Aminolipids, lipid nanoparticles and their use

Abstract

This invention provides aminolipids, lipid nanoparticles, and their uses. The aminolipids have a structure represented by general formula (I), or isomers thereof, pharmaceutically acceptable salts, prodrugs, or solvates. This invention further provides lipid nanoparticles containing these aminolipids. In this invention, an aminolipid having the structure represented by general formula (I) is used as an ionizable lipid compound and obtained LNP by self-assembling with a steroid, a neutral lipid, and a polymer-bound lipid. These LNPs improve the intracellular translational expression level of the supported nucleic acid, enhance the action of nucleic acid-LNP formulations, and provide a theoretical basis for personalized therapy with nucleic acid-LNP formulations. TIFF2026516582000075.tif28170

Description

[Technical Field]

[0001] This invention relates to the field of biochemistry, and more particularly to aminolipids, lipid nanoparticles, and their uses. [Background technology]

[0002] Lipid nanoparticles (LNPs) represent a novel nucleic acid biomolecule delivery technology. LNPs typically consist of four components: (1) ionizable lipids that can self-assemble with mRNA to form virus-sized particles and release mRNA from endosomes into the cytoplasm; (2) polymer-binding lipids that can extend the half-life of LNPs in the blood; (3) steroids that can improve the stability of the nanoparticles; and (4) neutral phospholipids that are favorable for the formation of a lipid bilayer structure.

[0003] LNPs not only protect mRNA from degradation by RNases but also protect mRNA molecules from recognition by TLRs, preventing overactivation of the innate immune system. The selection of ionizable lipids has the greatest impact on LNPs, as they not only promote uptake into cells but also facilitate the escape of drug molecules from endosomes, and influence the encapsulation rate, in vivo delivery efficiency, and cytotoxicity of nucleic acid drugs.

[0004] Currently, Moderna and BioNTech & Pfizer are developing vaccines against COVID-19. Both vaccines utilize LNP technology to deliver mRNA drugs, achieving preventive effects against COVID-19 and demonstrating the great potential of LNPs in the fields of mRNA medicine and vaccines. However, LNPs still have drawbacks, such as reduced inclusion rates, reduced escape rates from endosomes, reduced expression levels, and reduced safety. The development direction of LNP delivery systems is mainly focused on ionizable lipids and components.

[0005] Therefore, the development of novel ionizable lipid compounds is of great significance for the development of nucleic acid drugs.

Summary of the Invention

[0006] In a first aspect, the present invention provides an amino lipid having a structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof: TIFF2026516582000002.tif30170 wherein G is selected from H, OR, CN, -C(=O)OR’, -OC(=O)R’, -C(=O)NR’R’’, -NR’C(=O)R’’, NR’R’’, or a cyclic alkyl structure containing at least one heteroatom, and the substitutable carbon atom or heteroatom of the cyclic alkyl structure is unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups, M1, M2, M3, and M4 are the same as or different from each other and are each independently a C1-C 24 alkylene group, C3-C 24 cycloalkylene group, C2-C 24 alkenylene group, or C3-C 24 cycloalkenylene group, R 1 and R 2 are the same as or different from each other and are each independently H, C1-C 24 alkyl group, C3-C 24 cycloalkyl group, C2-C 24 alkenyl group, or C3-C 24 cycloalkenyl group, L1, L2, L3, and L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S-, or -S-S-, R, R’, and R’’ are the same as or different from each other and are each independently H, C1-C 10 alkyl group, C3-C10 Cycloalkyl groups, C3-C 10 Alkenyl group, C3-C 10 A cycloalkenyl group with a tertiary amine bonded to the terminal end (C1-C) 10 C3-C alkyl group with a tertiary amine bonded to the terminal. 10 Cycloalkyl group, C3-C with a tertiary amine bonded to the terminal. 10 Selected from an alkenyl group or a cyclic alkyl group containing at least one heteroatom, the cyclic alkyl group is either unsubstituted or substituted with one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups. M5 consists of independent single bonds, C1-C 16 Alkylene group, C2-C 16 Selected from an alkenylene group, a C3-C8 cycloalkylene group, or a C3-C8 cycloalkenylene group.

[0007] In a second aspect, the present invention provides a method for producing the above-mentioned aminolipids. The aminolipids are synthesized mainly by three reactions: the first reaction is a ring-opening reaction, the second reaction is a condensation reaction, and the third reaction is a substitution reaction.

[0008] In some embodiments of the present invention, the method for producing amino lipids is: S1: The epoxy compound and carboxylic acid undergo a ring-opening reaction to form intermediate 1R. 1 - The step of manufacturing L1-M1-OH, S2: Intermediate 1 and the carboxylic acid compound starting material are condensed in the presence of a condensing agent to produce intermediate 2R 1 -A step to produce an L1-M1-L2-M2-leaving group, S3: The step of producing the target product by performing a substitution reaction between intermediate 2 and an amino compound starting material one or more times, or Alternatively, the process involves protecting the S1':diol with TBS, then oxidizing it, and adding the oxidation product with a Grignard reagent to produce the intermediate 1'HO-M1-OTBS. S2': A step of condensing intermediate 1' with a carboxylic acid compound in the presence of a condensing agent to produce intermediate 2'TBSO-M1-L2-M2-leaving group, S3': The intermediate 2'TBSO-M1-L2-M2-leaving group is deprotected with TBS, and then condensed with a carboxylic acid compound in the presence of a coupling agent to form intermediate 2R 1 -A step of generating an L1-M1-L2-M2-leaving group, S4': The step of producing the target product by performing a substitution reaction between intermediate 2 and an amino compound starting material one or more times.

[0009] In a third embodiment, the present invention provides lipid nanoparticles comprising any one of the above aminolipids.

[0010] According to the lipid nanoparticles of the present invention, the lipid nanoparticles further comprise a steroid, a neutral lipid, and / or a polymer-bound lipid. The chemical formula for polymer-linked lipids is PYL, where P is the hydrophilic polymer portion, Y is an arbitrary linker, and L is the lipid portion.

[0011] In a fourth embodiment, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles and a pharmaceutically acceptable carrier.

[0012] In a fifth embodiment, the present invention further provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the above-mentioned aminolipids or lipid nanoparticles or pharmaceutical compositions.

[0013] The present invention further provides a method for performing gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0014] The present invention further provides the use of the above-mentioned aminolipids or lipid nanoparticles in the manufacture of agents for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0015] The present invention further provides the use of the above-mentioned aminolipids or lipid nanoparticles in the manufacture of agents for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy.

[0016] In a sixth embodiment, the present invention further provides a method for delivering a drug to a target, comprising administering the drug formulated in the lipid nanoparticles to the target.

[0017] The aminolipids of the present invention self-assemble with steroids, neutral lipids, and polymer-bound lipids to obtain LNPs. These LNPs enhance the intracellular translational expression level of the supported nucleic acid, thereby enhancing the effects of nucleic acid-LNP formulations and providing a theoretical basis for personalized therapy using nucleic acid-LNP formulations. [Brief explanation of the drawing]

[0018] [Figure 1] This is the 1H-NMR spectrum of E12LA6B6O3 in Example 2. [Figure 2] This is a tumor size monitoring curve for tumor-carrying mice after intramuscular injection of OVA mRNA vaccine in Example 39. [Figure 3] This is the survival curve of tumor-carrying mice after intramuscular injection of OVA mRNA vaccine in Example 39. [Modes for carrying out the invention]

[0019] definition For clarity and readability, the following scientific background information and definitions are provided. Any technical features described or disclosed herein may be part of any embodiment of the invention, or can be inferred from any embodiment of the invention. Additional definitions and explanations may be provided in the context of the invention.

[0020] Unless otherwise defined or required in a particular context, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0021] Unless otherwise indicated or required by context, the terms “equipped with,” “contains,” “includes,” and similar expressions in this specification and the claims should be interpreted in an open and comprehensive sense as “includes, but not limited to.”

[0022] Expressions such as "one embodiment," "embodiment," and "specific embodiment" mean that a particular characteristic, property, or feature, or a particular group or combination of characteristics, properties, or features referred to in combination with each expression, exists in at least one embodiment of the present invention. These expressions appearing in various places throughout the specification do not necessarily refer to the same embodiment. Furthermore, specific characteristics, properties, or features may be combined in any suitable manner in one or more embodiments.

[0023] Unless otherwise explicitly indicated in the context, the singular forms "one," "one item," and "the aforementioned" should be understood to include the plural referent.

[0024] The term "neutral," when used to describe compounds such as lipids and steroids, or to describe their groups or parts, means that they are neither cationic nor anionic, for example, that they do not possess functional groups that can be ionized under physiological conditions, like hydrocarbons, or that they are both cationic and anionic, i.e., amphoteric, under typical physiological conditions, such as typical natural phosphatidylcholine.

[0025] As used herein, "lipids" refers to a group of organic compounds characterized by being derivatives of fatty acids (such as esters), which are generally insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three types: (1) simple lipids, including fats, oils, and waxes; (2) complex lipids, including phospholipids and glycolipids; and (3) derived lipids, such as steroids. With respect to glycolipids, in certain embodiments, LNPs include glycolipids (e.g., monosialoganglioside GM1).

[0026] In this context, the prefix "poly" refers to multiple atoms or groups in a compound that each possess a specific characteristic. However, the absence of the prefix should not be interpreted as excluding multiple elements. For example, polycationic compounds are also cationic compounds and can be called cationic compounds.

[0027] The term "nucleic acid" refers to any compound containing or composed of DNA or RNA. This term may also be used for polynucleotides or oligonucleotides.

[0028] Immune System: The immune system can protect an organism from infection. When a pathogen breaches the organism's physical barriers and invades, the innate immune system provides an immediate but nonspecific response. If the pathogen evades this innate response, the vertebrate develops an adaptive immune system, which is a second layer of defense. Here, the immune system adapts its response to improve its recognition of the pathogen during infection. Thus, even after the pathogen has been eliminated, this improved response is retained in the form of immunological memory, allowing the adaptive immune system to launch a faster and more powerful attack each time it encounters the pathogen. According to this, the immune system includes the innate immune system and the adaptive immune system. Each of these two parts includes a so-called humoral component and a cellular component.

[0029] Adaptive Immune System: The adaptive immune system consists of highly specialized systemic cells and processes that eliminate or prevent the proliferation of pathogens. Adaptive immune responses provide the vertebrate immune system with the ability to recognize and remember (generate immunity to) specific pathogens and initiate a stronger attack each time it encounters them. Due to somatic hypermutation (a process of increased mutation frequency in somatic cells) and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments), this system is highly adaptable. This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on individual lymphocytes. Since gene rearrangement results in irreversible changes in the DNA of each cell, all of that cell's progeny (offspring) inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are key to longevity-specific immunity. Immune network theory is a theory of how the adaptive immune system works, based on the interactions between T cells, B cells, and the variable regions of receptors of molecules with variable regions produced by T cells and B cells.

[0030] The term "vaccine" is generally understood to refer to a preventative or therapeutic material that possesses at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0031] The term "antigen" usually refers to a substance that is recognized by the immune system, preferably the adaptive immune system, and can induce an antigen-specific immune response, such as through the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response.

[0032] The term "artificial mRNA" (sequence) can generally be understood as an mRNA molecule that does not exist in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such an mRNA molecule may be non-natural due to its individual sequences (which do not exist in nature) and / or other modifications (e.g., structural modifications of nucleotides that do not exist in nature). Generally, artificial mRNA molecules can be designed and / or fabricated by genetic engineering techniques to correspond to a desired artificial nucleotide sequence (heterogeneous sequence). In this context, the artificial sequence is usually a sequence that does not exist in nature, i.e., at least one nucleotide differs from the wild-type sequence. The term "wild-type" can be understood as a sequence that exists in nature.

[0033] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to the organism to which it is administered and does not negate the compound's biological activity and properties.

[0034] The compounds of the present invention may have polymorphic crystalline forms, i.e., different crystalline packing arrangements of the same elemental composition of the compound. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystalline forms, optical and electrical properties, stability, and solubility. Depending on various factors such as the recrystallization solvent, crystallization rate, and storage temperature, the single-crystal form may be the dominant recrystallized product. It is understood that the aminolipids of the present invention encompass all such crystalline forms.

[0035] The compounds of the present invention may have chiral centers and / or axial chirality, and therefore may exist in the form of racemates, racemic mixtures, single enantiomers, diastereomer compounds, single diastereomers, and cis-trans isomers. Each chiral center or axial chirality independently produces two optical isomers, and all possible optical isomers, diastereomer mixtures, and pure or partially pure compounds are within the scope of the present invention. The term "isomer" as defined in the present invention includes all such isomers of a compound.

[0036] The compounds of the present invention may have various hydrates or solvates. The solvates contain stoichiometric or non-stoichiometric amounts of solvent and are selectively formed during crystallization in pharmaceutically acceptable solvents such as water or other solvents such as ethanol. When the solvent is water, a hydrate is formed, and when the solvent is other solvents such as ethanol, a solvate is formed.

[0037] The term "prodrug," also known as a drug precursor, refers to a compound obtained after the chemical structure of a drug has been modified. While it exhibits inactivity or minimal activity in vitro, it releases the active drug in vivo through enzymatic or non-enzymatic transformation, thereby exerting pharmacological effects. There are two types of prodrugs: carrier prodrugs and bioprecursor prodrugs. A carrier prodrug is a compound in which the active compound and its transport carrier are covalently bonded. The carrier is easily removed by hydrolysis in the body, allowing the active compound to exert its pharmacological effects. Carrier prodrugs exhibit very weak or no activity compared to the parent compound. Because the carrier structure is primarily lipophilic, it must be harmless to the body and capable of releasing the active compound in a timely manner. Unlike carrier prodrugs, bioprecursor prodrugs do not require the active component to temporarily bind to a carrier; rather, they exert their effects through changes in their own molecular structure. While bioprecursor prodrugs themselves are inactive, their metabolites become active in the body.

