t,J=5.6Hz,4H),3.28(t,J=5.6Hz,4H),2.74(t,J=6.0Hz,4H),2.40-2 .45(m,1H),2.34(t,J=6.4Hz,4H),1.63-1.65(m,8H),1.25-1.35(m,36H),0.91(t,J=6.4Hz,6H).

[0414] 6. Preparation of Compound 6006

[0415] Structural formula:

[0416] Molecular weight: 553.48

[0417] 1.00g of oleylamine, 0.47g of hydroxyethyl acrylamide, and 8mL of ethanol were added to a reaction flask, stirred, and heated to 70°C. After 13 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. 1.5mL of n-butyl acrylate and 5mL of n-butanol were added, stirred, and heated to 100°C. After 3 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. A solution of 0.4g of sodium hydroxide, 10mL of methanol, and 1mL of water was added, stirred for 1h, and a solution of 1mL of concentrated hydrochloric acid and 8mL of methanol was added. The mixture was stirred for 30min, the solvent was dried and dissolved in 100mL of DCM, dried over anhydrous magnesium sulfate, filtered, and dried. 0.45g of 6-amino-1-hexanol, 0.27g of HOBT, 0.78g of EDCI, and 10mL of DCM were added, stirred. After 14 hours, the organic solvent was concentrated and passed through a methanol-DCM column to obtain 300mg of compound 6006. 1 HNMR(400MHz, CDCl3)δ7.83(s,1H),6.95(s,1H),5.43-5.28(m,2H),3.74-3 .67(m,2H),3.64(t,J=6.3Hz,2H),3.39(dd,J=10.0,5.3Hz,2H),3.24(dd,J= 12.9,6.7Hz,2H),2.74(t,J=5.8Hz,4H),2.48-2.42(m,2H),2.41-2.33(m,4 H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66-1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0418] 7. Compound 6007

[0419] Structural formula:

[0420] Molecular weight: 953.78

[0421] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h. The mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1-10:1) to obtain product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The mixture was concentrated under reduced pressure, and 30 mL of toluene was added. The mixture was heated to 60°C to dissolve, then slowly cooled to room temperature and evaporated to dryness under reduced pressure to obtain compound 3 (15 g). Compound 3 (2 g) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve. Methyl acrylate (2.5 g) was then slowly added and stirred to mix. The mixture was heated to 60°C overnight. The reaction was stopped, and the reaction solution was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1-20:1) to obtain compound 4 (2.5 g). Compound 4 (2.3 g) and ethylenediamine (20 mL) were added to the single-necked flask at room temperature and reacted overnight at room temperature. The reaction was stopped and the mixture was directly concentrated under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0422] 8. Compounds 6008-6018

[0423] Synthesis of compound 6009

[0424] Structural formula:

[0425] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise to a solution of stirred methyl methacrylate (6 mL) in methanol (20 mL) under a nitrogen atmosphere and the reaction was continued overnight. The reactant was spin-dried at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. The resulting product was then separated by column chromatography to obtain a colorless oil. 1H-NMR (300 MHz, CDCl 3 ): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0426] Then, a solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 ml) that was vigorously stirred at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, maintaining the temperature not higher than 40°C. Excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was repeatedly recrystallized from chloroform and cyclohexane to obtain a white solid. 1H-NMR (300MHz, CDCl3): 0.88(t,3H), 1.25(s,30H), 1.84(s,4H), 2.38(m,6H), 2.73(m,4H), 2.82(m,4H), 3.29(m,4H), 7.47(s,2H).

[0427] The preparation methods of 6008 and 6009 differ only in that an equal molar amount of tetradecylamine is used instead of hexadecylamine. 6010-6017 refer to the preparation method of 6009 and use an equal molar amount of the corresponding R a -NH2 is used to replace hexadecylamine.

[0428] 6018 was prepared by referring to the method of 6007, using an equimolar amount of the corresponding R a -NH2 replaces octadecylamine to prepare.

