Preparation method of lipid nanoparticles and compound thereof

Abstract

The invention provides a lipid nanoparticle and a preparation method of a compound thereof, the lipid nanoparticle comprises an amino lipid compound, and the amino lipid compound comprises at least one of a compound (I), a compound (II) and a compound (III). The amino lipid compound contains long non-polar residues, so that the whole amino lipid compound has a hydrophobic characteristic, and the amino group has a hydrophilic characteristic at the same time. Due to the amphiphilic characteristic, the amino lipid compound can be used for forming lipid nano-particles, the hydrophobic part of the amino lipid compound is beneficial to stabilizing the structure of the lipid nano-particles, and amino groups can generate relatively strong hydrophilicity in vivo, so that the water solubility and biocompatibility of the lipid nano-particles are improved; therefore, the loading capacity and the encapsulation efficiency of the lipid nanoparticles are improved, so that the drug or gene delivery is more efficient, the required administration dosage is reduced, and the side effect is reduced.

Description

Technical Field

[0001] The invention belongs to the field of synthetic chemistry, and in particular relates to a method for preparing lipid nanoparticles and a complex thereof. Background Art

[0002] Lipid nanoparticles (LNPs) are nanoscale particles made of lipid materials, which are usually used to encapsulate and deliver bioactive substances, such as nucleic acids (such as mRNA, siRNA), proteins, drugs, etc. These particles have good biocompatibility and biodegradability, and can effectively penetrate the cell membrane to deliver active ingredients into the cell. They are widely used in gene therapy, vaccine development, drug delivery and other fields. In recent years, with the rapid development of nucleic acid drugs and mRNA vaccines, the research and application of LNPs have received great attention.

[0003] Many LNPs will degrade during storage, especially when stored in liquid form, which may cause changes in encapsulation efficiency and particle size, thus affecting the efficacy and bioavailability of the drug. To overcome this problem, low-temperature storage or freeze-drying is usually required, but this increases storage costs and transportation difficulties. Especially in nucleic acid drug delivery, the loading capacity and encapsulation efficiency of existing LNPs technology are too low, resulting in larger dosages and more side effects. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing lipid nanoparticles and their complexes, aiming to solve the problems of difficulty in storing lipid nanoparticles and low loading capacity and encapsulation rate.

[0005] In order to solve the above technical problems, on the one hand, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises an amino lipid compound, wherein the amino lipid compound comprises at least one of compound (I), compound (II), and compound (III); wherein,

[0006] The structural formula of the compound (I) is The structural formula of the compound (II) is The structural formula of the compound (III) is

[0007] In the above structural formula, Z 1 , Z 2 , Z 3 , Z 4 and Z 5 Same or different from each other, Z 1 , Z 2 , Z 3 , Z 4 , Z 5Both include -CH(OR a )-, -C=C-, -C≡C-, -O-, -NH-, -C(=O)O-, -C(=O)-, -OC(=O)-, -N(R a )C(=O)-、-C(=O)N(R a )-、-N(R a )C(=O)N(R a )-、-OC(=O)N(R a )-、-N(R a )C(=O)O-, -C(=O)S-, -SC(=O)-, -SC=C-, -SS-, at least one of 1 , Z 2 , Z 3 , Z 4 , Z 5 Any R a Including H, C1-C 18 Hydrocarbyl, -OH, -O- optionally substituted C2-C 18 Hydrocarbon, -C=C-, -C≡C- optionally substituted C4-C 18 Hydrocarbon or C1-C 18 at least one of heteroalkyl groups;

[0008] A 1 and A 2 Same or different from each other, A 1 and A 2 All include C1-C 18 Alkylidene, C3-C 18 At least one of a cycloalkyl group, a phenyl group, a benzyl group, and a 4-7 membered heterocyclic ring, R 1 , R 2 , R 3 , R 4 All include H, C1-C 18 Alkyl, C1-C 18 Olefin, C1-C 18 Alkyne, C3-C 18 At least one of a cycloalkyl, a phenyl, a benzyl, and a 4-7 membered heterocyclic ring, wherein the heterocyclic ring in the 4-7 membered heterocyclic ring includes at least one of a substituent selected from the group consisting of a C1-C8 alkyl, a haloalkyl, -O-C1-C8 alkyl, a halogen, OH, CN, a nitro, NH2, -NH(C1-C6 alkyl), and N(C1-C6 alkyl)2;

[0009] m and n are the same or different from each other and are each independently an integer from 1 to 12;

[0010] G 1 and G 2 Same or different from each other, G1 and G 2 All include -C=C-, -C≡C-, -NH-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) p -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a 、-NR a At least one of C(=O)O-, p=0, 1 or 2, G 1 and G 2 R a Including H, C1-C 12 At least one of the hydrocarbon groups,

[0011] L 1 , L 2 , L 3 and L 4 Same or different from each other, L 1 , L 2 , L 3 , L 4 All include C1-C 24 Alkylene, C2-C 24 an optionally substituted 4- to 10-membered heterocyclic ring containing a heteroatom selected from nitrogen, sulfur and oxygen, wherein the C1-C 24 Alkylene, the C2-C 24 At least one of the alkenylene group, the C3-C8 cycloalkylene group, and the C3-C8 cycloalkenylene group is substituted with one or more substituents selected from the group consisting of a hydrocarbon group, a carboxyl group, an acyl group, and an alkoxy group;

[0012] R 1 Including H, OR 1a 、CN、-C(=O)OR 1a 、-OC(=O)R 1a 、-NR 1b C(=O)R 1a 、-NR 1a R 1b At least one of R 1a and R 1b Including H, C1-C 12 At least one hydrocarbon group, R 2 Including branched C6-C 24 Alkyl, branched C6-C 24 Alkenyl, branched C6-C24 At least one of the alkynyl groups.

[0013] On the other hand, the present invention provides a preparation method for preparing an mRNA-lipid nanoparticle complex comprising lipid nanoparticles, the preparation method comprising the steps of:

[0014] S1, synthetic amino lipid compounds;

[0015] S2, respectively dissolving the auxiliary lipid, the structural lipid, the polyethylene glycol-lipid, and the amino lipid compound in an organic solvent to obtain an auxiliary lipid solution, a structural lipid solution, a polyethylene glycol-lipid solution, and an amino lipid compound solution, respectively, and mixing the auxiliary lipid solution, the structural lipid solution, the polyethylene glycol-lipid solution, and the amino lipid compound solution to obtain an alcohol phase, wherein the organic solvent comprises at least one of anhydrous ethanol, chloroform, and ethyl acetate;

[0016] S3, mixing the mRNA stock solution, a pH adjuster and a solvent to obtain an aqueous phase, wherein the pH adjuster includes at least one of a phosphate buffer, a sodium acetate buffer and a sodium bicarbonate buffer, and the solvent includes at least one of anhydrous ethanol, pure water and dichloromethane;

[0017] S4, first mix the alcohol phase and the aqueous phase, then add a buffer to obtain an mRNA-lipid nanoparticle complex.

[0018] The method for preparing lipid nanoparticles and their complexes proposed by the present invention has the following advantages:

[0019] Amino lipid compounds all have hydrophobic characteristics due to their long non-polar residues, and they also have hydrophilic characteristics due to the amino group. Due to this amphiphilic characteristic, amino lipid compounds can be used to form lipid nanoparticles. The hydrophobic part of the amino lipid compound helps to stabilize the structure of the lipid nanoparticles, while the amino group can produce strong hydrophilicity in the body, increase the water solubility and biocompatibility of the lipid nanoparticles, thereby increasing the loading capacity and encapsulation rate of the lipid nanoparticles, which makes drug or gene delivery more efficient, helps to reduce the required dosage, and reduces side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a data graph showing the effects of different types of lipid nanoparticles on the release of cellular lactate dehydrogenase in one embodiment of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] The present invention provides a lipid nanoparticle, the lipid nanoparticle comprises an amino lipid compound, the amino lipid compound comprises at least one of compound (I), compound (II) and compound (III); wherein,

[0023] The structural formula of compound (I) is The structural formula of compound (II) is The structural formula of compound (III) is

[0024] In the above structural formula, Z 1 , Z 2 , Z 3 , Z 4 and Z 5 Same or different from each other, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 Both include -CH(OR a )-, -C=C-, -C≡C-, -O-, -NH-, -C(=O)O-, -C(=O)-, -OC(=O)-, -N(R a )C(=O)-、-C(=O)N(R a )-、-N(R a )C(=O)N(R a )-、-OC(=O)N(R a )-、-N(R a )C(=O)O-, -C(=O)S-, -SC(=O)-, -SC=C-, -SS-, at least one of 1 , Z 2 , Z 3 , Z 4 , Z 5 Any R a Including H, C1-C 18 Hydrocarbyl, -OH, -O- optionally substituted C2-C 18 Hydrocarbon, -C=C-, -C≡C- optionally substituted C4-C 18 Hydrocarbon or C1-C 18 at least one of heteroalkyl groups;

