Cationic lipid compound, composition containing the same, and uses thereof

Abstract

To provide cationic lipid compounds which can deliver nucleic acids with high cell transfection efficiency, low or no cell cytotoxicity, and high and sustained expression in animals.SOLUTION: The present disclosure provides a compound represented by formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. Further provided are a composition of the compound, and uses thereof in delivery of a therapeutic or prophylactic agent.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] This application claims priority to China Patent Application No. 202210034449.4, filed on January 13, 2022, the contents of which are incorporated herein by reference. [Technical field]

[0002] The present invention relates to the pharmaceutical field. In particular, the present invention relates to cationic lipid compounds, compositions containing same and uses thereof. [Background technology]

[0003] Effective targeted delivery of bioactive agents such as small molecule drugs, polypeptides, proteins, and nucleic acids, especially nucleic acid delivery, is a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to poor cell permeability and the high susceptibility of certain nucleic acid molecules (including RNA) to degradation.

[0004] Cationic lipid-containing compositions, liposomes and liposomal complexes (lipoplexes) have been demonstrated to be effective as delivery vehicles for delivering bioactive substances such as small molecule drugs, polypeptides, proteins and nucleic acids to cells and / or intracellular compartments. These compositions generally contain one or more "cationic" and / or amino (ionizable) lipids, as well as neutral lipids, structured lipids and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Although several types of such lipid-containing nanoparticle compositions have been demonstrated, there is still room for improvement in terms of safety, efficacy and specificity. It should be noted that the increased complexity of lipid nanoparticles (LNPs) can lead to increased production complexity and increased toxicity, which may be the main reasons limiting their clinical application. For example, LNP siRNA particles (e.g., patisiran) require prior administration of steroids and antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829). Therefore, there is a need to develop improved cationic lipid compounds and compositions containing the same that contribute to the delivery of therapeutic and / or prophylactic agents such as nucleic acids to cells. [Summary of the invention] [Problem to be solved by the invention]

[0005] The present disclosure is based at least on the finding that there is no clear correspondence between the structure of cationic lipid compounds and their intracellular transfection efficiency, cellular toxicity, and high and sustained expression in animals. Compounds with small structural differences have very large differences in transfection efficiency and / or cellular toxicity, and high intracellular expression. For example, compounds YK-009 and YK-010 of the present application have a nearly 60-fold difference in cell transfection efficiency and a 25% or greater difference in toxicity to transfected cells. Compounds YK-003 and YK-010 also have a nearly 50-fold difference in mouse in vivo expression and sustained expression.

[0006] Therefore, it is difficult to select suitable cationic lipid compounds that have high transfection efficiency and low toxicity to cells, and can be highly and persistently expressed in mouse body.The present disclosure has found through unique design that some compounds, such as YK-009, YK-003, YK-006, YK-008 and YK-011, can deliver nucleic acid with high cell transfection efficiency, low or no toxicity to cells, and high and persistent expression in animal body, compared with other compounds in the prior art, and obtain unexpected technical effects. [Means for solving the problem]

[0007] One aspect of the present disclosure provides novel cationic lipid compounds having formula (I) or an N-oxide, solvate, pharma-ceutically acceptable salt, or stereoisomer thereof:

[0008] [ka]

[0009] G1 is C 1~6 is an alkylene group, preferably an unsubstituted C 2~5 It is an alkylene group, more preferably an unsubstituted C3 alkylene group.

[0010] G2 is C 2~8 is an alkylene group, preferably an unsubstituted C 4~6 It is an alkylene group, more preferably an unsubstituted C5 alkylene group.

[0011] G3 is C 1~3 It is an alkylene group, preferably an unsubstituted C2 alkylene group.

[0012] L1 is C 6~15 A straight chain alkyl group, preferably an unsubstituted C 8~12 A straight chain alkyl group, more preferably an unsubstituted C 10 It is a straight chain alkyl group.

[0013] L2 is C 12~25 Branched alkyl groups, preferably unsubstituted C 14~22 Branched alkyl groups, more preferably unsubstituted C 18 It is a branched alkyl group. For example, L2 is

[0014] [ka] It is.

[0015] For example, a compound of formula (I) may have one of the following structures:

[0016] [ka] TIFF2025029048000004.tif251148

[0017] Another aspect of the present disclosure provides a composition comprising a carrier comprising the cationic lipid. For example, the molar ratio of the cationic lipid to the carrier is 30% to 70%.

[0018] In one embodiment, the carrier further comprises a neutral lipid. For example, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0019] In one embodiment, the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0020] For example, the neutral lipid may be 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycero (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0021] In a preferred embodiment, the neutral lipid is DOPE and / or DSPC.

[0022] In one embodiment, the carrier further comprises a structured lipid, for example, the molar ratio of the cationic lipid to the structured lipid is 1:1 to 5:1.

[0023] In one embodiment, the structured lipid is one or more selected from the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and adrenocortical hormones. In a preferred embodiment, the structured lipid is cholesterol.

[0024] In one embodiment, the carrier further comprises a polymer-conjugated lipid, for example, the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 5%.

[0025] In one embodiment, the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0026] For example, the polymer-conjugated lipid is one or more selected from the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0027] In one embodiment, the carrier comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5), preferably 50:10:38.5:1.5.

[0028] In one embodiment, the composition is a nanoparticle formulation, the nanoparticle formulation having an average particle size of 10 nm to 210 nm, preferably 100 nm to 205 nm, and the nanoparticle formulation having a polydispersity index of 50% or less, preferably 30% or less.

[0029] In one embodiment, the cationic lipid further comprises one or more other ionizable lipid compounds.

[0030] In one embodiment, the composition further comprises a therapeutic or prophylactic agent, for example, the mass ratio of the carrier to the therapeutic or prophylactic agent in the composition is 10:1 to 30:1.

[0031] In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1 to 25:1, preferably 16:1.

[0032] In one embodiment, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, and a protein. For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0033] In one embodiment, the therapeutic or prophylactic agent is a nucleic acid. For example, the therapeutic or prophylactic agent may be a deoxyribonucleic acid (DNA).

[0034] In one embodiment, the therapeutic or prophylactic agent is ribonucleic acid (RNA).

[0035] In one embodiment, the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof. For example, the RNA is mRNA.

[0036] In one embodiment, the composition further comprises one or more pharma- ceutically acceptable excipients or diluents.

[0037] Another aspect of the present disclosure provides a cationic lipid or composition as described above for delivery of a therapeutic or prophylactic agent to a patient in need thereof.

[0038] Another aspect of the present disclosure provides the use of the above cationic lipid or composition in the manufacture of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide drug, or a protein drug.

[0039] Another aspect of the present disclosure provides the use of the above cationic lipid or composition in the manufacture of a medicament for treating a disease or condition in a mammal in need thereof.

[0040] Another aspect of the present disclosure provides a cationic lipid or composition as described above for treating a disease or condition in a mammal in need thereof.

[0041] Another aspect of the present disclosure provides a method of treating or preventing a disease or condition comprising administering to a patient or subject in need thereof a therapeutically or prophylactically effective amount of the composition described above.

[0042] In one embodiment, the disease or condition is characterized by a dysfunction, an abnormal protein, or an abnormal polypeptide activity. For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. For example, the infectious disease is selected from the group consisting of a disease caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, or multiple types of herpes.

[0043] In a preferred embodiment, the mammal is a human. In one embodiment, the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. For example, the composition is administered subcutaneously.

[0044] In one embodiment, the therapeutic or prophylactic agent is administered to the mammal at a dose of about 0.001 mg / kg to about 10 mg / kg. In order to more clearly describe the technical solutions of the embodiments of the present disclosure, drawings of the embodiments are briefly introduced below. Of course, the drawings in the following description do not limit the present disclosure, but only relate to some examples of the present disclosure. [Brief description of the drawings]

[0045] [Figure 1] Figure 1 shows the results of cell transfection experiments in which different weight ratios of carriers (including YK-009) and mRNA were used to prepare LNP formulations, where a is carrier:mRNA=4:1, b is carrier:mRNA=16:1, and c is the blank control. [Diagram 2] Figure 2 shows the results of cell transfection experiments in which different molar ratios of cationic lipid YK-009 and neutral lipid DSPC were used to prepare LNP formulations: a, 1:1; b, 5:1; c, 10:1; and d, blank control. [Diagram 3] Figure 3 shows the results of cell transfection experiments with different molar ratios of polymer-conjugated lipid to carrier (containing YK-009) when preparing LNP formulations: a, 5%, b, 1.5%, and c, blank control. [Figure 4] Figure 4 shows the results of cell transfection experiments in which the ratios of each component of the carrier, cationic lipid YK-009, neutral lipid DSPC, structural lipid cholesterol, and polymer-conjugated lipid DMG-PEG2000, were different when preparing the prepared LNP formulation, a is 65:8:25:2, b is 50:10:38.5:1.5, and c is the blank control. [Diagram 5] FIG. 5 shows the fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared from different cationic lipids. [Figure 6]FIG. 6 shows the fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared from each cationic lipid YK-009 and compound 25, where the contents of Fluc-mRNA are 0.075 μg, 0.15 μg, 0.225 μg and 0.3 μg, respectively. [Figure 7] FIG. 7 shows the cell viability after 24 hours of culture in a cell culture medium containing LNP formulations of Fluc-mRNA prepared from different cationic lipids. [Figure 8] FIG. 8 shows the cell viability after 24 hours of culture in a cell culture medium containing LNP formulations of Fluc-mRNA prepared from each cationic lipid YK-009 and compound 25 (wherein the Fluc-mRNA contents were 0.375 μg, 0.75 μg, 1.125 μg, and 1.5 μg, respectively). [Figure 9] FIG. 9 shows the results of mouse in vivo imaging experiments for LNP formulations of Fluc-mRNA prepared from different cationic lipids. [Figure 10] FIG. 10 shows the results of a mouse bioimaging experiment on the LNP formulations of Fluc-mRNA prepared from each cationic lipid YK-009 and compound 25, in which the contents of Fluc-mRNA in the LNP formulations were different: a: 2.5 μg, b: 5 μg, c: 7.5 μg, and d: 10 μg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described below clearly and completely with reference to the drawings of the embodiments of the present disclosure. Of course, the described embodiments are only some of the embodiments of the present disclosure, and are not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by those skilled in the art without creative efforts shall be within the scope of protection of the present invention.

[0047] The present invention may be embodied in other specific forms without departing from the essential characteristics of the present invention. It is to be understood that any and all embodiments of the present invention may be combined with technical features of other embodiments or multiple other embodiments, unless inconsistent, to obtain another embodiment. The other embodiment obtained by such combination is encompassed by the present invention.

[0048] All publications and patents referred to in this disclosure are hereby incorporated by reference in their entirety, and in the event that the usage or terminology used in any incorporated publication or patent conflicts with the usage or terminology used in this disclosure, the usage and terminology in this disclosure shall control.

[0049] The section titles used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless otherwise stated, technical and scientific terms used herein have the ordinary meaning required to cover the field to which the subject belongs. In the event that there are multiple definitions for a term, the definition herein prevails.

