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

Abstract

The invention provides application of blank lipid nanoparticles in preparation of in-vivo delivery products, and belongs to the technical field of biological medicines. The application of the blank lipid nanoparticles in preparation of the in-vivo delivery product comprises the step of mixing the blank lipid nanoparticles with a biological active substance in a solvent to obtain a composition based on the blank lipid nanoparticles, the blank lipid nanoparticles comprise ionizable lipid, phospholipid, cholesterol and polyethylene glycol conjugated lipid. The blank lipid nanoparticles are adopted, the dosage of biological active substances can be flexibly adjusted according to the requirements of a user, drug delivery can be conducted in various ways such as intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection, and the ideal delivery effect can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of blank lipid nanoparticles in the preparation of in vivo delivery products. Background Art

[0002] Gene editing therapies have the potential to cure genetic diseases, but there is a need to be able 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). Successfully delivering 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 degradation of the drug; 2) Target specific cells; 3) Cross the cell membrane and enter the cell interior; 4) Release 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 major problem.

[0003] Existing in vivo introduction technologies for gene editing drugs still have many defects. For example, the most common introduction technology mediated by viral vectors 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 eye, liver, brain, myocardium, and skeletal muscle, and different types of AAV capsid serotypes produced naturally and synthetically in the laboratory can achieve different tissue targeting, but their loading capacity is limited (only 5 kb of DNA), and there are potential safety risks. Lentiviral vectors can accommodate up to 10 kb of DNA drugs, which is sufficient to load known gene editing drugs into a single vector, but there is a potential risk of genomic integration. Generally speaking, viral vectors face problems such as the immunogenicity of the vector, long-term expression of gene editing drugs, off-target gene editing, the possibility of genomic integration, manufacturing costs, and dose-limiting toxicity, and there are potential insecurities such as integration reactivation and causing an immune response in host cells, which limit their application.

[0004] And introduction technologies mediated by cationic polymers such as polyethyleneimine also have problems such as low transfection efficiency and high toxicity. Although there are many in vivo introduction methods for gene editing drugs at present, most of them are expensive, have safety risks, toxicity, or low transfection efficiency. Therefore, the current inefficient in vivo nucleic acid delivery is one of the bottlenecks restricting the further development of gene therapy and gene immunotherapy technologies. Therefore, there is an urgent need for an in vivo transfection reagent with high transfection efficiency and low toxicity to meet the current needs.

[0005] Lipid nanoparticle (LNP) delivery has the following advantages in the field of delivering gene editing drugs: 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 to the greatest extent; 2) The immunogenicity of LNP is much lower than that of viral vectors. In some cases, repeated dosing is possible, and it has good safety and biocompatibility, and can deliver a drug dose that meets the effective gene editing level; 3) Currently, the large-scale production process of LNP has been mature, which provides a basis for clinical trials using LNP to deliver in vivo gene editing drugs. Patents CN116832051A and CN116969850A have conducted research in this area. However, in the prior art, when using lipid nanoparticles (LNP) to deliver gene editing drugs, a special mixing process is usually required, otherwise good delivery effects cannot be achieved.

[0006] Based on this, the present invention provides the application of blank lipid nanoparticles in the preparation of in vivo delivery products. By simply mixing a biologically active substance with blank lipid nanoparticles in a solution state, safe and effective delivery of the biologically active substance can be achieved. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides the application of blank lipid nanoparticles in the preparation of in vivo delivery products. The application of the blank lipid nanoparticles in the preparation of in vivo delivery products includes the step 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 composition of the blank lipid nanoparticles includes: ionizable lipid, phospholipid, cholesterol, and polyethylene glycol-conjugated lipid. By using the blank lipid nanoparticles of the present invention, the dose of the biologically active substance can be flexibly adjusted according to the needs of the user, and ideal delivery effects can be obtained through various administration routes.

[0008] To achieve the above object, in a first aspect, the present invention provides the application of blank lipid nanoparticles in the preparation of in vivo delivery products, including the step 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 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 - 5 mol% of polyethylene glycol-conjugated lipid.

[0009] In a preferred embodiment, the product is a delivery system or a drug.

[0010] In a preferred embodiment, the application further includes the step of introducing the composition based on the blank lipid nanoparticles into an animal body.

[0011] In a preferred embodiment, the routes for introducing the blank lipid nanoparticle-based composition 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.

[0012] In a preferred embodiment, the animals are mammals and non-mammals.

[0013] In a preferred embodiment, the bioactive substances include nucleic acids, proteins, polypeptides, small molecule bioactive substances; the nucleic acids are DNA and / or RNA.

[0014] In a preferred embodiment, 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.

[0015] In a preferred embodiment, the dosage of the nucleic acid is 0.5%-50% of the total amount of the blank lipid nanoparticles and the nucleic acid; in the blank lipid nanoparticle-based composition, the concentration of the bioactive substance is 50-5000 ng / μl.

[0016] In a preferred embodiment, the first ionizable lipid is selected from compounds having the general formula (1) or pharmaceutically acceptable salts, stereoisomers, tautomers thereof;

[0017]

[0018] wherein R1, R2, and R3 are each independently H, C 5-40 a straight-chain or branched-chain alkyl group, C 5-40 a straight-chain or branched-chain alkenyl group, C 5-40 a straight-chain or branched-chain alkynyl group, a 3-6 membered saturated or partially unsaturated cycloalkyl group having 1-3 side chains, or a 6-10 membered aromatic group having 1-3 side chains; the side chains are independently selected from C 10-30 a straight-chain or branched-chain alkyl group, C 10-30 a straight-chain or branched-chain alkenyl group, C 10-30 a straight-chain or branched-chain alkynyl group; provided that at most one of R1, R2, and R3 is H;

[0019] 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;

[0020] 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 straight-chain or branched-chain alkyl group, C 2-6 straight-chain or branched-chain alkenyl group or C 2-6 straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, amino, amido, amidino, guanidino or halogen;

[0021] G1, G2, and 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-; where each R6 and R 13 are each independently selected from H, hydroxyl, C 1-30 a straight-chain or branched-chain alkyl or cycloalkyl group, C 2-30 a straight-chain or branched-chain alkenyl group;

[0022] 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 14C(=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, and R 7、 R8 are each independently 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 each independently selected from H, C 1-30 linear or branched alkyl or cycloalkyl, C 2-30 linear or branched alkenyl;

[0023] 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)(OR16 )- 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 each independently H, hydroxy, halogen, C 1-6 linear or branched alkyl or cycloalkyl, C 2-6 linear or branched alkenyl, each R 16 , R 17 are each independently selected from H, C 1-30 linear or branched alkyl or cycloalkyl, C 2-30 linear or branched alkenyl;

[0024] wherein the alkyl, cycloalkyl, alkenyl as described for R4 to R 17 is unsubstituted or substituted by one or more groups selected from hydroxy, mercapto, amino, substituted amino, halogen;

[0025] The salt does not include quaternary ammonium salts.

[0026] In a preferred embodiment, the R1, R2, R3 are each independently one of the following groups:

[0027]

[0028] wherein Y is absent or is C 1-30 linear or branched alkyl or cycloalkyl, C 2-20 linear or branched alkenyl, C 2-20 linear or branched alkynyl; R1', R2' are each independently H, C 1-30 linear or branched alkyl, C 2-30 linear or branched alkenyl, C 2-30 linear or branched alkynyl, and the total carbon chain length of Y, R1' and R2' is 8 - 40.

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

[0030]

[0031] wherein R1', R2' are each independently H, C 1-30 linear or branched alkyl, C 2-30 linear or branched alkenyl, C 2-30 linear or branched alkynyl, and the total carbon chain length of R1' and R2' is 8 - 30.

[0032] In a preferred embodiment, the R1, R2, R3 are each independently selected from any one of the following groups:

[0033]

[0034] In a preferred embodiment, G1, G2, and G3 are each independently -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-.

[0035] In a preferred embodiment, 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-.

[0036] In a preferred embodiment, 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-.

[0037] In a preferred embodiment, M is selected from the following structures:

[0038]

[0039] wherein m' and n' are independently of each other integers from 0 to 6, and R1'' and R2'' are independently of each other 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.

[0040] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1A):

[0041]

[0042] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1B):

[0043]

[0044] In a preferred embodiment, the compound of formula (1) is selected from the compounds of formula (1C):

[0045]

[0046] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1D):

[0047]

[0048] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1E)

[0049]

[0050] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1F):

[0051]

[0052] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1G):

[0053]

[0054] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1H):

[0055]

[0056] In a preferred embodiment, M is selected from any one of the following groups:

[0057]

[0058] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1I):

[0059]

[0060] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1J):

[0061]

[0062] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1K):

[0063]

[0064] In a preferred embodiment, Y is absent, and the compound of formula (1) is selected from the compounds represented by formula (1L):

[0065]

[0066] 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.

[0067] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1M):

[0068]

[0069] In a preferred embodiment, the compound of formula (1) is selected from:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] 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):

[0083]

[0084] 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.

[0085] 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):

[0086]

[0087] 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.

[0088] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0089]

[0090] 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.

[0091] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the steps of reacting a compound of formula (VII) successively with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X):

[0092]

[0093] Wherein Xg, Xh, Xi, Xj, Xk, 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.

[0094] 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):

[0095]

[0096] The step of reacting a compound of formula (XIII) with a compound of formula (XII):

[0097]

[0098] Wherein Xm is a group containing a nucleophilic group, and Xm and the C═C-L of the compound of formula (XII) 2a Form X1-L2 through an addition reaction.

[0099] In a preferred embodiment, R1, R2, and R3 are, independently of one another, the following groups:

[0100]

[0101] Wherein the meanings of Y, R1', and R2' are the same as defined above;

[0102] Wherein the method further comprises the step of forming the tail chains R1, R2, and R3:

[0103]

[0104] Wherein X is a leaving group.

[0105] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0106]

[0107] wherein N1 is NH or O;

[0108] R a is 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 is in 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 cycloalkane group, C6-C 12 aryl group, C1-C 12 alkyl alcohol, C1-C 12 heterocyclic group, alkylamine;

[0109] The alkylamine is wherein, 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 " is selected from C1-C6 alkyl which is unsubstituted or substituted by an amino group, and R c "'" is H,

[0110] provided that when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is C1-C6 alkyl; when A1' is -CO-, R c ' does not exist.