[0025] A 1 and A 2 Same or different from each other, A1 and A 2 All include C1-C 18 Alkylidene, C3-C 18 At least one of a cycloalkyl group, a phenyl group, a benzyl group, and a 4-7 membered heterocyclic ring, R 1 , R 2 , R 3 , R 4 All include H, C1-C 18 Alkyl, C1-C 18 Olefin, C1-C 18 Alkyne, C3-C 18 At least one of a cycloalkyl, a phenyl, a benzyl, and a 4-7 membered heterocyclic ring, wherein the heterocyclic ring in the 4-7 membered heterocyclic ring includes at least one of a substituent selected from the group consisting of a C1-C8 alkyl, a haloalkyl, -O-C1-C8 alkyl, a halogen, OH, CN, a nitro, NH2, -NH(C1-C6 alkyl), and N(C1-C6 alkyl)2;

[0026] m and n are the same as or different from each other, and are each independently an integer from 1 to 12;

[0027] G 1 and G 2 Same or different from each other, G 1 and G 2 All include -C=C-, -C≡C-, -NH-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) p -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、-NR a C(=O)NR a -、-OC(=O)NR a 、-NR a At least one of C(=O)O-, p=0, 1 or 2, G 1 and G 2 R a Including H, C1-C 12 At least one of the hydrocarbon groups,

[0028] L 1 , L 2 , L 3 and L 4 Same or different from each other, L 1 , L 2 , L 3 , L 4 All include C1-C 24 Alkylene, C2-C 24C3-C8 cycloalkylene, C3-C8 cycloalkenylene, an optionally substituted 4- to 10-membered heterocyclic ring having a heteroatom selected from nitrogen, sulfur and oxygen, C1-C 24 Alkylene, C2-C 24 At least one of the alkenylene group, the C3-C8 cycloalkylene group, and the C3-C8 cycloalkenylene group is substituted with one or more substituents selected from the group consisting of a hydrocarbon group, a carboxyl group, an acyl group, and an alkoxy group;

[0029] R 1 Including H, OR 1a 、CN、-C(=O)OR 1a 、-OC(=O)R 1a 、-NR 1b C(=O)R 1a 、-NR 1a R 1b At least one of R 1a and R 1b Including H, C1-C 12 At least one hydrocarbon group, R 2 Including branched C6-C 24 Alkyl, branched C6-C 24 Alkenyl, branched C6-C 24 At least one of the alkynyl groups.

[0030] Amino lipid compounds all have hydrophobic characteristics due to the long non-polar residues, and at the same time have hydrophilic characteristics due to the amino group. Due to this amphiphilic characteristic, amino lipid compounds can be used to form lipid nanoparticles, such as lipid bilayers, micelles, liposomes, etc. The hydrophobic part of the amino lipid compound helps to stabilize the structure of the lipid nanoparticles, while the amino group can produce strong hydrophilicity in the body, increasing the water solubility and biocompatibility of the lipid nanoparticles.

[0031] In the context of the present disclosure, the term "lipid nanoparticle" means a nanometer-sized substance obtained by placing an amino lipid compound in an aqueous solution. These particles are particularly lipid nanoparticles, lipid bilayer vesicles (liposomes), multilayer vesicles or micelles. In some preferred embodiments, the lipid nanoparticle is a liposome containing an amino lipid compound of the present disclosure, which is a microvesicle composed of a bilayer of lipid amphiphilic molecules enclosing an aqueous compartment. Liposome formation is not a spontaneous process. Lipid vesicles are first formed when lipids are placed in water, thereby forming a bilayer or a series of bilayers, each separated by water molecules. Liposomes can be formed by treating lipid vesicles in water with ultrasound. In the context of the present disclosure, the term "lipid bilayer" means a film formed by two layers of lipid molecules. The term "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solutions form aggregates with hydrophilic head regions when in contact with water, integrating the hydrophobic single tail region in the center of the micelle.

[0032] Therefore, lipid nanoparticles have excellent performance of encapsulating bioactive ingredients, and lipid nanoparticles containing bioactive ingredients can be used to deliver any of a variety of therapeutic agents to cells. The present invention discloses the use of lipid nanoparticles as described above to deliver bioactive ingredients to cells. The present invention discloses a method for delivering bioactive ingredients to cells, tissues or organs, which includes contacting lipid nanoparticles containing bioactive ingredients with cells, tissues or organs, providing new possibilities for therapeutic treatment of organisms.

[0033] The amphiphilic nature of lipid nanoparticles enables them to effectively encapsulate nucleic acid molecules (such as DNA or RNA) and enter cells through cell membranes. This efficient cellular uptake ability significantly improves the efficiency of nucleic acid transport. Lipid nanoparticles can form a protective coating that prevents nucleic acids from degrading in the in vivo environment and enhances their stability. By reducing exposure to external enzymes and other reactive molecules, lipid nanoparticles ensure that nucleic acids remain active.

[0034] Depending on the design of lipid nanoparticles, targeted release can be achieved by adjusting their surface properties. By modifying the affinity ligands of lipid molecules, nanoparticles can be made to bind to specific cell types more selectively, thereby achieving targeted transport. The construction of lipid nanoparticles can be designed to have controllable release characteristics to ensure the release of nucleic acids under appropriate time and environmental conditions. This controllability can improve the therapeutic effect and reduce side effects. The formation of lipid nanoparticles and their interaction with the cell membrane can promote their entry into specific subcellular structures within the cell and enhance the biological effects of nucleic acids. For example, they can help nucleic acids transport into the nucleus within the cell, thereby improving the efficiency of transcription and translation.

[0035] Lipid nanoparticles are based on natural lipids or their derivatives, and usually have good biocompatibility, can reduce toxic reactions to organisms, and help improve the safety of drugs. Lipid nanoparticles can not only be used to transport nucleic acids, but also can be added with other drug ingredients through surface modification to achieve combined therapy. This versatility gives it greater application potential in the treatment of various diseases (such as cancer, genetic defect diseases, etc.).

[0036] In one embodiment, the amino lipid compound disclosed in the present invention is used to prepare a carrier of an active ingredient. In some embodiments, the carrier is in the form of lipid nanoparticles, such as lipid bilayers, micelles, and liposomes.

[0037] In one embodiment, the lipid nanoparticles contain an amino lipid compound and a pharmaceutically acceptable carrier, diluent or excipient, which work together to ensure the stability, delivery efficiency and biocompatibility of the nanoparticles.

[0038] The carrier can be phospholipids, cholesterol, polyethylene glycol (PEG), lactose, etc. The carrier provides support to stabilize the structure of lipid nanoparticles and prevent nanoparticles from agglomerating or degrading in vivo or in vitro. Specific carriers (such as polyethylene glycol) can prolong the circulation time of drugs in the body and increase the probability of drugs reaching target tissues. Carriers can also optimize the biocompatibility of nanoparticles, reduce recognition and clearance by the immune system, and improve efficacy.

[0039] The diluent can be water, sterile saline, glucose solution, etc. The diluent is used to adjust the concentration of the lipid nanoparticle dispersion to an acceptable concentration level for easy injection or administration. Appropriate diluents can reduce interactions between particles, prolong the stability of the dispersion system, and prevent particle aggregation. The diluent helps to evenly distribute the nanoparticles in the solution, ensure consistent dosage, and facilitate accurate delivery.

[0040] Excipients can be glycerol, polysorbate 80 (such as Tween 80), propylene glycol, sorbitol, etc. Excipients can give lipid nanoparticles better physical properties, such as increasing their fluidity, lubricity, mouthfeel, etc., making the dosage form more suitable for clinical use. Excipients can protect the active drug ingredients in nanoparticles from degradation or oxidation. For example, antioxidant excipients can prolong drug stability. Certain excipients can interact with the outer layer of nanoparticles to regulate the release rate of the drug, thereby prolonging the drug's duration of action.

[0041] In some embodiments of the present invention, the lipid nanoparticles further include at least one of auxiliary lipids, structural lipids, and polyethylene glycol-lipids, and the molar ratio of amino lipid compound: auxiliary lipid: structural lipid: polyethylene glycol-lipid is 28-60: 5-42: 15.5-53.5: 0.5-3.5. For example, it can be 45:10:42.5:2.5, 45:11:41.5:2.5, 42.0:10.5:45.0:2.5, 42.0:16.0:39.5:2.5, 40.0:16.0:41.5:2.5, 40.0:18.0:39.5:2.5, 35.0:16.0:46.5:2.5, 35.0:25.0: 36.5:3.5、28.0:33.5:35.0:3.5、32.0:37.0:40.5:0.5、35.0:40.0:22.5:2.5、40.0:42.0:15.5:25、40.0:20.0:38.5:1.5、45.0:15.0:38.5:1.5、55.0:5.0:38.5:1.5、 60.0:5.0:33.5:1.5, 45.0:20.0:33.5:1.5, 50.0:20.0:28.5:1.5, 55.0:20.0:23.5:1.5, 60.0:20.0:18.5:1.5, 40.0:15.0:43.5:1.5, 50.0:15.0:33.5:1.5, 55.0:15. 0:28.5:1.5, 60.0:15.0:23.5:1.5, 40.0:10.0:48.5:15., 45.0:10.0:43.5:1.5, 55.0:10.0:33.5:1.5, 40.0:5.0:53.5:1.5, 45.0:5.0:48.5:1.5, 50.0:5.0:43.5:1.5.

