Carbamate compound, lipid nanoparticles constructed by carbamate compound, pharmaceutical composition and application

Abstract

The invention discloses a carbamate compound, lipid nanoparticles constructed by the carbamate compound, a pharmaceutical composition and application of the carbamate compound. A lipid nanoparticle carrier constructed by the series of cationic amino lipid compounds (formula I) can deliver mRNA to the spleen in a targeted manner on the premise of ensuring high efficiency and low toxicity, so that the immune response is effectively stimulated, the curative effect is remarkably improved, and the cationic amino lipid compound has important clinical application significance in cancer treatment.

Description

Technical Field

[0001] This invention relates to a carbamate compound and lipid nanoparticles thereof constructed therefrom, pharmaceutical compositions and uses thereof, and more particularly to a carbamate compound with low toxicity and spleen-targeting properties and lipid nanoparticles thereof constructed therefrom, pharmaceutical compositions and uses thereof. Background Technology

[0002] The spleen plays a central role in the human immune system, rich in various immune cells such as T lymphocytes, B lymphocytes, macrophages, and dendritic cells. These cells work together to play an irreplaceable role in the body's immune response, including defending against pathogen invasion and recognizing and eliminating tumor cells. In the field of tumor immunotherapy, stimulating splenic immune cells to recognize and attack tumor antigens can effectively activate a systemic anti-tumor immune response, inhibiting tumor growth and metastasis. For infectious diseases, especially certain viral or intracellular bacterial infections, immune cells in the spleen can respond rapidly, initiating immune defense mechanisms to eliminate pathogens.

[0003] Gene therapy involves artificially delivering genes with specific genetic information into target cells to produce proteins that can regulate, treat, or cure diseases caused by gene defects, or to achieve clinical therapeutic effects by regulating the expression of related genes. However, because nucleic acids and cell membranes carry negative charges, unmodified nucleic acids are difficult to directly enter cells and are easily destroyed by intracellular nucleases, thus hindering effective gene delivery and treatment. To address this issue, viral or non-viral vectors are typically used to assist in gene delivery.

[0004] Viral vectors are favored for their highly efficient transfection capabilities both in vivo and in vitro, but they also have some drawbacks, such as high toxicity, strong immune responses, limited gene-carrying capacity, poor targeting, and complex preparation processes. In contrast, non-viral vectors are receiving increasing attention in research and development due to their ease of preparation, transportation, and storage, as well as their advantages of high safety, efficacy, and non-immunogenicity.

[0005] Lipid nanoparticles (LNPs) are a widely used non-viral carrier, particularly suitable for vaccine development and nucleic acid drug delivery. Liposomes, as highly promising drug delivery carriers, utilize ionizable lipids as their core component. Their unique phospholipid bilayer structure effectively encapsulates both water-soluble and lipid-soluble drug molecules, improving pharmacokinetic properties and reducing drug toxicity. However, conventional liposomes lack tissue specificity in vivo and are easily recognized and cleared by the mononuclear macrophage system (MPS), primarily accumulating in macrophage-rich organs such as the liver and spleen. However, their targeting efficiency in the spleen is far from ideal. Excessive accumulation in the liver reduces the effective drug concentration in the spleen and increases the metabolic burden on the liver, potentially leading to liver dysfunction, all of which limit their potential for clinical application.

[0006] SM102 is a crucial component of mRNA vaccines. Although SM102 can improve the stability and delivery efficiency of mRNA, delivery efficiency can still be affected by various factors, such as the properties of the lipid components, the properties of the drug, and the characteristics of the target cells or tissues. These factors may prevent the effective delivery of mRNA to the target cells or tissues, leading to poor therapeutic effects. Furthermore, while existing targeting strategies, such as antibody or ligand-modified liposomes, have improved targeting in some tissues, specific modification techniques for the spleen remain incomplete. Existing spleen-targeting modification methods either have low targeting efficiency or are complex and costly, hindering large-scale clinical application. Therefore, developing a highly efficient, stable, and easily prepared spleen-targeting liposome to overcome the bottlenecks of existing drug delivery systems is crucial for improving the treatment of spleen-related diseases and is a key issue that urgently needs to be addressed in the current drug delivery field. Summary of the Invention

[0007] Purpose of the Invention: The first purpose of this invention is to provide a cationic amino lipid compound based on a carbamate structure; the second purpose is to provide lipid nanoparticles constructed from the cationic amino lipid compound and other lipid components; the third purpose is to provide a pharmaceutical composition constructed from the lipid nanoparticles that can effectively deliver nucleic acid molecules; and the fourth purpose is to provide pharmaceutical uses of the cationic amino lipid compound, the lipid nanoparticles constructed from it, and the pharmaceutical composition.

[0008] Technical solution: The carbamate compound or its N-oxide, solvate, isotopic compound, pharmaceutically acceptable salt or stereoisomer of the present invention, wherein the compound has the structure of Formula I,

[0009]

[0010] in,

[0011] R1 is selected from CH and N;

[0012] R2 is selected from CH, N, and O;

[0013] G1 and G2 are selected from H and OH;

[0014] G3 is selected from H and -(CH2). n OH or it may not be present;

[0015] n is selected from 1, 2, and 3.

[0016] The term "pharmaceutically acceptable" in this invention means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0017] The term "solvate" in this invention refers to a complex formed by combining a compound of Formula I or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a Formula I compound used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield a Formula I compound once absorbed into the subject, such that the use of a Formula I compound separately encompasses the use of any solvate of a Formula I compound.

[0018] The term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water.

[0019] It should be further understood that compounds of formula I or pharmaceutically acceptable salts thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this invention. For example, compounds of formula I or pharmaceutically acceptable salts thereof may exist in unsolvated forms as well as in solvated forms formed with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0020] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic addition salt of an inorganic or organic acid of the compounds of this invention. For example, see SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids include, for example, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, etc. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with compounds of Formula I.

[0021] The nitrogen-containing compounds of Formula I of the present invention can be converted into N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Therefore, provided that the valence state and structure permit, the compounds claimed by the present invention include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0022] Some compounds of the present invention can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in this invention also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, the compounds of the present invention can be salted by adding chiral acid resolving reagents such as chiral tartaric acid or chiral malic acid, and separation can be achieved by utilizing the physicochemical properties of the products, such as differences in solubility.