[0038] As a result of our research, we unexpectedly discovered that the use of novel aminolipids and / or lipid nanoparticles according to the present invention can effectively overcome the drawbacks of conventional LNPs, such as reduced encapsulation rates, reduced escape rates from endosomes, reduced expression levels, and reduced safety, thereby promoting the development of LNPs in the mRNA drug and vaccine fields.

[0039] Aminolipids Aminolipids are preferably cationizable; that is, when the pH becomes lower than the pKa of the ionizable group of the lipid, the aminolipid becomes protonated, and when positively charged, it can bind to negatively charged nucleic acids.

[0040] In one embodiment, the present invention provides an aminolipid having a structure represented by general formula (I), or an isomer thereof, a pharmaceutically acceptable salt, a prodrug, or a solvate thereof: In formula TIFF2026516582000003.tif30170, G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', NR'R'', or a cyclic alkyl structure (preferably a 3-10 membered cycloalkyl group, more preferably a 4-6 membered cycloalkyl group) containing at least one heteroatom (preferably the heteroatom is N or O, preferably one or two heteroatoms selected from N or O), wherein the substituted carbon atoms or heteroatoms of the cyclic alkyl structure are unsubstituted or substituted with one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups. M1, M2, M3, and M4 are either identical or different from each other, and each is independently C1-C 24 Alkylene group, C3-C 24 Cycloalkylene group, C2-C 24 Alkenylene group, or C3-C 24 Selected from cycloalkenylene groups, R 1 and R 2 These are either identical or different from each other, and each is independently H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl groups, C2-C 24 Alkenyl group, or C3-C 24 Selected from cycloalkenyl groups, L1, L2, L3, and L4 are either identical or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S-, or -SS-. R, R', and R'' are either identical or different from each other, and each is independently H, C1-C 10Alkyl (preferably C1-C6 alkyl, more preferably C1-C4 alkyl), C3-C 10 Cycloalkyl groups, C3-C 10 Alkenyl group, C3-C 10 A cycloalkenyl group with a tertiary amine bonded to the terminal end (C1-C) 10 C3-C alkyl group with a tertiary amine bonded to the terminal. 10 Cycloalkyl group, C3-C with a tertiary amine bonded to the terminal. 10 Selected from an alkenyl group or a cyclic alkyl group containing at least one heteroatom, the cyclic alkyl group is either unsubstituted or substituted with one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups, preferably with -N(C) at the terminal. 1-6 C1-C bonded to alkyl)2 10 C1-C 10 It is an alkyl group, C3-C 10 The cycloalkyl group and the 3- to 8-membered cyclic alkyl group (preferably a 4- to 6-membered cyclic alkyl group) may be substituted with a C1-C6 alkyl group. M5 has a single bond, C1-C 16 Alkylene group, C2-C 16 Selected from an alkenylene group, a C3-C8 cycloalkylene group, or a C3-C8 cycloalkenylene group.

[0041] Furthermore, the alkyl groups, alkenyl groups, alkylene groups, and alkenylene groups containing multiple carbon atoms mentioned in the present invention may be linear or branched unless explicitly stated otherwise. In addition, the alkyl groups, alkenyl groups, alkylene groups, alkenylene groups, and cycloalkyl groups, cycloalkenyl groups, cycloalkylene groups, and cycloalkenylene groups mentioned in the present invention may be unsubstituted or substituted, and the substituents may be any suitable substituents, i.e., any linear or branched alkyl groups, aryl groups, heteroalkyl groups, or heteroaromatic structures, and may optionally include other functional groups such as ester groups or amide groups.

[0042] In some preferred embodiments, G is selected from H, OR, NR'R'', or a cyclic alkyl structure containing at least one heteroatom, where the heteroatom is O or N, and R, R', and R'' are identical or different from each other and are independently selected from H, a C1-C8 alkyl group, a C3-C8 cycloalkyl group, a C3-C8 alkenyl group, or a C3-C8 cycloalkenyl group.

[0043] In a more preferred embodiment, G is selected from H, OH, or NR'R'', and R' and R'' are the same or different from each other and are independently selected from H or a C1-C4 alkyl group. That is, NR'R'' may be NH2, NHCH3, NHC2H5, NHC3H7, NHC4H9, N(CH3)2, CH3-N-C2H5, CH3-N-C3H7, CH3-N-C4H9, C2H5-N-C2H5, C2H5-N-C3H7, C2H5-N-C4H9, C3H7-N-C3H7, C3H7-N-C4H9, or C4H9-N-C4H9.

[0044] Alternatively, G is selected from substituted or unsubstituted oxa 5-membered cycloalkyl groups, aza 5-membered cycloalkyl groups, aza 6-membered cycloalkyl groups, diaza 6-membered cycloalkyl groups, or azaoxa 6-membered cycloalkyl groups. Preferably, the oxa 5-membered cycloalkyl group is TIFF2026516582000004.tif9170, aza 5-membered cycloalkyl is TIFF2026516582000005.tif9170, aza 6-membered cycloalkyl is TIFF2026516582000006.tif9170, diaza 6-membered cycloalkyl is TIFF2026516582000007.tif10170, azaoxa 6-membered cycloalkyl is The file is TIFF2026516582000008.tif9170, where the position of * is merged with M5.

[0045] In some embodiments, when G is a substituted diaza 6-membered cycloalkyl group, the substituent is a nitrogen atom not bonded to M5.

[0046] In some preferred embodiments, G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', NR'R'', or a cyclic alkyl structure (preferably a 3-10 membered cycloalkyl group, more preferably a 4-6 membered cycloalkyl group) containing one or two ring N atoms, and the substituted carbon atoms or heteroatoms of the cyclic alkyl structure are either unsubstituted or substituted with one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups. R, R', and R'' are identical or different from each other and are independently H, C1-C6 alkyl groups, C3-C 10 Cycloalkyl group, with -N(C) at the end. 1-6 C1-C bonded to alkyl)2 10 C1-C 10 Selected from alkyl groups, C3-C 10 The cycloalkyl groups and 4-6 membered cyclic alkyl groups may be substituted with C1-C6 alkyl groups.

[0047] In some embodiments, M5 is a single bond, C2-C 16 Alkylene group, C2-C 16 Selected from an alkenylene group, a C4-C8 cycloalkylene group, or a C3-C8 cycloalkenylene group.

[0048] More preferably, M5 is a single bond, C2-C 16 The alkylene group or the C4-C6 cycloalkylene group is selected. More preferably, M5 is selected from a single bond, a C2-C6 alkylene group, or a C4-C6 cycloalkylene group, or from a C3-C5 alkylene group.

[0049] In a preferred embodiment, in general formula (I), M5 and G are bonded together to form TIFF2026516582000009.tif9170 is one of the following selected from A1 to A38: TIFF2026516582000010.tif118170

[0050] more, TIFF2026516582000011.tif9170 is one of the following: A1-A18, A22-A24, or A28-A38.

[0051] More preferably, TIFF2026516582000012.tif9170 is one of the following selected from A15, A16, A17, A23, A29, A30, A33, A37, A38, and A42.

[0052] In some embodiments of the present invention, in general formula (I), M5 and G are bonded together and formed TIFF2026516582000013.tif9170 is one of the following selected from A39 to A52: TIFF2026516582000014.tif116170

[0053] In some embodiments of the present invention, L1, L2, L3, and L4 are identical or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, or -NRC(=O)-.

[0054] Here, if L1, L2, L3, and L4 are independently selected from -C(=O)NR- or -NRC(=O)-, then R is independently H or C1-C 10 It is selected from alkyl groups. Preferably, R is H.

[0055] In a preferred embodiment of the present invention, L1 and L4 are the same and are -C(=O)O- or -OC(=O)-.

[0056] In some embodiments of the present invention, M1, M2, M3, and M4 are identical or different from each other, and M1 and M4 each have independently branched chains C4-C 22 Alkylene group, branched chain C4-C 22 Cycloalkylene group, branched chain C4-C 22 Alkenylene group, or branched C4-C 22 Selected from cycloalkenylene groups, M2 and M3 are independently C4-C 22 Alkylene group, C4-C 22 Cycloalkylene group, C4-C 22 Alkenylene group, or C4-C 22 Selected from cycloalkenylene groups.

[0057] More preferably, M1, M2, M3, and M4 are identical or different from each other, and M1 and M4 each have independently branched chains in the C4-C 22 C4-C having an alkylene group or a branched chain 22 Selected from alkenylene groups, preferably C6-C having a branched chain. 16 These are alkylene groups, and M2 and M3 are independently C4-C 22 Alkylene group, or C4-C 22 A group selected from alkenylene groups, preferably a C3-C8 alkylene group.

[0058] More preferably, in some embodiments of the present invention, M2 and M3 are the same, and C4-C 22 It is an alkylene group.

[0059] In some embodiments of the present invention, M1 and M4 are the same, and C4-C has a branched chain. 22 It is an alkylene group.

[0060] In some embodiments of the present invention, R 1 and R 2 These are either identical or different from each other, and each is independently C4-C 22 Alkyl alkyl group or C4-C 22 Selected from alkenyl groups, preferably C5-C 12It is an alkyl group.

[0061] In a preferred embodiment, R 1 -L1-M1-L2-M2-fragments are R 1 -C(=O)O-M1-OC(=O)-M2- and R 2 -L4-M4-L3-M3-fragments are R 2 -C(=O)O-M4-OC(=O)-M3-. More preferably, M1, M2, M3, and M4 are the same or different from each other, and M1 and M4 each have independently branched chains C4-C 22 C4-C having an alkylene group or a branched chain 22 Selected from alkenylene groups, M2 and M3 are independently C4-C 22 Alkylene group or C4-C 22 Selected from alkenylene groups. 1 and R 2 These are either identical or different from each other, and each is independently C4-C 22 Alkyl alkyl group or C4-C 22 Selected from alkenyl groups.

[0062] Those skilled in the art can, in accordance with common sense, combine preferred embodiments relating to the different groups described above to obtain more preferred embodiments of the compounds of the present invention.

[0063] In a preferred embodiment of the present invention, the structure of general formula (I) is one selected from the following structures: TIFF2026516582000015.tif233170

[0064] In a preferred embodiment of the present invention, the structure of general formula (I) is one selected from the following structures: TIFF2026516582000016.tif235170TIFF2026516582000017.tif176170

[0065] A person skilled in the art can combine the preferred embodiments regarding the above different bases according to common knowledge to obtain more preferred embodiments of the compounds of the present invention.

[0066] Method for producing amino lipids The present invention provides a method for producing the above amino lipid. This method comprises S1: a step of subjecting an epoxy compound and a carboxylic acid to a ring-opening reaction to produce intermediate 1R 1 -L1-M1-OH; and S2: a step of subjecting intermediate 1 and a carboxylic acid compound raw material to a condensation reaction in the presence of a condensing agent to produce intermediate 2R 1 -L1-M1-L2-M2-eliminating group; and S3: a step of subjecting intermediate 2 and an amino compound raw material to a substitution reaction one or more times to produce a target product, or Alternatively, S1': a step of protecting a diol with TBS, then subjecting it to an oxidation reaction, and subjecting the oxidation product and a Grignard reagent to an addition reaction to produce intermediate 1' HO-M1-OTBS; and S2': a step of subjecting intermediate 1' and a carboxylic acid compound to a condensation reaction in the presence of a condensing agent to produce intermediate 2' TBSO-M1-L2-M2-eliminating group; and S3': a step of performing TBS deprotection of intermediate 2' TBSO-M1-L2-M2-eliminating group, and subjecting it to a condensation reaction in the presence of a carboxylic acid compound and a condensing agent to produce intermediate 2R 1 -L1-M1-L2-M2-eliminating group; and S4': a step of subjecting intermediate 2 and an amino compound raw material to a substitution reaction one or more times to produce a target product.

[0067] Preferably, the carboxylic acid in step S2' is H-L2-M2-eliminating group.

[0068] Preferably, the carboxylic acid in step S3' is R 1 -L1-H.

[0069] Preferably, the eliminating group is halogen.

[0070] Preferably, each intermediate and the target product produced is purified by column chromatography.

[0071] In some embodiments, the manufacturing method includes the following steps: Ring-opening reaction: A carboxylic acid compound (1.0 eq), epoxy compound starting material (0.6-3.0 eq), iron trichloride (0.5-10 mol%), and pyridine (0.2-20 mol%) were mixed and stirred overnight at room temperature. After the reaction was complete, the mixture was purified by column chromatography to obtain intermediate 1 (yield 56%-99.0%). Condensation reaction: Intermediate 1 (1.0 eq), halogen-containing carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM were sequentially mixed, stirred overnight at room temperature, and purified by column chromatography to obtain intermediate 2 (yield 46.0%-96.4%). Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), ammonia compound (1.0 eq), sodium iodide (1.0-3.0 eq), and acetonitrile were sequentially mixed, stirred overnight at 20-100°C, and purified by column chromatography to obtain aminolipids (yield 32.0%-92.1%).

[0072] In some embodiments, the manufacturing method includes the following steps: Addition reaction: After protecting the diol with TBS, it was oxidized with PCC, and 0.9 to 1.2 eqt of the oxidation product was stirred in anhydrous tetrahydrofuran at -20°C for 2 to 20 minutes. 1.2 to 1.5 eqt of Grignard reagent was added, and the mixture was stirred at -20°C to 0°C for 1 to 6 hours. The mixture was then purified by column chromatography to obtain intermediate 1. Condensation reaction 1: Intermediate 1' (1.0 eq), halogen-containing carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM were sequentially mixed, stirred overnight at room temperature, and purified by column chromatography to obtain intermediate 2'. Condensation reaction 2: Intermediate 2' (1.0 eq) was deprotected using TBS with ammonium fluoride (5.0-20.0 eq), mixed with carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM, stirred overnight at room temperature, and purified by column chromatography to obtain intermediate 2. Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), ammonia compound (1.0 eq), sodium iodide (1.0-3.0 eq), and acetonitrile were sequentially mixed, stirred overnight at 20-100°C, and purified by column chromatography to obtain amino lipids.