[0042] Optimizing the molar ratio can help obtain lipid nanoparticles of appropriate size, ensure uniform particle size distribution, and avoid particles that are too large or too small, which is beneficial for in vivo delivery and can improve drug loading and delivery effects. By adjusting the ratio of lipid components, lipid nanoparticles suitable for production on different scales can be obtained, making the technology highly scalable and suitable for large-scale industrial production. By adjusting the molar ratio of different lipids and optimizing the composition of lipid nanoparticles, the cell membrane penetration ability and endocytosis efficiency of liposomes can be improved, ensuring that mRNA can effectively enter the target cells for expression.

[0043] By adjusting the molar ratio of amino lipid compounds, auxiliary lipids, structural lipids and PEG-lipids, the particle size, morphology and surface properties of lipid nanoparticles can be further optimized to enhance their uptake and release efficiency in specific cells or tissues.

[0044] Through the appropriate molar ratio, lipid nanoparticles can better avoid recognition and clearance by the immune system in the body, thereby reducing immune responses and side effects. In addition, a reasonable lipid ratio helps prevent premature degradation of lipid particles in the body and enhances their long-term stability.

[0045] In summary, the composition and molar ratio of this lipid nanoparticle provides an optimized delivery system, which can effectively improve the stability, delivery efficiency, cell penetration ability and biocompatibility of mRNA, thereby providing a more efficient and safer delivery platform for the application of mRNA vaccines or gene therapy.

[0046] The auxiliary lipids include phospholipids, and the phospholipids include at least one of distearoylphosphatidylcholine, oleoylphosphatidylethanolamine, dioleoyl phosphatidylcholine, dioleoylphosphatidylserine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1.2-dipalmitoyl-sn-glycero-3'-O-4'-(N,N,N-trimethyl)homoserine, and blood phospholipids.

[0047] As an auxiliary component in the lipid system, auxiliary lipids can increase the stability of lipid particles, improve the morphology and size distribution of particles, and further improve the delivery efficiency of mRNA. At the same time, auxiliary lipids can also help improve the formation of lipid bilayer membranes and avoid premature degradation of lipid particles in the body.

[0048] The structural lipids include sterol substances, and the sterol substances include at least one of cholesterol, cholesterol ester, steroid hormones, steroid vitamins, bile acid, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives.

[0049] Structural lipids provide core support and stability for lipid particles, ensuring the integrity and functionality of lipid nanoparticles, thereby effectively encapsulating mRNA and preventing its degradation during transportation.

[0050] The polyethylene glycol-lipid includes at least one of polyethylene glycol-distearoylphosphatidylethanolamine and polyethylene glycol derivatives of 1,2-dimyristyl glyceride, and the average molecular weight of the polyethylene glycol in the polyethylene glycol-lipid is 2000 to 5000, for example, 2000, 3000, 4000, 5000, etc.

[0051] Polyethylene glycol-lipid (PEG-lipid) is often used to increase the blood half-life of lipid nanoparticles and reduce the clearance of the immune system. The hydrophilic part of PEG makes lipid particles more stable and can reduce the nonspecific adsorption of lipid nanoparticles to the cell surface, thereby improving the targeting and delivery efficiency of drugs.

[0052] The role of PEG-lipids: PEG-modified lipids can effectively reduce the interaction between lipid nanoparticles and proteins and immune cells in the blood, thereby extending the circulation time of lipid nanoparticles and reducing the distribution of particles in non-target tissues. By optimizing the ratio of PEG-lipids, the accumulation of lipid particles in target tissues (such as liver, muscle, etc.) can be improved, and the delivery efficiency can be improved.

[0053] The molecular weight of PEG has an important influence on the surface properties and particle size of lipid particles. The appropriate PEG molecular weight can adjust the size and surface properties of lipid particles, so that they have better distribution and delivery effects in the body. Too small PEG may not effectively reduce the immune response, while too large PEG may cause the particles to be too large and affect cell endocytosis. By optimizing the PEG molecular weight and lipid ratio, it is possible to ensure the stability and size uniformity of liposomes while avoiding cell rejection of particles and enhancing their delivery efficiency.

[0054] In one embodiment of the present invention, when the amino lipid compound comprises compound (I), step S1 comprises:

[0055] S1.1. Add a phosphorus source reagent to compound M11 and compound M12 to carry out a substitution reaction to obtain compound M13, wherein the phosphorus source reagent includes phosphorus oxychloride.

[0056] Phosphorus source reagents are used to initiate substitution reactions, usually replacing the position of a functional group or functional group. The selectivity of the phosphorus source reagent and the reaction conditions can affect the selectivity and yield of the reaction, so they are crucial when optimizing the synthetic route. The reaction in this step helps to introduce phosphate groups, giving the compound higher hydrophilicity or biological activity, and is widely used in drug development.

[0057] S1.2. Compound M13 and compound M14 are placed in an alkaline reagent for amidation reaction to obtain compound M15, wherein the alkaline reagent includes at least one of triethylamine, pyridine, N-methylmorpholine, N,N-diisopropylethylamine, potassium carbonate, and potassium hydroxide.

[0058] Amidation is one of the common bonding reactions in chemical synthesis. Through this reaction, amino and acyl groups can be efficiently connected to form biologically active amide bonds. Alkaline reagents usually help to deprotonate the amine group and increase its nucleophilicity, thereby promoting the amidation reaction. Different alkaline reagents will affect the reaction rate and selectivity. By precisely controlling the type and concentration of the alkaline reagent, the desired amidation product can be obtained in the synthesis and the occurrence of side reactions can be avoided.

[0059] S1.3. Place compound M16 and compound M17a in an alkaline reagent for condensation reaction to obtain compound M18; or place compound M16 and compound M17b in an alkaline reagent for substitution reaction to obtain compound M18.

[0060] This step generates new chemical bonds through condensation reactions, usually involving the removal of small molecules (such as water, alcohol, etc.), which is essential for the formation of large molecules or complex structures. Using different reagents or reaction conditions can selectively introduce new functional groups, further changing the properties or biological activity of the molecule. For example, substitution reactions can introduce different alkyl, aryl or other functional groups, providing more diversity for subsequent synthesis. By choosing different reaction pathways (condensation or substitution reactions), products with different structures but similar functions can be obtained. This provides greater flexibility for subsequent steps.

[0061] S1.4. Compound M15 and compound M18 are placed in an alkaline reagent to carry out a nucleophilic substitution reaction to obtain compound (I).

[0062] Nucleophilic substitution reactions involve the reaction of nucleophiles with carbon atoms or other electron-deficient centers in the reactants to form new chemical bonds. This reaction is often used to construct complex organic molecules and has high selectivity. Nucleophilic substitution reactions help introduce specific functional groups into the synthesis route, thereby optimizing the functional properties of the final product, such as improving the water solubility, stability or biological activity of the drug. By precisely controlling the reaction conditions, the yield and purity of the reaction can be improved, ensuring the quality of the synthesized target compound, which is particularly important in the synthesis of complex molecules.

[0063] The structural formula of the above compound is:

[0064]

[0065] The reaction process is as follows:

[0066]

[0067] In one embodiment of the present invention, when the amino lipid compound comprises compound (II), step S1 comprises:

[0068] S1.1. Add a phosphorus source reagent to compound M21 and compound M22 to carry out a substitution reaction to obtain compound M23, wherein the phosphorus source reagent includes phosphorus trichloride. The use of such a phosphorus source reagent can stably introduce phosphorus elements, so that the compound structure contains active phosphorus groups, which is helpful for further modification of subsequent reactions, improves the reactivity of the target compound and the efficiency of introducing specific functional groups, thereby causing a substitution reaction between the hydroxyl groups in compound M21 and compound M22 and the chlorine atoms.

[0069] S1.2, compound M23 and compound M24 are placed in an alkaline reagent, and a substitution reaction is performed to obtain compound M25, wherein the alkaline reagent includes at least one of triethylamine, pyridine, N-methylmorpholine, N, N-diisopropylethylamine, potassium carbonate, and potassium hydroxide. The alkaline environment in this step can effectively promote the substitution reaction of the chlorine atoms in compound M23 and the terminal hydrogen in compound M24, generate the required functional groups and ensure high selectivity. In addition, the alkaline environment can also suppress the occurrence of side reactions, improve product purity and reaction yield. This gives better structural stability to the final generated compound.

[0070] The bromine atom of compound M24 can also be replaced by an iodine atom, or by a sulfhydryl group (-SH). When the sulfhydryl group reacts with a strong reducing agent (such as hydrogen) or other reducing chemicals, a reduction reaction occurs to generate hydrogen sulfide.

