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

Abstract

The present invention provides the use of blank lipid nanoparticles in the preparation of in vivo delivery products, belonging to the field of biomedical technology. The use of the blank lipid nanoparticles in the preparation of in vivo delivery products includes the steps of mixing the blank lipid nanoparticles with biologically active substances in a solvent to obtain a composition based on blank lipid nanoparticles; the blank lipid nanoparticles are composed of: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids. The present invention adopts the blank lipid nanoparticles, and the dosage of biologically active substances can be flexibly adjusted according to the needs of users, and can be administered through multiple routes such as intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection, and an ideal delivery effect can be obtained.

Description

Technical Field

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

[0002] Gene editing therapy has the potential to cure genetic diseases, but it requires the ability to safely and effectively deliver gene editing drugs to the corresponding target organs and tissues in the body. Gene editing drugs can exist in the form of DNA, mRNA, protein or ribonucleoprotein (RNP). The successful delivery of such macromolecular drugs into cells requires breaking through multiple physiological barriers: 1) Before the drug enters the cell, it is necessary to avoid dissociation of the drug from the carrier or drug degradation; 2) Targeting specific cells; 3) Passing through the cell membrane to enter the cell: 4) Releasing the drug in a specific organelle. Therefore, how to effectively and safely introduce gene editing drugs into the corresponding target cells in the body is a big problem.

[0003] The existing in vivo introduction technology of gene-editing drugs still has 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. Different types of AAV capsid serotypes produced naturally and synthesized in the laboratory can achieve different tissue targeting, but their loading capacity is limited (only 5kb of DNA) and have potential safety risks. Lentivirus vectors can accommodate up to 10kb of DNA drugs, which is enough to load known gene-editing drugs into a single vector, but there is a potential risk of genome integration. In general, viral vectors face problems such as the immunogenicity of the vector, long-term expression of gene-editing drugs, non-target gene editing, the possibility of genome integration, manufacturing costs and dose-limiting toxicity, and potential unsafety such as integration renaturation and immune response in host cells, which limits their application.

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

[0005] Lipid nanoparticle (LNP) delivery has the following advantages in the field of gene editing drug delivery: 1) LNP delivery of gene editing drugs can achieve transient expression. Compared with the long-term expression of gene editing drugs brought by viral vectors, LNP can minimize the possibility of off-target effects; 2) The immunogenicity of LNP is much lower than that of viral vectors. In some cases, it can be repeatedly administered, has good safety and biocompatibility, and can deliver drug doses that meet the effective gene editing level; 3) The large-scale production process of LNP is currently mature, which provides a basis for clinical trials of using LNP to deliver gene editing drugs in vivo. Patents CN116832051A and CN116969850A have conducted research in this regard. However, in the prior art, the use of lipid nanoparticles (LNP) to deliver gene editing drugs usually requires a special mixing process, otherwise a good delivery effect cannot be achieved.

[0006] Based on this, the present invention provides the use of blank lipid nanoparticles in the preparation of in vivo delivery products. The biologically active substances are simply mixed with the blank lipid nanoparticles in a solution state to achieve safe and effective delivery of the biologically active substances. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides the use of blank lipid nanoparticles in the preparation of in vivo delivery products. The use of the blank lipid nanoparticles in the preparation of in vivo delivery products comprises the steps of mixing the blank lipid nanoparticles with biologically active substances in a solvent to obtain a composition based on the blank lipid nanoparticles; the blank lipid nanoparticles are composed of: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids. The present invention adopts the blank lipid nanoparticles, and the dosage of the biologically active substances can be flexibly adjusted according to the needs of the user, and can be administered through a variety of routes, all of which can achieve an ideal delivery effect.

[0008] To achieve the above objectives, in a first aspect, the present invention provides an application of blank lipid nanoparticles in the preparation of an in vivo delivery product, comprising the step of mixing the blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on the blank lipid nanoparticles; the blank lipid nanoparticles comprise: 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.

[0009] In a preferred embodiment, the blank lipid nanoparticle composition comprises: 10-60 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-30 mol% of a phospholipid, 15-70 mol% of cholesterol and 0-10 mol% of a polyethylene glycol-conjugated lipid.

[0010] In a preferred embodiment, the product is a delivery system or a medicament.

[0011] In a preferred embodiment, the use further comprises the step of introducing the blank lipid nanoparticle-based composition into an animal.

[0012] In a preferred embodiment, the route of introducing the blank lipid nanoparticle-based composition into an animal includes oral administration, intranasal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intraarticular administration, intralesional administration, intratracheal administration, subcutaneous administration or intradermal administration.

[0013] In a preferred embodiment, the animals are mammals and non-mammals.

[0014] In a preferred embodiment, the biologically active substances include nucleic acids, proteins, polypeptides, and small molecule active substances; the nucleic acids are DNA and / or RNA.

[0015] In a preferred embodiment, the nucleic acid comprises at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA minicircle, and msDNA.

[0016] In a preferred embodiment, the amount of the nucleic acid is 0.1%-50% of the total amount of blank lipid nanoparticles and nucleic acids; in the composition based on blank lipid nanoparticles, the concentration of the biologically active substance is 50-5000 ng / μl.

[0017] In a preferred embodiment, the first ionizable lipid is selected from a compound comprising the general formula (1) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof;

[0018]

[0019] Wherein, R1, R2, and R3 are independently H, C 5-40 Straight or branched alkyl, C 5-40 Straight or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10 -30 straight chain or branched alkyl, C 10-30 Straight or branched alkenyl, C10 -30 straight chain or branched alkynyl; provided that at most one of R1, R2, and R3 is H;

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

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

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

[0023] L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-、-OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -、-NR 14 -S(=O)2-、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-OR-OP(=O)(OR 14 )O-; wherein m is an integer of 2-6, R7 and R8 are independently H, hydroxyl, halogen, C 1-6 A straight or branched alkyl or cycloalkyl group, C 2-6 A straight-chain or branched alkenyl group, each R 14 , R 15 are independently selected from H, C 1-30 Straight or branched chain alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl;

[0024] L2 is -(CR9R 10 ) n -or-(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-、-OC(=O)NR 16 -、-NR 16 C(=O)NR 17 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16-、NR 16 -S(=O)2-、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-OR-OP(=O)(OR 16 )O-; n is an integer of 1-6; k is an integer of 1-6; R9, R 10 , R 11 , R 12 are independently H, hydroxyl, halogen, C 1-6 A straight or branched alkyl or cycloalkyl group, C 2-6 A straight-chain or branched alkenyl group, each R 16 , R 17 are independently selected from H, C 1-30 Straight or branched chain alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl;

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

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

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

[0028]

[0029] Where Y does not exist or is C 1-30 Straight or branched alkyl or cycloalkyl, C 2-20 Straight or branched alkenyl, C 2-20 Straight or branched alkynyl; R1', R2' are independently H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 A straight chain or branched alkynyl group, and the total carbon chain length of Y, R1' and R2' is 8-40.

[0030] In a preferred embodiment, R1, R2, and R3 are independently selected from the following groups:

[0031]

[0032] Wherein, R1' and R2' are independently H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

[0033] In a preferred embodiment, R1, R2, and R3 are independently selected from any one of the following groups:

[0034]

[0035] In a preferred embodiment, G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

[0036] In a preferred embodiment, L1 is selected from -(CR7R8) m -X1-, wherein X1 is selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-、-OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-OR-OP(=O)(OR 14 )O-.

[0037] In a preferred embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-、-OC(=O)NR 16 -、-NR 16 C(=O)NR17 -、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-OR-OP(=O)(OR 16 )O-.

[0038] In a preferred embodiment, M is selected from the following structures:

[0039]

[0040] wherein m' and n' are independently integers of 0-6, R1" and R2" are independently H, C 1-6 Alkyl, C 2-6 The alkenyl, guanidino, amidino, amide, fatty amine, 3-10 membered nitrogen-containing heterocyclic ring; the nitrogen-containing heterocyclic ring is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and tautomeric forms thereof, which are unsubstituted or optionally substituted with one or more selected from hydroxyl, thiol, amino, substituted amino, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 The aromatic group is substituted with an organic group.

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

[0042]

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

[0044]

[0045] In a preferred embodiment, the compound of formula (1) is selected from the compound of formula (1C):

[0046]

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

[0048]

[0049] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1E)

[0050]

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

[0052]

[0053] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1G):

[0054]

[0055] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1H):

[0056]

[0057] In a preferred embodiment, M is selected from any one of the following groups:

[0058]

[0059] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (II):

[0060]

[0061] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1J):

[0062]

[0063] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1K):

[0064]

[0065] In a preferred embodiment, Y is absent, and the compound of formula (1) is selected from the compounds represented by formula (1L):

[0066]

[0067] Wherein R1' and R2' are independently selected from H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-40.

[0068] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1M):

[0069]

[0070] The compound of formula (1) is selected from:

[0071]

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[0147]

[0148] 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):

[0149]

[0150] 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.

[0151] 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):

[0152]

[0153] 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.

[0154] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0155]

[0156] 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.

[0157] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the steps of reacting the compound of formula (VII) with the compound of formula (VIII), the compound of formula (IX), and the compound of formula (X) in sequence:

[0158]

[0159] Wherein Xg, Xh, Xi, Xj, Xk, and Xl are groups containing leaving groups or nucleophilic groups, and Xg and Xj form G1 through nucleophilic reaction or condensation reaction, Xh and Xk form G2 through nucleophilic reaction or condensation reaction, and Xi and Xl form G3 through nucleophilic reaction or condensation reaction.

[0160] 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):

[0161]

[0162] Steps for reacting a compound of formula (XIII) with a compound of formula (XII):

[0163]

[0164] Wherein Xm is a group containing a nucleophilic group, and Xm and C=CL of the compound of formula (XII) 2a X1-L2 is formed by addition reaction.

[0165] In a preferred embodiment, R1, R2, and R3 are independently the following groups:

[0166]

[0167] Wherein, Y, R1', and R2' have the same meanings as above;

[0168] It also includes the steps of forming tail chains R1, R2, and R3:

[0169]

[0170] Wherein X is a leaving group.