[0073] Lipid nanoparticles The present invention provides lipid nanoparticles containing any one of the above amino lipids.

[0074] It will be understood that all of the above selections and preferences regarding amino lipids also apply to the amino lipid-containing lipid nanoparticles of the present invention.

[0075] In some embodiments of the present invention, the lipid nanoparticles further comprise steroids, neutral lipids, and / or polymer-bound lipids.

[0076] steroid A "steroid" is an organic compound that has four rings arranged in a specific molecular configuration. Steroids contain the following carbon skeletons: TIFF2026516582000018.tif20170

[0077] Steroids and neutral steroids include naturally occurring steroids and their analogues (e.g., amphiphilic lipid cholesterol hemisuccinates (CHEMS) consisting of succinic acid esterified to the β-hydroxyl group of cholesterol as a cholesterol derivative). Neutral steroids may be steroids that do not have ionizable atoms or groups under physiological conditions, or they may be amphoteric steroids. In preferred embodiments, neutral steroids do not contain ionizable atoms or groups under physiological conditions. In some preferred embodiments, the steroid or steroid analogue is cholesterol. The terms “steroid” and “neutral steroid” are used interchangeably herein.

[0078] neutral lipid The “neutral lipids” of this invention are also called “helper lipids,” and are preferably phospholipids or neutral phospholipids. As used herein, “neutral phospholipids” are amphiphilic compounds consisting of molecules having two hydrophobic fatty acid “tails” and a hydrophilic “head” containing a phosphate group. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. Phospholipids are abundant in nature. The “phospholipids” or “neutral phospholipids” of this invention include both natural and synthetic phospholipids.

[0079] Polymer-bound lipids The term "polymer-bound lipid" refers to a molecule containing both a lipid portion and a polymer portion. Preferably, the polymer-bound lipid is polyethylene glycolated lipid or PEG-lipid. The terms "polyethylene glycolated lipid" or "PEG-lipid" refer to a molecule containing both a lipid portion and a polyethylene glycol portion. Polyethylene glycolated lipids are well known in the art and include PEG-DMG, etc.

[0080] In a specific embodiment, the chemical formula of the polymer-bound lipid is PYL, where P is the hydrophilic polymer portion, Y is an arbitrary linker, and L is the lipid portion.

[0081] Specifically, the hydrophilic polymer portion P may be polyethylene glycol PEG. In specific embodiments, the average molecular weight of the PEG portion is 1 kDa to 3 kDa, for example, 1.5 kDa to 2.5 kDa, 1.7 kDa to 2.3 kDa, 1.8 kDa to 2.2 kDa, 1.9 kDa to 2.1 kDa, or 2 kDa. Therefore, the PEG may be PEG commonly referred to as "PEG2000".

[0082] In another embodiment, the hydrophilic polymer moiety P in the polymer-bound lipid may also be a substantially hydrophilic polymer different from the hydrophilic polymer moiety described above. That is, the hydrophilic polymer moiety P in the polymer-bound lipid may be obtained based on poly(propylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, hydroxyethyl starch deposition (HESylation) (according to PMID24681396), PASylation (i.e., proline-alanine-serine), the art-known XTEN method (i.e., PEG-based peptide), polysarcosine, or poly(vinyl acetate).

[0083] Specifically, any linker Y may be any useful spacer structure. For example, it may be selected from spacers generally considered useful in polyethylene glycolated lipids, such as succinimide, amines, ethers, esters, anhydrides, aldehydes, ketones, amides, carbamate linkers, or combinations thereof (but not limited to these).

[0084] Specifically, the lipid portion L may be derived from phospholipids, sphingolipids, or ceramides. As used herein, the expression “phospholipid or ceramide-derived” includes free radicals of phospholipids and ceramides. Examples include polymer-bound lipids containing phosphatidylethanolamine or phosphatidylglycerol portions.

[0085] In preferred embodiments, the polymer-bound lipid is a polyethylene glycolated lipid. The polyethylene glycolated lipid includes, but is not limited to, polyethylene glycolated diacylglycerol lipids (PEG-DAG), polyethylene glycolated ceramide lipids (PEG-Cer), polyethylene glycolated phosphatidylethanolamine lipids (PEG-PE), polyethylene glycolated diacylglycerol succinate lipids (PEG-S-DAG), polyethylene glycolated dialkoxypropyl carbamate lipids, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG").

[0086] In a more preferred embodiment, the polymer-bound lipid is DMG-PEG2000.

[0087] Preferably, as used in the art, "DMG-PEG2000" is considered to be a mixture of 1,2-DMG PEG2000 and 1,3-DMG PEG2000 in a ratio of approximately 97:3.

[0088] In some embodiments of the present invention, the molar ratio of amino lipids, steroids, neutral lipids, and polymer-bound lipids in lipid nanoparticles is 30-70:30-65:0-30:0.2-5. More preferably, it is 30-60:35-60:0-20:0.3-3.

[0089] In a preferred embodiment of the present invention, in lipid nanoparticles, the amino lipid is the preferred amino lipid described above, the steroid is cholesterol, the neutral lipid is a phospholipid, and the polymer-bound lipid is a polyethylene glycolated lipid. The molar ratio of the amino lipid, cholesterol, phospholipid, and polyethylene glycolated lipid is 40-50:40-45:10-15:0.5-2.

[0090] In preferred embodiments, the polyethylene glycolated lipid in the lipid nanoparticles is DMG-PEG2000.

[0091] The lipid nanoparticles of the present invention are not limited to any particular form and should be interpreted as including any form produced when aminolipids and optionally one or more other lipids associate, for example, in an aqueous environment and / or in the presence of nucleic acid compounds. For example, liposomes, lipid complexes, lipoplexes, etc., fall within the scope of lipid nanoparticles.

[0092] The lipid nanoparticles of the present invention can be combined with at least one pharmaceutically acceptable carrier or excipient to obtain a pharmaceutical composition. Therefore, the composition may be a dry composition such as a powder or granules, or a solid unit such as a lyophilized form or a tablet. Alternatively, the composition may be in liquid form, with each excipient being incorporated independently in the form of dissolution or dispersion (e.g., suspension or emulsification). In preferred embodiments, the composition is formulated as a sterile solid composition, such as a powder or lyophilized form for reconstitution with an aqueous liquid carrier. Such formulations are also preferred for compositions containing bioactive ingredients, which are described in more detail below.

[0093] As used herein, “nanoparticles” are submicron particles having any structure or form. Submicron particles may also be called colloids or colloidal forms. With respect to the material on which nanoparticles are based and their structure or form, nanoparticles may be classified, to name only a few of the possible names for certain types of nanoparticles, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polyplexes, mixtures or hybrid complexes. “Lipid nanoparticles” (LNPs) are nanoparticles formed by lipids, typically comprising at least one amphiphilic membrane-forming lipid and possibly other lipids, and possibly cargo material such as nucleic acid compounds. As used herein, the expression “lipid nanoparticles” or “LNPs” includes any subtypes and forms of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes.

[0094] As defined above, lipid nanoparticles include any type of nanoparticles formed or co-formed by lipids. In particular, lipid nanoparticles can be co-formed by a combination of lipids, including at least one amphiphilic, vesicle-forming lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0095] Preferably, in some embodiments of the present invention, the lipid nanoparticles further comprise a bioactive component.

[0096] A bioactive component means any compound or material that possesses bioactivity. Due to its activity, the compound or material is useful for preventing, managing, improving, treating, or curing diseases or symptoms in a subject (e.g., animals, especially humans).

[0097] In some embodiments, the bioactive component is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA, or any combination thereof. Preferably, the bioactive component is mRNA or an mRNA compound.

[0098] In some embodiments, the bioactive component is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0099] In one embodiment of the present invention, the mRNA comprises one or more of the following: a stem-loop, a strand termination nucleoside, a poly(A) sequence, a polyadenylation signal, and / or a 5' cap structure.

[0100] In one embodiment of the present invention, the encapsulation rate of the bioactive component is at least 50-90%. More preferably, the encapsulation rate of the bioactive component is at least 60-80%.

[0101] In one embodiment of the present invention, the weight ratio of the lipid component to the bioactive component in the lipid nanoparticles is approximately 10:1 to approximately 60:1. More preferably, the weight ratio of the lipid component to the bioactive component is approximately 20:1.

[0102] In one embodiment of the present invention, the N:P ratio in the lipid nanoparticles is approximately 2:1 to approximately 30:1. More preferably, the N:P ratio is approximately 5.67:1.

[0103] In one embodiment of the present invention, the average size of lipid nanoparticles is approximately 70 nm to 100 nm.

[0104] In one embodiment of the present invention, the polydispersity index of lipid nanoparticles is approximately 0.10 to approximately 0.20.

[0105] In one embodiment of the present invention, the zeta potential of lipid nanoparticles is approximately -10mV to approximately +20mV.

[0106] In preferred embodiments, the bioactive component complexes or associates with one or more lipids (e.g., aminolipids and / or neutral lipids) to form liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. In this context, the terms “complexing” or “associating” mean that the bioactive component and one or more lipids combine substantially stably into a larger, non-covalent complex or aggregate.

[0107] use The present invention provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0108] Therefore, the present invention provides the use of the above-mentioned aminolipids or lipid nanoparticles or pharmaceutical compositions in the manufacture of agents for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0109] Here, infectious diseases include viral, bacterial, or protozoan infectious diseases. The viruses include, but are not limited to, SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), bunyavirus, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extraenteropathogenic Escherichia coli, lassa virus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, synovial respiratory virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, and Zika virus.

[0110] Cancer includes lung cancer, stomach cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, and prostate cancer.

[0111] The present invention further provides a method for performing gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0112] Therefore, the present invention further provides the use of the above-mentioned aminolipids or lipid nanoparticles in the manufacture of agents for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy.

[0113] The present invention further provides a method for delivering a drug to a target, which includes administering the drug formulated in the above-mentioned lipid nanoparticles to the target.

[0114] When administering a drug, the method of administration is determined by the drug's dosage form. The drug's dosage form is related to the excipients and / or carriers. Routes for systemic administration generally include transdermal, oral, and parenteral routes (including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injections, and / or intranasal routes). Routes for local administration generally include intradermal, transdermal, subcutaneous, or intramuscular injections, or intra-focal, intracranial, intrapulmonary, intracardiac, intratumoral, and sublingual injections. If the drug is in the form of a vaccine, the preferred routes of administration are intramuscular and intradermal injections.

[0115] The following examples are for illustrative purposes only and do not limit the scope of the present invention.

[0116] Unless specific techniques or conditions are stated in the examples, the procedures were followed according to the techniques and conditions described in the literature in this field, or according to the product's instruction manual. Unless the manufacturer is specified for the reagents or equipment used, they are all standard products that can be purchased through legitimate channels.

[0117] For illustrative purposes, the following examples illustrate a general method for producing the compounds according to the present invention. For a more detailed description of each reaction step, please refer to the following examples. Those skilled in the art will understand that other synthetic routes can be used to synthesize the compounds of the present invention. Specific raw materials and reagents are described and discussed below in the scheme, but various derivatives and / or reaction conditions can be obtained by readily substituting other raw materials and reagents. Furthermore, in conjunction with the disclosure of the following examples, many of the compounds produced by the following methods can be further modified using conventional chemical methods well known to those skilled in the art to obtain other compounds within the limited scope of the present invention.

[0118] The abbreviations used in this specification correspond to the following: Py Pyridine EDCI 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride DMAP 4-dimethylaminopyridine DIPEA Diisopropylethylamine DCM Dichloromethane MeCN acetonitrile DSPC Distearoylphosphatidylcholine DMG-PEG2000 1,2-Dimyristoyl-rac-Glycerol-3-Methoxypolyethylene Glycol 2000

[0119] Production of aminolipid compounds Example 1: Synthesis of aminolipid E8LA12B6O3 Step 1: Synthesis of E8LA12: In a 25 mL reaction tube, FeCl3 (16.2 mg, 2.5 mol%), Py (4 mg, 1.25 mmol%), lauric acid (200.32 g / mol, 800 mg, 4.0 mmol), and 1,2-epoxyoctane (128.22 g / mol, 1.02 g, 8 mmol) were sequentially added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was separated and purified by column chromatography to obtain the intermediate E8LA12 (colorless oily liquid) (1.2 g, yield 91%).

[0120] Step 2: Synthesis of E8LA12B6: In a 25 mL reaction tube, E8LA12 (328.54 g / mol, 1.2 g, 3.65 mmol), 6-bromohexanoic acid (195.06 g / mol, 855 mg, 4.38 mmol), EDCI (191.70 g / mol, 2.8 g, 14.6 mmol), DMAP (45 mg, 0.365 mmol), DIPEA (2.83 g, 21.9 mmol), and DCM (10 mL) were sequentially added and the mixture was stirred overnight at room temperature. After the reaction was complete, the intermediate E8LA12B6 (colorless oily liquid) (1.65 g, yield 89%) was obtained by separation and purification by column chromatography.

