Pulmonary delivery system containing ionizable lipids, preparation method and use thereof

Abstract

The present invention provides a pulmonary delivery system containing ionizable lipids, a preparation method thereof, and an application thereof, and belongs to the field of biomedical technology. The pulmonary delivery system containing ionizable lipids of the present invention comprises ionizable lipids, auxiliary lipids, and bioactive substances, and does not contain polymer-conjugated lipids. The pulmonary delivery system containing ionizable lipids can deliver bioactive substances to the lungs. The pulmonary delivery system has the characteristics of uniform particle size and high safety, and can efficiently deliver therapeutic agents to the lungs. Before and after atomization, the particle properties such as particle size, particle size distribution coefficient, potential, encapsulation efficiency and other particle characteristics remain unchanged, providing a safe and effective solution for the treatment of lung diseases, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pulmonary delivery system containing ionizable lipids, a preparation method and application thereof. Background Art

[0002] Pulmonary administration, also known as inhalation administration, is mainly achieved through inhalation into the airways through the throat, ultimately reaching the deep respiratory tract or the lungs, exerting a local or systemic therapeutic effect. Compared with other routes of administration, pulmonary administration has the advantages of a large absorption area, a thin alveolar epithelial cell membrane, and high permeability. The absorption site has abundant blood flow and relatively low enzyme activity, which can avoid the first-pass effect in the liver. Therefore, the bioavailability of pulmonary administration is high. For drugs that are easily destroyed in the gastrointestinal tract or have a strong first-pass effect in the liver, such as proteins or peptide drugs, pulmonary administration can significantly improve bioavailability.

[0003] Respiratory epithelial cells line the respiratory tract, and their primary function is to moisten the airways, protect them from potential pathogens, infections, and tissue damage, and facilitate gas exchange. Dysfunction of airway epithelial cells can lead to many conditions, including, for example, asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Delivery of payloads to respiratory epithelial cells can be used to induce immunity against antigens of interest and modulate the function of airway epithelial cells, such as replacing missing or mutated proteins or increasing or decreasing the functionality of such cells. Taking cystic fibrosis as an example, statistics show that the incidence of cystic fibrosis at birth in Caucasians is 1 in 2,500 to 3,500 people, and most current treatments for cystic fibrosis only prescribe antibiotics, anti-inflammatory drugs, bronchodilators, decongestants, high-protein and high-fat diets, and vitamin supplements to control symptoms, but cannot completely or reliably control the disease.

[0004] Therefore, there is an urgent need to develop new therapies for conditions associated with airway epithelial cell dysfunction or other difficult lung-related diseases. Inhaled aerosol formulations can deliver drugs directly to the lungs, significantly increasing drug concentration in the lungs and reducing systemic toxicity, an inherent advantage of respiratory drug delivery. However, the development of inhaled formulations, particularly inhaled nucleic acid LNP formulations, faces the challenge of poor aerosolization. This is due to the inability to withstand shear damage during aerosolization, leading to leakage of nucleic acid drugs during aerosolization, a significant increase in particle size and dispersion after aerosolization, and a significant decrease in transfection efficiency after aerosolization. Therefore, the primary key to developing aerosolized inhaled LNP nucleic acid drugs is to overcome the destructive effects of the aerosolization process on the LNPs. Currently, there are few patents and literature on aerosolized inhaled LNP nucleic acid drugs. Most reports have screened formulations for minimal changes in particle size, PDI, and encapsulation efficiency before and after aerosolization by varying the ratio of the four LNP components. However, this typically results in a significant increase in particle size and a significant decrease in encapsulation efficiency after aerosolization.

[0005] Patent CN116348147A discloses a composition for delivering payload molecules to the airway epithelium. The composition comprises a nanoparticle core formed from an ionizable lipid, phospholipid, structured lipid, PEG-lipid, polynucleic acid, or polypeptide, and a cationic agent located on the outer surface of the core. The specification discloses the use of administration procedure B (nasal aerosol exposure only), which uses a vibrating mesh nebulizer and a defined inlet air flow rate to generate aerosols for exposure to the animal's nose and mouth. The specification mentions monitoring the aerodynamic particle size distribution of the aerosol at the administration port before and after each administration, and evaluating the target respirable aerosol particle size, which is 1-4 μm for rats. It is speculated that the particle size of the composition provided in this patent increases to the micron level after atomization.

[0006] Patent CN115708818B discloses a universal formulation for aerosolized inhalation of lipid nanoparticles, comprising a lipid nanodispersion system and a surface tension-reducing modifier. While the universal formulation for lipid nanoparticles provided in this patent can maintain the particle size and encapsulation efficiency before and after aerosolization, it requires the addition of an organic solvent or surfactant as a surface tension modifier to reduce damage to the LNPs during aerosolization. However, these organic solvents or surfactants can increase cellular toxicity and reduce cellular tolerance.

[0007] Despite the success of lipid nanoparticles in delivering mRNA vaccines, inhaled mRNA therapies for lung diseases remain challenging. This is because LNPs are susceptible to shear stress during aerosolization, resulting in ineffective mRNA delivery. Therefore, there is an urgent need to improve the stability of LNP structures to meet the requirements of aerosolized drug delivery. Summary of the Invention

[0008] The present invention addresses the problems existing in the prior art and provides a pulmonary delivery system containing ionizable lipids, a preparation method thereof, and applications thereof. The pulmonary delivery system containing ionizable lipids of the present invention comprises ionizable lipids, auxiliary lipids, and bioactive substances, and does not contain polymer-conjugated lipids. The pulmonary delivery system containing ionizable lipids is delivered to the lungs by aerosol administration. The pulmonary delivery system has the characteristics of uniform particle size and high safety, and can efficiently deliver therapeutic agents to the lungs. Before and after aerosolization, the particle properties such as particle size, particle size distribution coefficient, potential, encapsulation efficiency and other particle characteristics remain unchanged, providing a safe and effective solution for the treatment of lung diseases.

[0009] To achieve the above objectives, in a first aspect, the present invention provides a pulmonary delivery system containing an ionizable lipid, comprising an ionizable lipid, a helper lipid, and a bioactive substance, wherein the pulmonary delivery system containing the ionizable lipid does not contain a polymer-conjugated lipid; the ionizable lipid is selected from at least one of a first ionizable lipid, a second ionizable lipid, and a third ionizable lipid;

[0010] The first ionizable lipid is selected from a compound comprising the general formula (1) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof;

[0011]

[0012] Wherein, R1, R2, and R3 are independently H, C 5-40 Straight or branched alkyl, C 5-40 Straight-chain or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10-30 Straight-chain or branched alkyl, C 10 -30 straight chain or branched alkenyl, C 10-30 Straight or branched alkynyl; provided that at most one of R1, R2, and R3 is H;

[0013] M is selected from -NR4R5, a saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, a 6-10 membered heteroaryl group containing at least one nitrogen atom, wherein the heterocyclic group and the heteroaryl group are unsubstituted or substituted with one or more -OH, carboxyl, amino, oxo or halogen;

[0014] R4 and R5 are independently H, C 1-6 Straight-chain or branched alkyl, C 2-6 Straight-chain or branched alkenyl or C 2-6 Straight chain or branched chain alkynyl, the C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, aminoamide, amidino, guanidino or halogen;

[0015] G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13-, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS (=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; where each R6, R 13 independently selected from H, hydroxyl, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0016] L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 14 -、-NR 14 -S(=O)2-、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-; wherein m is an integer of 2-6, R7, R8 are independently H, hydroxyl, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 14 、R 15 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0017] L2 is -(CR9R 10 )n -or-(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR 16 C(=O)NR 17 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR 16 -、NR 16 -S(=O)2-、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-; n is an integer from 1 to 6; k is an integer from 1 to 6; R9, R 10 、R 11 、R 12 are independently H, hydroxy, halogen, C 1-6 Straight or branched alkyl or cycloalkyl, C 2-6 A straight-chain or branched alkenyl group, each R 16 、R 17 independently selected from H, C 1-30 Straight-chain or branched alkyl or cycloalkyl, C 2-30 straight-chain or branched alkenyl;

[0018] Where R4 to R 17 The alkyl, cycloalkyl, and alkenyl groups are unsubstituted or substituted by one or more groups selected from hydroxyl, thiol, amino, substituted amino, and halogen;

[0019] The salts do not include quaternary ammonium salts.

[0020] In a preferred embodiment, the bioactive substance comprises at least one of DNA, RNA, protein, peptide and small molecule compound.

[0021] In a preferred embodiment, the amount of the bioactive substance is 0.1-50% (w / w), more preferably 1-50% (w / w), of the total amount of the lipid material and the bioactive substance.

[0022] In a preferred embodiment, R1, R2, and R3 are independently the following groups:

[0023]

[0024] Where Y does not exist or is C 1-30 Straight chain or branched chain alkyl or cycloalkyl, C 2-20 Straight-chain or branched alkenyl, C 2-20 Straight or branched alkynyl; R1', R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of Y, R1' and R2' is 8-40.

[0025] In a preferred embodiment, R1, R2, and R3 are independently selected from the following groups:

[0026]

[0027] Wherein, R1' and R2' are independently H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

[0028] In a preferred embodiment, R1, R2, and R3 are independently selected from any one of the following groups:

[0029]

[0030] In a preferred embodiment, G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

[0031] In a preferred embodiment, L1 is selected from -(CR7R8) m-X1-, wherein X1 is selected from -O-, -S-, -NR 14 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -、-NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -、-NR 14 C(=O)NR 15 -、-P(=O)(OR 14 )O-、-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-.

[0032] In a preferred embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, wherein X2 is selected from -O-, -S-, -NR 16 -, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -、-NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -、-NR 16 C(=O)NR 17 -、-P(=O)(OR 16 )O-、-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-.

[0033] In a preferred embodiment, M is selected from the following structures:

[0034]

[0035] wherein m' and n' are independently integers of 0-6, R1" and R2" are independently H, C 1-6 Alkyl, C 2-6The alkenyl, guanidino, amidino, amide, fatty amine, 3-10 membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and tautomeric forms thereof, which are unsubstituted or optionally substituted with one or more selected from hydroxyl, thiol, amino, substituted amino, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 The aromatic group is substituted with an organic group.

[0036] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1A):

[0037]

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

[0039]

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

[0041]

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

[0043]

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

[0045]

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

[0047]

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

[0049]

[0050] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1H):

[0051]

[0052] In a preferred embodiment, M is selected from any one of the following groups:

[0053]

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

[0055]

[0056] In a preferred embodiment, the compound of formula (1) is selected from the compound represented by formula (1J):

[0057]

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

[0059]

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

[0061]

[0062] Wherein R1', R2' are independently selected from H, C 1-30 Straight-chain or branched alkyl, C 2-30 Straight-chain or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-40.

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

[0064]

[0065] In a preferred embodiment, the compound of formula (1) is selected from:

[0066]

[0067]

[0068]

[0069]

[0070]

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[0140]

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[0142]

[0143]

[0144] 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):

[0145]

[0146] 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.

[0147] 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):

[0148]

[0149] 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.

[0150] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0151]

[0152] 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.

[0153] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the steps of reacting a compound of formula (VII) with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X) in sequence:

[0154]

[0155] wherein Xg, Xh, Xi, Xj, Xk, and Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G1 through a nucleophilic reaction or a condensation reaction, Xh and Xk form G2 through a nucleophilic reaction or a condensation reaction, and Xi and Xl form G3 through a nucleophilic reaction or a condensation reaction.

