Lipid compound and use thereof in delivery of nucleic acid

Abstract

Provided are an ionizable lipid compound with an adjacent cis-double bond structure of Formula (I), a preparation method therefor, and the use thereof in the delivery of an active therapeutic agent (e.g., a nucleic acid). The ionizable lipid compound can provide a higher encapsulation rate of active substances and a better cell or in vivo transfection rate, and is particularly suitable for preparing nanoparticles with a solid structure.

Description

Lipid compounds and their applications in nucleic acid delivery

[0001] This application claims priority to a prior application entitled “Compounds and Their Use in Nucleic Acid Delivery” filed with the State Intellectual Property Office of China on May 28, 2021, with patent application number 202110592439.8. The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the technical field of pharmaceutical compounds, and particularly relates to an ionizable lipid compound, a preparation method thereof, and an application thereof in the delivery of active therapeutic agents (such as nucleic acids). Background Art

[0003] Nucleic acids include small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), ribozymes, plasmids, and immunostimulatory nucleic acids, and they exert their effects through a variety of mechanisms. Taking mRNA as an example, mRNA is a type of RNA that transports genetic information from DNA to the ribosomes of the cytoplasm for protein translation. It does not need to enter the cell nucleus, so it does not pose the risk of genetic mutations and can be used to achieve the expression of specific cellular products. These nucleic acids are useful in treating diseases associated with protein or enzyme defects. However, nucleic acids encounter many problems in therapeutic settings. For example, mRNA, with its single-stranded structure, is very unstable and is quickly degraded by nucleases after entering the body. In addition, mRNA has a large molecular weight and carries a large amount of negative charge, making it difficult for it to pass through the negatively charged cell membrane and enter the target cell. Therefore, how to effectively deliver mRNA to cells is a technical key to its in vivo application. Similar problems exist with various therapeutic nucleic acids.

[0004] In gene therapy, ionizable lipid compounds have been shown to be excellent delivery vehicles for nucleic acids used to treat various diseases. The amine groups of these ionizable lipid compounds can be protonated under suitable acidic conditions to form a positively charged head group. The tail of these ionizable lipid compounds is composed of a hydrophobic carbon chain. The charged moiety is used to electrostatically bind to negatively charged RNA, while the hydrophobic tail enables them to self-assemble into lipophilic particles. Lipid nanoparticles (LNPs) formed by self-assembly of ionizable lipid compounds with three or four other lipids, such as DSPC (distearoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine), cholesterol (CHOL), and PEGylated lipids (DMG-PEG2000 (1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol 2000) or DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000)), are used to deliver nucleic acids, protecting them from degradation by nucleases and promoting cellular uptake. At present, ionizable lipid compounds have made great progress in nucleic acid delivery, but there is still a problem of low delivery efficiency, which is also one of the bottlenecks restricting the development of the industry.

[0005] Summary of the Invention

[0006] The present invention provides a novel ionizable lipid compound that can be used to deliver bioactive molecules (e.g., mRNA, siRNA, micRNA, proteins, peptides, etc.). Given that the amine structure can be protonated to form a positively charged cationic group, the ionizable lipid compound of the present invention is particularly suitable for delivering negatively charged active substances, such as DNA, RNA, or other nucleotide molecules.

[0007] The present invention also provides a bioactive substance delivery system comprising the ionizable lipid compound, which can be a microparticle, nanoparticle, liposome, lipid nanoparticle or microvesicle. In one embodiment of the present invention, the delivery system is a lipid nanoparticle. Such lipid nanoparticles can efficiently deliver bioactive substances (such as mRNA) to cells, tissues or organs, thereby achieving efficient regulation of bioactive substances. In the present invention, the ionizable lipid compound is combined with the bioactive substance delivered to the target cell or individual or further comprises other substances (e.g., other anions, cations or ionizable lipid compounds, synthetic or natural polymers, surfactants, cholesterol, carbohydrates, proteins, phospholipids, etc.) to form microparticles, nanoparticles, liposomes, lipid nanoparticles or microvesicles. The bioactive substance can be in the form of gas, liquid or solid, and can be a polynucleotide, protein, peptide or small molecule. In the present invention, the delivery system can then be optionally combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0008] The present invention also provides a method for synthesizing these novel ionizable lipid compounds.

[0009] The present invention also provides the use of the novel ionizable lipid compound in preparing a bioactive substance delivery system. The delivery system can be a microparticle, a nanoparticle, a liposome, a lipid nanoparticle, or a microbubble. In one embodiment of the present invention, the delivery system is a lipid nanoparticle.

