Cationic lipid compounds, compositions containing the same, and their uses

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 based on Chinese Patent Application No. 202210034449.4, filed on 13 January 2022, which incorporates the contents disclosed in the aforementioned Chinese Patent Application as part of this application. [Technical field]

[0002] This invention belongs to the pharmaceutical field. Specifically, this invention relates to cationic lipid compounds, compositions containing them, and their use. [Background technology]

[0003] The effective targeted delivery of bioactive substances such as small molecule drugs, polypeptides, proteins, and nucleic acids, particularly nucleic acid delivery, remains a persistent medical challenge. Nucleic acid therapies face significant challenges due to their low cellular permeability and high sensitivity to the degradation of certain nucleic acid molecules (including RNA).

[0004] Cationic lipid-containing compositions, liposomes, and liposome complexes (lipoplexes) have been demonstrated as delivery carriers for delivering bioactive substances such as small molecule drugs, polypeptides, proteins, and nucleic acids into cells and / or intracellular compartments. These compositions generally contain one or more "cationic" and / or amino (ionizable) lipids, as well as neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. While several 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 increasing complexity of lipid nanoparticles (LNPs) can lead to increased production complexity and potential toxicity, which may be a major limiting factor in their clinical application. For example, LNP siRNA particles (e.g., patisirane) 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 them that contribute to the delivery of therapeutic and / or prophylactic agents such as nucleic acids to cells. [Overview of the prefecture] [Problems the invention aims to solve]

[0005] This disclosure is based at least on the finding that there is no clear correspondence between the structure of cationic lipid compounds and their intracellular transfect efficiency, toxicity to cells, and high and sustained expression in animal bodies. Compounds with little structural difference can show very large differences in transfect efficiency and / or toxicity to cells and high intracellular expression. For example, compounds YK-009 and YK-010 in this application differ by nearly 60 times in cellular transfect efficiency and by more than 25% in toxicity to transfected cells. Furthermore, compounds YK-003 and YK-010 differ by nearly 50 times in expression and sustained expression in mice.

[0006] Therefore, it is difficult to select appropriate cationic lipid compounds that have high transfection efficiency and low toxicity to cells, as well as high and sustained expression in mice. In this disclosure, we have discovered, through unique design, that several compounds, such as YK-009, YK-003, YK-006, YK-008, and YK-011, can deliver nucleic acids with higher cell transfection efficiency, lower or no toxicity to cells, and high and sustained expression in animals compared to other compounds of the prior art, yielding unexpected technological benefits. [Means for solving the problem]

[0007] One aspect of this disclosure provides novel cationic lipid compounds, which are compounds represented by formula (I), or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers.

[0008] [ka]

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

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

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

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

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

[0014]

Chemical formula

[0015] For example, the compound represented by formula (I) has one of the following structures.

[0016]

Chemical formula

[0017] Another aspect of the present disclosure provides a composition comprising a carrier containing the above cationic lipid. For example, the molar ratio of the cationic lipid to the carrier is 30% - 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 - 10:1.

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

[0020] For example, the neutral lipids include 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), and 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-difytanoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (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 one or more selected from the group consisting of these.

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

[0022] In one embodiment, the carrier further comprises structural lipids. For example, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

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

[0024] In one embodiment, the carrier further comprises polymer-conjugated lipids. For example, the molar ratio of the polymer-conjugated lipids 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 structural lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural 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, wherein the average particle size of the nanoparticle formulation is 10 nm to 210 nm, preferably 100 nm to 205 nm, and the polydispersity index of the nanoparticle formulation is 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 preventive agent. For example, the mass ratio of the carrier in the composition to the therapeutic or preventive agent 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 preventive agent includes one or more of the following: nucleic acid molecules, small molecule compounds, polypeptides, and proteins. For example, the therapeutic or prophylactic agent is a vaccine or compound that can induce an immune response.

[0033] In one embodiment, the therapeutic or prophylactic agent is nucleic acid. For example, the therapeutic or prophylactic agent may be 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 pharmaceutically acceptable excipients or diluents.

[0037] Another aspect of this disclosure provides the cationic lipids or compositions for delivering therapeutic or prophylactic agents to patients in need.