[0156] 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):

[0157]

[0158] Steps for reacting the compound of formula (XIII) with the compound of formula (XII):

[0159]

[0160] wherein Xm is a group containing a nucleophilic group, and Xm and C=CL of the compound of formula (XII) 2a X1-L2 is formed by addition reaction.

[0161] In a preferred embodiment, R1, R2, and R3 are independently of one another the following groups:

[0162]

[0163] Wherein, Y, R1', and R2' have the same meanings as above;

[0164] It also includes the steps of forming tail chains R1, R2, and R3:

[0165]

[0166] Wherein X is a leaving group.

[0167] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0168]

[0169] Wherein N1 is NH or O;

[0170] R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short chain polyethylene oxide; the C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl, C6-C 24 Alcohol, C6-C 24 Short-chain polyoxyethylene is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C 12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0171] The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, Rc " is selected from C1-C6 alkyl groups which are unsubstituted or substituted with amino groups, R c ”' is H, or -R c '-A1'-R c ”-NH2;

[0172] Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

[0173] In a preferred embodiment, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines.

[0174] In a preferred embodiment, provided that, when R a For straight chain C6-C 24 When alkyl, N1 is not NH, or R b and R c Not C1-C 12 Alcohol; when R a C6-C 24 When alcohol, N1 is not O, or R b and R c Not C6-C 24 alkyl.

[0175] In a preferred embodiment, the condition is that when R a C6-C 24 In the case of alkenyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0176] When R a C6-C 24 When alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines;

[0177] R a For straight chain C6-C 24 When alkyl, A1 is NH or O, R b and R c Each independently selected from alkylamine; when R a For straight chain C6-C 24 When it is alkyl, A1 is O, R band R c Each independently selected from C1-C 12 Alkyl alcohol; when R a C6-C 24 In the case of short-chain polyoxyethylene, A1 is NH or O, R b and R c are each independently selected from alkylamines; or when R a C6-C 24 In the case of alkyl alcohol, A1 is NH or O, R b and R c Each is independently selected from alkylamines.

[0178] In a preferred embodiment, R a Selected from the following compound structures:

[0179]

[0180] In a preferred embodiment, R b and R c Selected from the following compound structures:

[0181]

[0182] R d Selected from C1-C6 alkanes or cycloalkanes.

[0183] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0184]

[0185]

[0186]

[0187] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0188] R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form ionizable lipid compound③:

[0189]

[0190] Among them, R a -NH2 is R a -NH2 or

[0191] R b ' and R b "At the same time H, or R b' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0192] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0193] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤;

[0194] 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③;

[0195]

[0196] Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a -NH2 is selected from R a -NH2 or

[0197]

[0198] R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

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

[0200] 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound② to form compound⑦;

[0201] 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨;

[0202] 3) Compound ⑨ reacts with nucleophile ⑩ to form an ionizable lipid compound

[0203]

[0204] Among them, R a-NH2 is R a -NH2, Z3 is a leaving group, A3 reacts with Z3 to obtain A1.

[0205] In a preferred embodiment, in Formula 2a-Formula 2c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to generate the ionizable lipid compound with two forks containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0206] In a preferred embodiment, Formula 2a-Formula 2b further comprises the step of using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a-Formula 2b as a starting material, and performing an iterative reaction according to Step 1 in Formula 2a or Steps 1-2 in Formula 2b;

[0207] The compound of formula (2) with a terminal amino group is

[0208] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0209] In a preferred embodiment, the reaction formula 2a-2c further comprises using the compound of formula (2) with a terminal amino group obtained by the reaction formula 2a-2c as a raw material, and The steps of the reaction;

[0210] The compound of formula (2) with a terminal amino group is

[0211] Among them, R b ' and R b "At the same time H, or R b ' and R b " is also a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2.

[0212] The leaving group mentioned above refers to the leaving part in the nucleophilic reaction or condensation reaction, including but not limited to: H,

[0213] OH, H2O, halogen (such as F, Cl, Br and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy (RO-), amino (-NHR, wherein R is an alkyl or aryl group with H removed), phenoxy, tertiary carbon cation (such as tert-butyl cation), carbon cation stabilized by an unsaturated system or a heteroatom, or the various protecting groups mentioned above.

[0214] The nucleophilic groups mentioned above refer to molecules or ions that can provide electron pairs to form new chemical bonds in chemical reactions. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, nucleophilic halogens (such as Cl - Br - or I - ), hydrogen ions (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxy anion (such as alcohol with hydroxyl hydrogen removed), amine group (including primary amine, secondary amine and tertiary amine) or amine anion, carbon anion (such as carbon anion in organometallic reagents such as Grignard reagent, organic lithium reagent, Gilman reagent), sulfhydryl or sulfhydryl anion, sulfide, enol or enol anion, alkenyl ether, enamine, carboxylic acid or carboxylic acid anion, alkyl or aryl phosphine (such as triphenylphosphine), aromatic heterocycle with lone pair electrons (such as pyridine), etc.

[0215] In a preferred embodiment, the raw materials used in the reaction process also contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0216] In a preferred embodiment, the third ionizable lipid is selected from at least one of the following compounds:

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] In a preferred embodiment, the helper lipid is a mixture of phospholipid and steroid or its derivatives.

[0223] In a preferred embodiment, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl at least one of 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0224] In a preferred embodiment, the steroid or its derivative comprises at least one of cholesterol, cholesterol stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, salinarate, tomatine, ursolic acid, and α-tocopherol.

[0225] In a preferred embodiment, the pulmonary delivery system comprising an ionizable lipid further comprises an alcohol compound.

[0226] In a preferred embodiment, the alcohol compound comprises at least one of C6-C18 alcohol and polyethylene glycol; the molar ratio of the alcohol compound to the ionizable lipid is 0-2:1, more preferably 0.01-0.5:1.

[0227] In a preferred embodiment, the amount of the ionizable lipid is 5-80 mol% of the total lipids in the prescription; the amount of the auxiliary lipid is 20-95 mol% of the total lipids in the prescription.

[0228] In a preferred embodiment, the molar ratio of phospholipid to steroid or its derivative in the helper lipid is (5-60):(10-80).

[0229] In a preferred embodiment, the ionizable lipid is the first ionizable lipid or the second ionizable lipid or the third ionizable lipid, or a mixture of the first ionizable lipid and the second ionizable lipid, or a mixture of the second ionizable lipid and the third ionizable lipid; in the mixture of the first ionizable lipid and the second ionizable lipid, the molar ratio of the first ionizable lipid to the second ionizable lipid is (5-90):(1-30); in the mixture of the second ionizable lipid and the third ionizable lipid, the molar ratio of the third ionizable lipid to the second ionizable lipid is (5-90):(1-30).

[0230] In a preferred embodiment, the particle size of the pulmonary delivery system containing ionizable lipids is 20-400 nm and the polydispersity index (PDI) is less than 0.5.

[0231] In a preferred embodiment, the surface potential of the pulmonary delivery system containing ionizable lipids is -20 mV to 60 mV.

[0232] In a preferred embodiment, the pulmonary delivery system containing ionizable lipids further comprises other excipients.

[0233] In a preferred embodiment, the excipient is a pharmaceutically acceptable excipient, including but not limited to buffer salts, carriers, diluents, absorbents, colorants, adjuvants, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, lubricants, dispersants, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0234] In a preferred embodiment, the pulmonary delivery system containing the ionizable lipid is in liquid form or solid form.

[0235] In a second aspect, the present invention provides a method for preparing the aforementioned pulmonary delivery system containing ionizable lipids, comprising the step of mixing a bioactive substance, an ionizable lipid, and an auxiliary lipid in a solvent.

[0236] In a preferred embodiment, the preparation method includes dissolving ionizable lipids and auxiliary lipids in a solvent to obtain a lipid solution; dissolving a bioactive substance in a solvent to obtain a bioactive substance solution; mixing the lipid solution and the bioactive substance solution, and ultrafiltration.

[0237] In a preferred embodiment, the solvent comprises at least one of alcohol, water, and a buffer solution.

[0238] In a third aspect, the present invention provides use of the aforementioned pulmonary delivery system containing ionizable lipids in the preparation of a medicament for delivering a bioactive substance via intranasal, intrabronchial or pulmonary administration.

[0239] In a preferred embodiment, the medicament is administered to a mammal.

[0240] In a preferred embodiment, the drug is administered to a cell.

[0241] In a preferred embodiment, the cell is at least one of a respiratory epithelial cell population, a nasal cell population, an alveolar epithelial cell population, a lung cell population, a bronchial epithelial cell population, and a human bronchial epithelial (HBE) cell population.

[0242] In a preferred embodiment, the drug is in liquid form.

[0243] In a preferred embodiment, the drug is in solid form

[0244] In a preferred embodiment, the medicament is suitable for inhalation.

[0245] In a preferred embodiment, the medicament is administered by a nebulizer or inhaler.

[0246] Compared with the prior art, the present invention has the following beneficial effects:

[0247] 1. The pulmonary delivery system containing ionizable lipids of the present invention comprises ionizable lipids, auxiliary lipids and bioactive substances, and does not contain polymer-conjugated lipids. The pulmonary delivery system containing ionizable lipids can be used for pulmonary administration, and the administration method can be intratracheal administration or nebulized inhalation administration. Both administration methods can efficiently deliver the bioactive substances to the lungs, and have good expression in the lungs, but no expression in other organs.

[0248] 2. The pulmonary delivery system containing ionizable lipids of the present invention has a particle size of 20-400 nm, a PDI < 0.5, and a potential of -20 to 60 mV, and is characterized by uniform particle size and high safety.

[0249] 3. The preparation process of the pulmonary delivery system containing ionizable lipids provided by the present invention is simple and has good process reproducibility.

[0250] 4. The pulmonary delivery system containing ionizable lipids of the present invention can efficiently deliver therapeutic agents to the lungs, and the particle properties such as particle size, particle size distribution coefficient, potential, encapsulation efficiency and other particle characteristics remain unchanged before and after atomization, providing a safe and effective solution for the treatment of lung diseases.

[0251] 5. The present invention discloses a pulmonary delivery system containing ionizable lipids and its aerosolized administration method, which has excellent safety and effectiveness. For example, disease treatment is achieved through cellular aggregation of nanolipids, expression of desired proteins and / or expression of mRNA in cells. This provides a new possibility for the treatment of pulmonary diseases and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0252] Figure 1 This is the in vivo imaging of mice administered intratracheal drug;

[0253] Figure 2 This is the in vivo imaging of mice receiving drug via aerosol inhalation;

[0254] Figure 3 Schematic diagram of IVIS imaging;

[0255] Figure 4 This is an IVIS imaging diagram of the lung distribution after spray administration of Example 6. DETAILED DESCRIPTION

[0256] The synthesis process of the present invention can accommodate multiple functional groups, so a variety of substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, but in some cases it may be necessary to further convert the compound into a pharmaceutically acceptable salt thereof. The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from easily prepared intermediates by using standard synthetic methods and procedures known to those skilled in the art or obvious to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for preparing organic molecules, as well as functional group transformations and manipulations, can be obtained from the relevant scientific literature or from standard textbooks in this field. The following description of the synthetic methods is designed to illustrate, but not to limit, the general procedures for preparing the compounds of the present invention.

[0257] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables in each general synthetic route (e.g., R1, R2, and R3, etc.) are as defined herein. One of ordinary skill in the art will note that in the reaction procedures and synthetic schemes described herein, the order of certain steps may be varied, such as the introduction and removal of protecting groups.