[0010] The ionizable lipid compound of the present invention has the structure shown in Formula I in:

[0011] Q is a substituted or unsubstituted straight-chain C2-20 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; or, Q is a substituted or unsubstituted, saturated or unsaturated 4-6 membered ring, wherein the ring atoms of the 4-6 membered ring optionally contain one or more heteroatoms independently selected from O, S and N; the substituted substituent is selected from halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5,

[0012] R1, R2, R3, and R4 may be the same or different and are independently selected from hydrogen, substituted or unsubstituted linear or branched C1-30 alkyl, substituted or unsubstituted linear or branched C2-30 alkenyl, substituted or unsubstituted linear or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl, or alkynyl group are optionally replaced by heteroatoms independently selected from O, S, and N, or -CH2CH(OH)R5; the substituted substituents are selected from halogen, -OH, linear or branched C1-10 alkyl, linear or branched C1-10 alkoxy;

[0013] The condition is that at least one of R1, R2, R3, and R4 is

[0014] R5 is selected from hydrogen, substituted or unsubstituted straight or branched C1-30 alkyl, substituted or unsubstituted straight or branched C2-30 alkenyl, substituted or unsubstituted straight or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl group are optionally replaced by heteroatoms independently selected from O, S and N; the substituted substituents are selected from halogen, -OH, straight or branched C1-10 alkyl, straight or branched C1-10 alkoxy;

[0015] R6 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH;

[0016] n is an integer selected from 1 to 8, m is an integer selected from 0 to 8, n and m are independent of each other and may be the same or different;

[0017] When at least two of R1, R2, R3, and R4 are When n and m in each of the groups are independent of each other, they may be the same or different.

[0018] In a preferred embodiment of the present invention, Q is a substituted or unsubstituted straight-chain C2-20 alkylene group, wherein one or more C atoms of the alkylene group are optionally replaced by heteroatoms independently selected from O, S and N;

[0019] Preferably, Q is wherein R8 and R9 are independently selected from substituted or unsubstituted straight-chain C1-10 alkylene, wherein one or more C atoms of the alkylene are optionally replaced by heteroatoms independently selected from O, S and N; R7 is hydrogen, halogen, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, or -CH2CH(OH)R5, or The substituted substituent group is halogen, -OH, linear or branched C1-10 alkyl, linear or branched C1-10 alkoxy;

[0020] Preferably, Q is wherein: x and y may be the same or different and independently selected from integers of 1 to 8; R7 is defined as above; preferably, x or y are the same or different and are selected from integers of 1 to 3, for example, 1, 2 or 3; preferably, R7 is a linear or branched C1-4 alkyl group, for example, methyl, ethyl, n-propyl, n-butyl, etc.

[0021] In some embodiments of the present invention, the saturated or unsaturated 4-6 membered ring is piperazinyl or cyclohexyl.

[0022] In a preferred embodiment of the present invention, R6 is -OH.

[0023] In a preferred embodiment of the present invention, n is selected from an integer of 4 to 8, and m is selected from an integer of 4 to 8.

[0024] In a preferred embodiment of the present invention, the compound of formula I is the following formula A, B, C or D:

[0025] wherein each n1 is independent of each other and may be the same or different, each n1 is selected from an integer of 1 to 8, each m1 is independent of each other and may be the same or different, each m1 is selected from an integer of 0 to 8; preferably, each n1 is selected from an integer of 4 to 8, each m1 is selected from an integer of 4 to 8; preferably, each n1 is the same as each other, and each m1 is the same as each other.

[0026] Wherein each n2 is independent of each other and may be the same or different, each n2 is selected from an integer of 1 to 8, each m2 is independent of each other and may be the same or different, each m2 is selected from an integer of 0 to 8; preferably, each n2 is selected from an integer of 4 to 8, each m2 is selected from an integer of 4 to 8; preferably, each n2 is the same as each other, and each m2 is the same as each other.

[0027] Wherein each n3 is independent of each other and may be the same or different, each n3 is selected from an integer of 1 to 8, each m3 is independent of each other and may be the same or different, each m3 is selected from an integer of 0 to 8; preferably, each n3 is selected from an integer of 4 to 8, each m3 is selected from an integer of 4 to 8; preferably, each n3 is the same as each other, and each m3 is the same as each other.

[0028] wherein each n4 is independent of each other and may be the same or different, each n4 is selected from an integer of 1 to 8, each m4 is independent of each other and may be the same or different, each m4 is selected from an integer of 0 to 8; preferably, each n4 is selected from an integer of 4 to 8, each m4 is selected from an integer of 4 to 8; preferably, each n4 is the same as each other, and each m4 is the same as each other.

[0029] In some embodiments of the present invention, the compound of formula I is selected from the following compounds shown in Table 1:

[0030] Table 1

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] The ionizable lipid compounds of the present invention can be synthesized using methods known in the art, for example, by reacting one or more equivalents of an amine with one or more equivalents of an epoxy-terminated compound under suitable conditions. The synthesis of the ionizable lipid compounds can be carried out with or without a solvent, and the synthesis can be carried out at elevated temperatures ranging from 25°C to 100°C. The resulting ionizable lipid compound can optionally be purified. For example, a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound. Alternatively, the mixture can be purified to obtain a specific stereoisomer or regioisomer. The epoxides can be purchased commercially or prepared synthetically.

[0043] In certain embodiments, all amino groups of the amine react completely with the epoxy-terminated compound to form tertiary amines. In other embodiments, not all amino groups of the amine react completely with the epoxy-terminated compound, thereby generating primary or secondary amines in the ionizable lipid compound. These primary or secondary amines may remain as is or may be reacted with another electrophilic reagent, such as a different epoxy-terminated compound. As will be appreciated by those skilled in the art, reacting an excess of amine with an epoxy-terminated compound will yield a variety of different ionizable lipid compounds with varying numbers of tails. For example, a diamine or polyamine may include one, two, three, or four epoxy-derived compound tails on various amino moieties of the molecule, thereby generating primary, secondary, and tertiary amines. In certain embodiments, two epoxy-terminated compounds of the same type are used. In other embodiments, two or more different epoxy-terminated compounds are used.