[0171] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0172]

[0173] Wherein N1 is NH or O;

[0174] R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short chain polyethylene oxide; the C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short-chain polyoxyethylene is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0175] The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, R c " is selected from C1-C6 alkyl which is unsubstituted or substituted with amino, R c ”' is H, or -R c '-A1'-R c ”-NH2;

[0176] 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.

[0177] In a preferred embodiment, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkyl amines.

[0178] In a preferred embodiment, the condition is that when R a C6-C 24 In the case of alkenyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0179] When R a For branched C6-C 24 When it is alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkyl amines;

[0180] R a For a straight chain C6-C 24 When it is alkyl, A1 is NH or O, R b and R c Each independently selected from alkylamines;

[0181] When R a For a straight chain C6-C 24 When it is alkyl, A1 is O, Rb and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0182] When R a C6-C 24 In the case of short-chain polyoxyethylene, A1 is NH or O, R b and R c Each independently selected from alkylamines;

[0183] Or when R a C6-C 24 In the case of alkyl alcohol, A1 is NH or O, R b and R c Each is independently selected from alkylamines.

[0184] In a preferred embodiment, R a Select from the following compound structures:

[0185]

[0186] In a preferred embodiment, R b and R c Select from the following compound structures:

[0187]

[0188] R d Selected from C1-C6 alkanes or cycloalkanes.

[0189] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0190]

[0191]

[0192]

[0193] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0194] R a ”-NH2① reacts with α,β-unsaturated carbonyl compound② to generate ionizable lipid compound③:

[0195]

[0196] Among them, R a -NH2 is R a -NH2 or

[0197] R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c :-NH2.

[0198] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0199] 1) R a ”-NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤;

[0200] 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③;

[0201]

[0202] Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a -NH2 is selected from R a -NH2 or

[0203]

[0204] R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0205] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0206] 1) R a ”-NH2① reacts with α,β-unsaturated carbonyl compound② to form compound⑦;

[0207] 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨;

[0208] 3) Compound ⑨ reacts with nucleophile ⑩ to generate ionizable lipid compound

[0209]

[0210] Among them, R a -NH2 is R a -NH2, Z3 is a leaving group, A3 reacts with Z3 to obtain A1.

[0211] In a preferred embodiment, in Formula 2a-Formula 2c, at least one nucleophilic reagent undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to generate the ionizable lipid compound with a bifurcated carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0212] In a preferred embodiment, Formula 2a-Formula 2b further comprises the step of using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a-Formula 2b as a raw material, and performing an iterative reaction according to Step 1 in Formula 2a or Steps 1-2 in Formula 2b;

[0213] The compound of formula (2) having a terminal amino group is

[0214] Among them, R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0215] In a preferred embodiment, the reaction formula 2a-2c further comprises using the compound of formula (2) with a terminal amino group obtained by the reaction formula 2a-2c as a raw material, and The steps of the reaction;

[0216] The compound of formula (2) having a terminal amino group is

[0217] Among them, R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0218] The leaving group mentioned above refers to the leaving part in the nucleophilic reaction or condensation reaction, including but not limited to: H,

[0219] OH, H2O, halogen (such as F, Cl, Br and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy (RO-), amino (-NHR, wherein R is an alkyl or aryl group from which H is removed), phenoxy, tertiary carbon cation (such as tert-butyl cation), carbon cation stabilized by an unsaturated system or a heteroatom, or the various protecting groups mentioned above.

[0220] The nucleophilic groups mentioned above refer to molecules or ions that can provide electron pairs to form new chemical bonds in chemical reactions. Common nucleophilic groups include: hydroxide (HO-), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, halogens with nucleophilicity (such as Cl-, Br - or I - ), hydrogen ion (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxy anion (for example, alcohol from which hydroxyl hydrogen is removed), amine (including primary amine, secondary amine and tertiary amine) or amine anion, carbon anion (for example, carbon anion in organometallic reagents such as Grignard reagent, organic lithium reagent and Gilman reagent), sulfhydryl or sulfhydryl anion, thioether, enol or enolate anion, alkenyl ether, enamine, carboxylic acid or carboxylic acid anion, alkyl or aryl phosphine (for example, triphenylphosphine), aromatic heterocycle with lone pair of electrons (for example, pyridine), etc.

[0221] In a preferred embodiment, the raw materials used in the reaction process also contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0222] In a preferred embodiment, the blank liposome-based in vivo delivery method further comprises 0-60 mol% of other ionizable lipids, wherein the other ionizable lipids are selected from at least one of the following compounds:

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] In a preferred embodiment, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl At least one of 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-oleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0229] In a preferred embodiment, the steroid or its derivative comprises at least one of cholesterol, cholesterol stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, salinarum sterol, tomatidine, ursolic acid, and α-tocopherol.

[0230] In a preferred embodiment, the polyethylene glycol-conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0231] In a preferred embodiment, the polyethylene glycol conjugated lipid includes at least one of PEG-distearyloxypropyl (PEG-DSA), PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate (PEG-DMA), 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroylphosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoylphosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0232] In a preferred embodiment, in the blank lipid nanoparticle-based composition, the biologically active substance is encapsulated inside the blank lipid nanoparticle and / or adsorbed on the surface of the blank lipid nanoparticle to form a complex.

[0233] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffered salt solution.

[0234] In a preferred embodiment, the pH of the buffered salt solution is 1-9, the concentration of the buffered salt is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration of <50%.

[0235] In a preferred embodiment, the alcohol is ethanol.

[0236] In a preferred embodiment, the buffered saline solution is selected from at least one of a citrate solution, an acetate solution, a tartrate solution, a phosphate solution, a carbonate solution, a Tris-HCl solution, and a sodium chloride solution.

[0237] In a preferred embodiment, at least one of sugar, glycerol, DMSO, salt, antibiotics, and surfactants is further added to the blank lipid nanoparticle-based composition.

[0238] In a second aspect, the present invention provides an in vitro gene delivery method, comprising the steps of mixing blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on blank lipid nanoparticles; the blank lipid nanoparticles comprise: 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.

[0239] Compared with the prior art, the present invention has the following beneficial effects:

[0240] 1. The use of the blank lipid nanoparticles in the preparation of an in vivo delivery product comprises the steps of mixing the blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on the blank lipid nanoparticles; the blank lipid nanoparticles comprise: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids.

[0241] 2. The present invention uses the blank lipid nanoparticles to flexibly adjust the dosage of biologically active substances according to user needs, and can be administered through a variety of routes to achieve ideal delivery effects.

[0242] 3. The preparation process of the present invention is simple and can be completed without the aid of equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0243] Figure 1 This is the whole-body in vivo imaging of blank lipid nanoparticle-luciferase mRNA complex in Example 5 3h / 6h after intravenous administration to mice;

[0244] Figure 2 For Example 10, blank lipid nanoparticle-luciferase mRNA complexes were injected intramuscularly into mice for 3 / 6 hours and then subjected to abdominal and lateral in vivo imaging;

[0245] Figure 3 In vivo imaging of blank lipid nanoparticle-luciferase mRNA complexes administered intraperitoneally and subcutaneously to mice for 3h / 6h. DETAILED DESCRIPTION

[0246] The synthesis process of the present invention can tolerate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, but in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or by easily prepared intermediates, by using standard synthetic methods and procedures known to those skilled in the art or obvious to the technician based on the teachings of this article. Standard synthetic methods and procedures for preparing organic molecules and functional group transformations and operations can be obtained from relevant scientific literature or from standard textbooks in the field. The following description of the synthetic method is designed to illustrate but not limit the general procedures for preparing the compounds of the present invention.

[0247] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables (e.g., R1, R2, and R3, etc.) in each general synthetic route are as defined herein. One of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of certain steps may vary, such as the introduction and removal of protecting groups.

[0248] In the reaction scheme described herein, multiple stereoisomers can be produced. When a specific stereoisomer is not indicated, this should be understood to include all possible stereoisomers produced by the reaction. It will be appreciated by those of ordinary skill in the art that the reaction can be optimized to preferentially obtain a type of isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC.

[0249] (1) Preparation Example 1 Synthesis of the First Ionizable Lipid

[0250] The general synthetic route for the first ionizable lipid is as shown in general synthetic route 1-5.

[0251] General synthetic route 1

[0252]

[0253] Wherein, M' is M or M containing a protecting group.

[0254] As described in the general synthetic route 1 above, Boc-aminotris(hydroxymethyl)methane reacts with an acid (compound 2) to form compound 3. Step 1 can be carried out in an organic solvent 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.

[0255] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0256] Next, compound 4 and compound 5 undergo condensation reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (eg, DCM or DMF) using EDCl and DMAP or dicyclohexylcarbodiimide (DCC) as a catalyst.

[0257] 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 selected protecting group removal reaction conditions.

[0258] General synthetic route 2

[0259]

[0260] Wherein M' is M or M containing a protecting group; A is O, NH or S.

[0261] As described in the above general synthetic route 2, compound 1 undergoes a condensation reaction with compound 2 to give compound 3. Step 1 can be carried out in an organic solvent (eg, DCM) in the presence of EDCl and DMAP.

[0262] Next, the tert-butyloxy protecting group of compound 3 is removed to obtain compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) in the presence of an acid (eg, trifluoroacetic acid) and a positive ion scavenger (eg, triisopropylsilane (TiPS)).

[0263] Next, compound 4 undergoes a condensation reaction with a compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent (eg, DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0264] 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.

[0265] General synthetic route 3

[0266]

[0267] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0268] As described in the above general synthetic route 3, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0269] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0270] Next, compound 4 is reacted with a halogen-substituted aldehyde (compound 5) by condensation and reduction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0271] Next, compound 6 reacts with amine (compound 7) to generate 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).

[0272] If the R4' and / or R5' groups of compound 8 have the above-mentioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0273] General synthetic route 4

[0274]

[0275] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 containing a protecting group; R5' is R5 or R5 containing a protecting group.

[0276] As described in the general synthetic route 4 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (eg, DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0277] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0278] Next, compound 4 undergoes a condensation reaction with a halogen-containing substituted compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF, catalyzed by EDCl and DMAP or DCC.

[0279] Next, compound 6 reacts with an amine (compound 7) to generate compound 8. Step 4 can be carried out in an organic solvent (e.g., DMF) in the presence of a base (e.g., a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0280] If the R4' and / or R5' groups of compound 8 have the above-mentioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0281] General synthetic route 5

[0282]

[0283] Among them, M pr o is M or M containing a protecting group.