[0429] 9. Compound 6019

[0430] Structural formula:

[0431] Molecular weight: 753.66

[0432] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask, followed by DCM (50 mL) and stirring. 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 5:1) to afford product 3 (4 g). At room temperature, product 3 (5.0 g), MeOH (50 mL), and Pd / C (1.0 g) were added to a three-necked flask. The hydrogen atmosphere was replaced, the temperature was raised to 50°C, and the mixture was stirred for 1 h. The reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was dried to afford product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. DMF (50 mL) was added and stirred to dissolve. Then, product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred. The mixture was allowed to react overnight at room temperature. Water (500 mL) and EA (500 mL) were added, and the mixture was separated and extracted. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 20:1-10:1) to obtain solid 6 (380 mg). Solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to the single-necked flask at room temperature and reacted at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0433] 10. Compound 6020

[0434] Structural formula:

[0435] Molecular weight: 810.19

[0436] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was added dropwise. The mixture was stirred and returned to room temperature for 4 h. The reaction was stopped, and the mixture was directly concentrated under reduced pressure. Purification by column chromatography (PE:EA = 20:1-10:1) afforded product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The reaction was stopped, the reaction solution was concentrated under reduced pressure, 30 mL of toluene was added, and the mixture was heated to 60°C for dissolution. The mixture was slowly cooled to room temperature, filtered, and evaporated to dryness under reduced pressure to afford compound 3 (15 g). At room temperature, compound 3 (1.3 g) was added to a single-necked flask, and DMF (20 mL) was added and stirred. Then, 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g) were added, stirred, and reacted at room temperature overnight. Water (200 mL) was added and the mixture was directly freeze-dried under reduced pressure to obtain compound 4 (4 g). Compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) were added to a single-necked flask at room temperature and reacted for 1 h. The reaction was stopped and the reaction solution was concentrated under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57-3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03-1.58 (m, 10H), 1.31 (s, 30H), 1.01-0.82 (m, 3H).

[0437] 11. Compound 6021

[0438] Structural formula:

[0439] Molecular weight: 1182.79

[0440] At room temperature, compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020) was added to a single-necked flask, MeOH (20 mL) was added and stirred, and then TREN (23.0 g) was added, stirred, and the temperature was raised to 60°C, stirred overnight, and concentrated under reduced pressure. The crude product was lyophilized to obtain product 3 (25 g). At room temperature, product 3 (22.0 g) and DCM (200 mL) were added to a single-necked flask, the temperature was lowered to 0°C, and (Boc)2O (71.7 g) was slowly added dropwise. After the temperature was restored to room temperature, the reaction was carried out for 3 h, water (200 mL) was added for extraction, and DCM (100 mL) was added for separation and extraction. The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain a yellow oil 4 (5.8 g). At room temperature, yellow oil 4 (1.4 g) was added to a single-necked flask. HCl / Dioxane (4 M) (15 mL) was slowly added under an ice-water bath and stirred. After addition, the mixture was returned to room temperature and reacted for 0.5 h. The mixture was then concentrated under reduced pressure to obtain compound 5 (800 mg). Compound 5 (1.0 g), (S)-2,6-di-tert-butyloxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g), and DMF (10 mL) were added to a single-necked flask at room temperature. The mixture was reacted overnight at room temperature. 100 mL of water and 100 mL of EA were added, and the mixture was separated and extracted. The EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). To a one-necked flask at room temperature, compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL) were added. The reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6021 (220 mg). 1H NMR(400MHz, Methanol-d4)δ3.96(t,J=6.6Hz,4H),3.75(dt,J=14.0,6.6Hz,4H),3.62-3.45(m,12H),3.36-3.33(m,14H),3.25-3.17(m,2H),3.04 -2.90(m,8H),2.83(t,J=6.9Hz,4H),2.05-1.82(m,8H),1.73(p,J=7.7Hz ,10H),1.53(qd,J=8.3,7.7,4.3Hz,8H),1.30(s,28H),0.97-0.87(m,3H).

