Use of a prefabricated carrier in the preparation of a product for in vitro gene delivery to immune cells and stem cells

Abstract

The present invention provides the use of a prefabricated vector in the preparation of a product for in vitro gene delivery to immune cells and stem cells, belonging to the field of biomedical technology. The use of the prefabricated vector in the preparation of a product for in vitro gene delivery to immune cells and stem cells includes the step of mixing the prefabricated vector and nucleic acid in a solvent to obtain a composition based on the prefabricated vector; the composition of the prefabricated vector includes: ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-conjugated lipids. The prefabricated vector of the present invention can be used as a vector tool for cell and gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of a prefabricated carrier in the preparation of a product for in vitro gene delivery to immune cells and stem cells. Background Art

[0002] Cell and gene therapies have very broad application prospects and are expected to cure various diseases from the source. Cell therapy refers to obtaining cells with specific functions and endowing these cells with functions such as enhanced immunity, killing pathogens and tumor cells through in vitro operations, and then inputting or implanting them into the human body for disease treatment, including immune cell and stem cell therapies. Gene therapy is to introduce exogenous normal genes into target cells to correct diseases caused by defective and abnormal genes, so that the target genes can be expressed in the cells to treat diseases. Gene therapy can be divided into in vivo and in vitro gene therapies.

[0003] In the field of biomedicine, the research and application of immune cells, stem cells, and nucleic acids have become the forefront and hotspots. Immune cells, such as T cells, B cells, NK cells, etc., play a key role in the human immune system and can recognize and attack foreign pathogens and cancer cells. Stem cells have the ability of self-renewal and multi-directional differentiation and are widely used in regenerative medicine and tissue engineering. Nucleic acids, including DNA and RNA, are carriers of genetic information in organisms and also have the function of regulating gene expression, so they have great potential in the fields of gene therapy and vaccine research and development.

[0004] To deliver biomacromolecules into cells for genome editing, the particles containing these biomacromolecules must first avoid extracellular barriers. These barriers include being phagocytosed by macrophages, or being degraded or hydrolyzed by enzymes, as well as the ability to induce immune responses and produce cytokines. If these biomacromolecules and nanoparticles can avoid being rejected by the body and enter the appropriate organs or bloodstream, they must also enter the target cells. Once inside the cell, the biomacromolecules and nanoparticles must escape from endosomes and localize to the mRNA in the cytoplasm or the nucleus to successfully edit the target gene.

[0005] Currently, cell and gene therapies mainly rely on chemical-mediated methods, physical-mediated methods, and virus-mediated methods to be completed. The chemical-mediated method uses carrier molecules to coat nucleic acids to make them exhibit a neutral or positive charge. For example, the DEAE-dextran method, calcium phosphate method, etc. are used to transfer nucleic acids into cells to achieve gene transfection and expression; the physical-mediated method uses physical means such as microinjection, electroporation, gene gun method, etc. to generate a transient pore on the cell membrane surface to allow nucleic acids to enter the cell; the virus-mediated method uses genetically engineered viruses to transfect non-viral genes into cells, such as retroviruses, adenoviruses, etc. These viruses can integrate nucleic acids into the cell genome to achieve long-term expression. However, the above methods all have their own limitations, such as low delivery efficiency, large cell damage, high cytotoxicity, and high technical difficulty, high operation complexity, poor reproducibility, etc. Therefore, there is an urgent need to develop a new delivery technology to meet the current needs of cell and gene therapies.

[0006] Based on this, the present invention provides the use of a prefabricated vector in the preparation of a product for in vitro gene delivery to immune cells and stem cells. By simply mixing a gene editing drug with the prefabricated vector in a solution state, safe and effective delivery of the gene editing drug can be achieved. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides the use of a prefabricated vector in the preparation of a product for in vitro gene delivery to immune cells and stem cells. The use of the prefabricated vector of the present invention in the preparation of a product for in vitro gene delivery to immune cells and stem cells includes the step of mixing the prefabricated vector with nucleic acids in a solvent to obtain a composition based on the prefabricated vector; the composition of the prefabricated vector includes: ionizable lipid, phospholipid, cholesterol, and polyethylene glycol-conjugated lipid. By using the prefabricated vector of the present invention, the dose of nucleic acids can be flexibly adjusted according to the needs of users, and ideal delivery effects can be obtained through various administration routes.

[0008] To achieve the above object, in the first aspect, the present invention provides the use of a prefabricated vector in the preparation of a product for in vitro gene delivery to immune cells and stem cells, including the step of mixing the prefabricated vector with nucleic acids in a solvent to obtain a composition based on the prefabricated vector; the composition of the prefabricated vector includes: 5-70 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-50 mol% of phospholipid, 10-70 mol% of cholesterol, and 0-15 mol% of polyethylene glycol-conjugated lipid.

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

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

[0011] In a preferred embodiment, the application further comprises the step of contacting the prefabricated carrier-based composition with immune cells or stem cells.

[0012] In a preferred embodiment, an adjuvant is further added to the product for in vitro gene delivery to immune cells and stem cells.

[0013] In a preferred embodiment, the adjuvant comprises at least one of lipids, polypeptides, lipopolyamines or synthetic polymers.

[0014] In a preferred embodiment, the nucleic acid is 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 microcircles, msDNA.

[0016] In a preferred embodiment, the dosage of the nucleic acid is 0.1%-50% (w / w) of the total amount of the prefabricated carrier and the nucleic acid; in the prefabricated carrier-based composition, the concentration of the nucleic acid is 5-1000 ng / μl.

[0017] In a preferred embodiment, the first ionizable lipid is selected from compounds having the general formula (1) or pharmaceutically acceptable salts, stereoisomers, tautomers thereof;

[0018]

[0019] wherein, R1, R2, R3 are independently of each other H, C 5-40 straight-chain or branched-chain alkyl, C 5-40 straight-chain or branched-chain alkenyl, C 5-40 straight-chain or branched-chain alkynyl, 3-6 membered saturated or partially unsaturated cycloalkyl group having 1-3 side chains, or 6-10 membered aromatic group having 1-3 side chains; the side chains are independently selected from C 10-30 straight-chain or branched-chain alkyl, C 10-30 straight-chain or branched-chain alkenyl, C 10-30 straight-chain or branched-chain alkynyl; provided that at most one of R1, R2, R3 is H;

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

[0021] R4 and R5 are each independently H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group, and the C 1-6 straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, amino amide group, amidino group, guanidine group or halogen;

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

[0023] L1 is selected from -X1- or -(CR7R8) m -X1-, where each X1 is independently selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -, -NR 14 -S(=O)2-, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-; where m is an integer from 2 to 6, and R7 and R8 are each independently H, hydroxy, halogen, a linear or branched alkyl or cycloalkyl of C 1-6 , a linear or branched alkenyl of C 2-6 , and each R 14 , R 15 is independently selected from H, a linear or branched alkyl or cycloalkyl of C 1-30 , a linear or branched alkenyl of C 2-30 ;

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

[0025] wherein the alkyl, cycloalkyl, alkenyl as described for R4 to R 17 is unsubstituted or substituted by one or more groups selected from hydroxy, mercapto, amino, substituted amino, halogen;

[0026] The salt does not include quaternary ammonium salts.

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

[0028]

[0029] wherein Y is absent or is a straight-chain or branched-chain alkyl or cycloalkyl of C 1-30 , a straight-chain or branched-chain alkenyl of C 2-20 , or a straight-chain or branched-chain alkynyl of C 2-20 ; R1', R2' are each independently H, a straight-chain or branched-chain alkyl of C 1-30 , a straight-chain or branched-chain alkenyl of C 2-30 , or a straight-chain or branched-chain alkynyl of C 2-30 ; and the total carbon chain length of Y, R1' and R2' is 8 - 40.

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

[0031]

[0032] wherein R1', R2' are each independently H, a straight-chain or branched-chain alkyl of C 1-30 , a straight-chain or branched-chain alkenyl of C 2-30 , or a straight-chain or branched-chain alkynyl of C 2-30 ; and the total carbon chain length of R1' and R2' is 8 - 30.

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

[0034]

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

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

[0037] In a preferred embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR16 )O-.

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

[0039]

[0040] wherein m' and n' are each independently an integer from 0 to 6, and R1" and R2" are each independently H, C 1-6 alkyl, C 2-6 alkenyl, guanidino, amidino, amido, aliphatic amine, 3- to 10-membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and their tautomeric forms, which are unsubstituted or optionally substituted by one or more organic groups selected from hydroxy, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

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

[0042]

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

[0044]

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

[0046]

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

[0048]

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

[0050]

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

[0052]

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

[0054]

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

[0060]

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

[0062]

[0063] In a preferred embodiment, the compound of formula (1) is selected from the compounds 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 a straight-chain or branched-chain alkyl group, C 2-30 a straight-chain or branched-chain alkenyl group, C 2-30 a straight-chain or branched-chain alkynyl group, and the total carbon chain length of R1' and R2' is 8 - 40.

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

[0069]

[0070] In a preferred embodiment, the compounds of formula (1) are selected from:

[0071] The compounds of formula (1) are selected from:

[0072]

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[0148]

[0149] 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):

[0150]

[0151] 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.

[0152] 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):

[0153]

[0154] 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.

[0155] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0156]

[0157] 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.

[0158] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VII) sequentially with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X):

[0159]

[0160] wherein Xg, Xh, Xi, Xj, Xk, Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G1 through a nucleophilic reaction or a condensation reaction, Xh and Xk form G2 through a nucleophilic reaction or a condensation reaction, and Xi and Xl form G3 through a nucleophilic reaction or a condensation reaction.

[0161] 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):

[0162]

[0163] Step of reacting the compound of formula (XIII) with the compound of formula (XII):

[0164]

[0165] wherein Xm is a group containing a nucleophilic group, and Xm reacts with C═C-L of the compound of formula (XII) 2a to form X1-L2 through an addition reaction.

[0166] In a preferred embodiment, R1, R2, and R3 are, independently of one another, the following groups:

[0167]

[0168] wherein the meanings of Y, R1', and R2' are as defined above;

[0169] which further includes the step of forming the tail chains R1, R2, and R3:

[0170]

[0171] wherein X is a leaving group.

[0172] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0173]

[0174] wherein N1 is NH or O;

[0175] R a is selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene; the C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene is of straight-chain or branched-chain structure; R b and R c each independently is selected from C1-C 12 alkyl, C2-C 12 alkenyl, C1-C 12 alkynyl, C3-C 12 cycloalkane group, C6-C 12 aryl group, C1-C12 Alkyl alcohol, C1-C 12 heterocyclic group, alkylamine;

[0176] The alkylamine is wherein, R a ’ is C1-C 12 alkyl group, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamine, R c ” is selected from unsubstituted or amino-substituted C1-C6 alkyl group, and R c ”’ is H, or -R c ’-A1’-R c ”-NH2;

[0177] Provided that when A1’ is -CO-NH-, -NH-CO- or -CO-O-, R c ’ is C1-C6 alkyl group; when A1’ is -CO-, R c ’ does not exist.