[0284] As described in the above general synthetic route 5, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0285] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0286] Next, compound 4 and compound 5 are subjected to condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0287] If the M of compound 6 pr If the o group contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0288] In addition, it should be understood that any specific embodiments of the present invention within the prior art may be explicitly 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 the exclusion is not explicitly stated herein.

[0289] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the cited source and the statement of this application, the statement of this application shall prevail.

[0290] (1) Synthesis of a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L) or (1M)

[0291] A. General considerations

[0292] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0293] The process routes described below can be used to synthesize compounds 1001-3422 of the present invention.

[0294] This article uses the following abbreviations:

[0295] THF: Tetrahydrofuran

[0296] MeCN: Acetonitrile

[0297] MeOH: Methanol

[0298] PE: Petroleum ether

[0299] EA: Ethyl acetate

[0300] DMF: N,N-dimethylformamide

[0301] EDCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0302] LAH: Lithium Aluminum Hydride

[0303] DCM: dichloromethane

[0304] DMAP: 4-dimethylaminopyridine

[0305] LDA: lithium diisopropylamide

[0306] rt: room temperature

[0307] DCE: 1,2-dichloroethane

[0308] n-BuLi: n-butyllithium

[0309] i-Pr2EtN: N,N-diisopropylethylamine

[0310] B. Intermediate Synthesis

[0311] Intermediate A:

[0312]

[0313] Intermediate A is obtained by the following synthetic process:

[0314]

[0315] Dissolve tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl carbonic anhydride (Boc2O) (99.1 g) in a mixed solvent of methanol (300 mL) / H2O (30 mL), react for 72 h at room temperature, and purify by silica gel column chromatography (DCM:MeOH=20:1-10:1) to obtain a white solid compound intermediate A (N-Boc-aminotrihydroxymethylmethane) (75.0 g). LCMS (ESI) calculation for C9H 19 NO5, [M+H] + m / z 222.13, found 222.25.

[0316] Intermediate B:

[0317]

[0318] Intermediate B is obtained by the following synthetic process:

[0319]

[0320] Add 2-ethyl-1,3-diethyl propanedioate (29.0 g), THF (90 mL), and DMF (30 mL) to a single-mouth bottle, add NaH (3.7 g) under ice bath, and stir at room temperature for 30 min under nitrogen protection; add pentadecane bromide (15.0 g), and react at 80° C. for 2 h under nitrogen protection; concentrate under reduced pressure to remove THF, add the reaction solution dropwise into ice water, add ethyl acetate, stir, and filter through diatomaceous earth, collect the organic phase and then separate the liquids, 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 the compound 2-ethyl-2-pentadecanyl maleic acid diethyl ester (16.5 g). EtOH (50 mL), H2O (50 mL), KOH (11.3 g) were added to 2-ethyl-2-pentadecyl maleic acid diethyl ester (8.0 g), and the mixture was reacted at 90° C. for 12 h; the mixture was concentrated under reduced pressure to remove EtOH, and the reaction system was adjusted to pH = 4-5 by adding dilute hydrochloric acid, and water and ethyl acetate were added for stirring and separation, and the organic phase was washed with saturated brine, and the organic phase was separated, and purified by column chromatography (PE: EA = 5: 1-1: 1) to obtain 2-ethyl-2-pentadecyl maleic acid (5.2 g). 2-ethyl-2-pentadecyl maleic acid (10.0 g) was reacted at 170° C. for 6 h under open conditions, and then cooled to room temperature, and water and ethyl acetate were added for stirring and separation, and the organic phase was washed with saturated brine, and the organic phase was separated, concentrated, and purified by column chromatography (DCM: MeOH = 20: 1-10: 1) to obtain intermediate B (2-ethyl heptadecanoic acid) (8.2 g). 1H NMR (400MHz, CDCl3) δ2.31 (tt, J = 8.6, 5.3Hz, 1H), 1.71-1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4Hz, 3H), 0.90 (t, J = 6.7Hz, 3H).

[0321] Intermediate C:

[0322]

[0323] Intermediate C is obtained by the following synthetic process:

[0324]

[0325] Add decanoic acid (50g) and solvent THF (500mL) to a three-necked flask, cool the system to 0°C, slowly add reactant NaH (23.22g), stir at 0°C, under nitrogen protection for 1h, slowly drop LDA (62.19g), continue stirring at 0°C, under nitrogen protection for 1h. Finally, drop reactant iodine nonane (88.52g), warm to room temperature and stir overnight, dilute with 1L DCM, wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, purify by silica gel chromatography (PE:EA=50:1), and finally obtain intermediate C (2-octyl undecanoic acid) (11g). 1H NMR (400MHz, Chloroform-d) δ2.37 (tt, J=8.7, 5.1Hz, 1H), 1.71-1.58 (m, 2H), 1.48 (dt, J=

[0326] 13.3, 6.7Hz, 2H), 1.29 (d, J = 9.4Hz, 25H), 0.90 (t, J = 6.8Hz, 6H).

[0327] Intermediate D:

[0328]

[0329] Intermediate D is obtained by the following synthetic process:

[0330]

[0331] Tridecanoic acid (79.5 g) and solvent THF (800 mL) were added to a three-necked flask, and NaH (22.25) was slowly added after the system was cooled to 0°C. After the reaction solution was stirred at 0°C under nitrogen protection for 1 hour, LDA (317.87 g) was slowly added dropwise to the reaction system; the reaction solution was stirred at 0°C under nitrogen protection for 1 hour. After the reactant iodinated n-hexane (94.39) was added dropwise, the temperature was raised to room temperature and stirred overnight, and the reaction solution was diluted with 1L DCM and washed with saturated NH4Cl solution and water respectively. The organic layer was dried with anhydrous Na2SO4, filtered and concentrated, and silica gel column chromatography (PE:EA=50:1) was performed to obtain intermediate D (2-hexyl tridecanoic acid) (26 g). 1H NMR (400MHz, Chloroform-d) δ2.36 (tt, J=8.7,5.4Hz,1H), 1.63 (ddd, J=

[0332] 14.3, 8.7, 5.5Hz, 2H), 1.49 (dq, J=13.5, 6.6Hz, 2H), 1.29 (d, J=10.1Hz, 26H), 0.90 (t, J=6.6Hz, 6H).

[0333] Intermediate E:

[0334]

[0335] Intermediate E is obtained by the following synthetic process:

[0336]

[0337] The compound tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1) were dissolved in EtOH (150 mL), reacted at 45°C for 30 h under nitrogen protection, and then concentrated under reduced pressure to remove EtOH. 100 mL × 3 of (PE:EA=20:1) solvent was added for slurrying, and filtered to obtain a white solid compound intermediate E (11.5 g). 1HNMR (400 MHz, CDCl3) δ3.60 (s, 6H), 2.84 (t, J=5.9 Hz, 2H), 2.47 (t, J=5.8 Hz, 2H), 1.48 (s, 9H).

[0338] Intermediate F:

[0339]

[0340] Intermediate F is obtained by the following synthetic process:

[0341]

[0342] To a single-mouth bottle, add tert-butyl (2-aminoethyl)carbamate (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), K2CO3 (11.65 g), react at 80°C overnight, add water and ethyl acetate, stir and separate, 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 dioxane hydrochloride solution (30 mL) to compound 2, stir at room temperature for 3 h, and concentrate under reduced pressure to obtain compound 3, i.e., intermediate F (6 g). LCMS (ESI) calculation for C 20 H 28 N2O2,[M+H] + m / z 329.22, found 329.24.

[0343] Intermediate G:

[0344]

[0345] Intermediate G is obtained by the following synthetic process:

[0346]

[0347] Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropane bromide (9.45 g), and K2CO3 (12.94 g) were added to a single-mouth bottle. After reacting at 80°C overnight, water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated and concentrated, and purified by column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (6.9 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.9 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate G (7.5 g). LCMS (ESI) calculation for C 22 H 32 N2O2,[M+H] + m / z 357.25, found 357.51.

[0348] Intermediate H:

[0349]

[0350] Intermediate H is obtained by the following synthetic process:

[0351]

[0352] Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl ether benzyl ester (5.02 g), K2CO3 (6.47 g) were added to a single-mouth bottle, and reacted at 80°C overnight. Water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated and concentrated, and purified by column chromatography (PE:EA=20:1-10:1) to obtain compound 2 (4.1 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (4.1 g.), stirred at room temperature for 3 h, and concentrated under reduced pressure to obtain intermediate H (4.9 g). LCMS (ESI) calculation for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56.

[0353] Intermediate I:

[0354]

[0355] Intermediate I is obtained by the following synthetic process:

[0356]

[0357] Compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), K2CO3 (15.46 g) were added to a single-mouth bottle, and reacted at 80°C overnight. Water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated and concentrated, and purified by column chromatography (PE:EA=20:1-10:1) to obtain compound 2 (6.8 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.8 g), stirred at room temperature for 3 h, and concentrated under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calculation for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0358] Intermediate J:

[0359]

[0360] Intermediate J is obtained by the following synthetic process:

[0361]

[0362] In a single-mouth bottle, add butyric acid (5.0 g), THF (100 mL), add NaH (2.73 g) at 0°C, slowly add LDA (56.8 mL), react at room temperature for 30 min, add 1-bromotridecane, continue to react at room temperature overnight, add ice water and ethyl acetate, stir and separate, wash the organic phase with saturated brine, separate the organic phase, concentrate, and purify by column chromatography (PE:EA=10:1-5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calculation for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0363] Intermediate K:

[0364]

[0365] Intermediate K is obtained by the following synthetic process:

[0366]

[0367] Add reactant 1 (24 g), Imidazole (19.00 g) and solvent DCM (200 mL) to a three-necked flask, cool to 0°C, slowly add reactant TBDMSCI (38.57 g), stir at 25°C for 4 h, dilute with 300 mL DCM, wash the organic phase twice with 1 L water and dry with anhydrous Na2SO4, filter and concentrate, add silica gel and pass through a column with 1:20 DCM:MeOH, collect and concentrate to obtain intermediate K (28.00 g). 1HNMR (400 MHz, CDCl3) δ4.89 (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).