[0071] S1.3, Compound M26 and Compound M27 are placed in an acidic reagent for imidization reaction to obtain Compound M28, wherein the acidic reagent includes at least one of acetic acid, hydrochloric acid, and p-toluenesulfonic acid. Under acidic conditions (such as acetic acid, hydrochloric acid, etc.), the aldehyde group of Compound M26 and the amino group of Compound M27 react to form an imine intermediate M28. The imidization reaction introduces double bonds and nitrogen elements to the target molecule by forming an imine group containing C=N. The technical effect of this step is that the introduction of the imine group increases the diversity of the functional groups of the compound, enhances the interaction between molecules, and creates conditions for further reduction reactions.

[0072] S1.4. Add a reducing agent to compound M28 to carry out a reduction reaction to obtain compound M29, wherein the reducing agent includes at least one of sodium borohydride, lithium aluminum hydride, and sodium cyanoborohydride.

[0073] In this step, the imine intermediate M28 is reduced to the amino intermediate M29 by adding a reducing agent (such as sodium cyanoborohydride). The reduction reaction ensures the complete conversion of the imine group to generate a relatively stable amino structure. The technical effect of this reduction step is to improve the chemical stability of the target compound and avoid the activity of the C=N double bond, thereby improving the controllability of subsequent reactions and the biocompatibility of the product.

[0074] S1.5. Compound M25 and compound M29 are placed in an alkaline reagent for nucleophilic substitution reaction to obtain compound (II). M25 and M29 are subjected to a nucleophilic substitution reaction in an alkaline reagent to obtain compound (II). The selection of nucleophilic reagent helps to optimize the reaction rate and selectivity, making the reaction more efficient. This reaction gives compound (II) a unique arrangement and stability of active functional groups, while ensuring the efficient generation of the final product. The technical effect of this substitution reaction is to enhance the biological activity, solubility and wide application of the compound.

[0075] The structural formula of the above compound is:

[0076]

[0077] The reaction process is as follows:

[0078]

[0079]

[0080] In one embodiment of the present invention, when the amino lipid compound comprises compound (III), step S1 comprises:

[0081] S1.1. Add an activator to compound M31 and compound M32, carry out chlorination reaction, and then place in an alkaline reagent to obtain M33, wherein the activator includes at least one of dichlorothionyl chloride, oxalyl chloride, thionyl chloride, and cyanogen chloride, and the alkaline reagent includes at least one of 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, pyridine, potassium carbonate, potassium hydroxide, sodium carbonate, and N-methylmorpholine.

[0082] The acyl chloride reaction increases the reactivity of the molecule by introducing an acyl chloride functional group, making it more selective and efficient in the subsequent amidation reaction. In addition, the acyl chloride treatment can also effectively reduce the generation of unnecessary by-products, thereby improving the overall reaction purity.

[0083] S1.2. Compound M33 and compound M34 are placed in an alkaline reagent for amidation reaction to obtain M35.

[0084] The amidation reaction converts the acyl chloride intermediate into an amide structure, which improves the molecular stability and increases the number of polar and hydrogen bond donors in the molecule, thereby improving the solubility of the final product in water or other polar solvents. At the same time, the introduction of amide bonds enhances the biocompatibility of the compound, which helps its performance in pharmaceutical or biological applications.

[0085] S1.3. Compound M36 and compound M37 are placed in an alkaline reagent to carry out a substitution reaction to obtain M38.

[0086] Substitution reactions introduce specific functional groups, which enhance the intermolecular interactions and potential functionality by changing the chemical structure of the compound. This process helps to improve the chemical stability and durability of the compound, making it more stable in different environments, thereby broadening its application areas.

[0087] S1.4. Place compound M35 and compound M38 in an alkaline reagent to carry out a cross-linking reaction to obtain compound (III).

[0088] The cross-linking reaction forms a multifunctional network structure, which makes compound (III) have higher mechanical strength and thermal stability, especially in materials science, showing excellent tensile strength and hydrolysis resistance. The cross-linking structure also gives the compound better chemical corrosion resistance, making it suitable for use under high-intensity or special environmental conditions.

[0089] The structural formula of the above compound is:

[0090]

[0091] The reaction process is as follows:

[0092]

[0093]

[0094] Specifically, several structural formulas of compound (I) are as follows:

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] Specifically, the steps for synthesizing I-1 are:

[0107]

[0108]

[0109] Tridecanol (1-1, 2.00 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C for 1 hour under nitrogen. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(2-aminoethyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours under nitrogen at room temperature. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain product 1-3 as a colorless oil (2.46 g, 90%).

[0110] 6-Bromohexanoyl chloride (1-4, 2.12 g, 10 mmol), n-undecanol (1-5, 1.72 g, 10 mmol) and dry DCM (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.10 g, 90% yield).

[0111] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-1 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 817 (M+H + ).

[0112] Steps for synthesizing I-2:

[0113]

[0114] 6-Bromohexanoyl chloride (2-1, 2.12 g, 10 mmol), n-tridecanol (2-2, 2.01 g, 10 mmol) and dry DCM (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.21 g, 90% yield).

[0115] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-2 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 80%, 0.63 g. MS (ES) m / z: 845 (M+H + ).

[0116] Steps for synthesizing I-3:

[0117]

[0118]

[0119] n-Undecyl alcohol (3-1, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C for 1 hour under nitrogen. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(2-aminoethyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours under nitrogen at room temperature. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain product 3-3 as a colorless oil (2.46 g, 90%).

[0120] Di-undecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (3-3, 0.256 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-3 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 761 (M+H + ).

[0121] Steps for synthesizing I-4:

[0122]

[0123] I-1 (0.016 g, 0.2 mmol), KOH (0.001 g, 0.2 mmol) and EtOH (5 ml) were added to a 5 ml round-bottom flask. Stirred at room temperature for 10 hours, and the organic solvent was removed under reduced pressure. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain the desired product I-4; Yield: 90%, 0.011 g. MS (ES) m / z: 691 (M+H+).

[0124] Steps for synthesizing I-6:

[0125]

[0126] Undecanoyl chloride (6-2, 2.05 g, 10 mmol), 6-bromohexanol (6-1, 1.81 g, 10 mmol) and dry DCM (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Then triethylamine (1.66 ml, 12 mmol) was added and stirred at room temperature for 3 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain a colorless oil (3.06 g, 90%).

[0127] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), undecyl 6-bromohexyl undecanoate (6-3, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-6 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 80%, 0.60 g. MS (ES) m / z: 817 (M+H + ).

[0128] Steps for synthesizing I-7:

[0129]

[0130]

[0131] 6-bromohexanoyl chloride (7-1, 2.12 g, 10 mmol), dec-2-yn-1-ol (7-2, 2.01 g, 10 mmol) and dry DCM (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. The reaction solution was stirred at room temperature for 3 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain a colorless oil (3.21 g, 90%).

[0132] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), dec-2-ynyl-1-yl 6-bromohexanoate (7-3, 0.18874 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-7 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 64%, 0.53 g. MS (ES) m / z: 799 (M+H + ).

[0133] Steps for synthesizing I-8:

[0134]

[0135] 6-Bromo-n-hexanol (8-1, 1.81 g, 10 mmol) and dichlorothionyl (0.73 ml, 10 mmol) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon and stirred at room temperature for 1 hour. The HCl gas generated by the reaction was extracted under reduced pressure and the nitrogen in the balloon was replaced. Dry dichloromethane (10 ml), diisopropylethylamine (2.68 ml, 15 mmol) and 2-hexyldecanoic acid (8-2, 2.56 g, 10 mmol) were added to the reaction flask under nitrogen protection and stirred at room temperature for 3 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain a colorless oil (3.97 g, 95%).

[0136] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), 6-bromohexyl 2-decanoate (8-3, 0.21 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-8 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 52%, 0.24 g. MS (ES) m / z: 887 (M+H + ).

[0137] Steps for synthesizing I-9:

[0138]

[0139] 6-Bromohexanoyl chloride (9-1, 2.12 g, 10 mmol), 2-hexyldecan-1-ol (9-2, 2.01 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (4.08 g, 96% yield).

[0140] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), 2-hexyldecyl 6-bromohexanoate (9-3, 0.21 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-9 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 52%, 0.24 g. MS (ES) m / z: 887 (M+H + ).

[0141] Steps for synthesizing I-10:

[0142]

[0143] Decyl-2-yn-1-ol (10-1, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.46 g, 90%).

[0144] Di(dec-2-yn-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (10-2, 0.256 g, 0.5 mmol), dec-2-yn-1-yl 6-bromohexanoate (7-3, 0.18874 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-10 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.16 g. MS (ES) m / z: 721 (M+H + ).

[0145] Steps for synthesizing I-11:

[0146]

[0147] Di(dec-2-yn-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (10-2, 0.256 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-11 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.18 g. MS (ES) m / z: 739 (M+H + ).