[0121] Step 3: Synthesis of E8LA12B6O3: In a 25 mL reaction tube, E8LA12B6 (505.58 g / mol, 1.65 g, 3.26 mmol), potassium carbonate (138.21 g / mol, 902 mg, 6.52 mmol), sodium iodide (149.89 g / mol, 489 mg, 3.26 mmol), aminopropanol (75.11 g / mol, 113 mg, 1.5 mmol), and acetonitrile (10 mL) were sequentially added, and the reaction was stirred overnight at 75 °C. After the reaction was complete, the mixture was separated and purified by column chromatography to obtain E8LA12B6O3 (colorless oily liquid) (0.94 g, yield 68%). 1 H NMR(400MHz,CDCl3):δ5.08-5.03(m,2H),4.22(dd,J1=11.6Hz,J2=3.6Hz,2H),4.02(dd,J1=12.0Hz,J2=6.4Hz,2H),3.82(t,J=5.6Hz,2H),2.96 (t,J=7.6Hz,2H),2.77(t,J=6.0Hz,4H),2.34-2.28(m,8H),1.89-1.88( m,2H),1.72-1.56(m,12H),1.40-1.25(m,56H),0.87(t,J=7.0Hz,12H). 13 C NMR(100MHz,CDCl3):δ173.1,70.7,65.5,59.0,58.5,57.2,34.2,33.9,31.9, 31.8,30.7,30.3,29.6,29.3,29.0,28.0,26.7,25.0,25.3,24.7,22.7,14.1. ESI-MS C 55 H 106 NO9 + [M+H] + The calculated value was 924.7862, and the measured value was 924.7850.

[0122] Example 2: Synthesis of aminolipid E12LA6B6O3 Step 1: Synthesis of E12LA6: In a 2217025 mL reaction tube, FeCl3 (16.2 mg, 2.5 mol%), Py (4 mg, 1.25 mmol%), n-hexanoic acid (116.1600 g / mol, 464 mg, 4.0 mmol), and 1,2-epoxydodecane (184.18 g / mol, 1.47 g, 8 mmol) were sequentially added, and the reaction was stirred overnight at room temperature. After the reaction was completed, it was separated and purified by column chromatography to obtain intermediate E12LA6 (colorless oily liquid) (1.02 g, yield 85%).

[0123] Step 2: Synthesis of E12LA6B6: In a 1817025 mL reaction tube, E12LA6 (300.48 g / mol, 901 mg, 3.0 mmol), 6-bromohexanoic acid (195.06 g / mol, 703 mg, 3.6 mmol), EDCI (191.70 g / mol, 2.3 g, 12.0 mmol), DMAP (37 mg, 0.3 mmol), DIPEA (2.33 g, 18 mmol), and DCM (10 mL) were sequentially added, and the reaction was stirred overnight at room temperature. After the reaction was completed, it was separated and purified by column chromatography to obtain intermediate E12LA6B6 (colorless oily liquid) (1.08 g, yield 76%).

[0124] Step 3: Synthesis of E12LA6B6O3: In a 2117025 mL reaction tube, E12LA6B6 (477.52 g / mol, 956 mg, 2.0 mmol), potassium carbonate (138.21 g / mol, 221 mg, 1.6 mmol), sodium iodide (149.89 g / mol, 120 mg, 0.8 mmol), aminopropanol (75.11 g / mol, 60 mg, 0.8 mmol), and acetonitrile (5 mL) were sequentially added, and the reaction was stirred overnight at 75 °C. After the reaction was completed, it was separated and purified by column chromatography to obtain E12LA6B6O3 (colorless oily liquid) (458 mg, yield 66%). 1H NMR(400MHz,CDCl3):δ5.08-5.06(m,2H),4.22(dd,J1=12.0Hz,J2=3.6Hz,2H),4.02(dd,J1=11.6Hz,J2=6.4Hz,2H),3.79(t,J=5 .2Hz,2H),2.73-2.70(m,2H),2.52-2.48(m,4H),2.33-2.28(m,8H),1.74-1.50(m,14H),1.42-1.25(m,48H),0.91-0.86(m,12H). ESI-MS C (as shown in Figure 1) 51 H 98 NO9 + [M+H] + The calculated value was 868.7236, and the measured value was 868.7234.

[0125] Example 3: Synthesis of aminolipid E8LA8B6O3 In Example 1, lauric acid was replaced with octanoic acid, and the amino lipid E8LA8B6O3 was obtained by the same method. The structural formula of this amino lipid is as follows. TIFF2026516582000025.tif31170 1 H NMR(400MHz,CDCl3):δ5.08-5.02(m,2H),4.22(dd,J1=12.0Hz,J2=3.6Hz,2H),4.01(dd,J1=11.6Hz,J2=6.0Hz,2H),3.81(t,J=5.2Hz,2H),2. 98(t,J=6.0Hz,2H),2.79(t,J=7.6Hz,4H),2.34-2.27(m,8H),1.92-1. 86(m,2H),1.73-1.56(m,12H),1.40-1.26(m,40H),0.88-0.85(m,12H). ESI-MS C 47 H 90 NO9 + [M+H] + The calculated value was 812.6610, and the measured value was 812.6598.

[0126] Example 4: Synthesis of aminolipid E8LA10B6O3 In Example 1, lauric acid was replaced with capric acid, and the aminolipid E8LA10B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000026.tif31170 1 H NMR(400MHz,CDCl3):δ0.85-0.89(m,12H),1.25-1.40(m,48H),1.56-1.72(m,12H),1.88-1.90(m,2H),2.28-2.33(m,8 H),2.77-2.79(m,4H),2.96-2.98(m,2H),3.79-3.80(m,2H),4.01-4.05(m,2H),4.21-4.24(m,2H),5.03-5.08(m,2H). ESI-MS C 51 H 98 NO9 + [M+H] + The calculated value was 868.7, and the measured value was 868.7.

[0127] Example 5: Synthesis of aminolipid E10LA6B6O3 In Example 2, 1,2-epoxydodecane was replaced with 1,2-epoxydecane, and the aminolipid E10LA6B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000027.tif30170 1 H NMR(400MHz,CDCl3):δ5.08-5.02(m,2H),4.22(dd,J1=12.0Hz,J2=3.2Hz,2H),4.01(dd,J1=11.6Hz,J2=6.4Hz,2H),3.79(t,J=5.2Hz,2H),2. 82(t,J=5.6Hz,2H),2.62(t,J=8.0Hz,4H),2.36-2.27(m,8H),1.81-1. 76 (m, 2H), 1.68-1.53 ​​(m, 12H), 1.37-1.24 (m, 40H), 0.90-0.84 (m, 12H). ESI-MS C 47 H 90 NO9 + [M+H] + The calculated value was 812.6610, and the measured value was 812.6634.

[0128] Example 6: Synthesis of aminolipid E10LA8B6O3 In Example 1, 1,2-epoxyoctane was replaced with 1,2-epoxydecane, and lauric acid was replaced with n-octanoic acid, and the aminolipid E10LA8B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000028.tif29170 1 H NMR (400MHz, CDCl3): δ0.84-0.89(m,12H),1.26-1.41(m,48H),1.56-1.73(m,12H),1.89-1.91(m,2H),2.26-2.32(m,8 H),2.78-2.80(m,4H),2.96-2.98(m,2H),3.80-3.81(m,2H),4.01-4.04(m,2H),4.20-4.24(m,2H),5.03-5.07(m,2H). ESI-MS C 51 H 98 NO9 + [M+H] + The calculated value was 868.7, and the measured value was 868.7.

[0129] Example 7: Synthesis of aminolipid E10LA10B6O3 In Example 1, 1,2-epoxyoctane was replaced with 1,2-epoxydecane, and lauric acid was replaced with capric acid, and the aminolipid E10LA10B6O3 was obtained in the same manner. The structural formula of this aminolipid is as follows. TIFF2026516582000029.tif28170 1 H NMR (400MHz, CDCl3): δ0.86-0.92(m,12H),1.25-1.40(m,56H),1.56-1.71(m,12H),1.87-1.89(m,2H),2.27-2.32(m,8 H),2.77-2.80(m,4H),2.97-2.99(m,2H),3.79-3.80,(m,2H),4.00-4.04(m,2H),4.21-4.25(m,2H),5.02-5.07(m,2H). ESI-MS C 55 H 106 NO9 + [M+H] +The calculated value was 924.8, and the measured value was 924.9.

[0130] Example 8: Synthesis of aminolipid E12LA8B6O3 In Example 2, n-hexanoic acid was replaced with n-octanoic acid, and aminolipid E12LA8B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000030.tif28170 1 H NMR(400MHz,CDCl3):δ5.07-5.04(m,2H),4.23(dd,J1=12.0Hz,J2=3.6Hz,2H),4.01(dd,J1=12.0Hz,J2=6.4Hz,2H),3.80(t,J=5.2Hz,2H),2. 86(t,J=5.6Hz,2H),2.66(t,J=7.6Hz,4H),2.33-2.28(m,8H),1.83-1. 80(m,2H),1.69-1.56(m,12H),1.38-1.25(m,56H),0.89-0.85(m,12H). ESI-MS C 55 H 106 NO9 + [M+H] + The calculated value was 924.7862, and the measured value was 924.7891.

[0131] Example 9: Synthesis of compound E12LA12B6O3 TIFF2026516582000031.tif72170

[0132] Step 1: Synthesis of compound E12LA12B6O3-2 In a 25 mL reaction tube, ferric trichloride (16.2 mg, 0.1 mmol, 0.025 eq), pyridine (4 mg, 0.05 mmol, 0.0125 eq), lauric acid (800 mg, 4.0 mmol, 1.0 eq), and 1,2-epoxydodecane (884 mg, 4.8 mmol, 1.2 eq) were added sequentially, and the reaction was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was concentrated and purified by flash column chromatography (n-heptane:ethyl acetate = 50:1~10:1) to obtain compound E12LA12B6O3-2 (1.2 g, 86.0%). ESI-MS C24 H 49 O3 + [M+H] + The calculated value was 385.4, and the measured value was 385.4.

[0133] Step 2: Synthesis of compound E12LA12B6O3-3 In a 25 mL reaction tube, E12LA12B6O3-2 (1.2 g, 3.65 mmol, 1.0 eq), 6-bromohexanoic acid (973 mg, 4.38 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.8 g, 14.6 mmol, 4.0 eq), 4-dimethylaminopyridine (45 mg, 0.365 mmol, 0.1 eq), N,N-diisopropylethylamine (2.83 g, 21.9 mmol, 6.0 eq), and 20 mL of dichloromethane were sequentially added and stirred overnight at room temperature. After the reaction was complete, the reaction mixture was concentrated and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~20:1) to obtain compound E12LA12B6O3-3 (1.65 g, 92.0%). ESI-MS C 30 H 58 BrO4 + [M+H] + The calculated value was 561.4, and the measured value was 561.4.

[0134] Step 3: Synthesis of E12LA12B6O3 In a 25 mL reaction tube, E12LA12B6O3-3 (1.65 g, 3.36 mmol, 2.2 eq), potassium carbonate (464 mg, 3.36 mmol, 2.2 eq), sodium iodide (150 mg, 1.0 mmol, 0.67 eq), 3-aminopropanol (113 mg, 1.5 mmol), and 10 mL of acetonitrile were added sequentially, and the reaction was stirred overnight at 70-80°C. After the reaction was complete, the mixture was separated by column chromatography to obtain E12LA12B6O3 (0.94 g, 68%). 1H NMR (400MHz, CDCl3): δ0.87-0.91(m,12H),1.24-1.38(m,68H),1.42-1.58(m,4H),1.55-1.69(m,14H),2.29-2.34(m,8 H),2.38-2.42(m,4H),2.61-2.64(m,2H),3.78-3.81,(m,2H),4.01-4.06(m,2H),4.20-4.24(m,2H),5.04-5.10(m,2H). ESI-MS C 63 H 122 NO9 + [M+H] + The calculated value was 1036.9, and the measured value was 1037.0.

[0135] Example 10: Synthesis of aminolipid E8CA5B6O3 In Example 9, 1,2-epoxydodecane was replaced with 1,2-epoxyoctane, and lauric acid was replaced with 2-pentylheptanoic acid, and the aminolipid E8CA5B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000032.tif28170 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,18H),1.25-1.37(m,48H),1.40-1.70(m,18H),2.25-2.36(m,6H),2.44- 2.48(m,4H),2.66-2.68(m,2H),3.77-3.79,(m,2H),4.00-4.05(m,2H),4.21-4.25(m,2H),5.03-5.08(m,2H). ESI-MS C 55 H 106 NO9 + [M+H] + The calculated value was 924.8, and the measured value was 924.8.

[0136] Example 11: Synthesis of aminolipid E12LA6B6O9 In Example 2, 3-aminopropanol was replaced with n-pentylamine, and aminolipid E12LA6B6O9 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000033.tif30170 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,15H),1.24-1.36(m,54H),1.53-1.74(m,16H),2. 27-2.33(m,8H),2.83(s,6H),3.98-4.03(m,2H),4.21-4.25(m,2H),5.02-5.07(m,2H). ESI-MS C 53 H 102 NO8 + [M+H] + The calculated value was 880.8, and the measured value was also 880.8.

[0137] Example 12: Synthesis of aminolipid E12LA6B6O10 In Example 2, 3-aminopropanol was replaced with trans-p-aminocyclohexanol, and aminolipid E12LA6B6O10 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000034.tif25170 1 H NMR(400MHz,CDCl3):δ0.84-0.90(m,12H),1.24-1.31(m,54H),1.53-1.65(m,14H),1.99-2.01(m,2 H),2.26-2.31(m,8H),2.42-2.46(m,4H),3.99-4.03(m,2H),4.19-4.22(m,2H),5.03-5.08(m,2H). ESI-MS C 54 H 102 NO9 + [M+H] + The calculated value was 908.8, and the measured value was 908.7.