[0023] This invention also includes all suitable isotopic variants of the compounds of this invention. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but whose atomic mass differs from that of atoms commonly or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively, for example… 2 H (deuterium) 3 H (tritium) 11 C 13 C 14 C 1 5N 17 O and 18 O.

[0024] Preferably, in the structure described above:

[0025] R1 is selected from CH and N, and R2 is selected from N and O, and R1 and R2 are different;

[0026] G1 and G2 are selected from H and OH, and G1 and G2 are the same;

[0027] G3 is selected from -(CH2)2OH or does not exist.

[0028] Preferably, in the structure described above:

[0029] When R1 is selected from CH, R2 is selected from O or N; when R1 is selected from N, R2 is selected from O.

[0030] Preferably, the carbamate compound is selected from any one of the following compounds:

[0031]

[0032] More preferably, the carbamate compound is selected from:

[0033]

[0034] The compounds designed in this invention are cationic lipids, which are lipids that carry a positive charge at a selected pH value.

[0035] Cationic liposomes readily bind to negatively charged nucleic acids, interacting with the negatively charged phosphate groups present in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery carriers.

[0036] In screening a large number of compounds, this invention found that identifying suitable cationic lipid compounds that meet the following criteria is extremely difficult: they must have a significantly different structure from existing representative cationic lipids, exhibit extremely high transfection efficiency and extremely low cytotoxicity, and demonstrate high and sustained expression in mice. Compounds such as R1, R2, and R6, which, compared to existing cationic lipids with significantly different chemical structures, can achieve significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression levels in the spleen of animals. These aminolipid compounds facilitate the release of target drugs or genes, thereby improving delivery efficiency, which has a positive effect on improving therapeutic efficacy and reducing side effects. The preparation method of the aminolipid compounds designed in this invention uses readily available raw materials, reducing production costs and enabling large-scale production. The reaction conditions during preparation are relatively mild, which helps protect the environment, reduces energy consumption, and lowers the requirements for high-end equipment. They also possess good stability, are easy to mass-produce, and have significant clinical implications.

[0037] In short, this invention is based on at least the following findings:

[0038] 1. A series of cationic amino lipid compounds were designed, including R1, R2, and R6.

[0039] 2. The LNP formulations prepared by R1, R2 and R6 significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly increased mRNA expression level and duration in mice, significantly increased expression level in mouse spleen and significantly reduced expression level in liver.

[0040] 3. Among a series of compounds with very small differences in chemical structure, the LNP preparations prepared by R1, R2 and R6 significantly improved cell transfection efficiency and significantly reduced cytotoxicity compared with other compounds; the expression level and duration of mRNA in mice were significantly increased.

[0041] 4. Through unique design and screening, some compounds, such as R1, R2 and R6, were discovered to significantly reduce cytotoxicity, significantly improve cell transfection efficiency, significantly increase expression levels and expression time in animals, significantly increase expression levels in the spleen and significantly decrease expression levels in the liver, thereby improving delivery efficiency and achieving excellent results.

[0042] 5. Through unique design and screening, several compounds, such as R1, R2, and R6, were discovered that can target mRNA delivery to the spleen while maintaining high efficiency and low toxicity. These compounds are not expressed in other organs, such as the lungs, heart, and kidneys, but are expressed in small amounts in the liver. The low efficiency of vaccine-induced immune responses is a reason why existing cancer therapeutic vaccines cannot achieve their maximum efficacy. The spleen is the largest secondary lymphoid organ in the body. LNP cancer vaccines targeting the spleen can effectively stimulate an immune response and significantly improve efficacy, holding important clinical application significance in cancer treatment.

[0043] The spleen is the largest secondary lymphoid organ in animals. By increasing the expression level of delivered mRNA in the spleen, mRNA vaccines can rapidly induce an immune response and produce antibodies in vivo. This can significantly improve the preventive effect without altering the vaccine composition, which has important clinical significance. It also shows good targeted efficacy for developing treatments for diseases caused by spleen damage or abnormalities, such as lymphoma and leukemia.

[0044] There is no direct correlation between the compound structure of cationic lipids and the expression level and duration of mRNA in animals. Compounds with very similar structures may produce LNP formulations with significantly different mRNA expression levels in animals. Therefore, it is impossible to predict the expression level and duration of mRNA in animals based on chemical structure. Screening for cationic lipids with high and sustained mRNA expression in animals is extremely difficult and requires a great deal of creative work.

[0045] This invention provides a novel cationic lipid compound for delivering therapeutic or preventative agents. It can be used to deliver nucleic acid molecules, small molecule compounds, peptides, or proteins, and exhibits high transfection efficiency, low cytotoxicity, and high expression in animals, thereby improving delivery efficiency and safety.

[0046] The lipid nanoparticles of the present invention are constructed from the carbamate compounds, neutral lipids, structural lipids and polymer conjugated lipids of the present invention, wherein the molar ratio between them is (25-75):(5-25):(15-65):(0.5-10).

[0047] The total molar percentage of the carbamate compounds, neutral lipids, structural lipids, and polymer-conjugated lipids is 100%.

[0048] Preferably, the molar ratio of carbamate compounds, neutral lipids, structural lipids and polymer conjugated lipids in the lipid nanoparticles is (35-49):(7.5-15):(35-55):(1-5).

[0049] Further preferably, the molar ratio of carbamate compounds, neutral lipids, structural lipids and polymer conjugated lipids in the lipid nanoparticles is 45:10:43.5:1.5 or 49:10:39.5:1.5, more preferably 49:10:39.5:1.5.

[0050] As used herein, the terms "comprising," "containing," or "including" mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms "containing" or "comprising (including)" as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include "consistently composed of" or "composed of."

[0051] Preferably, the carbamate compounds account for 25%-75% of the molar percentage in the lipid nanoparticles.

[0052] In one embodiment, the carbamate compound comprises 30%, 40%, 50%, 55%, 60%, 65%, or 70% in molar percentage.

[0053] The carrier may contain neutral lipids. In this invention, neutral lipids refer to lipids that are uncharged at a selected pH value or exist in a zwitterionic form and play an auxiliary role. These neutral lipids may modulate the flowability of nanoparticles to form a lipid bilayer structure and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0054] Preferably, in the lipid nanoparticles, the molar ratio of carbamate compounds to neutral lipids is 1:1 to 15:1, more preferably 4.9:1.