[0148] Steps for synthesizing I-12:

[0149]

[0150] 6-Bromohexanoyl chloride (12-1, 2.12 g, 10 mmol), undec-10-enyl-1-ol (12-2, 2.01 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.22 g, 90% yield).

[0151] Undecyl-10-enyl-1-ol (12-2, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and stirred at 50 ° C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.46 g, 90%)

[0152] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), undec-10-en-1-yl 6-bromohexanoate (12-3, 0.173 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-12 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 769 (M+H + ).

[0153] Steps for synthesizing I-13:

[0154]

[0155] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.18 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-13 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 799 (M+H + ).

[0156] Steps for synthesizing I-14:

[0157]

[0158] n-Undecyl alcohol (14-1, 1.72 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C for 1 hour under nitrogen. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 3-((4-aminobutyl)amino)propyl-1-ol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours under nitrogen at room temperature. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.46 g, 90%)

[0159] Di-undecyl (4-((3-hydroxypropyl)amino)butyl)phosphoramide (14-2, 70.26 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-14 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.21 g. MS (ES) m / z: 803 (M+H + ).

[0160] Steps for synthesizing I-15:

[0161]

[0162] Di-tridecyl (2-((2-hydroxyethyl)amino)ethyl)phosphoramide (1-3, 0.245 g, 0.5 mmol), undec-10-en-1-yl 6-bromohexanoate (12-3, 0.173 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-15 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.19 g. MS (ES) m / z: 815 (M+H + ).

[0163] Steps for synthesizing I-23:

[0164]

[0165] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), dec-2-ynyl-1-yl 6-bromohexanoate (7-3, 0.18874 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-23 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 753 (M+H + ).

[0166] Steps for synthesizing I-24:

[0167]

[0168] 6-Bromohexanoyl chloride (24-1, 2.12 g, 10 mmol), (E)-undec-2-ene-1-ol (24-2, 2.01 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.22 g, 90% yield).

[0169] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), (E)-undec-2-en-1-yl 6-bromohexanoate (24-3, 0.173 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-24 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.18 g. MS (ES) m / z: 769 (M+H + ).

[0170] Steps for synthesizing I-25:

[0171]

[0172] 8-Bromooctanoyl chloride (25-1, 2.40 g, 10 mmol), heptadecan-9-ol (25-2, 2.56 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.63 g, 90% yield).

[0173] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), heptadecan-9-yl 8-bromooctanoate (25-3, 0.23 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-25 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.23 g. MS (ES) m / z: 883 (M+H + ).

[0174] Steps for synthesizing I-26:

[0175]

[0176] 6-bromohexanoyl chloride (26-1, 2.12 g, 10 mmol), 2-pentyldecyl-1-ol (26-2, 2.28 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water 2 times. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.56 g, 90% yield).

[0177] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), 2-pentyldecyl-1-yl 6-bromohexanoate (26-3, 0.23 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-26 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.31 g. MS (ES) m / z: 827 (M+H + ).

[0178] Steps for synthesizing I-28:

[0179]

[0180] 6-bromohexanoyl chloride (28-1, 2.12 g, 10 mmol), (Z)-octadec-9-ene-1-ol (28-2, 2.68 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stirred for 3 hours at room temperature. Then diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.63 g, 90% yield).

[0181] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-28 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.20 g. MS (ES) m / z: 867 (M+H + ).

[0182] Steps for synthesizing I-29:

[0183]

[0184] Dodeca-11-ynyl-1-ol (29-1, 1.82 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C for 1 hour under nitrogen. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours under nitrogen at room temperature. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain a colorless oil (2.03 g, 80%).

[0185] Di(dodecan-11-ynyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (29-2, 0.26 g, 0.5 mmol), tridecyl 6-bromohexanoate (2-4, 0.174 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-29 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 823 (M+H + ).

[0186] Steps for synthesizing I-30:

[0187]

[0188] Di(dodecan-11-ynyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (29-2, 0.26 g, 0.5 mmol), undecyl 6-bromohexanoate (1-6, 0.16 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-30 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 795 (M+H + ).

[0189] Steps for synthesizing I-31:

[0190]

[0191] 6-bromohexanoyl chloride (31-1, 2.12 g, 10 mmol), dodeca-11-ynyl-1-ol (31-2, 1.82 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water 2 times. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.12 g, 90% yield).

[0192] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), dodecan-11-ynyl-1-yl 6-bromohexanoate (31-3, 0.18 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-31 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.16 g. MS (ES) m / z: 781 (M+H + ).

[0193] Steps for synthesizing I-32:

[0194]

[0195] (Z)-octadec-9-ene-1-ol (32-1, 2.68 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C for 1 hour under nitrogen. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(2-aminoethyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain a colorless oily product (6.12 g, 90%).

[0196] Di((Z)-octadec-9-en-1-yl)(2-((2-hydroxyethyl)amino)ethyl)phosphoramide (32-2, 0.34 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-32 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.31 g. MS (ES) m / z: 1077 (M+H + ).

[0197] Steps for synthesizing I-36:

[0198]

[0199] (Z)-Non-3-enyl-1-ol (36-1, 1.42 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50°C under nitrogen for 1 hour. After completion, the byproduct HCl gas was evacuated to nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.03 g, 80%).

[0200] Di((Z)-non-3-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (36-2, 0.23 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-36 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.19 g. MS (ES) m / z: 811 (M+H + ).

[0201] Steps for synthesizing I-38:

[0202]

[0203] 6-Bromohexanoyl chloride (38-1, 2.12 g, 10 mmol), dodeca-3-enyl-1-ol (38-2, 1.82 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water twice. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.12 g, 90% yield).

[0204] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), dodecan-3-en-1-yl 6-bromohexanoate (38-3, 0.18 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-38 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.16 g. MS (ES) m / z: 783 (M+H + ).

[0205] Steps for synthesizing I-40:

[0206]

[0207] (Z)-Dodeca-3-enyl-1-ol (40-1, 1.78 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50°C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.03 g, 80%).

[0208] Di((Z)-dodec-3-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (40-2, 0.23 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-40 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 895 (M+H + ).

[0209] Steps for synthesizing I-43:

[0210]

[0211] Undecyl-10-enyl-1-ol (43-1, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and stirred at 50 ° C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(4-aminobutyl)amino-ethanol (0.53 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.46 g, 90%)

[0212] Di(undec-10-en-1-yl)(4-((2-hydroxyethyl)amino)butyl)phosphoramide (43-2, 0.24 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-43 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.22 g. MS (ES) m / z: 881 (M+H + ).

[0213] Steps for synthesizing I-44:

[0214]

[0215] Undecyl-10-enyl-1-ol (43-1, 2.01 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and stirred at 50 ° C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 3-(4-aminobutyl)amino-propanol (0.54 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours at room temperature under nitrogen. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.46 g, 90%)

[0216] Di(undec-10-en-1-yl)(4-((3-hydroxypropyl)amino)butyl)phosphoramide (44-2, 0.24 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-44 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.22 g. MS (ES) m / z: 895 (M+H + ).

[0217] Steps for synthesizing I-45:

[0218]

[0219] 8-Bromooctanoyl chloride (45-1, 2.40 g, 10 mmol), (Z)-octadec-9-ene-1-ol (45-2, 2.68 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stirred for 3 hours at room temperature. Then diluted with DCM (10 mL) and washed with water 2 times. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.65 g, 90% yield).

[0220] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 8-bromooctanoate (45-3, 0.24 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-45 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.21 g. MS (ES) m / z: 895 (M+H + ).

[0221] Steps for synthesizing I-46:

[0222]

[0223] 6-bromohexanoyl chloride (46-1, 2.12 g, 10 mmol), (9Z, 12Z)-18-9,12-diene-1-ol (46-2, 2.68 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round-bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water 2 times. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.63 g, 90% yield).

[0224] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), (9Z,12Z)-octadec-9,12-dien-1-yl 6-bromohexanoate (46-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-46 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 45%, 0.20 g. MS (ES) m / z: 865 (M+H + ).

[0225] Steps for synthesizing I-47:

[0226]

[0227] Undec-10-ynyl-1-ol (47-1, 1.78 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50°C under nitrogen for 1 hour. After completion, the byproduct HCl gas was evacuated to nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred at room temperature under nitrogen for 3 hours. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to give a colorless oil (2.03 g, 80%).

[0228] Di(undec-10-ynyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (47-2, 0.23 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-47 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 863 (M+H + ).

[0229] Steps for synthesizing I-48:

[0230]

[0231] Dodeca-11-ynyl-1-ol (48-1, 1.92 g, 10 mmol) and phosphorus oxychloride (0.77 g, 5 mmol) were added to a 50 mL round-bottom flask equipped with a stirring bar and a nitrogen balloon. The solution was stirred at room temperature for 1 hour and then stirred at 50 ° C under nitrogen for 1 hour. After completion, the byproduct HCl gas was replaced with nitrogen. Dry DCM (5 ml) was added to the colorless liquid, and DIPEA (2.68 ml, 15 mmol) and 2-(3-aminopropyl)amino-ethanol (0.52 g, 5 mmol) were added under nitrogen. The solution was stirred for 3 hours under nitrogen at room temperature. It was then diluted with DCM (10 mL) and washed with water twice. The organic phase was separated and dried over anhydrous MgSO4. The desired product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain a colorless oil (2.03 g, 80%).