[0138] Example 13: Synthesis of aminolipid E12LA6B6O12 In Example 2, 3-aminopropanol was replaced with 3-dimethylaminopropylamine, and the aminolipid E12LA6B6O12 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000035.tif27170 11H NMR (400 MHz, CDCl3): δ 0.86 - 0.94 (m, 12H), 1.26 - 1.35 (m, 42H), 1.41 - 1.48 (m, 6H), 1.55 - 1.72 (m, 18H), 2.29 - 2.36 (m, 8H), 2.70 - 2.76 (m, 2H), 3.37 - 3.41 (m, 4H), 3.99 - 4.04 (m, 2H), 4.24 - 4.34 (m, 6H), 5.00 - 5.06 (m, 2H). ESI-MS C 53 H 103 N2O8 + [M + H] + The calculated value of

[0139] Example 14: Synthesis of Amino Lipid E12LA6B6O13 Replace 3 - aminopropanol in Example 2 with 1-(2 - aminoethyl)piperidine, and obtain amino lipid E12LA6B6O13 in the same method. The structural formula of this amino lipid is as follows. TIFF2026516582000036.tif28170 1 1H NMR (400 MHz, CDCl3): δ 0.82 - 0.92 (m, 12H), 1.24 - 1.36 (m, 50H), 1.44 - 1.69 (m, 18H), 2.27 - 2.31 (m, 8H), 2.47 - 2.50 (m, 4H), 2.57 - 2.61 (m, 4H), 2.72 - 2.74 (m, 2H), 3.99 - 4.04 (m, 2H), 4.19 - 4.23 (m, 2H), 5.03 - 5.08 (m, 2H). ESI-MS C 55 H 105 N2O8 + [M + H] + The calculated value of

[0140] Example 15: Synthesis of Amino Lipid E12LA6B6O15 Replace 3 - aminopropanol in Example 2 with N-(2 - aminoethyl)-4 - hydroxypiperidine, and obtain amino lipid E12LA6B6O15 in the same method. The structural formula of this amino lipid is as follows. TIFF2026516582000037.tif251701 1H NMR (400 MHz, CDCl3): δ 0.86 - 0.90 (m, 12H), 1.26 - 1.37 (m, 50H), 1.48 - 1.67 (m, 16H), 2.28 - 2.33 (m, 8H), 2.54 - 2.61 (m, 6H), 2.84 - 2.89 (m, 4H), 3.76 (s, 1H), 4.00 - 4.07 (m, 2H), 4.20 - 4.24 (m, 2H), 5.04 - 5.09 (m, 2H). ESI-MS C 55 H 105 N2O9 + [M + H] + The calculated value of [M + H] was 937.8, and the measured value was 937.9.

[0141] Example 16: Synthesis of Amino Lipid E12LA12B6O30 The 3 - aminopropanol in Example 2 was replaced with β - alanine, and amino lipid E12LA12B6O30 was obtained in the same manner. The structural formula of this amino lipid is as follows. TIFF2026516582000038.tif29170 1 1H NMR (400 MHz, CDCl3): δ 0.86 - 0.92 (m, 12H), 1.24 - 1.37 (m, 68H), 1.44 - 1.60 (m, 4H), 1.54 - 1.70 (m, 14H), 2.29 - 2.44 (m, 12H), 2.61 - 2.64 (m, 2H), 3.78 - 3.81, (m, 2H), 4.01 - 4.05 (m, 2H), 4.21 - 4.24 (m, 2H), 5.02 - 5.08 (m, 2H). ESI-MS C 64 H 122 NO 10 + [M + H] + The calculated value of [M + H] was 1064.9, and the measured value was 1065.0.

[0142] Example 17: Synthesis of Amino Lipid E12LA12B6O31 The 3 - aminopropanol in Example 2 was replaced with 4 - aminobutyronitrile, and amino lipid E12LA12B6O31 was obtained in the same manner. The structural formula of this amino lipid is as follows. TIFF2026516582000039.tif301701 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.25-1.39(m,68H),1.42-1.60(m,4H),1.58-1.72(m,12H),1.78-1.82(m,2H) ),2.31-2.42(m,12H),2.60-2.64(m,2H),3.79-3.82,(m,2H),4.00-4.04(m,2H),4.18-4.22(m,2H),5.02-5.09(m,2H). ESI-MS C 64 H 121 N2O8 + [M+H] + The calculated value was 1045.9, and the measured value was 1045.9.

[0143] Example 18: Synthesis of aminolipid K3LA6B6O3 TIFF2026516582000040.tif89170

[0144] Step 1: Synthesis of compound K3LA6B6O3-2 Imidazole (6.54 g, 96 mmol, 1.2 eq) was added to a 250 mL round-bottom flask. The flask was then filled with nitrogen gas under vacuum, and dichloromethane (60 mL), N,N-dimethylformamide (30 mL), and 1,3-propylene glycol (6.09 g, 80 mmol, 1.0 eq) were added sequentially. The mixture was stirred at 0°C for 10 minutes. Tert-butyldimethylsilyl chloride (12.06 g, 80 mmol, 1.0 eq) was dissolved in dichloromethane (60 mL) and added dropwise to the mixture. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was washed three times with water, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~10:1) to obtain compound K3LA6B6O3-2 (10.96 g, 72%). ESI-MS C9H 23 O2Si + [M+H] + The calculated value was 191.1, and the measured value was 191.1.

[0145] Step 2: Synthesis of compound K3LA6B6O3-3 A solution of pyridinium chlorochromate (6.09 g, 80 mmol, 1.0 eq) and compound K3LA6B6O3-2 (10.96 g, 57.6 mmol, 1.0 eq) in dichloromethane (100 mL) was stirred at room temperature for 5 hours. The reaction mixture was filtered, and the filtrate was concentrated and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~20:1) to obtain compound K3LA6B6O3-3 (8.69 g, 80%). ESI-MS C9H 21 O2Si + [M+H] + The calculated value was 189.1, and the measured value was 189.1.

[0146] Step 3: Synthesis of compound K3LA6B6O3-4 A 500 mL round-bottom flask was filled with nitrogen gas by vacuum, and compound K3LA6B6O3-3 (8.69 g, 46.14 mmol, 1.0 eq) and anhydrous tetrahydrofuran (150 mL) were added. The mixture was stirred at -20°C for 10 minutes, and 70 mL of decyl magnesium bromide solution (1 mol / L anhydrous tetrahydrofuran solution, 1.5 eq) was added dropwise to the mixture. The mixture was stirred at -20°C to 0°C for 3 hours, and 50 mL of saturated ammonium chloride solution was added to stop the reaction. The mixture was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound K3LA6B6O3-4 (9.93 g, 65%). ESI-MS C 19 H 43 O2Si + [M+H] + The calculated value was 331.3, and the measured value was 331.4.

[0147] Step 4: Synthesis of compound K3LA6B6O3-5 A solution of K3LA6B6O3-4 (9.93 g, 30 mmol), 6-bromohexanoic acid (7.02 g, 36 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.50 g, 60 mmol, 2.0 eq), N,N-diisopropylethylamine (15.51 g, 120 mmol, 4.0 eq), and 4-dimethylaminopyridine (0.37 g, 3 mmol, 0.1 eq) in dichloromethane (100 mL) was stirred overnight at room temperature. Once TLC indicated the completion of the reaction, the reaction mixture was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~20:1) to obtain compound K3LA6B6O3-5 (12.79 g, 84%). ESI-MS C 25 H 52 BrO3Si + [M+H] + The calculated value was 507.3, and the measured value was 507.2.

[0148] Step 5: Synthesis of compound K3LA6B6O3-6 K3LA6B6O3-5 (12.79 g, 25.2 mmol, 1.0 eq), ammonium fluoride (9.33 g, 252 mmol, 10.0 eq), and methanol (75 mL) were stirred under reflux at 65°C for 3 hours. Once TLC indicated the completion of the reaction, the reaction mixture was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 50:1~5:1) to obtain compound K3LA6B6O3-6 (8.72 g, 88%). ESI-MS C 19 H 38 BrO3 + [M+H] + The calculated value was 393.2, and the measured value was 393.2.

[0149] Step 6: Synthesis of compound K3LA6B6O3-7 A solution of K3LA6B6O3-6 (8.72 g, 22.2 mmol), hexanoic acid (3.09 g, 26.64 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.51 g, 44.4 mmol, 2.0 eq), N,N-diisopropylethylamine (11.48 g, 88.8 mmol, 4.0 eq), and 4-dimethylaminopyridine (0.27 g, 2.2 mmol, 0.1 eq) in dichloromethane (50 mL) was stirred overnight at room temperature. Once TLC indicated the completion of the reaction, the reaction mixture was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~20:1) to obtain compound K3LA6B6O3-7 (7.86 g, 72%). ESI-MS C 25 H 48 BrO4 + [M+H] + The calculated value was 491.3, and the measured value was 491.2.

[0150] Step 7: Synthesis of compound K3LA6B6O3 A solution of compound K3LA6B6O3-7 (7.86 g, 15.99 mmol, 3.0 eq), 3-aminopropanol (0.40 g, 5.33 mmol, 3.0 eq), potassium carbonate (1.47 g, 10.66 mmol, 2.0 eq), and sodium iodide (0.80 g, 5.33 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred overnight at 75°C. After concentrating the reaction mixture, it was purified by flash column chromatography (dichloromethane:methanol = 100:1 to 20:1) to obtain compound K3LA6B6O3-7 (3.58 g, 75%). 1 H NMR(400MHz,CDCl3):δ0.85-0.90(m,12H),1.24-1.36(m,48H),1.56-1.73(m,18H),2.25-2.29(m,8 H),2.48-2.52(m,4H),2.70-2.72(m,2H),3.76-3.79(m,2H),4.03-4.11(m,4H),4.93-4.96(m,2H). ESI-MS C 53 H 102NO9 + [M+H] + The calculated value was 896.8, and the measured value was 896.9.

[0151] Example 19: Synthesis of aminolipid K4LA6B6O3 In Example 18, 1,3-propylene glycol was replaced with 1,4-butanediol, and the aminolipid K4LA6B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000041.tif29170 1 H NMR(400MHz,CDCl3):δ0.85-0.91(m,12H),1.26-1.41(m,48H),1.56-1.72(m,18H),2.27-2.33(m,8 H),2.81-2.84(m,4H),3.00-3.04(m,2H),3.81-3.84(m,2H),4.04-4.07(m,4H),4.87-4.90(m,2H). ESI-MS C 55 H 106 NO9 + [M+H] + The calculated value was 924.8, and the measured value was 924.8.

[0152] Example 20: Synthesis of aminolipid K5LA6B6O3 In Example 18, 1,3-propylene glycol was replaced with 1,5-pentanediol, and the aminolipid K5LA6B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000042.tif22170 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.26-1.41(m,54H),1.50-1.67(m,18H),2.27-2.34(m,8 H),2.91-2.96(m,4H),3.10-3.14(m,2H),3.83-3.85(m,2H),4.03-4.06(m,4H),4.85-4.89(m,2H). ESI-MS C 57 H 110 NO9 + [M+H] +The calculated value was 952.8, and the measured value was 952.8.

[0153] Example 21: Synthesis of amino lipid E12LA6B6O3A2 TIFF2026516582000043.tif20170

[0154] Step 1: Synthesis of compound E12LA6B6O3 E12LA6B6O3 was synthesized by the method of Example 2.

[0155] Step 2: Synthesis of compound E12LA6B6O3A2 A dichloromethane solution of E12LA6B6O3 (0.87 g, 1 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.41 g, 1 mmol, 2.0 eq), DMAP (0.12 g, 0.1 mmol, 0.1 eq), and 3-(4-phenylpiperazin-1-yl)propionic acid (0.26 g, 1.5 mmol, 1.5 eq) was stirred at room temperature for 4 hours. When TLC indicated that the reaction was complete, the reaction solution was filtered, the filtrate was concentrated, and then purified by flash column chromatography (dichloromethane:methanol = 100:1 to 10:1) to obtain compound E12LA6B6O3A2 (0.80 g, 78%). 1 H NMR (400 MHz, CDCl3): δ0.85 - 0.90 (m, 12H), 1.25 - 1.32 (m, 48H), 1.36 - 1.49 (m, 4H), 1.53 - 1.66 (m, 12H), 2.27 - 2.50 (m, 25H), 2.67 - 2.71 (m, 2H), 4.00 - 4.04 (m, 2H), 4.08 - 4.11 (m, 2H), 4.19 - 4.23 (m, 2H), 5.04 - 5.09 (m, 2H). ESI-MS C 59 H 112 N3O 10 + [M + H] + The calculated value was 1022.8, and the measured value was 1022.9.

[0156] Example 22: Synthesis of amino lipid E12LA6B6O3A3 In Example 21, 3-(4-phenylpiperazin-1-yl)propionic acid was replaced with 4-(4-methyl-1-piperazinyl)butyric acid, and aminolipid E12LA6B6O3A3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000044.tif26170 1 H NMR(400MHz,CDCl3):δ0.85-0.90(m,12H),1.25-1.45(m,50H),1.55-1.66(m,12H),1.69-1.83(m,4 H),2.27-2.47(m,27H),4.00-4.04(m,2H),4.07-4.10(m,2H),4.19-4.23(m,2H),5.04-5.10(m,2H). ESI-MS C 60 H 114 N3O 10 + [M+H] + The calculated value was 1036.8, and the measured value was 1036.8.

[0157] Example 23: Synthesis of aminolipid E12LA6B6O3A4 In Example 21, 3-(4-phenylpiperazin-1-yl)propionic acid was replaced with 3-(dimethylamino)propionic acid, and aminolipid E12LA6B6O3A4 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000045.tif26170 1 H NMR(400MHz,CDCl3):δ0.84-0.89(m,12H),1.24-1.35(m,50H),1.41-1.48(m,6H),1.52-1.65(m,12H),2.23(s,6H),2.26-2.30(m,8H) ,2.35-2.39(m,4H),2.44-2.48(m,4H),2.58-2.62(m,2H),3.99-4.03(m,2H),4.08-4.11(m,2H),4.18-4.22(m,2H),5.03-5.09(m,2H). ESI-MS C 56 H 107 N2O 10 + [M+H] +The calculated value was 967.8, and the measured value was 967.8.