[0258] In the reaction schemes described herein, a variety of stereoisomers can be produced. When a specific stereoisomer is not indicated, this is understood to include all possible stereoisomers produced by the reaction. One of ordinary skill in the art will recognize that the reaction can be optimized to preferentially obtain a single isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0259] (1) Synthesis of the first ionizable lipid in Example 1

[0260] The general synthetic route for the first ionizable lipid is as shown in general synthetic route 1-5.

[0261] General synthetic route 1

[0262]

[0263] Wherein, M' is M or M containing a protecting group.

[0264] As described in the general synthetic route 1 above, Boc-aminotris(hydroxymethyl)methane reacts with an acid (Compound 2) to form Compound 3. Step 1 can be carried out in an organic solvent (e.g., dichloromethane (DCM)) in the presence of, for example, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 4-dimethylaminopyridine (DMAP). Step 1 can be carried out at room temperature for 24 hours.

[0265] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0266] Next, compound 4 and compound 5 undergo condensation reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) using EDCl and DMAP or dicyclohexylcarbodiimide (DCC) as a catalyst.

[0267] If the M' group of compound 6 contains the aforementioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the selected protecting group removal reaction conditions.

[0268] General synthetic route 2

[0269]

[0270] Wherein M' is M or M containing a protecting group; A is O, NH or S.

[0271] As described in the above general synthetic route 2, compound 1 and compound 2 undergo condensation reaction to give compound 3. Step 1 can be carried out in an organic solvent (eg, DCM) in the presence of EDCl and DMAP.

[0272] Next, the tert-butyloxy protecting group of compound 3 is removed to obtain compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) in the presence of an acid (eg, trifluoroacetic acid) and a positive ion scavenger (eg, triisopropylsilane (TiPS)).

[0273] Next, compound 4 undergoes a condensation reaction with a compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0274] 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.

[0275] General synthetic route 3

[0276]

[0277] 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.

[0278] As described in the general synthetic route 3 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0279] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0280] Next, compound 4 is reacted with a halogen-substituted aldehyde (compound 5) through condensation and reduction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0281] Next, compound 6 reacts with an amine (compound 7) to produce compound 8. Step 4 can be carried out in an organic solvent (such as DMF) in the presence of a base (such as a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0282] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0283] General synthetic route 4

[0284]

[0285] 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.

[0286] As described in the general synthetic route 4 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0287] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0288] Next, compound 4 undergoes a condensation reaction with a halogen-substituted compound 5 to obtain compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF using EDCl and DMAP or DCC as catalyst.

[0289] Next, compound 6 reacts with an amine (compound 7) to form compound 8. Step 4 can be carried out in an organic solvent (e.g., DMF) in the presence of a base (e.g., a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0290] If the R4' and / or R5' groups of compound 8 contain the aforementioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting groups.

[0291] General synthetic route 5

[0292]

[0293] Among them, M pro is M or M containing a protecting group.

[0294] As described in the general synthetic route 5 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to produce compound 3. Step 1 can be carried out in an organic solvent (e.g., DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0295] Next, the Boc protecting group of compound 3 is removed to generate compound 4. Step 2 can be carried out in an organic solvent (eg, DCM) under the catalysis of an acid (eg, trifluoroacetic acid).

[0296] Next, compound 4 and compound 5 are subjected to condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetylborohydride (NaBH(OAc)3)).

[0297] If the M of compound 6 pro If the group contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0298] Furthermore, it is to be understood that any particular embodiment of the present invention that is within the prior art may be expressly excluded from any one or more claims. Such embodiments may be excluded because they are deemed to be known to one of ordinary skill in the art, even if such exclusion is not expressly stated herein.

[0299] All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the present application, the present application shall prevail.

[0300] (1) Synthesis of a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L) or (1M)

[0301] A. General considerations

[0302] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0303] The process routes described below can be used to synthesize compounds 1001-3422 of the present invention.

[0304] This article uses the following abbreviations:

[0305] THF: Tetrahydrofuran

[0306] MeCN: acetonitrile

[0307] MeOH: methanol

[0308] PE: Petroleum ether

[0309] EA: ethyl acetate

[0310] DMF: N,N-dimethylformamide

[0311] EDCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0312] LAH: lithium aluminum hydride

[0313] DCM: dichloromethane

[0314] DMAP: 4-dimethylaminopyridine

[0315] LDA: lithium diisopropylamide

[0316] rt: room temperature

[0317] DCE: 1,2-dichloroethane

[0318] n-BuLi: n-butyllithium

[0319] i-Pr2EtN:N,N-diisopropylethylamine

[0320] B. Intermediate Synthesis

[0321] Intermediate A:

[0322]

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

[0324]

[0325] Tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl carbonic anhydride (Boc2O) (99.1 g) were dissolved in a methanol (300 mL) / H2O (30 mL) mixed solvent, reacted at room temperature for 72 h, and purified by silica gel column chromatography (DCM:MeOH=20:1-10:1) to obtain a white solid compound intermediate A (N-Boc-aminotrihydroxymethylmethane) (75.0 g). LCMS (ESI) calculated for C9H 19 NO5, [M+H] + m / z 222.13, found 222.25.

[0326] Intermediate B:

[0327]

[0328] Intermediate B is obtained by the following synthetic process:

[0329]

[0330] To a single-necked flask, diethyl 2-ethyl-1,3-propanedioate (29.0 g), THF (90 mL), and DMF (30 mL) were added, and NaH (3.7 g) was added under ice bath. The mixture was stirred at room temperature for 30 min under nitrogen protection; pentadecane bromide (15.0 g) was added, and the mixture was reacted at 80° C. for 2 h under nitrogen protection; THF was concentrated under reduced pressure to remove THF, and the reaction solution was added dropwise to ice water. Ethyl acetate was added and stirred, and the mixture was filtered through diatomaceous earth. The organic phase was collected and then separated. The organic phase was washed with saturated brine, separated, and purified by silica gel column chromatography (PE:EA=100:1-50:1) to obtain the compound diethyl 2-ethyl-2-pentadecylamealate (16.5 g). To diethyl 2-ethyl-2-pentadecylmaleate (8.0 g) was added EtOH (50 mL), H2O (50 mL), and KOH (11.3 g), and the mixture was reacted at 90°C for 12 h; the EtOH was removed by concentration under reduced pressure, and the reaction system was adjusted to pH 4-5 by adding dilute hydrochloric acid. Water and ethyl acetate were added, stirred, and separated, and the organic phase was washed with saturated brine, separated, and purified by column chromatography (PE:EA=5:1-1:1) to obtain 2-ethyl-2-pentadecylmaleic acid (5.2 g). 2-ethyl-2-pentadecylmaleic acid (10.0 g) was reacted at 170°C for 6 h under open conditions, cooled to room temperature, and water and ethyl acetate were added, stirred, separated, and the organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (DCM:MeOH=20:1-10:1) to obtain intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1HNMR (400MHz, CDCl3) δ2.31 (tt, J = 8.6, 5.3Hz, 1H), 1.71-1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4Hz, 3H), 0.90 (t, J = 6.7Hz, 3H).

[0331] Intermediate C:

[0332]

[0333] Intermediate C is obtained by the following synthetic process:

[0334]

[0335] To a three-necked flask, add decanoic acid (50 g) and THF (500 mL). After cooling the system to 0°C, slowly add NaH (23.22 g). After stirring at 0°C under nitrogen for 1 hour, slowly add LDA (62.19 g) dropwise. Stirring at 0°C under nitrogen for another 1 hour is then continued. Finally, add iodonane (88.52 g) dropwise, warm to room temperature, stir overnight, then dilute with 1 L of DCM and wash with saturated NH4Cl solution and water, respectively. The organic layer is dried over anhydrous Na2SO4, filtered, and concentrated. Purify by silica gel chromatography (PE:EA = 50:1) to obtain intermediate C (2-octylundecanoic acid) (11 g). 1HNMR(400MHz,Chloroform-d)δ2.37(tt,J=8.7,5.1Hz,1H),1.71-1.58(m,2H),1.48(dt,J=

[0336] 13.3, 6.7Hz, 2H), 1.29 (d, J = 9.4Hz, 25H), 0.90 (t, J = 6.8Hz, 6H).

[0337] Intermediate D:

[0338]

[0339] Intermediate D is obtained by the following synthetic route:

[0340]

[0341] Tridecanoic acid (79.5 g) and THF (800 mL) were added to a three-necked flask. The temperature was lowered to 0°C, and NaH (22.25 g) was slowly added. The reaction mixture was stirred at 0°C under nitrogen for 1 hour, and then LDA (317.87 g) was slowly added dropwise to the reaction system. The reaction mixture was stirred at 0°C under nitrogen for another 1 hour. After adding n-hexane iodide (94.3 g) dropwise, the mixture was warmed to room temperature and stirred overnight. The reaction mixture was then diluted with 1 L of DCM and washed with saturated NH4Cl solution and water, respectively. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The mixture was then purified by 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=

[0342] 14.3, 8.7, 5.5Hz, 2H), 1.49 (dq, J=13.5, 6.6Hz, 2H), 1.29 (d, J=10.1Hz, 26H), 0.90 (t, J=6.6Hz, 6H).

[0343] Intermediate E:

[0344]

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

[0346]

[0347] Tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1 1) were dissolved in EtOH (150 mL) and reacted at 45°C under nitrogen for 30 h. The mixture was then concentrated under reduced pressure to remove the EtOH. Three 100 mL portions of (PE:EA=20:1) were added for slurrying, and the mixture was filtered to obtain Intermediate E (11.5 g) as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.60 (s, 6H), 2.84 (t, J=5.9 Hz, 2H), 2.47 (t, J=5.8 Hz, 2H), 1.48 (s, 9H).

[0348] Intermediate F:

[0349]

[0350] Intermediate F is obtained by the following synthetic route:

[0351]

[0352] To a single-necked flask, tert-butyl (2-aminoethyl)carbamate (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) were added. After reacting at 80°C overnight, water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (5.5 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2, stirred at room temperature for 3 h, and then concentrated under reduced pressure to obtain compound 3, intermediate F (6 g). LCMS (ESI) calculated for C 20 H 28 N2O2,[M+H] + m / z329.22, found 329.24.

[0353] Intermediate G:

[0354]

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

[0356]

[0357] Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropane bromide (9.45 g), and K2CO3 (12.94 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=10:1-5:1) to obtain compound 2 (6.9 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.9 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate G (7.5 g). LCMS (ESI) calculated for C 22 H 32 N2O2,[M+H] + m / z 357.25, found357.51.

[0358] Intermediate H:

[0359]

[0360] Intermediate H is obtained using the following synthetic process:

[0361]

[0362] Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), and K2CO3 (6.47 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 20:1-10:1) to obtain compound 2 (4.1 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (4.1 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate H (4.9 g). LCMS (ESI) calculated for C 24 H 36 N2O2, [M+H] + m / z 385.28, found385.56.