[0038] Other aspects of this disclosure provide the use of the cationic lipids or compositions in the manufacture of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs, or protein drugs.

[0039] Another aspect of this disclosure provides the use of the cationic lipids or compositions in the manufacture of pharmaceuticals for treating diseases or conditions in mammals that require such use.

[0040] Other aspects of this disclosure provide cationic lipids or compositions for treating mammalian diseases or conditions requiring treatment.

[0041] Another aspect of this disclosure provides a method for treating or preventing a disease or condition, comprising administering the composition in a therapeutically effective or preventively effective dose to a patient or subject in need.

[0042] In one embodiment, the disease or condition is characterized by dysfunction, abnormal proteins, or abnormal polypeptide activity. For example, the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases and renovascular diseases, and metabolic diseases. For example, 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, 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 in a dose of approximately 0.001 mg / kg to approximately 10 mg / kg. To more clearly illustrate the technical solutions of the embodiments of this disclosure, some drawings of the embodiments are briefly shown below. Of course, the drawings in the following description are not limiting to this disclosure and relate only to some of the embodiments of this disclosure. [Brief explanation of the drawing]

[0045] [Figure 1] Figure 1 shows the results of cell transfection experiments using different weight ratios of carrier (including YK-009) and mRNA when preparing LNP formulations: a is carrier:mRNA=4:1, b is carrier:mRNA=16:1, and c is a blank control. [Figure 2] Figure 2 shows the results of cell transfection experiments using cationic lipid YK-009 and neutral lipid DSPC in different molar ratios when preparing LNP formulations: a is 1:1, b is 5:1, c is 10:1, and d is a blank control. [Figure 3] Figure 3 shows the results of cell transfection experiments in which different molar ratios of polymer-conjugated lipids to the carrier (containing YK-009) were used when preparing LNP formulations: a is 5%, b is 1.5%, and c is a blank control. [Figure 4] Figure 4 shows the results of cell transfection experiments in which the proportions of the carrier components—cationic lipid YK-009, neutral lipid DSPC, structural lipid cholesterol, and polymer-conjugated lipid DMG-PEG2000—were different when preparing the LNP formulations. a is 65:8:25:2, b is 50:10:38.5:1.5, and c is a blank control. [Figure 5] Figure 5 shows the fluorescence absorption intensities of LNP formulations of Fluc-mRNA prepared from different cationic lipids. [Figure 6]Figure 6 shows the fluorescence absorption intensities of LNP formulations of Fluc-mRNA prepared from the cationic lipid YK-009 and compound 25, respectively, where the Fluc-mRNA content is 0.075 μg, 0.15 μg, 0.225 μg, and 0.3 μg, respectively. [Figure 7] Figure 7 shows the cell viability after 24 hours of culture in cell culture media supplemented with LNP preparations of Fluc-mRNA prepared from different cationic lipids. [Figure 8] Figure 8 shows the cell viability after 24 hours of incubation in cell culture media to which LNP preparations of Fluc-mRNA prepared from each cationic lipid YK-009 and compound 25 were added (the Fluc-mRNA content was 0.375 μg, 0.75 μg, 1.125 μg, and 1.5 μg, respectively). [Figure 9] Figure 9 shows the results of mouse in vivo imaging experiments for LNP formulations of Fluc-mRNA prepared from different cationic lipids. [Figure 10] Figure 10 shows the results of mouse in vivo imaging experiments for LNP formulations of Fluc-mRNA prepared from each cationic lipid YK-009 and compound 25. The Fluc-mRNA content differs in the LNP formulations: a is 2.5 μg, b is 5 μg, c is 7.5 μg, and d is 10 μg. [Modes for carrying out the invention]

[0046] To further clarify the purpose, technical proposal, and advantages of the embodiments relating to this disclosure, the technical proposal of the embodiments relating to this disclosure will be described clearly and completely below with reference to the drawings of the embodiments relating to this disclosure. Of course, the embodiments described are only some embodiments of this disclosure, not all embodiments. All other embodiments that a person skilled in the art could obtain without creative effort based on the embodiments relating to this disclosure described shall be included within the scope of protection of the present invention.