[0441] 12. Compound 6023

[0442] Structural formula:

[0443] Molecular weight: 670

[0444] At room temperature, compound dodecylamine (10 g) was added to a three-necked flask, followed by MeOH (100 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (10.2 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h, then stopped, concentrated under reduced pressure, and purified by column chromatography (PE:EA=20:1-10:1) to obtain product 2 (19 g). At room temperature, product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) were added to a single-necked flask, heated to 60°C, maintained at this temperature for overnight, and concentrated under reduced pressure to obtain compound 3 (20 g). 500 mg of the product was purified and lyophilized to obtain compound 6023-1 (218 mg). At room temperature, raw material 6023-1 (1.4 g) was added to a single-necked flask. DCM (20 mL) was added and stirred to dissolve. (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was separated, extracted, dried, and concentrated under reduced pressure to obtain compound 4 (3.0 g). Compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) were added to the single-necked flask at room temperature and reacted for 2 h. The mixture was then concentrated under reduced pressure, purified, and lyophilized to obtain compound 6023 (123 mg). 1H NMR(400MHz, Methanol-d4)δ3.88(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.36-3.33(m,2H),3.31-3.14(m,4H),3.04-2.93(m,4H),2. 77(t,J=6.5Hz,4H),2.00-1.82(m,4H),1.82-1.66(m,6H),1.50(qd,J=8.2,7.8,4.2Hz,4H),1.45-1.25(m,18H),0.96-0.87(m,3H).

[0445] 13. Compound 6024

[0446] Structural formula:

[0447] Molecular weight: 870.24

[0448] Product 6023-1 (1.4 g, synthesized by the same method as 6023-1 in compound 6023) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.8 g) was slowly added and stirred to mix. The temperature was raised to 60°C, and the reaction was allowed to proceed overnight at this temperature. The mixture was concentrated under reduced pressure to obtain compound 4 (2.0 g). Compound 4 (2.4 g) and ethylenediamine (20 mL) were added to a single-necked flask at room temperature, and the reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6024 (191 mg). 1HNMR (400MHz, Methanol-d4) δ3.69(t,J=6.0Hz,4H),3.60-3.48(m,20H),3.42(t,J=6.1Hz,4H),3.25-3.19(m,2H),3 .12(t,J=5.8Hz,8H),2.83(t,J=6.5Hz,12H),1.78(tt,J=11.0,6.4Hz,2H),1.51-1.18(m,20H),0.91(t,J=6.7Hz,3H).

[0449] 14. Compound 6026

[0450] Structural formula:

[0451] Molecular weight: 443.67

[0452] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the following differences: an equal molar amount of compound 1 was used instead of starting material 5 of compound 6019, and an equal molar amount of N-(tert-butyloxycarbonyl)ethanolamine was used instead of intermediate 4 of compound 6019. 1H NMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36-1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0453] Compounds 6025-6034 were obtained by referring to the synthetic route of the aforementioned compounds, except that hydroxyethylamine was used instead of the corresponding ethylenediamine, and the amino group of the hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc. After the reaction was completed, the protecting group was removed according to conventional methods.

[0454] (3) First Ionizable Lipid Example Compounds and Their Properties

[0455] The exemplary compounds of the present application and their properties are listed in Table 1, which were synthesized according to the process routes in Preparation Example 1.

[0456] The calculated c-pKa (molnetwork), LogP (cLogP driver) of the exemplary compounds of this application, c-pKa and cLogP values ​​were generated by the ChemDraw module of Chemoffice.