[0111] In a preferred embodiment, R b and R c are each independently selected from C1-C 12 alkyl alcohol, alkylamine.

[0112] In a preferred embodiment, the condition is: when R a is C6-C 24 alkenyl, A1 is NH, and R b and Rc Each independently selected from C1-C 12 alkyl alcohol;

[0113] When R a is a branched C6-C 24 alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 alkyl alcohol, alkylamine;

[0114] R a is a straight-chain C6-C 24 alkyl, A1 is NH or O, R b and R c Each independently selected from alkylamine;

[0115] When R a is a straight-chain C6-C 24 alkyl, A1 is O, R b and R c Each independently selected from C1-C 12 alkyl alcohol;

[0116] When R a is C6-C 24 short-chain polyoxyethylene, A1 is NH or O, R b and R c Each independently selected from alkylamine; or when R a is C6-C 24 alkyl alcohol, A1 is NH or O, R b and R c Each independently selected from alkylamine.

[0117] In a preferred embodiment, R a is selected from the following compound structures:

[0118]

[0119] In a preferred embodiment, R b and R c are selected from the following compound structures:

[0120]

[0121] R d is selected from C1-C6 alkanes or cycloalkanes.

[0122] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0123]

[0124]

[0125]

[0126] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0127] R a ”-NH2 ① reacts with an α,β-unsaturated carbonyl compound ② to form an ionizable lipid compound ③:

[0128]

[0129] wherein, R a ”-NH2 is R a -NH2 or

[0130] R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0131] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0132] 1) R a ”-NH2 ① reacts with an α,β-unsaturated carbonyl compound ④ to form a compound ⑤;

[0133] 2) The compound ⑤ reacts with a nucleophile ⑥ to form an ionizable lipid compound ③;

[0134]

[0135] wherein, the nucleophile ⑥ is R b -NH2 or R b -OH; R a ”-NH2 is selected from R a -NH2 or

[0136] R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c’-A1’-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0137] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0138] 1) R a ”-NH2① reacts with an α,β-unsaturated carbonyl compound② to form a compound⑦;

[0139] 2) The compound⑦ reacts with an α,β-unsaturated carbonyl compound⑧ to form a compound⑨;

[0140] 3) The compound⑨ reacts with a nucleophile⑩ to form an ionizable lipid compound

[0141]

[0142] wherein, R a ”-NH2 is R a -NH2, Z3 is a leaving group, and A3 reacts with Z3 to obtain A1.

[0143] 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 form the bifurcated ionizable lipid compound containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond, or a carbon-selenium bond.

[0144] In a preferred embodiment, Formula 2a - Formula 2b further comprises the step of performing an iterative reaction using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a - Formula 2b as a raw material, according to Step 1 in Formula 2a or Steps 1 - 2 in Formula 2b;

[0145] The compound of Formula (2) with a terminal amino group is

[0146] wherein, R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1 - C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2.

[0147] In a preferred embodiment, the reaction formula of Formula 2a - Formula 2c further comprises the step of reacting with using the compound of Formula (2) with a terminal amino group obtained by the reaction formula of Formula 2a - Formula 2c as a raw material;

[0148] The compound of formula (2) with an amino group at the end is

[0149] wherein R b ’ and R b ” are both H at the same time, or R b ’ and R b ” are both C1-C6 amines at the same time, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0150] The leaving group mentioned above refers to the leaving part in a nucleophilic reaction or a condensation reaction, including but not limited to: H,

[0151] 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 (R-O-), amino (-NHR, where R is an alkyl or aryl group from which H is removed), phenoxy, tertiary carbocation (such as tert-butyl cation), carbocation stabilized by an unsaturated system or a heteroatom, or various protecting groups mentioned above.

[0152] The nucleophilic group mentioned above refers to a molecule or ion that can provide an electron pair to form a new chemical bond in a chemical reaction. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazine, nucleophilic halogen (such as Cl - ), Br - or I - ), hydride ion (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxide anion (such as alcohol from which the hydroxyl hydrogen is removed), amino group (including primary amine, secondary amine and tertiary amine) or amine anion, carbanion (such as carbanion in organometallic reagents such as Grignard reagent, organolithium reagent, Gilman reagent, etc.), mercapto or mercapto anion, thioether, enol or enolate anion, vinyl ether, enamine, carboxylic acid or carboxylate anion, alkyl or aryl phosphine (such as triphenylphosphine), aromatic heterocycle with lone pair electrons (such as pyridine), etc.

[0153] In a preferred embodiment, the raw materials used in the reaction process further contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0154] In a preferred embodiment, the in vivo delivery method based on blank liposomes further comprises 0 - 60 mol% of other ionizable lipids, and the other ionizable lipids are selected from at least one of the following compounds:

[0155]

[0156]

[0157]

[0158]

[0159] In a preferred embodiment, the phospholipids include at least one of 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE).

[0160] In a preferred embodiment, the steroid or its derivative includes at least one of cholesterol, cholesteryl stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, halocynthia sterol, tomatine, ursolic acid, α-tocopherol.

[0161] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0162] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises 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), dipalmitin-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroylphosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoylphosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0163] In a preferred embodiment, 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.

[0164] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

[0165] In a preferred embodiment, 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%.

[0166] In a preferred embodiment, the alcohol is ethanol.

[0167] In a preferred embodiment, 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.

[0168] In a preferred embodiment, at least one of sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the composition based on blank lipid nanoparticles.

[0169] In a second aspect, the present invention provides an in vitro gene delivery method, which 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 cholesterol, and 0-5 mol% of a polyethylene glycol-conjugated lipid

[0170] Compared with the prior art, the present invention has the following beneficial effects:

[0171] 1. The application of the blank lipid nanoparticles in the preparation of an in vivo delivery product includes the step 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 composition of the blank lipid nanoparticles includes: an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol-conjugated lipid.

[0172] 2. By using the blank lipid nanoparticles of the present invention, the dose of the biologically active substance can be flexibly adjusted according to the needs of the user, and it can be administered through various routes to obtain an ideal delivery effect.

[0173] 3. The preparation process of the present invention is simple and can be completed without the aid of equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0174] Figure 1 Whole-body in vivo imaging at 3 h / 6 h after intravenous administration of the blank lipid nanoparticle-luciferase mRNA complex in Example 5 to mice;

[0175] Figure 2 Abdominal and lateral in vivo imaging at 3 / 6 h after intramuscular injection of the blank lipid nanoparticle-luciferase mRNA complex in Example 10 to mice;

[0176] Figure 3 In vivo imaging at 3 h / 6 h after intraperitoneal and subcutaneous administration of the blank lipid nanoparticle-luciferase mRNA complex in Example 16 to mice. DETAILED DESCRIPTION OF THE INVENTION

[0177] The synthesis processes of the present invention can accommodate multiple functional groups, and thus various substituted starting materials can be used. These processes generally provide the desired final compounds at or near the end of the overall process, although in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or intermediates that are readily prepared, by employing standard synthetic methods and procedures known to those skilled in the art or that are obvious to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for preparing organic molecules, as well as functional group transformations and manipulations, can be obtained from relevant scientific literature or from standard textbooks in the field. The following descriptions of the synthetic methods are designed to illustrate rather than limit the general procedures for preparing the compounds of the present invention.

[0178] 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 procedures according to the processes illustrated in the corresponding general synthetic routes. The variables (such as R1, R2, and R3, etc.) in each general synthetic route are defined as herein. Those of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of certain steps can be varied, such as the introduction and removal of protecting groups.

[0179] In the reaction schemes described herein, multiple stereoisomers can be produced. When no specific stereoisomer is indicated, this should be understood to include all possible stereoisomers produced by the reaction. Those of ordinary skill in the art should recognize that the reaction can be optimized to preferentially obtain one isomer, or new schemes can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0180] (1) Synthesis of the first ionizable lipid of Preparation Example 1

[0181] The general synthetic route of the first ionizable lipid is as in General Synthetic Routes 1-5.

[0182] General Synthetic Route 1

[0183]

[0184] Wherein, M' is M or M with a protecting group.

[0185] As described in the general synthetic route 1 above, Boc-amino tris(hydroxymethyl)methane reacts with an acid (Compound 2) through a condensation reaction to form Compound 3. Step 1 can be carried out in an organic solvent (such as 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.

[0186] Then, the Boc protecting group of Compound 3 is removed to generate Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0187] Then, Compound 4 undergoes a condensation reaction with Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) under the catalysis of EDCl and DMAP or dicyclohexylcarbodiimide (DCC).

[0188] 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 reaction conditions for the removal of the selected protecting group.

[0189] General Synthetic Route 2

[0190]

[0191] Wherein M' is M or M with a protecting group; A is O, NH or S.

[0192] As described in the general synthetic route 2 above, Compound 1 undergoes a condensation reaction with Compound 2 to obtain Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of EDCl and DMAP.

[0193] Then, the tert-butoxy protecting group of Compound 3 is removed to obtain Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) in the presence of an acid (such as trifluoroacetic acid) and a cation scavenger (such as triisopropylsilane (TiPS)).

[0194] Then, 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.

[0195] 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.

[0196] General Synthetic Route 3

[0197]

[0198] Among them, 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.

[0199] As described in the above general synthetic route 3, Boc-amino tris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to form compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0200] Then, the Boc protecting group of compound 3 is removed to form compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0201] Then, compound 4 reacts with a halogen-substituted aldehyde (compound 5) through a 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 triacetoxyborohydride (NaBH(OAc)3)).

[0202] Then, compound 6 undergoes a nucleophilic reaction with an amine (compound 7) to form 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 (such as triethylamine, iPr2EtN) or an inorganic base (such as K2CO3)) and a catalyst (KI or NaI).

[0203] If there are the above-mentioned protecting groups in the R4’ and / or R5’ groups of compound 8, 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 group.

[0204] General synthetic route 4

[0205]

[0206] Among them, 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.