[0047] The present invention may be implemented in other specific forms without departing from the essential characteristics of the invention. It should be understood that any and all embodiments of the invention may be combined with the technical features of other embodiments or more other embodiments, to obtain alternative embodiments, insofar as they do not contradict each other. Such alternative embodiments obtained by combination are incorporated into the invention.

[0048] All publications and patents referenced in this disclosure are incorporated herein by reference. In the event of any conflict between the uses and terms used in any incorporated publication or patent and those used herein, the uses and terms of this disclosure shall prevail.

[0049] The section titles used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described. Unless otherwise specified, technical and scientific terms used herein have their usual meanings, which are required to protect the field to which the subject matter pertains. If multiple definitions exist for a term, the definition provided herein shall prevail.

[0050] It should be understood that all numerical values, such as dose amounts, in this specification and in the claims, are modified with "approximately" unless otherwise described in the embodiments or otherwise specified. It should also be understood that the numerical ranges illustrated in this application are intended to include all subranges encompassed within that range, and any combination of the extreme values ​​within that range or subrange.

[0051] Similar terms used in this disclosure, such as “includes,” “contains,” or “includes,” mean that the element preceding the term covers the elements listed after the term and their equivalents, and do not exclude any elements not described. The terms “contains” or “includes” as used herein may be open, semi-closed, or closed. In other words, the terms also include “substantially consist of” or “composed of.”

[0052] In this application, the term “pharmaceutically acceptable” means that the compound or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or with the human or mammal used for prevention or treatment.

[0053] In this application, the terms "subject" or "patient" include humans and mammals.

[0054] As used herein, the term “treatment” means administering one or more drugs to a patient or subject suffering from a disease or symptoms of a disease in order to treat, alleviate, mitigate, improve or influence the disease. In the context of this application, unless otherwise stated, the term “treatment” may also include prevention.

[0055] In this application, the term "solvate" refers to a complex formed by a compound represented by formula (I) or a pharmaceutically acceptable salt with a solvent (e.g., ethanol or water). It is understood that the use of any solvate of a compound of formula (I), used to treat a disease or condition, encompasses the use of any solvate of the compound of formula (I), since, although the solvates may impart different properties (including pharmacokinetic properties), they yield the compound of formula (I) upon absorption by the subject.

[0056] The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water.

[0057] It should be further understood that the compounds represented by formula I or their pharmaceutically acceptable salts can be isolated as solvates, and for this reason, any of these solvates are within the scope of the present invention. For example, the compounds of formula I or their pharmaceutically acceptable salts may exist in a non-solvated form and in a solvated form with a pharmaceutically acceptable solvent (e.g., water, ethanol, etc.).

[0058] The term "pharmaceutically acceptable salt" means an inorganic or organic acid addition salt that is non-toxic to the compound relating to this disclosure. See, for example, SMBerge 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, and nitric acid. 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, sulfamic acid. Examples include methanesulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, 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, glycerophosphate, aspartic acid, and sulfosalicylic acid. For example, a pharmaceutically acceptable salt can be formed using the compound represented by formula I 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) relating to this disclosure can be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperbenzoic acid, hydrogen peroxide, ozone). Therefore, under conditions permitted by the valence state and structure, the compounds claimed in this application include not only the nitrogen-containing compounds shown in the structural formulas but also their N-oxide derivatives.

[0060] Certain compounds of this disclosure may exist as one or more stereoisomers. Stereoiomers include geometric isomers, diastereomers, and enantiomers. Thus, the compounds claimed in this disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art will understand that one stereoisomer may have better effects 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. Racemic mixtures can be chiralized by chromatographic or chemical resolution. For example, by adding chiral tartaric acid, chiral malic acid, or other chiral acid resolution reagents to form salts with the compounds of this disclosure, the products can be separated by their differing physicochemical properties, such as solubility.

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

[0062] The term "alkyl group" in this disclosure includes branched and linear saturated aliphatic monovalent hydrocarbon groups having a predetermined number of carbon atoms. The term "alkylene group" in this disclosure includes branched and linear saturated aliphatic divalent hydrocarbon groups having a predetermined number of carbon atoms. n~m This 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. Alternatively, the alkyl group (or alkylene group) may be substituted such that at least one hydrogen is replaced by another chemical group.