[0457] Table 1 Properties of exemplary compounds of the first ionizable lipid

[0458] The above properties of the first ionizable lipid exemplary compounds of the present application indicate that they can serve as excellent surfactants and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0459] (4) Preparation of compositions based on blank lipid nanoparticles

[0460] 4.1 Preparation of blank lipid nanoparticles

[0461] Blank lipid nanoparticles were prepared according to the above molar ratios, and the specific methods are as follows:

[0462] The lipid raw material was dissolved in ethanol to obtain a lipid ethanol solution, and the total concentration of all lipid raw materials in ethanol was 8 mg / mL. The lipid ethanol solution was mixed with 50 mM citrate buffer saline (pH 4.0) solution in a nanopreparation equipment at a volume ratio of 1:3, and then ultrafiltered and collected to obtain blank lipid nanoparticles. The lipid concentration of the composition was 2 mg / mL.

[0463] 4.2 Preparation of blank lipid nanoparticle-based compositions

[0464] Nucleic acid is used as the active ingredient, and the nucleic acid is prepared into a solution with a concentration twice that of the solution in each embodiment using nuclease-free water as solution 1; blank lipid nanoparticles are diluted with nuclease-free water according to the proportion of the prescription in each embodiment to obtain solution 2; equal volumes of solution 1 and solution 2 are mixed and vortexed for 2-3 seconds to obtain a composition based on blank lipid nanoparticles.

[0465] (5) Particle size and zeta potential measurement of blank lipid nanoparticle compositions

[0466] 1. Particle Size and Polydispersity Index (PDI) Determination: The average particle size and PDI of the nanoparticle sample solutions in the examples were determined by dynamic light scattering using a Malvern ZetaSizerNano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0467] 2. The encapsulation efficiency test method is as follows:

[0468] The mRNA encapsulation efficiency in the blank lipid nanoparticle-based composition was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions.

[0469] The pDNA encapsulation efficiency in the blank lipid nanoparticle-based composition was determined using a dsDNA HS kit (Novozymes / EQ121) according to the manufacturer's instructions.

[0470] Follow the manufacturer's instructions for use. microRNA Reagent Kit, used to determine the siRNA encapsulation efficiency in blank lipid nanoparticle-based compositions.

[0471] The average particle size, PDI, and encapsulation efficiency data of the blank lipid nanoparticle-based composition prepared in the examples are shown in Table 2.

[0472] Table 2 Summary of particle size, PDI, and encapsulation efficiency of compositions based on blank lipid nanoparticles

[0473] As can be seen from Table 2, the composition based on blank lipid nanoparticles provided in the present application has a small and uniform particle size and a high encapsulation efficiency (>80%).

[0474] (6) Evaluation of in vivo delivery effects of compositions based on blank lipid nanoparticles

[0475] 6.1 Evaluation of the in vivo delivery effect of blank lipid nanoparticle-mRNA complexes injected intravenously into mice

[0476] The in vivo delivery efficiency of the blank lipid nanoparticle-firefly luciferase mRNA (Luciferase mRNA) complex of Example 5 was determined using 6-8 week old female Balb / c mice. Blank lipid nanoparticle-Luciferase mRNA complexes were administered via tail vein injection at a single dose of 0.3 mpk. At specific time points (e.g., 3 h and 6 h) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. In vivo imaging of the animals was performed using a PerkinElmer small animal imaging system, and the bioluminescent signal was measured. The results are shown in Table 3 and Figure 1.

[0477] Table 3 Evaluation of in vivo activity of blank lipid nanoparticle-mRNA complexes in Example 5

[0478] The results show that after the blank lipid nanoparticles provided in this application are combined with luciferase mRNA, a higher luciferase protein expression rate can be achieved in mice.

[0479] The in vivo delivery efficiency of the blank lipid nanoparticle-human erythropoietin mRNA (hEPO mRNA) complexes of Examples 6 and 12 was evaluated using 6-8 week old female Balb / c mice. The blank lipid nanoparticle-hEPO mRNA complexes were administered via tail vein injection at single doses of 1.5 mpk and 3 mpk, respectively. The hEPO expression levels in the mice were measured at specific time points (24 h) after administration. The results are shown in Table 4.

[0480] Table 4 hEPO expression (pg / ml) after 24 h administration of blank lipid nanoparticle-human erythropoietin mRNA complexes of Examples 6 and 12

[0481] It can be seen that after the blank lipid nanoparticles provided in the present application are combined with human erythropoietin mRNA, a higher hEPO expression rate can be achieved in mice through intravenous injection.