[0044] In some embodiments of the present invention, the ionizable lipid compound of the present invention can be prepared using the following general preparation method.

[0045]

[0046] Step 1: Restore

[0047] In the presence of a reducing agent, the carboxyl group of compound A1 is reduced to a hydroxyl group to obtain compound A2. Examples of reducing agents include, but are not limited to, lithium aluminum hydride, diisobutylaluminum hydride, and the like. Examples of solvents used in the reaction include, but are not limited to, ethers (such as diethyl ether, tetrahydrofuran, and dioxane), halogenated hydrocarbons (such as chloroform, dichloromethane, and dichloroethane), hydrocarbons (such as n-pentane, n-hexane, benzene, and toluene), and mixed solvents of two or more of these solvents.

[0048] Step 2: Oxidation

[0049] In the presence of an oxidizing agent, the hydroxyl group of compound A2 is oxidized to an aldehyde group to obtain compound A3. Examples of oxidizing agents include, but are not limited to, 2-iodoxybenzoic acid (IBX), pyridinium chlorochromate (PCC), pyridinium dichlorochromate (PDC), Dess-Martin periodinane, manganese dioxide, and the like. Examples of solvents used in the reaction include, but are not limited to, halogenated hydrocarbons (such as chloroform, dichloromethane, and dichloroethane), hydrocarbons (such as n-pentane, n-hexane, benzene, and toluene), nitriles (such as acetonitrile), and mixed solvents of two or more of these solvents.

[0050] Step 3: Halogenation-reduction

[0051] First, under acidic conditions, the aldehyde α-hydrogen of compound A3 is halogenated with a halogenating agent to obtain an α-halogenated aldehyde intermediate, and then in the presence of a reducing agent, the aldehyde group of the α-halogenated aldehyde is reduced to a hydroxyl group to obtain compound A4. Examples of providing acidic conditions include, but are not limited to, DL-proline. Examples of halogenating agents include, but are not limited to, N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS). Examples of reducing agents include, but are not limited to, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0052] Step 4: Epoxidation

[0053] Compound A4 is subjected to an intramolecular nucleophilic substitution reaction in the presence of a base to obtain epoxy compound A5. Examples of bases include, but are not limited to, alkali metal hydroxides or hydrides, such as sodium hydroxide, potassium hydroxide, and sodium hydride. Examples of solvents used in the reaction include, but are not limited to, a mixture of dioxane and water.

[0054] Step 5: Ring-opening reaction

[0055] Compound A5 is subjected to a ring-opening reaction with an amine (e.g., N,N-bis(2-aminoethyl)methylamine) to obtain the final compound. Examples of reaction solvents include, but are not limited to, ethanol, methanol, isopropanol, tetrahydrofuran, chloroform, hexane, toluene, ether, and the like.

[0056] The raw material A1 in the preparation method can be purchased commercially or synthesized using conventional methods.

[0057] The ionizable lipid molecules of the present invention contain two adjacent cis double bonds, which enable them to have high encapsulation efficiency and good cell transfection efficiency when subsequently used in delivery systems to encapsulate active substances (e.g., nucleic acids such as mRNA). In addition, when preparing lipid nanoparticles, the resulting lipid nanoparticles have a more uniform particle size. The ionizable lipid compounds of the present invention are particularly suitable for preparing solid nanoparticles.

[0058] The ionizable lipid compounds of the present invention, when used in drug delivery systems, can encapsulate pharmaceutical agents, including polynucleotides, small molecules, proteins, peptides, metals, etc. The ionizable lipid compounds have several properties suitable for preparing drug delivery systems: 1) the ability to lipid-complex and "protect" unstable pharmaceutical agents; 2) the ability to buffer pH in vivo; 3) the ability to act as a "proton sponge" and induce dissolution in vivo; and 4) the ability to neutralize the charge on negatively charged active substances.

[0059] The drug delivery system can be in the form of particles. In certain embodiments, the particle diameter is in the range of 1 μm to 1000 μm. In certain embodiments, the particle diameter is in the range of 1 nm to 1000 nm. For example, the particle diameter is in the range of 1 μm to 100 μm, or in the range of 1 μm to 10 μm, or in the range of 10 μm to 100 μm, or in the range of 20 nm to 800 nm, or in the range of 50 nm to 500 nm, or in the range of 80 nm to 200 nm, or in the range of 1 nm to 100 nm, or in the range of 1 nm to 100 nm. When the particle size range is in the range of 1 nm to 1000 nm, it is nanoparticles generally described in the art. The particles can be prepared using any method known in the art. These methods include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex coagulation, and other methods well known to those of ordinary skill in the art.

[0060] The drug delivery system may also be microbubbles, liposomes or lipid nanoparticles, which are well suited for delivering pharmaceutical agents.

[0061] The active substance that can be delivered by the delivery system formed by the ionizable lipid compound of the present invention can be a therapeutic agent, a diagnostic agent or a prophylactic agent. The nature of the active substance can be a small molecule compound, a nucleic acid, a protein, a peptide, a metal, an isotope-labeled compound, a vaccine, etc.