[0178] In a preferred embodiment, R b and R c are each independently selected from C1-C 12 alkyl alcohol, alkylamine.

[0179] In a preferred embodiment, the condition is: when R a is C6-C 24 alkenyl group, A1 is NH, and R b and R c are each independently selected from C1-C 12 alkyl alcohol;

[0180] When R a is branched C6-C 24 alkyl group, A1 is NH, and R b and R c are each independently selected from C1-C 12 alkyl alcohol, alkylamine;

[0181] R a is straight-chain C6-C 24 alkyl group, A1 is NH or O, and R b and R c are each independently selected from alkylamine;

[0182] When R a is straight-chain C6-C 24 alkyl group, A1 is O, and R b and R cEach independently selected from C1-C 12 alkyl alcohol;

[0183] When R a is C6-C 24 short-chain polyoxyethylene, A1 is NH or O, R b and R c are each independently selected from alkylamines;

[0184] Or when R a is C6-C 24 alkyl alcohol, A1 is NH or O, R b and R c are each independently selected from alkylamines.

[0185] In a preferred embodiment, R a is selected from the following compound structures:

[0186]

[0187] In a preferred embodiment, R b and R c are selected from the following compound structures:

[0188]

[0189] R d is selected from C1-C6 alkanes or cycloalkanes.

[0190] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0191]

[0192]

[0193]

[0194] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0195] R a ”-NH2① reacts with α,β-unsaturated carbonyl compound ② to form an ionizable lipid compound ③:

[0196]

[0197] wherein, R a ”-NH2 is R a -NH2 or

[0198] R b’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0199] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0200] 1) R a ”-NH2① reacts with an α,β-unsaturated carbonyl compound ④ to form a compound ⑤;

[0201] 2) The compound ⑤ reacts with a nucleophile ⑥ to form an ionizable lipid compound ③;

[0202]

[0203] wherein the nucleophile ⑥ is R b -NH2 or R b -OH; R a ”-NH2 is selected from R a -NH2 or

[0204]

[0205] R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0206] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0207] 1) R a ”-NH2① reacts with an α,β-unsaturated carbonyl compound ② to form a compound ⑦;

[0208] 2) The compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form a compound ⑨;

[0209] 3) The compound ⑨ reacts with a nucleophile ⑩ to form an ionizable lipid compound

[0210]

[0211] Among them, R a ”-NH2 is R a -NH2, Z3 is a leaving group, and A3 reacts with Z3 to obtain A1.

[0212] In a preferred embodiment, in Formula 2a-Formula 2c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to form the ionizable lipid compound with two branches containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0213] In a preferred embodiment, Formula 2a-Formula 2b further includes the step of performing an iterative reaction using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a-Formula 2b as a raw material according to Step 1 in Formula 2a or Steps 1-2 in Formula 2b;

[0214] The compound of Formula (2) with a terminal amino group is

[0215] Among them, R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1-C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2.

[0216] In a preferred embodiment, the reaction formula of Formula 2a-Formula 2c further includes the step of reacting with the compound of Formula (2) with a terminal amino group obtained by the reaction formula of Formula 2a-Formula 2c as a raw material ;

[0217] The compound of Formula (2) with a terminal amino group is

[0218] Among them, R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1-C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2.

[0219] The leaving group described above refers to the leaving part in a nucleophilic reaction or a condensation reaction, including but not limited to: H,

[0220] OH, H2O, halogens (such as F, Cl, Br, and I), cyanate anions, inorganic acids (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acids (such as acetic acid, trifluoroacetic acid, and benzoic acid, etc.), sulfonic acids (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy groups (R-O-), amino groups (-NHR, where R is an alkyl or aryl group from which H is removed), phenoxy groups, tertiary carbocations (such as tert-butyl cation), carbocations stabilized by unsaturated systems or heteroatoms, or various protecting groups described above.

[0221] The nucleophilic groups described above refer to molecules or ions that can provide electron pairs to form new chemical bonds in chemical reactions. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, nucleophilic halogens (such as Cl - , Br - or I - ), hydride ion (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxide anions (such as alcohols from which the hydroxyl hydrogen is removed), amino groups (including primary amines, secondary amines, and tertiary amines) or amine anions, carbanions (such as carbanions in organometallic reagents such as Grignard reagents, organolithium reagents, and Gilman reagents), thiol or thiolate anions, thioethers, enols or enolate anions, vinyl ethers, enamines, carboxylic acids or carboxylate anions, alkyl or aryl phosphines (such as triphenylphosphine), heteroaromatic rings with lone pairs of electrons (such as pyridine), etc.

[0222] In a preferred embodiment, the starting materials used in the reaction process also contain protecting groups, and the reaction steps include protection and / or deprotection steps.

[0223] In a preferred embodiment, the composition based on the preformed carrier further comprises 0 - 60 mol% of other ionizable lipids selected from at least one of the following compounds:

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] In a preferred embodiment, the phospholipids include at least one of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diuvacenoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE).

[0230] In a preferred embodiment, the steroid or its derivative includes at least one of cholesterol, cholesteryl stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatine, ursolic acid, α-tocopherol.

[0231] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0232] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises at least one of PEG-distearyloxypropyl (PEG-DSA), PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate (PEG-DMA), 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), dipalmitoyl glycerol-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroyl phosphatidylethanolamine-polyethylene glycol (PEG-DLPE), dimyristoyl phosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0233] In a preferred embodiment, in the preformed carrier-based composition, the nucleic acid is encapsulated inside the preformed carrier and / or adsorbed on the surface of the preformed carrier to form a complex.

[0234] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffered salt solution.

[0235] In a preferred embodiment, the buffered salt solution has a pH of 1-9, the buffered salt concentration is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration <50%.

[0236] In a preferred embodiment, the alcohol is ethanol.

[0237] In a preferred embodiment, the buffered salt solution is selected from at least one of citrate solution, acetate solution, tartrate solution, phosphate solution, carbonate solution, Tris-HCl solution, and sodium chloride solution.

[0238] In a preferred embodiment, at least one of sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the preformed carrier-based composition.

[0239] In a second aspect, the present invention provides an in vitro gene delivery method for immune cells and stem cells, which includes the step of mixing a prefabricated vector and nucleic acid in a solvent to obtain a composition based on the prefabricated vector; the composition of the prefabricated vector includes: 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.

[0240] Compared with the prior art, the present invention has the following beneficial effects:

[0241] 1. The application of the prefabricated vector of the present invention in the preparation of products for in vitro gene delivery of immune cells and stem cells includes the step of mixing the prefabricated vector and nucleic acid in a solvent to obtain a composition based on the prefabricated vector; the composition of the prefabricated vector includes: an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol-conjugated lipid.

[0242] 2. By using the prefabricated vector of the present invention, the dosage of nucleic acid can be flexibly adjusted according to the needs of users, and it has a high protein expression efficiency in various cells such as human NK cells, human MSC cells, T cells, and primary T cells, and has an ideal delivery effect.

[0243] 3. The preparation process of the present invention is simple and can be completed without the aid of equipment. Detailed implementation manners

[0244] The synthesis process of the present invention can accommodate 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. However, in some cases, the compound may need to be further converted into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in various ways using commercially available starting materials, compounds known in the literature, or intermediates that are easily prepared, by adopting standard synthesis methods and procedures known to those skilled in the art or that are obvious to those skilled in the art based on the teachings herein. Standard synthesis methods and procedures for preparing organic molecules, as well as functional group transformations and operations, can be obtained from relevant scientific literature or from standard textbooks in the field. The following description of the synthesis method is designed to illustrate rather than limit the general procedures for preparing the compounds of the present invention.

[0245] 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 (such as R1, R2, and R3, etc.) in each general synthetic route are defined as herein. Those of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of some steps can be changed, such as the introduction and removal of protecting groups.

[0246] In the reaction schemes described herein, multiple stereoisomers can be produced. When no specific stereoisomer is indicated, this should be understood to include all possible stereoisomers produced by the reaction. Those of ordinary skill in the art should recognize that the reaction can be optimized to preferentially obtain one isomer, or new schemes can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0247] (1) Synthesis of the First Ionizable Lipid in Preparation Example 1

[0248] The general synthetic route of the first ionizable lipid is as shown in General Synthetic Routes 1 - 5.

[0249] General Synthetic Route 1

[0250]

[0251] Wherein, M' is M or M with a protecting group.

[0252] As illustrated in General Synthetic Route 1 above, Boc - aminotris(hydroxymethyl)methane reacts with an acid (Compound 2) in a condensation reaction to form Compound 3. Step 1 can be carried out in an organic solvent (such as dichloromethane (DCM)) in the presence of, for example, 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 4 - dimethylaminopyridine (DMAP). Step 1 can be carried out at room temperature for 24 hours.

[0253] Next, the Boc protecting group of Compound 3 is removed to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0254] Next, Compound 4 undergoes a condensation reaction with Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) under the catalysis of EDCl and DMAP or dicyclohexylcarbodiimide (DCC).

[0255] If the M' group of Compound 6 contains the above - mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the reaction conditions for removing the selected protecting group.

[0256] General Synthetic Route 2

[0257]

[0258] Wherein M' is M or M with a protecting group; A is O, NH or S.

[0259] As described in General Synthetic Route 2 above, Compound 1 undergoes a condensation reaction with Compound 2 to obtain Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of EDCl and DMAP.

[0260] Subsequently, the tert-butoxycarbonyl protecting group is removed from Compound 3 to obtain Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) in the presence of an acid (such as trifluoroacetic acid) and a cation scavenger (such as triisopropylsilane (TiPS)).

[0261] Subsequently, Compound 4 undergoes a condensation reaction with a Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0262] 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.

[0263] General Synthetic Route 3

[0264]

[0265] Among them, X is a halogen, such as Cl, Br or I; R4’ is R4 or R4 containing a protecting group; R5’ is R5 or R5 containing a protecting group.

[0266] As described in General Synthetic Route 3 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with Compound 2 to form Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0267] Subsequently, the Boc protecting group is removed from Compound 3 to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0268] Subsequently, Compound 4 undergoes a condensation and reduction reaction with a halogen-substituted aldehyde (Compound 5) to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetoxyborohydride (NaBH(OAc)3)).

[0269] Subsequently, Compound 6 undergoes a nucleophilic reaction with an amine (Compound 7) to form Compound 8. Step 4 can be carried out in an organic solvent (such as in 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).

[0270] If there are the above-mentioned protecting groups in the R4’ and / or R5’ groups of Compound 8, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting group.

[0271] General Synthetic Route 4

[0272]

[0273] 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.

[0274] As described in the above General Synthetic Route 4, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with Compound 2 to form Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0275] Then, the Boc protecting group of Compound 3 is removed to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0276] Then, Compound 4 undergoes a condensation reaction with a halogen-substituted Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF under the catalysis of EDCl and DMAP or DCC.

[0277] Then, Compound 6 undergoes a nucleophilic reaction with an amine (Compound 7) to form Compound 8. Step 4 can be carried out in an organic solvent (such as DMF) in the presence of a base (such as a non-nucleophilic organic base (such as triethylamine, iPr2EtN) or an inorganic base (such as K2CO3)) and a catalyst (KI or NaI).