[0363] Intermediate I:

[0364]

[0365] Intermediate I is obtained by the following synthetic route:

[0366]

[0367] Compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), and K2CO3 (15.46 g) were added to a single-necked flask and reacted overnight at 80°C. Water and ethyl acetate were then added for separation by stirring. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA=20:1-10:1) to obtain compound 2 (6.8 g). Dioxane (30 mL) and dioxane hydrochloride solution (30 mL) were added to compound 2 (6.8 g). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calculated for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0368] Intermediate J:

[0369]

[0370] Intermediate J is obtained using the following synthetic route:

[0371]

[0372] To a single-necked flask, butyric acid (5.0 g) and THF (100 mL) were added. NaH (2.73 g) was added at 0°C, and LDA (56.8 mL) was slowly added dropwise. The mixture was reacted at room temperature for 30 min. 1-Bromotridecane was added and the reaction was continued at room temperature overnight. Ice water and ethyl acetate were added and stirred for separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by column chromatography (PE:EA = 10:1-5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calculated for C 17 H 34 O2, [M+H] + m / z 271.26, found 271.46.

[0373] Intermediate K:

[0374]

[0375] Intermediate K is obtained by the following synthetic process:

[0376]

[0377] To a three-necked flask, reactant 1 (24 g), imidazole (19.00 g), and DCM (200 mL) were added. The temperature was lowered to 0°C, and then TBDMSCI (38.57 g) was slowly added. After stirring at 25°C for 4 h, the mixture was diluted with 300 mL of DCM. The organic phase was washed twice with 1 L of water and dried over anhydrous Na2SO4. After filtration and concentration, silica gel was added and column chromatography with 1:20 DCM:MeOH was performed. The sample was collected and concentrated to yield intermediate K (28.00 g). 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 3.58 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.67-1.40 (m, 4H), 0.84 (s, 9H).

[0378] Intermediate L:

[0379]

[0380] Intermediate L is obtained by the following synthetic process:

[0381]

[0382] Compound DMSO (3.58 g) and anhydrous DCM (30 mL) were added to a three-necked flask. The system was cooled to -78°C and oxalyl chloride (2.91 g) was slowly added. The reaction solution was stirred at -78°C under N2 protection for 10 minutes, and compound 1 (3 g) was slowly added dropwise. Stirring was continued at -78°C under N2 protection for 1 hour. Finally, TEA (9.28 g) was added dropwise. Stirring was continued at -78°C under N2 protection for 0.5 hour, and the mixture was diluted with 100 mL DCM and washed with saturated NH4Cl solution and water, respectively. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to obtain intermediate L (3.0 g). LCMS (ESI) calculated for C 11 H 14 O3, [M+H] + m / z 195.09, found195.23.

[0383] Intermediate M:

[0384]

[0385] Intermediate M is obtained by the following synthetic process:

[0386]

[0387] To a sealed tube, intermediate K (2.0 g), compound 1 (1.79 g), and ACN (20 mL) were added. The reactants, KCO (3.81 g) and KI (1.52 g), were then added. The mixture was stirred overnight at 70°C under N2 protection, then diluted with 100 mL of EA and washed with saturated NH4Cl solution and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The mixture was then added to a silica gel column and filtered with 15:1 DCM:MeOH. The sample was collected and concentrated to yield compound 2 (2.0 g). Reactant 2 (1.80 g) and solvent DCM (20 mL) were added to a three-necked flask. The system was purged with nitrogen and the temperature was lowered to -78°C. DIBAL-H (1.00 g) was then added dropwise. After stirring at -78°C for 4 h, methanol and sodium carbonate solution were added dropwise to the reaction solution to quench the mixture. The mixture was then extracted with DCM (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate M (1.36 g). LCMS (ESI) calculated for C 15 H 33 NO2Si,[M+H] + m / z 288.52, found 288.23.

[0388] C. Compound 1003 was synthesized according to general synthetic route 1

[0389] Structural formula:

[0390] Chemical formula: C 63 H 122 N2O7

[0391] Molecular weight: 1019.68

[0392] Step 1: Synthesis of compound 3 in general synthetic route 1

[0393]

[0394] Compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) were added to compound 2 (intermediate B) (4.3 g). The mixture was stirred at room temperature for 12 h under nitrogen protection, and then water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated. The organic phase was purified by silica gel column chromatography (PE:EA=40:1-30:1) to give compound 3 (2.6 g).

[0395] Step 2: Synthesis of compound 4 in general synthetic route 1

[0396]

[0397] DCM (15 mL) and TFA (5 mL) were added to compound 3 (2.6 g), and the mixture was stirred at room temperature for 3 h, concentrated, and purified by silica gel column chromatography (PE:EA=10:1-5:1) to give compound 4 (3.2 g).

[0398] Step 3: Synthesis of compound 6 in general synthetic route 1

[0399]

[0400] To compound 4 (3.2 g) were added DCM (20 mL), Boc-glycine (compound 5, 578 mg), and DCC (6.8 g). The mixture was stirred at room temperature under nitrogen for 12 h, and then water and ethyl acetate were added for stirring and separation. The organic phase was washed with saturated brine, separated, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA=5:1-2:1) to give compound 6 (2.2 g).

[0401] Step 4: Compound 1003

[0402]

[0403] To compound 6 (2.2 g) were added DCM (15 mL) and TFA (5 mL), and the mixture was stirred at room temperature for 12 h and concentrated. The mixture was purified by column chromatography (DCM:MeOH=30:1-20:1) to give compound 1003 (1.3 g). 1 H NMR (400MHz, CDCl3) δ4.49 (d, J=2.6Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J=8.6, 5. 5Hz, 3H), 1.62 (q, J = 7.1Hz, 12H), 1.27 (d, J = 3.4Hz, 78H), 0.94-0.86 (m, 18H).

[0404] D. Compound 1002 was synthesized according to general synthetic route 1

[0405] Structural formula:

[0406] Chemical formula: C 60 H 116 N2O7

[0407] Molecular weight: 977.60

[0408] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that intermediate C was used as compound 2 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5Hz, 3H), 1 .64(d,J=6.9Hz,6H),1.50-1.42(m,6H),1.27(s,72H),0.90(t,J=6.7Hz,18H).

[0409] E. Compound 1004 was synthesized according to general synthetic route 1

[0410] Structural formula:

[0411] Chemical formula: C 54 H 104 N2O7

[0412] Molecular weight: 893.43

[0413] The compound was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that palmitic acid was used as compound 2 in the general synthetic route 1. 1 H NMR (400MHz, CDCl3) δ7.55 (s, 1H), 4.45 (s, 6H), 3.74 (s, 2H), 2.35 (t, J = 7.6Hz, 6H), 1.60 (p, J = 6.9Hz, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7Hz, 9H).

[0414] F. Compound 1001 was synthesized according to general synthetic route 1

[0415] Structural formula:

[0416] Chemical formula: C 59 H 108 N2O7

[0417] Molecular weight: 957.52

[0418] The product was synthesized according to the general synthetic route 1, which is similar to the synthetic process of compound 1003, except that (9Z)-9-hexadecenoic acid was used as compound 2 in the general synthetic route 1, and 5-(N,N-dimethylamino)pentanoic acid was used as compound 5 in the general synthetic route 1. 1H NMR (400MHz, CDCl3) δ5.42-5.30(m,6H),4.51-4.38(m,6H),3.19(dt,J=11.3,5 .6Hz,2H),2.97(t,J=5.9Hz,6H),2.46(t,J=6.6Hz,1H),2.35(td,J=7.6,3.1Hz, 5H),2.20(t,J=7.6Hz,2H),2.03(d,J=6.2Hz,12H),1.89-1.81(m,3H),1.74(q, J=7.8Hz,2H),1.67-1.51(m,6H),1.31(d,J=7.8Hz,48H),0.90(t,J=6.7Hz,9H).

[0419] G. Compound 1014 was synthesized according to general synthetic route 2

[0420] Structural formula:

[0421] Chemical formula: C 70 H 137 N3O9

[0422] Molecular weight: 1164.88

[0423] Step 1: Synthesis of Intermediate 3 in General Synthesis Route 2

[0424]

[0425] Intermediate 3 was synthesized according to the method shown in step 1 of general synthetic route 1, except that intermediate E was used as compound 1 in general synthetic route 1.

[0426] Step 2: Synthesis of compound 4 of general synthetic route 2

[0427]

[0428] DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added to compound 3 (4.8 g). After reacting at room temperature for 12 h, water and dichloromethane were added for stirring and separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH=20:1-10:1) to give compound 4 (3.3 g).

[0429] Step 3: Synthesis of compound 6 of general synthetic route 2

[0430]

[0431] Compound 4 (4.0 g), DCM (80 mL), EDCI (1.1 g), 1-hydroxybenzotriazole (HOBt) (0.78 g), and DIEA (2.5 g) were added to compound 5 (1.9 g). After stirring at room temperature for 12 h, water and dichloromethane were added, stirred and separated, and the organic phase was washed with saturated brine. The organic phase was separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH=30:1-20:1) to give compound 6 (4.0 g).

[0432] Step 4: Compound 1014

[0433]

[0434] MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added to compound 6 (4.0 g). The mixture was stirred at room temperature overnight under hydrogen atmosphere, filtered through celite, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1-

[0435] 15:1) to give compound 1014 (2 g). 1 H(400MHz, CDCl3)δ7.38(t,J=5.8Hz,1H),4.14-4.05(m,6H),3.58(t,J=4.9Hz,4H),3.31(q,J=5.7Hz,2H),2.89(t,J=6.3Hz, 2H), 2.64 (q, J=5.2Hz, 6H), 2.29 (dq, J=10.8, 4.4Hz, 6H), 1.66-1.37 (m, 12H), 1.24 (d, J=4.3Hz, 78H), 0.87 (t, J=7.1Hz, 18H).

[0436] H. Compound 1015 was synthesized according to general synthetic route 2

[0437] Structural formula:

[0438] Chemical formula: C 72 H 141 N3O9

[0439] Molecular weight: 1192.93

[0440] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate G was used as compound 5 in general synthetic route 2. 1H(400MHz, CDCl3)δ7.58(t,J=5.7Hz,1H),4.15-4.01(m,6H),3.70(t,J=5.4Hz,4H),3.39(q,J=5.7Hz,2H),2.86(t,J=6.2Hz,2H),2 .57(dt,J=20.5,5.8Hz,6H),2.36-2.22(m,5H),1.76-1.67(m,4H),1.62-1.35(m,13H),1.23(d,J=4.6Hz,78H),0.92-0.80(m,18H).

[0441] I. Compound 1020, synthesized according to general synthetic route 2

[0442] Structural formula:

[0443] Chemical formula: C 74 H 145 N3O9

[0444] Molecular weight: 1220.99

[0445] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate H was used as compound 5 in the general synthetic route 2.

[0446] J. Compound 1025 was synthesized according to general synthetic route 2

[0447] Structural formula:

[0448] Chemical formula: C 69 H 135 N3O8

[0449] Molecular weight: 1134.85

[0450] The compound was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate I was used as compound 5 in the general synthetic route 2. 1 H(400MHz, CDCl3)δ6.98(t,J=5.7Hz,1H),4.10(m,J=3.8Hz,6H),3.63(t,J=5.2Hz,2H),3.35(q,J=5.8Hz,2H),2.89(t,J=6.1Hz,2 H), 2.57 (q, J = 5.3Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1Hz, 5H), 1.65-1.41 (m, 13H), 1.24 (d, J = 4.4Hz, 78H), 0.87 (t, J = 7.1Hz, 18H).

[0451] K. Compound 1005, synthesized according to general synthetic route 2

[0452] Structural formula:

[0453] Chemical formula: C 68 H 133 N3O7

[0454] Molecular weight: 1104.83

[0455] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-dimethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.19-4.07(m,6H),3.40(d,J=5.7Hz,2H),2.95-2.87(m,2H),2.82(s,4H),2.64-2.55(m,2H),2.38 (s, 6H), 2.33-2.29 (m, 3H), 1.63-1.55 (m, 6H), 1.49-1.44 (m, 2H), 1.26 (d, J = 5.4Hz, 78H), 0.89 (td, J = 7.0, 2.8Hz, 18H).