[0158] Example 24: Synthesis of aminolipid E12LA6B6O3A5 In Example 21, 3-(4-phenylpiperazin-1-yl)propionic acid was replaced with 5-(dimethylamino)pentanoic acid, and aminolipid E12LA6B6O3A5 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000046.tif28170 1 H NMR(400MHz,CDCl3):δ0.84-0.89(m,12H),1.24-1.35(m,48H),1.41-1.75(m,18H),2.21(s,6H),2.22-2.30(m,12H) ,2.35-2.37(m,4H),2.42-2.45(m,2H),3.98-4.03(m,2H),4.05-4.09(m,2H),4.18-4.22(m,2H),5.03-5.09(m,2H). ESI-MS C 58 H 111 N2O 10 + [M+H] + The calculated value was 995.8, and the measured value was 995.9.

[0159] Example 25: Synthesis of aminolipid E12LA6B6O3A6 In Example 21, 3-(4-phenylpiperazin-1-yl)propionic acid was replaced with 7-(dimethylamino)heptanoic acid, and aminolipid E12LA6B6O3A6 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000047.tif27170 1H NMR (400MHz, CDCl3): δ0.84-0.91(m,12H),1.23-1.37(m,50H),1.39-1.49(m,6H),1.52-1.64(m,14H),2.21(s,6H),2.25-2.3 0(m,12H),2.33-2.37(m,4H),2.42-2.45(m,2H),3.98-4.03(m,2H),4.05-4.08(m,2H),4.18-4.22(m,2H),5.03-5.08(m,2H). ESI-MS C 60 H 115 N2O 10 + [M+H] + The calculated value was 1023.8, and the measured value was 1023.8.

[0160] Example 26: Synthesis of aminolipid E8LA6B6O3A6 In Example 21, E12LA6B6O3 was replaced with E8LA6B6O3, and the amino lipid E8LA6B6O3A6 was obtained by the same method. The structural formula of this amino lipid is as follows. TIFF2026516582000048.tif23170 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.26-1.43(m,36H),1.39-1.49(m,6H),1.56-1 .79(m,12H),2.23-2.48(m,24H),4.00-4.11(m,4H),4.20-4.24(m,2H),5.07-5.11(m,2H). ESI-MS C 52 H 99 N2O 10 + [M+H] + The calculated value was 911.7, and the measured value was 911.9.

[0161] Example 27: Synthesis of aminolipid E12LA6B6O3A7 In Example 21, 3-(4-phenylpiperazine-1-yl)propionic acid was replaced with 1-methylpiperidine-4-carboxylic acid, and aminolipid E12LA6B6O3A7 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000049.tif23170 1 H NMR (400MHz, CDCl3): δ0.87-0.92(m,12H),1.26-1.41(m,48H),1.57-1.83(m,20H),2.24-2.40(m,16H),2.45 -2.48(m,2H),2.80-2.84(m,2H),4.01-4.06(m,2H),4.10-4.13(m,2H),4.21-4.25(m,2H),5.06-5.11(m,2H). ESI-MS C 58 H 109 N2O 10 + [M+H] + The calculated value was 993.8, and the measured value was 993.7.

[0162] Example 28: Synthesis of aminolipid E12LA6B6O3A8 In Example 21, 3-(4-phenylpiperazin-1-yl)propionic acid was replaced with 1-piperidinepropionic acid, and aminolipid E12LA6B6O3A8 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000050.tif22170 1 H NMR (400MHz, CDCl3): δ0.85-0.92(m,12H),1.24-1.36(m,44H),1.40-1.45(m,6H),1.54-1.69(m,12H),2.27-2.30(m,8 H),2.34-2.52(m,12H),2.63-2.67(m,2H),4.00-4.04(m,2H),4.07-4.10(m,8H),4.19-4.23(m,2H),5.03-5.09(m,2H). ESI-MS C 59 H 111 N2O 10 + [M+H] + The calculated value was 1007.8, and the measured value was 1007.9.

[0163] Example 29: Synthesis of aminolipid E12LA12B6O30A9 TIFF2026516582000051.tif27170

[0164] Step 1: Synthesis of compound E12LA12B6O30A9-1 E12LA12B6O30A9-1 was synthesized using the method of Example 16.

[0165] Step 2: Synthesis of compound E12LA12B6O30A9 A dichloromethane solution of E12LA12B6O30A9-1 (1.06 g, 1 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.41 g, 2 mmol, 2.0 eq), DMAP (0.12 g, 0.1 mmol, 0.1 eq), and 3-dimethylaminopropanol (0.16 g, 1.5 mmol, 1.5 eq) was stirred at room temperature for 4 hours. Once TLC indicated the completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated, and then purified by flash column chromatography (dichloromethane:methanol = 50:1 to 10:1) to obtain compound E12LA12B6O30A9 (0.92 g, 80%). 1 H NMR(400MHz,CDCl3):δ0.86-0.91(m,12H),1.24-1.42(m,68H),1.45-1.72(m,20H),1.84-1.86(m,2H),2.26(s,6H), 2.30-2.42(m,12H),2.60-2.63(m,2H),3.78-3.81,(m,2H),4.01-4.15(m,4H),4.22-4.26(m,2H),5.06-5.12(m,2H). ESI-MS C 69 H 133 N2O 10 + [M+H] + The calculated value was 1050.0, and the measured value was 1050.3.

[0166] Example 30: Synthesis of aminolipid E12LA12B6O30A10 In Example 29, 3-dimethylaminopropanol was replaced with 3-dimethylaminopropylamine, and aminolipid E12LA12B6O30A10 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000052.tif291701 H NMR(400MHz,CDCl3):δ0.86-0.91(m,12H),1.24-1.42(m,68H),1.45-1.84(m,22H),2.28(s,6H),2.32-2.44(m,12H) ,2.60-2.63(m,2H),3.50-3.54,(m,2H),3.78-3.82(m,2H),4.00-4.04(m,2H),4.22-4.26(m,2H),5.06-5.12(m,2H). ESI-MS C 69 H 134 N3O9 + [M+H] + The calculated value was 1149.0, and the measured value was 1149.0.

[0167] Example 31: Synthesis of aminolipid E12LA12B6O3A11 TIFF2026516582000053.tif72170

[0168] Step 1: Synthesis of compound E12LA12B6O3A11-1 E12LA6B6O3A11-1 was synthesized using the method of Example 9.

[0169] Step 2: Synthesis of compound E12LA6B6O3A11-1-2 A 50 mL round-bottom flask was filled with nitrogen gas by vacuum, and compound E12LA6B6O3A11-1 (5.18 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added. The mixture was stirred at 0°C for 10 minutes, and trifluoromethanesulfonic acid anhydride (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise. The mixture was stirred at 0°C for 10 minutes, and 2,6-dimethylpyridine (0.80 g, 7.5 mmol, 1.5 eq) was added. The mixture was stirred at room temperature overnight. The reaction solution was washed once each with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (dichloromethane:methanol = 50:1~20:1) to obtain compound E12LA6B6O3A11-2 (4.38 g, 75%). ESI-MS C 64 H 121 F3NO 11 S+ [M+H] + The calculated value was 1168.9, and the measured value was 1169.0.

[0170] Step 3: Synthesis of compound E12LA6B6O3A11-3 In a 100 mL pressure bottle, E12LA6B6O3A11-2 (4.38 g, 3.75 mmol, 1.0 eq), 20 mL of tetrahydrofuran, and 5 mL of aqueous ammonia (25% by mass aqueous solution) were added. The mixture was stirred overnight at 100°C, and the reaction solution was washed once each with water and saturated sodium chloride solution. After drying over anhydrous sodium sulfate and concentration, the mixture was purified by flash column chromatography (dichloromethane:methanol = 20:1~5:1) to obtain compound E12LA6B6O3A11-3 (1.71 g, 44%). ESI-MS C 63 H 123 N2O8 + [M+H] + The calculated value was 1035.9, and the measured value was 1035.9.

[0171] Step 4: Synthesis of compound E12LA6B6O3A11 A dichloromethane solution of E12LA6B6O3A11-3 (1.71 g, 1.65 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.68 g, 3.3 mmol, 2.0 eq), DMAP (0.20 g, 0.16 mmol, 0.1 eq), and 4-dimethylaminobutyric acid (0.32 g, 2.48 mmol, 1.5 eq) was stirred at room temperature for 4 hours. Once TLC indicated the completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated, and then purified by flash column chromatography (dichloromethane:methanol = 50:1~10:1) to obtain compound E12LA6B6O3A11 (1.48 g, 78%). 1H NMR(400MHz,CDCl3):δ0.85-0.91(m,12H),1.24-1.41(m,68H),1.45-1.82(m,22H),2.27(s,6H),2.32-2.42(m,12H) ,2.61-2.63(m,2H),3.36-3.42,(m,2H),3.77-3.79(m,2H),4.01-4.04(m,2H),4.23-4.26(m,2H),5.04-5.10(m,2H). ESI-MS C 69 H 134 N3O9 + [M+H] + The calculated value was 1049.0, and the measured value was 1049.1.

[0172] Example 32: Synthesis of aminolipid E12N1LA6B6O3 TIFF2026516582000054.tif73170

[0173] Step 1: Synthesis of compound E12N1LA6B6O3-2 A mixture of 1,2-epoxydodecane (3.69 g, 20 mmol, 1.0 eq), 20 mL of aqueous ammonia (25% by mass aqueous solution), 10 mL of ethanol, and 10 mL of water was stirred at 60°C for 16 hours. The reaction mixture was spin-dried, 3 mL of ethanol and 30 mL of n-heptane were added, and the mixture was slurryed at 60°C for 4 hours. The mixture was filtered, and the filter cake was dried at 50°C for 10 hours to obtain compound E12N1LA6B6O3-2 (3.46 g, 86%). ESI-MS C 12 H 28 NO + [M+H] + The calculated value was 202.2, and the measured value was 202.2.

[0174] Step 2: Synthesis of compound E12N1LA6B6O3-3 A solution of compound E12N1LA6B6O3-2 (3.46 g, 17.2 mmol, 1.5 eq), hexanoic acid (1.34 g, 11.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.30 g, 17.2 mmol, 1.5 eq), N,N-diisopropylethylamine (2.22 g, 17.2 mmol, 1.5 eq), and 4-dimethylaminopyridine (2.10 g, 17.2 mmol, 1.5 eq) in dichloromethane (40 mL) was stirred overnight at room temperature. The reaction mixture was extracted three times with dichloromethane, washed once with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 10:1~3:1) to obtain compound E12N1LA6B6O3-3 (1.10 g, 32%). ESI-MS C 18 H 38 NO2 + [M+H] + The calculated value was 300.3, and the measured value was 300.2.

[0175] Step 3: Synthesis of compound E12N1LA6B6O3-4 A solution of compound E12N1LA6B6O3-3 (1.10 g, 3.68 mmol, 1.0 eq), hexanoic acid (0.51 g, 4.42 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.41 g, 7.36 mmol, 2.0 eq), N,N-diisopropylethylamine (1.90 g, 14.2 mmol, 4.0 eq), and 4-dimethylaminopyridine (0.04 g, 0.37 mmol, 0.1 eq) in dichloromethane (20 mL) was stirred overnight at room temperature. Once TLC indicated the completion of the reaction, the reaction mixture was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~20:1) to obtain compound E12N1LA6B6O3-4 (1.42 g, 81%). ESI-MS C 24 H 47 BrNO3 + [M+H] +The calculated value was 476.3, and the measured value was 476.3.

[0176] Step 4: Synthesis of compound E12N1LA6B6O3 A solution of compound E12N1LA6B6O3-4 (1.42 g, 2.99 mmol, 3.0 eq), 3-aminopropanol (0.075 g, 1.00 mmol, 1.0 eq), potassium carbonate (0.28 g, 2.00 mmol, 2.0 eq), and sodium iodide (0.15 g, 1.00 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred overnight at 75°C. After concentrating the reaction mixture, it was purified by flash column chromatography (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12N1LA6B6O3 (0.68 g, 78%). 1 H NMR(400MHz,CDCl3):δ0.86-0.93(m,12H),1.25-1.37(m,48H),1.41-1.69(m,16 H),2.14-2.18(m,8H),2.30-2.43(m,6H),3.34-3.48(m,4H),4.90-4.96(m,2H). ESI-MS C 51 H 100 N3O7 + [M+H] + The calculated value was 866.8, and the measured value was 866.7.

[0177] Example 33: Synthesis of aminolipid E12N1LA8B6O3 In Example 32, hexanoic acid was replaced with octanoic acid, and the aminolipid E12N1LA8B6O3 was obtained by the same method. The structural formula of this aminolipid is as follows. TIFF2026516582000055.tif24170 1 H NMR(400MHz,CDCl3):δ0.86-0.93(m,12H),1.25-1.37(m,48H),1.42-1.70(m,18 H),2.14-2.18(m,8H),2.31-2.43(m,6H),3.35-3.48(m,4H),4.90-4.96(m,2H). ESI-MS C 55 H 108 N3O7 + [M+H]+ The calculated value was 922.8, and the measured value was 923.0.

[0178] Example 34: Synthesis of aminolipid E12N2LA6B6O3 TIFF2026516582000056.tif64170

[0179] Step 1: Synthesis of compound E12N2LA6B6O3-1 E12N2LA6B6O3-1 was synthesized according to steps 1 and 2 of Example 32.