[0482] 6.2 Evaluation of in vivo delivery of lipid nanoparticle-mRNA complexes by intramuscular injection in mice

[0483] The in vivo delivery efficiency of the lipid nanoparticle-luciferase mRNA complex in Example 10 was determined using 6-8 week old female Balb / c mice. Blank lipid nanoparticle-luciferase mRNA complexes were administered intramuscularly at a single dose of 0.3 mpk. At specific time points (3 h and 6 h) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. In vivo imaging of the animals was performed using a PerkinElmer small animal imaging system, and the bioluminescent signal was measured. The results are shown in Table 5 and Figure 2.

[0484] Table 5 In vivo activity evaluation after administration of blank lipid nanoparticle-mRNA complex of Example 10 for 3 / 6 hours

[0485] The results showed that after the blank lipid nanoparticles provided in this application were combined with luciferase mRNA, a high luciferase protein expression rate could be achieved in mice through intramuscular injection.

[0486] 6.3 Intraperitoneal (IP) and subcutaneous (SC) administration

[0487] The in vivo delivery efficiency of the lipid nanoparticle-firefly luciferase mRNA (Luciferase mRNA) complex of Example 16 was measured using 6-8 week old female Balb / c mice. Blank lipid nanoparticle-Luciferase mRNA complexes were administered intraperitoneally and subcutaneously, with a single dose of 0.3 mpk. At specific time points (3 h and 6 h) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. The animals were imaged in vivo using a PerkinElmer small animal imaging system, and the bioluminescent signal was measured. The results are shown in Table 6 and Figure 3.

[0488] Table 6 Evaluation of in vivo activity of blank lipid nanoparticle-mRNA complexes in Example 16

[0489] The results showed that after the blank lipid nanoparticles provided in the present application were combined with luciferase mRNA, a high luciferase protein expression rate could be achieved in mice through intraperitoneal injection and subcutaneous injection.

[0490] The present application is not limited to the technical means disclosed by the above technical means, but also includes technical solutions composed of any combination of the above technical features. The above is a specific embodiment of the present application. It should be pointed out that for ordinary technicians in the field of this application, several improvements and modifications can be made without departing from the principles of this application. These improvements and modifications are also considered to be within the scope of protection of this application.

Claims

1. Use of blank lipid nanoparticles in the preparation of in vivo delivery products, wherein, The application includes the step of mixing blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on the blank lipid nanoparticles; The composition of the blank lipid nanoparticles includes: 10-60 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-30 mol% of a phospholipid, 15-70 mol% of a steroid or its derivative, and 0-10 mol% of a polyethylene glycol-conjugated lipid.

2. The application according to claim 1, wherein The delivery method further includes the step of introducing the composition based on the blank lipid nanoparticles into an animal.

3. The application according to claim 2, wherein The routes for introducing the composition based on the blank lipid nanoparticles into an animal include oral administration, intranasal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intra-articular administration, intralesional administration, intratracheal administration, subcutaneous administration, or intradermal administration.

4. The application according to claim 2, wherein, The animals are mammals and non-mammals.

5. The application according to claim 1, wherein, The biologically active substances include nucleic acids, proteins, polypeptides, small molecule compounds; the nucleic acids are DNA and / or RNA.

6. The application according to claim 5, wherein, The nucleic acids include at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA microcircles, msDNA.

7. The application according to claim 1, wherein, The dosage of the nucleic acid is 0.5%-50% (w / w) of the total amount of the blank lipid nanoparticles and the nucleic acid; in the composition based on the blank lipid nanoparticles, the concentration of the biologically active substance is 50-5000 ng / μl.