[0456] L. Compound 1006 was synthesized according to general synthetic route 2

[0457] Structural formula:

[0458] Chemical formula: C 53 H 103 N3O7

[0459] Molecular weight: 894.42

[0460] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that tetradecanoic acid was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 HNMR (400MHz, CDCl3) δ7.99(t,J=5.8Hz,1H),4.13(s,6H),3.60(q,J=5.3Hz,2H),3.38-3.34(m,2H),3.00(s,6H),2.94(t, J=5.8Hz,2H),2.45(t,J=5.8Hz,2H),2.35(t,J=7.6Hz,6H),1.66-1.56(m,6H),1.33-1.23(m,60H),0.90(t,J=6.8Hz,9H).

[0461] M. Compound 1007, synthesized according to general synthetic route 2

[0462] Structural formula:

[0463] Chemical formula: C 68 H 133 N3O7

[0464] Molecular weight: 1104.83

[0465] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate D was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 HNMR(400MHz,Chloroform-d)δ7.13(t,J=5.0Hz,1H),4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=6.0Hz,2H),2.47-2.28(m,7H ), 2.24 (s, 6H), 1.60 (dq, J = 14.7, 7.2Hz, 6H), 1.47 (dd, J = 14.2, 6.7Hz, 6H), 1.27 (d, J = 3.4Hz, 78H), 0.89 (td, J = 6.7, 2.5Hz, 18H).

[0466] N. Compound 1008, synthesized according to general synthetic route 2

[0467] Structural formula:

[0468] Chemical formula: C 68 H 133 N3O7

[0469] Molecular weight: 1104.01

[0470] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1HNMR(400MHz,Chloroform-d)δ4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=5.9Hz,2H),2.51-2.26(m,7H),2.24 (s, 6H), 1.59 (ddt, J = 14.8, 10.9, 6.4Hz, 6H), 1.47 (tq, J = 11.0, 5.4Hz, 6H), 1.26 (s, 77H), 0.89 (t, J = 6.7Hz, 18H).

[0471] O. Compound 1009, synthesized according to general synthetic route 2

[0472] Structural formula:

[0473] Chemical formula: C 62 H 121 N3O7

[0474] Molecular weight: 1020.66

[0475] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate J was used as compound 2 in general synthetic route 2 and N,N-dimethylethylenediamine was used as compound 5 in general synthetic route 2. 1 H(400MHz, CDCl3)δ7.04(t,J=5.0Hz,1H),4.12(m,J=3.3Hz,6H),3.30(q,J=5.6Hz,2H),2.91(t,J=6.0Hz,2H),2.39 (t,J=6.0Hz,2H),2.33-2.25(m,5H),2.22(s,6H),1.76-1.54(m,12H),1.24(m,J=3.7Hz,66H),0.90-0.84(m,18H).

[0476] P. Compound 1011 was synthesized according to general synthetic route 2

[0477] Structural formula:

[0478] Chemical formula: C 70 H 137 N3O7

[0479] Molecular weight: 1132.88

[0480] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.19-4.08(m,6H),3.31(q,J=5.8Hz,2H),2.92(t,2H),2.57(t,J=6.9Hz,6H),2.37-2.26( m,5H),1.62(ddd,J=18.3,9.0,4.3Hz,12H),1.27(d,J=4.7Hz,78H),1.04(t,J=7.1Hz,6H),0.94-0.86(m,18H).

[0481] Q. Compound 1012, synthesized according to general synthetic route 2

[0482] Structural formula:

[0483] Chemical formula: C 72 H 141 N3O7

[0484] Molecular weight: 1160.93

[0485] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ6.59 (s, 1H), 4.18-4.07 (m, 6H), 3.29 (q, J = 5.7Hz, 2H), 2.91 (t, J = 6.2Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5Hz, 4H), 2.35-2.27 (m, 5H), 1.63-1.42 (m, 16H), 1.27 (m, J = 4.5Hz, 78H), 0.93-0.85 (m, 24H).

[0486] R. Compound 1013, synthesized according to general synthetic route 2

[0487] Structural formula:

[0488] Chemical formula: C 74 H 145 N3O7

[0489] Molecular weight: 1188.99

[0490] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethylethylenediamine was used as compound 5 in the general synthetic route 2. 1H NMR (400MHz, CDCl3) δ4.10(d,J=4.1Hz,6H),3.26(q,J=5.8Hz,2H),2.89(t,J=6.2Hz,2H),2.52(d,J=6.6Hz,2H) ,2.41(t,J=7.4Hz,4H),2.33-2.25(m,5H),1.60(d,J=11.8Hz,16H),1.24(d,J=4.5Hz,82H),0.96-0.79(m,24H).

[0491] S. Compound 1029, synthesized according to general synthetic route 2

[0492] Structural formula:

[0493] Chemical formula: C 68 H 132 N2O8

[0494] Molecular weight: 1105.81

[0495] The compound 5 was synthesized according to the general synthetic route 2, which is similar to the synthetic process of compound 1014, except that N,N-diethyl-2-hydroxyethylamine was used as compound 5 in the general synthetic route 2. 1 H NMR (400MHz, CDCl3) δ4.16(t,J=5.8Hz,2H),4.13-4.05(m,6H),2.86(t,J=6.3Hz,2H),2.55(t,J=5.8Hz,2H) ,2.44(t,J=6.3Hz,2H),2.35-2.20(m,9H),1.60-1.39(m,11H),1.24(d,J=4.5Hz,80H),1.02-0.79(m,18H).

[0496] T. Compound 1111, synthesized according to general synthetic route 2

[0497] Structural formula:

[0498] Chemical formula: C 70 H 136 N4O7

[0499] Molecular weight: 1145.88

[0500] The product was synthesized according to general synthetic route 2, which was similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate was used as compound 5 in general synthetic route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97-2.80 (m, 5H), 2.70 (s, 5H), 2.46-2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54-1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0501] U. Compound 1118, synthesized according to general synthetic route 2

[0502] Structural formula:

[0503] Chemical formula: C 71 H 139 N3O8

[0504] Molecular weight: 1162.91

[0505] The compound was synthesized according to general synthetic route 2, which is similar to the synthetic process of compound 1014, except that intermediate C was used as compound 2 in general synthetic route 2 and intermediate K was used as compound 5 in general synthetic route 2. 1 HNMR(400MHz,Chloroform-d)δ4.11(s,6H),3.61(s,2H),3.42(d,J=33.5Hz,2H),2.91(t,J=6.2Hz,3 H), 2.36 (dd, J = 9.2, 3.6Hz, 6H), 1.47 (dd, J = 13.9, 6.2Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7Hz, 18H).

[0506] V. Compound 1010, synthesized according to general synthetic route 3

[0507] Structural formula:

[0508] Chemical formula: C 67 H 133 N3O6

[0509] Molecular weight: 1076.82

[0510] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 3

[0511]

[0512] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0513] Step 3: Synthesis of compound 6 of general synthetic route 3

[0514]

[0515] Compound 4 (600 mg), chloroacetaldehyde (compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) were added to DCE (60 mL). After reacting at room temperature overnight, water and dichloromethane were added, stirred, and the liquids were separated. The organic phase was washed with saturated brine, separated, and concentrated. Compound 6 (270 mg) was obtained by silica gel column chromatography (DCM:MeOH=30:1-15:1).

[0516] Step 4: Synthesis of Compound 1010

[0517]

[0518] MeCN (20 mL), N,N-dimethylethylenediamine (compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) were added to compound 6 (270 mg). The mixture was reacted at 70°C under nitrogen protection overnight, and then silica gel column chromatography was used to obtain compound 1010 (16 mg). 1 H(400MHz, CDCl3)δ4.10(m,J=2.9Hz,6H),2.76-2.64(m,6H),2.40(t,J=6.2Hz,2H),2.29(ddd,J=8.5,5.6 ,2.9Hz,3H),2.22(s,6H),1.97(s,6H),1.50-1.41(m,6H),1.24(d,J=4.2Hz,78H),0.87(t,J=7.1Hz,18H).

[0519] W. Compound 1059, synthesized according to general synthetic route 3

[0520] Structural formula:

[0521] Chemical formula: C 67 H 132 N2O7

[0522] Molecular weight: 1077.80

[0523] The synthesis was carried out according to the general synthetic route 3, which is similar to the synthesis process of compound 1010, except that intermediate C and intermediate L were used as compound 2 and compound 5 in the general synthetic route 3, respectively. In addition, compound 6 in the general synthetic route 3 was subjected to the following process steps for functional group transformation:

[0524]

[0525] To intermediate 6a (1.5 g) was added MeOH (5 mL) and THF (5 mL), and the reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g) were added. The mixture was reacted under H2 at 25°C overnight, filtered through celite, and the filtrate was concentrated and chromatographed on a silica gel column (PE:EA 30:1-10:1) to afford intermediate 6b (520 mg). To intermediate 6b (470 mg) was added DCM (5 mL), and SOCl2 (1064.26 mg, 8.946 mmol) was added dropwise at 0°C. The mixture was allowed to react at room temperature for 3 hours, and then the reaction mixture was extracted with water and DCM. The organic phase was concentrated and chromatographed on a silica gel column (PE:EA 20:1) to afford intermediate 6c (181 mg). To intermediate 6c (170 mg) were added dimethylamine (143.39 mg) and DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg). The reaction solution was stirred at 70°C, filtered, concentrated, and purified by silica gel column chromatography (DCM:MeOH 20:1) to give compound 1059 (91 mg, 53.22% yield). 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.51(dt,J=18.3,5.6Hz,4H),2.81(dd,J=10.3,5.1Hz,2H),2.50(t,J=6.0 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 = 6.7Hz, 18H).

[0526] X. Compound 1112, synthesized according to general synthetic route 3

[0527] Structural formula:

[0528] Chemical formula: C 69 H 136 N2O6

[0529] Molecular weight: 1089.85

[0530] The compound was synthesized according to the general synthetic route 3, which is similar to the synthetic process of compound 1010, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in the general synthetic route 3, respectively. 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.11(s,1H),2.57(t,J=6.8Hz,2H),2.44-2.19 (m,12H),2.04(q,J=6.5Hz,2H),1.53-1.38(m,13H),1.27(s,92H),0.90(t,J=6.7Hz,20H).

[0531] Y. Compound 1113, synthesized according to general synthetic route 4

[0532] Structural formula:

[0533] Chemical formula: C 71 H 138 N2O8

[0534] Molecular weight: 1147.89

[0535] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 4

[0536]

[0537] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0538] Step 3: Synthesis of compound 6 of general synthetic route 4

[0539]

[0540] DIEA (0.30 g) and DCM (20 mL) were added to compound 4. Under nitrogen, the temperature was cooled to 0°C, and 5-bromovaleryl chloride (compound 5, 0.27 g) was added. The mixture was stirred at 25°C for 2 h. After dilution with 100 mL of DCM, the organic phase was washed twice with 100 mL of water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (PE:EA 20:1) to obtain compound 6 (800 mg).