[0055] In one embodiment, the molar ratio of the carbamate compound to the neutral lipid is 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5:1 (for example, it can be 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, or any range of any two of the above ratios and any intermediate ratio therein).

[0056] Carriers containing carbamic acid lipid compounds may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can assemble into one or more lipid bilayers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0057] Preferably, in the lipid nanoparticles, the neutral lipids are selected from one or more of sphingomyelin, ceramide, phosphatidylcholine, phosphatidylethanolamine, and sterols.

[0058] The neutral lipid moiety may be selected from the non-restricted group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-restricted group consisting of lauric acid, myristic acid, myristenoic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzanoic acid, docosapentaenoic acid, and docosahexaenoic acid. It also encompasses non-natural species including natural species with modifications and substitutions, such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of a composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components such as targeting or imaging components (e.g., dyes).

[0059] Further preferably, in the lipid nanoparticles, the neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1,15-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl-sa-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG) Palmitoyl oleoyl phosphatidyl ethanolamine (POPE), distearate phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), 1-stearoyl-2-oleoyl stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl ethanolamine (LPE).

[0060] More preferably, in the lipid nanoparticles, the neutral lipid is selected from one or more of the following: 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) and 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0061] In some embodiments, neutral lipids include DSPC. In some embodiments, neutral lipids include DOPE. In some embodiments, neutral lipids include both DSPC and DOPE.

[0062] The carrier containing carbamate compounds may also include one or more structural lipids. In this invention, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0063] Preferably, in the lipid nanoparticles, the molar ratio of carbamate compounds to structural lipids is 0.6:1 to 3:1.

[0064] In one embodiment, the molar ratio of the carbamate compound to the structural lipid is 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, or 3:1.

[0065] Preferably, the structural lipids in the lipid nanoparticles are selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0066] Further preferably, the structural lipid in the lipid nanoparticles is cholesterol.

[0067] In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0068] Carriers containing carbamate compounds may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages.

[0069] Preferably, the molar percentage of polymer-conjugated lipids in the lipid nanoparticles is 0.5%-10%, more preferably 1.5%.

[0070] Preferably, in the lipid nanoparticles, the polymer-conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0071] In one embodiment, the molecular weight of the PEG used for modification is typically 350-5000 Da.

[0072] Further preferably, in the lipid nanoparticles, the polymer-conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0073] In one embodiment of the present invention, the polymeric conjugated lipid is DMG-PEG2000.

[0074] The pharmaceutical composition of the present invention is constructed by loading a drug onto lipid nanoparticles as described in the present invention, wherein the mass ratio of the lipid nanoparticles to the drug is 10:1-30:1.

[0075] The lipid nanoparticles (carriers) described above can be used to deliver active ingredients, such as therapeutic or preventative agents. The active ingredients may be encapsulated within or bound to the carrier, and may include one or more therapeutic and / or preventative agents.

[0076] Preferably, in the pharmaceutical composition, the mass ratio of lipid nanoparticles to the drug is 12.5:1 to 20:1.

[0077] More preferably, in the pharmaceutical composition, the mass ratio of lipid nanoparticles to the drug is 15:1.

[0078] In one embodiment, the mass ratio of the carrier to the therapeutic or preventative agent is 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0079] In one embodiment, the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, or 20:1.

[0080] Preferably, in the pharmaceutical composition, the drug is selected from one or more of the following: nucleic acids, small molecule compounds, polypeptides, and proteins. Therefore, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of therapeutic or preventative agents described above, the compositions of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0081] Preferably, in the pharmaceutical composition, the drug is selected from vaccines or small molecule compounds capable of inducing an immune response.

[0082] Further preferably, in the pharmaceutical composition, the drug is a nucleic acid.

[0083] More preferably, in the pharmaceutical composition, the drug is selected from one or more of the following: ribonucleic acid and deoxyribonucleic acid.

[0084] More preferably, in the pharmaceutical composition, the drug is selected from one or more of the following: single-stranded DNA, double-stranded DNA, and RNA.

[0085] In a further preferred embodiment, the drug in the pharmaceutical composition is selected from one or more of the following: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), and messenger RNA (mRNA).

[0086] Most preferably, in the pharmaceutical composition, the drug is messenger RNA.

[0087] Preferably, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients or diluents.

[0088] Preferably, the pharmaceutical composition has an average particle size of 10-250 nm and a polydispersity index ≤50%.

[0089] More preferably, the pharmaceutical composition has an average particle size of 100-240 nm and a polydispersity index of ≤45%.

[0090] In one embodiment, the polydispersity factor of the pharmaceutical composition is more preferably ≤30%.

[0091] The purpose of the carbamate compounds or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or lipid nanoparticles or pharmaceutical compositions thereof described in this invention is to prepare nucleic acid drugs or gene vaccine drugs.

[0092] Preferably, the drug is a drug for treating diseases or conditions characterized by dysfunction or abnormal protein or polypeptide activity.

[0093] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of said disease, in order to cure, alleviate, reduce, improve, or affect said disease or its symptoms. In the context of this invention, unless specifically stated otherwise, the term "treatment" may also include prevention.

[0094] More preferably, the drug is a drug for treating one or more diseases or conditions selected from the following: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

[0095] More preferably, the infectious disease is selected from one or more of the following: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes diseases.

[0096] More preferably, the drug is a drug for treating metabolic diseases.

[0097] Preferably, the drug is administered to a human.

[0098] The terms “subject” or “patient” in this invention include humans and mammals.

[0099] More preferably, the route of administration of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation, and more preferably subcutaneous.

[0100] The pharmaceutical composition of the present invention is administered in a therapeutically effective amount, which can vary not only with the specific reagent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician.

[0101] More preferably, the dosage of the drug is 0.001-10 mg / kg.

[0102] "Therapeutic effective amount" is the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" is the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can conventionally determine the therapeutic and preventive effective amounts based on their knowledge and the present invention.

[0103] The pharmaceutical compositions of the present invention may include one or more components other than those described in the foregoing sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0104] The pharmaceutical composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0105] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, and eprazinone). The composition may contain mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, streptococcal DNase α (domasealfa), neltenexine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0106] The pharmaceutical composition may also contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0107] In addition to these components, the pharmaceutical composition may include any substance that can be used in the pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients include, for example, starch, lactose, or dextrin.