[0062] The delivery system formed by the ionizable lipid compound of the present invention can also be modified with a targeting molecule to make it a targeting agent that can target specific cells, tissues or organs. The targeting molecule can be included in the entire delivery system or can be located only on its surface. The targeting molecule can be a protein, peptide, glycoprotein, lipid, small molecule, nucleic acid, etc., examples of which include (but are not limited to) antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialoglycoprotein (asialycoprotein), receptor ligands, sialic acid, aptamers, etc.

[0063] The delivery system formed by the ionizable lipid compound of the present invention can be combined with one or more pharmaceutical excipients to form a pharmaceutical composition suitable for administration to animals (including humans). The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, etc., including but not limited to sugars such as lactose, trehalose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; gelatin; talc; oils such as peanut oil, cottonseed oil, safflower oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; surfactants such as Tween 80; buffers such as phosphate buffer solution, acetate buffer and citrate buffer; colorants, sweeteners, flavorings and fragrances, preservatives and antioxidants, etc.

[0064] The pharmaceutical composition of the present invention can be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, vaginally, intranasally, intraperitoneally, buccally, or in the form of an oral or nasal spray.

[0065] As used herein, the term "alkyl" refers to a saturated hydrocarbon radical derived from a hydrocarbon moiety containing 1 to 30 carbon atoms by removing a single hydrogen atom. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and n-dodecyl.

[0066] The term "alkenyl" refers to a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by removing a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0067] The term "alkynyl" refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by removing a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0068] The term "alkoxy" refers to an alkyl group as defined above attached to the parent molecular group through an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.

[0069] The terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0070] The term "saturated or unsaturated 4-6 membered ring" refers to a ring having 4-6 ring atoms, which may be C, N, S, or O. Examples include, but are not limited to, 4-6 membered saturated cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; 4-6 membered aryl groups, such as phenyl; 4-6 membered heterocyclic groups, such as pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl; and 4-6 membered heteroaryl groups, such as triazolyl, oxazolyl, isoxazolyl, and thiazolyl. In some embodiments of the present invention, the saturated or unsaturated 4-6 membered ring is preferably piperazinyl or cyclohexyl.

[0071] The terms "substituted" (whether or not preceded by the term "optionally") and "substituent" refer to the ability to change one functional group to another, provided that the valence of all atoms is maintained. When more than one position in any particular structure can be substituted with more than one substituent selected from a specified group, the substituent may be the same or different at each position. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 pKa curve of ionizable lipid II-37

[0073] Figure 2 Cell transfection efficiency of mRNA-encapsulated LNPs formed by ionizable lipid II-37

[0074] Figure 3 Cell transfection efficiency of pDNA-encapsulated LNPs formed by ionizable lipid II-37

[0075] Figure 4 Cell transfection efficiency of siRNA-encapsulated LNPs formed by ionizable lipid II-37

[0076] Figure 5 Comparison of cell transfection efficiency of mRNA-encapsulated LNPs formed by ionizable lipid II-37 and commercial molecule MC3

[0077] Figure 6: Cy5-labeled loaded mRNA, ionizable lipid II-37 and commercial molecule MC3 were used to form LNPs encapsulating mRNA, and the efficiency of LNP delivery of mRNA into cells was observed by fluorescence staining.

[0078] Figure 7 Comparison of cell transfection efficiency of mRNA-encapsulated LNPs formed by ionizable lipids II-37 and C14-113

[0079] Figure 8 Cytotoxicity of II-37-LNP and C14-113-LNP determined by MTT assay DETAILED DESCRIPTION

[0080] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0081] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0082] Example 1 Synthesis of ionizable lipid II-37

[0083]

[0084] Synthesis of Linoleic Alcohol (a2): LiAlH₄ (7.20 g) and linoleic acid (50 g, a1) were added to 950 mL of tetrahydrofuran at 0°C, and the mixture was stirred at 25°C for 2 h. After completion of the reaction as determined by thin-layer chromatography (TLC), the reaction mixture was quenched by the addition of water (7.2 mL), aqueous NaOH (7.2 mL, 15% by mass), and water (21.6 mL). An appropriate amount of Na₂SO₄ was added, and the mixture was stirred for 15 minutes. After filtration through a Buchner funnel, the filter cake was washed with ethyl acetate. The filtrate was collected and concentrated by evaporation to yield 47.4 g of the desired product, linoleic alcohol (a2).

[0085] 1 H NMR (400MHz, CDCl3): δ5.27-5.44(m,4H),3.63(t,J=6.63Hz,2H),2.77(t,J=6.44H z,2H),1.97-2.12(m,4H),1.57-1.63(m,1H),1.20-1.46(m,18H),0.83-0.95(m,3H)

[0086] Synthesis of (9Z,12Z)-octadeca-9,12-dienal (a3): Linolenic alcohol (25.0 g, a2) and 2-iodoacylbenzoic acid (39.4 g) were added to 170 mL of acetonitrile at room temperature, and the mixture was stirred at 85°C for 4 h. The reaction mixture was filtered through a Buchner funnel, and the filter cake was washed with dichloromethane. The filtrate was collected and concentrated by evaporation to obtain 24.0 g of the desired product, (9Z,12Z)-octadeca-9,12-dienal (a3).