[0368] Intermediate L:

[0369]

[0370] Intermediate L is obtained by the following synthetic process:

[0371]

[0372] Add compound DMSO (3.58 g) and anhydrous solvent DCM (30 mL) to a three-necked flask, cool the system to -78°C, then slowly add oxalyl chloride (2.91 g), stir the reaction solution at -78°C under N2 protection for 10 minutes, slowly drop compound 1 (3 g), continue stirring at -78°C under N2 protection for 1 hour, finally drop TEA (9.28 g), continue stirring at -78°C under N2 protection for 0.5 hour, dilute with 100 mL DCM, and wash with saturated NH4Cl solution and water, respectively. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated to obtain intermediate L (3.0 g). LCMS (ESI) calculated for C 11 H 14 O3, [M+H] + m / z 195.09, found 195.23.

[0373] Intermediate M:

[0374]

[0375] The intermediate M is obtained by the following synthetic process:

[0376]

[0377] Add intermediate K (2.0 g), compound 1 (1.79 g) and solvent ACN (20 mL) to the sealed tube, add reactants K2CO3 (3.81 g) and KI (1.52 g), stir overnight at 70°C under N2 protection, dilute with 100 mL EA, wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, add silica gel and pass through a column with 15:1 DCM:MeOH, collect the sample and concentrate to obtain compound 2 (2.0 g). Reactant 2 (1.80 g) and solvent DCM (20 mL) were added to a three-necked flask, the system was replaced with nitrogen and the temperature was lowered to -78°C, then DIBAL-H (1.00 g) was added dropwise, and after stirring at -78°C for 4 h, methanol and sodium carbonate solution were added dropwise to the reaction solution to quench, and then extracted with DCM (100 mL), the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain intermediate M (1.36 g). LCMS (ESI) calculated for C 15 H 33 NO2Si,[M+H] + m / z 288.52, found 288.23.

[0378] C. Compound 1003 was synthesized according to general synthetic route 1

[0379] Structural formula:

[0380] Chemical formula: C 63 H 122 N2O7

[0381] Molecular weight: 1019.68

[0382] Step 1: Synthesis of compound 3 in general synthetic route 1

[0383]

[0384] Compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) were added to compound 2 (intermediate B) (4.3 g). The mixture was stirred at room temperature for 12 h under nitrogen protection. Water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated. The organic phase was purified by silica gel column chromatography (PE:EA=40:1-30:1) to give compound 3 (2.6 g).

[0385] Step 2: Synthesis of compound 4 in general synthetic route 1

[0386]

[0387] DCM (15 mL) and TFA (5 mL) were added to compound 3 (2.6 g). The mixture was stirred at room temperature for 3 h, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to give compound 4 (3.2 g).

[0388] Step 3: Synthesis of compound 6 in general synthetic route 1

[0389]

[0390] DCM (20 mL), Boc-glycine (compound 5, 578 mg), and DCC (6.8 g) were added to compound 4 (3.2 g). The mixture was stirred at room temperature under nitrogen for 12 h, and then water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA=5:1-2:1) to obtain compound 6 (2.2 g).

[0391] Step 4: Compound 1003

[0392]

[0393] DCM (15 mL) and TFA (5 mL) were added to compound 6 (2.2 g), and the mixture was stirred at room temperature for 12 h and concentrated. The mixture was purified by column chromatography (DCM:MeOH=30:1-20:1) to give compound 1003 (1.3 g). 1 H NMR (400MHz, CDCl3) δ4.49 (d, J=2.6Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J=8.6, 5. 5Hz, 3H), 1.62 (q, J = 7.1Hz, 12H), 1.27 (d, J = 3.4Hz, 78H), 0.94-0.86 (m, 18H).

[0394] D. Compound 1002 was synthesized according to general synthetic route 1

[0395] Structural formula:

[0396] Chemical formula: C 60 H 116 N2O7

[0397] Molecular weight: 977.60

[0398] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that intermediate C was used as compound 2 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5Hz, 3H), 1 .64(d,J=6.9Hz,6H),1.50-1.42(m,6H),1.27(s,72H),0.90(t,J=6.7Hz,18H).

[0399] E. Compound 1004, synthesized according to general synthetic route 1

[0400] Structural formula:

[0401] Chemical formula: C 54 H 104 N2O7

[0402] Molecular weight: 893.43

[0403] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that palmitic acid was used as compound 2 in the general synthetic route 1. 1 H NMR (400MHz, CDCl3) δ7.55 (s, 1H), 4.45 (s, 6H), 3.74 (s, 2H), 2.35 (t, J = 7.6Hz, 6H), 1.60 (p, J = 6.9Hz, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7Hz, 9H).

[0404] F. Compound 1001, synthesized according to general synthetic route 1

[0405] Structural formula:

[0406] Chemical formula: C 59 H 108 N2O7

[0407] Molecular weight: 957.52

[0408] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that (9Z)-9-hexadecenoic acid was used as compound 2 in the general synthetic route 1, and 5-(N,N-dimethylamino)pentanoic acid was used as compound 5 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ5.42-5.30(m,6H),4.51-4.38(m,6H),3.19(dt,J=11.3,5 .6Hz,2H),2.97(t,J=5.9Hz,6H),2.46(t,J=6.6Hz,1H),2.35(td,J=7.6,3.1Hz, 5H),2.20(t,J=7.6Hz,2H),2.03(d,J=6.2Hz,12H),1.89-1.81(m,3H),1.74(q, J=7.8Hz,2H),1.67-1.51(m,6H),1.31(d,J=7.8Hz,48H),0.90(t,J=6.7Hz,9H).

[0409] G. Compound 1014, synthesized according to general synthetic route 2

[0410] Structural formula:

[0411] Chemical formula: C 70 H 137 N3O9

[0412] Molecular weight: 1164.88

[0413] Step 1: Synthesis of Intermediate 3 of General Synthesis Route 2

[0414]

[0415] Intermediate 3 was synthesized according to the method shown in step 1 of general synthetic route 1, except that intermediate E was used as compound 1 in general synthetic route 1.

[0416] Step 2: Synthesis of compound 4 of general synthetic route 2

[0417]

[0418] DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added to compound 3 (4.8 g). After reacting at room temperature for 12 h, water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine, separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH=20:1-10:1) to obtain compound 4 (3.3 g).

[0419] Step 3: Synthesis of compound 6 of general synthetic route 2

[0420]

[0421] 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 for stirring and separation. The organic phase was washed with saturated brine, separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH=30:1-20:1) to obtain compound 6 (4.0 g).

[0422] Step 4: Compound 1014

[0423]

[0424] MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added to compound 6 (4.0 g). The mixture was stirred at room temperature overnight under hydrogen atmosphere, filtered through celite, concentrated, and purified by silica gel column chromatography (DCM: MeOH 30:1-

[0425] 15:1) to give compound 1014 (2 g). 1 H(400MHz, CDCl3)δ7.38(t,J=5.8Hz,1H),4.14-4.05(m,6H),3.58(t,J=4.9Hz,4H),3.31(q,J=5.7Hz,2H),2.89(t,J=6.3Hz, 2H), 2.64 (q, J=5.2Hz, 6H), 2.29 (dq, J=10.8, 4.4Hz, 6H), 1.66-1.37 (m, 12H), 1.24 (d, J=4.3Hz, 78H), 0.87 (t, J=7.1Hz, 18H).

[0426] H. Compound 1015, synthesized according to general synthetic route 2

[0427] Structural formula:

[0428] Chemical formula: C 72 H 141 N3O9

[0429] Molecular weight: 1192.93

[0430] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate G was used as compound 5 in general synthetic route 2. 1H(400MHz, CDCl3)δ7.58(t,J=5.7Hz,1H),4.15-4.01(m,6H),3.70(t,J=5.4Hz,4H),3.39(q,J=5.7Hz,2H),2.86(t,J=6.2Hz,2H),2 .57(dt,J=20.5,5.8Hz,6H),2.36-2.22(m,5H),1.76-1.67(m,4H),1.62-1.35(m,13H),1.23(d,J=4.6Hz,78H),0.92-0.80(m,18H).

[0431] I. Compound 1020, synthesized according to general synthetic route 2

[0432] Structural formula:

[0433] Chemical formula: C 74 H 145 N3O9

[0434] Molecular weight: 1220.99

[0435] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate H was used as compound 5 in general synthetic route 2.

[0436] J. Compound 1025, synthesized according to general synthetic route 2

[0437] Structural formula:

[0438] Chemical formula: C 69 H 135 N3O8

[0439] Molecular weight: 1134.85

[0440] The synthesis was carried out according to the general synthetic route 2, which was similar to the synthetic process of compound 1014, except that intermediate I was used as compound 5 in the general synthetic route 2. 1 H(400MHz, CDCl3)δ6.98(t,J=5.7Hz,1H),4.10(m,J=3.8Hz,6H),3.63(t,J=5.2Hz,2H),3.35(q,J=5.8Hz,2H),2.89(t,J=6.1Hz,2 H), 2.57 (q, J = 5.3Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1Hz, 5H), 1.65-1.41 (m, 13H), 1.24 (d, J = 4.4Hz, 78H), 0.87 (t, J = 7.1Hz, 18H).

[0441] K. Compound 1005, synthesized according to general synthetic route 2

[0442] Structural formula:

[0443] Chemical formula: C 68 H 133 N3O7

[0444] Molecular weight: 1104.83

[0445] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-dimethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.19-4.07(m,6H),3.40(d,J=5.7Hz,2H),2.95-2.87(m,2H),2.82(s,4H),2.64-2.55(m,2H),2.38 (s, 6H), 2.33-2.29 (m, 3H), 1.63-1.55 (m, 6H), 1.49-1.44 (m, 2H), 1.26 (d, J = 5.4Hz, 78H), 0.89 (td, J = 7.0, 2.8Hz, 18H).

[0446] L. Compound 1006, synthesized according to general synthetic route 2

[0447] Structural formula:

[0448] Chemical formula: C 53 H 103 N3O7

[0449] Molecular weight: 894.42

[0450] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that tetradecanoic acid was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 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).