[0207] As described in the above general synthetic route 4, Boc-amino tris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to form compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0208] Subsequently, the Boc protecting group of compound 3 was removed to form compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0209] Subsequently, compound 4 underwent 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 under the catalysis of EDCl and DMAP or DCC.

[0210] Subsequently, compound 6 underwent a nucleophilic reaction with an amine (compound 7) to form 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 (such as triethylamine, iPr2EtN) or an inorganic base (such as K2CO3)) and a catalyst (KI or NaI).

[0211] If the R4’ and / or R5’ groups of compound 8 have the above-mentioned protecting groups, the protecting groups were removed to obtain the target lipid compound. Step 5 was carried out under the deprotection reaction conditions of the selected protecting group.

[0212] General synthetic route 5

[0213]

[0214] Where M pro is M or M with a protecting group.

[0215] As described in the above general synthetic route 5, Boc-aminotris(hydroxymethyl)methane underwent a condensation reaction with compound 2 to form compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0216] Subsequently, the Boc protecting group of compound 3 was removed to form compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0217] Subsequently, compound 4 and compound 5 underwent a 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 triacetoxyborohydride (NaBH(OAc)3)).

[0218] If the M of compound 6 pro group contains the above-mentioned protecting group, the protecting group was removed to obtain the target lipid compound. Step 4 was carried out under the deprotection reaction conditions of the selected protecting group.

[0219] In addition, it should be understood that any specific embodiment of the present invention within the scope of the prior art may be expressly excluded from any one or more claims. Since these embodiments are considered to be known to those of ordinary skill in the art, they may be excluded, even if such exclusion is not expressly stated herein.

[0220] All cited sources, such as the references, publications, databases, database entries, and technologies cited herein, are incorporated herein by reference, even if not expressly stated in the citation. In the event of a conflict between the cited source and the statements of the present application, the statements of the present application shall prevail.

[0221] (1) Synthesize a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L), or (1M)

[0222] A. General considerations

[0223] It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is imposed on their sources.

[0224] The process routes described below can be used to synthesize the compounds 1001 - 3422 of the present invention.

[0225] The following abbreviations are used herein:

[0226] THF: Tetrahydrofuran

[0227] MeCN: Acetonitrile

[0228] MeOH: Methanol

[0229] PE: Petroleum ether

[0230] EA: Ethyl acetate

[0231] DMF: N,N - Dimethylformamide

[0232] EDCl: 1 - Ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride

[0233] LAH: Lithium aluminum hydride

[0234] DCM: Dichloromethane

[0235] DMAP: 4 - Dimethylaminopyridine

[0236] LDA: Lithium diisopropylamide

[0237] rt: Room temperature

[0238] DCE: 1,2 - Dichloroethane

[0239] n-BuLi: n-Butyllithium

[0240] i-Pr2EtN: N,N-Diisopropylethylamine

[0241] B. Intermediate Synthesis

[0242] Intermediate A:

[0243]

[0244] Intermediate A was obtained through the following synthetic process route:

[0245]

[0246] Dissolve tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl dicarbonate (Boc2O) (99.1 g) in a mixed solvent of methanol (300 mL) / H2O (30 mL), react at room temperature for 72 h, and purify by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain the white solid compound Intermediate A (N-Boc-aminotrimethylolmethane) (75.0 g). LCMS (ESI) calcd for C9H 19 NO 5， [M+H] + m / z 222.13, found 222.25.

[0247] Intermediate B:

[0248]

[0249] Intermediate B was obtained through the following synthetic process route:

[0250]

[0251] Add diethyl 2-ethyl-1,3-propanedicarboxylate (29.0 g), THF (90 mL), DMF (30 mL) to a single-necked flask, add NaH (3.7 g) under an ice bath, stir at room temperature for 30 min under nitrogen protection; add pentadecyl bromide (15.0 g), react at 80 °C for 2 h under nitrogen protection; concentrate under reduced pressure to remove THF, add the reaction solution dropwise to ice water, add ethyl acetate and stir, filter through diatomaceous earth, collect the organic phase and then separate the layers, wash the organic phase with saturated brine, separate the organic phase, and purify by silica gel column chromatography (PE:EA = 100:1 - 50:1) to obtain diethyl 2-ethyl-2-pentadecylmalonate (16.5 g). Add EtOH (50 mL), H2O (50 mL), KOH (11.3 g) to diethyl 2-ethyl-2-pentadecylmalonate (8.0 g), react at 90 °C for 12 h; concentrate under reduced pressure to remove EtOH, add dilute hydrochloric acid to adjust the reaction system to pH = 4 - 5, add water and ethyl acetate and stir to separate the layers, wash the organic phase with saturated brine, separate the organic phase, and purify by column chromatography (PE:EA = 5:1 - 1:1) to obtain 2-ethyl-2-pentadecylmalonic acid (5.2 g). 2-Ethyl-2-pentadecylmalonic acid (10.0 g) is reacted at 170 °C for 6 h under open conditions, cooled to room temperature, add water and ethyl acetate and stir to separate the layers, wash the organic phase with saturated brine, separate the organic phase, concentrate, and purify by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1H NMR (400 MHz, CDCl3) δ 2.31 (tt, J = 8.6, 5.3 Hz, 1H), 1.71 - 1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 6.7 Hz, 3H).

[0252] Intermediate C:

[0253]

[0254] Intermediate C is obtained by the following synthetic process route:

[0255]

[0256] Add capric acid (50 g) and the solvent THF (500 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add the reactant NaH (23.22 g). Stir for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (62.19 g). Continue to stir for 1 h at 0 °C under nitrogen protection. Finally, after dropwise adding the reactant iodo-nonane (88.52 g), raise the temperature to room temperature and stir overnight. 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, and then purified by silica gel column chromatography (PE:EA = 50:1) to finally obtain intermediate C (2-octylundecanoic acid) (11 g). 1H NMR (400 MHz, Chloroform-d) δ 2.37 (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).

[0257] Intermediate D:

[0258]

[0259] Intermediate D is obtained by the following synthetic process route:

[0260]

[0261] Add tridecanoic acid (79.5 g) and the solvent THF (800 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add NaH (22.25). Stir the reaction solution for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (317.87 g) to the reaction system; continue to stir the reaction solution for 1 h at 0 °C under nitrogen protection. After dropwise adding the reactant iodo-n-hexane (94.39), raise the temperature to room temperature and stir overnight. Dilute the reaction solution 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, and silica gel column chromatography (PE:EA = 50:1) gives intermediate D (2-hexyltridecanoic acid) (26 g). 1H NMR (400 MHz, Chloroform-d) δ 2.36 (tt, J = 8.7, 5.4 Hz, 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).

[0262] Intermediate E:

[0263]

[0264] Intermediate E is obtained through the following synthetic process route:

[0265]

[0266] Dissolve tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1) in EtOH (150 mL). Under nitrogen protection, react at 45 °C for 30 h, then concentrate under reduced pressure to remove EtOH. Add 100 mL × 3 of solvent (PE:EA = 20:1) for pulping, filter to obtain white solid compound Intermediate E (11.5 g). 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).

[0267] Intermediate F:

[0268]

[0269] Intermediate F is obtained through the following synthetic process route:

[0270]

[0271] Add (2-aminoethyl)carbamic acid tert-butyl ester (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) to a single-necked flask. React at 80 °C overnight, then add water and ethyl acetate for stirring and liquid separation. Wash the organic phase with saturated brine, separate the organic phase, concentrate, and purify by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain compound 2 (5.5 g). Add dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) to compound 2, stir at room temperature for 3 h, then concentrate under reduced pressure to obtain compound 3, namely Intermediate F (6 g). LCMS (ESI) calcd for C 20 H 28 N2O2, [M + H] + m / z 329.22, found 329.24.

[0272] Intermediate G:

[0273]

[0274] Intermediate G is obtained through the following synthetic process route:

[0275]

[0276] To a single-necked flask, add Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropyl bromide (9.45 g), K2CO3 (12.94 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 2 (6.9 g). To Compound 2 (6.9 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). Stir at room temperature for 3 h, then concentrate under reduced pressure to obtain Intermediate G (7.5 g). LCMS (ESI) calcd for C 22 H 32 N2O2, [M+H] + m / z 357.25, found 357.51。

[0277] Intermediate H:

[0278]

[0279] Intermediate H was obtained by the following synthetic route:

[0280]

[0281] To a single-necked flask, add Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), K2CO3 (6.47 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain Compound 2 (4.1 g). To Compound 2 (4.1 g.), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). Stir at room temperature for 3 h, then concentrate under reduced pressure to obtain Intermediate H (4.9 g). LCMS (ESI) calcd for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56。

[0282] Intermediate I:

[0283]

[0284] Intermediate I was obtained by the following synthetic route:

[0285]

[0286] To a single-necked flask, add compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), K2CO3 (15.46 g). After reacting overnight at 80 °C, add water and ethyl acetate and stir to separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain compound 2 (6.8 g). To compound 2 (6.8 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). After stirring at room temperature for 3 h, concentrate under reduced pressure to obtain intermediate I (6.5 g). LCMS(ESI) calcd for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0287] Intermediate J:

[0288]

[0289] Intermediate J was obtained by the following synthetic route:

[0290]

[0291] To a single-necked flask, add butyric acid (5.0 g), THF (100 mL). At 0 °C, add NaH (2.73 g), and slowly dropwise add LDA (56.8 mL). React at room temperature for 30 min, add 1-bromotridecane, and continue to react overnight at room temperature. Then add ice water and ethyl acetate and stir to separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain intermediate J (5.0 g). LCMS(ESI) calcd for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0292] Intermediate K:

[0293]

[0294] Intermediate K was obtained by the following synthetic route:

[0295]

[0296] Add reactant 1 (24 g), Imidazole (19.00 g) and solvent DCM (200 mL) to a three-necked flask. After cooling to 0 °C, slowly add reactant TBDMSCI (38.57 g). Stir at 25 °C for 4 h, then dilute with 300 mL of DCM. Wash the organic phase twice with 1 L of water and dry over anhydrous Na2SO4. After filtration and concentration, add silica gel and elute with 1:20 DCM:MeOH by column chromatography. Collect the sample and concentrate to obtain 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).