[0087] 1H NMR (400MHz, CDCl3): δ9.76(t,J=1.76Hz,1H),5.25-5.43(m,4H),2.76(t,J=6.17Hz,2H),2.41(td,J=7 .33,1.87Hz,2H),2.04(q,J=6.84Hz,4H),1.56-1.68(m,2H),1.22-1.36(m,14H),0.88(t,J=6.73Hz,3H)

[0088] Synthesis of (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4): To 246 mL of acetonitrile was added (9Z,12Z)-octadeca-9,12-dienal (43.0 g, a3), DL-proline (5.62 g), and N-chlorosuccinimide at 0°C, followed by stirring at 0°C for 2 h. After completion, the reaction mixture was diluted with anhydrous ethanol (246 mL), sodium borohydride (8.8 g) was added, and the mixture was stirred at 0°C for 4 h. The reaction mixture was quenched with water (120 mL) and extracted with methyl tert-butyl ether. The combined organic phases were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation to afford the desired product, (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (a4, 46 g), which was used directly in the next step.

[0089] 1 H NMR (400MHz, CDCl3): δ5.25-5.51(m,4H),3.97-4.07(m,1H),3.79(dd,J=12.01,3.63Hz,1H),3.59- 3.70(m,1H),2.67-2.90(m,2H),1.96-2.15(m,5H),1.64-1.82(m,1H),1.20-1.49(m,15H),0.89(br t,J=6.75Hz,3H)

[0090] Synthesis of 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (a5): To 450 mL of 1,4-dioxane were added (9Z,12Z)-2-chloro-octadeca-9,12-dien-1-ol (45 g, a4) and a sodium hydroxide solution (120 g of sodium hydroxide dissolved in 585 mL of water) at room temperature. After the addition was complete, the mixture was stirred at 35°C for 2 h. After TLC indicated the reaction was complete, the reaction solution was separated using a separatory funnel, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated by evaporation. The residue was then purified by flash column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 29.11 g of the desired product, 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (a5).

[0091] 1 H NMR (400MHz, CDCl3): δ5.27-5.46(m,4H),2.87-2.98(m,1H),2.70-2.85(m,3H),2.4 6(dd,J=5.00,2.75Hz,1H),1.94-2.21(m,4H),1.24-1.58(m,17H),0.78-1.00(m,3H)

[0092] Synthesis of II-37: 2-[(7Z,10Z)-hexadecane-7,10-diene]oxirane (5 g) and N,N-bis(2-aminoethyl)methylamine (739 mg) were added to 10 mL of ethanol at room temperature, and the mixture was stirred at 90°C for 36 h. The reaction solution was concentrated by evaporation, and the residue was purified by flash column chromatography with dichloromethane / methanol to obtain crude product II-37 (4 g). The target product was further purified by flash column chromatography with dichloromethane / methanol to obtain II-37 (2.2 g).

[0093] 1 H NMR (400MHz, CDCl3): δ5.27-5.44(m,12H),3.48-3.79(m,3H),2.63-3.00(m,12H),2. 16-2.61(m,12H),2.05(q,J=6.80Hz,12H),1.18-1.57(m,51H),0.89(t,J=6.88Hz,9H)

[0094] ESI-MS: m / z 910.8[M+H] + ,911.8[M+2H] + ,912.8[M+3H] +

[0095] Example 2 Dissociation Constant (pKa) of Ionizable Lipid II-37

[0096] Ionizable lipids have two primary functions: binding nucleic acids and allowing for their release within cells. The lipid's pKa is a crucial factor, as it must be positively charged at low pH to bind nucleic acids but uncharged at neutral pH to prevent toxicity in the resulting LNPs. The pKa of ionizable lipid II-37 was determined to be 6.81 by a TNS dye-binding assay, as shown in Figure 1.

[0097] Example 3 Preparation of lipid nanoparticles by encapsulating mRNA in II-37

[0098] An ethanol solution of ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 at a molar ratio of 35%:15%:48.5%:1.5% was prepared as the organic phase. Lucferase mRNA (LucRNA) was dissolved in a pH 4 aqueous solution as the aqueous phase. Nanoparticle suspensions were prepared using microfluidics on a nanomedicine fabricator (PNI, Canada, Ignite model) at a volume ratio of 3:1 between the aqueous and organic phases. The final LucRNA-LNP lipid nanoparticles were concentrated by ultrafiltration and stored at 2-8°C until ready for use.

[0099] The particle size and zeta potential of LucRNA-LNP were characterized using a Zetasizer Pro nanoparticle size analyzer (Malvern Panalytical). The encapsulation efficiency of LucRNA-LNP was determined using the Ribogreen method using an F-280 fluorescence spectrophotometer (Tianjin Gangdong). The transfection efficiency of the prepared LucRNA-LNP CHO cells was determined using a fluorescein reporter gene assay using a multifunctional microplate reader (BioTek, model SLXFATS). The in vitro transcription of LucRNA was performed as follows: CHO-K1 cells were plated at a cell density of 2.5 × 10 5 Cells were transfected at a confluency of 30%-50% at 40% cells / mL. Each well was transfected with 2 μg of LucRNA. A positive control was transfected using the transfection reagent Lipofectamine MessagerMAX (ThermoFisher Scientific) according to the manufacturer's instructions. Protein expression was measured using a multi-function microplate reader 48 hours after transfection. A negative control consisted of cell culture medium without LucRNA-LNP. The test results for Example 3 are shown in Table 2.