[0278] If there are the above-mentioned protecting groups in the R4’ and / or R5’ groups of Compound 8, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting group.

[0279] General Synthetic Route 5

[0280]

[0281] Where M pro is M or M containing a protecting group.

[0282] As described in the general synthetic route 5 above, Boc-amino tris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to form compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0283] Then, the Boc protecting group of compound 3 is removed to form compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0284] Then, compound 4 and compound 5 undergo a condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetoxyborohydride (NaBH(OAc)3)).

[0285] If the M pro group of compound 6 contains the above 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.

[0286] In addition, it should also be understood that any specific embodiment of the present invention within the scope of the prior art can 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 can be excluded even if such exclusion is not explicitly stated herein.

[0287] All cited sources, such as references, publications, databases, database entries, and technologies cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the case of a conflict between the cited source and the statements of the present application, the statements of the present application shall prevail.

[0288] (1) Synthesize the compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L), or (1M)

[0289] A. General considerations

[0290] It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is imposed on their sources.

[0291] The process routes described below can be used to synthesize the compounds 1001 - 3422 of the present invention.

[0292] The following abbreviations are used herein:

[0293] THF: Tetrahydrofuran

[0294] MeCN: Acetonitrile

[0295] MeOH: Methanol

[0296] PE: Petroleum ether

[0297] EA: Ethyl acetate

[0298] DMF: N,N-Dimethylformamide

[0299] EDCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0300] LAH: Lithium aluminum hydride

[0301] DCM: Dichloromethane

[0302] DMAP: 4-Dimethylaminopyridine

[0303] LDA: Lithium diisopropylamide

[0304] rt: Room temperature

[0305] DCE: 1,2-Dichloroethane

[0306] n-BuLi: n-Butyllithium

[0307] i-Pr2EtN: N,N-Diisopropylethylamine

[0308] B. Intermediate synthesis

[0309] Intermediate A:

[0310]

[0311] Intermediate A was obtained by the following synthetic route:

[0312]

[0313] Dissolve tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl dicarbonate (Boc2O) (99.1 g) in a mixed solvent of methanol (300 mL) / H2O (30 mL), react at room temperature for 72 h, and purify by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain the white solid compound Intermediate A (N-Boc-aminotrimethylolmethane) (75.0 g). LCMS (ESI) calcd for C9H 19 NO5, [M+H] + m / z 222.13, found 222.25.

[0314] Intermediate B:

[0315]

[0316] Intermediate B is obtained through the following synthetic process route:

[0317]

[0318] Add diethyl 2-ethyl-1,3-propanedicarboxylate (29.0 g), THF (90 mL), and DMF (30 mL) into a single-neck flask. Add NaH (3.7 g) under an ice bath. Stir at room temperature for 30 min under nitrogen protection. Add pentadecyl bromide (15.0 g). React at 80 °C for 2 h under nitrogen protection. Concentrate under reduced pressure to remove THF. Drop the reaction solution into ice water, add ethyl acetate and stir, then filter through diatomaceous earth. Collect the organic phase and separate it. Wash the organic phase with saturated brine, separate out the organic phase, and purify it by silica gel column chromatography (PE:EA = 100:1 - 50:1) to obtain diethyl 2-ethyl-2-pentadecylmalonate (16.5 g). Add EtOH (50 mL), H2O (50 mL), and KOH (11.3 g) into diethyl 2-ethyl-2-pentadecylmalonate (8.0 g). React at 90 °C for 12 h. Concentrate under reduced pressure to remove EtOH. Add dilute hydrochloric acid to adjust the reaction system to pH = 4 - 5. Add water and ethyl acetate and stir to separate. Wash the organic phase with saturated brine, separate out the organic phase, and purify it by column chromatography (PE:EA = 5:1 - 1:1) to obtain 2-ethyl-2-pentadecylmalonic acid (5.2 g). React 2-ethyl-2-pentadecylmalonic acid (10.0 g) under open conditions at 170 °C for 6 h. Cool to room temperature, add water and ethyl acetate and stir to separate. Wash the organic phase with saturated brine, separate out the organic phase, concentrate, and purify it by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain Intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1HNMR (400 MHz, CDCl3) δ 2.31 (tt, J = 8.6, 5.3 Hz, 1H), 1.71 - 1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 6.7 Hz, 3H).

[0319] Intermediate C:

[0320]

[0321] Intermediate C is obtained through the following synthetic process route:

[0322]

[0323] Add capric acid (50 g) and the solvent THF (500 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add the reactant NaH (23.22 g). Stir for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (62.19 g). Continue to stir for 1 h at 0 °C under nitrogen protection. Finally, after dropwise adding the reactant iodo-nonane (88.52 g), raise the temperature to room temperature and stir overnight. Dilute with 1 L of DCM and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, and purify by silica gel column chromatography (PE:EA = 50:1) to finally obtain intermediate C (2-octylundecanoic acid) (11 g). 1H NMR (400 MHz, Chloroform-d) δ 2.37 (tt, J = 8.7, 5.1 Hz, 1H), 1.71 - 1.58 (m, 2H), 1.48 (dt, J = 13.3, 6.7 Hz, 2H), 1.29 (d, J = 9.4 Hz, 25H), 0.90 (t, J = 6.8 Hz, 6H).

[0324] Intermediate D:

[0325]

[0326] Intermediate D is obtained by the following synthetic process route:

[0327]

[0328] Add tridecanoic acid (79.5 g) and the solvent THF (800 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add NaH (22.25). Stir the reaction solution for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (317.87 g) to the reaction system; continue to stir the reaction solution for 1 h at 0 °C under nitrogen protection. After dropwise adding the reactant iodo-n-hexane (94.39), raise the temperature to room temperature and stir overnight. Dilute the reaction solution with 1 L of DCM and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, and perform silica gel column chromatography (PE:EA = 50:1) to obtain intermediate D (2-hexyltridecanoic acid) (26 g). 1H NMR (400 MHz, Chloroform-d) δ 2.36 (tt, J = 8.7, 5.4 Hz, 1H), 1.63 (ddd, J = 14.3, 8.7, 5.5 Hz, 2H), 1.49 (dq, J = 13.5, 6.6 Hz, 2H), 1.29 (d, J = 10.1 Hz, 26H), 0.90 (t, J = 6.6 Hz, 6H).

[0329] Intermediate E:

[0330]

[0331] The intermediate E is obtained through the following synthetic process route:

[0332]

[0333] Dissolve tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1) in EtOH (150 mL). Under nitrogen protection, react at 45 °C for 30 h, then concentrate under reduced pressure to remove EtOH. Add 100 mL × 3 of the solvent (PE:EA = 20:1) for pulping, filter, and obtain the white solid compound intermediate E (11.5 g). 1HNMR (400 MHz, CDCl3) δ 3.60 (s, 6H), 2.84 (t, J = 5.9 Hz, 2H), 2.47 (t, J = 5.8 Hz, 2H), 1.48 (s, 9H).

[0334] Intermediate F:

[0335]

[0336] Intermediate F is obtained through the following synthetic process route:

[0337]

[0338] Add (2-aminoethyl)carbamic acid tert-butyl ester (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) to a single-necked flask. React at 80 °C overnight, then add water and ethyl acetate for stirring and liquid separation. Wash the organic phase with saturated brine, separate the organic phase, concentrate, and purify by silica gel column chromatography (PE:EA =

[0339] 10:1 - 5:1) to obtain compound 2 (5.5 g). Add dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) to compound 2. Stir at room temperature for 3 h, then concentrate under reduced pressure to obtain compound 3, which is intermediate F (6 g). LCMS (ESI) calcd for C 20 H 28 N2O2, [M + H] + m / z 329.22, found 329.24.

[0340] Intermediate G:

[0341]

[0342] Intermediate G is obtained through the following synthetic process route:

[0343]

[0344] To a single-necked flask, add Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropyl bromide (9.45 g), K2CO3 (12.94 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 2 (6.9 g). To Compound 2 (6.9 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). Stir at room temperature for 3 h, then concentrate under reduced pressure to obtain Intermediate G (7.5 g). LCMS (ESI) calcd for C 22 H 32 N2O2, [M+H] + m / z 357.25, found 357.51.

[0345] Intermediate H:

[0346]

[0347] Intermediate H was obtained by the following synthetic route:

[0348]

[0349] To a single-necked flask, add Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), K2CO3 (6.47 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain Compound 2 (4.1 g). To Compound 2 (4.1 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). Stir at room temperature for 3 h, then concentrate under reduced pressure to obtain Intermediate H (4.9 g). LCMS (ESI) calcd for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56.

[0350] Intermediate I:

[0351]

[0352] Intermediate I was obtained by the following synthetic route:

[0353]

[0354] To a single-necked flask, add compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), K2CO3 (15.46 g). After reacting overnight at 80 °C, add water and ethyl acetate and stir to separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain compound 2 (6.8 g). To compound 2 (6.8 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). After stirring at room temperature for 3 h, concentrate under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calcd for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0355] Intermediate J:

[0356]

[0357] Intermediate J was obtained by the following synthetic route:

[0358]

[0359] To a single-necked flask, add butyric acid (5.0 g), THF (100 mL). Add NaH (2.73 g) at 0 °C, slowly dropwise add LDA (56.8 mL), react at room temperature for 30 min, add 1-bromotridecane, continue to react overnight at room temperature, then add ice water and ethyl acetate and stir to separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calcd for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0360] Intermediate K:

[0361]

[0362] Intermediate K was obtained by the following synthetic route:

[0363]

[0364] Add reactant 1 (24 g), Imidazole (19.00 g) and solvent DCM (200 mL) to a three-necked flask. After cooling to 0 °C, slowly add reactant TBDMSCI (38.57 g). Stir at 25 °C for 4 h, then dilute with 300 mL of DCM. Wash the organic phase twice with 1 L of water and dry over anhydrous Na2SO4. After filtration and concentration, add silica gel and column chromatograph with 1:20 DCM:MeOH to collect the sample and concentrate to obtain intermediate K (28.00 g). 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 3.58 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.67 - 1.40 (m, 4H), 0.84 (s, 9H).

[0365] Intermediate L:

[0366]

[0367] Intermediate L was obtained through the following synthetic process route:

[0368]

[0369] Add compound DMSO (3.58 g) and anhydrous solvent DCM (30 mL) to a three-necked flask. Cool the system to -78 °C and slowly add oxalyl chloride (2.91 g). Stir the reaction solution at -78 °C under N2 protection for 10 minutes, then slowly add dropwise compound 1 (3 g). Continue to stir at -78 °C under N2 protection for 1 h. Finally, add dropwise TEA (9.28 g). Continue to stir at -78 °C under N2 protection for 0.5 h, then dilute with 100 mL of DCM and wash with saturated NH4Cl solution and water respectively. Dry the organic layer over anhydrous Na2SO4, filter and concentrate to obtain intermediate L (3.0 g). LCMS (ESI) calcd for C 11 H 14 O3, [M + H] + m / z 195.09, found 195.23.