[0050] It should be understood that all numerical values ​​of quantitative properties, such as dosage amounts, described in the specification and claims are modified in all cases by the word "about" unless otherwise specified in the embodiments or otherwise specified. It should also be understood that the numerical ranges exemplified in this application are intended to include all subranges subsumed within the ranges, and any combination of the end values ​​of the ranges or subranges.

[0051] As used in this disclosure, the terms "comprise", "contain", "include" and similar terms mean that the elements before the term cover the elements listed after the term and their equivalents, and do not exclude elements not listed. As used herein, the terms "comprise" or "include" may refer to open, semi-closed and closed systems. In other words, the terms also include "consisting essentially of" or "consisting of".

[0052] The term "pharmaceutical acceptable" in this application means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with the human or mammalian animal in which it is used for the prevention or treatment.

[0053] As used herein, the term "subject" or "patient" includes humans and mammals.

[0054] The term "treatment" as used herein refers to the administration of one or more drugs to a patient or subject suffering from a disease or a symptom of a disease to cure, alleviate, relieve, improve or affect the disease. In the context of this application, unless specifically stated to the contrary, the term "treat" may also include prophylaxis.

[0055] The term "solvate" as used herein refers to a complex formed between a compound of formula (I) or a pharma- ceutically acceptable salt and a solvent (e.g., methanol or water). Although solvates of compounds of formula I used to treat a disease or condition may confer different properties (including pharmacokinetic properties), when absorbed by a subject, the compound of formula I is obtained, and it is understood that the use of a compound of formula I includes the use of any solvate of the compound of formula I.

[0056] The term "hydrate" refers to the above term "solvate" in which the solvent is water.

[0057] It should be further understood that the compounds of formula I, or a pharma- ceutically acceptable salt thereof, can be isolated as solvates, and thus all such solvates are within the scope of the present invention. For example, the compounds of formula I, or a pharma- ceutically acceptable salt thereof, can exist in unsolvated forms as well as solvated forms with pharma- ceutically acceptable solvents (e.g., water, ethanol, and the like).

[0058] The term "pharmaceutically acceptable salt" refers to a non-toxic inorganic or organic acid addition salt of the compounds of the present disclosure. See, for example, SM Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Examples of organic acids include formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butanoic acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, D-glucaric acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfaminic acid, and the like. Acids that may be used include, for example, carboxylic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenesulfonic 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, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, and sulfosalicylic acid. For example, a compound of formula I can be used to form a pharma- ceutically acceptable salt with HCl (or hydrochloric acid), HBr (or hydrobromic acid), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid.

[0059] The nitrogen-containing compounds of formula (I) of the present disclosure can be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperbenzoic acid, hydrogen peroxide, ozone). Thus, where valence states and structure permit, the compounds claimed in this application include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0060] Certain compounds of the present disclosure may exist as one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Thus, the compounds claimed in the present disclosure further include racemic mixtures, single stereoisomers, and optically active mixtures. It will be understood by those skilled in the art that one stereoisomer may have better efficacy and / or fewer side effects than another stereoisomer. Single stereoisomers and optically active mixtures can be obtained by methods such as asymmetric synthesis, asymmetric catalysis, and chiral resolution. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral tartaric acid, chiral malic acid, or other chiral acid resolving agents can be added to form salts with the compounds of the present disclosure, which can be separated due to the different physicochemical properties of the products, such as solubility.

[0061] The present invention further includes all suitable isotopic variants of the compounds of the present disclosure. An isotopic variant is defined as a compound in which at least one atom has the same atomic number but is replaced by an atom having an atomic mass different from the atomic mass commonly or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of the present disclosure include, for example, 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N, 17 O and 18 Included are isotopes of hydrogen, carbon, nitrogen, and oxygen, such as O.

[0062] The term "alkyl group" is intended in this disclosure to include both branched and straight-chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. The term "alkylene group" is intended in this disclosure to include both branched and straight-chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. n~m refers to a group having n to m carbon atoms. For example, C 2~5 Alkylene groups include C2 alkylene groups, C3 alkylene groups, C4 alkylene groups, and C5 alkylene groups. The alkyl group (or alkylene group) may be unsubstituted, or the alkyl group (or alkylene group) may be substituted such that at least one hydrogen has been replaced with another chemical group.

[0063] A "therapeutically effective amount" is the amount of a therapeutic agent that is capable of improving a disease or condition when administered to a patient. A "prophylactically effective amount" is the amount of a prophylactic agent that is capable of preventing a disease or condition when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" will vary depending on the therapeutic / prophylactic agent, the disease state and its severity, and the age and weight of the patient / subject to be treated. Therapeutically effective amounts and prophylactically effective amounts can be routinely determined by one of ordinary skill in the art based on their knowledge and this disclosure.

[0064] In this application, when the name and structural formula of a compound are inconsistent, the structural formula takes precedence.

[0065] As used herein, the term "compounds according to the present disclosure" may include compounds of formula I, N-oxides, solvates, pharma- ceutically acceptable salts, stereoisomers, and mixtures thereof, depending on the context.

[0066] As used herein, the term "cationic lipid" refers to a lipid that is positively charged at a selected pH value. Cationic lipid bodies are easily combined with negatively charged nucleic acids, i.e., they interact with the negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs), which are currently one of the main delivery carriers.

[0067] The present inventors found that when screening a large number of compounds, it is very difficult to select a suitable cationic lipid compound that simultaneously meets the conditions of high transfection efficiency, low cytotoxicity, and high and sustained expression in mice. The present inventors found that some compounds, such as YK-009, YK-003, YK-006, YK-008, and YK-011, can deliver nucleic acids with high transfection efficiency in cells, low or no toxicity to cells, and high and sustained expression in animals, compared with the compounds of the prior art. Even compounds with small structural differences have very large differences in transfection efficiency and / or toxicity to cells, and high expression in cells. For example, the compounds YK-009 and YK-010 of the present application have a nearly 60-fold difference in cell transfection efficiency and a 25% or more difference in toxicity to transfected cells. In addition, compounds YK-003 and YK-010 have a nearly 50-fold difference in expression and sustained expression in mice.

[0068] Here, the cationic lipid DLin-MC3-DMA (MC3) disclosed in CN102625696B by Alnylam Pharmaceuticals, Inc. (NASDAQ: ALNY) was compared with the compound designed in the present invention. DLin-MC3-DMA (MC3) is now widely used for the delivery of mRNA in addition to efficient and safe delivery of siRNA. Structure of DLin-MC3-DMA (MC3) cationic lipid:

[0069] [ka]

[0070] In addition, the compounds disclosed herein were compared with structurally similar compound 23, compound 25 (i.e., SM-102, CAS No.: 2089251-47-6, a cationic lipid used in Moderna's COVID-19 vaccine mRNA-1273), and compound 27 in Patent CN110520409A filed by Moderna.

[0071] [ka]

[0072] As a result, it was found that the compounds according to the present disclosure are significantly improved in terms of transfection efficiency and / or toxicity to cells. For example, the cell transfection efficiency of compound YK-009 when transfected with mRNA is 40 times, 8 times, and 13 times or more higher than that of MC3, compound 23, and compound 27, respectively, and the toxicity to the transfected cells is 12%, 13%, and 16% lower than that of MC3, compound 23, and compound 27, respectively. Furthermore, it was found that the expression in mice was 25 times, 7 times, and 6 times higher than that of MC3, compound 23, and compound 27, respectively, and that compound YK-003 was 18 times, 8 times, and 7 times higher than that of MC3, compound 23, and compound 27.

[0073] Compared with compound 25, compound YK-009 of the present disclosure showed a 7-fold increase in cell transfection efficiency when transfected with mRNA, a 9% decrease in toxicity to transfected cells, and a more than 7-fold increase in expression in mice.

[0074] One aspect of the present disclosure provides a cationic lipid compound for delivering a novel therapeutic or prophylactic agent. The cationic lipid compound of the present disclosure can be used to deliver a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. Compared to known cationic lipid compounds, the cationic lipid compound of the present disclosure has high transfection efficiency, low cytotoxicity, and improved delivery efficiency and safety.

[0075] The present disclosure provides a cationic lipid that is a compound according to formula (I), or an N-oxide, solvate, pharma- ceutically acceptable salt, or stereoisomer thereof:

[0076] [ka] Here, G1 is C 1~6 is an alkylene group, preferably an unsubstituted C 2~5 It is an alkylene group, more preferably an unsubstituted C3 alkylene group. G2 is C 2~8 is an alkylene group, preferably an unsubstituted C 4~6 It is an alkylene group, more preferably an unsubstituted C5 alkylene group. G3 is C 1~3 It is an alkylene group, preferably an unsubstituted C2 alkylene group. L1 is C 6~15 A straight chain alkyl group, preferably an unsubstituted C 8~12 A straight chain alkyl group, more preferably an unsubstituted C 10 It is a straight chain alkyl group. L2 is C 12~25 Branched alkyl groups, preferably unsubstituted C 14~22 Branched alkyl groups, more preferably unsubstituted C 18 It is a branched alkyl group.

[0077] In one embodiment, G is unsubstituted C 2~5 It is an alkylene group, preferably an unsubstituted C3 alkylene group, such as -(CH2)3-.

[0078] In one embodiment, G2 is unsubstituted C 4~6 It is an alkylene group, preferably an unsubstituted C5 alkylene group, such as -(CH2)5-.

[0079] In one embodiment, G3 is an unsubstituted C2 alkylene group, ie, -(CH2)2-.

[0080] In one embodiment, L is unsubstituted C 8~12 A straight chain alkyl group, preferably an unsubstituted C 10 A straight chain alkyl group, for example -(CH2)9CH3.

[0081] In one embodiment, L2 is unsubstituted C 14~22 A branched alkyl group, preferably an unsubstituted C 18 It is a branched alkyl group. For example, L2 is

[0082] [ka] It is.

[0083] In one embodiment, G1 is -(CH2)3-, G2 is -(CH2)5-, G3 is -(CH2)2-, L1 is -(CH2)9CH3, and L2 is

[0084] [ka] It is.

[0085] In exemplary embodiments, the compound is selected from the group consisting of the following compounds or an N-oxide, solvate, pharma- ceutically acceptable salt, or stereoisomer thereof:

[0086] [ka] TIFF2025029048000011.tif249134

[0087] Another aspect of the present disclosure provides a composition comprising a carrier, the carrier comprising a cationic lipid, the cationic lipid comprising a compound represented by formula (I) above or an N-oxide, solvate, pharma-ceutically acceptable salt, or stereoisomer thereof.

[0088] In one embodiment, the composition is a nanoparticle formulation. The nanoparticle formulation has an average size of 10 nm to 210 nm, preferably 100 nm to 205 nm. The nanoparticle formulation has a polydispersity index of 50% or less, preferably 30% or less, more preferably 25% or less.