[0451] M. Compound 1007, synthesized according to general synthetic route 2

[0452] Structural formula:

[0453] Chemical formula: C 68 H 133 N3O7

[0454] Molecular weight: 1104.83

[0455] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate D was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 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 = 3.4Hz, 78H), 0.89 (td, J = 6.7, 2.5Hz, 18H).

[0456] N. Compound 1008, synthesized according to general synthetic route 2

[0457] Structural formula:

[0458] Chemical formula: C 68 H 133 N3O7

[0459] Molecular weight: 1104.01

[0460] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1HNMR(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).

[0461] O. Compound 1009, synthesized according to general synthetic route 2

[0462] Structural formula:

[0463] Chemical formula: C 62 H 121 N3O7

[0464] Molecular weight: 1020.66

[0465] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate J was used as compound 2 in the general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H(400MHz, CDCl3)δ7.04(t,J=5.0Hz,1H),4.12(m,J=3.3Hz,6H),3.30(q,J=5.6Hz,2H),2.91(t,J=6.0Hz,2H),2.39 (t,J=6.0Hz,2H),2.33-2.25(m,5H),2.22(s,6H),1.76-1.54(m,12H),1.24(m,J=3.7Hz,66H),0.90-0.84(m,18H).

[0466] P. Compound 1011, synthesized according to general synthetic route 2

[0467] Structural formula:

[0468] Chemical formula: C 70 H 137 N3O7

[0469] Molecular weight: 1132.88

[0470] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.19-4.08(m,6H),3.31(q,J=5.8Hz,2H),2.92(t,2H),2.57(t,J=6.9Hz,6H),2.37-2.26( m,5H),1.62(ddd,J=18.3,9.0,4.3Hz,12H),1.27(d,J=4.7Hz,78H),1.04(t,J=7.1Hz,6H),0.94-0.86(m,18H).

[0471] Q. Compound 1012, synthesized according to general synthetic route 2

[0472] Structural formula:

[0473] Chemical formula: C 72 H 141 N3O7

[0474] Molecular weight: 1160.93

[0475] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ6.59 (s, 1H), 4.18-4.07 (m, 6H), 3.29 (q, J = 5.7Hz, 2H), 2.91 (t, J = 6.2Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5Hz, 4H), 2.35-2.27 (m, 5H), 1.63-1.42 (m, 16H), 1.27 (m, J = 4.5Hz, 78H), 0.93-0.85 (m, 24H).

[0476] R. Compound 1013, synthesized according to general synthetic route 2

[0477] Structural formula:

[0478] Chemical formula: C 74 H 145 N3O7

[0479] Molecular weight: 1188.99

[0480] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.10(d,J=4.1Hz,6H),3.26(q,J=5.8Hz,2H),2.89(t,J=6.2Hz,2H),2.52(d,J=6.6Hz,2H) ,2.41(t,J=7.4Hz,4H),2.33-2.25(m,5H),1.60(d,J=11.8Hz,16H),1.24(d,J=4.5Hz,82H),0.96-0.79(m,24H).

[0481] S. Compound 1029, synthesized according to general synthetic route 2

[0482] Structural formula:

[0483] Chemical formula: C 68 H 132 N2O8

[0484] Molecular weight: 1105.81

[0485] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethyl-2-hydroxyethylamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.16(t,J=5.8Hz,2H),4.13-4.05(m,6H),2.86(t,J=6.3Hz,2H),2.55(t,J=5.8Hz,2H) ,2.44(t,J=6.3Hz,2H),2.35-2.20(m,9H),1.60-1.39(m,11H),1.24(d,J=4.5Hz,80H),1.02-0.79(m,18H).

[0486] T. Compound 1111, synthesized according to general synthetic route 2

[0487] Structural formula:

[0488] Chemical formula: C 70 H 136 N4O7

[0489] Molecular weight: 1145.88

[0490] The product was synthesized according to general synthetic route 2, which was similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate was used as compound 5 in general synthetic route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97-2.80 (m, 5H), 2.70 (s, 5H), 2.46-2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54-1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0491] U. Compound 1118, synthesized according to general synthetic route 2

[0492] Structural formula:

[0493] Chemical formula: C 71 H 139 N3O8

[0494] Molecular weight: 1162.91

[0495] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and intermediate K was used as compound 5 in general synthetic route 2. 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.61(s,2H),3.42(d,J=33.5Hz,2H),2.91(t,J=6.2Hz,3H ), 2.36 (dd, J = 9.2, 3.6Hz, 6H), 1.47 (dd, J = 13.9, 6.2Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7Hz, 18H).

[0496] V. Compound 1010, synthesized according to general synthetic route 3

[0497] Structural formula:

[0498] Chemical formula: C 67 H 133 N3O6

[0499] Molecular weight: 1076.82

[0500] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 3

[0501]

[0502] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0503] Step 3: Synthesis of compound 6 of general synthetic route 3

[0504]

[0505] Compound 4 (600 mg), chloroacetaldehyde (compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) were added to DCE (60 mL). After reacting at room temperature overnight, water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine, separated, concentrated, and subjected to silica gel column chromatography (DCM:MeOH=30:1-15:1) to obtain compound 6 (270 mg).

[0506] Step 4: Synthesis of Compound 1010

[0507]

[0508] MeCN (20 mL), N,N-dimethylethylenediamine (compound 7, 229 mg), KI (43 mg), K2CO3 (179 mg) were added to compound 6 (270 mg), and the mixture was reacted at 70°C overnight under nitrogen protection, and then silica gel column chromatography was performed to obtain compound 1010 (16 mg). 1H (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).

[0509] W. Compound 1059, synthesized according to general synthetic route 3

[0510] Structural formula:

[0511] Chemical formula: C 67 H 132 N2O7

[0512] Molecular weight: 1077.80

[0513] The synthesis was carried out according to the general synthetic route 3, which was similar to the synthetic process of compound 1010, except that intermediate C and intermediate L were used as compound 2 and compound 5 in the general synthetic route 3, respectively. In addition, compound 6 in the general synthetic route 3 was required to undergo functional group transformation through the following process steps:

[0514]

[0515] Solvent MeOH (5 mL), THF (5 mL) were added to the intermediate 6a (1.5 g), and reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g) were added. After reacting overnight at 25°C under H2 conditions, the mixture was filtered through diatomaceous earth, the filtrate was concentrated, and silica gel column chromatography (PE:EA 30:1-10:1) was performed to obtain the intermediate 6b (520 mg). DCM (5 mL) was added to the intermediate 6b (470 mg), and SOCl2 (1064.26 mg, 8.946 mmol) was added dropwise at 0°C. After reacting at room temperature for 3 hours, the reaction solution was extracted with water and DCM, the organic phase was concentrated, and silica gel column chromatography (PE:EA 20:1) was performed to obtain the intermediate 6c (181 mg). Dimethylamine (143.39 mg) and DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg) were added to intermediate 6c (170 mg). The reaction solution was stirred at 70°C, filtered, concentrated, and purified by silica gel column chromatography (DCM:MeOH 20:1) to give compound 1059 (91 mg, yield 53.22%). 1H NMR(400M Hz,Chloroform-d)δ4.11(s,6H),3.51(dt,J=18.3,5.6Hz,4H),2.81(dd,J=10.3,5.1Hz,2H),2.50(t,J=6.0Hz,2 H),2.42-2.32(m,3H),2.28(s,6H),2.08-1.98(m,1H),1.53-1.38(m,7H),1.27(s,77H),0.90(t,J=6.7Hz,18H).

[0516] X. Compound 1112, synthesized according to general synthetic route 3

[0517] Structural formula:

[0518] Chemical formula: C 69 H 136 N2O6

[0519] Molecular weight: 1089.85

[0520] The compound was synthesized according to the general synthetic route 3, which is similar to the synthetic process of compound 1010, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in the general synthetic route 3, respectively. 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).

[0521] Y. Compound 1113, synthesized according to general synthetic route 4

[0522] Structural formula:

[0523] Chemical formula: C 71 H 138 N2O8

[0524] Molecular weight: 1147.89

[0525] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 4

[0526]

[0527] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0528] Step 3: Synthesis of compound 6 of general synthetic route 4

[0529]

[0530] DIEA (0.30 g) and solvent DCM (20 mL) were added to compound 4. After cooling to 0°C under nitrogen protection, 5-bromovaleryl chloride (compound 5, 0.27 g) was added, and the mixture was stirred at 25°C for 2 h. After dilution with 100 mL DCM, the organic phase was washed twice with 100 mL water. The organic phase was dried over anhydrous Na2SO4, filtered and concentrated, and then chromatographed on a silica gel column (PE:EA 20:1) to obtain compound 6 (800 mg).

[0531] Step 4: Synthesis of Compound 8 of General Synthesis Route 4

[0532]

[0533] Compound 6 (750.00 mg), intermediate K (compound 7, 217.31 mg) and solvent DMF (20 mL) were added to a three-necked flask, and reactants K2CO3 (184.16 mg) and KI (110.60 mg) were added and 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 EA, and then the organic phase was washed twice with 300 mL saturated brine; the organic phase was dried over anhydrous Na2SO4, filtered and concentrated, and then silica gel was added and passed through a column with 20:1 DCM:MeOH, and the sample was collected and concentrated to obtain compound 8 (700.00 mg).

[0534] Step 5: Synthesis of compound 1113

[0535]

[0536] Compound 8 (700.00 mg) and solvent THF (5 mL) were added to a three-necked flask, and a 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 EA, and then the organic phase was washed with a saturated NaHCO3 solution and water, and then dried over anhydrous Na2SO4, filtered and concentrated, and then chromatographed on a silica gel column (MeOH: DCM 1:20) to obtain compound 1113 (201.1 mg, yield 31.59%). 1 H NMR (400MHz, CDCl3) δ4.50-4.39(m,6H),3.59(t,J=4.6Hz,2H),2.49(s,3H),2.35-2.26(m,6H),2.14 (t,J=7.5Hz,2H),1.75-1.69(m,4H),1.66-1.43(m,17H),1.27(d,J=3.7Hz,78H),0.94-0.87(m,18H).