[0063] The "therapeutic dose" is the amount of therapeutic agent administered to a patient that can improve a disease or symptom. The "preventive dose" is the amount of prophylactic agent administered to a subject that can prevent a disease or symptom. The amount of therapeutic agent that constitutes the "therapeutic dose" or the amount of prophylactic agent that constitutes the "preventive dose" varies depending on the therapeutic / preventive agent, the disease state and its severity, and the age and weight of the patient / subject being treated. The therapeutic and preventive doses can be commonly determined by those skilled in the art based on knowledge and this disclosure.

[0064] In this application, if the name of a compound and its structural formula do not match, the structural formula shall take precedence.

[0065] The term "compounds relating to the disclosure" as used in this application may, depending on the context, include compounds of formula I, N-oxides, solvates, pharmaceutically acceptable salts, stereoisomers, and mixtures thereof.

[0066] As used herein, the term "cationic lipid" refers to a lipid that is positively charged at a selected pH value. Cationic lipids readily bind to negatively charged nucleic acids, and thus form lipid nanoparticles (LNPs) by interacting with negatively charged phosphate groups in nucleic acids through electrostatic forces. Currently, LNPs are one of the main delivery carriers.

[0067] The inventors have found that when selecting a large number of compounds, it is extremely difficult to select a suitable cationic lipid compound that simultaneously satisfies the conditions of high transfection efficiency, low cytotoxicity, and high and sustained expression in mice. The inventors have found that several compounds, such as YK-009, YK-003, YK-006, YK-008, and YK-011, can deliver nucleic acids with higher transfection efficiency in cells, low or no toxicity to cells, and high and sustained expression in animals, compared to conventional compounds. Even compounds with little structural difference show very large differences in transfection efficiency and / or toxicity to cells and high intracellular expression. For example, compounds YK-009 and YK-010 of this application show a difference of nearly 60 times in cell transfection efficiency and a difference of more than 25% in toxicity to transfected cells. Furthermore, compounds YK-003 and YK-010 show nearly a 50-fold difference in in vivo expression and sustained expression in mice.

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

[0069] [ka]

[0070] Furthermore, the compounds relating to this disclosure were compared with structurally similar compounds 23, 25 (i.e., SM-102, CAS No.: 2089251-47-6, a cationic lipid used in Moderna's COVID-19 vaccine mRNA-1273), and 27 in patent CN110520409A filed by Moderna Corporation.

[0071] [ka]

[0072] As a result, the compounds relating to this disclosure were found to be significantly improved in terms of transfection efficiency and / or toxicity to cells. For example, when compound YK-009 was transfected with mRNA, the cell transfection efficiency was 40-fold, 8-fold, and 13-fold or more compared to MC3, compound 23, and compound 27, respectively, and the toxicity to transfected cells was reduced by 12%, 13%, and 16%, respectively, compared to MC3, compound 23, and compound 27. Furthermore, it was found that expression in mice was 25-fold, 7-fold, and 6-fold or more for compound YK-009 than for MC3, compound 23, and compound 27, respectively, and 18-fold, 8-fold, and 7-fold or more for compound YK-003 than for MC3, compound 23, and compound 27.

[0073] Compared to compound 25, compound YK-009 according to this disclosure showed a 7-fold improvement in cell transfection efficiency when mRNA was transfected, a 9% reduction in toxicity to transfected cells, and a more than 7-fold improvement in expression in mice.

[0074] One aspect of this disclosure provides cationic lipid compounds for delivering novel therapeutic or prophylactic agents. The cationic lipid compounds of this disclosure can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds of this disclosure exhibit higher transfection efficiency, lower cytotoxicity, and improved delivery efficiency and safety.

[0075] This disclosure provides cationic lipids that are compounds represented by formula (I), or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers.

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

[0077] In one embodiment, G1 is an 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 an 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, i.e., -(CH2)2-.

[0080] In one embodiment, L1 is an unsubstituted C 8~12 A linear alkyl group, preferably an unsubstituted C 10 A linear alkyl group, for example, -(CH2)9CH3.

[0081] In one embodiment, L2 is an 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] That is the case.

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

[0084] [ka] That is the case.