[0541] Step 4: Synthesis of compound 8 of general synthetic route 4

[0542]

[0543] Compound 6 (750.00 mg), intermediate K (compound 7, 217.31 mg), and DMF (20 mL) were added to a three-necked flask. KCO (184.16 mg) and KI (110.60 mg) were then added and stirred at 25°C for 16 h. The temperature was then raised to 100°C and stirred for 3 h. The reaction solution was diluted with 200 mL of EA, and the organic phase was washed twice with 300 mL of saturated brine. The organic phase was dried over anhydrous NaSO, filtered, and concentrated. Silica gel was added and the mixture was filtered through a column using 20:1 DCM:MeOH. The sample was collected and concentrated to yield compound 8 (700.00 mg).

[0544] Step 5: Synthesis of Compound 1113

[0545]

[0546] Compound 8 (700.00 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (5.55 mL). The reaction mixture was stirred at 25°C for 2 h, diluted with 50 mL of EA, and the organic phase was washed with a saturated NaHCO solution and water. The organic phase was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1113 (201.1 mg, 31.59% yield). 1 H NMR (400MHz, CDCl3) δ4.50-4.39(m,6H),3.59(t,J=4.6Hz,2H),2.49(s,3H),2.35-2.26(m,6H),2.14 (t,J=7.5Hz,2H),1.75-1.69(m,4H),1.66-1.43(m,17H),1.27(d,J=3.7Hz,78H),0.94-0.87(m,18H).

[0547] Z. Compound 1115, synthesized according to general synthetic route 4

[0548] Structural formula:

[0549] Chemical formula: C 67 H 131 N3O7

[0550] Molecular weight: 1090.80

[0551] The compound was synthesized according to general synthetic route 4, which is similar to the synthetic process of compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine were used to synthesize compound 5 and compound 7 in general synthetic route 4, respectively. 1 HNMR (400

[0552] MHz, CDCl3) δ7.42(s,1H),4.48(tt,J=11.9,5.7Hz,6H),3.27(s,2H),3.13(s,2H),3.01(s,2H),2.83(s,6H),2 .38-2.25(m,3H),1.55(dtt,J=36.3,14.1,7.3Hz,12H),1.27(d,J=3.3Hz,78H),0.90(td,J=7.2,3.4Hz,18H).

[0553] AA. Compound 1114, synthesized according to general synthetic route 5

[0554] Structural formula:

[0555] Chemical formula: C 70 H 138 N2O7

[0556] Molecular weight: 1119.88

[0557] Step 1 and Step 2: Synthesis of Compound 3 and Compound 4 of General Synthesis Route 5

[0558]

[0559] According to steps 1 and 2 of general synthetic route 1, the synthesis process is the same as that of compound 1003.

[0560] Step 3: Synthesis of compound 6 of general synthetic route 5

[0561]

[0562] Compound 4 (500 mg), intermediate M (compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) were added to a three-necked flask. The reaction solution was stirred under N2 at 25°C for 16 h, then 100 mL of water was added to the reaction solution. The mixture was extracted twice with EA (100 mL). The organic phases were combined and concentrated. Silica gel was added and the mixture was passed through a column with 1:20 DCM:MeOH. The sample was collected and concentrated to give compound 6 (500 mg).

[0563] Step 4: Synthesis of Compound 1114

[0564]

[0565] Compound 6 (450 mg) and THF (5 mL) were added to a three-necked flask, followed by a 1 M dioxane hydrochloride solution (3.65 mL). After stirring at 25°C for 2 h, the mixture was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO solution and water. The organic layer was dried over anhydrous NaSO, filtered, concentrated, and then purified by silica gel column chromatography (MeOH:DCM 1:20) to afford compound 1114 (107 mg, 24.84% yield). 1 H NMR (400MHz, CDCl3) δ4.16-4.07(m,6H),3.60(t,J=4.8Hz,2H),2.62(t,J=7.0Hz,2H),2.49(s,3H),2.31(tt, J=8.4,5.5Hz,6H),1.70(d,J=4.6Hz,3H),1.64-1.39(m,18H),1.27(d,J=4.0Hz,78H),0.90(t,J=7.1Hz,18H).

[0566] Z. Compound 1120, synthesized according to general synthetic route 5

[0567] Structural formula:

[0568] Chemical formula: C 72 H 142 N2O7

[0569] Molecular weight: 1147.90

[0570] The product was synthesized according to general synthetic route 5, which was similar to the synthetic process of compound 1114, except that intermediate C and 6-bromohexanal were used to synthesize compound 2 and compound 5 in general synthetic route 5, respectively. 1H NMR (400 MHz, Chloroform-d) δ4.11 (s, 6H), 3.63 (s, 2H), 2.57 (t, J = 6.8 Hz, 4H), 2.37 (ddd, J = 8.5, 7.1, 4.3 Hz, 5H), 1.67-1.41 (m, 24H), 1.27 (s, 79H), 0.90 (t, J = 6.7 Hz, 18H).

[0571] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0572] 1. Preparation of Compound 6001

[0573] Structural formula:

[0574] Molecular weight: 499.43

[0575] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h and then passed through a reverse column to obtain compound 6002.

[0576] 0.7 g of compound 6002, 0.4 g of palladium on carbon and 28 mL of tetrahydrofuran were added to a reaction flask and stirred. The gas was replaced with hydrogen and the pressure was maintained with a hydrogen balloon. After 16 h, the mixture was filtered and passed through a column (methanol-DCM system) in the forward direction to obtain 200 mg of compound 6001. 1 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).

[0577] 2. Preparation of Compound 6002

[0578] Structural formula:

[0579] Molecular weight: 497.42

[0580] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. The reaction was continued for 16 h, and the organic solvent was concentrated. The mixture was passed through a reverse phase column and freeze-dried to obtain 400 mg of compound 6002. 1 H NMR(400MHz, CDCl3) δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79-3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2. 82-2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10-1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0581] 3. Preparation of Compound 6003

[0582] Structural formula:

[0583] Molecular weight: 445.34

[0584] 4.00 g of tetradecylamine, 6.53 g of hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70°C. After 29 h, the organic solvent was concentrated, passed through a reverse phase column and freeze-dried to obtain 1.5 g of compound 6003. 1 H NMR(400MHz, CDCl3)δ4.29(t,J=7.5Hz,4H),3.89-3.75(m,4H),2.81(t,J=6.2Hz,4H), 2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0585] 4. Preparation of Compound 6004

[0586] Structural formula:

[0587] Molecular weight: 553.48

[0588] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate, and 5.6 mL of n-butanol were added to a reaction flask, stirred, and heated to 100°C. After 4 h, 1 mL of n-butyl acrylate was added, and the reaction was continued for 1 h. The mixture was passed through a normal phase column (petroleum ether-ethyl acetate system), and 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added, and stirred for hydrolysis for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to obtain a mixed solution, which was adjusted to neutral pH, dried by rotation, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was dried by rotation. 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT, and 2.3 g of EDCI were added, and the reaction was allowed to react at room temperature for 18 h. After concentrating the organic solvent, the mixture was passed through a reverse phase column and freeze-dried to obtain 240 mg of compound 6004. 1 HNMR (400MHz, CDCl3) δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17( s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0589] 5. Preparation of Compound 6005

[0590] Structural formula:

[0591] Molecular weight: 597.54

[0592] 25.00 g of 11-henicosone, 62.05 g of ammonium acetate, and 500 mL of methanol were added to a reaction flask and stirred. 6.55 g of sodium cyanoborohydride was added and reacted for 16 h. 250 mL of water and 250 mL of DCM were added, and the layers were separated. The aqueous phase was extracted with 50 mL of DCM, and the combined organic phases were passed through a normal phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0593] 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex were stirred in a reaction flask and heated to 100° C. 10 mL of n-butyl acrylate was added. After 16 h, the mixture was passed through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0594] A solution prepared by mixing 31.00 g of compound 6005-B with 6.55 g of sodium hydroxide, 310 mL of methanol, and 31 mL of water was added to a reaction flask and stirred. After 20 minutes, 150 mL of THF was added, and the temperature was raised to 50°C with stirring. After 30 minutes, 16.13 g of concentrated hydrochloric acid and 160 mL of methanol were added and stirred. After 30 minutes, the solvent was dried by spin-drying, and 300 mL of DCM was added to dissolve the mixture. The mixture was dried over anhydrous magnesium sulfate, filtered, and dried to obtain 27.00 g of compound 6005-C as an oil.

[0595] 27.00 g of compound 6005-C, 25.00 g of 4-amino-1-butanol, 41.85 g of EDCI, 14.74 g of HOBT, and 310 mL of DCM were added to a reaction flask and stirred. After 18 h, the organic solvent was concentrated and filtered through a column (methanol-DCM system) to remove excess 4-amino-1-butanol, yielding 6.80 g of compound 6005. 1 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).

[0596] 6. Preparation of Compound 6006

[0597] Structural formula:

[0598] Molecular weight: 553.48

[0599] 1.00g of oleylamine, 0.47g of hydroxyethyl acrylamide, and 8mL of ethanol were added to a reaction flask, stirred, and heated to 70°C. After 13 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. 1.5mL of n-butyl acrylate and 5mL of n-butanol were added, stirred, and heated to 100°C. After 3 hours, the solvent was dried and passed through a methanol-DCM column to obtain 800mg of intermediate 1. A solution of 0.4g of sodium hydroxide, 10mL of methanol, and 1mL of water was added, stirred for 1h, and a solution of 1mL of concentrated hydrochloric acid and 8mL of methanol was added. The mixture was stirred for 30min, the solvent was dried and dissolved in 100mL of DCM, dried over anhydrous magnesium sulfate, filtered, and dried. 0.45g of 6-amino-1-hexanol, 0.27g of HOBT, 0.78g of EDCI, and 10mL of DCM were added, stirred. After 14 hours, the organic solvent was concentrated and passed through a methanol-DCM column to obtain 300mg of compound 6006. 1 HNMR(400MHz, CDCl3)δ7.83(s,1H),6.95(s,1H),5.43-5.28(m,2H),3.74-3 .67(m,2H),3.64(t,J=6.3Hz,2H),3.39(dd,J=10.0,5.3Hz,2H),3.24(dd,J= 12.9,6.7Hz,2H),2.74(t,J=5.8Hz,4H),2.48-2.42(m,2H),2.41-2.33(m,4 H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66-1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0600] 7. Compound 6007

[0601] Structural formula:

[0602] Molecular weight: 953.78

[0603]

[0604] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h. The mixture was concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1-10:1) to obtain product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The mixture was concentrated under reduced pressure, and 30 mL of toluene was added. The mixture was heated to 60°C to dissolve, then slowly cooled to room temperature and evaporated to dryness under reduced pressure to obtain compound 3 (15 g). Compound 3 (2 g) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve. Methyl acrylate (2.5 g) was then slowly added and stirred to mix. The mixture was heated to 60°C overnight. The reaction was stopped, and the reaction solution was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1-20:1) to obtain compound 4 (2.5 g). Compound 4 (2.3 g) and ethylenediamine (20 mL) were added to the single-necked flask at room temperature and reacted overnight at room temperature. The reaction was stopped and the mixture was directly concentrated under reduced pressure to obtain compound 6007 (24 mg). 1HNMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H ), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9Hz, 4H), 1.73 ( p, J = 7.7Hz, 4H), 1.49 ( tdd, J = 9.2, 7.9, 7.1, 4.0Hz, 4H), 1.31 ( s, 28H), 0.92 ( t, J = 6.8Hz, 3H).