[0370] Intermediate M:

[0371]

[0372] Intermediate M was obtained through the following synthetic process route:

[0373]

[0374] 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, then dilute with 100 mL of EA and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, add silica gel and column chromatograph with 15:1 DCM:MeOH to collect the sample and concentrate to obtain compound 2 (2.0 g). Add reactant 2 (1.80 g) and solvent DCM (20 mL) to the three-necked flask. Replace the system with nitrogen and cool to -78 °C, then add DIBAL-H (1.00 g) dropwise, stir at -78 °C for 4 h, quench with methanol and sodium carbonate solution into the reaction solution, then extract with DCM (100 mL), dry the organic layer with anhydrous sodium sulfate, filter and concentrate to obtain intermediate M (1.36 g). LCMS (ESI) calcd for C 15 H 33 NO2Si, [M+H] + m / z 288.52, found 288.23.

[0375] C, Compound 1003, synthesized according to General Synthetic Route 1

[0376] Structural formula:

[0377] Chemical formula: C 63 H 122 N2O7

[0378] Molecular weight: 1019.68

[0379] Step 1: Synthesize compound 3 in General Synthetic Route 1

[0380]

[0381] Add compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) to compound 2 (intermediate B) (4.3 g). Stir at room temperature for 12 h under nitrogen protection, then add water and dichloromethane and stir for liquid separation. Wash the organic phase with saturated brine, separate the organic phase, dry the organic phase with anhydrous sodium sulfate again, concentrate, and purify by silica gel column chromatography (PE:EA = 40:1 - 30:1) to obtain compound 3 (2.6 g).

[0382] Step 2: Synthesize compound 4 in General Synthetic Route 1

[0383]

[0384] DCM (15 mL) and TFA (5 mL) were added to Compound 3 (2.6 g). After stirring at room temperature for 3 h, the mixture was concentrated and purified by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 4 (3.2 g).

[0385] Step 3: Synthesis of Compound 6 in General Synthetic Route 1

[0386]

[0387] DCM (20 mL), Boc-glycine (Compound 5, 578 mg), and DCC (6.8 g) were added to Compound 4 (3.2 g). After stirring at room temperature under nitrogen protection for 12 h, water and ethyl acetate were added and the mixture was stirred and separated. The organic phase was washed with saturated brine, the organic phase was 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).

[0388] Step 4: Compound 1003

[0389]

[0390] DCM (15 mL) and TFA (5 mL) were added to Compound 6 (2.2 g). After stirring at room temperature for 12 h, the mixture was concentrated and purified by column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain Compound 1003 (1.3 g). 1 H NMR (400 MHz, CDCl3) δ 4.49 (d, J = 2.6 Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J = 8.6, 5.5 Hz, 3H), 1.62 (q, J = 7.1 Hz, 12H), 1.27 (d, J = 3.4 Hz, 78H), 0.94 - 0.86 (m, 18H).

[0391] D. Compound 1002, synthesized according to General Synthetic Route 1

[0392] Structural formula:

[0393] Chemical formula: C 60 H 116 N2O7

[0394] Molecular weight: 977.60

[0395] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that Intermediate C was used as Compound 2 in General Synthetic Route 1. 11H NMR (400 MHz, CDCl3) δ 4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5 Hz, 3H), 1.64 (d, J = 6.9 Hz, 6H), 1.50 - 1.42 (m, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 18H).

[0396] E. Compound 1004 was synthesized according to General Synthetic Route 1

[0397] Structural formula:

[0398] Chemical formula: C 54 H 104 N2O7

[0399] Molecular weight: 893.43

[0400] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that palmitic acid was used as Compound 2 in General Synthetic Route 1 1 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 4.45 (s, 6H), 3.74 (s, 2H), 2.35 (t, J = 7.6 Hz, 6H), 1.60 (p, J = 6.9 Hz, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 9H).

[0401] F. Compound 1001 was synthesized according to General Synthetic Route 1

[0402] Structural formula:

[0403] Chemical formula: C 59 H 108 N2O7

[0404] Molecular weight: 957.52

[0405] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that (9Z)-9-hexadecenoic acid was used as Compound 2 and 5-(N,N-dimethylamino)valeric acid was used as Compound 5 in General Synthetic Route 1 11H NMR (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 6H), 4.51 - 4.38 (m, 6H), 3.19 (dt, J = 11.3, 5.6 Hz, 2H), 2.97 (t, J = 5.9 Hz, 6H), 2.46 (t, J = 6.6 Hz, 1H), 2.35 (td, J = 7.6, 3.1 Hz, 5H), 2.20 (t, J = 7.6 Hz, 2H), 2.03 (d, J = 6.2 Hz, 12H), 1.89 - 1.81 (m, 3H), 1.74 (q, J = 7.8 Hz, 2H), 1.67 - 1.51 (m, 6H), 1.31 (d, J = 7.8 Hz, 48H), 0.90 (t, J = 6.7 Hz, 9H).

[0406] G. Compound 1014 was synthesized according to General Synthetic Route 2

[0407] Structural formula:

[0408] Chemical formula: C 70 H 137 N3O9

[0409] Molecular weight: 1164.88

[0410] Step 1: Synthesis of Intermediate 3 of General Synthetic Route 2

[0411]

[0412] Intermediate 3 was synthesized according to the method described in Step 1 of General Synthetic Route 1, except that Intermediate E was used as Compound 1 in General Synthetic Route 1.

[0413] Step 2: Synthesis of Compound 4 of General Synthetic Route 2

[0414]

[0415] To Compound 3 (4.8 g), DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added. After reacting at room temperature for 12 h, water and dichloromethane were added and stirred for liquid separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain Compound 4 (3.3 g).

[0416] Step 3: Synthesis of Compound 6 of General Synthetic Route 2

[0417]

[0418] Compound 4 (4.0 g), DCM (80 mL), EDCI (1.1 g), 1-hydroxybenzotriazole (HOBt) (0.78 g), and DIEA (2.5 g) were added to compound 5 (1.9 g). After stirring at room temperature for 12 h, water and dichloromethane were added and stirred, followed by liquid separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain compound 6 (4.0 g).

[0419] Step 4: Compound 1014

[0420]

[0421] MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added to compound 6 (4.0 g). After stirring overnight at room temperature under hydrogen, it was filtered through diatomaceous earth, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1 - 15:1) to obtain compound 1014 (2 g). 1 H (400 MHz, CDCl3) δ 7.38 (t, J = 5.8 Hz, 1H), 4.14 - 4.05 (m, 6H), 3.58 (t, J = 4.9 Hz, 4H), 3.31 (q, J = 5.7 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.64 (q, J = 5.2 Hz, 6H), 2.29 (dq, J = 10.8, 4.4 Hz, 6H), 1.66 - 1.37 (m, 12H), 1.24 (d, J = 4.3 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0422] H. Compound 1015 was synthesized according to the general synthetic route 2

[0423] Structural formula:

[0424] Chemical formula: C 72 H 141 N3O9

[0425] Molecular weight: 1192.93

[0426] Synthesized according to the general synthetic route 2, similar to the synthesis process of compound 1014, except that intermediate G was used as compound 5 in the general synthetic route 2. 1H (400 MHz, CDCl3) δ 7.58 (t, J = 5.7 Hz, 1H), 4.15 - 4.01 (m, 6H), 3.70 (t, J = 5.4 Hz, 4H), 3.39 (q, J = 5.7 Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.57 (dt, J = 20.5, 5.8 Hz, 6H), 2.36 - 2.22 (m, 5H), 1.76 - 1.67 (m, 4H), 1.62 - 1.35 (m, 13H), 1.23 (d, J = 4.6 Hz, 78H), 0.92 - 0.80 (m, 18H).

[0427] I. Compound 1020, synthesized according to General Synthetic Route 2

[0428] Structural formula:

[0429] Chemical formula: C 74 H 145 N3O9

[0430] Molecular weight: 1220.99

[0431] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate H is used as Compound 5 in General Synthetic Route 2.

[0432] J. Compound 1025, synthesized according to General Synthetic Route 2

[0433] Structural formula:

[0434] Chemical formula: C 69 H 135 N3O8

[0435] Molecular weight: 1134.85

[0436] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate I is used as Compound 5 in General Synthetic Route 2. 1 H (400 MHz, CDCl3) δ 6.98 (t, J = 5.7 Hz, 1H), 4.10 (m, J = 3.8 Hz, 6H), 3.63 (t, J = 5.2 Hz, 2H), 3.35 (q, J = 5.8 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.57 (q, J = 5.3 Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1 Hz, 5H), 1.65 - 1.41 (m, 13H), 1.24 (d, J = 4.4 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0437] Compound 1005, synthesized according to General Synthetic Route 2

[0438] Structural formula:

[0439] Chemical formula: C 68 H 133 N3O7

[0440] Molecular weight: 1104.83

[0441] Synthesized according to General Synthetic Route 2, with a synthetic process similar to that of Compound 1014, except that N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 H NMR (400 MHz, CDCl3) δ 4.19 - 4.07 (m, 6H), 3.40 (d, J = 5.7 Hz, 2H), 2.95 - 2.87 (m, 2H), 2.82 (s, 4H), 2.64 - 2.55 (m, 2H), 2.38 (s, 6H), 2.33 - 2.29 (m, 3H), 1.63 - 1.55 (m, 6H), 1.49 - 1.44 (m, 2H), 1.26 (d, J = 5.4 Hz, 78H), 0.89 (td, J = 7.0, 2.8 Hz, 18H).

[0442] L. Compound 1006, synthesized according to General Synthetic Route 2

[0443] Structural formula:

[0444] Chemical formula: C 53 H 103 N3O7

[0445] Molecular weight: 894.42

[0446] Synthesized according to General Synthetic Route 2, with a synthetic process similar to that of Compound 1014, except that myristic acid is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR (400 MHz, CDCl3) δ 7.99 (t, J = 5.8 Hz, 1H), 4.13 (s, 6H), 3.60 (q, J = 5.3 Hz, 2H), 3.38 - 3.34 (m, 2H), 3.00 (s, 6H), 2.94 (t, J = 5.8 Hz, 2H), 2.45 (t, J = 5.8 Hz, 2H), 2.35 (t, J = 7.6 Hz, 6H), 1.66 - 1.56 (m, 6H), 1.33 - 1.23 (m, 60H), 0.90 (t, J = 6.8 Hz, 9H).

[0447] Compound 1007, synthesized according to General Synthetic Route 2

[0448] Structural formula:

[0449] Chemical formula: C 68 H 133 N3O7

[0450] Molecular weight: 1104.83

[0451] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, with the difference that Intermediate D is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(400MHz, Chloroform-d)δ7.13(t, J = 5.0Hz, 1H), 4.13(s, 6H), 3.33(q, J = 5.6Hz, 2H), 2.92(t, J = 6.0Hz, 2H), 2.47 - 2.28(m, 7H), 2.24(s, 6H), 1.60(dq, J = 14.7, 7.2Hz, 6H), 1.47(dd, J = 14.2, 6.7Hz, 6H), 1.27(d, J = 3.4Hz, 78H), 0.89(td, J = 6.7, 2.5Hz, 18H).