[0297] Intermediate L:

[0298]

[0299] Intermediate L is obtained by the following synthetic route:

[0300]

[0301] Add compound DMSO (3.58 g) and anhydrous solvent DCM (30 mL) to a three-necked flask. Cool the system to -78 °C and slowly add oxalyl chloride (2.91 g). Stir the reaction solution at -78 °C under N2 protection for 10 minutes, then slowly dropwise add compound 1 (3 g). Continue to stir at -78 °C under N2 protection for 1 h. Finally, add TEA (9.28 g) dropwise and continue to stir at -78 °C under N2 protection for 0.5 h. Dilute with 100 mL of DCM and wash with saturated NH4Cl solution and water respectively. Dry the organic layer over anhydrous Na2SO4, filter and concentrate to obtain intermediate L (3.0 g). LCMS (ESI) calcd for C 11 H 14 O3, [M + H] + m / z 195.09, found 195.23.

[0302] Intermediate M:

[0303]

[0304] Intermediate M is obtained by the following synthetic route:

[0305]

[0306] Add intermediate K (2.0 g), compound 1 (1.79 g) and solvent ACN (20 mL) to a sealed tube. Add reactants K2CO3 (3.81 g) and KI (1.52 g). Stir overnight at 70 °C under N2 protection, then dilute with 100 mL of EA and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, then add silica gel and elute through a column with 15:1 DCM:MeOH to collect the sample and concentrate to obtain compound 2 (2.0 g). Add reactant 2 (1.80 g) and solvent DCM (20 mL) to a three-necked flask. Replace the system with nitrogen and cool to -78 °C, then add DIBAL-H (1.00 g) dropwise. Stir at -78 °C for 4 h, then quench by dropping methanol and sodium carbonate solution into the reaction solution. Then extract with DCM (100 mL), dry the organic layer with anhydrous sodium sulfate, filter and concentrate to obtain intermediate M (1.36 g). LCMS (ESI) calcd for C 15 H 33 NO2Si,[M+H] + m / z 288.52,found 288.23。

[0307] Compound 1003, synthesized according to general synthetic route 1

[0308] Structural formula:

[0309] Chemical formula: C 63 H 122 N2O7

[0310] Molecular weight: 1019.68

[0311] Step 1: Synthesize compound 3 in general synthetic route 1

[0312]

[0313] Add compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) to compound 2 (intermediate B) (4.3 g). Stir at room temperature for 12 h under N2 protection, then add water and dichloromethane and stir to separate the layers. Wash the organic phase with saturated brine, separate the organic phase, dry the organic phase with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (PE:EA = 40:1 - 30:1) to obtain compound 3 (2.6 g).

[0314] Step 2: Synthesize compound 4 in general synthetic route 1

[0315]

[0316] To compound 3 (2.6 g), DCM (15 mL) and TFA (5 mL) were added. After stirring at room temperature for 3 h, it was concentrated and purified by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain compound 4 (3.2 g).

[0317] Step 3: Synthesis of compound 6 in General Synthetic Route 1

[0318]

[0319] To compound 4 (3.2 g), DCM (20 mL), Boc-glycine (compound 5, 578 mg) and DCC (6.8 g) were added. After stirring at room temperature under nitrogen protection for 12 h, water and ethyl acetate were added for stirring and liquid separation. The organic phase was washed with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate and concentrated, and purified by column chromatography (PE:EA = 5:1 - 2:1) to obtain compound 6 (2.2 g).

[0320] Step 4: Compound 1003

[0321]

[0322] To compound 6 (2.2 g), DCM (15 mL) and TFA (5 mL) were added. After stirring at room temperature for 12 h, it was concentrated and purified by column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain compound 1003 (1.3 g). 1 H NMR (400 MHz, CDCl3) δ 4.49 (d, J = 2.6 Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J = 8.6, 5.5 Hz, 3H), 1.62 (q, J = 7.1 Hz, 12H), 1.27 (d, J = 3.4 Hz, 78H), 0.94 - 0.86 (m, 18H).

[0323] D. Compound 1002 was synthesized according to General Synthetic Route 1

[0324] Structural formula:

[0325] Chemical formula: C 60 H 116 N2O7

[0326] Molecular weight: 977.60

[0327] It was synthesized according to General Synthetic Route 1, and the synthesis process was similar to that of compound 1003, except that intermediate C was used as compound 2 in General Synthetic Route 1. 11H NMR (400 MHz, CDCl3) δ 4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5 Hz, 3H), 1.64 (d, J = 6.9 Hz, 6H), 1.50 - 1.42 (m, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 18H).

[0328] E. Compound 1004 was synthesized according to General Synthetic Route 1.

[0329] Structural formula:

[0330] Chemical formula: C 54 H 104 N2O7

[0331] Molecular weight: 893.43

[0332] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that palmitic acid was used as Compound 2 in General Synthetic Route 1. 1 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 4.45 (s, 6H), 3.74 (s, 2H), 2.35 (t, J = 7.6 Hz, 6H), 1.60 (p, J = 6.9 Hz, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 9H).

[0333] F. Compound 1001 was synthesized according to General Synthetic Route 1.

[0334] Structural formula:

[0335] Chemical formula: C 59 H 108 N2O7

[0336] Molecular weight: 957.52

[0337] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that (9Z)-9-hexadecenoic acid was used as Compound 2 in General Synthetic Route 1, and 5-(N,N-dimethylamino)valeric acid was used as Compound 5 in General Synthetic Route 1. 1HNMR (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 6H), 4.51 - 4.38 (m, 6H), 3.19 (dt, J = 11.3, 5.6 Hz, 2H), 2.97 (t, J = 5.9 Hz, 6H), 2.46 (t, J = 6.6 Hz, 1H), 2.35 (td, J = 7.6, 3.1 Hz, 5H), 2.20 (t, J = 7.6 Hz, 2H), 2.03 (d, J = 6.2

[0338] Hz, 12H), 1.89 - 1.81 (m, 3H), 1.74 (q, J = 7.8 Hz, 2H), 1.67 - 1.51 (m, 6H), 1.31 (d, J = 7.8 Hz, 48H), 0.90 (t, J = 6.7 Hz, 9H).

[0339] G. Compound 1014 was synthesized according to General Synthetic Route 2

[0340] Structural formula:

[0341] Chemical formula: C 70 H 137 N3O9

[0342] Molecular weight: 1164.88

[0343] Step 1: Synthesis of Intermediate 3 in General Synthetic Route 2

[0344]

[0345] Intermediate 3 was synthesized according to the method described in Step 1 of General Synthetic Route 1, with the difference that Intermediate E was used as Compound 1 in General Synthetic Route 1

[0346] Step 2: Synthesis of Compound 4 in General Synthetic Route 2

[0347]

[0348] To Compound 3 (4.8 g), DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added. After reacting at room temperature for 12 h, water and dichloromethane were added and stirred for liquid 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 obtain Compound 4 (3.3 g).

[0349] Step 3: Synthesis of Compound 6 in General Synthetic Route 2

[0350]

[0351] 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 and stirred, followed by liquid separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain compound 6 (4.0 g).

[0352] Step 4: Compound 1014

[0353]

[0354] MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added to compound 6 (4.0 g). After stirring overnight at room temperature under hydrogen gas, it was filtered through diatomaceous earth, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1 - 15:1) to obtain compound 1014 (2 g). 1 H (400 MHz, CDCl3) δ 7.38 (t, J = 5.8 Hz, 1H), 4.14 - 4.05 (m, 6H), 3.58 (t, J = 4.9 Hz, 4H), 3.31 (q, J = 5.7 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.64 (q, J = 5.2 Hz, 6H), 2.29 (dq, J = 10.8, 4.4 Hz, 6H), 1.66 - 1.37 (m, 12H), 1.24 (d, J = 4.3 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0355] H. Compound 1015 was synthesized according to General Synthetic Route 2

[0356] Structural formula:

[0357] Chemical formula: C 72 H 141 N3O9

[0358] Molecular weight: 1192.93

[0359] Synthesized according to General Synthetic Route 2, similar to the synthesis process of compound 1014, except that intermediate G was used as compound 5 in General Synthetic Route 2 1H (400 MHz, CDCl3) δ 7.58 (t, J = 5.7 Hz, 1H), 4.15 - 4.01 (m, 6H), 3.70 (t, J = 5.4 Hz, 4H), 3.39 (q, J = 5.7 Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.57 (dt, J = 20.5, 5.8 Hz, 6H), 2.36 - 2.22 (m, 5H), 1.76 - 1.67 (m, 4H), 1.62 - 1.35 (m, 13H), 1.23 (d, J = 4.6 Hz, 78H), 0.92 - 0.80 (m, 18H).

[0360] I. Compound 1020, synthesized according to General Synthetic Route 2

[0361] Structural formula:

[0362] Chemical formula: C 74 H 145 N3O9

[0363] Molecular weight: 1220.99

[0364] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate H is used as Compound 5 in General Synthetic Route 2.

[0365] J. Compound 1025, synthesized according to General Synthetic Route 2

[0366] Structural formula:

[0367] Chemical formula: C 69 H 135 N3O8

[0368] Molecular weight: 1134.85

[0369] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate I is used as Compound 5 in General Synthetic Route 2. 1H (400 MHz, CDCl3) δ 6.98 (t, J = 5.7 Hz, 1H), 4.10 (m, J = 3.8 Hz, 6H), 3.63 (t, J = 5.2 Hz, 2H), 3.35 (q, J = 5.8 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.57 (q, J = 5.3 Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1 Hz, 5H), 1.65 - 1.41 (m, 13H), 1.24 (d, J = 4.4 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0370] K. Compound 1005 was synthesized according to General Synthetic Route 2.

[0371] Structural formula:

[0372] Chemical formula: C 68 H 133 N3O7

[0373] Molecular weight: 1104.83

[0374] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-dimethylethylenediamine was used as Compound 5 in General Synthetic Route 2. 1 H NMR (400 MHz, CDCl3) δ 4.19 - 4.07 (m, 6H), 3.40 (d, J = 5.7 Hz, 2H), 2.95 - 2.87 (m, 2H), 2.82 (s, 4H), 2.64 - 2.55 (m, 2H), 2.38 (s, 6H), 2.33 - 2.29 (m, 3H), 1.63 - 1.55 (m, 6H), 1.49 - 1.44 (m, 2H), 1.26 (d, J = 5.4 Hz, 78H), 0.89 (td, J = 7.0, 2.8 Hz, 18H).