[0108] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0109] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0110] The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions of the present invention can be prepared by methods well known in the pharmaceutical industry. For example, a sterile injectable solution can be prepared by incorporating the desired amount of the therapeutic or preventative agent with the various other desired ingredients described above into a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Surfactants may also be added to promote the formation of a homogeneous solution or suspension.

[0111] The present invention also provides methods for treating diseases or conditions in mammals in need, including administering a composition comprising a therapeutic agent and / or a preventive agent to the mammal and / or contacting mammalian cells with the composition.

[0112] Therapeutic agents and / or preventive agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic agents and / or preventive agents can be substances that, upon delivery to a cell or organ, induce a desired change in that cell or organ or other body tissue or system. Such species can be used to treat one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventive agents are small molecule drug substances that can be used to treat a specific disease, condition, or illness.Examples of pharmaceuticals that can be used in a composition include, but are not limited to, anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside). Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, cvotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0113] In some implementations, the therapeutic and / or prophylactic agent is a cytotoxic agent, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that induces an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxic agents or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, and ethidium bromide. bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine...). 125 or iodine 131 ),strontium 89 Phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90 ,samarium 153Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, and may include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and / or novel epitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, lactamase (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), and cyclophosphamides). Phosphoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP, cisplatin), anthracyclines (e.g. daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), and antimitotic agents (e.g. vincristine, vinblastine, paclitaxel and levothyroxine).

[0114] In other embodiments, the therapeutic and / or preventative agents are proteins. Therapeutic proteins that can be used in the nanoparticles of this invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0115] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), hybrids thereof; RNAi inducible factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribonuclease; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or preventive agent is RNA. RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribonuclease, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0116] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any polypeptide that is naturally occurring or non-naturally present or otherwise modified. The polypeptide encoded by the mRNA may have any size and may possess any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells.

[0117] In other embodiments, the therapeutic and / or preventative agent is siRNA. siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA may be chosen such that, upon administration of a composition comprising the siRNA to a subject in need, a gene associated with a specific disease, symptom, or condition is silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0118] In some implementations, the therapeutic and / or preventative agents are sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0119] In some implementations, the therapeutic and / or prophylactic agent is shRNA or its encoding vector or plasmid. shRNA can be generated within the target cell after delivery of an appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant field.

[0120] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0121] This invention designs a series of cationic amino lipid compounds, and the lipid nanoparticle carriers constructed from them can deliver mRNA to the spleen in a targeted manner while ensuring high efficiency and low toxicity, thereby effectively stimulating the immune response and significantly improving the therapeutic effect. This has important clinical application significance in cancer treatment. Attached Figure Description

[0122] Figure 1 Cell transfection rate of the LNP formulation containing Fluc-mRNA prepared for this invention;

[0123] Figure 2 Cell viability of the LNP formulation containing Fluc-mRNA prepared for this invention. Detailed Implementation

[0124] The technical solution of the present invention will be further described below with reference to the embodiments.

[0125] The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications. Implementation conditions not specified are standard conditions in this industry. In the specific embodiments of this invention, all raw materials used are commercially available. Unless otherwise stated, percentages in the context are by weight, and all temperatures are given in degrees Celsius.

[0126] Example 1: Synthesis of cationic amino lipid compounds

[0127] 1. Synthesis of R1

[0128]

[0129] Compound A (23.8 g, 100 mmol) and compound B (25.6 g, 100 mmol) were added sequentially to a 250 mL reaction flask, followed by 100 mL of dichloromethane. After stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), and N,N-diisopropylethylamine (25.8 g, 200 mmol) were added. The mixture was reacted at room temperature for 2 h. After washing three times with water, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a rapid column chromatography system (petroleum ether: ethyl acetate = 10:1 to 2:1) to give intermediate C (40.1 g, 90% yield).

[0130]

[0131] Take a 50 mL dry round-bottom flask and add compound D (2.75 g, 11.69 mmol), potassium carbonate (4.83 g, 35.08 mmol), copper chloride (99 mg, 0.58 mmol), and compound E (16 mL) sequentially. Under nitrogen protection, react in an oil bath at 130 °C with stirring for 12 h. Cool to room temperature, adjust the pH to 3 with dilute hydrochloric acid, extract with ethyl acetate, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate F-1 (950 mg, yield 35.15%).

[0132]

[0133] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-1 (950 mg, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-1 (732.5 mg, yield 65%).

[0134]

[0135] Compound I (97.23 mg, 0.267 mmol) and compound H-1 (732.5 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, followed by 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate G-1 (999 mg, yield 67%).

[0136]

[0137] Compound G-1 (999 mg, 1.79 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-1 (613.6 mg, yield 63%).

[0138]

[0139] In a 100 mL reaction flask, compound C (428.4 mg, 0.9 mmol), compound K-1 (587.9 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R1 (691 mg, yield 68%).

[0140] 2. Synthesis of R2

[0141]

[0142] In a 100 mL reaction flask, compound M (1 g, 9.6 mmol) and anhydrous potassium carbonate (1.6 g, 11.4 mmol) were dissolved in anhydrous acetone (15 mL). Compound D (2.4 g, 10.4 mmol) was added dropwise to the reaction solution. The reaction solution was refluxed and stirred for 16 h. After the reaction was complete, the reaction solution was diluted with cold water and extracted three times with dichloromethane / methanol (10:1). The extract was then washed with brine, dried, and concentrated under reduced pressure to give compound F-2 (2.3 g, yield 78%).

[0143]

[0144] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-2 (1.18 g, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-2 (953 mg, yield 70%).

[0145]

[0146]

[0147] Compound I (97.23 mg, 0.267 mmol) and compound H-2 (884.4 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, followed by 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give intermediate G-2 (1.08 g, yield 66%).

[0148]

[0149] Compound G-2 (923 mg, 1.5 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-2 (502 mg, yield 57%).

[0150]

[0151] In a 100 mL reaction flask, compound C (428.4 mg, 0.9 mmol), compound K-2 (634.4 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R2 (443 mg, yield 50%).

[0152] 3. Synthesis of R3

[0153] The synthesis route is as follows:

[0154]

[0155] Take a 50 mL dry round-bottom flask and add compound N (2.43 g, 11.69 mmol), potassium carbonate (4.83 g, 35.08 mmol), copper chloride (99 mg, 0.58 mmol), and compound E (16 mL) sequentially. Under nitrogen protection, react in an oil bath at 130 °C with stirring for 12 h. Cool to room temperature, adjust the pH to 3 with dilute hydrochloric acid, extract with ethyl acetate, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate F-3 (1 g, yield 42%).