[0089] Cationic lipids In one embodiment of the composition / carrier according to the present disclosure, the cationic lipid is one or more selected from the group consisting of a compound represented by the above formula (I) or an N-oxide, solvate, pharma-ceutically acceptable salt, or stereoisomer thereof. In one embodiment, the cationic lipid is selected from the compound represented by the above formula (I). For example, the cationic lipid is compound YK-001, YK-002, YK-003, YK-004, YK-005, YK-006, YK-007, YK-008, YK-009, YK-010, or YK-011. In one preferred embodiment, the cationic lipid is compound YK-009.

[0090] In another embodiment of the composition / carrier of the present disclosure, the cationic lipid comprises one or more (a) selected from the group consisting of the compound represented by formula (I) above or its N-oxide, solvate, pharma-ceutically acceptable salt, or stereoisomer, and one or more other ionizable lipid compounds (b) different from (a). The (b) cationic lipid compound may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the (b) cationic lipid compound may be DLin-MC3-DMA (MC3). For example, the (b) cationic lipid compound may be compounds 23, 25, 27, etc. in CN110520409A.

[0091] In one embodiment, the molar ratio of the cationic lipid to the carrier is 30% to 70%, for example, 35%, 45%, 50%, 55%, 60%, or 65%.

[0092] The carrier is used to deliver an active ingredient, such as a therapeutic or prophylactic agent, The active ingredient may be encapsulated within the carrier or may be associated with the carrier.

[0093] For example, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNA includes, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0094] neutral lipid The carrier may comprise a neutral lipid, which in the present disclosure refers to a supplementary lipid that is uncharged or exists as a zwitterion at a selected pH value, which may improve efficacy by facilitating lipid phase transition, but may also affect target organ specificity.

[0095] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 10:1, for example, about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5:1.

[0096] For example, neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0097] The carrier component of the composition comprising cationic lipid can comprise one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids.Phospholipids can be assembled into one or more lipid bilayers.In general, phospholipids can comprise a phospholipid moiety and one or more fatty acid moieties.

[0098] The neutral lipid moiety may be optionally selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycero, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin, but is not limited thereto. The fatty acid moiety may be optionally selected from the group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid, but is not limited thereto. Also included are non-natural products consisting of natural products with modifications and substitutions such as branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds) or crosslinked with one or more alkynes. Under appropriate reaction conditions, alkynyl groups can undergo copper-catalyzed cycloaddition reactions when exposed to azides. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components, such as targeting or imaging moieties (e.g., dyes).

[0099] The neutral lipids used in these compositions are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycero (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSP) E), dipalmitoylphosphatidylethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0100] In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0101] structural lipids The carrier of the composition containing the cationic lipid may further contain one or more types of structured lipid. In the present disclosure, the structured lipid refers to a lipid that enhances the stability of the nanoparticle by filling the gaps between the lipids.

[0102] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 1.0:1, 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, 2.0:1.

[0103] The structured lipid may be selected from the group consisting of, but is not limited to, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0104] Polymer-conjugated lipids The carrier of the composition containing cationic lipid may further contain one or more polymer-conjugated lipids. Polymer-conjugated lipids mainly refer to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, controls nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing non-specific interactions with macrophages.

[0105] In one embodiment, the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG modification is usually 350 to 5000 Da.

[0106] For example, the polymer-conjugated lipid is one or more selected from the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0107] In one embodiment of the composition / carrier of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0108] In one embodiment of the composition / carrier according to the present disclosure, the carrier comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5), for example, (45-55):(9-11):(34-43):(0.5-2.5).

[0109] In one embodiment of the composition / carrier of the present disclosure, the carrier comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid to the neutral lipid to the structured lipid to the polymer-conjugated lipid is 50:10:38.5:1.5.

[0110] Therapeutic and / or prophylactic agents The composition may contain one or more therapeutic and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, for example, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

[0111] In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1 to 25:1, preferably 16:1.

[0112] The therapeutic or prophylactic agent may include, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0113] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0114] The carriers of the present disclosure enable delivery of therapeutic and / or prophylactic agents to mammalian cells or organs, and therefore the present disclosure also provides methods of treating a disease or condition in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and / or prophylactic agent and / or contacting a mammalian cell with the composition.

[0115] Therapeutic and / or prophylactic agents include biologically active substances, alternatively referred to as "active agents." Therapeutic and / or prophylactic agents are substances that, after delivery to a cell or organ, cause a desired change in the cell or organ, or in other body tissues or systems. Such substances can be used to treat one or more diseases, symptoms, or conditions. In some embodiments, the therapeutic and / or prophylactic agents are small molecule pharmaceutical agents for treating a particular disease, symptom, or condition. Examples of drugs that can be used in the compositions include anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, and the like), anti-inflammatory agents (e.g., cyclophosphamide, methotrexate, and streptozotocin ...arabinosides, anthracyclines, alkylating agents, platinum-based compounds, antimetabolites, and nucleoside analogues, such as methotrexate, and purine and pyrimidine analogues; anti-infectives; local anesthetics (such as dibucaine and chlorpromazine); beta-adrenergic blocking agents (such as propranolol, timolol, and labetalol); antihypertensives (such as clonidine and hydralazine); antidepressants (such as imipramine, amitriptyline, and doxepin); anticonvulsants (such as phenytoin); antihistamines (such as phenytoin); amines (e.g. diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g. gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g. miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B). B)) antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and contrast media.

[0116] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or other therapeutic and / or prophylactic agents. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, and the like. 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-153, and praseodymium. Vaccines include compounds and preparations that confer immunity against one or more symptoms associated with infectious diseases, such as influenza, measles, human papilloma virus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis, as well as mRNAs that encode antigens and / or epitopes from infectious diseases. Vaccines also include compounds and preparations that elicit an immune response against cancer cells, as well as mRNAs that encode antigens, epitopes, and / or neoepitopes from tumor cells.Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and / or compounds include those that can elicit an immune response by intramuscular administration of a composition comprising a compound of formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (III) (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122). Other therapeutic and / or prophylactic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanidine, cytarabine, and 5-dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptomycin, cyclosulfate, cyclopentasiloxane, cyclohexyl ether ... These include, but are not limited to, zotocin, mitomycin C and cis-dichlorodiaminoplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly called actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

[0117] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factors, luteinizing hormone-releasing hormone (LHRH) analogs, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0118] In some embodiments, the therapeutic agent is a polynucleotide or a nucleic acid (e.g., a ribonucleic acid or a deoxyribonucleic acid). The term "polynucleotide" in its broadest sense includes any compound and / or substance that can be presented in or incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA) and hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, and the like. In some embodiments, the therapeutic and / or prophylactic agent is RNA. The RNA that can be used in the compositions and methods described herein can be any selected from the group consisting of, but not limited to, shortmers, antagomirs, antisense RNA, ribozymes, 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.

[0119] In some embodiments, the therapeutic and / or prophylactic agent is an mRNA. The mRNA can encode a polypeptide of interest, including any natural, non-natural, or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0120] In other embodiments, the therapeutic and / or prophylactic agent is an siRNA. The siRNA can selectively reduce or downregulate the expression of a gene of interest. For example, the siRNA can be selected such that, when a composition comprising the siRNA is administered to a subject in need thereof, a gene associated with a particular disease, condition, or pathology is silenced. The siRNA can include a sequence complementary to an mRNA sequence that codes for a gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0121] In some embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, sgRNA-cas9 complex can affect the translation of cellular genetic mRNA.

[0122] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or carrier or plasmid encoded therein.shRNA is produced in target cell after appropriate construct is delivered to nucleus.The construct and mechanism of shRNA are well known in the relevant technical field.

[0123] Disease or condition The composition / carrier of the present disclosure can deliver a therapeutic or preventive agent to a subject or patient. The therapeutic or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. Therefore, the composition of the present disclosure can be used to manufacture a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide drug, or a protein drug. Since the therapeutic or preventive agent is diverse, the composition of the present disclosure can be used to treat or prevent various diseases or pathologies.

[0124] In one embodiment, the disease or condition is characterized by a dysfunction, an abnormal protein or an abnormal polypeptide activity.

[0125] For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0126] In one embodiment, the infectious disease is selected from the group consisting of a disease caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, and multiple types of herpes.

[0127] Other Ingredients The composition may include one or more ingredients other than those described in the previous 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.

[0128] The composition may also include one or more of a permeability enhancing molecule, a carbohydrate, a polymer, a surface-altering agent, or other moiety. The permeability enhancing molecule may be, for example, a molecule described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrate may include monosaccharides, (e.g., glucose), and polysaccharides, (e.g., glycogen and its derivatives and analogs).

[0129] Surface-altering agents include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., the cationic surfactant dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alpha, etc.). alfa, neltenexine and erdosteine, and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent may be disposed (e.g., by coating, adsorption, covalent attachment, or other methods) within and / or on the nanoparticles of the composition.

[0130] The composition may also include one or more functionalized lipids. For example, lipids can be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, lipid bilayers can be functionalized in this way with one or more groups that effectively facilitate membrane permeation, cell recognition, or imaging. The surface of the composition may further be conjugated with one or more useful antibodies. Functional groups and conjugates used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0131] In addition to these ingredients, the composition may contain any material used in pharmaceutical compositions. The composition may contain one or more pharma- ceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonicity agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives, flavors, and coloring agents. Excipients are, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st ed., AR Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0132] Examples of diluents 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.

[0133] In some embodiments, a composition comprising one or more lipids described herein further comprises one or more adjuvants, such as, for example, glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotide (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0134] The compositions of the present disclosure can be formulated as 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 disclosure can be prepared by methods well known in the pharmaceutical field.For example, sterile injections can be prepared by mixing the required amount of therapeutic or prophylactic agent and the other necessary components described above in a suitable solvent such as sterile distilled water, followed by filtration sterilization.Furthermore, surfactants can be added to promote the formation of a uniform solution or suspension.

[0135] For example, compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.

[0136] The composition of the present disclosure is administered in a therapeutically effective amount. The therapeutically effective amount may vary depending on the specific drug selected, the route of administration, the nature of the disease being treated, the age and condition of the patient, and is ultimately determined by the attending physician or clinician. For example, the therapeutic or prophylactic agent may be administered to a mammal (e.g., a human) at a dose of about 0.001 mg / kg to about 10 mg / kg. EXAMPLES

[0137] The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples. If no specific conditions are given in the examples, the examples may be carried out according to standard conditions or conditions recommended by the manufacturer. If no manufacturer specifies the reagents or equipment used, they may be conventional products available on the market.

[0138] Example 1: Synthesis of cationic lipid compounds 1. Synthesis of 2-octyldecyl 6-((4-(undecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)caproate (YK-001) The synthetic route is as follows:

[0139] [ka]

[0140] Step 1: Synthesis of n-undecyl 4-bromobutyrate (YK-001-PM1) n-Undecanol (5.00g, 29.02mmol) and 4-bromobutyric acid (5.14g, 30.78mmol) were dissolved in methylene chloride (40mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.67g, 34.82mmol) and 4-dimethylaminopyridine (177mg, 1.45mmol) were added to the above solution, and the mixture was reacted for 8 hours with stirring at 30-35°C. After the reaction was completed, the reaction solution was washed with saturated sodium carbonate and saturated saline, and dried with Na2SO4. The mixture was filtered, and the filtrate was concentrated under vacuum reduced pressure. The residue was purified by silica gel chromatography to obtain n-undecyl 4-bromobutyrate (6.68g, 20.79mmol, 71.64%). 1 H NMR (400MHz, CDCl3) δ4.08(t, J=6.8Hz, 2H), 3.47(t, J=6.5Hz, 2H), 2.50(t, J=7.2Hz, 2H), 2. 18(p, J=6.8Hz, 2H), 1.61(dd, J=14.2, 7.0Hz, 2H), 1.39-1.19(m, 16H), 0.88(t, J=6.9Hz, 3H).