[0375] L. Compound 1006 was synthesized according to General Synthetic Route 2.

[0376] Structural formula:

[0377] Chemical formula: C 53 H 103 N3O7

[0378] Molecular weight: 894.42

[0379] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that myristic acid is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(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).

[0380] M. Compound 1007, synthesized according to General Synthetic Route 2

[0381] Structural formula:

[0382] Chemical formula: C 68 H 133 N3O7

[0383] Molecular weight: 1104.83

[0384] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate D is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(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=3.4

[0385] Hz,78H),0.89(td,J=6.7,2.5Hz,18H).

[0386] N. Compound 1008, synthesized according to General Synthetic Route 2

[0387] Structural formula:

[0388] Chemical formula: C68 H 133 N3O7

[0389] Molecular weight: 1104.01

[0390] Synthesized according to General Synthetic Route 2, similar to the synthetic process of Compound 1014, with the difference that Intermediate C is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(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).

[0391] O. Compound 1009, synthesized according to General Synthetic Route 2

[0392] Structural formula:

[0393] Chemical formula: C 62 H 121 N3O7

[0394] Molecular weight: 1020.66

[0395] Synthesized according to General Synthetic Route 2, similar to the synthetic process of Compound 1014, with the difference that Intermediate J is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 H(400

[0396] MHz,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=3.7

[0397] Hz,66H),0.90-0.84(m,18H).

[0398] P. Compound 1011, synthesized according to General Synthetic Route 2

[0399] Structural formula:

[0400] Chemical formula: C 70 H 137 N3O7

[0401] Molecular weight: 1132.88

[0402] Synthesized according to General Synthesis Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine is used as Compound 5 in General Synthesis Route 2. 1 H NMR(400MHz,CDCl3)δ4.19 - 4.08(m,6H),3.31(q,J=5.8

[0403] Hz,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).

[0404] Q. Compound 1012, synthesized according to General Synthesis Route 2

[0405] Structural formula:

[0406] Chemical formula: C 72 H 141 N3O7

[0407] Molecular weight: 1160.93

[0408] Synthesized according to General Synthesis Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine is used as Compound 5 in General Synthesis 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=6.1Hz,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).

[0409] R. Compound 1013, synthesized according to General Synthesis Route 2

[0410] Structural formula:

[0411] Chemical formula: C 74 H 145 N3O7

[0412] Molecular weight: 1188.99

[0413] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-diethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 H NMR(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=

[0414] 11.8Hz,16H),1.24(d,J=4.5Hz,82H),0.96-0.79(m,24H).

[0415] S. Compound 1029, synthesized according to General Synthetic Route 2

[0416] Structural formula:

[0417] Chemical formula: C 68 H 132 N2O8

[0418] Molecular weight: 1105.81

[0419] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-diethyl-2-hydroxyethylamine is used as Compound 5 in 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).

[0420] T. Compound 1111, synthesized according to General Synthetic Route 2

[0421] Structural formula:

[0422] Chemical formula: C 70 H 136 N4O7

[0423] Molecular weight: 1145.88

[0424] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate is 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).

[0425] U. Compound 1118, synthesized according to General Synthetic Route 2

[0426] Structural formula:

[0427] Chemical formula: C 71 H 139 N3O8

[0428] Molecular weight: 1162.91

[0429] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and Intermediate K is used as Compound 5 in General Synthetic Route 2. 1 1H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.61 (s, 2H), 3.42 (d, J = 33.5 Hz, 2H), 2.91 (t, J = 6.2 Hz, 3H), 2.36 (dd, J = 9.2, 3.6 Hz, 6H), 1.47 (dd, J = 13.9, 6.2 Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7 Hz, 18H).

[0430] V. Compound 1010, synthesized according to General Synthetic Route 3

[0431] Structural formula:

[0432] Chemical formula: C 67 H 133 N3O6

[0433] Molecular weight: 1076.82

[0434] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 3

[0435]

[0436] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003.

[0437] Step 3: Synthesize Compound 6 of General Synthetic Route 3

[0438]

[0439] Add Compound 4 (600 mg), chloroacetaldehyde (Compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) to DCE (60 mL). After reacting overnight at room temperature, add water and dichloromethane and stir to separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and perform silica gel column chromatography (DCM:MeOH = 30:1 - 15:1) to obtain Compound 6 (270 mg).

[0440] Step 4: Synthesize Compound 1010

[0441]

[0442] Add MeCN (20 mL), N,N - dimethylethylenediamine (Compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) to Compound 6 (270 mg). React overnight at 70°C under nitrogen protection, and then perform silica gel column chromatography to obtain Compound 1010 (16 mg). 1 H (400 MHz, CDCl3) δ 4.10 (m, J = 2.9 Hz, 6H), 2.76 - 2.64 (m, 6H), 2.40 (t, J = 6.2 Hz, 2H), 2.29 (ddd, J = 8.5, 5.6, 2.9 Hz, 3H), 2.22 (s, 6H), 1.97 (s, 6H), 1.50 - 1.41 (m, 6H), 1.24 (d, J = 4.2 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0443] W. Compound 1059, synthesized according to General Synthetic Route 3

[0444] Structural formula:

[0445] Chemical formula: C 67 H 132 N2O7

[0446] Molecular weight: 1077.80

[0447] Synthesized according to General Synthetic Route 3, similar to the synthesis process of Compound 1010, except that Intermediate C and Intermediate L are used as Compound 2 and Compound 5 in General Synthetic Route 3 respectively. In addition, the functional group transformation of Compound 6 in General Synthetic Route 3 needs to be carried out through the following process steps:

[0448]

[0449] Add solvent MeOH (5 mL) and THF (5 mL) to Intermediate 6a (1.5 g), add reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g), react overnight at 25 °C under H2 condition, filter with diatomaceous earth, concentrate the filtrate, and carry out silica gel column chromatography (PE:EA 30:1 - 10:1) to obtain Intermediate 6b (520 mg). Add DCM (5 mL) to Intermediate 6b (470 mg), dropwise add SOCl2 (1064.26 mg, 8.946 mmol) at 0 °C, transfer to room temperature and react for 3 hours, then extract the reaction solution with water and DCM, concentrate the organic phase, and carry out silica gel column chromatography (PE:EA 20:1) to obtain Intermediate 6c (181 mg). Add dimethylamine (143.39 mg), DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg) to Intermediate 6c (170 mg), stir the reaction solution at 70 °C and then filter, concentrate, and carry out silica gel column chromatography (DCM:MeOH 20:1) to obtain Compound 1059 (91 mg, yield 53.22%). 1 H NMR (400 MHz, 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,2H),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).

[0450] X. Compound 1112, synthesized according to General Synthetic Route 3

[0451] Structural formula:

[0452] Chemical formula: C 69 H 136 N2O6

[0453] Molecular weight: 1089.85

[0454] Synthesized according to General Synthetic Route 3, similar to the synthesis process of Compound 1010, with the difference that Intermediate C and 6-bromohexanal are used instead of Compound 2 and Compound 5 in General Synthetic Route 3 respectively. 1 H NMR(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).

[0455] Y. Compound 1113, synthesized according to General Synthetic Route 4

[0456] Structural formula:

[0457] Chemical formula: C 71 H 138 N2O8

[0458] Molecular weight: 1147.89

[0459] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 4

[0460]

[0461] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003.

[0462] Step 3: Synthesize Compound 6 of General Synthetic Route 4

[0463]

[0464] DIEA (0.30 g) and solvent DCM (20 mL) were added to Compound 4. Under nitrogen protection, the temperature was lowered to 0 °C, and then 5-bromovaleryl chloride (Compound 5, 0.27 g) was added. The mixture was stirred at 25 °C for 2 h, diluted with 100 mL of DCM, and then the organic phase was washed twice with 100 mL of water. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (PE:EA 20:1) to obtain Compound 6 (800 mg).

[0465] Step 4: Synthesis of Compound 8 in General Synthetic Route 4

[0466]

[0467] Compound 6 (750.00 mg), intermediate K (Compound 7, 217.31 mg) and solvent DMF (20 mL) were added to a three-necked flask. Reactants K2CO3 (184.16 mg) and KI (110.60 mg) were added and the mixture was stirred at 25 °C for 16 h, then heated to 100 °C and stirred for 3 h. The reaction solution was diluted with 200 mL of EA, and then the organic phase was washed twice with 300 mL of saturated brine; the organic phase was dried over anhydrous Na2SO4, filtered, concentrated, silica gel was added, and the mixture was chromatographed on a column with 20:1 DCM:MeOH. The collected sample was concentrated to obtain Compound 8 (700.00 mg).

[0468] Step 5: Synthesis of Compound 1113

[0469]

[0470] Compound 8 (700.00 mg) and solvent THF (5 mL) were added to a three-necked flask, and hydrochloric acid dioxane solution (1 M) (5.55 mL) was added. The reaction solution was stirred at 25 °C for 2 h, diluted with 50 mL of EA, and then the organic phase was washed with saturated NaHCO3 solution and water. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (MeOH:DCM 1:20) to obtain Compound 1113 (201.1 mg, yield 31.59%). 1 H NMR (400 MHz, CDCl3) δ 4.50 - 4.39 (m, 6H), 3.59 (t, J = 4.6 Hz, 2H), 2.49 (s, 3H), 2.35 - 2.26 (m, 6H), 2.14 (t, J = 7.5 Hz, 2H), 1.75 - 1.69 (m, 4H), 1.66 - 1.43 (m, 17H), 1.27 (d, J = 3.7 Hz, 78H), 0.94 - 0.87 (m, 18H).