[0100] Table 2

[0101] Particle size (nm) PDI Zeta potential (mV) Encapsulation efficiency (%) RLU (2μg / mL) LucRNA-LNP 108.66 0.13 17.87 96.4% 1088 112

[0102] The results of Example 3 show that the lipid nanoparticles LucRNA-LNPs prepared using the novel lipid compound have a particle size of approximately 108 nm, a narrow particle size distribution (small PDI), and an encapsulation efficiency of up to 96%. The in vitro cell transfection efficiency reached 1 million. This demonstrates that LNPs encapsulated with mRNA prepared using the ionizable lipid II-37 not only exhibit excellent physicochemical parameters but also exhibit extremely high cell transfection efficiency.

[0103] Furthermore, the same LNP was used to detect the transfection efficiency of the prepared LucRNA-LNP HEK293T cells using a multifunctional microplate reader (BioTek, model SLXFATS) using a fluorescein reporter gene assay. The transfected LucRNA amounts were 0.5 μg, 1.0 μg, and 2.0 μg, respectively. The in vitro transcription of LucRNA was performed as follows: HEK293T cells were plated at a cell density of 2.5 × 10 5 cells / mL, and transfection was performed when the cell confluency was 30%-50%. For the positive control, 0.5 μg of LucRNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was performed according to the transfection reagent product instructions. Protein expression was detected using a multifunctional microplate reader 48 hours after transfection. The negative control was cell culture medium without the addition of LucRNA-LNP. The in vitro cell transfection efficiency is shown in Figure 2, indicating that the LNP-encapsulated mRNA prepared by the ionizable lipid II-37 has an extremely high cell transfection efficiency, which is about 10 times higher than that of commercial Lipofectamine 2000 when transfecting the same amount of mRNA.

[0104] Example 4 II-37 Preparation of Lipid Nanoparticles by Encapsulating DNA

[0105] An ethanol solution of ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 at a molar ratio of 45%:10%:43.5%:1.5% was prepared as the organic phase. Lucferase DNA (pDNA) was dissolved in a pH 4 aqueous solution as the aqueous phase. Nanoparticle suspensions were prepared using microfluidics on a nanomedicine fabricator (PNI, Canada, Ignite model) at a volume ratio of 3:1 between the aqueous and organic phases. The final pDNA-LNP lipid nanoparticles were concentrated by ultrafiltration and stored at 2-8°C until ready for use.

[0106] The pDNA-LNP particle size and zeta potential were characterized using a Zetasizer Pro nanoparticle size analyzer (Malvern Panalytical). The test results of Example 4 are shown in Table 3. The lipid nanoparticles pDNA-LNP prepared by combining the novel lipid compound have a particle size of approximately 173 nm and a narrow particle size distribution (PDI).

[0107] Table 3

[0108] Particle size (nm) PDI Zeta potential (mV) pDNA-LNP 173.20.21324.1

[0109] The transfection efficiency of the prepared pDNA-LNP293T cells was detected by the fluorescein reporter gene method using a multifunctional microplate reader (BioTek, model SLXFATS). The transfected pDNA amounts were 0.5 μg, 1.0 μg, and 2.0 μg, respectively. The in vitro transcription method was as follows: 293T cells were plated at a cell density of 2.0 × 10 5 cells / mL, and transfection was performed when the cell confluency was 30%-50%. For the positive control, 2 μg of pDNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was performed according to the transfection reagent product instructions. Protein expression was detected using a multifunctional microplate reader 48 hours after transfection. The negative control was cell culture medium without the addition of pDNA-LNP. The in vitro cell transfection efficiency is shown in Figure 3, indicating that the LNP-encapsulated DNA prepared by the ionizable lipid II-37 has an extremely high cell transfection efficiency: the protein expression level of 1.0 μg of pDNA transfected with LNP prepared by II-37 was higher than that of 2 μg of pDNA transfected with Lipofectamine 2000; when the same 2 μg of pDNA was transfected, the in vitro cell transfection efficiency of II-37 was approximately 3 times higher than that of commercial Lipofectamine 2000.

[0110] Example 5 Preparation of lipid nanoparticles by encapsulating siRNA in II-37

[0111] An ethanol solution of ionizable lipid II-37, DSPC, CHOL, and DMG-PEG2000 at a molar ratio of 45%:15%:38.5%:1.5% was prepared as the organic phase. Lucferase siRNA (siRNA) was dissolved in a pH 4 aqueous solution as the aqueous phase. Nanoparticle suspensions were prepared using microfluidics on a nanomedicine fabricator (PNI, Canada, Ignite model) at a volume ratio of 3:1 between the aqueous and organic phases. The final siRNA-LNP lipid nanoparticles were concentrated by ultrafiltration and stored at 2-8°C until ready for use.

[0112] The particle size and zeta potential of siRNA-LNPs were characterized using a Zetasizer Pro nanoparticle size analyzer (Malvern Panalytical). The test results of Example 5 are shown in Table 4. The particle size of the lipid nanoparticles siRNA-LNPs prepared by combining the novel lipid compound was approximately 294 nm.