8. The application according to any one of claims 1-7, wherein, The first ionizable lipid is selected from compounds having the general formula (1) or pharmaceutically acceptable salts, stereoisomers, and tautomers thereof; wherein, R1, R2, and R3 are each independently H, C 5-40 a linear or branched alkyl group, C 5-40 a linear or branched alkenyl group, C 5-40 a linear or branched alkynyl group, a 3- to 6-membered saturated or partially unsaturated cycloalkyl group having 1 to 3 side chains, or a 6- to 10-membered aromatic group having 1 to 3 side chains; the side chains are independently selected from C 10-30 a linear or branched alkyl group, C 10-30 a linear or branched alkenyl group, C 10-30 a linear or branched alkynyl group; provided that at most one of R1, R2, and R3 is H; M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, a 6-10 membered heteroaryl group containing at least one nitrogen atom, and the heterocyclic group and heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amino, oxo, or halogen; R4 and R5 are each independently H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group, and the C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, aminoamide, amidino, guanidine or halogen groups; G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; wherein each R6, R 13 is independently selected from H, hydroxy, C 1-30 linear or branched alkyl or cycloalkyl, C 2-30 linear or branched alkenyl; L1 is selected from -X1- or -(CR7R8) m -X1-, where each X1 is independently selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -, -NR 14 -S(=O)2-, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-; where m is an integer from 2 to 6, and R7 and R8 are independently of each other H, hydroxy, halogen, C 1-6 linear or branched alkyl or cycloalkyl, C 2-6 linear or branched alkenyl, each R 14 , R 15 are independently of each other selected from H, C 1-30 linear or branched alkyl or cycloalkyl, C 2-30 linear or branched alkenyl; L2 is -(CR9R 10 ) n - or -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16 -, NR 16 -S(=O)2-, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-; n is an integer from 1 to 6; k is an integer from 1 to 6; R9, R 10 , R 11 , R 12 are independently of each other H, hydroxy, halogen, a straight-chain or branched alkyl or cycloalkyl of C 1-6 , a straight-chain or branched alkenyl of C 2-6 , and each R 16 , R 17 is independently selected from H, a straight-chain or branched alkyl or cycloalkyl of C 1-30 , a straight-chain or branched alkenyl of C 2-30 ; wherein R4 to R 17 the alkyl, cycloalkyl, or alkenyl groups described therein are unsubstituted or substituted by one or more groups selected from hydroxy, mercapto, amino, substituted amino, and halogen; The salt does not include quaternary ammonium salts.

9. The application according to claim 8, wherein R1, R2, and R3 are, independently of one another, groups as follows: wherein, Y does not exist, or is C 1-30 a linear or branched alkyl or cycloalkyl group, C 2-20 a linear or branched alkenyl group, C 2-20 a linear or branched alkynyl group; R1' and R2' are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of Y, R1' and R2' is 8-40.

10. The application according to claim 8, wherein R1, R2, and R3 are each independently selected from the following groups: wherein, R1’ and R2’ are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of R1’ and R2’ is 8-30.

11. The application according to claim 8, wherein, R1, R2, and R3 are each independently selected from any one of the following groups:

12. The application according to claim 8, wherein, G1, G2, G3 are, independently of one another, -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

13. The application according to claim 8, wherein, L1 is selected from -(CR7R8) m -X1-, where X1 is selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-.

14. The application according to claim 8, wherein, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-.

15. The application according to claim 8, wherein, M is selected from the following structures: wherein m' and n' are each independently an integer from 0 to 6, and R1'' and R2'' are each independently H, C 1-6 alkyl, C 2-6 alkenyl, guanidyl, amidino, amido, aliphatic amine, 3- to 10-membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine, and pyrimidine and their tautomeric forms, which are unsubstituted or optionally substituted by one or more organic groups selected from hydroxy, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

16. The application according to claim 8, wherein The compound of formula (1) is selected from the compounds represented by formula (1A):

17. The application according to claim 8, wherein, The compound of formula (1) is selected from the compounds represented by formula (1B):

18. The application according to claim 8, wherein, The compound of formula (1) is selected from the compounds of formula (1C):