[0537] Z. Compound 1115, synthesized according to general synthetic route 4

[0538] Structural formula:

[0539] Chemical formula: C 67 H 131 N3O7

[0540] Molecular weight: 1090.80

[0541] The compound was synthesized according to general synthetic route 4, which is similar to the synthetic process of compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine were used to synthesize compound 5 and compound 7 in general synthetic route 4, respectively. 1 HNMR (400

[0542] MHz, 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=36.3,14.1,7.3Hz,12H),1.27(d,J=3.3Hz,78H),0.90(td,J=7.2,3.4Hz,18H).

[0543] AA. Compound 1114, synthesized according to general synthetic route 5

[0544] Structural formula:

[0545] Chemical formula: C 70 H 138 N2O7

[0546] Molecular weight: 1119.88

[0547] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 5

[0548]

[0549] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0550] Step 3: Synthesis of compound 6 of general synthetic route 5

[0551]

[0552] Compound 4 (500 mg), intermediate M (compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) were added to a three-necked flask. The reaction solution was stirred under N2 at 25°C for 16 h, then 100 mL of water was added to the reaction solution. The mixture was extracted twice with EA (100 mL). The organic phases were combined and concentrated. Silica gel was added and the mixture was filtered through a column with 1:20 DCM:MeOH. The sample was collected and concentrated to obtain compound 6 (500 mg).

[0553] Step 4: Synthesis of compound 1114

[0554]

[0555] Compound 6 (450 mg) and solvent THF (5 mL) were added to a three-necked flask, and a hydrochloric acid dioxane solution (1 M) (3.65 mL) was added. After stirring at 25° C. for 2 h, the mixture was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO 3 solution and water. The organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated, and then chromatographed on a silica gel column (MeOH: DCM 1:20) to obtain compound 1114 (107 mg, yield 24.84%). 1 H NMR (400MHz, CDCl3) δ4.16-4.07(m,6H),3.60(t,J=4.8Hz,2H),2.62(t,J=7.0Hz,2H),2.49(s,3H),2.31(tt, J=8.4,5.5Hz,6H),1.70(d,J=4.6Hz,3H),1.64-1.39(m,18H),1.27(d,J=4.0Hz,78H),0.90(t,J=7.1Hz,18H).

[0556] Z. Compound 1120, synthesized according to general synthetic route 5

[0557] Structural formula:

[0558] Chemical formula: C 72 H 142 N2O7

[0559] Molecular weight: 1147.90

[0560] The product was synthesized according to general synthetic route 5, which was similar to the synthetic process of compound 1114, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in general synthetic route 5, respectively. 1H NMR (400 MHz, Chloroform-d) δ4.11 (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).

[0561] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0562] 1. Preparation of Compound 6001

[0563] Structural formula:

[0564] Molecular weight: 499.43

[0565] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, and the mixture was heated to 70°C with stirring. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added, and the reaction was continued for 16 h. The mixture was passed through a reverse column to obtain compound 6002.

[0566] 0.7 g of compound 6002, 0.4 g of palladium on carbon and 28 mL of tetrahydrofuran were added to a reaction bottle and stirred. The gas was replaced with hydrogen and the pressure was maintained with a hydrogen balloon. After 16 h, the mixture was filtered and passed through a column (methanol-DCM system) in the forward direction to obtain 200 mg of compound 6001. 1 H NMR (400MHz, CDCl3) δ7.54(t,J=5.5Hz,2H),3.79-3.68(m,4H),3.42(dd,J=10.0,5.4Hz,4H),2.89(t,J =6.0Hz, 4H), 2.55 (dt, J = 11.8, 6.8Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2Hz, 32H), 0.91 (t, J = 6.8Hz, 3H).

[0567] 2. Preparation of Compound 6002

[0568] Structural formula:

[0569] Molecular weight: 497.42

[0570] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. After the reaction was continued for 16 h, the organic solvent was concentrated, the mixture was passed through a reverse column and freeze-dried to obtain 400 mg of compound 6002. 1 H NMR(400MHz, CDCl3) δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79-3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2. 82-2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10-1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0571] 3. Preparation of Compound 6003

[0572] Structural formula:

[0573] Molecular weight: 445.34

[0574] 4.00 g of tetradecylamine, 6.53 g of hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70°C. After 29 h, the organic solvent was concentrated, passed through a reverse column and freeze-dried to obtain 1.5 g of compound 6003. 1 H NMR(400MHz, CDCl3)δ4.29(t,J=7.5Hz,4H),3.89-3.75(m,4H),2.81(t,J=6.2Hz,4H), 2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0575] 4. Preparation of Compound 6004

[0576] Structural formula:

[0577] Molecular weight: 553.48

[0578] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol were added to the reaction flask, stirred and heated to 100°C. After 4 hours, 1 mL of n-butyl acrylate was added, and the reaction was continued for 1 hour. Then, the mixture was passed through a normal phase column (petroleum ether-ethyl acetate system), and 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added, and stirred for hydrolysis for 30 minutes. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to obtain a mixed solution, and the pH was adjusted to neutral. The solution was spin-dried, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was spin-dried. 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT and 2.3 g of EDCI were added, and the reaction was carried out at room temperature for 18 hours. After concentrating the organic solvent, the mixture was passed through a reverse phase column and freeze-dried to obtain 240 mg of compound 6004. 1 HNMR (400MHz, CDCl3) δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17( s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0579] 5. Preparation of Compound 6005

[0580] Structural formula:

[0581] Molecular weight: 597.54

[0582] 25.00 g of 11-henicosone, 62.05 g of ammonium acetate and 500 mL of methanol were added to a reaction flask and stirred. 6.55 g of sodium cyanoborohydride was added. After reacting for 16 h, 250 mL of water and 250 mL of DCM were added, the liquids were separated, the aqueous phase was extracted with 50 mL of DCM, and the organic phases were combined and passed through a normal phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0583] 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex were stirred in a reaction bottle and heated to 100° C. 10 mL of n-butyl acrylate was added and after 16 hours, the mixture was passed through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0584] A solution prepared by mixing 31.00 g of compound 6005-B and 6.55 g of sodium hydroxide, 310 mL of methanol and 31 mL of water was added to a reaction flask and stirred. After 20 minutes, 150 mL of THF was added, and the temperature was raised to 50°C with stirring. After 30 minutes, 16.13 g of concentrated hydrochloric acid and 160 mL of methanol were added and stirred. After 30 minutes, the solvent was dried by spin drying, 300 mL of DCM was added to dissolve, the mixture was dried over anhydrous magnesium sulfate, filtered and dried to obtain 27.00 g of oily compound 6005-C.

[0585] 27.00 g of compound 6005-C, 25.00 g of 4-amino-1-butanol, 41.85 g of EDCI, 14.74 g of HOBT and 310 mL of DCM were added to a reaction flask and stirred. After 18 hours, the organic solvent was concentrated and passed through a column (methanol-DCM system) to remove excess 4-amino-1-butanol compound to obtain 6.80 g of compound 6005. 1 HNMR (400MHz, CDCl3) δ3.70(t,J=5.6Hz,4H),3.28(t,J=5.6Hz,4H),2.74(t,J=6.0Hz,4H),2.40- 2.45(m,1H),2.34(t,J=6.4Hz,4H),1.63-1.65(m,8H),1.25-1.35(m,36H),0.91(t,J=6.4Hz,6H).

[0586] 6. Preparation of Compound 6006

[0587] Structural formula:

[0588] Molecular weight: 553.48

[0589] Add 1.00g oleylamine, 0.47g hydroxyethyl acrylamide and 8mL ethanol to the reaction bottle, stir and heat to 70°C. After 13h, spin dry the solvent and pass through a column (methanol-DCM system) to obtain 800mg of intermediate 1. Add 1.5mL n-butyl acrylate and 5mL n-butanol, stir and heat to 100°C. After 3h, spin dry the solvent and pass through a column (methanol-DCM system). Add 0.4g sodium hydroxide and 10mL methanol, 1mL water to make a solution, stir for 1h, add 1mL concentrated hydrochloric acid and 8mL methanol to make a solution, stir for 30min, spin dry the solvent and add 100mL DCM to dissolve, dry over anhydrous magnesium sulfate, filter,

[0590] The mixture was spin dried, 0.45 g of 6-amino-1-hexanol, 0.27 g of HOBT, 0.78 g of EDCI and 10 mL of DCM were added and stirred. After 14 h, the organic solvent was concentrated and passed through a column (methanol-DCM system) to obtain 300 mg of compound 6006. 1 HNMR(400MHz, CDCl3)δ7.83(s,1H),6.95(s,1H),5.43-5.28(m,2H),3.74-3 .67(m,2H),3.64(t,J=6.3Hz,2H),3.39(dd,J=10.0,5.3Hz,2H),3.24(dd,J= 12.9,6.7Hz,2H),2.74(t,J=5.8Hz,4H),2.48-2.42(m,2H),2.41-2.33(m,4 H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66-1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0591] 7. Compound 6007

[0592] Structural formula:

[0593] Molecular weight: 953.78

[0594]

[0595] At room temperature, add compound octadecylamine (50 g) to a three-necked flask, add MeOH (200 mL) and stir to mix, cool to 5 degrees Celsius in an ice-water bath, slowly drop methyl acrylate (31.5 g), stir to mix, return to room temperature, and react at room temperature for 4 hours; 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 keep the 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, compound 3 (2 g) was added to a single-mouth bottle, and MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.5 g) was slowly added and stirred to mix, and the temperature was raised to 60°C and left overnight; the reaction was stopped, and the reaction solution was concentrated under reduced pressure and purified by column chromatography (DCM: MeOH =

[0596] 50:1-20:1) to obtain compound 4 (2.5 g). At room temperature, compound 4 (2.3 g) and ethylenediamine (20 mL) were added to a single-mouth bottle and reacted overnight at room temperature; the reaction was stopped and concentrated under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0597] 8. Compounds 6008-6018

[0598] Synthesis of compound 6009

[0599] Structural formula:

[0600] At room temperature, the methanol (20mL) solution of freshly recrystallized hexadecylamine (0.03mol) was added dropwise to a solution in the methanol (20mL) of stirred methyl methacrylate (6mL) under a nitrogen atmosphere, and the reaction was overnight. The reactant was spin-dried at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. Then a colorless oil was obtained by column chromatography. 1H-NMR (300MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0601] Then, a solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 ml) which was stirred vigorously at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, keeping the temperature not higher than 40°C. Excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was repeatedly recrystallized with chloroform and cyclohexane to obtain a white solid. 1H-NMR (300MHz, CDCl3): 0.88(t,3H), 1.25(s,30H), 1.84(s,4H), 2.38(m,6H), 2.73(m,4H), 2.82(m,4H), 3.29(m,4H), 7.47(s,2H).