[0141] Step 2: Synthesis of n-undecyl 4-((2-hydroxyethyl)amino)butyrate (YK-001-PM2) 4-Bromobutyric acid n-undecyl (2.71g, 8.43mmol) and ethanolamine (1.40g, 22.92mmol) were dissolved in acetonitrile (50mL). Potassium carbonate (3.17g, 22.92mmol) was added to the above system, heated to 70℃, and reacted for 2 hours with stirring. After the reaction was completed, the reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure in vacuum to remove the solvent. The residue was purified by silica gel chromatography to obtain 4-((2-hydroxyethyl)amino)butyric acid n-undecyl (1.47g, 4.88mmol, 57.89%). C 17 H 35 NO3, MS(ES): m / z(M+H + )302.2. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.67-3.60(m, 3H), 2.82-2.77(m, 2H), 2.69(t, J=7.0Hz, 2H), 2.38(t, J=7. 3Hz, 2H), 2.13-2.02(m, 3H), 1.84(p, J=7.2Hz, 2H), 1.66-1.53(m, 2H), 1.28(d, J=15.5Hz, 14H), 0.89(d, J=6.7Hz, 3H).

[0142] Step 3: Synthesis of 2-octyldecyl 6-bromohexanoate (YK-001-PM3) Using 6-bromohexanoic acid (2.60 g, 13.33 mmol) and 2-octyldecanol (3.00 g, 11.09 mmol) as raw materials, 2-octyldecyl 6-bromohexanoate (3.05 g, 6.82 mmol, 61.50%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ3.97(d, J=5.8Hz, 2H), 3.40(t, J=6.8Hz, 2H), 2.33(t, J=7.4Hz, 2H), 1.93-1.84(m, 2H) ), 1.66(dt, J=20.5, 7.4Hz, 3H), 1.48(m, J=8.6, 6.9, 4.2Hz, 2H), 1.35-1.19(m, 28H), 0.88(t, J=6.9Hz, 6H).

[0143] Step 4: Synthesis of 2-octyldecyl 6-((4-(undecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)caproate (YK-001) 2-Octyldecyl 6-bromohexanoate (200 mg, 0.45 mmol) and n-undecyl 4-((2-hydroxyethyl)amino)butyrate (102 mg, 0.34 mmol) were dissolved in acetonitrile (10 mL). Potassium carbonate (188 mg, 1.36 mmol) and potassium iodide (5 mg, 0.03 mmol) were added to the above system, and the mixture was heated to 70° C. and reacted for 20 hours with stirring. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under reduced pressure in vacuum to remove the solvent. The residue was purified by silica gel chromatography to obtain the target compound (36 mg, 0.054 mmol, 14.7%). C 41 H 81 NO5, MS(ES): m / z(M+H + )668.6. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 3.84-3.72(m, 1H), 3.53-3.48(m, 1H), 3.46-3.40(m, 1H), 2.42 (d, J=8.3Hz, 1H), 2.32(dd, J=15.2, 7.6Hz, 4H), 2.13-2.04(m, 1H), 1.70-1.57(m, 6H), 1.39-1.18(m, 53H), 0.88(t, J=6.8Hz, 9H).

[0144] 2. Synthesis of 2-octyldecyl 6-((5-(decyloxy)-5-oxopentyl)(2-hydroxyethyl)amino)caproate (YK-002) The synthetic route is as follows:

[0145] [ka]

[0146] Step 1: Synthesis of n-decyl 5-bromopentanoate (YK-002-PM1) Using 5-bromopentanoic acid (1.81 g, 10.00 mmol) and n-decanol (1.45 g, 9.16 mmol) as raw materials, n-decyl 5-bromopentanoate (2.50 g, 7.78 mmol, 84.93%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ4.07(t, J=6.8Hz, 2H), 3.41(t, J=6.6Hz, 2H), 2.34(t, J=7.3Hz, 2H), 1.96-1.85 (m, 2H), 1.83-1.73(m, 2H), 1.69-1.54(m, 2H), 1.31(dd, J=18.9, 15.6Hz, 14H), 0.88(t, J=6.9Hz, 3H).

[0147] Step 2: Synthesis of n-decyl 5-((2-hydroxyethyl)amino)pentanoate (YK-002-PM2) Using n-decyl 5-bromopentanoate (1.92 g, 5.98 mmol) and ethanolamine (0.31 g, 5.07 mmol) as raw materials, n-decyl 5-((2-hydroxyethyl)amino)pentanoate (0.34 g, 1.13 mmol, 18.90%) was obtained by the preparation method of YK-001-PM2. 17 H 35 NO3, MS(ES): m / z(M+H + )302.3. 1 H NMR (400MHz, CDCl3) δ4.05(dd, J=8.9, 4.7Hz, 1H), 4.00-3.92(m, 1H), 3.82-3.77(m, 2H), 3.63(t, J=6.7Hz, 2H), 3.58-3.51(m, 2H), 3.37(t, J=5.3Hz, 2H), 2.41(m, 2H), 1.84-1.76(m, 4H), 1.66-1.51(m, 4H), 1.28(d, J=14.2Hz, 12H), 0.88(t, J=6.8Hz, 3H).

[0148] Step 3: Synthesis of 2-octyldecyl 6-((5-(decyloxy)-5-oxopentyl)(2-hydroxyethyl)amino)caproate (YK-002) Using n-decyl 5-((2-hydroxyethyl)amino)pentanoate (151 mg, 0.50 mmol) and 2-octyldecyl 6-bromohexanoate (226 mg, 0.50 mmol) as raw materials, the target compound (216 mg, 0.32 mmol, 64.0%) was obtained by the preparation method of YK-001. 41 H 81 NO5, MS(ES): m / z(M+H + )668.5. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 3.66-3.64(m, 2H), 2.42(t, J=6.2Hz, 6H ), 2.32(q, J=7.1Hz, 4H), 1.88-1.76(m, 5H), 1.68-1.58(m, 7H), 1.37-1.20(m, 44H), 0.88(t, J=6.8Hz, 9H).

[0149] 3. Synthesis of 2-octyldecyl 6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)caproate (YK-003) The synthetic route is as follows:

[0150] [ka]

[0151] Step 1: Synthesis of n-decyl 6-bromohexanoate (YK-003-PM1) Using 6-bromohexanoic acid (1.95 g, 10.00 mmol) and n-decanol (1.45 g, 9.16 mmol) as raw materials, n-decyl 6-bromohexanoate (2.41 g, 7.19 mmol, 78.49%) was obtained by the preparation method of YK-001-PM1. 1H NMR (400MHz, CDCl3) δ4.06(t, J=6.7Hz, 2H), 3.41(t, J=6.8Hz, 2H), 2.32(t, J=7.4Hz, 2H), 1.94 -1.81(m, 2H), 1.73-1.56(m, 3H), 1.54-1.41(m, 2H), 1.39-1.19(m, 15H), 0.88(t, J=6.9Hz, 3H).

[0152] Step 2: Synthesis of n-decyl 6-((2-hydroxyethyl)amino)caproate (YK-003-PM2) 6-bromohexanoic acid n-decyl (1.12 g, 3.34 mmol) and ethanolamine (8.20 g, 134.25 mmol) were dissolved in ethanol (15 mL) and reacted at room temperature with stirring for 16 hours. The reaction solution was concentrated under reduced pressure in vacuum to remove the solvent, then diluted with ethyl acetate (80 mL) and washed with saturated saline (50 mL x 3). The organic phase was concentrated under reduced pressure and purified by silica gel chromatography to obtain 6-((2-hydroxyethyl)amino)caproic acid n-decyl (0.66 g, 2.09 mmol, 62.6%). C 18 H 37 NO3, MS(ES): m / z(M+H + )316.3. 1 H NMR (400MHz, CDCl3) δ4.05(t, J=6.8Hz, 2H), 3.93-3.88(m, 1H), 3.72-3.70(m, 1H), 3.66-3.56(m, 1H), 3.55-3.50(m, 1H), 3.34(s, 2 H), 2.83(d, J=5.1Hz, 1H), 2.72-2.68(m, 1H), 2.31(t, J=7.4Hz, 2H), 1.69-1.54(m, 6H), 1.35-1.21(m, 16H), 0.88(t, J=6.9Hz, 3H).

[0153] Step 3: Synthesis of 2-octyldecyl 6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)caproate (YK-003) Using 6-((2-hydroxyethyl)amino)n-decyl caproate (157 mg, 0.50 mmol) and 6-bromohexanoate-2-octyldecyl (226 mg, 0.50 mmol) as raw materials, the target compound (140 mg, 0.21 mmol, 42.00%) was obtained by the preparation method of YK-001. 42 H 83 NO5, MS(ES): m / z(M+H + )682.8. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 3.72(q, J=7.0Hz, 1H), 3.64(t, J=6 .6Hz, 1H), 2.32(td, J=7.4, 2.4Hz, 5H), 1.73-1.54(m, 14H), 1.41-1.19(m, 49H), 0.89(d, J=6.6Hz, 9H).

[0154] 4. Synthesis of 2-octyldecyl 4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyrate (YK-004) The synthetic route is as follows.

[0155] [ka]

[0156] Step 1: Synthesis of n-decyl 4-bromobutyrate (YK-004-PM1) Using 4-bromobutyric acid (15.00 g, 89.82 mmol) and 1-decanol (12.90 g, 81.50 mmol) as raw materials, 4-bromobutyric acid n-decyl ester (10.52 g, 34.2 mmol, 42.0%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ4.07(d, J=6.8Hz, 2H), 3.47(t, J=6.5Hz, 2H), 2.50(t, J=7.2Hz, 2H), 2. 18(dd, J=12.5, 5.5Hz, 2H), 1.62(t, J=7.2Hz, 2H), 1.30-1.20(m, 14H), 0.88(t, J=6.9Hz, 3H).

[0157] Step 2: Synthesis of n-decyl 4-((2-hydroxyethyl)amino)butyrate (YK-004-PM2) Using 4-bromobutyric acid n-decyl (8.00 g, 26.04 mmol) and ethanolamine (4.78 g, 78.26 mmol) as raw materials, 4-((2-hydroxyethyl)amino)butyric acid n-decyl (4.40 g, 15.3 mmol, 58.8%) was obtained by the preparation method of YK-001-PM2. 16 H 33 NO3, MS(ES): m / z(M+H + )288.2. 1 H NMR (400MHz, CDCl3) δ3.76(t, J=5.2Hz, 2H), 3.62(dd, J=8.3, 4.9Hz, 4H), 3.50(t, J=7.1Hz, 2H), 3.45-3.36(m, 2H), 2.91- 2.76(m, 2H), 2.44(d, J=8.0Hz, 2H), 2.08(dd, J=15.1, 7.5Hz, 2H), 1.67-1.51(m, 2H), 1.27(s, 12H), 0.88(t, J=6.5Hz, 3H).