[0232] Di(dode-11-ynyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (48-2, 0.24 g, 0.5 mmol), (Z)-octadec-9-en-1-yl 6-bromohexanoate (28-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-48 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 48%, 0.21 g. MS (ES) m / z: 891 (M+H + )

[0233] Steps for synthesizing I-50:

[0234]

[0235] 8-Bromooctanoyl chloride (50-1, 2.40 g, 10 mmol), dodeca-11-yn-1-ol (50-2, 2.24 g, 10 mmol) and dry dichloromethane (10 ml) were added to a 50 ml round bottom flask equipped with a nitrogen balloon. Triethylamine (1.66 ml, 12 mmol) was slowly added under nitrogen. Stir at room temperature for 3 hours. Then dilute with DCM (10 mL) and wash with water 2 times. The organic phase was concentrated under reduced pressure to obtain a colorless oily compound (3.65 g, 90% yield).

[0236] Di(undec-10-en-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (12-4, 0.25 g, 0.5 mmol), dodecan-11-yn-1-yl 8-bromooctanoate (50-3, 0.21 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-50 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 42%, 0.21 g. MS (ES) m / z: 809 (M+H + ).

[0237] Steps for synthesizing I-51:

[0238]

[0239] Di((Z)-dodec-3-enyl-1-yl)(3-((2-hydroxyethyl)amino)propyl)phosphoramide (40-2, 0.23 g, 0.5 mmol), (9Z,12Z)-octadec-9,12-dien-1-yl 6-bromohexanoate (46-3, 0.22 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were placed in a 5 ml vial. The mixture was stirred at 60°C for 24 hours. The solvent was removed under reduced pressure. The desired product I-51 was purified by silica gel column chromatography (DCM / MeOH=20 / 1); Yield: 50%, 0.21 g. MS (ES) m / z: 893 (M+H + ).

[0240] Synthesis steps of I-34:

[0241]

[0242]

[0243] 8-Bromooctanoic acid (34-1, 2.23 g, 10 mmol) and dichlorothionyl (1.3 g, 0.79 ml, 11 mmol) were added to a 50 mL round bottom flask equipped with a nitrogen balloon and stirred at room temperature for 1 hour. The by-product hydrogen chloride gas was extracted and replaced with nitrogen. Dry dichloromethane (10 ml), triethylamine (2.02 g, 2.84 ml, 20 mmol) and heptadecan-9-ol (34-2, 2.56 g, 10 mmol) were added to the bottle and stirred at room temperature for 1 hour. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (3.71 g, 80% yield).

[0244] Heptadecan-9-yl 8-bromooctanoate (34-3, 2.3 g, 5 mmol), ethanolamine (1.22 g, 1.22 ml, 20 mmol) and MeCN (10 ml) were added to a 50 mL round-bottom flask and stirred at 70° C. for 12 hours. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave a colorless liquid (1.62 g, 85% yield).

[0245] 6-bromohexanoyl chloride (34-5, 1.06 g, 5 mmol), dodeca-11-ynyl-1-ol (34-6, 0.91 g, 5 mmol) and dry dichloromethane (10 ml) were added to a 25 mL round-bottom flask equipped with a nitrogen balloon, and triethylamine (1.66 ml, 12 mmol) was slowly added to the reaction, and stirred at room temperature for 2 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (1.03 g, 80% yield).

[0246] Heptadecyl-9-yl 8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), dodecan-11-yn-1-yl 6-bromohexanoate (34-7, 0.18 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were added to a 10 ml reaction bottle. The reaction was stirred at 70°C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave a colorless liquid I-34; Yield: 50%, 0.18 g. MS (ES) m / z: 720 (M+H + ).

[0247] Synthesis steps of I-39:

[0248]

[0249] 6-Bromohexanoyl chloride (39-1, 1.06 g, 5 mmol), 10-undecyl-1-ol (39-2, 0.91 g, 5 mmol) and dry dichloromethane (10 ml) were added to a 25 mL round-bottom flask equipped with a nitrogen balloon, and triethylamine (1.66 ml, 12 mmol) was slowly added to the reaction, and stirred at room temperature for 2 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (1.03 g, 80% yield).

[0250] Heptadecyl-9-yl 8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), undec-10-yn-1-yl 6-bromohexanoate (39-3, 0.18 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were added to a 10 ml reaction bottle. The reaction was stirred at 70°C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave colorless liquid I-39; Yield: 50%, 0.18 g. MS (ES) m / z: 706 (M+H + ).

[0251] Synthesis steps of I-41:

[0252]

[0253] 8-Bromooctanoyl chloride (41-1, 1.20 g, 5 mmol), dodeca-11-ynyl-1-ol (41-2, 0.91 g, 5 mmol) and dry dichloromethane (10 ml) were added to a 25 mL round-bottom flask equipped with a nitrogen balloon, triethylamine (1.66 ml, 12 mmol) was slowly added to the reaction, and stirred at room temperature for 2 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (1.83 g, 80% yield).

[0254] Heptadecyl-9-yl 8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), dodecan-11-yn-1-yl 8-bromooctanoate (41-3, 0.19 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were added to a 10 ml reaction bottle. The reaction was stirred at 70°C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave colorless liquid I-41; Yield: 48%, 0.18 g. MS (ES) m / z: 748 (M+H + ).

[0255] Synthesis steps of I-42:

[0256]

[0257] 8-Bromooctanoyl chloride (42-1, 1.20 g, 5 mmol), hex-5-ynyl-1-ol (42-2, 0.49 g, 5 mmol) and dry dichloromethane (10 ml) were added to a 25 mL round-bottom flask equipped with a nitrogen balloon, triethylamine (1.66 ml, 12 mmol) was slowly added to the reaction, and stirred at room temperature for 2 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (1.21 g, 80% yield).

[0258] Heptadecyl-9-yl 8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), hex-5-ynyl-1-yl 8-bromooctanoate (42-3, 0.15 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were added to a 10 ml reaction bottle. The reaction was stirred at 70°C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave a colorless liquid I-42; Yield: 48%, 0.14 g. MS (ES) m / z: 664 (M+H + ).

[0259] Synthesis steps of I-49:

[0260]

[0261] 8-Bromooctanoyl chloride (49-1, 1.20 g, 5 mmol), non-8-ynyl-1-ol (49-2, 0.70 g, 5 mmol) and dry dichloromethane (10 ml) were added to a 25 mL round-bottom flask equipped with a nitrogen balloon, and triethylamine (1.66 ml, 12 mmol) was slowly added to the reaction, and stirred at room temperature for 2 hours. Then it was diluted with DCM (10 mL) and washed with water twice. The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate=20 / 1) to obtain a colorless oil (1.41 g, 80% yield).

[0262] Heptadecyl-9-yl 8-((2-hydroxyethyl)amino)octanoate (34-4, 0.22 g, 0.5 mmol), non-8-ynyl-1-8-bromooctanoate (42-3, 0.15 g, 0.5 mmol), K2CO3 (0.138 g, 1 mmol), KI (0.0032 g 0.02 mmol) and acetonitrile (4 ml) were added to a 10 ml reaction bottle. The reaction was stirred at 70°C for 24 hours and then cooled to room temperature. The solvent was removed under reduced pressure. Purification by silica gel column chromatography (DCM / MeOH=20 / 1) gave colorless liquid I-49; Yield: 48%, 0.18 g. MS (ES) m / z: 706 (M+H + ).

[0263] In one embodiment of the present invention, the steps for preparing lipid nanoparticles encapsulating luciferase mRNA (FucmRNA) include:

[0264] 3 μl of CleanCapFluc mRNA stock solution (1 mg / mL), 5.63 μl of 0.1 M sodium acetate buffer and 13.87 μl were added to the container and mixed to prepare an aqueous phase.

[0265] The amino lipid compound, auxiliary lipid, structural lipid and PEG-lipid disclosed in the present invention were dissolved in anhydrous ethanol to prepare respective solutions with concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL and 25 mg / mL, respectively. In a molar ratio, Lipid: DSPC: Cholesterol: DMG-PEG2000 was 48: 10: 40.5: 1.5, and the above 4 solutions were respectively pipetted to prepare an alcohol phase with a Lipid concentration of 7.3 mmol / L.

[0266] Take the above 7.5 μl ethanol solution and the aqueous solution and mix them quickly, then add 570 μl 25 mM Trisbuffer-10% sucrose (pH 7.0, filtered) solution and mix quickly to obtain the mRNA-LNP complex sample.