[0085] In an exemplary embodiment, the compound is selected from the group consisting of the following compounds or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers.

[0086] [ka] TIFF0007863167000011.tif249134

[0087] Another aspect of this disclosure provides compositions comprising a carrier, the carrier comprising a cationic lipid, the cationic lipid comprising a compound represented by formula (I) above, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

[0088] In one embodiment, the composition is a nanoparticle formulation. The average size of the nanoparticle formulation is 10 nm to 210 nm, preferably 100 nm to 205 nm. The polydispersity index of the nanoparticle formulation is 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 compounds selected from the group consisting of the compound represented by formula (I) above, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer. In one embodiment, the cationic lipid is selected from the compounds represented by formula (I) above. 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 according to this disclosure, the cationic lipid includes one or more compounds (a) selected from the group consisting of the compound represented by formula (I) above or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, and one or more other ionizable lipid compounds (b) different from (a). The cationic lipid compound (b) may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the cationic lipid compound (b) may be DLin-MC3-DMA(MC3). For example, the cationic lipid compound (b) may be compounds 23, 25, 27 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] This carrier is used, for example, to deliver active ingredients such as therapeutic or prophylactic agents. The active ingredients may be encapsulated within the carrier or bound to the carrier.

[0093] For example, the therapeutic or prophylactic agent includes one or more of the following: nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are 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 contain neutral lipids. In this disclosure, neutral lipids refer to auxiliary lipids that are uncharged or exist as amphoteric ions at a selected pH value. Such neutral lipids may improve efficiency by promoting lipid phase transitions, 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, or 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 the following: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and their derivatives.

[0097] The carrier component of a composition containing cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (polyunsaturated) lipids. The phospholipids can be assembled into one or more lipid bilayers. Generally, the phospholipids may include a phospholipid portion and one or more fatty acid portions.

[0098] The neutral lipid portion may be arbitrarily selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin, but is not limited to these. The fatty acid portion may be arbitrarily 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 to these. Non-natural products consisting of natural products that have modifications and substitutions such as branching, oxidation, cyclization, and alkynes are also included. For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds), or crosslinked with such one or more alkynes. Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions when exposed to azides. These reactions can be used to functionalize the lipid bilayer of a composition to promote membrane permeability or cell recognition, or to conjugate a composition with a useful component such as a targeting or imaging moiety (e.g., a dye).

[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), and 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-difytanoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoylphosphatidylethanolamine (DSP E) The following can be selected, but are not limited to, the group consisting of 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 lipids include DSPC. In certain embodiments, the neutral lipids include DOPE. In some embodiments, the neutral lipids include both DSPC and DOPE.

[0101] structured lipids The carrier of the composition containing cationic lipids may further contain one or more structural lipids. In this disclosure, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

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

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

[0104] Polymer-conjugated lipids The support for a composition containing cationic lipids may further contain one or more polymer-conjugated lipids. Polymer-conjugated lipids refer mainly to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, controls nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific 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 PEG molecular weight of the PEG modification is typically 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 according to this 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 structural lipid, and a polymer-conjugated lipid. The molar ratio of the cationic lipid, the neutral lipid, the structural 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 according to this disclosure, the carrier comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid. 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.

[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, or 25:1.

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

[0112] The therapeutic or prophylactic agent includes, but is not limited to, one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0113] For example, the therapeutic or prophylactic agent is a vaccine or compound that can induce an immune response.

[0114] The carriers of this disclosure can deliver therapeutic and / or prophylactic agents to mammalian cells or organs, and therefore the disclosure also provides a method for treating a mammalian disease or condition of interest, which includes administering a composition containing a therapeutic and / or prophylactic agent to a mammal and / or bringing mammalian cells into contact with the composition.

[0115] Therapeutic and / or prophylactic agents include bioactive substances, sometimes referred to as “activators.” These are substances that, after being delivered to cells or organs, cause desired changes within those cells or organs, or in other body tissues or systems. Such substances can be used to treat one or more diseases, symptoms, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small-molecule pharmaceuticals for treating specific diseases, symptoms, or conditions. Examples of drugs that can be used in the composition include antiproliferative agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside)Arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs (e.g., methotrexate, and purine and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., dibucaine and chlorpromazine), β-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines Mins (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B) B) Examples include, but are not limited to, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and contrast agents.