[0452] Compound 1008, synthesized according to General Synthetic Route 2

[0453] Structural formula:

[0454] Chemical formula: C 68 H 133 N3O7

[0455] Molecular weight: 1104.01

[0456] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, with the difference that Intermediate C is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1HNMR (400 MHz, Chloroform-d) δ 4.13 (s, 6H), 3.33 (q, J = 5.6 Hz, 2H), 2.92 (t, J = 5.9 Hz, 2H), 2.51 - 2.26 (m, 7H), 2.24 (s, 6H), 1.59 (ddt, J = 14.8, 10.9, 6.4 Hz, 6H), 1.47 (tq, J = 11.0, 5.4 Hz, 6H), 1.26 (s, 77H), 0.89 (t, J = 6.7 Hz, 18H).

[0457] O. Compound 1009 was synthesized according to General Synthetic Route 2.

[0458] Structural formula:

[0459] Chemical formula: C 62 H 121 N3O7

[0460] Molecular weight: 1020.66

[0461] It was synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate J was used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine was used as Compound 5 in General Synthetic Route 2. 1 1H (400 MHz, CDCl3) δ 7.04 (t, J = 5.0 Hz, 1H), 4.12 (m, J = 3.3 Hz, 6H), 3.30 (q, J = 5.6 Hz, 2H), 2.91 (t, J = 6.0 Hz, 2H), 2.39 (t, J = 6.0 Hz, 2H), 2.33 - 2.25 (m, 5H), 2.22 (s, 6H), 1.76 - 1.54 (m, 12H), 1.24 (m, J = 3.7 Hz, 66H), 0.90 - 0.84 (m, 18H).

[0462] P. Compound 1011 was synthesized according to General Synthetic Route 2.

[0463] Structural formula:

[0464] Chemical formula: C 70 H 137 N3O7

[0465] Molecular weight: 1132.88

[0466] It was synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-diethylethylenediamine was used as Compound 5 in General Synthetic Route 2. 11H NMR (400 MHz, CDCl3) δ 4.19 - 4.08 (m, 6H), 3.31 (q, J = 5.8 Hz, 2H), 2.92 (t, 2H), 2.57 (t, J = 6.9 Hz, 6H), 2.37 - 2.26 (m, 5H), 1.62 (ddd, J = 18.3, 9.0, 4.3 Hz, 12H), 1.27 (d, J = 4.7 Hz, 78H), 1.04 (t, J = 7.1 Hz, 6H), 0.94 - 0.86 (m, 18H).

[0467] Q. Compound 1012 was synthesized according to General Synthetic Route 2

[0468] Structural formula:

[0469] Chemical formula: C 72 H 141 N3O7

[0470] Molecular weight: 1160.93

[0471] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine was used as Compound 5 in General Synthetic Route 2 1 1H NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 4.18 - 4.07 (m, 6H), 3.29 (q, J = 5.7 Hz, 2H), 2.91 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5 Hz, 4H), 2.35 - 2.27 (m, 5H), 1.63 - 1.42 (m, 16H), 1.27 (m, J = 4.5 Hz, 78H), 0.93 - 0.85 (m, 24H).

[0472] R. Compound 1013 was synthesized according to General Synthetic Route 2

[0473] Structural formula:

[0474] Chemical formula: C 74 H 145 N3O7

[0475] Molecular weight: 1188.99

[0476] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine was used as Compound 5 in General Synthetic Route 2 11H NMR (400 MHz, CDCl3) δ 4.10 (d, J = 4.1 Hz, 6H), 3.26 (q, J = 5.8 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.52 (d, J = 6.6 Hz, 2H), 2.41 (t, J = 7.4 Hz, 4H), 2.33 - 2.25 (m, 5H), 1.60 (d, J = 11.8 Hz, 16H), 1.24 (d, J = 4.5 Hz, 82H), 0.96 - 0.79 (m, 24H).

[0477] S. Compound 1029 was synthesized according to General Synthetic Route 2

[0478] Structural formula:

[0479] Chemical formula: C 68 H 132 N2O8

[0480] Molecular weight: 1105.81

[0481] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethyl - 2 - hydroxyethylamine was used as Compound 5 in General Synthetic Route 2 1 1H NMR (400 MHz, CDCl3) δ 4.16 (t, J = 5.8 Hz, 2H), 4.13 - 4.05 (m, 6H), 2.86 (t, J = 6.3 Hz, 2H), 2.55 (t, J = 5.8 Hz, 2H), 2.44 (t, J = 6.3 Hz, 2H), 2.35 - 2.20 (m, 9H), 1.60 - 1.39 (m, 11H), 1.24 (d, J = 4.5 Hz, 80H), 1.02 - 0.79 (m, 18H).

[0482] T. Compound 1111 was synthesized according to General Synthetic Route 2

[0483] Structural formula:

[0484] Chemical formula: C 70 H 136 N4O7

[0485] Molecular weight: 1145.88

[0486] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate is used as Compound 5 in General Synthetic Route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97 - 2.80 (m, 5H), 2.70 (s, 5H), 2.46 - 2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54 - 1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0487] U. Compound 1118, synthesized according to General Synthetic Route 2

[0488] Structural formula:

[0489] Chemical formula: C 71 H 139 N3O8

[0490] Molecular weight: 1162.91

[0491] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and Intermediate K is used as Compound 5 in General Synthetic Route 2. 1 HNMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.61 (s, 2H), 3.42 (d, J = 33.5 Hz, 2H), 2.91 (t, J = 6.2 Hz, 3H), 2.36 (dd, J = 9.2, 3.6 Hz, 6H), 1.47 (dd, J = 13.9, 6.2 Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7 Hz, 18H).

[0492] V. Compound 1010, synthesized according to General Synthetic Route 3

[0493] Structural formula:

[0494] Chemical formula: C 67 H 133 N3O6

[0495] Molecular weight: 1076.82

[0496] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 3

[0497]

[0498] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003.

[0499] Step 3: Synthesize Compound 6 of General Synthetic Route 3

[0500]

[0501] Add Compound 4 (600 mg), chloroacetaldehyde (Compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) to DCE (60 mL). After reacting overnight at room temperature, add water and dichloromethane and stir to separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and perform silica gel column chromatography (DCM:MeOH = 30:1 - 15:1) to obtain Compound 6 (270 mg).

[0502] Step 4: Synthesize Compound 1010

[0503]

[0504] Add MeCN (20 mL), N,N-dimethylethylenediamine (Compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) to Compound 6 (270 mg). React overnight at 70 °C under nitrogen protection, and perform silica gel column chromatography to obtain Compound 1010 (16 mg). 1 H (400 MHz, CDCl3) δ 4.10 (m, J = 2.9 Hz, 6H), 2.76 - 2.64 (m, 6H), 2.40 (t, J = 6.2 Hz, 2H), 2.29 (ddd, J = 8.5, 5.6, 2.9 Hz, 3H), 2.22 (s, 6H), 1.97 (s, 6H), 1.50 - 1.41 (m, 6H), 1.24 (d, J = 4.2 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0505] W. Compound 1059 is synthesized according to General Synthetic Route 3

[0506] Structural formula:

[0507] Chemical formula: C 67 H 132 N2O7

[0508] Molecular weight: 1077.80

[0509] Synthesized according to General Synthetic Route 3, similar to the synthesis process of Compound 1010, with the difference that Intermediate C and Intermediate L are used as Compound 2 and Compound 5 in General Synthetic Route 3 respectively. In addition, the functional group transformation of Compound 6 in General Synthetic Route 3 needs to be carried out through the following process steps:

[0510]

[0511] To Intermediate 6a (1.5 g), add solvents MeOH (5 mL) and THF (5 mL), add reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g), react overnight at 25 °C under H2 condition, filter with diatomaceous earth, concentrate the filtrate, and perform silica gel column chromatography (PE:EA 30:1 - 10:1) to obtain Intermediate 6b (520 mg). To Intermediate 6b (470 mg), add DCM (5 mL), dropwise add SOCl2 (1064.26 mg, 8.946 mmol) at 0 °C, transfer to room temperature and react for 3 hours, then extract the reaction solution with water and DCM, concentrate the organic phase, and perform silica gel column chromatography (PE:EA 20:1) to obtain Intermediate 6c (181 mg). To Intermediate 6c (170 mg), add dimethylamine (143.39 mg), DMF (2 mL), potassium carbonate (43.96 mg), and potassium iodide (26.39 mg), stir the reaction solution at 70 °C, filter, concentrate, and perform silica gel column chromatography (DCM:MeOH 20:1) to obtain Compound 1059 (91 mg, yield 53.22%). 1 H NMR (400 MHz, Chloroform - d) δ4.11 (s, 6H), 3.51 (dt, J = 18.3, 5.6 Hz, 4H), 2.81 (dd, J = 10.3, 5.1 Hz, 2H), 2.50 (t, J = 6.0 Hz, 2H), 2.42 - 2.32 (m, 3H), 2.28 (s, 6H), 2.08 - 1.98 (m, 1H), 1.53 - 1.38 (m, 7H), 1.27 (s, 77H), 0.90 (t, J = 6.7 Hz, 18H).

[0512] X. Compound 1112, synthesized according to General Synthetic Route 3

[0513] Structural formula:

[0514] Chemical formula: C 69 H 136 N2O6

[0515] Molecular weight: 1089.85

[0516] Synthesized according to the general synthetic route 3, which is similar to the synthesis process of compound 1010, except that intermediate C and 6-bromohexanal are used instead of 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).

[0517] Y. Compound 1113, synthesized according to the general synthetic route 4

[0518] Structural formula:

[0519] Chemical formula: C 71 H 138 N2O8

[0520] Molecular weight: 1147.89

[0521] Steps 1 and 2: Synthesize compound 3 and compound 4 of the general synthetic route 4

[0522]

[0523] According to steps 1 and 2 of the general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0524] Step 3: Synthesize compound 6 of the general synthetic route 4

[0525]

[0526] Add DIEA (0.30 g) and solvent DCM (20 mL) to compound 4. Under nitrogen protection, after cooling to 0 °C, add 5-bromovaleryl chloride (compound 5, 0.27 g), stir at 25 °C for 2 h, dilute with 100 mL of DCM, and then wash the organic phase twice with 100 mL of water. The organic phase is dried over anhydrous Na2SO4, filtered and concentrated, and then subjected to silica gel column chromatography (PE:EA 20:1) to obtain compound 6 (800 mg).

[0527] Step 4: Synthesize compound 8 of the general synthetic route 4

[0528]

[0529] Compound 6 (750.00 mg), intermediate K (Compound 7, 217.31 mg) and solvent DMF (20 mL) were added to a three-necked flask. Reactants K2CO3 (184.16 mg) and KI (110.60 mg) were added. After stirring at 25 °C for 16 h, the temperature was raised to 100 °C and stirred for 3 h. The reaction solution was diluted with 200 mL of EA, and the organic phase was washed twice with 300 mL of saturated brine; the organic phase was dried over anhydrous Na2SO4, filtered and concentrated, silica gel was added, and column chromatography was carried out with 20:1 DCM:MeOH, and the sample was collected and concentrated to obtain Compound 8 (700.00 mg).