[0180] Step 2: Synthesis of compound E12N2LA6B6O3-2 A 50 mL round-bottom flask was filled with nitrogen gas under vacuum. Compound E12N2LA6B6O3-1 (1.50 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added, and the mixture was stirred at 0°C for 10 minutes. Trifluoromethanesulfonic acid anhydride (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise, and the mixture was stirred at 0°C for 10 minutes. 2,6-dimethylpyridine (0.80 g, 7.5 mmol, 1.5 eq) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was washed once each with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (dichloromethane:methanol = 50:1~20:1) to obtain compound E12N2LA6B6O3-2 (1.51 g, 70%). ESI-MS C 19 H 37 F3NO4S + [M+H] + The calculated value was 432.2, and the measured value was 432.2.

[0181] Step 3: Synthesis of compound E12N2LA6B6O3-3 In a 100 mL pressure bottle, E12N2LA6B6O3-2 (1.51 g, 3.50 mmol, 1.0 eq), 20 mL of tetrahydrofuran, and 5 mL of aqueous ammonia (25% by mass aqueous solution) were added. The mixture was stirred overnight at 100°C, and the reaction solution was washed once each with water and saturated sodium chloride solution. After drying over anhydrous sodium sulfate and concentration, the mixture was purified by flash column chromatography (dichloromethane:methanol = 20:1~5:1) to obtain compound E12N2LA6B6O3-3 (0.47 g, 45%). ESI-MS C 18 H 39 N2O + [M+H] + The calculated value was 299.3, and the measured value was 299.3.

[0182] Step 4: Synthesis of compound E12N2LA6B6O3-4 A dichloromethane solution of compound E12N2LA6B6O3-3 (0.74 g, 1.58 mmol, 3.0 eq), N,N'-dicyclohexylcarbodiimide (0.65 g, 3.15 mmol, 2.0 eq), DMAP (0.20 g, 0.16 mmol, 0.1 eq), N,N-diisopropylethylamine (0.82 g, 6.32 mmol, 4.0 eq), and 6-bromohexanoic acid (0.46 g, 2.37 mmol, 1.5 eq) was stirred at room temperature for 4 hours. After TLC indicated completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 50:1~1:1) to obtain compound E12N2LA6B6O3-4 (1.48 g, 88%). ESI-MS C 24 H 48 BrN2O2 + [M+H] + The calculated value was 475.3, and the measured value was 475.3.

[0183] Step 5: Synthesis of compound E12N2LA6B6O3 A solution of compound E12N2LA6B6O3-4 (1.48 g, 1.39 mmol, 3.0 eq), 3-aminopropanol (0.035 g, 0.46 mmol, 1.0 eq), potassium carbonate (0.13 g, 0.92 mmol, 2.0 eq), and sodium iodide (0.069 g, 0.46 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred overnight at 75°C. After concentrating the reaction mixture, it was purified by flash column chromatography (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12N2LA6B6O3 (0.97 g, 81%). 1 H NMR (400MHz, CDCl3): δ0.86-0.92(m,12H),1.25-1.37(m,42H),1.41-1.69(m,18H),2.06- 2.18(m,12H),2.30-2.42(m,4H),3.32-3.48(m,4H),3.70-3.74(m,2H),4.90-4.96(m,2H). ESI-MS C 51 H 102 N5O5 + [M+H] + The calculated value was 864.8, and the measured value was 864.7.

[0184] Example 35: Synthesis of aminolipid E12S1LA6B6O3 TIFF2026516582000057.tif65170

[0185] Step 1: Synthesis of compound E12S1LA6B6O3-2 In a 25 mL reaction tube, ferric trichloride (16.2 mg, 0.1 mmol, 0.025 eq), pyridine (4 mg, 0.05 mmol, 0.0125 eq), hexanoic acid (464 mg, 4.0 mmol, 1.0 eq), and 1,2-epoxydodecane (884 mg, 4.8 mmol, 1.2 eq) were added sequentially, and the reaction was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was concentrated and purified by flash column chromatography (n-heptane:ethyl acetate = 50:1~10:1) to obtain compound E12S1LA6B6O3-2 (1.2 g, 90.0%). ESI-MS C 18 H 37 O3 + [M+H]+ The calculated value was 301.3, and the measured value was 301.3.

[0186] Step 2: Synthesis of compound E12S1LA6B6O3-3 In a 25 mL reaction tube, E12S1LA6B6O3-2 (1.2 g, 3.6 mmol, 1.0 eq), triethylamine (0.36 g, 3.6 mmol, 1.0 eq), methanesulfonyl chloride (0.49 g, 4.32 mmol, 1.2 eq), and 10 mL of dichloromethane were added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was washed once each with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. 10 mL of N,N-dimethylformamide and sodium hydrosulfide (0.24 g, 4.32 mmol, 1.2 eq) were added, and the mixture was stirred overnight at 45°C. The reaction mixture was extracted three times with ethyl acetate, washed once with saturated saline solution, dried over anhydrous sodium sulfate, concentrated, and then purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~10:1) to obtain compound E12S1LA6B6O3-3 (0.69 g, 61%). ESI-MS C 18 H 37 O2S + [M+H] + The calculated value was 317.3, and the measured value was 317.2.

[0187] Step 3: Synthesis of compound E12S1LA6B6O3-4 In a 25 mL reaction tube, E12S1LA6B6O3-3 (0.69 g, 2.2 mmol, 1.0 eq), dibromohydantoin (0.13 g, 0.44 mmol, 0.2 eq), 3-bromopropane-1-thiol (0.41 g, 2.64 mmol, 1.2 eq), and 10 mL of dichloromethane were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1~10:1) to obtain compound E12S1LA6B6O3-4 (0.70 g, 68%). ESI-MS C 21 H 42 BrO2S2+ [M+H] + The calculated value was 469.2, and the measured value was 469.2.

[0188] Step 4: Synthesis of compound E12S1LA6B6O3 A solution of compound E12S1LA6B6O3-4 (0.70 g, 1.50 mmol, 3.0 eq), 3-aminopropanol (0.038 g, 0.50 mmol, 1.0 eq), potassium carbonate (0.14 g, 1.00 mmol, 2.0 eq), and sodium iodide (0.075 g, 0.50 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred overnight at 75°C. After concentrating the reaction mixture, it was purified by flash column chromatography (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12S1LA6B6O3 (1.09 g, 80%). 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.25-1.35(m,40H),1.41-1.54(m,12H),1.59-1.67(m,10 H),2.09-2.18(m,10H),2.34-2.40(m,4H),2.50-2.54(m,2H),3.64-3.72(m,2H),4.00-4.06(m,4H). ESI-MS C 49 H 98 NO5S4 + [M+H] + The calculated value was 908.6, and the measured value was 908.7.

[0189] Manufacturing of lipid nanoparticles Example 36: Production of lipid nanoparticles The aminolipid compound of the present invention (or DLin-MC3 or SM-102 (purchased from Aweituo (Shanghai) Pharmaceutical Technology Co., Ltd.)), phospholipid (DSPC), cholesterol, and polyethylene glycolated lipid (DMG-PEG2000) were mixed in a molar ratio of 47.5:10:41:1.5 and dissolved in anhydrous ethanol. Using a microfluidic system, the obtained ethanol solution was mixed in a 1:3 volume ratio in a microfluidic chip with citrate buffer (50 mM, pH=4.0) containing dissolved Luc-mRNA (TriLink) to obtain a crude solution of lipid nanoparticles. Subsequently, the solution was dialyzed with 1×PBS at 4°C for 6 hours using a dialysis cassette (Fisher, MWCO20,000). Before use, the solution was filtered through a 0.22 μm microporous membrane. The mass ratio of the aminolipid compound to luciferase mRNA (Luc mRNA) was approximately 40:1.

[0190] The properties of the obtained lipid nanoparticles were evaluated, and the results are shown in Table 1.

[0191] Here, the particle size and PDI (polydispersion index) of the fabricated lipid nanoparticles were measured using Nano-ZSZEN3600 (Malvern). The particle size was measured three times using 20 μL of lipid nanoparticle (LNP) solution (30 seconds each time).

[0192] Inclusion rates were measured according to the standard procedure for the Quant-iT RiboGreen RNA kit.

[0193] TIFF2026516582000058.tif250170TIFF2026516582000059.tif10170

[0194] The results above show that the encapsulation rates of the lipid nanoparticles according to the present invention are all higher than those of DLin-MC3, and that the encapsulation rates of most lipid nanoparticles are higher than those of SM-102.

[0195] Example 37: Introduction of lipid nanoparticles produced with aminolipid compounds into primary BMDC cells Animal preparation: Female C57BL / 6 mice weighing approximately 20g and 6 weeks old were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics standards.

[0196] Cell collection: C57BL / 6 mice were sacrificed by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and the femurs and tibias of the mice were dissected. The attached muscles were removed to expose the bone. Then, bone marrow was aspirated from the tibia using a 1 mL syringe containing PBS, and the obtained bone marrow was dispersed and filtered through a 50 μm filter to remove impurities. Erythrocyte lysate (3-4 mL) was added to the obtained filtrate and allowed to stand for 5 minutes, then centrifuged at 800 g for 5 minutes and the supernatant was removed. The obtained cells were resuspended in 1640 medium (containing 10% fetal bovine serum, 20 ng / mL GMCSF, and 10 ng / mL IL-4), seeded at a seeding density of 100,000 cells / mL in 6-well plates, and transferred to an incubator at 37°C and 5% CO2, with half of the medium being changed every 2 days. On day 7, suspension and adherent cells were collected and seeded into 96-well white ELISA plates at a density of 20,000 cells / well / 100 μL.

[0197] Cellular introduction: Lipid nanoparticles containing luciferase mRNA were added to a 96-well white ELISA plate containing primary cells at a volume of 10 μL per well. The plate was then incubated at 37°C in a 5% CO2 incubator for 12 hours.

[0198] Measurement of introduction efficiency: Substrate ONE-Glo is introduced into each well of a 96-well white ELISA plate. TM Luciferase was added in 20 μL, and the expression levels were measured after 1 minute using a multifunctional microplate reader (Biorek SynergyH1). Table 2 shows the expression levels of Luc mRNA introduced into BMDCs using lipid nanoparticles (LNPs) consisting of representative aminolipid compounds, with DLin-MC3 and SM0102 used as controls.

[0199] TIFF2026516582000060.tif246170TIFF2026516582000061.tif12170

[0200] The expression intensity of amino lipids according to the present invention is significantly superior to that of DLin-MC3, and it has been shown that the expression intensity of some amino lipids is superior to that of SM-102.

[0201] Example 38: Evaluation of the in vivo delivery performance of luciferase mRNA using lipid nanoparticles. Manufacturing of lipid nanoparticles: Same as in Example 36.

[0202] Animal experiments Animal preparation: Female C57BL / 6 mice weighing approximately 20g and 6 weeks old were selected and housed in an SPF-grade enclosure. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics standards.

[0203] In vivo delivery: Five C57BL / 6 mice were randomly selected from each group and intramuscularly injected with a lipid nanoparticle solution at a mRNA dose of 0.5 mg / kg. Twelve hours later, each mouse was intraperitoneally injected with 200 μL of 10 mg / mL D-luciferin potassium salt. Five minutes later, the mice were placed in an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded. Table 3 shows the expression intensity of Luc mRNA delivered by intramuscular injection of LNPs consisting of representative aminolipid compounds, with DLin-MC3 and SM-102 used as controls.

[0204] TIFF2026516582000062.tif107170

[0205] Example 39: Evaluation of the in vivo immunological and tumor therapeutic effects of lipid nanoparticles produced with aminolipid compounds. Manufacturing Method: The aminolipid compound of the present invention was mixed with DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 47.5:10:41:1.5 and dissolved in anhydrous ethanol. Using a microfluidic system, the obtained ethanol solution was mixed with citrate buffer (50 mM, pH=4.0) containing dissolved OVA-mRNA in a microfluidic chip in a volume ratio of 1:3 to obtain lipid nanoparticles. Subsequently, the solution was dialyzed with 1×PBS at 4°C for 6 hours using a dialysis cassette (Fisher, MWCO20,000), and filtered through a 0.22 μm microporous membrane before use. The mass ratio of the aminolipid compound to ovalbumin mRNA (OVA mRNA) was approximately 40:1.

[0206] Animal preparation: Female C57BL / 6 mice weighing approximately 18-20g and aged 5-6 weeks were selected and housed in SPF-grade housing. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics standards.

[0207] In vivo delivery: B16-OVA melanoma cells (1.5 × 10⁻⁶) 5 The drug was subcutaneously injected into the outer thigh of mice. The tumor was 50 mm. 3 Vaccination was initiated when the animals reached approximately 6 or 7 days post-inoculation. Animals were immunized twice by intramuscular injection of an LNP preparation containing 1 μg of OVA-mRNA, with the second injection administered 7 days later. Tumor growth was measured three times a week using a digital caliper and calculated using the formula 0.5 × length × width × width. The tumor volume was 1500 mm³. 3 The mice were euthanized when they reached a certain stage. The tumor growth rate in the E12LA6B6O3 and E10LA6B6O3 groups was significantly slower than in the MC3 group (shown in Figure 2), and 100% (E12LA6B6O3 group) and 80% (E10LA6B6O3 group), respectively, achieved complete remission, which was significantly better than in the MC3 group (shown in Figure 3).

[0208] Example 40: Evaluation of the in vivo delivery performance of luciferase mRNA from LNPs composed of different phospholipid ratios. Production of lipid nanoparticles: While maintaining an ionizable lipid (E12LA6B6O3) content of 42.5% and a polyethylene glycolated lipid DMG-PEG2000 content of 1.5%, the phospholipid ratio was adjusted to 15, 12.5, 10, 7.5, 5.0, 2.5, and 0. By increasing or decreasing the molar ratio of the adjusted phospholipids relative to the cholesterol ratio, the quality control evaluation of lipid nanoparticles and the effect on the in vivo delivery performance of luciferase mRNA were investigated. The group numbers are B1 to B7, and the specific ratios are shown in Table 4. ALC-0315 and SM-102 were used as positive controls.