[0602] The preparation methods of 6008 and 6009 differ only in that an equimolar amount of tetradecylamine is used to replace hexadecylamine. 6010-6017 refer to the preparation method of 6009, using an equimolar amount of the corresponding R a It is prepared by replacing hexadecylamine with -NH2.

[0603] 6018 was prepared by referring to the method of 6007, using an equimolar amount of the corresponding R a -NH2 is used to replace octadecylamine.

[0604] 9. Compound 6019

[0605] Structural formula:

[0606] Molecular weight: 753.66

[0607]

[0608] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked bottle, DCM (50 mL) was added and stirred, and then 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, stirred, and reacted overnight at room temperature; the mixture was 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 bottle, hydrogen was replaced, the temperature was raised to 50°C, and stirred for 1 h; the reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was dried to obtain product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-mouth bottle, DMF (50 mL) was added and stirred to dissolve, and then product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred to mix, and the mixture was reacted overnight at room temperature; water (500 mL) and EA (500 mL) were added, and the mixture was separated and extracted, and the organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM: MeOH = 20: 1-10: 1) to obtain solid 6 (380 mg). At room temperature, solid 6 (380 mg) and HCl / Dioxane (4M) (1.6 mL) were added to a single-mouth bottle, and the mixture was reacted for 2 h at room temperature; the mixture was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1HNMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H ), 1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0609] 10. Compound 6020

[0610] Structural formula:

[0611] Molecular weight: 810.19

[0612]

[0613] At room temperature, add compound octadecylamine (50 g) to a three-necked flask, add MeOH (200 mL) and stir to mix, cool to 5 degrees Celsius in an ice-water bath, then drop methyl acrylate (31.5 g), stir to mix, return to room temperature, and react for 4 hours; stop the reaction, directly concentrate under reduced pressure, purify by column chromatography (PE: EA = 20: 1-10: 1), and 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 keep the 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, compound 3 (1.3 g) was added to a single-necked bottle, DMF (20 mL) was added and stirred, and then 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g) were added, stirred, and reacted at room temperature overnight; water (200 mL) was added and the mixture was directly freeze-dried under reduced pressure to obtain compound 4 (4 g). Compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) were added to a single-necked bottle at room temperature and reacted for 1 h. The reaction was stopped and the reaction solution was concentrated under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57-3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03-1.58 (m, 10H), 1.31 (s, 30H), 1.01-0.82 (m, 3H).

[0614] 11. Compound 6021

[0615] Structural formula:

[0616] Molecular weight: 1182.79

[0617]

[0618] At room temperature, compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020) was added to a single-necked bottle, MeOH (20 mL) was added and stirred, TREN (23.0 g) was added, the mixture was stirred, the temperature was raised to 60°C, the mixture was stirred overnight, and the mixture was concentrated under reduced pressure. The crude product was lyophilized to obtain product 3 (25 g). At room temperature, product 3 (22.0 g) and DCM (200 mL) were added to a single-necked bottle, the temperature was lowered to 0°C, (Boc)2O (71.7 g) was slowly added dropwise, the temperature was restored to room temperature after the addition was completed, the reaction was carried out for 3 h, water (200 mL) was added for extraction, and DCM (100 mL) was added for separation and extraction. The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column to obtain yellow oil 4 (5.8 g). At room temperature, yellow oil 4 (1.4 g) was added to a single-mouth bottle, HCl / Dioxane (4M) (15 mL) was slowly added under an ice-water bath and stirred and mixed, the temperature was restored to room temperature after addition, the reaction was continued for 0.5 h, and the mixture was concentrated under reduced pressure to obtain compound 5 (800 mg). At room temperature, compound 5 (1.0 g), (S)-2,6-di-tert-butyloxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL) were added to a single-mouth bottle, the reaction was continued overnight at room temperature, 100 mL of water and 100 mL of EA were added, the mixture was separated and extracted, the EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM: MeOH = 10: 1) to obtain compound 6 (1.1 g). Into a single-necked bottle at room temperature, compound 6 (1.2 g), HCl / Dioxane (4M) (10 mL) and solvent DCM (10 mL) were added. The reaction was allowed to react overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6021 (220 mg). 1H NMR(400MHz, Methanol-d4)δ3.96(t,J=6.6Hz,4H),3.75(dt,J=14.0,6.6Hz,4H),3.62-3.45(m,12H),3.36-3.33(m,14H),3.25-3.17(m,2H),3.04 -2.90(m,8H),2.83(t,J=6.9Hz,4H),2.05-1.82(m,8H),1.73(p,J=7.7Hz ,10H),1.53(qd,J=8.3,7.7,4.3Hz,8H),1.30(s,28H),0.97-0.87(m,3H).

[0619] 12. Compound 6023

[0620] Structural formula:

[0621] Molecular weight: 670

[0622]

[0623] At room temperature, compound dodecylamine (10 g) was added to a three-necked flask, MeOH (100 mL) was added and stirred, then cooled to 5°C in an ice-water bath, methyl acrylate (10.2 g) was slowly added dropwise, stirred, and returned to room temperature. The reaction solution was allowed to react at room temperature for 4 hours, then the reaction was stopped, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 20: 1-10: 1) to obtain product 2 (19 g). At room temperature, product 2 (19.0 g), MeOH (200 mL) and ethylenediamine (127.7 g) were added to a single-necked flask, the temperature was raised to 60°C, the temperature was maintained for overnight reaction, and the compound 3 (20 g) was obtained after concentration under reduced pressure. 500 mg was purified and lyophilized to obtain compound 6023-1 (218 mg). At room temperature, add raw material 6023-1 (1.4 g) to a single-mouth bottle, add DCM (20 mL) and stir to dissolve, then add (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), DMAP (1.2 g) and stir to mix, keep at room temperature, react overnight, separate and extract, dry, and concentrate under reduced pressure. Compound 4 (3.0 g) is obtained. At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4M) (20 mL) to a single-mouth bottle, react for 2 h at room temperature; concentrate under reduced pressure, purify, and freeze-dry to obtain compound 6023 (123 mg). 1H NMR(400MHz, Methanol-d4)δ3.88(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.36-3.33(m,2H),3.31-3.14(m,4H),3.04-2.93(m,4H),2. 77(t,J=6.5Hz,4H),2.00-1.82(m,4H),1.82-1.66(m,6H),1.50(qd,J=8.2,7.8,4.2Hz,4H),1.45-1.25(m,18H),0.96-0.87(m,3H).

[0624] 13. Compound 6024

[0625] Structural formula:

[0626] Molecular weight: 870.24

[0627]

[0628] At room temperature, product 6023-1 (1.4 g, the synthesis method is the same as 6023-1 in compound 6023) was added to a single-mouth bottle, MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.8 g) was slowly added and stirred to mix. The temperature was raised to 60°C, and the reaction was carried out overnight at this temperature. The mixture was concentrated under reduced pressure to obtain compound 4 (2.0 g). At room temperature, compound 4 (2.4 g) and ethylenediamine (20 mL) were added to a single-mouth bottle, and the reaction was carried out overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6024 (191 mg). 1HNMR (400MHz, Methanol-d4) δ3.69(t,J=6.0Hz,4H),3.60-3.48(m,20H),3.42(t,J=6.1Hz,4H),3.25-3.19(m,2H),3 .12(t,J=5.8Hz,8H),2.83(t,J=6.5Hz,12H),1.78(tt,J=11.0,6.4Hz,2H),1.51-1.18(m,20H),0.91(t,J=6.7Hz,3H).

[0629] 14. Compound 6026

[0630] Structural formula:

[0631] Molecular weight: 443.67

[0632]

[0633] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, except that an equal molar amount of compound 1 was used to replace the raw material 5 of compound 6019, and an equal molar amount of N-(tert-butyloxycarbonyl)ethanolamine was used to replace the intermediate 4 of compound 6019. 1H NMR (300MHz, 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).

[0634] Compounds 6025-6034 were obtained by referring to the synthetic route of the aforementioned compounds, except that hydroxyethylamine was used instead of the corresponding ethylenediamine, and the amino group of the hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc, and the protecting group was removed by conventional methods after the reaction was completed.

[0635] (3) Exemplary compounds of the first ionizable lipid and their properties

[0636] The exemplary compounds of the present invention and their properties are listed in Table 1, which are synthesized according to the process routes in Preparation Example 1.

[0637] The calculated c-pKa (molnetwork), LogP (cLogP driver) of the exemplary compounds of the present invention, c-pKa and cLogP values ​​were generated by the ChemDraw module of Chemoffice.

[0638] Table 1 Properties of exemplary compounds of the first ionizable lipid

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666] The above properties of the exemplary compounds of the first ionizable lipid of the present invention indicate that they can serve as excellent surfactants and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0667] (4) Preparation of compositions based on blank lipid nanoparticles

[0668] 4.1 Preparation of blank lipid nanoparticles

[0669]

[0670] Blank lipid nanoparticles were prepared according to the above molar ratios, and the specific methods are as follows:

[0671] The lipid raw material is dissolved in ethanol to obtain a lipid ethanol solution, and the total concentration of all lipid raw materials in ethanol is 8 mg / mL. The lipid ethanol solution is mixed with 50 mM citrate buffer saline (pH 4.0) solution in a volume ratio of 1:3 in a nanopreparation device, and then ultrafiltered and collected to obtain blank lipid nanoparticles. The lipid concentration of the composition is 2 mg / mL.

[0672] 4.2 Preparation of blank lipid nanoparticle-based compositions

[0673]

[0674] Nucleic acid is used as an active ingredient, and nuclease-free water is used to prepare the nucleic acid into a solution with a concentration twice that of the concentration in each embodiment as solution 1; nuclease-free water is used to dilute blank lipid nanoparticles according to the prescription ratio of each embodiment to obtain solution 2; solution 1 and solution 2 are mixed in equal volumes, and vortexed for 2-3 seconds to obtain a composition based on blank lipid nanoparticles.