[0605] 8. Compounds 6008-6018

[0606] Synthesis of compound 6009

[0607] Structural formula:

[0608] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise to a solution of stirred methyl methacrylate (6 mL) in methanol (20 mL) under a nitrogen atmosphere, and the reaction was continued overnight. The reactant was spin-dried at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. The resulting product was then separated by column chromatography to obtain a colorless oil. 1H-NMR (300 MHz, CDCl 3 ): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0609] Then, a solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 ml) that was vigorously stirred at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, maintaining the temperature not higher than 40°C. Excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was repeatedly recrystallized from chloroform and cyclohexane to obtain a white solid. 1H-NMR (300MHz, CDCl3): 0.88(t,3H), 1.25(s,30H), 1.84(s,4H), 2.38(m,6H), 2.73(m,4H), 2.82(m,4H), 3.29(m,4H), 7.47(s,2H).

[0610] The preparation methods of 6008 and 6009 differ only in that an equal molar amount of tetradecylamine is used instead of hexadecylamine. 6010-6017 refer to the preparation method of 6009 and use an equal molar amount of the corresponding R a -NH2 is used to replace hexadecylamine.

[0611] 6018 was prepared by referring to the method of 6007, using an equimolar amount of the corresponding R a -NH2 replaces octadecylamine to prepare.

[0612] 9. Compound 6019

[0613] Structural formula:

[0614] Molecular weight: 753.66

[0615]

[0616] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask, followed by DCM (50 mL) and stirring. 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 5:1) to afford product 3 (4 g). At room temperature, product 3 (5.0 g), MeOH (50 mL), and Pd / C (1.0 g) were added to a three-necked flask. The hydrogen atmosphere was replaced, the temperature was raised to 50°C, and the mixture was stirred for 1 h. The reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was dried to afford product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. DMF (50 mL) was added and stirred to dissolve. Then, product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred. The mixture was allowed to react overnight at room temperature. Water (500 mL) and EA (500 mL) were added, and the mixture was separated and extracted. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM:MeOH = 20:1-10:1) to obtain solid 6 (380 mg). Solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to the single-necked flask at room temperature and reacted at room temperature for 2 h. The mixture was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1HNMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.33(s,8H),3.27-3.16(m,2H),3.06-2.90(m,4H),2.77(t,J=6.5Hz,4H ), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9Hz, 4H), 1.73 ( p, J = 7.7Hz, 4H), 1.49 ( tdd, J = 9.2, 7.9, 7.1, 4.0Hz, 4H), 1.31 ( s, 28H), 0.92 ( t, J = 6.8Hz, 3H).

[0617] 10. Compound 6020

[0618] Structural formula:

[0619] Molecular weight: 810.19

[0620]

[0621] At room temperature, octadecylamine (50 g) was added to a three-necked flask, followed by MeOH (200 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (31.5 g) was added dropwise. The mixture was stirred and returned to room temperature for 4 h. The reaction was stopped, and the mixture was directly concentrated under reduced pressure. Purification by column chromatography (PE:EA = 20:1-10:1) afforded product 2 (68 g). At room temperature, product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) were added to a single-necked flask, heated to 60°C, and maintained at this temperature for overnight reaction. The reaction was stopped, the reaction solution was concentrated under reduced pressure, 30 mL of toluene was added, and the mixture was heated to 60°C for dissolution. The mixture was slowly cooled to room temperature, filtered, and evaporated to dryness under reduced pressure to afford compound 3 (15 g). At room temperature, compound 3 (1.3 g) was added to a single-necked flask, and DMF (20 mL) was added and stirred. Then, 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g) were added, stirred, and reacted at room temperature overnight. Water (200 mL) was added and the mixture was directly freeze-dried under reduced pressure to obtain compound 4 (4 g). Compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) were added to a single-necked flask at room temperature and reacted for 1 h. The reaction was stopped and the reaction solution was concentrated under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57-3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03-1.58 (m, 10H), 1.31 (s, 30H), 1.01-0.82 (m, 3H).

[0622] 11. Compound 6021

[0623] Structural formula:

[0624] Molecular weight: 1182.79

[0625]

[0626] At room temperature, compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020) was added to a single-necked flask, MeOH (20 mL) was added and stirred, and then TREN (23.0 g) was added, stirred, and the temperature was raised to 60°C, stirred overnight, and concentrated under reduced pressure. The crude product was lyophilized to obtain product 3 (25 g). At room temperature, product 3 (22.0 g) and DCM (200 mL) were added to a single-necked flask, the temperature was lowered to 0°C, and (Boc)2O (71.7 g) was slowly added dropwise. After the temperature was restored to room temperature, the reaction was carried out for 3 h, water (200 mL) was added for extraction, and DCM (100 mL) was added for separation and extraction. The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain a yellow oil 4 (5.8 g). At room temperature, yellow oil 4 (1.4 g) was added to a single-necked flask. HCl / Dioxane (4 M) (15 mL) was slowly added under an ice-water bath and stirred. After addition, the mixture was returned to room temperature and reacted for 0.5 h. The mixture was then concentrated under reduced pressure to obtain compound 5 (800 mg). Compound 5 (1.0 g), (S)-2,6-di-tert-butyloxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g), and DMF (10 mL) were added to a single-necked flask at room temperature. The mixture was reacted overnight at room temperature. 100 mL of water and 100 mL of EA were added, and the mixture was separated and extracted. The EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). To a one-necked flask at room temperature, compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL) were added. The reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6021 (220 mg). 1H NMR(400MHz, Methanol-d4)δ3.96(t,J=6.6Hz,4H),3.75(dt,J=14.0,6.6Hz,4H),3.62-3.45(m,12H),3.36-3.33(m,14H),3.25-3.17(m,2H),3.04 -2.90(m,8H),2.83(t,J=6.9Hz,4H),2.05-1.82(m,8H),1.73(p,J=7.7Hz ,10H),1.53(qd,J=8.3,7.7,4.3Hz,8H),1.30(s,28H),0.97-0.87(m,3H).

[0627] 12. Compound 6023

[0628] Structural formula:

[0629] Molecular weight: 670

[0630]

[0631] At room temperature, compound dodecylamine (10 g) was added to a three-necked flask, followed by MeOH (100 mL) and stirring. The mixture was then cooled to 5°C in an ice-water bath, and methyl acrylate (10.2 g) was slowly added dropwise. The mixture was stirred and returned to room temperature. The reaction mixture was allowed to react at room temperature for 4 h, then stopped, concentrated under reduced pressure, and purified by column chromatography (PE:EA=20:1-10:1) to obtain product 2 (19 g). At room temperature, product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) were added to a single-necked flask, heated to 60°C, maintained at this temperature for overnight, and concentrated under reduced pressure to obtain compound 3 (20 g). 500 mg of the product was purified and lyophilized to obtain compound 6023-1 (218 mg). At room temperature, raw material 6023-1 (1.4 g) was added to a single-necked flask. DCM (20 mL) was added and stirred to dissolve. (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g) were then added and stirred. The mixture was allowed to react overnight at room temperature. The mixture was separated, extracted, dried, and concentrated under reduced pressure to obtain compound 4 (3.0 g). Compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) were added to the single-necked flask at room temperature and reacted for 2 h. The mixture was then concentrated under reduced pressure, purified, and lyophilized to obtain compound 6023 (123 mg). 1H NMR(400MHz, Methanol-d4)δ3.88(t,J=6.6Hz,2H),3.55-3.36(m,8H),3.36-3.33(m,2H),3.31-3.14(m,4H),3.04-2.93(m,4H),2. 77(t,J=6.5Hz,4H),2.00-1.82(m,4H),1.82-1.66(m,6H),1.50(qd,J=8.2,7.8,4.2Hz,4H),1.45-1.25(m,18H),0.96-0.87(m,3H).

[0632] 13. Compound 6024

[0633] Structural formula:

[0634] Molecular weight: 870.24

[0635]

[0636] Product 6023-1 (1.4 g, synthesized by the same method as 6023-1 in compound 6023) was added to a single-necked flask at room temperature. MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.8 g) was slowly added and stirred to mix. The temperature was raised to 60°C, and the reaction was allowed to proceed overnight at this temperature. The mixture was concentrated under reduced pressure to obtain compound 4 (2.0 g). Compound 4 (2.4 g) and ethylenediamine (20 mL) were added to a single-necked flask at room temperature, and the reaction was allowed to proceed overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6024 (191 mg). 1HNMR (400MHz, Methanol-d4) δ3.69(t,J=6.0Hz,4H),3.60-3.48(m,20H),3.42(t,J=6.1Hz,4H),3.25-3.19(m,2H),3 .12(t,J=5.8Hz,8H),2.83(t,J=6.5Hz,12H),1.78(tt,J=11.0,6.4Hz,2H),1.51-1.18(m,20H),0.91(t,J=6.7Hz,3H).

[0637] 14. Compound 6026

[0638] Structural formula:

[0639] Molecular weight: 443.67

[0640]

[0641] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the following differences: an equal molar amount of compound 1 was used instead of starting material 5 of compound 6019, and an equal molar amount of N-(tert-butyloxycarbonyl)ethanolamine was used instead of intermediate 4 of compound 6019. 1H NMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36-1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0642] Compounds 6025-6034 were obtained by referring to the synthetic route of the aforementioned compounds, except that hydroxyethylamine was used instead of the corresponding ethylenediamine, and the amino group of the hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc. After the reaction was completed, the protecting group was removed according to conventional methods.

[0643] (3) First Ionizable Lipid Example Compounds and Their Properties

[0644] The exemplary compounds of the present invention and their properties are listed in Table 1, which were synthesized according to the process routes in Preparation Example 1.

[0645] The calculated c-pKa (molnetwork), LogP (cLogP driver) of the exemplary compounds of the present invention, c-pKa and cLogP values ​​were generated by the ChemDraw module of Chemoffice.

[0646] Table 1 Properties of exemplary compounds of the first ionizable lipid

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674] The above properties of the exemplary compounds of the first ionizable lipid of the present invention indicate that they can serve as excellent surfactants and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0675] (4) Preparation of pulmonary delivery systems containing ionizable lipids

[0676]

[0677]

[0678] The pulmonary delivery systems containing ionizable lipids were prepared according to the above molar ratios, and the specific methods are as follows:

[0679] The lipid component was dissolved in ethanol, and the total concentration of all lipid raw materials in ethanol was 3 mg / mL. Firefly luciferase mRNA (Luciferase mRNA) was dissolved in 50 mM citrate buffer (pH 4.0) solution to prepare a Luciferase mRNA solution (wherein, the concentration of the Luciferase mRNA solution in Example 4 was 0.19 mg / ml, and the concentrations of other examples were adjusted according to the ratio). The lipid ethanol solution and the Luciferase mRNA solution were mixed in a nanopreparation device at a volume ratio of 1:3, and then ultrafiltration was performed. The sample was collected to obtain a pulmonary delivery system (nanoparticle) containing ionizable lipids.

[0680] Comparative Example 2

[0681] Based on Example 12, an additional 1.5% (based on the total molar amount of lipid used in Example 12 as 100%) of conjugated lipid (ALC-0159) was added as a lipid raw material, and all lipid components were dissolved in ethanol. The total concentration of all lipid raw materials in ethanol was 3.045 mg / mL. Luciferase mRNA was dissolved in 50 mM citrate buffer (pH 4.0) solution to prepare a Luciferase mRNA solution with a concentration of 0.19 mg / ml. The lipid ethanol solution and the Luciferase mRNA solution were mixed in a nanopreparation device at a volume ratio of 1:3, and then ultrafiltered and the sample was collected to obtain a pulmonary delivery system (nanoparticles) containing ionizable lipids.