[0530] Step 5: Synthesis of Compound 1113

[0531]

[0532] Compound 8 (700.00 mg) and solvent THF (5 mL) were added to a three-necked flask. Hydrochloric acid dioxane solution (1 M) (5.55 mL) was added. After the reaction solution was stirred at 25 °C for 2 h, it was diluted with 50 mL of EA, and then the organic phase was washed with saturated NaHCO3 solution and water. The organic phase was dried over anhydrous Na2SO4, filtered and concentrated, and silica gel column chromatography (MeOH:DCM 1:20) was carried out to obtain Compound 1113 (201.1 mg, yield 31.59%). 1 H NMR (400 MHz, CDCl3) δ 4.50 - 4.39 (m, 6H), 3.59 (t, J = 4.6 Hz, 2H), 2.49 (s, 3H), 2.35 - 2.26 (m, 6H), 2.14 (t, J = 7.5 Hz, 2H), 1.75 - 1.69 (m, 4H), 1.66 - 1.43 (m, 17H), 1.27 (d, J = 3.7 Hz, 78H), 0.94 - 0.87 (m, 18H).

[0533] Z. Compound 1115, synthesized according to General Synthetic Route 4

[0534] Structural formula:

[0535] Chemical formula: C 67 H 131 N3O7

[0536] Molecular weight: 1090.80

[0537] Synthesized according to General Synthetic Route 4, similar to the synthesis process of Compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine were used instead of Compound 5 and Compound 7 in General Synthetic Route 4 respectively. 11H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H), 4.48 (tt, J = 11.9, 5.7 Hz, 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, 7.3 Hz, 12H), 1.27 (d, J = 3.3 Hz, 78H), 0.90 (td, J = 7.2, 3.4 Hz, 18H).

[0538] AA. Compound 1114 was synthesized according to General Synthetic Route 5

[0539] Structural formula:

[0540] Chemical formula: C 70 H 138 N2O7

[0541] Molecular weight: 1119.88

[0542] Steps 1 and 2: Synthesize Compounds 3 and 4 of General Synthetic Route 5

[0543]

[0544] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003

[0545] Step 3: Synthesize Compound 6 of General Synthetic Route 5

[0546]

[0547] Add Compound 4 (500 mg), Intermediate M (Compound 5, 298.68 mg), Reactant STAB (328.68 mg) and DCE (10 mL) to a three-necked flask. After stirring the reaction solution at N2, 25 °C for 16 h, add 100 mL of water to the reaction solution, extract twice with EA (100 mL), combine the organic phases, concentrate, add silica gel, and then use 1:20

[0548] DCM:MeOH for column chromatography, collect the sample and concentrate to obtain Compound 6 (500 mg).

[0549] Step 4: Synthesize Compound 1114

[0550]

[0551] Compound 6 (450 mg) and the solvent THF (5 mL) were added to a three-necked flask, and a dioxane hydrochloride solution (1 M) (3.65 mL) was added. After stirring at 25 °C for 2 h, it was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO3 solution and water. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated, and then subjected to silica gel column chromatography (MeOH:DCM 1:20) to obtain compound 1114 (107 mg, yield 24.84%). 1 H NMR (400 MHz, CDCl3) δ 4.16 - 4.07 (m, 6H), 3.60 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 2.49 (s, 3H), 2.31 (tt, J = 8.4, 5.5 Hz, 6H), 1.70 (d, J = 4.6 Hz, 3H), 1.64 - 1.39 (m, 18H), 1.27 (d, J = 4.0 Hz, 78H), 0.90 (t, J = 7.1 Hz, 18H).

[0552] Compound 1120 was synthesized according to General Synthetic Route 5

[0553] Structural formula:

[0554] Chemical formula: C 72 H 142 N2O7

[0555] Molecular weight: 1147.90

[0556] Synthesized according to General Synthetic Route 5, similar to the synthesis process of compound 1114, except that intermediate C and 6-bromohexanal were used instead of compound 2 and compound 5 in General Synthetic Route 5 respectively. 1H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.63 (s, 2H), 2.57 (t, J = 6.8 Hz, 4H), 2.37 (ddd, J = 8.5, 7.1, 4.3 Hz, 5H), 1.67 - 1.41 (m, 24H), 1.27 (s, 79H), 0.90 (t, J = 6.7 Hz, 18H).

[0557] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0558] 1. Preparation of compound 6001

[0559] Structural formula:

[0560] Molecular weight: 499.43

[0561] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide and continue the reaction for 16 h. Then pass through a reverse column to obtain Compound 6002.

[0562] Add 0.7 g of Compound 6002, 0.4 g of palladium on carbon, and 28 mL of tetrahydrofuran to a reaction flask and stir. Replace with hydrogen and maintain the pressure with a hydrogen balloon. After 16 h, filter and pass through a normal column (methanol - DCM system) to obtain 200 mg of Compound 6001. 1 HNMR(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).

[0563] 2. Preparation of Compound 6002

[0564] Structural formula:

[0565] Molecular weight: 497.42

[0566] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide and continue the reaction for 16 h. Then concentrate the organic solvent, pass through a reverse column, and lyophilize to obtain 400 mg of Compound 6002 in total. 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).

[0567] 3. Preparation of Compound 6003

[0568] Structural formula:

[0569] Molecular weight: 445.34

[0570] 4.00 g of tetradecylamine, 6.53 g of 2-hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask and stirred while heating to 70 °C. After 29 h, the organic solvent was concentrated, passed through a reverse column, and freeze-dried to obtain 1.5 g of compound 6003. 1 1H NMR (400 MHz, CDCl3) δ 4.29 (t, J = 7.5 Hz, 4H), 3.89 - 3.75 (m, 4H), 2.81 (t, J = 6.2 Hz, 4H), 2.50 (ddd, J = 23.2, 14.1, 7.1 Hz, 6H), 1.46 (s, 2H), 1.28 (s, 24H), 0.91 (t, J = 6.3 Hz, 3H).

[0571] 4. Preparation of compound 6004

[0572] Structural formula:

[0573] Molecular weight: 553.48

[0574] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol were added to a reaction flask and stirred while heating to 100 °C. After 4 h, 1 mL of n-butyl acrylate was added, and after continuing the reaction for 1 h, it was passed through a normal-phase column (petroleum ether - ethyl acetate system). 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added and stirred for hydrolysis for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to the resulting mixed solution to adjust the pH to neutral, and the solvent was rotary evaporated. It was dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, the solvent was rotary evaporated, 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 h. After concentrating the organic solvent, it was passed through a reverse column and freeze-dried to obtain 240 mg of compound 6004. 1 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 16.6 Hz, 2H), 5.44 - 5.31 (m, 2H), 3.72 (d, J = 16.5 Hz, 4H), 3.31 (d, J = 5.6 Hz, 4H), 3.17 (s, 4H), 2.84 (s, 2H), 2.69 (s, 4H), 2.09 - 2.01 (m, 4H), 1.67 (d, J = 2.7 Hz, 10H), 1.37 - 1.26 (m, 24H), 0.91 (t, J = 6.8 Hz, 3H).

[0575] 5. Preparation of compound 6005

[0576] Structural formula:

[0577] Molecular weight: 597.54

[0578] Add 25.00 g of 11 - hentriacontanone, 62.05 g of ammonium acetate and 500 mL of methanol to a reaction flask and stir. Then add 6.55 g of sodium cyanoborohydride. After reacting for 16 h, add 250 mL of water and 250 mL of DCM. Separate the layers. Extract the aqueous phase with 50 mL of DCM and combine the organic phases. Pass through a normal phase column (methanol - DCM system) to obtain 21.00 g of compound 6005 - A.

[0579] Add 21.00 g of compound 6005 - A, 58 mL of n - butyl acrylate and 100 mL of n - butanol latex to a reaction flask and stir. Heat the mixture to 100 °C and add an additional 10 mL of n - butyl acrylate. After 16 h, pass through a column (petroleum ether - ethyl acetate system) to obtain 31.00 g of compound 6005 - B.

[0580] Prepare a solution by adding 31.00 g of compound 6005 - B, 6.55 g of sodium hydroxide, 310 mL of methanol and 31 mL of water to a reaction flask and stir. After 20 min, add 150 mL of THF and stir while heating to 50 °C. After 30 min, add a solution prepared by mixing 16.13 g of concentrated hydrochloric acid and 160 mL of methanol and stir. After 30 min, evaporate the solvent. Dissolve the residue in 300 mL of DCM, dry over anhydrous magnesium sulfate, filter, and evaporate the solvent to obtain 27.00 g of an oily compound 6005 - C.

[0581] Add 27.00 g of compound 6005 - C, 25.00 g of 4 - amino - 1 - butanol, 41.85 g of EDCI, 14.74 g of HOBT and 310 mL of DCM to a reaction flask and stir. After 18 h, concentrate the organic solvent and pass through a column (methanol - DCM system) to remove the 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).

[0582] 6. Preparation of compound 6006

[0583] Structural formula:

[0584] Molecular weight: 553.48

[0585] Add 1.00 g of oleylamine, 0.47 g of 2-(acryloyloxy)ethyl acrylamide, and 8 mL of ethanol to a reaction flask, stir and heat to 70 °C. After 13 h, rotary evaporate the solvent and perform column chromatography (methanol-DCM system) to obtain 800 mg of intermediate 1. Add 1.5 mL of n-butyl acrylate and 5 mL of n-butanol, stir and heat to 100 °C. After 3 h, rotary evaporate the solvent and perform column chromatography (methanol-DCM system). Add a solution prepared from 0.4 g of sodium hydroxide, 10 mL of methanol, and 1 mL of water, stir for 1 h, add a solution prepared from 1 mL of concentrated hydrochloric acid and 8 mL of methanol, stir for 30 min, rotary evaporate the solvent, add 100 mL of DCM to dissolve, dry over anhydrous magnesium sulfate, filter,

[0586] rotary evaporate, add 0.45 g of 6-amino-1-hexanol, 0.27 g of HOBT, 0.78 g of EDCI, and 10 mL of DCM, stir. After 14 h, concentrate the organic solvent and perform column chromatography (methanol-DCM system) to obtain 300 mg of compound 6006. 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,4H),2.01(dd,J=13.6,6.8Hz,4H),1.66-1.17(m,34H),0.89(t,J=6.7Hz,3H).