[0158] Step 3: Synthesis of 2-heptylnonyl 4-bromobutyrate (YK-004-PM3) Using 2-heptylnonanol (390 mg, 1.45 mmol) and 4-bromobutyric acid (265 mg, 1.59 mmol) as raw materials, 4-bromobutyric acid-2-heptylnonyl acid (500 mg, 1.19 mmol, 82.07%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ3.99(d, J=5.8Hz, 2H), 3.47(t, J=6.5Hz, 2H), 2.51(t, J=7. 2Hz, 2H), 2.18(t, J=6.9Hz, 2H), 1.29(d, J=21.2Hz, 29H), 0.88(t, J=6.8Hz, 6H).

[0159] Step 4: Synthesis of 2-octyldecyl 4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyrate (YK-004) Using 4-bromobutyrate-2-heptylnonyl (250 mg, 0.60 mmol) and 4-((2-hydroxyethyl)amino)butyrate n-decyl (172 mg, 0.60 mmol) as raw materials, the target compound (50 mg, 0.081 mmol, 13.3%) was obtained by the preparation method of YK-001. 38 H 75 NO5, MS(ES): m / z(M+H + )626.6. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.9Hz, 2H), 3.61-3.58(m, 2H), 2.33( t, J=6.9Hz, 4H), 1.81(s, 3H), 1.65-1.55(m, 3H), 1.35-1.18(m, 50H), 0.87(d, J=7.0Hz, 9H).

[0160] 5. Synthesis of 8-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octane 9-heptadecyl (YK-005) The synthetic route is as follows:

[0161] [ka]

[0162] Step 1: Synthesis of 8-bromooctanoic acid-9-heptadecyl (YK-005-PM1) Using 8-bromooctanoic acid (2.87 g, 12.86 mmol) and 9-heptadecyl alcohol (3.00 g, 11.70 mmol) as raw materials, 8-bromooctanoic acid-9-heptadecyl (3.15 g, 6.82 mmol, 58.3%) was obtained by the preparation method of YK-001-PM1.

[0163] Step 2: Synthesis of 8-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octane 9-heptadecyl (YK-005) Using n-decyl 4-((2-hydroxyethyl)amino)butyrate (150 mg, 0.52 mmol) and 9-heptadecyl 8-bromooctanoate (285 mg, 0.62 mmol) as raw materials, the target compound (140 mg, 0.21 mmol, 40.4%) was obtained by the preparation method of YK-001. 41 H 81 NO5, MS(ES): m / z(M+H + )668.6. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.63(t, J=5.0Hz, 2H), 2.71(t, J=4.8Hz, 2H), 2.67-2.54(m, 4H), 2.35(t, J=7.1Hz, 2H), 2 .28(t, J=7.5Hz, 2H), 1.89-1.80(m, 2H), 1.61(dd, J=12.7, 5.9Hz, 5H), 1.50(d, J=5.9Hz, 6H), 1.38-1.18(m, 45H), 0.90-0.84(m, 9H).

[0164] 6. Synthesis of 2-octyldecyl 5-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)pentanoate (YK-006) The synthetic route is as follows:

[0165] [ka]

[0166] Step 1: Synthesis of 2-heptylnonyl 5-bromopentanoate (YK-006-PM1) Using 2-heptylnonanol (300 mg, 1.11 mmol) and 5-bromopentanoic acid (220 mg, 1.22 mmol) as raw materials, 5-bromopentanoic acid-2-heptylnonyl acid (438 mg, 1.01 mmol, 91.0%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ3.98(d, J=5.8Hz, 2H), 3.41(t, J=6.6Hz, 2H), 2.35(t, J=7.2Hz, 2H), 1.90( ddd, J=13.6, 7.7, 3.7Hz, 2H), 1.84-1.72(m, 2H), 1.29(d, J=21.6Hz, 29H), 0.88(t, J=6.8Hz, 6H).

[0167] Step 2: Synthesis of 2-octyldecyl 5-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)pentanoate (YK-006) Using 2-heptylnonyl 5-bromopentanoate (200 mg, 0.46 mmol) and n-decyl 4-((2-hydroxyethyl)amino)butyrate (133 mg, 0.46 mmol) as raw materials, the target compound (65 mg, 0.10 mmol, 21.7%) was obtained by the preparation method of YK-001. 39 H 77 NO5, MS(ES): m / z(M+H + )640.6. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 3.55-3.50(m, 2H), 2.65-2.45(m, 3H) )2.33(t, J=6.7Hz, 4H), 2.01(s, 1H), 1.61(d, J=6.8Hz, 10H), 1.39-1.10(m, 44H), 0.88(t, J=6.8Hz, 9H).

[0168] 7. Synthesis of 2-octyldecyl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)caproate (YK-007) The synthetic route is as follows:

[0169] [ka]

[0170] Step 1: Synthesis of 1-undecyl 6-bromohexanoate (YK-007-PM1) Using 6-bromohexanoic acid (2.50 g, 12.82 mmol) and 1-undecanol (2.00 g, 11.61 mmol) as raw materials, 1-undecyl 6-bromohexanoate (2.40 g, 6.87 mmol, 59.2%) was obtained by the preparation method of YK-001-PM1.

[0171] Step 2: Synthesis of undecyl 6-((2-hydroxyethyl)amino)hexanoate (YK-007-PM2) Using 1-undecyl 6-bromohexanoate (2.25 g, 6.44 mmol) and ethanolamine (1.18 g, 19.29 mmol) as raw materials, undecyl 6-((2-hydroxyethyl)amino)hexanoate (855 mg, 2.59 mmol, 40.2%) was obtained by the preparation method of YK-001-PM2. 19 H 39 NO3, MS(ES): m / z(M+H + )330.3.

[0172] Step 3: Synthesis of 2-octyldecyl 6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)caproate (YK-007) Using undecyl 6-((2-hydroxyethyl)amino)hexanoate (300 mg, 0.91 mmol) and 2-octyldecyl 6-bromohexanoate (488 mg, 1.09 mmol) as raw materials, the target compound (260 mg, 0.37 mmol, 41.1%) was obtained by the preparation method of YK-001. 43 H 85 NO5, MS(ES): m / z(M+H + )696.6. 1H NMR (400MHz, CDCl3) δ4.09-4.02 (m, 2H), 3.97 (d, J=5.8Hz, 2H), 3.53 -3.48(m, 2H), 2.35-2.29(m, 4H), 2.01(dd, J=12.6, 6.9Hz, 2H), 1.74-1.55(m, 6H), 1.28(d, J=14.7Hz, 58H), 0.89(d, J=6.5Hz, 9H).

[0173] 8. Synthesis of 2-octyldecyl 6-((4-(nonyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-008) The synthetic route is as follows:

[0174] [ka]

[0175] Step 1: Synthesis of n-nonyl 4-bromobutyrate (YK-008-PM1) Using n-nonanol (5.00 g, 34.66 mmol) and 4-bromobutyric acid (6.11 g, 36.59 mmol) as raw materials, n-nonyl 4-bromobutyrate (3.66 g, 12.5 mmol, 36.0%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ4.08(t, J=6.8Hz, 2H), 3.47(t, J=6.5Hz, 2H), 2.50(t, J=7.2Hz, 2H), 2. 18(p, J=6.7Hz, 2H), 1.61(dd, J=14.1, 7.0Hz, 2H), 1.40-1.19(m, 12H), 0.88(t, J=6.9Hz, 3H).

[0176] Step 2: Synthesis of n-nonyl 4-((2-hydroxyethyl)amino)butyrate (YK-008-PM2) Using 4-bromobutyric acid n-nonyl (2.46 g, 8.39 mmol) and ethanolamine (1.28 g, 20.96 mmol) as raw materials, 4-((2-hydroxyethyl)amino)butyric acid n-nonyl (1.22 g, 4.46 mmol, 53.2%) was obtained by the preparation method of YK-001-PM2. 15 H 31 NO3, MS(ES): m / z(M+H + )274.2. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.53-3.47(m, 3H), 2.68(t, J=7.0Hz, 2H), 2.43(t, J=8.1Hz, 2H) , 2.37(s, 1H), 2.12-2.01(m, 2H), 1.83(m, 2H), 1.67-1.51(m, 2H), 1.40-1.21(m, 10H), 0.88(t, J=6.9Hz, 3H).

[0177] Step 3: Synthesis of 2-octyldecyl 6-((4-(nonyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-008) Using 2-octyldecyl 6-bromohexanoate (200 mg, 0.45 mmol) and n-nonyl 4-((2-hydroxyethyl)amino)butyrate (102 mg, 0.37 mmol) as raw materials, the target compound (30 mg, 0.047 mmol, 13.5%) was obtained by the preparation method of YK-001. 39 H 77 NO5, MS(ES): m / z(M+H + )640.6. 1 H NMR (400MHz, CDCl3) δ4.06(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 3.68-3.62(m, 2H), 2.32(dd , J=15.2, 7.6Hz, 4H), 1.63(dd, J=15.1, 7.6Hz, 6H), 1.39-1.17(m, 52H), 0.87(d, J=7.0Hz, 9H).

[0178] 9. Synthesis of 2-octyldecyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-009) The synthetic route is as follows:

[0179] [ka]

[0180] Step 1: Synthesis of 2-octyldecyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-009) Using n-decyl 4-((2-hydroxyethyl)amino)butyrate (1.00 g, 3.48 mmol) and 2-octyldecyl 6-bromohexanoate (1.87 g, 4.18 mmol) as raw materials, the target compound (0.92 g, 1.41 mmol, 40.5%) was obtained by the preparation method of YK-001. 40 H 79 NO5, MS(ES): m / z(M+H + )654.6. 1 H NMR (400MHz, CDCl3) δ4.07(t, J=6.8Hz, 4H), 3.96(d, J=5.8Hz, 2H), 2.49(t, J=5.7Hz, 2H), 2.34(t, J=7.3Hz, 2H), 2.27 -2.17(m, 2H), 2.01(d, J=5.7Hz, 2H), 1.68-1.62(m, 9H), 1.46-1.43(m, 3H), 1.36-1.15(m, 44H), 0.87(d, J=7.0Hz, 9H).

[0181] 10. Synthesis of 2-heptylnonyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-010) The synthetic route is as follows:

[0182] [ka]

[0183] Step 1: Synthesis of 2-heptylnonyl 6-bromohexanoate (YK-010-PM1) Using 6-bromohexanoic acid (290 mg, 1.49 mmol) and 2-heptylnonanol (300 mg, 1.24 mmol) as raw materials, 2-heptylnonyl 6-bromohexanoate (280 mg, 0.67 mmol, 54.0%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ3.97(d, J=5.8Hz, 2H), 3.41(t, J=6.8Hz, 2H), 2.33(t, J=7.4Hz, 2H), 1.93-1.84 (m, 2H), 1.66(dt, J=20.5, 7.4Hz, 3H), 1.52-1.43(m, 2H), 1.36-1.20(m, 24H), 0.88(t, J=6.9Hz, 6H).