[0209] TIFF2026516582000063.tif61170

[0210] E12LA6B6O3, phospholipid (DSPC), cholesterol, and polyethylene glycolated lipid DMG-PEG2000 were mixed in the molar ratios shown in Table 4 and dissolved in anhydrous ethanol. Using a microfluidic system, the resulting ethanol solution was mixed with citrate buffer (50 mM, pH=4.0) containing dissolved Luc-mRNA (TriLink) in a 1:3 volume ratio in a microfluidic chip to obtain a crude solution of lipid nanoparticles. Subsequently, the solution was dialyzed with 1×PBS at 4°C for 6 hours using a dialysis cassette (Fisher, MWCO20,000). Before use, the solution was filtered through a 0.22 μm microporous membrane. The molar ratio of aminolipid compounds to luciferase mRNA (Luc mRNA) bases was approximately 6.5:1. The quality control of the lipid nanoparticles was measured using a Malvern particle size analyzer and microplate reader, with SM-102 and ALC-0315 (purchased from Aweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) used as control groups.

[0211] Animal preparation: Female Balb / C mice weighing approximately 20g and 6 weeks old were selected and housed in SPF-grade housing. Animal experiments were conducted strictly in accordance with national health agency guidelines and animal ethics standards.

[0212] In vivo delivery: Three Balb / C mice were randomly selected from each group and injected via tail vein with a lipid nanoparticle solution at a dose of 0.1 mg / kg mRNA. Six hours later, each mouse was intraperitoneally injected with 150 μL of 15 mg / mL D-luciferin potassium salt. Ten minutes later, the mice were placed in an in vivo imaging system (IVIS Spectrum), and the total fluorescence intensity of each mouse was observed and recorded. Table 5 shows the expression intensity of Luc mRNA delivered by tail vein injection of LNPs consisting of different DSPC content.

[0213] TIFF2026516582000064.tif74170

[0214] Finally, it should be noted that the above embodiments are merely for illustrating the technical means of the present invention and do not limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical means described in each of the above embodiments can be modified or some of their technical features can be replaced with equivalents. Such modifications or substitutions will not cause the essence of the corresponding technical means to deviate from the spirit and scope of the technical means in each embodiment of the present invention.

Claims

1. Aminolipids having a structure represented by general formula (I), or its isomers, pharmaceutically acceptable salts, prodrugs, or solvates: In the formula, G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', -NR'R'', or a cyclic alkyl structure containing at least one heteroatom, wherein the substituted carbon atoms or heteroatoms of the cyclic alkyl structure are unsubstituted or one or more hydroxyl groups, C １ -C ４ alkyl group, C ２ -C ４ Alkenyl group, C ３ -C ８ Cycloalkyl groups, or C ３ -C ８ Substituted with a cycloalkenyl group, M １ 、 M ２ 、 M ３ 、 and M ４ are the same as or different from each other and are each independently selected from a C １ -C ２４ alkylene group, a C ３ -C ２４ cycloalkylene group, a C ２ -C ２４ alkenylene group, or a C ３ -C ２４ cycloalkenylene group, R １ and R ２ H and C are either identical or different from each other, and are independent of each other. １ -C ２４ alkyl group, C ３ -C ２４ Cycloalkyl groups, C ２ -C ２４ Alkenyl group, or C ３ -C ２４ Selected from cycloalkenyl groups, L １ , L ２ , L ３ , and L ４ These are either identical or different from each other, and independently -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O) ２ -, -S (=O) ２ Selected from O-, -O-, -S-, or -S-S-, R, R', and R'' are either identical or different from each other, and each is independently H, C １ -C １０ alkyl group, C ３ -C １０ Cycloalkyl groups, C ３ -C １０ Alkenyl group, C ３ -C １０ A cycloalkenyl group, with a tertiary amine bonded to the terminal end. １ -C １０ C with alkyl group and tertiary amine bonded to the terminal. ３ -C １０ Cycloalkyl groups, C with a tertiary amine bonded to the terminal. ３ -C １０ Selected from an alkenyl group or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or has one or more C １ -C ４ alkyl group, C ２ -C ４ Alkenyl group, C ３ -C ８ Cycloalkyl groups, C ３ -C ８ Substituted with a cycloalkenyl group, M ５ Each is independently a single bond, C １ -C １６ Alkylene group, C ２ -C １６ Alkenylene group, C ３ -C ８ Cycloalkylene group, or C ３ -C ８ Selected from cycloalkenylene groups.

2. G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', -NR'R'', or a cyclic alkyl structure containing at least one heteroatom, and the replaceable carbon atom or heteroatom of the cyclic alkyl structure is unsubstituted or has one or more C １ -C ４ alkyl group, C ２ -C ４ alkenyl group, C ３ -C ８ cycloalkyl group, or C ３ -C ８ substituted with a cycloalkenyl group, and R, R', and R'' are the same as or different from each other and are each independently H, C １ -C １０ alkyl group, C ３ -C １０ cycloalkyl group, C ３ -C １０ alkenyl group, C ３ -C １０ cycloalkenyl group, or selected from a cyclic alkyl containing at least one heteroatom, and the cyclic alkyl is unsubstituted or has one or more C １ -C ４ alkyl group, C ２ -C ４ alkenyl group, C ３ -C ８ cycloalkyl group, C ３ -C ８ substituted with a cycloalkenyl group, and the amino lipid according to claim 1, characterized in that.

3. In general formula (I), G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R'', -NR'C(=O)R'', -NR'R'', or a cyclic alkyl structure containing at least one heteroatom, the heteroatom is O or N, and the cyclic alkyl is unsubstituted or is substituted with one or more C １ -C ４ alkyl groups, C ３ -C ８ cycloalkyl groups, or hydroxyl groups, and R, R', and R'' are the same as or different from each other and are each independently H, C １ -C ８ alkyl groups, C ３ -C ８ cycloalkyl groups, C ３ -C ８ alkenyl groups, C ３ -C ８ cycloalkenyl groups, C with a tertiary amine bonded to the end １ -C １０ alkyl groups, C with a tertiary amine bonded to the end ３ -C １０ cycloalkyl groups, or are selected from cyclic alkyl containing at least one heteroatom, and the cyclic alkyl is unsubstituted or is substituted with one or more C １ -C ４ alkyl groups, C ３ -C ８ cycloalkyl groups, and is characterized in that it is the amino lipid according to claim 1 or 2.

4. In general formula (I), G is selected from H, OH, or NR'R'', and R' and R'' are either the same or different from each other, and each is independently H or C １ -C ４ Selected from alkyl groups, Alternatively, the aminolipid according to claim 3, characterized in that G is selected from a substituted or unsubstituted oxa 5-membered cycloalkyl group, aza 5-membered cycloalkyl group, aza 6-membered cycloalkyl group, diaza 6-membered cycloalkyl group, or azaoxa 6-membered cycloalkyl group.

5. In general formula (I), if G is a substituted diaza 6-membered cycloalkyl group, the position of the substituent is M. ５ The aminolipid according to claim 4, characterized in that it is a nitrogen atom that is not bonded to a nucleotide.

6. In general formula (I), M ５ C is a single bond. ２ -C １６ Alkylene group, C ２ -C １６ Alkenylene group, C ４ -C ８ Cycloalkylene group, or C ３ -C ８ The aminolipid according to claim 3, characterized in that it is selected from cycloalkenylene groups.

7. In general formula (I), M ５ C is a single bond. ２ -C １６ Alkylene group, or C ４ -C ６ The aminolipid according to claim 6, characterized in that it is selected from cycloalkylene groups.

8. In general formula (I), M ５ G was formed by the combination of these two. The aminolipid according to claim 7, characterized in that is one selected from A1 to A38:

9. In general formula (I), M ５ G was formed by the combination of these two. The aminolipid according to claim 7, characterized in that is one selected from A39 to A52:

10. The aforementioned The aminolipid according to claim 8, characterized in that is one selected from A1 to A18, A22 to A24, and A28 to A38.

11. The aforementioned The aminolipid according to claim 10, characterized in that is one selected from A15, A16, A17, A23, A29, A30, A33, A37, A38, and A42.

12. In general formula (I), L １ , L ２ , L ３ , and L ４ These are either identical or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, or -S-S-. Preferably, L １ and L ４ The aminolipid according to any one of claims 1 to 11, characterized in that the two are identical and are -C(=O)O- or -OC(=O)-.

13. L １ , L ２ , L ３ , and L ４ If R is independently selected from -C(=O)NR- or -NRC(=O)-, then R is independently selected from H or C １ -C １０ The aminolipid according to claim 12, characterized in that it is selected from alkyl groups.

14. In general formula (I), M １ M ２ M ３ , and M ４ They are either identical or different from each other, M １ and M ４ Each of these C has an independently branched chain. ４ -C ２２ C with alkylene group and branched chain ４ -C ２２ Cycloalkylene group, branched chain C ４ -C ２２ C having an alkenylene group or a branched chain ４ -C ２２ Selected from cycloalkenylene groups, M ２ and M ３ Each of them is independently C ４ -C ２２ Alkylene group, C ４ -C ２２ Cycloalkylene group, C ４ -C ２２ Alkenylene group, or C ４ -C ２２ The aminolipid according to any one of claims 1 to 13, characterized in that it is selected from cycloalkenylene groups.

15. In general formula (I), M ２ and M ３ They are identical, C ４ -C ２２ It is an alkylene group, and / or, M １ and M ４ They are identical and have a branched chain C ４ -C ２２ The aminolipid according to claim 14, characterized by being an alkylene group.

16. In general formula (I), R １ and R ２ These are either identical or different from each other, and each is independently C ４ -C ２２ Alkyl alkyl group or C ４ -C ２２ The aminolipid according to any one of claims 2 to 15, characterized in that it is selected from alkenyl groups.

17. In general formula (I), R １ -L １ -M １ -L ２ -M ２ - Fragment is R １ -C(=O)O-M １ -OC(=O)-M ２ - and R ２ -L ４ -M ４ -L ３ -M ３ - Fragment is R ２ -C(=O)O-M ４ The aminolipid according to claim 16, characterized in that it is -OC(=O)-M3-.

18. The aminolipid according to claim 17, characterized in that the structure of general formula (I) is one selected from the following structures:

19. A method for producing amino lipids according to any one of claims 1 to 18, S1: The epoxy compound and the carboxylic acid are subjected to a ring-opening reaction to form intermediate 1R. １ -L １ -M １ - Steps for manufacturing OH, S2: Intermediate 1 and the carboxylic acid compound starting material are condensed in the presence of a condensing agent to produce intermediate 2R １ -L １ -M １ -L ２ -M ２ - A step to produce a leaving group, S3: The step of producing the target product by performing a substitution reaction between intermediate 2 and an amino compound starting material one or more times, Alternatively, S1': After protecting the diol with TBS, it is oxidized, and the oxidation product is added to the Grignard reagent to obtain the intermediate 1'HO-M １ - Steps for manufacturing OTBS, S2': Intermediate 1' and a carboxylic acid compound starting material are condensed in the presence of a condensing agent to produce intermediate 2'TBSO-M １ -L ２ -M ２ - A step to produce a leaving group, S3': Intermediate 2'TBSO-M １ -L ２ -M ２ - The leaving group is deprotected using TBS, and the mixture is reacted with a carboxylic acid to form intermediate 2R. １ -L １ -M １ -L ２ -M ２ - A step of generating a leaving group, A method for producing a target product, comprising the step of S4': reacting intermediate 2 with an amino compound raw material one or more times by substitution.

20. Lipid nanoparticles characterized by containing an aminolipid according to any one of claims 1 to 18.

21. The lipid nanoparticles further comprise a steroid, a neutral lipid, and / or a polymer-bound lipid. The lipid nanoparticle according to claim 20, characterized in that the chemical formula of the polymer-bound lipid is P-Y-L, where P is a hydrophilic polymer portion, Y is an arbitrary linker, and L is a lipid portion.

22. The aforementioned steroid is cholesterol, and / or, the neutral lipid is a phospholipid, The lipid nanoparticle according to claim 21, characterized in that and / or the polymer-bound lipid is a polyethylene glycolated lipid.

23. The lipid nanoparticles according to claim 21, characterized in that the molar ratio of amino lipids, steroids, neutral lipids, and polymer-bound lipids in the lipid nanoparticles is 30-70:30-65:0-30:0.2-5.

24. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 20 to 23 and a pharmaceutically acceptable carrier.

25. A method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using an aminolipid according to any one of claims 1 to 18, or a lipid nanoparticle according to any one of claims 20 to 23, or a pharmaceutical composition according to claim 24.

26. The method according to claim 25, characterized in that the cancer includes lung cancer, stomach cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, brain cancer, lymphoma, hematological cancer, and prostate cancer.

27. A method for performing gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy using an aminolipid according to any one of claims 1 to 18, or a lipid nanoparticle according to any one of claims 20 to 23, or a pharmaceutical composition according to claim 24.

28. Use of an aminolipid according to any one of claims 1 to 18, or a lipid nanoparticle according to any one of claims 20 to 23, or a pharmaceutical composition according to claim 24, in the manufacture of a drug for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

29. The use according to claim 28, characterized in that the cancer includes lung cancer, stomach cancer, liver cancer, esophageal cancer, colorectal cancer, pancreatic cancer, brain cancer, lymphoma, hematological cancer, and prostate cancer.

30. Use of an aminolipid according to any one of claims 1 to 18, or a lipid nanoparticle according to any one of claims 20 to 23, or a pharmaceutical composition according to claim 24, in the manufacture of a drug for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or RNA interference therapy.

31. A method for delivering a drug to a target, characterized by comprising administering a drug formulated in lipid nanoparticles according to any one of claims 20 to 23 to the target.

32. The aminolipid according to any one of claims 1 to 18 for treating or preventing infectious diseases, cancer, genetic disorders, allergies, toxicity, and autoimmune diseases.

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