[0587] 7. Compound 6007

[0588] Structural formula:

[0589] Molecular weight: 953.78

[0590]

[0591] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then slowly add methyl acrylate (31.5 g) dropwise, stir to mix, and restore to room temperature. The reaction solution is reacted at room temperature for 4 h; concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL) and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight; concentrate under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (2 g) to a single-necked flask. After adding MeOH (20 mL) and stirring to dissolve, then slowly add methyl acrylate (2.5 g) and stir to mix. Heat to 60 °C and react overnight; stop the reaction, concentrate the reaction solution under reduced pressure, and purify by column chromatography (DCM:MeOH =

[0592] 50:1 - 20:1) to obtain compound 4 (2.5 g). At room temperature, add compound 4 (2.3 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature; stop the reaction and directly concentrate under reduced pressure to obtain compound 6007 (24 mg). 1HNMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0593] 8. Compounds 6008 - 6018

[0594] Synthesis of Compound 6009

[0595] Structural formula:

[0596] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise, under a nitrogen atmosphere, to a stirred solution of methyl methacrylate (6 mL) in methanol (20 mL). The reaction was carried out overnight. The reactants were dried by rotary evaporation at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. Then, a colorless oil was obtained by column chromatography. 1H-NMR (300 MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0597] Then, a solution of the above-mentioned colorless oil (11.05 g) in methanol (20 mL) was added to a vigorously stirred solution of 1,2-diaminoethane (75 g) in methanol (100 mL) at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure while maintaining the temperature not higher than 40 °C. The excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained and repeatedly recrystallized from chloroform and cyclohexane to give a white solid. 1H-NMR (300 MHz, CDCl3): 0.88 (t, 3H), 1.25 (s, 30H), 1.84 (s, 4H), 2.38 (m, 6H), 2.73 (m, 4H), 2.82 (m, 4H), 3.29 (m, 4H), 7.47 (s, 2H).

[0598] The preparation methods of 6008 and 6009 are different only in that: equimolar amounts of tetradecylamine are used instead of hexadecylamine. For 6010 - 6017, refer to the preparation method of 6009 and use equimolar amounts of the corresponding R a -NH2 to replace hexadecylamine for preparation.

[0599] For 6018, refer to the preparation method of 6007 and use equimolar amounts of the corresponding R a -NH2 to replace octadecylamine for preparation.

[0600] 9. Compound 6019

[0601] Structural formula:

[0602] Molecular weight: 753.66

[0603]

[0604] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask. After adding DCM (50 mL) and stirring to mix, compound 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, and the mixture was stirred and mixed. The reaction was carried out overnight at room temperature; it 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 flask. The hydrogen was replaced, the temperature was raised to 50 °C, and it was stirred for 1 h; the reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated by rotary evaporation to obtain product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. After adding DMF (50 mL) and stirring to dissolve, then product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred and mixed. The reaction was carried out overnight at room temperature; water (500 mL) and EA (500 mL) were added, and liquid-liquid extraction was carried out. The organic phase was dried over Na2SO4, concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain solid 6 (380 mg). At room temperature, solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to a single-necked flask. The reaction was carried out at room temperature for 2 h; it was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0605] 10. Compound 6020

[0606] Structural formula:

[0607] Molecular weight: 810.19

[0608]

[0609] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then, dropwise add methyl acrylate (31.5 g), stir to mix, restore to room temperature, and react for 4 h. Stop the reaction, directly concentrate under reduced pressure, and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight. Stop the reaction, concentrate the reaction solution under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, filter, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (1.3 g) to a single-necked flask. After adding DMF (20 mL) and stirring to mix, then add 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g), stir to mix, and react overnight at room temperature. Add water (200 mL) and directly freeze-dry under reduced pressure to obtain compound 4 (4 g). At room temperature, add compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) to a single-necked flask, and react at room temperature for 1 h. Stop the reaction, concentrate and purify the reaction solution under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57 - 3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03 - 1.58 (m, 10H), 1.31 (s, 30H), 1.01 - 0.82 (m, 3H).

[0610] 11. Compound 6021

[0611] Structural formula:

[0612] Molecular weight: 1182.79

[0613]

[0614] At room temperature, into a single-necked flask, add compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020), add MeOH (20 mL), stir and mix, then add TREN (23.0 g), stir and mix, heat up to 60 °C, stir overnight, concentrate under reduced pressure, and lyophilize the obtained crude product to obtain product 3 (25 g); at room temperature, into a single-necked flask, add product 3 (22.0 g), DCM (200 mL), cool down to 0 °C, slowly dropwise add (Boc)2O (71.7 g), after dropping, restore to room temperature, react for 3 h, add water (200 mL) to quench, then add DCM (100 mL) for liquid-liquid extraction, dry the organic phase with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography to obtain yellow oil 4 (5.8 g). At room temperature, into a single-necked flask, add yellow oil 4 (1.4 g), slowly add HCl / Dioxane (4M) (15 mL) under an ice-water bath, stir and mix, after adding, restore to room temperature, react for 0.5 h, concentrate under reduced pressure to obtain compound 5 (800 mg). At room temperature, into a single-necked flask, add compound 5 (1.0 g), (S)-2,6-di-tert-butoxycarbonylaminocaproic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL), react overnight at room temperature, add 100 mL of water and 100 mL of EA, perform liquid-liquid extraction, wash the EA phase once with 100 mL of saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). At room temperature, into a single-necked flask, add compound 6 (1.2 g), HCl / Dioxane (4M) (10 mL) and solvent DCM (10 mL), react overnight at room temperature, concentrate under reduced pressure, and purify to obtain compound 6021 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.96 (t, J = 6.6 Hz, 4H), 3.75 (dt, J = 14.0, 6.6 Hz, 4H), 3.62 - 3.45 (m, 12H), 3.36 - 3.33 (m, 14H), 3.25 - 3.17 (m, 2H), 3.04 - 2.90 (m, 8H), 2.83 (t, J = 6.9 Hz, 4H), 2.05 - 1.82 (m, 8H), 1.73 (p, J = 7.7 Hz, 10H), 1.53 (qd, J = 8.3, 7.7, 4.3 Hz, 8H), 1.30 (s, 28H), 0.97 - 0.87 (m, 3H).

[0615] 12. Compound 6023

[0616] Structural formula:

[0617] Molecular weight: 670

[0618]

[0619] At room temperature, add dodecylamine (10 g) to a three-necked flask. After adding MeOH (100 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Slowly add methyl acrylate (10.2 g) dropwise, stir to mix, and then restore to room temperature. Stop the reaction after the reaction solution has reacted at room temperature for 4 h. Concentrate under reduced pressure and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (19 g). At room temperature, add product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) to a single-necked flask. Heat the mixture to 60 °C and maintain this temperature to react overnight. Concentrate under reduced pressure to obtain compound 3 (20 g). Take 500 mg for purification and freeze-dry to obtain compound 6023-1 (218 mg). At room temperature, add starting material 6023-1 (1.4 g) to a single-necked flask. After adding DCM (20 mL) and stirring to dissolve, add (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g), stir to mix, and maintain at room temperature to react overnight. Separate by liquid-liquid extraction, dry, and concentrate under reduced pressure to obtain compound 4 (3.0 g). At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) to a single-necked flask and react at room temperature for 2 h; concentrate under reduced pressure, purify, and freeze-dry to obtain compound 6023 (123 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.88 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.36 - 3.33 (m, 2H), 3.31 - 3.14 (m, 4H), 3.04 - 2.93 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 2.00 - 1.82 (m, 4H), 1.82 - 1.66 (m, 6H), 1.50 (qd, J = 8.2, 7.8, 4.2 Hz, 4H), 1.45 - 1.25 (m, 18H), 0.96 - 0.87 (m, 3H).

[0620] Compound 6024

[0621] Structural formula:

[0622] Molecular weight: 870.24

[0623]

[0624] At room temperature, to a single-necked flask, add product 6023-1 (1.4 g, synthesized in the same method as 6023-1 in compound 6023). After adding MeOH (20 mL) and stirring until dissolved, slowly add methyl acrylate (2.8 g) and stir to mix. Heat up to 60 °C and react overnight at this temperature. Concentrate under reduced pressure to obtain compound 4 (2.0 g). At room temperature, add compound 4 (2.4 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature. Concentrate under reduced pressure and purify to obtain compound 6024 (191 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.69 (t, J = 6.0 Hz, 4H), 3.60 - 3.48 (m, 20H), 3.42 (t, J = 6.1 Hz, 4H), 3.25 - 3.19 (m, 2H), 3.12 (t, J = 5.8 Hz, 8H), 2.83 (t, J = 6.5 Hz, 12H), 1.78 (tt, J = 11.0, 6.4 Hz, 2H), 1.51 - 1.18 (m, 20H), 0.91 (t, J = 6.7 Hz, 3H).

[0625] 14. Compound 6026

[0626] Structural formula:

[0627] Molecular weight: 443.67

[0628]

[0629] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the only difference being that equimolar amounts of compound 1 were used to replace the raw material 5 of 6019, and equimolar amounts of N-(tert-butoxycarbonyl)ethanolamine were used to replace intermediate 4 of compound 6019. 1H NMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36 - 1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0630] Compounds 6025 - 6034 were obtained by referring to the synthetic routes of the aforementioned compounds, with the difference being that hydroxyethylamine was used to replace the corresponding ethylenediamine, and the amino group of hydroxyethylamine was first protected with protecting groups such as Fmoc or Boc, and the protecting group was removed by conventional methods after the reaction was completed.

[0631] (3) Exemplary compounds of the first ionizable lipid and their properties

[0632] 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, respectively.

[0633] The calculated c-pKa (molnetwork) and LogP (cLogP driver) of the exemplary compounds of the present invention, and the c-pKa and cLogP values are generated by the ChemDraw module of Chemoffice.

[0634] Properties of exemplary compounds of the first ionizable lipid in Table 1

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661] The above properties of the exemplary compounds of the first ionizable lipid of the present invention indicate that they can be used as excellent surfactants, especially suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0662] (4) Preparation of the application of the prefabricated carrier in the preparation of products for in vitro gene delivery to immune cells and stem cells

[0663] 4.1 Preparation of the prefabricated carrier (LNP)

[0664]

[0665]

[0666] The prefabricated carrier is prepared respectively according to the above molar ratios, and the specific method is as follows:

[0667] Dissolve the lipid raw materials in ethanol to obtain a lipid ethanol solution. The total concentration of all lipid raw materials in ethanol is 8 mg / mL. After mixing the lipid ethanol solution with a 50 mM citric acid buffer (pH 4.0) solution in a volume ratio of 1:3 in a nano-preparation device, ultrafiltration is carried out, and the sample is collected to obtain the prefabricated carrier with a lipid concentration of 2 mg / mL.

[0668] 4.2 Preparation of the composition based on the prefabricated carrier

[0669]

[0670] Using nucleic acid as the active ingredient, configure the nucleic acid into a solution with a concentration 2 times that of each example using nuclease-free water as Solution 1; dilute the prefabricated carrier according to the prescription ratio of each example using nuclease-free water as Solution 2; mix Solution 1 and Solution 2 in equal volume and vortex for 2 - 3 s to obtain the composition based on the prefabricated carrier.