[0156]

[0157] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-3 (950 mg, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-3 (629.8 mg, yield 62%).

[0158]

[0159] Compound I (97.23 mg, 0.267 mmol) and compound H-3 (659.9 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, followed by 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate G-3 (993 mg, yield 70%).

[0160]

[0161] Compound G-3 (951 mg, 1.79 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-3 (564 mg, yield 61%).

[0162]

[0163] In a 100 mL reaction flask, compound C (428.4 mg, 0.9 mmol), compound K-3 (559 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R3 (671 mg, yield 68%).

[0164] 4. Synthesis of R4

[0165]

[0166] In a 250 mL reaction flask, starting material O (22.3 g, 100 mmol) and starting material B (25.6 g, 100 mmol) were added sequentially, followed by 100 mL of dichloromethane. After stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), and N,N-diisopropylethylamine (25.8 g, 200 mmol) were added. The mixture was reacted at room temperature for 2 h. After washing three times with water, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a rapid column chromatography system (petroleum ether: ethyl acetate = 10:1 to 2:1) to give intermediate P (41.5 g, 90% yield).

[0167]

[0168] Take a 50 mL dry round-bottom flask and add compound D (2.75 g, 11.69 mmol), potassium carbonate (4.83 g, 35.08 mmol), copper chloride (99 mg, 0.58 mmol), and compound E (16 mL) sequentially. Under nitrogen protection, react in an oil bath at 130 °C with stirring for 12 h. Cool to room temperature, adjust the pH to 3 with dilute hydrochloric acid, extract with ethyl acetate, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate F-4 (950 mg, yield 35.15%).

[0169]

[0170] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-4 (950 mg, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-4 (732.5 mg, yield 65%).

[0171]

[0172] Compound Q (92.96 mg, 0.267 mmol) and compound H-4 (732.5 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, along with 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate G-4 (970 mg, yield 67%).

[0173]

[0174] Compound G-4 (970 mg, 1.79 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-4 (596 mg, yield 63%).

[0175]

[0176]

[0177] In a 100 mL reaction flask, compound S (414 mg, 0.9 mmol), compound K-4 (571 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R4 (667 mg, yield 68%).

[0178] 5. Synthesis of R5

[0179]

[0180] In a 100 mL reaction flask, compound M (1 g, 9.6 mmol) and anhydrous potassium carbonate (1.6 g, 11.4 mmol) were dissolved in anhydrous acetone (15 mL). Compound D (2.4 g, 10.4 mmol) was added dropwise to the reaction solution. The reaction solution was refluxed and stirred for 16 h. After the reaction was complete, the reaction solution was diluted with cold water and extracted three times with dichloromethane / methanol (10:1). The extract was then washed with brine, dried, and concentrated under reduced pressure to give compound F-5 (2.3 g, yield 78%).

[0181]

[0182] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-5 (1.18 g, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-5 (953 mg, yield 70%).

[0183]

[0184]

[0185] Compound Q (92.96 mg, 0.267 mmol) and compound H-5 (884.4 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, followed by 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate G-5 (1.06 g, yield 66%).

[0186]

[0187] Compound G-5 (899 mg, 1.5 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-5 (489 mg, yield 57%).

[0188]

[0189] In a 100 mL reaction flask, compound S (414 mg, 0.9 mmol), compound K-5 (617 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R5 (514 mg, yield 50%).

[0190] 6. Synthesis of R6

[0191]

[0192] Take a 50 mL dry round-bottom flask and add compound N (2.43 g, 11.69 mmol), potassium carbonate (4.83 g, 35.08 mmol), copper chloride (99 mg, 0.58 mmol), and compound E (16 mL) sequentially. Under nitrogen protection, react in an oil bath at 130 °C with stirring for 12 h. Cool to room temperature, adjust the pH to 3 with dilute hydrochloric acid, extract with ethyl acetate, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate F-6 (1 g, yield 42%).

[0193]

[0194] Take a 50 mL dry round-bottom flask and add FeBr2 (11.86 mg, 0.055 mmol), compound G (164.4 mg, 2.74 mmol), and compound F-6 (950 mg, 4.11 mmol) sequentially. Under nitrogen protection, react in an oil bath at 150 °C with stirring for 6 h. Cool to room temperature, extract with dichloromethane, combine the organic phases, dry and filter, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 10:1) to give intermediate H-6 (629.8 mg, yield 62%).

[0195]

[0196] Compound Q (92.96 mg, 0.267 mmol) and compound H-6 (659.9 mg, 2.67 mmol) were added sequentially to a 100 mL reaction flask, followed by 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (68.5 mg, 0.53 mmol) was added, and the mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate G-6 (993 mg, yield 70%).

[0197]

[0198] Compound G-6 (923 mg, 1.79 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL reaction flask under ice bath conditions. Then, diluted BBr3 (1.5 mL) was added dropwise, and the mixture was stirred in an ice bath for 1 h. The reaction was then moved to room temperature and stirred overnight. After the reaction was completed, the reaction solution was transferred to water and the solid was collected to give compound K-6 (547 mg, yield 61%).

[0199]

[0200] In a 100 mL reaction flask, compound S (414 mg, 0.9 mmol), compound K-6 (541 mg, 1.08 mmol), and 20 mL of acetonitrile were added sequentially and stirred until dissolved. Then, potassium carbonate (248.4 mg, 1.8 mmol) and potassium iodide (149.4 mg, 0.9 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to give compound R6 (6736 mg, yield 68%).

[0201] Example 2: Preparation of lipid nanoparticles (LNP formulation)

[0202] Cell transfection assay using LNP formulation of eGFP-mRNA:

[0203] Cell resuscitation and passage: 293T cells were resuscitated and passaged in culture dishes to the required number of cells.

[0204] Seeding: Digest and count the cells in the culture dish, seed 10,000 cells per well into a 96-well plate or 150,000 cells per well into a 12-well plate, and culture overnight until the cells adhere.

[0205] Cell transfection experiment: LNP preparation containing less than 1.5 μg of eGFP-mRNA prepared under experimental conditions (the cationic lipids in the vector are R1, R2 and R6) and Lipofectamine 3000 preparation containing eGFP-mRNA were added to the cell culture medium of 12-well plates. After culturing for 24 h, the transfection efficiency of the samples was evaluated by fluorescence microscopy based on the fluorescence intensity.