[0184] Step 2: Synthesis of 2-heptylnonyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-010) Using n-decyl 4-((2-hydroxyethyl)amino)butyrate (150 mg, 0.52 mmol) and 2-heptylnonyl 6-bromohexanoate (260 mg, 0.62 mmol) as raw materials, the target compound (150 mg, 0.24 mmol, 46.2%) was obtained by the preparation method of YK-001. 38 H 75 NO5, MS(ES): m / z(M+H + )626.7. 1 H NMR (400MHz, CDCl3) δ4.27(qd, J=11.0, 5.8Hz, 2H), 4.13-4.01(m, 2H), 3.97(d, J=5.8Hz, 2H), 2.33(dt, J=12.5, 7.3Hz, 4H), 1.86-1.81(m, 1H), 1.78-1.68(m, 2H), 1.67-1.59(m, 4H), 1.33-1.17(m, 49H), 0.88(t, J=4.6Hz, 9H).

[0185] 11. Synthesis of 2-hexyloctyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-011) The synthetic route is as follows:

[0186] [ka]

[0187] Step 1: Synthesis of 2-hexyloctyl 6-bromohexanoate (YK-011-PM1) Using 6-bromohexanoic acid (290 mg, 1.49 mmol) and 2-hexyloctanol (300 mg, 1.40 mmol) as raw materials, 2-hexyloctyl 6-bromohexanoate (240 mg, 0.61 mmol, 41.6%) was obtained by the preparation method of YK-001-PM1. 1 H NMR (400MHz, CDCl3) δ3.97(d, J=5.8Hz, 2H), 3.41(t, J=6.8Hz, 2H), 2.33(t, J=7.4Hz, 2H), 1.92-1.83(m, 2H) , 1.66(dt, J=20.5, 7.4Hz, 3H), 1.48(ddd, J=8.6, 6.8, 4.2Hz, 2H), 1.37-1.20(m, 20H), 0.88(t, J=6.8Hz, 6H).

[0188] Step 2: Synthesis of 2-hexyloctyl 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-011) Using n-decyl 4-((2-hydroxyethyl)amino)butyrate (150 mg, 0.52 mmol) and 2-hexyloctyl 6-bromohexanoate (240 mg, 0.61 mmol) as raw materials, the target compound (120 mg, 0.20 mmol, 38.5%) was obtained by the preparation method of YK-001. 36 H 71 NO5, MS(ES): m / z(M+H + )598.5. 1H NMR (400MHz, CDCl3) δ4.09-4.03(m, 4H), 3.56-3.53(m, 2H), 2.41(t, J=6.8Hz, 2H), 2.22(t, J= 7.1Hz, 2H), 1.71-1.63(m, 3H), 1.58-1.54(m, 2H), 1.42-1.19(m, 47H), 0.88(t, J=6.8Hz, 9H).

[0189] 12. Synthesis of 8-((2-hydroxyethyl)(10-((9-heptadecyl)oxy)-10-oxodecyl)amino)nonyl octanoate (Compound 23) The synthetic route is as follows:

[0190] [ka]

[0191] Step 1: Synthesis of n-nonyl 8-bromooctanoate (compound 23-PM1) Using 8-bromooctanoic acid (2.50 g, 11.21 mmol) and n-nonanol (1.47 g, 10.19 mmol) as raw materials, 8-bromooctanoic acid n-nonyl ester (1.85 g, 5.30 mmol, 52.0%) was obtained by the preparation method of YK-001-PM1.

[0192] Step 2: Synthesis of 8-((2-hydroxyethyl)amino)nonyl octanoate (compound 23-PM2) Using the above-prepared 8-bromooctanoate (1.50 g, 4.29 mmol) and ethanolamine (7.96 g, 130.32 mmol) as raw materials, 8-((2-hydroxyethyl)amino)nonyl octanoate (610 mg, 1.85 mmol, 43.1%) was obtained by the method of YK-001-PM2. 19 H 39 NO3, MS(ES): m / z(M+H + )330.3.

[0193] Step 3: Synthesis of 10-bromodecanoic acid-9-heptadecyl (compound 23-PM3) Using 10-bromodecanoic acid (3.23 g, 12.86 mmol) and 9-heptadecanol (3.00 g, 11.70 mmol) as raw materials, 10-bromodecanoic acid-9-heptadecyl (3.10 g, 6.72 mmol, 57.4%) was obtained by the preparation method of YK-001-PM1.

[0194] Step 4: Synthesis of nonyl 8-(10-((9-heptadecyl)oxy)-10-oxodecyl)amino)octanoate (compound 23) Using 8-((2-hydroxyethyl)amino)nonyl octanoate (500 mg, 1.52 mmol) and 10-bromodecanoate-9-heptadecyl (840 mg, 1.72 mmol) as raw materials, the target compound (520 mg, 0.70 mmol, 46.1%) was obtained by the preparation method of YK-001. 46 H 91 NO5, MS(ES): m / z(M+H + )738.8. 1 H NMR (400MHz, CDCl3) δ4.88-4.86(m, 1H), 4.05(t, J=6.8Hz, 2H), 3.97(d, J=5.8Hz, 2H), 2.77-2.69(m ,5H), 2.25(m,4H), 1.61(dd, J=13.5, 6.6Hz, 13H), 1.38-1.18(m, 55H), 0.88(t, J=6.8Hz, 9H).

[0195] 13. Synthesis of 8-((2-hydroxyethyl)(6-((9-heptadecyl)oxy)-6-oxohexyl)amino)nonyl octanoate (Compound 27) The synthetic route is as follows:

[0196] [ka]

[0197] Step 1: Synthesis of 6-bromohexanoic acid-9-heptadecyl (compound 27-PM1) Using 6-bromohexanoic acid (2.51 g, 12.87 mmol) and 9-heptadecanol (3.00 g, 11.70 mmol) as raw materials, 6-bromohexanoic acid-9-heptadecyl (2.77 g, 6.39 mmol, 54.6%) was obtained by the preparation method of YK-001-PM1.

[0198] Step 2: Synthesis of nonyl 8-(6-((9-heptadecyl)oxy)-6-oxohexyl)amino)octanoate (compound 27) Using 8-((2-hydroxyethyl)amino)nonyl octanoate (500 mg, 1.52 mmol) and 6-bromohexanoate-9-heptadecyl (760 mg, 1.75 mmol) as raw materials, the target compound (471 mg, 0.69 mmol, 45.4%) was obtained by the preparation method of YK-001. 42 H 83 NO5, MS(ES): m / z(M+H + )682.6. 1 H NMR (400MHz, CDCl3) δ4.83-4.81(m, 1H), 4.05-4.00(m, 2H), 3.77-3.69(m, 2H), 2.75-2.59(m, 5H ), 2.25-2.19(m, 4H), 1.59(dd, J=13.3, 6.3Hz, 13H), 1.34-1.03(m, 47H), 0.87(t, J=6.7Hz, 9H).

[0199] Example 2: Optimization of preparation conditions for nano lipid particles (LNP formulation) 1. Optimization of the ratio of carrier (liposome) to mRNA

[0200] [ka]

[0201] The cationic lipid compound YK-009 synthesized in Example 1, DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution. The ethanol lipid solution was quickly added with citrate buffer (pH = 4-5) by ethanol injection method and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Origin Experimental Reagents Co., Ltd.) was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared using fixed volumes of liposome solution and mRNA aqueous solution so that the weight ratio of total lipid to mRNA was 4:1, 10:1, 16:1, 24:1, and 30:1, respectively. The solution was sonicated at 25 °C for 15 minutes (ultrasonic frequency 40 kHz, ultrasonic output 800 W). The resulting liposomes were diluted 10 times with PBS, then ultrafiltered in a 300KDa ultrafiltration tube to remove ethanol, and then adjusted to a fixed volume with PBS to obtain an LNP formulation encapsulating eGFP-mRNA using the cationic lipid YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar percentage 50:10:38.5:1.5).

[0202] The results of the cell transfection experiment showed that when the weight ratio of the carrier to the mRNA was in the range of 10:1 to 30:1, all the transfection effects were good. Among them, the transfection effect was the best when the weight ratio was in the range of 16:1, but when the weight ratio was 4:1, the transfection effect was low, so this ratio could not be used for carrying the mRNA (Figure 1).

[0203] 2. Optimizing the ratio of cationic lipids to neutral lipids According to the method in section 1, LNP formulations encapsulating eGFP-mRNA were prepared, in which the molar ratios of cationic lipid YK-009 and neutral lipid DSPC were 1:1, 5:1, and 10:1, respectively. Cell transfection experiments showed that when the molar ratio of cationic lipid to neutral lipid was between 1:1 and 10:1, all of them had a transfection effect, and among them, when the molar ratio was 5:1, the transfection efficiency was the highest (Figure 2).

[0204] 3. Optimization of the ratio of polymer-conjugated lipid to carrier (liposome) Using the method described in Section 1, LNP formulations encapsulating eGFP-mRNA were prepared, in which the cationic lipid in the carrier was YK-009, and the molar ratios of the polymer-conjugated lipid DMG-PEG2000 to the carrier were 0.5%, 1.5%, 2.5%, 3.5%, and 5%, respectively.

[0205] Cell transfection experiments showed that all molar ratios of polymer-conjugated lipid to carrier in the range of 0.5% to 5% were effective for transfection, and the transfection efficiency was highest when the molar ratio was 1.5% and lowest when the molar ratio was 0.5% (Figure 3).

[0206] 4. Optimization of the ratio of each component in the carrier (liposome) Using the method described in Section 1, LNP formulations encapsulating eGFP-mRNA were prepared, with the molar ratios of cationic lipid YK-009, neutral lipid DSPC, structural lipid cholesterol, and polymer-conjugated lipid DMG-PEG2000 being 65:8:25:2, 50:10:38.5:1.5, 40:17.5:40:2.5, and 25:35:35:5, respectively.

[0207] Cell transfection experiments showed that the highest transfection efficiency was achieved when the molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids was 50:10:38.5:1.5, whereas the transfection efficiency was slightly worse when the molar ratio was 65:8:25:2, but transfection was still possible (Figure 4).

[0208] Example 3: Cell transfection experiments with LNP formulations of eGFP-mRNA Cell recovery and passaging: 293T cells were recovered, cultured in culture dishes, and passaged to the required cell number. Seeding plates: The cells in the culture dishes were digested and counted. The cells were plated at 10,000 per well in 96-well plates and 150,000 per well in 12-well plates, and then cultured overnight until the cells attached to the walls.

[0209] Cell transfection experiment: The LNP formulation containing 1.5 μg of eGFP-mRNA prepared in Example 2 (the cationic lipid in the carrier is YK-009) and the Lipofectamin2000 formulation containing eGFP-mRNA were added to the cell culture medium of a 12-well plate, respectively. After continuing the culture for 24 h, the cells were observed under a fluorescent microscope, and the transfection efficiency of various samples was investigated by the fluorescence intensity.