[0671] (5) Determination of the particle size and Zeta potential of the composition based on the prefabricated carrier

[0672] 1. Determination of particle size and polydispersity index (PDI): The average particle size and PDI of the sample solution in the prefabricated carrier-based composition of the examples were determined by dynamic light scattering using a Malvern ZetaSizer Nano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0673] 2. The encapsulation efficiency test method is as follows:

[0674] According to the manufacturer's instructions, the encapsulation efficiency of mRNA was determined using a Quant-it Ribogreen RNA quantification assay kit (ThermoFisher Scientific, UK).

[0675] According to the manufacturer's instructions, the encapsulation efficiency of pDNA in the prefabricated carrier-based composition was determined using a dsDNA HS kit (Novoprotein / EQ121).

[0676] According to the manufacturer's instructions, using the microRNA Reagent kit, the encapsulation efficiency of siRNA in the prefabricated carrier-based composition was determined.

[0677] The average particle size, PDI, and encapsulation efficiency data of the prefabricated carrier-based composition prepared in the examples are shown in Table 2.

[0678] Table 2 Summary of particle size, PDI, and encapsulation efficiency of the prefabricated carrier-based composition

[0679] Example number Particle size nm PDI Entrapment efficiency % 1 190.6 0.146 82.1 2 246.7 0.156 90.5 3 160.4 0.168 96.2 4 124.3 0.118 84.6 5 156.1 0.137 89.4 6 124.8 0.117 94.6 7 114.3 0.105 96.7 8 448.7 0.201 82.3 9 172.6 0.189 85.8 10 215.6 0.179 84.2 11 346.7 0.193 85.6 12 120.7 0.137 85.1 13 426.7 0.204 80.8 14 246.7 0.173 88.7 15 147.4 0.156 83.7 16 190.6 0.172 90.7

[0680] As can be seen from Table 2, the prefabricated carrier-based composition provided by the present invention has a small and uniform particle size and a high encapsulation efficiency (>80%).

[0681] (6) Evaluation of the in vitro immune cell delivery effect of the prefabricated carrier-based composition

[0682] 6.1 Human NK cells (NK-92)

[0683] The human NK cells (NK-92) were used to evaluate the in vitro delivery efficiency of the prefabricated carrier-eGFP mRNA complexes in Example 3 and Example 6. The cells were cultured in a special medium for NK-92 to ensure that the cells were in the logarithmic growth phase. One day before delivery, the cells were seeded onto a 24-well culture plate at an appropriate cell density and allowed to grow overnight. At the time of delivery, the prefabricated carrier-nucleic acid complexes prepared in Example 3 and Example 6 were diluted with buffer and added to the 24-well cell culture plate so that the amount of eGFP-mRNA in each well reached 500 ng and 1000 ng respectively. Lipofectamine 2000 was used to deliver eGFP mRNA as a control. After culturing in a 37 °C, 5% CO2 incubator for 24 h, the cell viability and delivery efficiency were detected by flow cytometry. The results are shown in Table 3.

[0684] Table 3 In vitro delivery efficiency of prefabricated carrier-nucleic acid complexes to human NK cells

[0685]

[0686] The results showed that the prefabricated carrier-eGFP mRNA complexes provided by the present invention all had high protein expression efficiency in NK-92 cells.

[0687] 6.2 Application in human MSC cells

[0688] The human MSC cells were used to evaluate the in vitro transfection efficiency of the prefabricated carrier-eGFP mRNA complexes in Example 9. The cells were cultured in a special medium to ensure that the cells were in the logarithmic growth phase. One day before delivery, the cells were seeded onto a 24-well culture plate at an appropriate cell density and allowed to grow overnight. At the time of delivery, the prefabricated carrier-eGFP mRNA complexes prepared in Example 20 were diluted with buffer and added to the 24-well cell culture plate so that the amount of eGFP-mRNA in each well reached 250 ng / 500 ng respectively. Lipofectamine 2000 was used to deliver eGFP mRNA as a control. After culturing in a 37 °C, 5% CO2 incubator for 24 h, the cell viability and delivery efficiency were detected by flow cytometry. The results are shown in Table 4.

[0689] Table 4 In vitro delivery efficiency of prefabricated carrier-nucleic acid complexes to human MSC cells

[0690]

[0691] The results showed that the prefabricated carrier-eGFP mRNA complexes provided by the present invention all had high protein expression efficiency in human MSC cells.

[0692] 6.3 Application in T cells

[0693] The human T lymphocyte leukemia cells (Jurkat) were used to evaluate the in vitro delivery efficiency of the prefabricated carrier-eGFP mRNA complexes in Examples 7, 11 and 14. The cells were cultured in RPMI-1640 medium to ensure that the cells were in the logarithmic growth phase. One day before delivery, the cells were seeded onto a 24-well culture plate at an appropriate cell density and grown overnight. At the time of delivery, the prefabricated carrier-eGFP mRNA complexes of Examples 7, 11 and 14 were diluted with buffer and added to the 24-well cell culture plate, so that the amount of eGFP-mRNA per well reached 500 ng / 1000 ng respectively. After culturing in a 37 °C, 5% CO2 incubator for 24 h, the cell viability and delivery efficiency were detected by flow cytometry, and the results are shown in Table 5.

[0694] Table 5 In vitro delivery efficiency of prefabricated carrier-nucleic acid complexes to T cells

[0695]

[0696]

[0697] The results showed that the prefabricated carrier-eGFP mRNA complex provided by the present invention had a high protein expression efficiency in T cells and could be applied to T cell delivery.

[0698] 6.4 Primary T cells

[0699] The primary T cells isolated from human peripheral blood were used to evaluate the in vitro delivery efficiency of the prefabricated carrier-eGFP mRNA complex in Example 11. The cells were cultured in a special medium and activated with CD3 / CD28 to ensure that the cells were in the logarithmic growth phase. The cells were seeded onto a 24-well culture plate at an appropriate cell density and grown overnight. The CD25 positive rate of the cells before delivery must be greater than 60%. At the time of delivery, the prefabricated carrier-eGFP mRNA complex of Example 11 was diluted with buffer and added to the 24-well cell culture plate, so that the amount of eGFP-mRNA per well reached 500 ng / 1000 ng respectively. After culturing in a 37 °C, 5% CO2 incubator for 24 h, the cell viability and delivery efficiency were detected by flow cytometry, and the results are shown in Table 6.

[0700] Table 6 In vitro delivery efficiency of prefabricated carrier-nucleic acid complexes to primary T cells

[0701]

[0702] The results showed that the prefabricated carrier-eGFP mRNA complex provided by the present invention had a high protein expression efficiency in primary T cells and could be applied to primary T cell delivery.

[0703] The solution of the present invention is not limited to the technical means disclosed above, but also includes technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art of the present invention, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. Use of a prefabricated carrier in the preparation of a product for in vitro gene delivery to immune cells and stem cells, characterized in that, Comprising the step of mixing a prefabricated carrier and a nucleic acid in a solvent to obtain a prefabricated carrier-based composition; the composition of the prefabricated carrier includes: 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 compounds having the general formula (1E) or pharmaceutically acceptable salts, stereoisomers, and tautomers thereof; wherein, R1, R2, and R3 are each independently H, C 5-40 a straight-chain or branched-chain alkyl group, C 5-40 a straight-chain or branched-chain alkenyl group, C 5-40 a straight-chain or branched-chain alkynyl group, a 3- to 6-membered saturated or partially unsaturated cycloalkyl group having 1 to 3 side chains, or a 6- to 10-membered aromatic group having 1 to 3 side chains; the side chains are independently selected from C 10-30 a straight-chain or branched-chain alkyl group, C 10-30 a straight-chain or branched-chain alkenyl group, C 10-30 a straight-chain or branched-chain alkynyl group; provided that at most one of R1, R2, and R3 is H; M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, and a 6-10 membered heteroaryl group containing at least one nitrogen atom, and the heterocyclic group and heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amine, oxo, or halogen groups; R4 and R5 are each independently H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group, where the C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, amino, amide, amidino, guanidine or halogen groups; G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 is independently selected from H, hydroxy, C 1-30 a linear or branched alkyl or cycloalkyl, C 2-30 a linear or branched alkenyl; R 9、 R 10 Each is independently H; X2 is selected from -O-, -S-, NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-; R 11、 R 12 each independently is H, C 1-6 a linear or branched alkyl or cycloalkyl group; Each R 16 , R 17 are each independently selected from H, C 1-30 a linear or branched alkyl or cycloalkyl, C 2-30 a linear or branched alkenyl; Among them, the alkyl, cycloalkyl, and alkenyl groups described for R4, R5, R6, R 11 , R 12 , R 13 , R 16 , R 17 are unsubstituted or substituted by one or more groups selected from hydroxyl, mercapto, amino, substituted amino, and halogen; n is an integer from 2 to 6; k is an integer from 1 to 6; The salt does not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof: Wherein, A1 is NH; R a Selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkyl alcohol, C6-C 24 cycloalkyl; the C6-C 24 alkyl is a branched structure; the C6-C 24 alkenyl, C6-C 24 alkyl alcohol is a straight-chain or branched structure; R b and R c are each independently selected from C1-C 12 alkyl alcohol.

2. The application according to claim 1, characterized in that The application further includes the step of contacting the prefabricated carrier-based composition with immune cells or stem cells.

3. The application according to claim 2, wherein An adjuvant is further added to the product for in vitro gene delivery to immune cells and stem cells.

4. The application according to claim 3, characterized in that, The adjuvant includes at least one of a lipid, a polypeptide, a lipopolyamine, or a synthetic polymer.

5. The application according to claim 1, characterized in that, The nucleic acid is DNA and / or RNA.

6. The application 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 microcircles, msDNA.

7. The application according to claim 1, characterized in that The dosage of the nucleic acid is 0.1%-50% (w / w) of the total amount of the prefabricated carrier and the nucleic acid; in the prefabricated carrier-based composition, the concentration of the nucleic acid is 5-1000 ng / μl.

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

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

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

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

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

13. The application according to claim 1, characterized in that, The compound of formula (1E) is selected from the compounds shown in formula (1F):

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

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

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

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

18. The application according to claim 1, wherein The prefabricated carrier-based composition further includes 0-60 mol% of other ionizable lipids, and the other ionizable lipids are selected from at least one of the following compounds:

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

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

21. The application according to claim 20, wherein The PEG-conjugated lipids include at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, glycerol dipalmitate-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosanylethanamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol lipids.

22. The application according to claim 1, wherein In the preformed carrier-based composition, the nucleic acid is encapsulated inside the preformed carrier and / or adsorbed on the surface of the preformed carrier to form a complex.

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

24. The application according to claim 23, wherein The pH of the buffer salt solution is 1-9, the concentration of the buffer salt is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration <50%.

25. The application according to claim 24, characterized in that, The buffer salt solution is selected from at least one of citrate solution, acetate solution, tartrate solution, phosphate solution, carbonate solution, Tris-HCl solution, and sodium chloride solution.

26. The application according to claim 1, characterized in that, At least one of sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the preformed carrier-based composition.

27. An in vitro gene delivery method for immune cells and stem cells, characterized in that, It includes the step of mixing the preformed carrier and the nucleic acid in a solvent to obtain the preformed carrier-based composition; the preformed carrier is the preformed carrier described in claim 1.

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