[0471] Z. Compound 1115, synthesized according to General Synthetic Route 4

[0472] Structural formula:

[0473] Chemical formula: C 67 H 131 N3O7

[0474] Molecular weight: 1090.80

[0475] Synthesized according to General Synthetic Route 4, similar to the synthesis process of Compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine are used instead of Compound 5 and Compound 7 in General Synthetic Route 4 respectively. 1 H 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,6H),2.38-2.25(m,3H),1.55(dtt,J=

[0476] 36.3,14.1,7.3Hz,12H),1.27(d,J=3.3Hz,78H),0.90(td,J=7.2,3.4Hz,18H).

[0477] AA. Compound 1114, synthesized according to General Synthetic Route 5

[0478] Structural formula:

[0479] Chemical formula: C 70 H 138 N2O7

[0480] Molecular weight: 1119.88

[0481] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 5

[0482]

[0483] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003.

[0484] Step 3: Synthesize Compound 6 of General Synthetic Route 5

[0485]

[0486] Add compound 4 (500 mg), intermediate M (compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) into a three-necked flask. After stirring the reaction solution in N2 at 25 °C for 16 h, add 100 mL of water to the reaction solution, extract it twice with EA (100 mL), combine the organic phases, concentrate, add silica gel, and then column-chromatograph it with 1:20 DCM:MeOH to collect the sample and concentrate to obtain compound 6 (500 mg).

[0487] Step 4: Synthesis of compound 1114

[0488]

[0489] Add compound 6 (450 mg) and solvent THF (5 mL) into a three-necked flask, and add dioxane hydrochloride solution (1 M) (3.65 mL). After stirring at 25 °C for 2 h, dilute it with 50 mL of EA, wash the organic phase with 50 mL of saturated NaHCO3 solution and water, dry the organic layer with anhydrous Na2SO4, filter and concentrate, and then perform silica gel column chromatography (MeOH:DCM 1:20) to obtain compound 1114 (107 mg, yield 24.84%). 1 H NMR (400 MHz, CDCl3) δ 4.16 - 4.07 (m, 6H), 3.60 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 2.49 (s, 3H), 2.31 (tt, J = 8.4, 5.5 Hz, 6H), 1.70 (d, J = 4.6 Hz, 3H), 1.64 - 1.39 (m, 18H), 1.27 (d, J = 4.0 Hz, 78H), 0.90 (t, J = 7.1 Hz, 18H).

[0490] Z. Compound 1120, synthesized according to the general synthetic route 5

[0491] Structural formula:

[0492] Chemical formula: C 72 H 142 N2O7

[0493] Molecular weight: 1147.90

[0494] Synthesized according to the general synthetic route 5, similar to the synthesis process of compound 1114, with the difference that intermediate C and 6-bromohexanal are used instead of compound 2 and compound 5 in the 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).

[0495] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0496] 1. Preparation of compound 6001

[0497] Structural formula:

[0498] Molecular weight: 499.43

[0499] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide and continue the reaction for 16 h. Then pass through a reverse column to obtain compound 6002.

[0500] Add 0.7 g of compound 6002, 0.4 g of palladium on carbon, and 28 mL of tetrahydrofuran to a reaction flask, stir, displace with hydrogen and maintain the pressure with a hydrogen balloon. After 16 h, filter and pass through a normal column (methanol - DCM system) to obtain 200 mg of compound 6001. 1 HNMR (400 MHz, CDCl3) δ

[0501] 7.54 (t, J = 5.5 Hz, 2H), 3.79 - 3.68 (m, 4H), 3.42 (dd, J = 10.0, 5.4 Hz, 4H), 2.89 (t, J = 6.0 Hz, 4H), 2.55 (dt, J = 11.8, 6.8 Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2 Hz, 32H), 0.91 (t, J = 6.8 Hz, 3H).

[0502] 2. Preparation of compound 6002

[0503] Structural formula:

[0504] Molecular weight: 497.42

[0505] 0.8 g of oleylamine and 0.8 g of 2 - hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70 °C. After 3.5 h, 0.8 g of 2 - hydroxyethyl acrylamide was added, and the reaction continued for 16 h. Then the organic solvent was concentrated, passed through a reverse column, and freeze - dried to obtain 400 mg of compound 6002. 1 1H NMR (400 MHz, CDCl3) δ 7.31 (s, 2H), 5.38 (dd, J = 13.0, 7.4 Hz, 2H), 3.79 - 3.66 (m, 4H), 3.41 (dd, J =

[0506] 10.1, 5.4 Hz, 4H), 2.82 - 2.72 (m, 4H), 2.44 (dd, J = 15.1, 9.2 Hz, 6H), 2.10 - 1.99 (m, 4H), 1.46 (s, 2H), 1.29 (s, 24H), 0.90 (t, J = 6.8 Hz, 3H).

[0507] 3. Preparation of compound 6003

[0508] Structural formula:

[0509] Molecular weight: 445.34

[0510] 4.00 g of tetradecylamine, 6.53 g of 2 - 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 column, and freeze - dried to obtain 1.5 g of compound 6003. 1 1H NMR (400 MHz, CDCl3) δ 4.29 (t, J = 7.5 Hz, 4H), 3.89 - 3.75 (m, 4H), 2.81 (t, J = 6.2 Hz, 4H), 2.50 (ddd, J = 23.2, 14.1, 7.1 Hz, 6H), 1.46 (s, 2H), 1.28 (s, 24H), 0.91 (t, J = 6.3 Hz, 3H).

[0511] 4. Preparation of compound 6004

[0512] Structural formula:

[0513] Molecular weight: 553.48

[0514] Add 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol to a reaction flask, stir and heat to 100 °C. After 4 h, add 1 mL of n-butyl acrylate. Continue the reaction for 1 h, then pass through a normal-phase column (petroleum ether-ethyl acetate system). Add 0.4 g of sodium hydroxide, 1 mL of water and 10 mL of methanol, stir and hydrolyze for 30 min. Add 1 mL of concentrated hydrochloric acid and 10 mL of methanol to obtain a mixed solution, adjust the pH to neutral, spin-dry, dissolve in DCM, dry with anhydrous magnesium sulfate, filter, spin-dry the solvent, add 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT and 2.3 g of EDCI, react at room temperature for 18 h. After concentrating the organic solvent, pass through a reverse-phase column and lyophilize to obtain 240 mg of compound 6004. 1 HNMR(400 MHz, CDCl3) δ 7.58 (d, J = 16.6 Hz, 2H), 5.44 - 5.31 (m, 2H), 3.72 (d, J = 16.5 Hz, 4H), 3.31 (d, J = 5.6 Hz, 4H), 3.17 (s, 4H), 2.84 (s, 2H), 2.69 (s, 4H), 2.09 - 2.01 (m, 4H), 1.67 (d, J = 2.7 Hz, 10H), 1.37 - 1.26 (m, 24H), 0.91 (t, J = 6.8 Hz, 3H).

[0515] 5. Preparation of Compound 6005

[0516] Structural formula:

[0517] Molecular weight: 597.54

[0518] Add 25.00 g of 11-heneicosanone, 62.05 g of ammonium acetate and 500 mL of methanol to a reaction flask, stir, add 6.55 g of sodium cyanoborohydride. After reacting for 16 h, add 250 mL of water and 250 mL of DCM, separate the layers. Extract the aqueous phase with 50 mL of DCM and combine the organic phases. Pass through a normal-phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0519] Add 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex to a reaction flask, stir and heat to 100 °C. Add 10 mL of n-butyl acrylate. After 16 h, pass through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0520] Dissolve 31.00 g of Compound 6005-B, 6.55 g of sodium hydroxide, 310 mL of methanol, and 31 mL of water in a reaction flask and stir. After 20 min, add 150 mL of THF, stir and heat up to 50 °C. After 30 min, add a solution prepared from 16.13 g of concentrated hydrochloric acid and 160 mL of methanol, stir. After 30 min, evaporate the solvent under reduced pressure, dissolve in 300 mL of DCM, dry over anhydrous magnesium sulfate, filter, and evaporate the solvent under reduced pressure to obtain 27.00 g of an oily Compound 6005-C.

[0521] Add 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 to a reaction flask and stir. After 18 h, concentrate the organic solvent, and perform column chromatography (methanol-DCM system) to remove the excess 4-amino-1-butanol compound, obtaining 6.80 g of Compound 6005. 1 H NMR (400 MHz, CDCl3) δ 3.70 (t, J = 5.6 Hz, 4H), 3.28 (t, J = 5.6 Hz, 4H), 2.74 (t, J = 6.0 Hz, 4H), 2.40 - 2.45 (m, 1H), 2.34 (t, J = 6.4 Hz, 4H), 1.63 - 1.65 (m, 8H), 1.25 - 1.35 (m, 36H), 0.91 (t, J = 6.4 Hz, 6H).

[0522] 6. Preparation of Compound 6006

[0523] Structural formula:

[0524] Molecular weight: 553.48

[0525] Add 1.00 g of oleylamine, 0.47 g of hydroxyethyl acrylamide, and 8 mL of ethanol to a reaction flask, stir and heat up to 70 °C. After 13 h, evaporate the solvent under reduced pressure and perform column chromatography (methanol-DCM system) to obtain 800 mg of Intermediate 1. Add 1.5 mL of n-butyl acrylate and 5 mL of n-butanol, stir and heat up to 100 °C. After 3 h, evaporate the solvent under reduced pressure and perform column chromatography (methanol-DCM system). Add a solution prepared from 0.4 g of sodium hydroxide, 10 mL of methanol, and 1 mL of water, stir for 1 h, add a solution prepared from 1 mL of concentrated hydrochloric acid and 8 mL of methanol, stir for 30 min, evaporate the solvent under reduced pressure, dissolve in 100 mL of DCM, dry over anhydrous magnesium sulfate, filter, evaporate the solvent under reduced pressure, add 0.45 g of 6-amino-1-hexanol, 0.27 g of HOBT, 0.78 g of EDCI, and 10 mL of DCM, stir. After 14 h, concentrate the organic solvent, and perform column chromatography (methanol-DCM system) to obtain 300 mg of Compound 6006. 1HNMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 6.95 (s, 1H), 5.43 - 5.28 (m, 2H), 3.74 - 3.67 (m, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.39 (dd, J = 10.0, 5.3 Hz, 2H), 3.24 (dd, J = 12.9, 6.7 Hz, 2H), 2.74 (t, J = 5.8 Hz, 4H), 2.48 - 2.42 (m, 2H), 2.41 - 2.33 (m, 4H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66 - 1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0526] 7. Compound 6007