[0267] Furthermore, the in vitro mRNA-LNP delivery efficiency detection tool includes:

[0268] Among them, cell line: HEK-293 cells (Procell, CL-0001), culture medium: DMEM supplemented with 10% fetal bovine serum (Invitrogen), screening format: 24-well plate cell transfection

[0269] Detection (readout): The absolute value of the reporter gene firefly luciferase activity relative to the total protein expression of the cells (using the BCA protein concentration assay kit to detect the total protein content of each well of cells, Beyotime, P0012) (using the Luciferase Assay Kit to detect the bioluminescence intensity and then indirectly determine the firefly luciferase activity, Promega E1483). According to the manufacturer's instructions, TransIT-mRNA Transfection Kit (Mirusbio, MIR2225) was used as a positive control group.

[0270] The steps include:

[0271] 1. One day before mRNA transfection, HEK293 cells were seeded in a 24-well plate at 25,000 cells / ml, 0.4 ml per well, and cultured overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere.

[0272] 2. On the day of transfection, prepare TransIT-mRNAReagent / mRNABoost / RNA complex and mRNA-LNP complex respectively.

[0273] a) Take 240ul preheated Opti-MEM Reduced Serum Medium and put it into a 1.5ml centrifuge tube. First, add 1.2ug mRNA and gently pipette to mix. Then add 2.4ul mRNA Boost and gently pipette to mix. Finally, add 2.4ul mRNA Reagent and gently pipette to mix. Let it stand at room temperature for 5 minutes to form a complex. Take 100ul of the complex and evenly add it to each well of cells. Add it to 2 wells in total.

[0274] b) Take 100ul of the prepared mRNA-LNP complex and evenly add it to each well of cells. Each LNP sample is added to 2 wells.

[0275] c) Gently shake the 24-well plate and place it in a 37°C, 5% CO2 incubator for overnight culture.

[0276] 3. On the second day of transfection, remove the 24-well plate from the incubator, remove the cell supernatant, add 150ul of cell lysis buffer (Passive lysis 5X buffer, Promega, E1941) to each well, repeatedly blow with a pipette tip, collect the cells into a new 1.5ml centrifuge tube, centrifuge at 14000rpm for 30s, take 20ul of cell lysis supernatant for BCA assay, take 2ul of cell lysis supernatant for luciferase assay, and use VarioskanLUX multifunctional microplate reader (Thermo, VLBL00D1) for absorbance detection at 562nm and chemiluminescence detection, respectively.

[0277]

[0278] The test results are shown in Table 1:

[0279] Table 1:

[0280]

[0281]

[0282] The TransIT control group showed a higher luciferase activity (about 1.65E+06), indicating that the transfection efficiency of this control group was higher, so it can be used as a positive control to evaluate the relative efficiency of other LNP samples. The numerical differences between different LNP samples (such as I-1, I-2, etc.) in the table indicate that different formulations have different delivery efficiencies for mRNA in HEK-293 cells. For example, some samples have higher luciferase activity (such as I-29, I-37, etc.), while other samples have lower activity (such as I-6, I-12, etc.), indicating that these samples have significant differences in delivery efficiency.

[0283] The two repeated measurements of each sample are usually close, indicating good experimental repeatability. The two data of some samples are quite different, which may indicate that the stability or uniformity of the sample is poor and deserves further optimization. By comparing the luciferase activity of the TransIT control group and the LNP sample, the delivery efficiency of different LNP samples can be evaluated. The high value of the control group provides a benchmark for the LNP sample. If the value of the sample is close to or higher than the control group, the delivery effect is more ideal; otherwise, the delivery effect needs to be improved.

[0284] In one embodiment, the in vitro toxicity detection kit of LNP nanoparticles comprises:

[0285] (1) Ingredients:

[0286] Add the specified amount of RNAse-free pure water and sodium acetate buffer to the container and mix well to prepare a 0.25M sodium acetate solution. Dissolve the amino lipid compound, auxiliary lipid, structural lipid and PEG-lipid disclosed in the present invention in anhydrous ethanol to prepare respective solutions with concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL and 25 mg / mL, respectively. In molar ratio, Lipid:DSPC:CHO-HPM-DMG-2000 is 48:10:40.5:1.5. Pipette the above 4 solutions respectively to prepare an alcohol phase with a Lipid concentration of 7.3 mmol / L.

[0287] (2) Microfluidic preparation:

[0288] Use an automatic syringe pump to inject the water phase into the microfluidic chip at a flow rate of 7.5 mL / min and the alcohol phase at a flow rate of 4 mL / min to obtain a blank lipid nanoparticle sample.

[0289] (3) Dialysis: The product of step (2) was loaded into a dialysis bag and placed in a TrisBuffer-10% sucrose solution for displacement to remove residual ethanol, unassembled lipids, etc. Dialysis was performed for 24 hours at room temperature in the dark under magnetic stirring (the dialysate was replaced every 12 hours).

[0290] (4) Concentration:

[0291] The product of step (3) was concentrated 5 times by centrifugation to obtain a sample to be tested.

[0292] (5) Particle size test

[0293] Part of the samples were tested for particle size using the Zetasizer Pro instrument from Malvern Panalytical. The particle size data are shown in Table 2:

[0294] Table 2:

[0295] sample Particle size (nM) I-29 183 I-39 220 SM-102 (control) 210

[0296] In one embodiment, the cytotoxicity assessment tool comprises:

[0297] Cell line: HEK-293 cells (Procell, CL-0001)

[0298] Culture medium: DMEM supplemented with 10% fetal bovine serum (Invitrogen)

[0299] Screening format: 96-well cell culture plate

[0300] Detection (readout): Cytotoxicity assay kit (CyQUANT TMLDHCytotoxicity Assay Kit (Invitrogen, C20300) converts lactate dehydrogenase (LDH) in the cell culture supernatant into a red formazan product through a coupled enzymatic reaction. The product can be measured spectrophotometrically at 490 nm. The level of formazan formed is proportional to the amount of LDH released into the culture medium, thereby evaluating the degree of cytotoxicity.

[0301] The steps of cytotoxicity assessment include:

[0302] 1. On the first day, HEK293 cells were seeded at 100,000 cells / ml in a 96-well plate, 0.1 ml per well, and cultured overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere.

[0303] 2. The next day, the prepared blank LNP lipid nanoparticles were diluted into 3 concentrations (62.5ug / ml, 125ug / ml, 250ug / ml), added to the cells, 10ul was added to each well, and 3 wells of cells were added for each concentration; 10ul sterile water was added to the spontaneous LDH release control well, and a total of 3 wells of cells were added; the maximum LDH release control well was not treated. The 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator and incubated overnight.

[0304] 3. On the third day, take out the 96-well plate from the incubator, add 10ul of cell lysate to the maximum LDH release control well, add 3 wells of cells in total, and put them back into the incubator at 37°C and 5% CO2 for incubation for 45 minutes; after the time is up, take out the 96-well plate, pipette 50ul of cell culture supernatant from each well of cells into a new 96-well plate, add 50ul of reaction mixture to each well, mix well and incubate at room temperature in the dark for 30min; then add 50ul of reaction stop solution to each well and immediately measure the absorbance at 490nm and 680nm (VarioskanLUX multifunctional microplate reader, Thermo, VLBL00D1), and subtract the absorbance at 680nm (background) from the absorbance at 490nm. Calculate cytotoxicity using the following formula:

[0305]

[0306] Figure 1 To measure the effects of three different types of LNPs (lipid nanoparticles) on the release of cellular lactate dehydrogenase (LDH). LDH release is usually used to evaluate cell membrane damage and cytotoxicity, so the results of this experiment can reflect the toxicity of different concentrations of LNPs to cells. Experimental grouping: The figure shows three different types of LNPs - I-29, I-39, and SM-102. In the legend, I-29 is marked in blue, I-39 is orange, and SM-102 is gray.

[0307] The experiment used three different concentrations of LNP, which can be 62.5μg / mL, 125μg / mL and 250μg / mL. The percentage change of LDH release of different LNPs was measured at each concentration. The percentage data shown in the figure indicates the LDH release level of different LNPs at each concentration. All LDH release values ​​are negative (for example, -7.51%, -4.01%, etc.), which usually means a reduction (inhibition) of LDH release. The lower the value, the smaller the effect on LDH release, or the lower the toxicity to cells.

[0308] All experimental groups had negative LDH release at all concentrations, indicating that these LNPs had little or no significant effect on LDH release from cells, suggesting lower cytotoxicity. The three LNPs (I-29, I-39, SM-102) had slightly different effects on LDH release at different concentrations, but the overall differences were small. This may indicate that the cytotoxicity of these LNPs is not much different. : As the concentration increases (from 62.5μg / mL to 250μg / mL), the negative value of LDH release does not change much, indicating that these LNPs do not significantly increase cytotoxicity within the measured concentration range.