[0682] (5) Particle size and zeta potential measurement of pulmonary delivery systems containing ionizable lipids

[0683] 1. Particle Size and Polydispersity Index (PDI) Determination: The average particle size and PDI of the nanoparticle sample solutions in the examples were determined by dynamic light scattering using a Malvern ZetaSizer Nano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0684] 2. Zeta potential: The zeta potential of the nanoparticle sample solution in the examples was measured using a Malvern ZetaSizer Nano ZS90 equipped with electrophoretic light scattering (ELS) technology. The refractive index of the dispersant was 1.330 and the test temperature was 25°C.

[0685] The average particle size, PDI, and Zeta potential data of the pulmonary delivery system containing ionizable lipids prepared in the examples are shown in Table 2.

[0686] Table 2 Summary of particle size, PDI, and Zeta potential of pulmonary delivery systems containing ionizable lipids

[0687]

[0688]

[0689] As can be seen from Table 2, the pulmonary delivery system containing ionizable lipids provided by the present invention has small and uniform particle size and is neutral or weakly electrically charged.

[0690] (6) Investigation of nanoparticle characteristics after atomization

[0691] The pulmonary delivery system containing ionizable lipids was atomized using an Omron compression nebulizer, and the atomized samples were collected for determination of properties such as particle size, potential, and encapsulation efficiency. The test method was the same as (5), and the results are shown in Table 3.

[0692] Table 3 Characteristic data of pulmonary delivery system containing ionizable lipids before and after aerosolization

[0693]

[0694]

[0695] As can be seen from Table 3, the particle properties of the pulmonary delivery system containing ionizable lipids of the present invention, such as particle size, particle size distribution coefficient, potential, encapsulation efficiency, etc., remain unchanged before and after atomization, and have good stability.

[0696] (7) In vivo evaluation in mice

[0697] 7.1 Intratracheal Administration

[0698] The pulmonary transfection efficiency and safety of the pulmonary delivery systems containing ionizable lipids prepared in each example were evaluated using 6-8 week old female Balb / c mice. The pulmonary delivery systems containing ionizable lipids of Examples 5 and 15 were administered intratracheally at a single dose of 0.375 mpk. At a specific time point (6 h) after administration, the animals were imaged in vivo using a PerkinElmer small animal imaging system to measure the bioluminescent signals in various organ tissues. The results are shown in Table 4 and the attached Figure 4. Figure 1 .

[0699] Table 4 In vivo imaging results of mice 6 hours after intratracheal administration

[0700]

[0701] * ND means not detected.

[0702] The results showed that the fluorescence expression was only in the lungs of mice dissected 6 hours ago, and there was basically no expression in other organs.

[0703] 7.2 Nebulized Inhalation

[0704] The pulmonary transfection efficiency and safety of the pulmonary delivery systems containing ionizable lipids prepared in each example were evaluated using 6-8 week old female Balb / c mice. The pulmonary delivery systems containing ionizable lipids of Examples 4, 7, and 12 and Comparative Examples 1 and 2 were administered by aerosol inhalation at a single dose of 1.125 mpk. At a specific time point (6 h) after administration, the animals were imaged in vivo using a PerkinElmer small animal imaging system to measure the bioluminescent signals in various organ tissues. The results are shown in Table 5 and the attached Figure 2 .

[0705] Table 5 In vivo imaging results of mice 6 hours after aerosol administration

[0706]

[0707] *ND means not detected.

[0708] The results showed that the fluorescence expression was only in the lungs of mice dissected 6 hours ago, and there was basically no expression in other organs.

[0709] 7.3 Pulmonary Distribution of Intratracheal Spray

[0710] The pulmonary transfection efficiency of the pulmonary delivery system containing ionizable lipids prepared in Example 6 was evaluated in female Balb / c mice aged 6-8 weeks. The pulmonary delivery system containing ionizable lipids in Example 6 and normal saline (blank control) were administered by intratracheal spray administration at a single dose of 0.375 mpk. Lungs were harvested at specific time points (e.g., 6 h, 24 h, 48 h, 72 h) after administration, and the five lobes of the lungs (according to the attached figure) were imaged using a PerkinElmer small animal imaging system. Figure 3 IVIS imaging was performed and the bioluminescent signal intensity in each lung lobe was measured. The results are shown in the attached Figure 4 .

[0711] It can be seen that after the mice were given the pulmonary delivery system containing ionizable lipids by intratracheal spray administration, all lobes of the mouse lungs had strong luciferase expression.

[0712] 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 a specific embodiment of the present invention. It should be noted that for those skilled in the art in the field of the present invention, various improvements and modifications can be made without departing from the principles of the present invention. Such improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A pulmonary delivery system comprising an ionizable lipid, characterized in that The invention relates to a pulmonary delivery system comprising an ionizable lipid, an auxiliary lipid and a bioactive substance, wherein the pulmonary delivery system comprising the ionizable lipid does not contain a polymer-conjugated lipid; the ionizable lipid is selected from at least one of a first ionizable lipid, a second ionizable lipid and a third ionizable lipid; The first ionizable lipid is selected from a compound comprising the general formula (1E) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof; （1E） Wherein, R1, R2, and R3 are independently selected from any one of the following groups: ； M is selected from -NR4R5; R4 and R5 are independently H, C 1-6 Straight-chain or branched alkyl, C 2-6 Straight-chain or branched alkenyl or C 2-6 Straight chain or branched chain alkynyl, the C 1-6 Straight-chain or branched alkyl, C 2-6 Straight chain or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, amine, amide, amidino, guanidino or halogen; G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -; wherein each R6, R 13 are independently selected from H, hydroxyl; R9, R 10 Independently of each other are 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 -; R11 and R12 are independently H, C1-6 straight or branched alkyl; Each R 16 、R 17 are independently selected from H; wherein the alkyl and alkenyl groups described in R4, R5, R6, R11, R12, R13, and R16 are unsubstituted or substituted with one or more groups selected from hydroxyl, thiol, amine, substituted amine, and halogen; n is an integer from 2 to 6; k is an integer from 1 to 6; Said salts do not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof: （2）； Wherein A1 is NH or O; When R a Selected from C6-C 24 Alkyl, C6-C 24 When alkenyl, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkylamines; Or when R a Selected from C6-C 24 When alcohol, R b and R c Each independently selected from C1-C 12 Alkyl, alkylamine; The C6-C 24 Alkyl, C6-C 24 The alkenyl group is a straight chain or branched structure; the alkylamine is , where R a ' is C1-C 12 Alkyl, the R b ' and R b ''Each independently selected from H, C1-C6 alkylamine, or ; R c '' is selected from C1-C6 alkyl groups which are unsubstituted or substituted with amino groups, R c ''' is H, or ; Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'does not exist; The third ionizable lipid is selected from at least one of the following compounds: 。 2. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The bioactive substance includes at least one of DNA, RNA, protein, peptide and small molecule compound.

3. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The amount of the bioactive substance used is 0.1-50% w / w of the total amount of the lipid raw material and the bioactive substance.

4. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein M is selected from the following structures: ； wherein m' and n' are independently integers of 0-6, R1'' and R2'' are independently H, C 1-6 Alkyl, C 2-6 alkenyl, guanidine, amidine, amide, and fatty amine groups.

5. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The compound of formula (1E) is selected from the compound represented by formula (1F): （1F）。 6. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The compound of formula (1E) is selected from the compound represented by formula (1G): （1G）。 7. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The compound of formula (1E) is selected from the compound represented by formula (1H): （1H）。 8. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The compound of formula (1E) is selected from: 。 9. The pulmonary delivery system containing ionizable lipids according to claim 1, wherein The compound of formula (2) is selected from at least one of the following compounds: 。 10. The pulmonary delivery system containing ionizable lipids according to any one of claims 1 to 7, wherein The helper lipid is a mixture of phospholipids and steroids.

11. The pulmonary delivery system containing ionizable lipids according to claim 10, wherein The phospholipids include at least one of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

12. The pulmonary delivery system containing ionizable lipids according to claim 10, wherein The phospholipid includes at least one of palmitoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine.

13. The pulmonary delivery system containing ionizable lipids according to claim 10, wherein The phospholipid includes at least one of lysophosphatidylcholine and lysophosphatidylethanolamine.

14. The pulmonary delivery system containing ionizable lipids according to claim 10, wherein The phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholestylsuccinyl-sn-glycero-3-phosphocholine, Phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-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-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, At least one of 2-diachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, and 1-stearoyl-2-oleoyl-phosphatidylcholine.

15. The pulmonary delivery system containing ionizable lipids according to claim 10, wherein The steroid includes at least one of cholesterol, cholesterol stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, salinarate, tomatine, ursolic acid, and α-tocopherol.

16. The pulmonary delivery system containing an ionizable lipid according to any one of claims 1 to 9, wherein The ionizable lipid-containing pulmonary delivery system further comprises an alcohol compound.

17. The pulmonary delivery system comprising an ionizable lipid according to claim 16, wherein The alcohol compound includes at least one of C6-C18 alcohol and polyethylene glycol; the molar ratio of the alcohol compound to the ionizable lipid is 0-2:

1.

18. The pulmonary delivery system containing an ionizable lipid according to claim 1, wherein The amount of the ionizable lipid is 5-80 mol% of the total lipid in the prescription; the amount of the auxiliary lipid is 20-95 mol% of the total lipid in the prescription.

19. The pulmonary delivery system comprising an ionizable lipid according to claim 1, wherein In the auxiliary lipid, the molar ratio of phospholipid to steroid is (5-60): (10-80).

20. The pulmonary delivery system comprising an ionizable lipid according to claim 1, wherein The ionizable lipid is a first ionizable lipid, a second ionizable lipid, or a third ionizable lipid, or a mixture of the first ionizable lipid and the second ionizable lipid, or a mixture of the second ionizable lipid and the third ionizable lipid; in the mixture of the first ionizable lipid and the second ionizable lipid, the molar ratio of the first ionizable lipid to the second ionizable lipid is (5-90): (1-30); in the mixture of the second ionizable lipid and the third ionizable lipid, the molar ratio of the third ionizable lipid to the second ionizable lipid is (5-90): (1-30).

21. The pulmonary delivery system comprising an ionizable lipid according to claim 1, wherein The particle size of the pulmonary delivery system containing ionizable lipids is 20-400 nm, the polydispersity index is less than 0.5, and the surface potential is -20 mV to 60 mV.

22. The pulmonary delivery system containing an ionizable lipid according to any one of claims 1 to 7, wherein Also included are excipients.

23. The pulmonary delivery system comprising an ionizable lipid according to claim 21, wherein The pulmonary delivery system containing ionizable lipids is in liquid form or solid form.

24. The method for preparing a pulmonary delivery system containing ionizable lipids according to any one of claims 1 to 23, wherein: include: The step of mixing the bioactive substance, the ionizable lipid and the helper lipid in a solvent.

25. Use of the pulmonary delivery system containing ionizable lipids according to any one of claims 1 to 23 in the preparation of a medicament for delivering a biologically active substance via intranasal, intrabronchial or pulmonary administration.

26. The use according to claim 25, characterized in that The drug is administered to a mammal.

27. The use according to claim 25, characterized in that The drug is administered to the cells.

28. The use according to claim 25, characterized in that The drug is administered via a nebulizer or inhaler.

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