[0527] Structural formula:

[0528] Molecular weight: 953.78

[0529]

[0530] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then slowly add methyl acrylate (31.5 g) dropwise, stir to mix, and restore to room temperature. The reaction solution is reacted at room temperature for 4 h; concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL) and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight; concentrate under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (2 g) to a single-necked flask. After adding MeOH (20 mL) and stirring to dissolve, then slowly add methyl acrylate (2.5 g) and stir to mix. Heat to 60 °C and react overnight; stop the reaction, concentrate the reaction solution under reduced pressure, and purify by column chromatography (DCM:MeOH = 50:1 - 20:1) to obtain compound 4 (2.5 g). At room temperature, add compound 4 (2.3 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature; stop the reaction and directly concentrate under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 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 = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0531] 9. Compounds 6008 - 6018

[0532] Synthesis of Compound 6009

[0533] Structural formula:

[0534] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise under a nitrogen atmosphere to a solution of methyl methacrylate (6 mL) in methanol (20 mL) with stirring, and the reaction was carried out overnight. The reactant was dried by rotary evaporation 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. Then, a colorless oil was obtained by column chromatography separation. 1H-NMR (300 MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0535] Then, a solution of the above-mentioned colorless oil (11.05 g) in methanol (20 mL) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 mL) with vigorous stirring at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure while maintaining the temperature not higher than 40 °C. The 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 recrystallized repeatedly from chloroform and cyclohexane to finally obtain a white solid. 1H-NMR (300 MHz, 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).

[0536] The preparation methods of 6008 and 6009 are only different in that: equimolar amount of tetradecylamine is used instead of hexadecylamine. For 6010 - 6017, referring to the preparation method of 6009, they are prepared by using equimolar amount of the corresponding R a -NH2 to replace hexadecylamine.

[0537] For 6018, referring to the preparation method of 6007, it is prepared by using equimolar amount of the corresponding R a -NH2 to replace octadecylamine.

[0538] 9. Compound 6019

[0539] Structural formula:

[0540] Molecular weight: 753.66

[0541]

[0542] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask. After adding DCM (50 mL) and stirring to mix, 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, and the mixture was stirred and mixed, and reacted overnight at room temperature; concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 5:1) to obtain 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 was displaced, 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 concentrated by rotation to obtain product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. After adding DMF (50 mL) and stirring to dissolve, then product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred and mixed, and the reaction was carried out overnight at room temperature; water (500 mL) and EA (500 mL) were added, and liquid-liquid extraction was carried out. The organic phase was dried over Na2SO4, concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain solid 6 (380 mg). At room temperature, solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to a single-necked flask, and the reaction was carried out for 2 h at room temperature; concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 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 = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0543] 10. Compound 6020

[0544] Structural formula:

[0545] Molecular weight: 810.19

[0546]

[0547] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then, dropwise add methyl acrylate (31.5 g), stir to mix, restore to room temperature, and react for 4 h. Stop the reaction, directly concentrate under reduced pressure, and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight. Stop the reaction, concentrate the reaction solution under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, filter, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (1.3 g) to a single-necked flask. After adding DMF (20 mL) and stirring to mix, add 35897 - 34 - 8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g), stir to mix, and react overnight at room temperature. Add water (200 mL) and directly freeze-dry under reduced pressure to obtain compound 4 (4 g). At room temperature, add compound 4 (100 mg) and HCl / Dioxane (4M) (2 mL) to a single-necked flask, and react at room temperature for 1 h. Stop the reaction, concentrate and purify the reaction solution 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).

[0548] 11. Compound 6021

[0549] Structural formula:

[0550] Molecular weight: 1182.79

[0551]

[0552] At room temperature, into a single-necked flask, add compound 2 (3.5 g, synthesized by the same process as product 2 in compound 6020), add MeOH (20 mL), stir and mix, then add TREN (23.0 g), stir and mix, heat up to 60 °C, stir overnight, concentrate under reduced pressure, and lyophilize the obtained crude product to obtain product 3 (25 g); at room temperature, into a single-necked flask, add product 3 (22.0 g) and DCM (200 mL), cool down to 0 °C, slowly dropwise add (Boc)2O (71.7 g), after dropping, restore to room temperature, react for 3 h, add water (200 mL) to quench, then add DCM (100 mL) for liquid-liquid extraction, dry the organic phase with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography to obtain yellow oily substance 4 (5.8 g). At room temperature, into a single-necked flask, add yellow oily substance 4 (1.4 g), slowly add HCl / Dioxane (4 M) (15 mL) under an ice-water bath, stir and mix, after adding, restore to room temperature, react for 0.5 h, concentrate under reduced pressure to obtain compound 5 (800 mg). At room temperature, into a single-necked flask, add compound 5 (1.0 g), (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL), react overnight at room temperature, add 100 mL of water and 100 mL of EA, perform liquid-liquid extraction, wash the EA phase once with 100 mL of saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). At room temperature, into a single-necked flask, add compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL), react overnight at room temperature, concentrate under reduced pressure, and purify to obtain compound 6021 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.96 (t, J = 6.6 Hz, 4H), 3.75 (dt, J = 14.0, 6.6 Hz, 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 = 6.9 Hz, 4H), 2.05 - 1.82 (m, 8H), 1.73 (p, J = 7.7 Hz, 10H), 1.53 (qd, J = 8.3, 7.7, 4.3 Hz, 8H), 1.30 (s, 28H), 0.97 - 0.87 (m, 3H).

[0553] 12. Compound 6023

[0554] Structural formula:

[0555] Molecular weight: 670

[0556]

[0557]

[0558] At room temperature, add dodecylamine (10 g) to a three-necked flask. After adding MeOH (100 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath, slowly add methyl acrylate (10.2 g) dropwise, stir to mix, and then return to room temperature. Stop the reaction after the reaction solution has reacted at room temperature for 4 h. Concentrate under reduced pressure and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (19 g). At room temperature, add product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight. Concentrate under reduced pressure to obtain compound 3 (20 g). Take 500 mg for purification and freeze-dry to obtain compound 6023-1 (218 mg). At room temperature, add starting material 6023-1 (1.4 g) to a single-necked flask. After adding DCM (20 mL) and stirring to dissolve, add (S)-2,6-di-tert-butoxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g), stir to mix, maintain at room temperature, and react overnight. Separate and extract, dry, and concentrate under reduced pressure to obtain compound 4 (3.0 g). At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) to a single-necked flask, and react at room temperature for 2 h; concentrate under reduced pressure, purify, and freeze-dry to obtain compound 6023 (123 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.88 (t, J = 6.6 Hz, 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 = 6.5 Hz, 4H), 2.00 - 1.82 (m, 4H), 1.82 - 1.66 (m, 6H), 1.50 (qd, J = 8.2, 7.8, 4.2 Hz, 4H), 1.45 - 1.25 (m, 18H), 0.96 - 0.87 (m, 3H).

[0559] Compound 6024

[0560] Structural formula:

[0561] Molecular weight: 870.24

[0562]

[0563] At room temperature, to a single-necked flask, add product 6023-1 (1.4 g, synthesized in the same method as 6023-1 in compound 6023). After adding MeOH (20 mL) and stirring until dissolved, slowly add methyl acrylate (2.8 g) and stir to mix. Heat up to 60 °C and react overnight at this temperature. Concentrate under reduced pressure to obtain compound 4 (2.0 g). At room temperature, add compound 4 (2.4 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature. Concentrate under reduced pressure and purify to obtain compound 6024 (191 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.69 (t, J = 6.0 Hz, 4H), 3.60 - 3.48 (m, 20H), 3.42 (t, J = 6.1 Hz, 4H), 3.25 - 3.19 (m, 2H), 3.12 (t, J = 5.8 Hz, 8H), 2.83 (t, J = 6.5 Hz, 12H), 1.78 (tt, J = 11.0, 6.4 Hz, 2H), 1.51 - 1.18 (m, 20H), 0.91 (t, J = 6.7 Hz, 3H).

[0564] 13. Compound 6026

[0565] Structural formula:

[0566] Molecular weight: 443.67

[0567]

[0568] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the only difference being that an equimolar amount of compound 1 was used to replace raw material 5 of 6019 and an equimolar amount of N-(tert-butoxycarbonyl)ethanolamine was used to replace 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).

[0569] Compounds 6025 - 6034 were obtained by referring to the synthetic routes of the aforementioned compounds, with the difference being that hydroxyethylamine was used to replace the corresponding ethylenediamine, and the amino group of hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc, and the protecting group was removed by a conventional method after the reaction was completed.

[0570] (3) Exemplary Compounds of the First Ionizable Lipids and Their Properties

[0571] Exemplary compounds of the present invention and their properties are listed in Table 1, and they are synthesized according to the process routes in Preparation Example 1 respectively.

[0572] The calculated c-pKa (molnetwork) and LogP (cLogP driver) of the exemplary compounds of the present invention, as well as the c-pKa and cLogP values, are generated by the ChemDraw module of Chemoffice.

[0573] Properties of exemplary compounds of the first ionizable lipid in Table 1

[0574]

[0575]

[0576]

[0577]

[0578]

[0579] The above properties of the exemplary compounds of the first ionizable lipid of the present invention indicate that they can be used as excellent surfactants, and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0580] (4) Preparation of compositions based on blank lipid nanoparticles

[0581] 4.1 Preparation of blank lipid nanoparticles

[0582]

[0583] Blank lipid nanoparticles are prepared respectively according to the above molar ratios, and the specific method is as follows:

[0584] Dissolve the lipid raw materials in ethanol to obtain a lipid ethanol solution. The total concentration of all lipid raw materials in ethanol is 8 mg / mL. After mixing the lipid ethanol solution with a 50 mM citrate buffer (pH 4.0) solution in a volume ratio of 1:3 in a nanoparticle preparation device, ultrafiltration is carried out, and the sample is collected to obtain blank lipid nanoparticles, and the lipid concentration of the composition is 2 mg / mL.