[0309] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A lipid nanoparticle, characterized in that: The lipid nanoparticles include an amino lipid compound, and the amino lipid compound includes at least one of compound (I), compound (II), and compound (III); wherein, The structural formula of the compound (I) is The structural formula of the compound (II) is The structural formula of the compound (III) is In the above structural formula, Z 1 , Z 2 , Z 3 , Z 4 and Z 5 Same or different from each other, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 Both include -CH(OR a )-, -C=C-, -C≡C-, -O-, -NH-, -C(=O)O-, -C(=O)-, -OC(=O)-, -N(R a )C(=O)-、-C(=O)N(R a )-、-N(R a )C(=O)N(R a )-、-OC(=O)N(R a )-、-N(R a )C(=O)O-, -C(=O)S-, -SC(=O)-, -SC=C-, -SS-, at least one of 1 , Z 2 , Z 3 , Z 4 , Z 5 Any R a Including H, C1-C 18 Hydrocarbyl, -OH, -O- optionally substituted C2-C 18 Hydrocarbon, -C=C-, -C≡C- optionally substituted C4-C 18 Hydrocarbon or C1-C 18 at least one of heteroalkyl groups; A 1 and A 2 Same or different from each other, A 1 and A 2 All include C1-C 18 Alkylidene, C3-C 18 At least one of a cycloalkyl group, a phenyl group, a benzyl group, and a 4-7 membered heterocyclic ring, R 1 , R 2 , R 3 , R 4 All include H, C1-C 18 Alkyl, C1-C 18 Olefin, C1-C 18 Alkyne, C3-C 18 At least one of a cycloalkyl, a phenyl, a benzyl, and a 4-7 membered heterocyclic ring, wherein the heterocyclic ring in the 4-7 membered heterocyclic ring includes at least one of a substituent selected from the group consisting of a C1-C8 alkyl, a haloalkyl, -O-C1-C8 alkyl, a halogen, OH, CN, a nitro, NH2, -NH(C1-C6 alkyl), and N(C1-C6 alkyl)2; m and n are the same as or different from each other, and are each independently an integer from 1 to 12; G 1 and G 2 Same or different from each other, G 1 and G 2 All include -C=C-, -C≡C-, -NH-, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) p -, -SS-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa, -NRaC(=O)O-, p=0, 1 or 2, G 1 and G 2 Ra includes H, C1-C 12 At least one of the hydrocarbon groups, L 1 , L 2 , L 3 and L 4 Same or different from each other, L 1 , L 2 , L 3 , L 4 All include C1-C 24 Alkylene, C2-C 24 an optionally substituted 4- to 10-membered heterocyclic ring containing a heteroatom selected from nitrogen, sulfur and oxygen, wherein the C1-C 24 Alkylene, the C2-C 24 At least one of the alkenylene group, the C3-C8 cycloalkylene group, and the C3-C8 cycloalkenylene group is substituted with one or more substituents selected from the group consisting of a hydrocarbon group, a carboxyl group, an acyl group, and an alkoxy group; R 1 including H, OR 1a , CN, -C(=O)OR 1a , -OC(=O)R 1a , -NR 1b C(=O)R 1a , -NR 1a R 1b At least one of R 1a and R 1b Including H, C1-C 12 At least one hydrocarbon group, R 2 Including branched C6-C 24 Alkyl, branched C6-C 24 Alkenyl, branched C6-C 24 At least one of the alkynyl groups.

2. The lipid nanoparticle according to claim 1, characterized in that Several structural formulas of the amino lipid compounds are as follows:

3. The lipid nanoparticle according to claim 1, characterized in that The lipid nanoparticles also include at least one of auxiliary lipids, structural lipids, and polyethylene glycol-lipids, and the molar ratio of the amino lipid compound: the auxiliary lipid: the structural lipid: the polyethylene glycol-lipid is 28-60: 5-42: 15.5-53.5: 0.5-3.

5.

4. The lipid nanoparticle according to claim 3, characterized in that The auxiliary lipids include phospholipids, and the phospholipids include at least one of distearoylphosphatidylcholine, oleoylphosphatidylethanolamine, dioleoyl phosphatidylcholine, dioleoylphosphatidylserine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1.2-dipalmitoyl-sn-glycero-3'-O-4'-(N,N,N-trimethyl)homoserine, and blood phospholipids.

5. The lipid nanoparticle according to claim 3, characterized in that The structural lipids include sterol substances, and the sterol substances include at least one of cholesterol, cholesterol ester, steroid hormones, steroid vitamins, bile acid, cholesterol, ergosterol, β-sitosterol, and oxidized cholesterol derivatives.

6. The lipid nanoparticle according to claim 3, characterized in that The polyethylene glycol-lipid comprises at least one of polyethylene glycol-distearoylphosphatidylethanolamine and polyethylene glycol derivatives of 1,2-dimyristyl glyceride, and the average molecular weight of the polyethylene glycol in the polyethylene glycol-lipid is 2000-5000.

7. A preparation method, characterized in that: For preparing an mRNA-lipid nanoparticle complex comprising the lipid nanoparticles according to any one of claims 1 to 6, the preparation method comprising the steps of: S1, synthetic amino lipid compounds; S2, respectively dissolving the auxiliary lipid, the structural lipid, the polyethylene glycol-lipid, and the amino lipid compound in an organic solvent to obtain an auxiliary lipid solution, a structural lipid solution, a polyethylene glycol-lipid solution, and an amino lipid compound solution, respectively, and mixing the auxiliary lipid solution, the structural lipid solution, the polyethylene glycol-lipid solution, and the amino lipid compound solution to obtain an alcohol phase, wherein the organic solvent comprises at least one of anhydrous ethanol, chloroform, and ethyl acetate; S3, mixing the mRNA stock solution, a pH adjuster and a solvent to obtain an aqueous phase, wherein the pH adjuster includes at least one of a phosphate buffer, a sodium acetate buffer and a sodium bicarbonate buffer, and the solvent includes at least one of anhydrous ethanol, pure water and dichloromethane; S4, first mix the alcohol phase and the aqueous phase, then add a buffer to obtain an mRNA-lipid nanoparticle complex.

8. The preparation method according to claim 7, characterized in that: When the amino lipid compound comprises compound (I), step S1 comprises: S1.1, adding a phosphorus source reagent to compound M11 and compound M12 to carry out a substitution reaction to obtain compound M13, wherein the phosphorus source reagent includes phosphorus oxychloride; S1.2, placing the compound M13 and the compound M14 in an alkaline reagent to carry out an amidation reaction to obtain a compound M15, wherein the alkaline reagent includes at least one of triethylamine, pyridine, N-methylmorpholine, N,N-diisopropylethylamine, potassium carbonate, and potassium hydroxide; S1.3, placing compound M16 and compound M17a in the alkaline reagent to carry out a condensation reaction to obtain compound M18; or, placing compound M16 and compound M17b in the alkaline reagent to carry out a substitution reaction to obtain compound M18; S1.4, placing the compound M15 and the compound M18 in the alkaline reagent to carry out a nucleophilic substitution reaction to obtain compound (I); The structural formula of the above compound is:

9. The preparation method according to claim 7, characterized in that: When the amino lipid compound comprises compound (II), step S1 comprises: S1.1, adding a phosphorus source reagent to compound M21 and compound M22 to carry out a substitution reaction to obtain compound M23, wherein the phosphorus source reagent includes phosphorus trichloride; S1.2, placing the compound M23 and the compound M24 in an alkaline reagent to carry out a substitution reaction to obtain a compound M25, wherein the alkaline reagent includes at least one of triethylamine, pyridine, N-methylmorpholine, N,N-diisopropylethylamine, potassium carbonate, and potassium hydroxide; S1.3, placing compound M26 and compound M27 in an acidic reagent to carry out imidization reaction to obtain compound M28, wherein the acidic reagent includes at least one of acetic acid, hydrochloric acid, and p-toluenesulfonic acid; S1.4, adding a reducing agent to the compound M28 to carry out a reduction reaction to obtain a compound M29, wherein the reducing agent comprises at least one of sodium borohydride, lithium aluminum hydride, and sodium cyanoborohydride; S1.5, placing the compound M25 and the compound M29 in the alkaline reagent to carry out a nucleophilic substitution reaction to obtain compound (II); The structural formula of the above compound is:

10. The preparation method according to claim 7, characterized in that: When the amino lipid compound comprises compound (III), step S1 comprises: S1.

1. Add an activator to compound M31 and compound M32, perform chlorination reaction, and then place in an alkaline reagent to obtain M33, wherein the activator includes at least one of thionyl chloride, oxalyl chloride, thionyl chloride, and cyanogen chloride, and the alkaline reagent includes at least one of 4-dimethylaminopyridine, triethylamine, N,N-diisopropylethylamine, pyridine, potassium carbonate, potassium hydroxide, sodium carbonate, and N-methylmorpholine; S1.2, placing the compound M33 and the compound M34 in the alkaline reagent to carry out an amidation reaction to obtain M35; S1.3, placing compound M36 and compound M37 in the alkaline reagent to carry out a substitution reaction to obtain M38; S1.

4. Place the compound M35 and the compound M38 in the alkaline reagent to carry out a cross-linking reaction to obtain compound (III). The structural formula of the above compound is:

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