[0206] 1. Screening of the ratio of vector (liposome) to mRNA

[0207] The cationic lipid compounds R1, R2, and R6 synthesized in Example 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5, respectively, to prepare ethanol lipid solutions. The ethanol lipid solutions were rapidly added to citrate buffer (pH = 4-5) using the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA was diluted in citrate buffer (pH = 4-5) to obtain an aqueous mRNA solution. A certain volume of liposome solution and the aqueous mRNA solution were mixed to prepare liposomes at total lipid to mRNA weight ratios of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1, respectively. The liposomes were sonicated at 25°C for 15 min (ultrasound frequency 40 kHz, sonic power 800 W). The resulting liposomes were diluted 10 times with PBS and then ultrafiltered in a 300 kDa ultrafiltration tube to remove ethanol. The volume was then adjusted to a certain level with PBS to obtain an LNP formulation in which eGFP-mRNA was encapsulated using cationic lipids R1 (or R2, R6) / DSPC / cholesterol / DMG-PEG2000 (molar ratio of 49:10:39.5:1.5).

[0208] Cell transfection experiments showed that a vector-to-mRNA weight ratio of 10:1 to 30:1 resulted in good transfection effects, with 15:1 being the most effective. Ratios of 5:1 and 35:1 showed poor transfection effects and should not be used to transport mRNA.

[0209]

[0210]

[0211] 2. Screening of the ratio of cationic lipids to neutral lipids

[0212] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, wherein the molar ratios of cationic lipid R1 (or R2, R6) to neutral lipid DSPC were 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

[0213] Cell transfection experiments showed that transfection was effective at molar ratios of cationic lipids to neutral lipids ranging from 1:1 to 15:1, with the highest transfection efficiency observed at a ratio of 4.9:1.

[0214] molar ratio of cationic lipids to neutral lipids Relative light unit (RLU) 1：1 83443 3：1 106577 3.5：1 162598 4：1 258588 4.9：1 354576 10　1 275447 15：1 150744 20：1 13858

[0215] 3. Screening of the ratio of polymer-conjugated lipids to carriers (liposomes)

[0216] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1. The cationic lipid in the carrier was R1 (or R2, R6), and the molar ratio of polymeric conjugated lipid DMG-PEG2000 in the carrier was 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10%, and 15%, respectively.

[0217] Cell transfection experiments showed that transfection was effective when the molar ratio of polymer conjugated lipids to carrier was in the range of 0.5%-10%, with the highest transfection efficiency at 1.5% and the lowest at 10%.

[0218] Polymer conjugated lipid molar ratio to carrier Relative light unit (RLU) 0.5％ 213436 1.5％ 467326 2.5％ 367590 3.5％ 246268 5％ 154576 10％ 32447 15％ 1730

[0219] 4. Screening of the proportions of each component in the carrier (liposome)

[0220] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, wherein the molar ratios of cationic lipid R1 (or R2, R6), neutral lipid DSPC, structural lipid cholesterol, and polymeric conjugated lipid DMG-PEG2000 were 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.

[0221] Cell transfection experiments showed that transfection was achieved at molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer conjugated lipids of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5. Good transfection results were observed within the range of (35-49):(7.5-15):(35-55):(1-5), with the best transfection effect observed at a molar ratio of 49:10:39.5:1.5.

[0222] Molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids Relative light unit (RLU) 75：5：15：5 26737 65：8：25：2 34622 49：10：39.5：1.5 578284 45：10：43.5：1.5 246268 45：25：20：10 267904 40：10：48.5：1.5 378903 35：10：53.5：1.5 346727 25：5：65：5 47279

[0223] Based on the experimental results, the preparation conditions for the lipid nanoparticles (LNP formulation) were finally determined as follows: the ratio of carrier to mRNA was 15:1; the molar ratio of cationic lipids to neutral lipids was 4.9:1; the proportion of polymer-conjugated lipids in liposomes was 1.5%; and the molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids was 49:10:39.5:1.5. Subsequent experiments were conducted to prepare lipid nanoparticles (LNP formulation) under these conditions.

[0224] Example 3: Preparation of lipid nanoparticles (LNP formulation) (optimal conditions)

[0225]

[0226] The cationic lipids, SM102, and MC3 prepared in Example 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1 to prepare ethanol lipid solutions. The ethanol lipid solutions were rapidly added to citrate buffer (pH = 4-5) using the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA, Flux-mRNA, or Cy5-labeled Leqvio were diluted in citrate buffer (pH = 4-5) to obtain an aqueous mRNA solution. A certain volume of liposome solution was mixed with the aqueous mRNA solution at a total lipid to mRNA weight ratio of 15:1 to prepare liposomes. The liposomes were sonicated at 25°C for 15 min (ultrasound frequency 40 kHz, sonic power 800 W). The resulting liposomes were diluted 10 times with PBS and then ultrafiltered in a 300 kDa ultrafiltration tube to remove ethanol. The volume was then adjusted to a certain level with PBS to obtain an LNP formulation in which eGFP-mRNA or Fluc-mRNA was encapsulated using cationic lipids / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 49:10:39.5:1.5).

[0227] Example 4: Evaluation of Particle Size and Polydispersity Index (PDI) of Nanolipid Particles

[0228] Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering.

[0229] Take 10 μL of the liposome solution prepared in Example 3, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and measure each sample three times. The measurement conditions are: 90° scattering angle, 25℃. The detection results are shown in Table 1.

[0230] Table 1. Particle size and polydispersity index (PDI) of nanolipid particles

[0231]

[0232]

[0233] As shown in Table 1, the nanolipid particles prepared in Example 3 have particle sizes between 150 and 190 nm, all of which can be used to deliver mRNA. Smaller particle sizes are more easily taken up by cells, thereby improving drug delivery efficiency. A lower PDI value indicates a more uniform particle size distribution, implying a more stable drug delivery system. Among them, the particles prepared by R6 have the smallest particle size at 157 nm, while the particles prepared by R4 have the largest particle size at 170 nm. The polydispersity index (PDI) of all nanolipid particles ranges from 5% to 25%, with R6 having the smallest at 7.5% and R4 having the largest at 12.5%.

[0234] Example 5: In vitro evaluation of the performance of LNP delivery vector

[0235] Cell resuscitation and passage: The method is the same as in Example 2.