[0113] Table 4

[0114] Diameter (nm) PDI Zeta potential (mV) siRNA-LNP 294.00.31820.3

[0115] The transfection efficiency of the prepared siRNA-LNP 293T cells was detected by luciferase reporter gene assay using a multifunctional microplate reader (BioTek, model SLXFATS). The transfected siRNA amounts were 0.5 μg, 1.0 μg, and 2.0 μg, respectively. The in vitro transcription method was as follows: 293T cells stably transfected with Lucferase reporter were plated at a cell density of 2.0 × 10 5 cells / mL, and transfection was performed when the cell confluency was 30%-50%. For the positive control, 1.0 μg of siRNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection procedure was performed according to the transfection reagent product instructions. Protein expression was detected using a multifunctional microplate reader 24 hours after transfection. The negative control was cell culture medium without the addition of siRNA-LNP. The in vitro cell transfection efficiency is shown in Figure 4, indicating that the LNP-encapsulated siRNA prepared by the ionizable lipid II-37 has extremely high protein knockdown efficiency.

[0116] Example 6 Comparison of the effects of II-37 and commercial ionizable cationic lipid molecule MC3

[0117] The molecular formula of MC3 is: 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl ester.

[0118] According to the method described in Example 3, lipid nanoparticles were prepared using II37 and MC3, respectively, with the specific molar ratios being: II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; MC3:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; and the N / P ratio being 5:1.

[0119] The physical and chemical quality control data of the prepared lipid nanoparticles are shown in the following table:

[0120] Sample information Particle size (nm) PDI Zeta potential Encapsulation efficiency mRNA-LNP (II-37) 154.58 0.1068 22.079 0.5 mRNA-LNP (MC3) 234.08 0.1259 2.444 0.7

[0121] As can be seen from the above table, the encapsulation efficiency of lipid nanoparticles prepared by II-37 is as high as 90.5%, which is much higher than that of MC3 lipid nanoparticles, and the particle size is smaller and more uniform, and the potential is higher.

[0122] The same transfection method as in Example 3 was used to transfect CHO-K1 cells with the prepared lipid nanoparticles to understand the protein expression. The results are shown in Figure 5. When the amount of transfected mRNA was the same, the protein expression level in the cells after the lipid nanoparticles prepared by II-37 (represented by C2 in the figure) carrying mRNA were transfected into the cells was much higher than that of MC3, indicating that the cell transfection efficiency of the lipid nanoparticles prepared by II-37 was very high.

[0123] In addition, loaded mRNA was labeled with Cy5 to produce Cy5-mRNA-LNP (II-37) and Cy5-mRNA-LNP (MC3). After incubation with 293T cells for 2 and 6 hours, lysosomes were stained with LysoSensor™ Green to observe the cellular entry of Cy5-mRNA. As shown in Figure 6, after 6 hours of incubation with cells for Cy5-mRNA-LNP (II-37), the majority of Cy5-mRNA reached lysosomes, with a colocalization coefficient of 0.626. However, after 6 hours of incubation with cells for Cy5-mRNA-LNP (MC3), less Cy5-mRNA entered the lysosomes. This comparison shows that the nucleic acid delivery efficiency of II-37 is superior to that of MC3.

[0124] From the results of Example 6, it can be seen that the lipid nanoparticles prepared by combining the novel lipid compounds are superior to the MC3 molecules in nucleic acid delivery efficiency and in vitro cell transfection efficiency.

[0125] Example 7 Comparison of II-37 and its structural analog C14-113

[0126] The structural formula of C14-113 is:

[0127]

[0128] According to the method described in Example 3, lipid nanoparticles were prepared using II37 and C14-113, respectively, with the specific molar ratios being: II-37:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; C14-113:DSPC:CHOL:DMG-PEG2000=45:15:38.5:1.5; and the N / P ratio being 10:1.

[0129] The physical and chemical quality control data of the prepared lipid nanoparticles are shown in the following table:

[0130] Sample information Particle size (nm) PDI Zeta potential mRNA-LNP (II-37-LNP) 136.68 0.142 0.07 mRNA-LNP (C14-113-LNP) 152.65 0.122 4.1

[0131] The same transfection method as in Example 3 was used to transfect the prepared lipid nanoparticles into 293T cells to understand the protein expression. The results are shown in Figure 7. When the amount of transfected mRNA was the same, the protein expression level in the cells after the lipid nanoparticles prepared by II-37 (represented by II-37-LNP in the figure) carrying mRNA were transfected into the cells was much higher than that of C14-113, indicating that the cell transfection efficiency of lipid nanoparticles made of II-37 is very high.

[0132] In addition, the cytotoxicity of II-37-LNP and C14-113-LNP was determined using the MTT assay to examine the effects of factors such as carrier dose and exposure time on the proliferation of normal 293T cells. As shown in Figure 8, lipid nanoparticles prepared with II-37 (represented in the figure as II-37-LNP) carrying mRNA maintained good cell viability even at a higher dose (2 μg / mL) 48 hours after transfection of cells, indicating that lipid nanoparticles prepared with II-37 have very low cytotoxicity.

[0133] From the results of Example 7, it can be seen that the lipid nanoparticles prepared by combining the novel lipid compound have low cytotoxicity and are more efficient in mRNA transfection than the structural analog molecule C14-113.