[0210] From the results of the experiment, the preparation conditions of nanolipid particles (LNP formulation) were finally determined. That is, the ratio of carrier to mRNA was 16:1, the molar ratio of cationic lipid to neutral lipid was 5:1, the polymer-conjugated lipid in the liposome was 1.5%, and the molar ratio of cationic lipid to neutral lipid to structural lipid to polymer-conjugated lipid was 50:10:38.5:1.5. The nanolipid particles (LNP formulation) were prepared in the following experiment.

[0211] Example 4: Preparation of Nano-Lipid Particles (LNP Formulation) (Optimal Ratio)

[0212] [Table 1] TIFF2025029048000027.tif243170TIFF2025029048000028.tif106170The cationic lipids listed in Table 1, DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution. The ethanol lipid solution was quickly added with citrate buffer (pH = 4-5) by the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Origin Experimental Reagent Co., Ltd.) or Fluc-mRNA (purchased from Shanghai Origin Experimental Reagent Co., Ltd.) was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared using a fixed volume of liposome solution and an aqueous mRNA solution so that the weight ratio of total lipid to mRNA was 16:1. The solution was sonicated at 25°C for 15 min (ultrasonic frequency 40 kHz, ultrasonic output 800 W). The obtained liposomes were diluted to 10 times the volume with PBS, and then ultrafiltered with a 300 KDa ultrafiltration tube to remove ethanol. Then, the volume was fixed with PBS to obtain LNP formulations encapsulating eGFP-mRNA or Fluc-mRNA using cationic lipid YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar percentage 50:10:38.5:1.5).

[0213] Example 5: Measurement of particle size and polydispersity index (PDI) of nanolipid particles Particle size and polydispersity index (PDI) were measured by dynamic light scattering using a Malvern laser particle size analyzer. 10 μL of liposome solution was taken out, diluted to 1 mL with RNase-free deionized water, and added to the sample pool. Each sample was measured in triplicate. The measurement conditions were a scattering angle of 90° and 25°C. The measurement results are shown in the table below.

[0214] [Table 2]

[0215] The particle diameter of the nanolipid particles prepared in Example 4 was 110-210 nm, and all of them could be used for delivery of mRNA. And the particle diameter of the particles prepared from compound 23 and YK-003 was the smallest, 114.12 nm and 119.91 nm, respectively, while the particle diameter of the particles prepared from YK-008 and MC3 was the largest, 205.00 nm and 205.20 nm, respectively. The polydispersity index of all nanolipid particles was 5%-30%, with the polydispersity index of YK-004 being the smallest at 9.7% and the polydispersity index of YK-002 being the largest at 27.7%.

[0216] Example 6: Validation of the performance of LNP delivery vehicles in vitro Cell recovery and passaging: The same as in Example 3. Seeding plate: same as in Example 3.

[0217] 1.Fluorescence measurement of Fluc-mRNA The LNP formulation containing 0.3 μg of Fluc-mRNA (the carrier components of the LNP formulation are cationic lipids, neutral lipids, structural lipids and polymer-conjugated lipids in a molar ratio of 50:10:38.5:1.5, where the cationic lipids are the cationic lipids listed in Table 1) was added to the 96-well plate cell culture medium, and after further culturing for 24 hours, the corresponding reagent was added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence measurement system. In this experiment, the transfection efficiency of the LNP formulation in cells was verified. The results are shown in Table 3.

[0218] In addition, to compare the transfection efficiency of LNP formulations prepared from cationic lipid YK-009 and compound 25 in cells, LNP formulations with Fluc-mRNA contents of 0.3 μg, 0.225 μg, 0.15 μg and 0.075 μg, respectively, were prepared (the carrier components of the LNP formulation are cationic lipid, neutral lipid, structured lipid and polymer-conjugated lipid in a molar ratio of 50:10:38.5:1.5, where the cationic lipid is YK-009 or compound 25), and the intracellular transfection activity of the prepared LNP formulations was detected by the same method as above. The results are shown in Table 4.

[0219] [Table 3]

[0220] [Table 4]

[0221] As can be seen from Table 3 and Figure 5, among the LNP formulations of Fluc-mRNA prepared from different cationic lipids, YK-009 had the strongest fluorescence absorption, with an RLU value of 5479373, and YK-001, YK-002, YK-005, YK-006 and YK-008 also had very strong fluorescence absorption, with an RLU value of 10 6 ~10 7 The RLU values ​​of YK-009, YK-006, YK-001, YK-005, YK-002 and YK-008 were 8.4, 4.1, 3.4, 2.2, 2.0 and 1.9 times that of compound 23, respectively. YK-010 had the weakest fluorescence absorption, with an RLU value of 93801. The fluorescence absorption of YK-004, compound 23, compound 27 and MC3 was also very weak. The RLU values ​​of YK-009 were 58, 39, 8, 13 and 42 times that of YK-010, YK-004, compound 23, compound 27 and MC3, respectively.

[0222] From Table 4 and Figure 6, it can be seen that there was a significant difference in the fluorescence absorption of the LNP formulations prepared from YK-009 and compound 25, and the fluorescence absorption of the LNP formulations containing 0.075 μg, 0.15 μg, 0.225 μg and 0.3 μg of Fluc-mRNA and the YK-009 formulation was 3.7 times, 4.0 times, 7.0 times and 4.4 times that of the compound 25 formulation, respectively.

[0223] The data was analyzed using GraphPad Prism software. Here, YK-009, YK-006, YK-001, YK-005, YK-002 and YK-008 were significantly different from compound 23, and YK-009, YK-006, YK-001, YK-005, YK-002 and YK-008 were significantly different from compound 27. All compounds except YK-004 and YK-010 were significantly different from MC3. The LNP formulations containing 0.075 μg, 0.15 μg, 0.225 μg and 0.3 μg of Fluc-mRNA, YK-009, were all significantly different from compound 25.

[0224] In terms of structure, compared with YK-009, YK-001 has one more C in the L1 group but the other structures are completely the same, YK-002 has one more C in the G1 group but the other structures are completely the same, YK-004 has two less C in the G2 group but the other structures are completely the same, YK-006 has one less C in the G2 group but the other structures are completely the same, YK-010 has one less C in each of the two strands of the L2 group but the other structures are completely the same, and compound 23 has one less C in the G1 group. Compound 25 has 2 more C's in the G1 group, 1 more C's in the L1 group, 2 more C's in the G2 group, and 1 less C's in the single strand of the L2 group, but the other structures are exactly the same; compound 27 has 4 more C's in the G1 group, 1 less C's in the L1 group, and 1 less C's in the single strand of the L2 group, but the other structures are exactly the same.

[0225] This shows that there is no correspondence between the structure of a compound and its transfection efficiency in cells, and compounds with small structural differences are likely to have very large differences in transfection efficiency. For example, compared with YK-009, YK-010, YK-004, compound 23, and compound 27 have slightly different structures, but the cell transfection efficiency of YK-009 was 58 times, 39 times, 8 times, and 13 times that of these cationic lipid compounds, respectively. YK-009 and compound 25 have only slight structural differences, and the difference in cell transfection efficiency was as much as 7 times. Therefore, it is not true that compounds with similar structures must have similar transfection efficiency, and the transfection efficiency may be significantly different. It is not easy to select cationic lipid compounds with high transfection efficiency, and various designs and many creative efforts are required.

[0226] 2. Measurement of Cell Viability LNP formulation containing 1.5 μg of Fluc-mRNA (the carrier components of the LNP formulation are cationic lipids, neutral lipids, structural lipids and polymer-conjugated lipids in a molar ratio of 50:10:38.5:1.5, where the cationic lipids are the cationic lipids listed in Table 1) was added to the cell culture medium of a 96-well plate and further cultured for 24 hours, after which 10 μL of CCK-8 solution was added to each well and the culture plate was incubated in an incubator for 1 hour. The absorbance at 450 nm was measured with a microplate reader. The results are shown in Table 4.

[0227] To compare the cytotoxicity of LNP formulations prepared from cationic lipid YK-009 and compound 25, LNP formulations with Fluc-mRNA contents of 1.5 μg, 1.125 μg, 0.75 μg and 0.375 μg were prepared (the carrier components of the LNP formulation are cationic lipid, neutral lipid, structured lipid and polymer-conjugated lipid in a molar ratio of 50:10:38.5:1.5, where the cationic lipid is YK-009 or compound 25). The method for measuring cell viability is the same as above. The results are shown in Table 6.

[0228] [Table 5]

[0229] [Table 6]

[0230] From Table 5 and Figure 7, it can be seen that the LNP formulations of Fluc-mRNA prepared by different cationic lipids have very different cytotoxicity. The cell viability was the highest for YK-009 (100%), followed by YK-011 (98%), but was 77% for YK-010, 84% for YK-003, 85% for YK-001, 87% for compound 23, 84% for compound 27, and 88% for MC3. These were obviously lower than YK-009.

[0231] From Table 6 and Figure 8, it can be seen that the LNP formulations prepared from YK-009 and compound 25 had very different inhibitory activities against cells. Here, the LNP formulations and YK-009 formulations with Fluc-mRNA contents of 1.5 μg, 1.125 μg, 0.75 μg and 0.375 μg all had 100% cell viability, but the inhibitory activity of the compound 25 formulation against cells was up to 9% lower than that of the YK-009 formulation. The data was analyzed using GraphPad Prism software, where YK-009, YK-011, YK-006 and YK-007 had significant differences in cytotoxicity compared to compound 23, compound 27 and MC3.

[0232] From the above experimental results, it was found that there is no corresponding relationship between the structure of cationic lipid compounds and cytotoxicity, and compounds with small structural differences have very large differences in cytotoxicity. For example, compared with YK-009, YK-010 has one less C in each double chain of the L2 group, and the other structures are completely identical, but the cell viability decreased by 23%. YK-002 has one more C in the G1 group, and the other structures are completely identical, but the cell viability decreased by 11%. Compound 23 has four more C in the G1 group, one less C in the L1 group, four more C in the G2 group, and one less C in the single chain of the L2 group, and the other structures are completely identical, but the cell viability decreased by 13%. Compound 25 has two more Cs in the G1 group, one more Cs in the L1 group, two more Cs in the G2 group, and one less Cs in the single chain of the L2 group, and the other structures are completely identical, but the cell viability was reduced by 9%. Compound 27 has four more Cs in the G1 group, one less Cs in the L1 group, and one less Cs in the single chain of the L2 group, and the other structures are completely identical, but the cell viability was reduced by 16%. YK-009 has a similar structure to YK-010, YK-002, compound 23, compound 27, and compound 25, but its cytotoxicity was low at 23%, 11%, 13%, 16%, and 9%, respectively. Thus, it was found that cationic lipids with small structural differences may cause significantly different cytotoxicity.

[0233] Example 7: Verification of the performance of cationic lipids (LNPs) as delivery carriers in vivo In addition, we also verified the protein expression and duration of the mRNA delivered to mice by the cationic lipid delivery carrier designed by the present inventors.In vivo experiments further demonstrated that the LNP delivery carrier of the present invention can effectively deliver mRNA into the body and efficiently and sustainably express it.