19. The application according to claim 17, wherein, The compound of formula (1) is selected from the compounds of formula (1D):

20. The application according to claim 19, wherein, The compound of formula (1) is selected from the compounds of formula (1E) 21. The application according to claim 20, wherein, The compound of formula (1) is selected from the compounds represented by formula (1F):

22. The application according to claim 21, wherein, The compound of formula (1) is selected from the compounds represented by formula (1G):

23. The application according to claim 21, wherein, The compound of formula (1) is selected from the compounds represented by formula (1H):

24. The application according to claim 8, wherein, The compound of formula (1) is selected from the compounds shown in formula (1I):

25. The application according to claim 17, wherein The compound of formula (1) is selected from the compounds represented by formula (1J):

26. The application according to claim 24, wherein, The compound of formula (1) is selected from the compounds represented by formula (1K):

27. The application according to claim 9, wherein, Y does not exist, and the compound of formula (1) is selected from the compounds represented by formula (1L): Wherein R1’ and R2’ are independently selected from H, C1-30 straight or branched alkyl, C2-30 straight or branched alkenyl, C2-30 straight or branched alkynyl, and the total carbon chain length of R1’ and R2’ is 8-40.

28. The application according to claim 9, wherein, The compound of formula (1) is selected from the compounds represented by formula (1M):

29. The application according to claim 8, wherein, The compound of formula (1) is selected from:

30. The application according to claim 8, wherein, The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof: Wherein A1 is NH or O; R a selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene; the C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene has a straight-chain or branched-chain structure; R b and R c are each independently selected from C1-C 12 alkyl, C2-C 12 alkenyl, C1-C 12 alkynyl, C3-C 12 cycloalkanyl, C6-C 12 aryl, C1-C 12 alkyl alcohol, C1-C 12 heterocyclic group, alkylamine; The alkylamine is Among them, R a ’ is C1-C 12 alkyl, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamine, R c "selected from unsubstituted or amino-substituted C1-C6 alkyl, R c "’ is H, Provided that when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl; when A1' is -CO-, R c ' does not exist.

31. The application according to claim 30, wherein, The compound of formula (2) is selected from at least one of the following compounds:

32. The application according to claim 8, wherein The blank lipid nanoparticles further comprise 0-60 mol% of other ionizable lipids, and the other ionizable lipids are selected from at least one of the following compounds:

33. The application according to claim 1, wherein The phospholipids include at least one of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl stearoylethanolamine, 1-stearoyl-2-oleoyl phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine.

34. The application according to claim 1, wherein The steroid or its derivative includes at least one of cholesterol, cholesteryl stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatine, ursolic acid, α-tocopherol.

35. The application according to claim 1, wherein, The polyethylene glycol conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

36. The application according to claim 35, wherein, The PEG-conjugated lipids include at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, glyceryl dipalmitate-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol lipids.

37. The application according to claim 1, wherein In the composition based on blank lipid nanoparticles, the biologically active substance is encapsulated inside the blank lipid nanoparticles and / or adsorbed on the surface of the blank lipid nanoparticles to form a complex.

38. The application according to claim 37, wherein, The solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

39. The application according to claim 38, wherein, The pH of the buffer salt solution is 1-9, the concentration of the buffer salt is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration <50%.

40. The application according to claim 40, wherein, The buffer salt solution is selected from at least one of citrate solution, acetate solution, tartrate solution, phosphate solution, carbonate solution, Tris-HCl solution, and sodium chloride solution.

41. The application according to claim 1, wherein, At least one of sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the composition based on blank lipid nanoparticles.

42. An in vitro gene delivery method, wherein, The in vitro gene delivery method includes the step of mixing blank lipid nanoparticles and a biologically active substance in a solvent to obtain a composition based on blank lipid nanoparticles; The composition of the blank lipid nanoparticles includes: 10-60 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-30 mol% of phospholipid, 15-70 mol% of cholesterol, and 0-10 mol% of PEG-conjugated lipid.

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