[0134] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A structure having the structure shown in Formula I The compounds, wherein: Q is a substituted or unsubstituted straight-chain C2-20 alkylene group, wherein one or more C atoms of the alkylene group are optionally replaced by heteroatoms independently selected from O, S, and N; or, Q is a substituted or unsubstituted, saturated or unsaturated 4-6 membered ring, wherein the ring atom of the 4-6 membered ring optionally contains one or more heteroatoms independently selected from O, S, and N; the substituent group is selected from halogens, -OH, straight-chain or branched C1-20 alkyl, straight-chain or branched C1-20 alkoxy, straight-chain or branched C2-20 alkenyl, straight-chain or branched C2-20 alkynyl, -CH2CH(OH)R5, R1, R2, R3, and R4 may be the same or different, and each is independently selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl, substituted or unsubstituted straight-chain or branched C2-30 alkenyl, substituted or unsubstituted straight-chain or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl, or alkynyl group are optionally replaced by heteroatoms independently selected from O, S, and N, or -CH2CH(OH)R5; the substituted substituents are selected from halogens, -OH, straight-chain or branched C1-10 alkyl, and straight-chain or branched C1-10 alkoxy groups; The condition is that at least one of R1, R2, R3, and R4 is R5 is selected from hydrogen, substituted or unsubstituted straight-chain or branched C1-30 alkyl, substituted or unsubstituted straight-chain or branched C2-30 alkenyl, substituted or unsubstituted straight-chain or branched C2-30 alkynyl, wherein one or more C atoms of the alkyl, alkenyl or alkynyl group are optionally replaced by heteroatoms independently selected from O, S and N; wherein the substituted substituents are selected from halogens, -OH, straight-chain or branched C1-10 alkyl, straight-chain or branched C1-10 alkoxy. R6 is selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, -OH; n is selected from integers from 1 to 8, and m is selected from integers from 0 to 8. n and m are independent of each other and can be the same or different. When at least two of R1, R2, R3, and R4 are In this case, n and m in each of the groups are independent of each other and can be the same or different.

2. The compound according to claim 1, characterized in that, Q is The substituted group is a halogen, -OH, straight-chain or branched C1-10 alkyl, or straight-chain or branched C1-10 alkoxy group.

3. The compound according to claim 1 or 2, characterized in that, Q is Where x and y are the same or different, and are independently selected from integers from 1 to 8; Preferably, x and y are the same or different, and are integers selected from 1 to 3; Preferably, R7 is a straight-chain or branched C1-4 alkyl group.

4. The compound according to any one of claims 1-3, characterized in that, R6 is -OH.

5. The compound according to any one of claims 1-4, characterized in that, n is an integer selected from 4 to 8, and m is an integer selected from 4 to 8.

6. The compound according to any one of claims 1-5, selected from formula A, B, C or D: Each n1 is independent of each other, either the same or different, and each n1 is selected from an integer from 1 to 8. Each m1 is independent of each other, either the same or different, and each m1 is selected from an integer from 0 to 8. Preferably, each n1 is selected from an integer from 4 to 8, and each m1 is selected from an integer from 4 to 8. Preferably, each n1 is the same as each other, and each m1 is the same as each other. Each n2 is independent of each other, either identical or different, and each n2 is selected from integers from 1 to 8; each m2 is independent of each other, either identical or different, and each m2 is selected from integers from 0 to 8; preferably, each n2 is selected from integers from 4 to 8, and each m2 is selected from integers from 4 to 8; preferably, each n2 is identical to each other, and each m2 is identical to each other. Each n3 is independent of each other, either identical or different, and each n3 is selected from integers from 1 to 8; each m3 is independent of each other, either identical or different, and each m3 is selected from integers from 0 to 8; preferably, each n3 is selected from integers from 4 to 8, and each m3 is selected from integers from 4 to 8; preferably, each n3 is identical to each other, and each m3 is identical to each other. Each n4 is independent of each other, either identical or different, and each n4 is selected from integers from 1 to 8; each m4 is independent of each other, either identical or different, and each m4 is selected from integers from 0 to 8; preferably, each n4 is selected from integers from 4 to 8, and each m4 is selected from integers from 4 to 8; preferably, each n4 is identical to each other, and each m4 is identical to each other. Preferred, 7. The use of the compound according to any one of claims 1-6 in the preparation of a bioactive substance delivery system; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

8. A bioactive substance delivery system, characterized in that, The product contains a compound according to any one of claims 1-6; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

9. A pharmaceutical composition, characterized in that, The bioactive substance delivery system of claim 8 is included.

10. A method for preparing the compound according to any one of claims 1-6, characterized in that: The process involves: 1) reducing the carboxyl group of compound A1 to a hydroxyl group in the presence of a reducing agent to obtain compound A2; 2) oxidation, oxidizing the hydroxyl group of compound A2 to an aldehyde group in the presence of an oxidizing agent to obtain compound A3; 3) halogenation-reduction, first reacting the α-hydrogen of the aldehyde in compound A3 with a halogenating agent under acidic conditions to obtain an α-haloaldehyde intermediate, and then reducing the aldehyde group of the α-haloaldehyde to a hydroxyl group in the presence of a reducing agent to obtain compound A4; 4) epoxidation, subjecting compound A4 to an intramolecular nucleophilic substitution reaction in the presence of a base to obtain epoxide compound A5; and 5) ring-opening reaction, causing compound A5 to undergo a ring-opening reaction with an amine to obtain the final compound.

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