[0236] Seed plate: The method is the same as in Example 2.

[0237] 1. Fluorescence detection of Fluc-mRNA (transfection efficiency)

[0238] Lipofectamine 3000 transfection reagent is widely used for cell transfection due to its excellent transfection performance and efficiency, as well as its ability to improve cell viability. It is also suitable for difficult-to-transfect cell types. Using Lipofectamine 3000 transfection reagent as a control, eGFP-mRNA or Fluc-mRNA preparations were prepared according to the method described in the Lipofectamine 3000 (Invictus (Shanghai) Trading Co., Ltd.) instructions.

[0239] LNP formulations were prepared according to the method in Example 3. An LNP formulation containing 0.3 μg Fluc-mRNA (the LNP formulation carrier components were cationic lipids, DSPC, cholesterol, and DMG-PEG2000, with a molar ratio of 49:10:39.5:1.5, wherein the cationic lipids were those listed in Example 1) was added to the cell culture medium of a 96-well plate. After culturing for 24 h, the appropriate reagents were added according to the Gaussian Luciferase Assay Kit instructions, and the fluorescence expression intensity of each well was detected using an IVIS fluorescence detection system. The transfection efficiency of LNP formulations prepared from a series of cationic lipid compounds designed in this invention and existing cationic lipids, including MC3, SM102, and Lipofectamine 3000, in cells is shown in the results below. Figure 1 And Table 2.

[0240] Table 2 lists the fluorescence detection results of LNP formulations containing Fluc-mRNA prepared from different cationic lipids.

[0241] Table 2 Fluorescence detection results of Fluc-mRNA

[0242] Serial Number cationic lipids Relative light unit (RLU) R1 is a multiple thereof R2 is a multiple thereof R6 is a multiple thereof 1 R1 5695757 1.00 0.67 0.84 2 R2 3836537 1.48 1.00 1.24 3 R3 75447 75.53 5.08 63.12 4 R4 98678 57.72 38.88 48.26 5 R5 143570 39.73 26.72 33.17 6 R6 4762198 1.19 0.81 1.00 7 MC3 105631 53.92 36.32 45.08 8 SM102 146923 38.76 26.11 32.41 9 Lipofectamine 3000 805681 7.07 4.76 5.91

[0243] Depend on Figure 1 As shown in Table 2, the LNP formulations containing Fluc-mRNA prepared by R1, R2 and R6 had the strongest fluorescence absorption, with RLU values ​​of 5695757, 3836537 and 4762198, respectively.

[0244] SM102 and MC3 are representative cationic lipids with good transfection performance. The LNP formulations prepared by R1, R2, and R6 showed significantly improved cell transfection efficiency compared to these representative cationic lipids. Specifically, R1 achieved a transfection efficiency 38.76 times that of SM102, 53.92 times that of MC3, and 7.07 times that of Lipofectamine 3000. R2 achieved a transfection efficiency 26.11 times that of SM-102, 36.32 times that of MC3, and 4.76 times that of Lipofectamine 3000, demonstrating a significant improvement. R6 achieved a transfection efficiency 32.41 times that of SM-102, 45.08 times that of MC3, and 5.91 times that of Lipofectamine 3000, also showing a significant improvement.

[0245] 2. Cell viability evaluation

[0246] LNP formulations were prepared according to the method in Example 3. An LNP formulation containing 1.5 μg Fluc-mRNA (the LNP formulation carrier components were cationic lipids, DSPC, cholesterol, and DMG-PEG2000, with a molar ratio of 49:10:39.5:1.5, wherein the cationic lipids were those listed in Example 1) was added to the cell culture medium in a 96-well plate. After culturing for 24 h, 10 μL of CCK-8 solution was added to each well. The plate was incubated in an incubator for 1 h, and the absorbance at 450 nm was measured using a microplate reader. Cell viability results are shown below. Figure 2 And Table 3.

[0247] Table 3 Cell viability

[0248] Serial Number cationic lipids Cell viability (%) 1 R1 88 2 R2 90 3 R3 54 4 R4 49 5 R5 55 6 R6 89 7 MC3 75 8 SM102 81 9 Lipofectamine 3000 30

[0249] Depend on Figure 2 As shown in Table 3, the cationic lipids designed in this invention have significantly different chemical structures compared to existing representative cationic lipids SM102 and MC3. The LNP formulations prepared from R1, R2, and R6 exhibited the lowest cytotoxicity and significantly improved cell viability. Specifically, the cell viability of R1 was 7% higher than that of SM102, 13% higher than that of MC3, and 58% higher than that of Lipofectamine 3000.

[0250] Example 6: In vivo evaluation of the performance of cationic lipid (LNP) delivery carriers

[0251] 1. Expression of delivered mRNA in mice

[0252] This experiment verified the expression and duration of mRNA delivered by the cationic lipids designed in this invention in mice. In vivo experiments further demonstrated that the LNP delivery vector designed in this invention, compared with existing representative cationic lipids, (1) can effectively deliver mRNA into the animal body and express it efficiently and continuously; (2) the delivered mRNA can be highly expressed in the spleen and lowly expressed in the liver; and (3) the delivered small nucleic acid drug is highly expressed in the spleen.

[0253] The LNP formulation was prepared according to the method in Example 3. The LNP formulation containing 10 μg of Fluc-mRNA was injected intravenously into 4-6 week old female BALB / c mice weighing 17-19 g. At specific time points after administration (6 h, 24 h, 48 h, and 7 d), the mice were intraperitoneally injected with a fluorescence imaging substrate. The mice were allowed free movement for 5 min, and then the mean radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed by the LNP-carrying mRNA in the mice was detected using an IVIS Spectrum small animal in vivo imaging system. The mean radiation intensity (corresponding to fluorescence expression intensity) of the protein expressed by the LNP-carrying mRNA in the mice, as detected by the IVIS Spectrum small animal in vivo imaging system, is shown in Table 4.

[0254] Table 4. Mouse in vivo imaging experimental data

[0255]

[0256]

[0257] As shown in Table 4, the LNP formulations prepared by R1, R2, and R6 significantly increased and sustained the expression of mRNA in mice compared to existing representative cationic lipids. For example, the mRNA expression level of the LNP formulation prepared by R6 in animals reached 5.07 times that of SM102 at 24 h, 7.69 times at 48 h, and 13.76 times at 7 days. The mRNA expression in mice was consistent with the cell transfection activity.