[0585] 4.2 Preparation of compositions based on blank lipid nanoparticles

[0586]

[0587]

[0588] Using nucleic acid as the active ingredient, configure the nucleic acid into a solution with a concentration twice that of each example using nuclease-free water as Solution 1; dilute the blank lipid nanoparticles according to the prescription ratio of each example using nuclease-free water as Solution 2; mix Solution 1 and Solution 2 in equal volumes and vortex for 2 - 3 s to obtain the composition based on blank lipid nanoparticles.

[0589] (5) Determination of the particle size and Zeta potential of the composition based on blank lipid nanoparticles

[0590] 1. Determination of particle size and polydispersity index (PDI): Use a Malvern ZetaSizer Nano ZS90 to measure the average particle size and PDI of the nanoparticle sample solution in the examples by dynamic light scattering. The measurement angle is 90°, the refractive index of the dispersant is 1.330, and the test temperature is 25 °C.

[0591] 2. The encapsulation efficiency test method is as follows:

[0592] According to the manufacturer's instructions, use the Quant-it Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK) to measure the encapsulation efficiency of mRNA in the composition based on blank lipid nanoparticles.

[0593] According to the manufacturer's instructions, use the dsDNA HS Kit (Novizan / EQ121) to measure the encapsulation efficiency of pDNA in the composition based on blank lipid nanoparticles.

[0594] According to the manufacturer's instructions, use the microRNA Reagent Kit to measure the encapsulation efficiency of siRNA in the composition based on blank lipid nanoparticles.

[0595] The average particle size, PDI, and encapsulation efficiency data of the composition based on blank lipid nanoparticles prepared in the examples are shown in Table 2.

[0596] Table 2 Summary of particle size, PDI, and encapsulation efficiency of the composition based on blank lipid nanoparticles

[0597] Example number Particle size nm PDI Entrapment efficiency % 1 198.5 0.156 88.6 2 176.8 0.145 90.5 3 150.7 0.189 95.7 4 204.8 0.198 80.5 5 156.7 0.124 94.8 6 187.8 0.146 96.7 7 105.9 0.124 89.7 8 118.7 0.186 84.3 9 168.5 0.201 90.7 10 170.8 0.157 94.3 11 216.4 0.197 85.5 12 172.4 0.136 92.3 13 189.1 0.201 80.4 14 203.4 0.096 82.1 15 152.8 0.168 82.8 16 179.4 0.149 86.7 17 126.7 0.125 89.1 18 465.7 0.204 82.4

[0598] It can be seen from Table 2 that the composition based on blank lipid nanoparticles provided by the present invention has a small and uniform particle size and a high encapsulation efficiency (> 80%).

[0599] (6) Evaluation of the in vivo delivery effect of the composition based on blank lipid nanoparticles

[0600] 6.1 Evaluation of the in vivo delivery effect of blank lipid nanoparticle - mRNA complexes by intravenous injection in mice

[0601] Female Balb / c mice aged 6 - 8 weeks were used to evaluate the in vivo delivery efficiency of the blank lipid nanoparticles - firefly luciferase mRNA complex in Example 5. The blank lipid nanoparticles - luciferase mRNA complex was administered by tail vein injection at a single dose of 0.3 mpk. At specific time points after administration (such as 3 h, 6 h), the fluorescence imaging substrate was intraperitoneally injected into the mice, and in vivo imaging of the animals was performed using a PerkinElmer small animal imaging system to measure the bioluminescence signal. The results are shown in Table 3 and Figure 1 .

[0602] Table 3 In vivo activity evaluation of the blank lipid nanoparticles - mRNA complex in Example 5

[0603]

[0604] The results showed that after the blank lipid nanoparticles provided by the present invention were combined with luciferase mRNA, a relatively high luciferase protein expression rate could be achieved in mice in vivo.

[0605] Female Balb / c mice aged 6 - 8 weeks were used to evaluate the in vivo delivery efficiency of the blank lipid nanoparticles - human erythropoietin mRNA (hEPO mRNA) complex in Example 6 and Example 12. The blank lipid nanoparticles - hEPO mRNA complex was administered by tail vein injection at single doses of 1.5 mpk and 3 mpk respectively. At a specific time point (24 h) after administration, the hEPO expression level in the mice was measured. The results are shown in Table 4.

[0606] Table 4 hEPO expression level (pg / ml) 24 h after administration of the blank lipid nanoparticles - human erythropoietin mRNA complex in Example 6 and 12

[0607]

[0608] It can be seen that after the blank lipid nanoparticles provided by the present invention were combined with human erythropoietin mRNA and administered by intravenous injection, a relatively high hEPO expression rate could be achieved in mice in vivo.

[0609] 6.2 Evaluation of the in vivo delivery effect of lipid nanoparticles - mRNA complex by intramuscular injection in mice

[0610] Female Balb / c mice aged 6 - 8 weeks were used to evaluate the in vivo delivery efficiency of the lipid nanoparticle - luciferase mRNA complex in Example 10. The blank lipid nanoparticle - luciferase mRNA complex was administered by intramuscular injection at a single dose of 0.3 mpk. At specific time points after administration (3 h, 6 h), the fluorescence imaging substrate was intraperitoneally injected into the mice, and in vivo imaging of the animals was performed using a PerkinElmer small animal imaging system to measure the bioluminescence signal. The results are shown in Table 5 and Figure 2 。

[0611] Table 5 In vivo activity evaluation after 3 / 6 h of administration of the blank lipid nanoparticle - mRNA complex in Example 10

[0612]

[0613] The results showed that after the blank lipid nanoparticles provided by the present invention were combined with luciferase mRNA and administered by intramuscular injection, a relatively high luciferase protein expression rate could be achieved in mice.

[0614] 6.3 Intraperitoneal administration (IP) and subcutaneous administration (SC)

[0615] Female Balb / c mice aged 6 - 8 weeks were used to evaluate the in vivo delivery efficiency of the lipid nanoparticle - firefly luciferase mRNA (Luciferase mRNA) complex in Example 16. The blank lipid nanoparticle - Luciferase mRNA complex was administered by intraperitoneal injection and subcutaneous injection respectively at a single dose of 0.3 mpk. At specific time points after administration (3 h, 6 h), the fluorescence imaging substrate was intraperitoneally injected into the mice, and in vivo imaging of the animals was performed using a PerkinElmer small animal imaging system to measure the bioluminescence signal. The results are shown in Table 6 and Figure 3 。

[0616] Table 6 In vivo activity evaluation of the blank lipid nanoparticle - mRNA complex in Example 16

[0617]

[0618] The results showed that after the blank lipid nanoparticles provided by the present invention were combined with Luciferase mRNA and administered by intraperitoneal injection and subcutaneous injection, a relatively high luciferase protein expression rate could be achieved in mice.

[0619] The solution of the present invention is not limited to the technical means disclosed above, but also includes technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art of the present invention, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. Use of blank lipid nanoparticles in the preparation of in vivo delivery products, characterized in that, Comprising 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 comprises: 5-70 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-50 mol% of a phospholipid, 10-70 mol% of cholesterol, and 0-15 mol% of a polyethylene glycol-conjugated lipid; The first ionizable lipid is selected from compounds having the general formula (1J) or pharmaceutically acceptable salts, stereoisomers, 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, wherein 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, amino, amide, 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; X1 is 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-; wherein each R 14 , R 15 is independently selected from H, C 1-30 linear or branched alkyl or cycloalkyl, C 2-30 linear or branched alkenyl; R9, R 10 each independently is 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 ; n is an integer from 1 to 6; Among them, the alkyl, cycloalkyl, and alkenyl groups described in R4, R5, R6, R9, R 10 , R 13 , R 14 , R 15 are unsubstituted or substituted by one or more groups selected from hydroxyl, mercapto, amino, substituted amino, and halogen; The salt does not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, 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 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 wherein R a ’ is a C1-C 12 alkyl group, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamines, R c ” is selected from C1-C6 alkyl groups which are unsubstituted or substituted by an amino group, and R c ”’ is H, or -R c ’—A1'-R c ”—NH2; 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.

2. The application according to claim 1, characterized in that The delivery method further comprises the step of introducing the composition based on the blank lipid nanoparticles into an animal body.

3. The application according to claim 2, characterized in that The routes for introducing the composition based on the blank lipid nanoparticles into an animal body 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, characterized in that The animals are mammals and non-mammals.

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

6. The application according to claim 5, characterized in that, 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 0.1%-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 claim 1, characterized in that, R1, R2, and R3 are each independently the following groups: wherein, Y does not exist, or is C 1-30 a straight-chain or branched-chain alkyl or cycloalkyl group, C 2-20 a straight-chain or branched-chain alkenyl group, C 2-20 a straight-chain or branched-chain alkynyl group; R1' and R2' are each independently H, C 1-30 a straight-chain or branched-chain alkyl group, C 2-30 a straight-chain or branched-chain alkenyl group, C 2-30 a straight-chain or branched-chain alkynyl group, and the total carbon chain length of Y, R1' and R2' is 8-40.

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

10. The application according to claim 1, characterized in that R1, R2, and R3 are each independently selected from any one of the following groups:

11. The application according to claim 1, wherein G1, G2, G3 are each independently -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-.

12. The application according to claim 1, characterized in that, 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-.

13. The application according to claim 1, characterized in that 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, guanidino, amidino, amido, aliphatic amine group, 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 hydroxyl, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

14. The application according to claim 1, characterized in that The compounds of formula (1J) are selected from:

15. The application according to claim 1, characterized in that, The compounds of formula (2) are selected from at least one of the following compounds:

16. The application according to claim 1, characterized in that, 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:

17. The application according to claim 1, characterized in that, 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.

18. The application according to claim 1, characterized in that The polyethylene glycol-conjugated lipids include at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

19. The application according to claim 18, characterized in that, 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.

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

21. The application according to claim 1, characterized in that The solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

22. The application according to claim 21, 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%.

23. The application according to claim 21, 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.

24. 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.

25. An in vitro gene delivery method, characterized in that, It includes the step 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 the blank lipid nanoparticles described in claim 1.

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