[0675] (5) Particle size and zeta potential measurement of blank lipid nanoparticle compositions

[0676] 1. Particle size and polydispersity index (PDI) determination: Malvern ZetaSizer Nano ZS90 was used to determine the average particle size and PDI of the nanoparticle sample solution in the embodiment by dynamic light scattering. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0677] 2. The encapsulation efficiency test method is as follows:

[0678] The mRNA encapsulation efficiency in the blank lipid nanoparticle-based composition was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions.

[0679] The pDNA encapsulation efficiency in the blank lipid nanoparticle-based composition was determined using a dsDNA HS kit (Novozyme / EQ121) according to the manufacturer's instructions.

[0680] According to the manufacturer's instructions, use microRNA Reagent Kit, to determine the encapsulation efficiency of siRNA in blank lipid nanoparticle-based compositions.

[0681] The average particle size, PDI, and encapsulation efficiency data of the composition based on blank lipid nanoparticles prepared in the example are shown in Table 2.

[0682] Table 2 Particle size, PDI, and encapsulation efficiency of compositions based on blank lipid nanoparticles

[0683] Example No. Particle size nm PDI Encapsulation rate% 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

[0684] As can be seen from Table 2, 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%).

[0685] (6) Evaluation of in vivo delivery effects of compositions based on blank lipid nanoparticles

[0686] 6.1 Evaluation of in vivo delivery effect of blank lipid nanoparticle-mRNA complexes injected intravenously into mice

[0687] Female Balb / c mice aged 6-8 weeks were used to test the in vivo delivery efficiency of the blank lipid nanoparticle-firefly luciferase mRNA complex in Example 5. Blank lipid nanoparticle-Luciferase mRNA complex was administered by tail vein injection, with a single dose of 0.3 mpk. At a specific time point (such as 3h, 6h) after administration, the mice were intraperitoneally injected with fluorescent imaging substrates, and the animals were imaged in vivo using the PerkinElmer small animal imaging system to measure the bioluminescent signal. The results are shown in Tables 3 and Figure 1 .

[0688] Table 3 Evaluation of in vivo activity of blank lipid nanoparticle-mRNA complexes in Example 5

[0689]

[0690] The results show that after the blank lipid nanoparticles provided by the present invention are combined with Luciferase mRNA, a higher luciferase protein expression rate can be achieved in mice.

[0691] Female Balb / c mice aged 6-8 weeks were used to test the in vivo delivery efficiency of blank lipid nanoparticle-human erythropoietin mRNA (hEPO mRNA) complexes in Example 6 and Example 12. Blank lipid nanoparticle-hEPO mRNA complexes were administered by tail vein injection, with single doses of 1.5 mpk and 3 mpk, respectively, and the hEPO expression in the mice was measured at a specific time point (24 h) after administration. The results are shown in Table 4.

[0692] Table 4 hEPO expression (pg / ml) after 24h administration of blank lipid nanoparticle-human erythropoietin mRNA complexes of Examples 6 and 12

[0693]

[0694] It can be seen that after the blank lipid nanoparticles provided by the present invention are combined with human erythropoietin mRNA, a higher hEPO expression rate can be achieved in mice through intravenous injection.

[0695] 6.2 Evaluation of in vivo delivery effect of lipid nanoparticle-mRNA complexes injected intramuscularly into mice

[0696] Female Balb / c mice aged 6-8 weeks were used to test the in vivo delivery efficiency of the lipid nanoparticle-luciferase mRNA complex in Example 10. Blank lipid nanoparticle-luciferase mRNA complex was administered by intramuscular injection, with a single dose of 0.3 mpk. At specific time points (3 h, 6 h) after administration, the mice were intraperitoneally injected with fluorescent imaging substrates. The animals were imaged in vivo using the PerkinElmer small animal imaging system to measure the bioluminescent signal. The results are shown in Tables 5 and Figure 2 .

[0697] Table 5 In vivo activity evaluation after administration of blank lipid nanoparticle-mRNA complex of Example 10 for 3 / 6 hours

[0698]

[0699] The results show that after the blank lipid nanoparticles provided by the present invention are combined with luciferase mRNA, a higher luciferase protein expression rate can be achieved in mice through intramuscular injection.

[0700] 6.3 Intraperitoneal (IP) and subcutaneous (SC) administration

[0701] Female Balb / c mice aged 6-8 weeks were used to test the in vivo delivery efficiency of the lipid nanoparticle-firefly luciferase mRNA complex of Example 16. Blank lipid nanoparticle-Luciferase mRNA complex was administered by intraperitoneal injection and subcutaneous injection, respectively, with a single dose of 0.3 mpk. At specific time points (3h, 6h) after administration, the mice were intraperitoneally injected with fluorescent imaging substrates, and the animals were imaged in vivo using the PerkinElmer small animal imaging system to measure the bioluminescent signal. The results are shown in Tables 6 and Figure 3 .

[0702] Table 6 Example 16 Evaluation of in vivo activity of blank lipid nanoparticle-mRNA complex

[0703]

[0704] The results show that after the blank lipid nanoparticles provided by the present invention are combined with Luciferase mRNA, a high luciferase protein expression rate can be achieved in mice through intraperitoneal injection and subcutaneous injection.

[0705] The solutions of the present invention are not limited to the technical means disclosed by the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific implementation method of the present invention. It should be pointed out that for ordinary technicians in the field of the present invention, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered 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: The method comprises the steps of mixing blank lipid nanoparticles with a biologically active substance in a solvent to obtain a composition based on blank lipid nanoparticles; The blank lipid nanoparticle composition 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, wherein the second ionizable lipid is not 0; The first ionizable lipid is selected from a compound containing the general formula (1E) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof; Wherein, R1, R2, and R3 are independently H, C 5-40 Straight or branched alkyl, C 5-40 Straight-chain or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10-30 Straight or branched alkyl, C 10-30 Straight or branched alkenyl, C 10-30 Straight or branched alkynyl; 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, wherein the heterocyclic group and the heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amine, oxo or halogen; R4 and R5 are independently H, C 1-6 Straight or branched alkyl, C 2-6 Straight or branched alkenyl or C 2-6 A straight chain or branched chain alkynyl group, the C 1-6 Straight or branched alkyl, C 2-6 Straight or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, amine, amide, amidino, guanidino or halogen; G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS (=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 are independently selected from H, hydroxyl, C 1-30 Straight or branched chain alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl; R 9、 R 10 Independently of each other are H; X2 is selected from -O-, -S-, NR 16 -, -SS-, -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-; R 11、 R 12 Independently of each other are H, C 1-6 A straight or branched alkyl or cycloalkyl group; Each R 16 , R 17 are independently selected from H, C 1-30 Straight or branched chain alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl; Among them, R4, R5, R6, R 11 , R 12 , R 13 , R 16 , R 17 The alkyl, cycloalkyl, alkenyl described in is unsubstituted or substituted by one or more groups selected from hydroxyl, thiol, amine, substituted amine, and halogen; n is an integer from 2 to 6; k is an integer from 1 to 6; The salts do not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof: Among them, A1 is NH; R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol, C6-C 24 The cycloalkyl group of C6-C 24 The alkyl group is a branched structure; the C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl alcohol.

2. The use according to claim 1, characterized in that The use further comprises the step of introducing the blank lipid nanoparticle-based composition into an animal.

3. The use according to claim 2, characterized in that Routes for introducing the blank lipid nanoparticle-based composition into an animal include oral administration, intranasal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intraarticular administration, intralesional administration, intratracheal administration, subcutaneous administration or intradermal administration.

4. The use according to claim 2, characterized in that The animals are mammals and non-mammals.

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

6. The use according to claim 5, characterized in that The nucleic acid includes at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA minicircle, and msDNA.

7. The use according to claim 1, characterized in that The dosage of the nucleic acid is 0.1%-50% (w / w) of the total amount of blank lipid nanoparticles and nucleic acid; in the composition based on blank lipid nanoparticles, the concentration of the biologically active substance is 50-5000 ng / μl.

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

9. The use according to claim 1, characterized in that The R1, R2, and R3 are independently selected from the following groups: Wherein, R1' and R2' are independently H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

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

11. The use according to claim 1, characterized in that G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

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

13. The use according to claim 1, characterized in that The compound of formula (1E) is selected from the compound represented by formula (1F):

14. The use according to claim 13, characterized in that The compound of formula (1E) is selected from the compound represented by formula (1G):

15. The use according to claim 13, characterized in that The compound of formula (1E) is selected from the compound represented by formula (1H):

16. The use according to claim 1, characterized in that The compound of formula (1E) is selected from:

17. The use according to claim 1, characterized in that The compound of formula (2) is selected from at least one of the following compounds:

18. The use according to claim 1, characterized in that The blank lipid nanoparticles further include 0-60 mol% of other ionizable lipids, wherein the other ionizable lipids are selected from at least one of the following compounds:

19. The use according to claim 1, characterized in that The phospholipids include 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-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0 -Octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphyton Alkanoyl-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) At least one of sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

20. The use according to claim 1, characterized in that The polyethylene glycol-conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

21. The use according to claim 20, characterized in that The polyethylene glycol conjugated lipid includes at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, dipalmitoylglycerol-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol lipids.

22. The use according to claim 1, characterized in that 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.

23. The use according to claim 22, characterized in that The solvent is at least one of water, an aqueous solution of an organic solvent, and a buffered saline solution.

24. The use according to claim 23, characterized in that 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 of less than 50%.

25. The use according to claim 24, characterized in that The buffered saline solution is selected from at least one of a citrate solution, an acetate solution, a tartrate solution, a phosphate solution, a carbonate solution, a Tris-HCl solution, and a sodium chloride solution.

26. The use according to claim 1, characterized in that At least one of sugar, glycerol, DMSO, salt, antibiotics and surfactant is also added to the composition based on blank lipid nanoparticles.

27. An in vitro gene delivery method, characterized in that: The method comprises the steps of mixing blank lipid nanoparticles with biologically active substances in a solvent to obtain a composition based on blank lipid nanoparticles; the blank lipid nanoparticles are the blank lipid nanoparticles according to claim 1.

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