[0258] Furthermore, compared to compound R5, which has a similar structure but differs slightly in a few functional groups, the LNP formulations prepared from R1, R2, and R6 showed significantly increased mRNA expression intensity and duration in mice. For example, the mRNA expression level of the LNP formulation prepared from R6 in animals reached 115.70 times that of R5 at 24 hours, 203.38 times at 48 hours, and 116.77 times at 7 days. The mRNA expression in mice was consistent with its cell transfection activity.

[0259] 2. Distribution of liposomes in mice

[0260] This experiment further verified that the delivery vectors prepared by the cationic lipids designed in this invention, such as R1, R2 and R6, can be enriched in the mouse spleen. The delivered mRNA significantly enhances the protein expression level in the mouse spleen compared with existing representative cationic lipids, such as SM102, while the expression in the liver is very weak.

[0261] The specific experimental procedure is as follows:

[0262] The LNP formulation was prepared according to the method in Example 3. The LNP formulation containing 10 μg of Fluc-mRNA was injected intravenously into 4-6 week old female BALB / c mice weighing 17-19 g. At a specific time point (6 h) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. The mice were allowed free movement for 5 min. The mean radiation intensity (corresponding to fluorescence expression intensity) of the proteins expressed by the LNP-carrying mRNA in the mice was then detected using an IVIS Spectrum small animal in vivo imaging system. After sampling, the mice were euthanized by cervical dislocation, and their internal organs—heart, liver, spleen, lung, and kidney—were precisely isolated. The mean radiation intensity (corresponding to fluorescence expression intensity) of the proteins expressed by the LNP-carrying mRNA in each mouse organ was detected using an IVIS Spectrum small animal in vivo imaging system. The mouse in vivo imaging results are shown in Table 5.

[0263] Table 5. Organ imaging data of mice at specific time points (6 h) after drug administration.

[0264]

[0265] As shown in Table 5, the LNP formulations prepared from R1, R2, and R6 significantly increased mRNA expression levels in mouse spleen compared to the existing representative cationic lipid SM102. Specifically, the expression levels of R1, R2, and R6 in the spleen were 12.97-fold, 8.60-fold, and 7.19-fold, respectively, higher than those of SM102. The mRNA expression in the mouse spleen was consistent with the cell transfection results in Example 5. The expression levels of R1, R2, and R6 in the liver were very weak, at 0.08-fold, 0.02-fold, and 0.04-fold, respectively, higher than those of SM102. The ratio of delivered mRNA expression levels in the spleen and liver was only 0.09-fold for SM102, 13.82-fold for R1, 32.84-fold for R2, and 13.82-fold for R6.

[0266] The LNP formulation containing Fluc-mRNA prepared by the cationic lipids designed in this invention showed very large expression differences in different organs of mice. R1, R2 and R6 were expressed almost only in the spleen, a small amount in the liver, and none in other organs such as the heart, lungs and kidneys. SM102 was expressed in the liver and spleen, but none in the heart, lungs and kidneys.

[0267] In summary, this invention designs a series of cationic lipid compounds, among which R1, R2, and R6 significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly increase mRNA expression levels and duration in mice, and significantly increase expression levels in the spleen while significantly reducing expression levels in the liver, thereby improving delivery efficiency and achieving unexpected technical effects. This enables rapid in vivo induction of immune responses and antibody production from mRNA vaccines. It significantly enhances preventative efficacy without altering the vaccine composition, which has important clinical significance.

[0268] This invention, through unique design and screening, discovered a series of compounds, such as R1, R2, and R6, that can target mRNA delivery to the spleen while maintaining high efficiency and low toxicity. These compounds are not expressed in other organs, such as the lungs, heart, and kidneys, but are expressed in small amounts in the liver. The low efficiency of vaccine-induced immune responses is a reason why existing cancer therapeutic vaccines cannot achieve their maximum efficacy. The spleen is the largest secondary lymphoid organ in the body. LNP cancer vaccines targeting the spleen can effectively stimulate an immune response and significantly improve efficacy, thus having important clinical application significance in cancer treatment.

Claims

1. A carbamate compound or its N-oxide, solvate, isotopic compound, pharmaceutically acceptable salt or stereoisomer, characterized in that, The compound has the structure of Formula I. in, R1 is selected from CH and N; R2 is selected from CH, N, and O; G1 and G2 are selected from H and OH; G3 is selected from H and -(CH2). n OH or it may not be present; n is selected from 1, 2, and 3.

2. The carbamate compound or its N-oxide, solvate, isotopic compound, pharmaceutically acceptable salt or stereoisomer according to claim 1, characterized in that, In the aforementioned structure: R1 is selected from CH and N, and R2 is selected from N and O, and R1 and R2 are different; G1 and G2 are selected from H and OH, and G1 and G2 are the same; G3 is selected from -(CH2)2OH or does not exist.

3. The carbamate compound or its N-oxide, solvate, isotopic compound, pharmaceutically acceptable salt or stereoisomer according to claim 1, characterized in that, In the aforementioned structure: When R1 is selected from CH, R2 is selected from O or N; when R1 is selected from N, R2 is selected from O.

4. The carbamate compound or its N-oxide, solvate, isotopic compound, pharmaceutically acceptable salt or stereoisomer according to claim 1, characterized in that, The compound is selected from any one of the following compounds:

5. A lipid nanoparticle, characterized in that, It is constructed from the carbamate compound, neutral lipid, structural lipid and polymer conjugated lipid as described in claim 1, wherein the molar ratio between them is (25-75):(5-25):(15-65):(0.5-10).

6. A pharmaceutical composition, characterized in that, The drug is constructed from lipid nanoparticles according to claim 5, wherein the mass ratio of the lipid nanoparticles to the drug is 10:1 to 30:

1.

7. The pharmaceutical composition according to claim 6, characterized in that, The drug is selected from one or more of the following: nucleic acids, small molecule compounds, polypeptides, and proteins.

8. The pharmaceutical composition according to claim 7, characterized in that, The drug is selected from one or more of the following: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA.

9. The pharmaceutical composition according to claim 6, characterized in that, Its average particle size is 10-250 nm, and its polydispersity index is ≤50%.

10. Use of a carbamate compound of claim 1 or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, lipid nanoparticle of claim 5 or pharmaceutical composition of claim 6 in the preparation of a nucleic acid drug or gene vaccine drug.

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