[0234] An LNP formulation containing 10 μg of Fluc-mRNA was intramuscularly injected into female BALB / C mice aged 4 to 6 weeks and weighing 17 to 19 g. After administration, the mice were intraperitoneally injected with a fluorescent imaging substrate at predetermined time points (3 h, 6 h, 24 h, 48 h, and 72 h). After allowing the mice to move freely for 5 min, a small animal live imager was used to detect the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed in the mice by the mRNA carried by the LNPs. The detection results are shown in Table 5 and Figure 7.

[0235] To compare the protein expression and duration in mice of the LNP formulations prepared from cationic lipid YK-009 and compound 25, LNP formulations with Fluc-mRNA contents of 10 μg, 7.5 μg, 5 μg and 2.5 μg were prepared, respectively (the carrier components of the LNP formulation are cationic lipid, neutral lipid, structured lipid and polymer-conjugated lipid in a molar ratio of 50:10:38.5:1.5, where the cationic lipid is YK-009 or compound 25). The experimental method of mouse bioimaging is the same as above. The results are shown in Table 8.

[0236] [Table 7]

[0237] [Table 8]

[0238] From Table 7 and Figure 9, it can be seen that the expression intensity of the LNP formulations of Fluc-mRNA prepared from different cationic lipids in mice is significantly different. From 3h to 72h, the expression intensity of YK-003 and YK-009 was the highest, indicating that the LNP formulations prepared by them were highly and persistently expressed in vivo. At 3h, the average radiation intensity of YK-003 and YK-009 was 1500820 and 1234280, respectively, while that of YK-004 and YK-010 was the lowest at 69640 and 60100, respectively. The expression of Fluc-mRNA differed by more than 20-fold between the highest and lowest expression values ​​in animals for the mRNAs contained in these four. At 72 h, the average radiation intensities of YK-003 and YK-009 were 39538 and 29435, respectively, while those of YK-004 and YK-010 were the lowest at 2066 and 810.2. The mRNAs of these four compounds showed more than 50-fold difference in Fluc-mRNA expression between the highest and lowest expression values ​​in the animal body. The values ​​of compounds 23 and 27 were 554600 and 632450, respectively, at 3 h, but rapidly decreased between 6 h and 48 h, and were only 4801 and 5512 at 72 h, indicating that the mRNAs of the LNP preparations prepared by them were rapidly degraded or metabolized in the mouse body. The expression of the mRNA of MC3 was very low at all time points, indicating that its mRNA was lowly expressed in vivo and the expression was not sustained.

[0239] From Table 8 and Figure 10, there is a large difference in the expression of LNP formulations prepared from YK-009 and compound 25 in mice. In the LNP formulations with Fluc-mRNA contents of 2.5 μg, 5.0 μg, 7.5 μg and 10 μg, the expression of YK-009 was 1.6 to 2.1 times that of compound 25 at 3 h, 1.4 to 1.7 times at 6 h, 5.2 to 5.5 times at 24 h, 6.5 to 7.1 times at 48 h, and 6.1 to 6.8 times at 72 h. In the LNP formulations with different Fluc-mRNA contents, the YK-009 formulations were all highly expressed in the body for a long time, and the compound 25 formulation could maintain higher expression at 3 h and 6 h, but rapidly decreased at 6 to 48 h.

[0240] The data was analyzed using GraphPad Prism software. Here, in terms of high expression and sustained expression of mRNA possessed by LNP in animals, YK-003 and YK-009 were significantly different from compound 23, YK-003 and YK-009 were significantly different from compound 25, YK-003 and YK-009 were significantly different from compound 27, and YK-003 and YK-009 were significantly different from MC3.

[0241] From the above experimental results, it was found that there is no corresponding relationship between the structure of cationic lipid compounds and the high and sustained expression of mRNA possessed by LNP in the animal body. Cationic lipid compounds with small structural differences have a large difference in promoting mRNA expression in the mouse body. For example, compared with YK-009, YK-004 has two fewer Cs in the G2 group, but the other structures are completely identical; YK-010 has one fewer C in each double strand of the L2 group, but the other structures are completely identical; compound 23 has four more Cs in the G1 group, one fewer C in the L1 group, four more Cs in the G2 group, one fewer C in the single strand of the L2 group, but the other structures are completely identical; compound 27 has four more Cs in the G1 group, one fewer C in the L1 group, and one fewer C in the single strand of the L2 group, but the other structures are completely identical. However, at 3 h, the mRNAs contained in these four genes had a greater than 20-fold difference between the highest and lowest levels of expression in vivo, and at 72 h, the mRNAs contained in these four genes had a greater than 50-fold difference between the highest and lowest levels of expression in vivo.

[0242] Compared with YK-009, compound 25 has two more Cs in the G1 group, one more Cs in the L1 group, two more Cs in the G2 group, and one less Cs in the single strand of the L2 group, and the other structures are completely identical. However, in the LNP formulations prepared from YK-009 and compound 25 with Fluc-mRNA contents of 2.5 μg, 5.0 μg, 7.5 μg, and 10 μg, at 3 h, YK-009 was up to 2.1 times that of compound 25, at 6 h, YK-009 was up to 1.7 times that of compound 25, at 24 h, YK-009 was up to 5.5 times that of compound 25, at 48 h, YK-009 was up to 7.1 times that of compound 25, and at 72 h, YK-009 was up to 7.2 times that of compound 25.

[0243] conclusion The above experiments to verify the performance of LNP delivery carriers in vitro and in vivo revealed that there is no clear correspondence between the structure of cationic lipid compounds and their intracellular transfection efficiency, toxicity to cells, and high and sustained expression in animals. Compounds with small structural differences may have very large differences in transfection efficiency and / or toxicity to cells, and high expression in cells. For example, compounds YK-009 and YK-010 according to the present application have a nearly 60-fold difference in cell transfection efficiency and a 25% or more difference in toxicity to transfected cells. Compounds YK-003 and YK-010 also have a nearly 50-fold difference in mouse in vivo expression and sustained expression. Therefore, it is difficult to select a suitable cationic lipid compound that has high transfection efficiency and toxicity to cells, as well as high and sustained expression in mice.

[0244] Through unique design and extensive screening, the present application has discovered several compounds, such as YK-009, YK-003, YK-006, YK-008 and YK-011, which, compared with other compounds in the prior art, can deliver nucleic acids with high cell transfection efficiency, low or no toxicity to cells, and high and sustained expression in animals, achieving unexpected technical effects.

[0245] The above description is merely exemplary embodiments of the present disclosure, and is not intended to limit the protection scope of the present disclosure, which is determined by the appended claims.

Claims

1. A compound of formula (I): or an N-oxide, solvate, pharma- ceutically acceptable salt or stereoisomer thereof. 【Chemistry 1】 (In formula (I), G １ is C ３～５ Alkylene group, G ２ is C ２～５ Alkylene group, G ３ is C ２ Alkylene group, L １ is C ８～１２ Linear alkyl group, L ２ is C １８ It is a branched alkyl group.

2. L ２ but, 【Chemistry 2】 That is, 2. A compound of formula (I) according to claim 1, or an N-oxide, solvate, pharma- ceutically acceptable salt or stereoisomer thereof.

3. 2. The compound of formula (I) according to claim 1, wherein the compound of formula (I) has one of the following structures: 【Chemistry 3】

4. A composition comprising a carrier containing the cationic lipid comprising the compound of formula (I) according to any one of claims 1 to 3, or an N-oxide, solvate, pharma-ceutically acceptable salt or stereoisomer thereof.

5. The composition of claim 4, wherein the molar ratio of said cationic lipid to carrier is from 30% to 70%.

6. (1) The carrier further comprises a neutral lipid; (2) The carrier further comprises a structured lipid; (3) The condition in which the carrier further contains a polymer-conjugated lipid The composition according to claim 4, which satisfies any one of the above requirements.

7. (1) The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1; (2) The molar ratio of the cationic lipid to the structured lipid is 1:1 to 5:1; (3) The molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 5%; The composition according to claim 6, which satisfies any one of the above requirements.

8. (1) The neutral lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof; (2) The structured lipid is one or more selected from the group consisting of cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and adrenocortical hormones; (3) The polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The composition according to claim 6, which satisfies any one of the above requirements.

9. (1) The neutral lipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycero (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), 1,The condition is that the glycerol is one or more selected from the group consisting of 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof, (2) The structural lipid is cholesterol; (3) The polymer-conjugated lipid is one or more selected from the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159). The composition according to claim 6, which satisfies any one of the above requirements.

10. The composition of claim 9, wherein the neutral lipid is DOPE and / or DSPC.

11. (1) The carrier contains a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25 to 65):(5 to 25):(25 to 45):(0.5 to 5); (2) the composition is a nanoparticle formulation having an average particle size of 10 nm to 210 nm and a polydispersity coefficient of 50% or less; (3) The composition further comprises a therapeutic or prophylactic agent. (4) The composition further comprises one or more pharma- ceutically acceptable excipients or diluents. The composition according to claim 4, which satisfies any one of the above requirements.

12. (1) The molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5; (2) The composition is a nanoparticle formulation having an average particle size of 100 nm to 205 nm and a polydispersity coefficient of 30% or less; (3) The mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:

1. The composition according to claim 11, which satisfies any one of the above requirements.

13. (1) The cationic lipid further comprises one or more other ionizable lipid compounds; (2) The mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1 to 25:1; (3) The therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. The composition according to claim 11, which satisfies any one of the above requirements.

14. (1) the mass ratio of the carrier to the therapeutic or prophylactic agent is 16:1; (2) The condition that the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response; (3) The condition that the therapeutic or prophylactic agent is a nucleic acid. The composition according to claim 13, which satisfies any one of the above requirements.

15. 15. The composition of claim 14, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA) or the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

16. 16. The composition of claim 15, wherein the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

17. The composition of claim 16 , wherein the RNA is mRNA.

18. Use of a compound represented by formula (I) according to any one of claims 1 to 3, or an N-oxide, solvate, pharma- ceutically acceptable salt or stereoisomer thereof, or a composition according to any one of claims 4 to 17, in the manufacture of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide drug, or a protein drug.

19. Use of a compound of general formula (I) or an N-oxide, solvate, pharma- ceutically acceptable salt or stereoisomer thereof as defined in any one of claims 1 to 3, or a composition as defined in any one of claims 4 to 17, in the manufacture of a medicament for the treatment of a disease or condition in a mammal in need thereof.

20. 20. The use according to claim 19, wherein the disease or condition is characterised by a dysfunction, an abnormal protein or an abnormal polypeptide activity.

21. 20. The use according to claim 19, wherein the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

22. 22. The use according to claim 21, wherein the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, and multiple types of herpes.

23. (1) The condition that the mammal is a human. (2) The condition in which the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation; (3) The condition of administering the therapeutic or prophylactic agent to the mammal at a dose of about 0.001 mg / kg to about 10 mg / kg. The use according to any one of claims 19 to 22, which satisfies any one of the following requirements:

24. 24. The use according to claim 23, wherein the composition is administered subcutaneously.

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