[0116] In some embodiments, therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that induce an immune response, and / or other therapeutic and / or prophylactic agents. Cytotoxins or cytotoxic agents include any reagents that are harmful to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthracine dione. Examples of radioactive ions include, but are not limited to, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogs or homologs. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate ions, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines contain compounds and formulations that provide immunity to one or more symptoms associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, as well as mRNA encoding antigens and / or epitopes derived from infectious diseases. Furthermore, vaccines contain compounds and formulations that elicit an immune response against cancer cells, as well as mRNA encoding antigens, epitopes, and / or neoepitopes derived from tumor cells.Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other types. In some embodiments, vaccines and / or compounds include those that can induce an immune response by intramuscular administration of a composition containing compounds represented by 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-thioguani, cytarabine, and 5-dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, racermycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptomycin), This includes, but is not limited to, zotosin, mitomycin C, and cis-dichlorodiaminoplatinum(II) (DDP, cisplatin), anthracyclines (e.g., daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (sometimes called actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and meitansinoids).

[0117] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of this 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 factor, luteinizing hormone-releasing hormone (LHRH) analogues, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0118] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest sense of the term "polynucleotide" includes any compound and / or substance that can be represented by an oligonucleotide chain or incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with this disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA) and its hybrids, RNAi inducers, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, etc. In some embodiments, the therapeutic and / or prophylactic agent is RNA. The RNAs that can be used in the compositions and methods described herein are, but are not limited to, those arbitrarily selected from the group consisting of shortmers, antagomirs, antisense RNAs, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and mixtures thereof. In some embodiments, the RNA is mRNA.

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

[0120] In other embodiments, the therapeutic and / or prophylactic agent is siRNA. siRNA can selectively reduce or downregulate the expression of a gene of interest. For example, siRNA can be selectively silenced when administered to a subject requiring a composition containing the siRNA, thereby silencing a gene associated with a specific disease, symptom, or condition. siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be 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, the sgRNA-cas9 complex may affect the translation of cytogenetic mRNA.

[0122] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or a carrier or plasmid encoded therein. The shRNA is generated within the target cell after a suitable construct has been delivered to the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant technical field.

[0123] Disease or condition The compositions / carriers of this disclosure can deliver therapeutic or prophylactic agents to subjects or patients. The therapeutic or prophylactic agents include, but are not limited to, one or more nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compositions of this disclosure can be used in the manufacture of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs, or protein drugs. Because the therapeutic or prophylactic agents are diverse, the compositions of this disclosure can be used to treat or prevent a variety of diseases or conditions.

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

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

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

[0127] Other ingredients The composition may contain one or more components other than those described in the preceding section. For example, the composition may contain one or more hydrophobic low-molecular-weight molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0128] The composition may also contain one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. The permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and their derivatives and analogs).

[0129] Surface modifiers include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., the cationic surfactant dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clorodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, thiopronin, gelsolin, thymosin β4, dornase alpha) This includes, but is not limited to, alfa, neltenexine, and erdosteine, and DNA enzymes (e.g., rhDNA enzymes). Surface modifiers may be placed inside and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

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

[0131] Aside from these components, the composition may contain any other materials used in pharmaceutical compositions. The composition may contain, but is not limited to, one or more pharmaceutically acceptable excipients or auxiliary components, such as solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, flow accelerators, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, fragrances, and colorants. Examples of excipients include starch, lactose, or dextrin. Pharmaceutically acceptable excipients are those well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; 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, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0133] In some embodiments, compositions comprising one or more of the lipids described herein further comprise one or more adjuvants, such as glucopyranosyllipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., type A or type B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0134] The compositions of this disclosure can be formulated in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions of this disclosure can be prepared by methods well known in the field of pharmacy. For example, a sterile injection can be prepared by mixing the required amount of therapeutic or prophylactic agent and the other required components in a suitable solvent such as sterile distilled water, and then sterilizing by filtration. Furthermore, surfactants may be added to promote the formation of a homogeneous solution or suspension.

[0135] For example, the 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 compositions disclosed herein are administered in therapeutically effective doses. The therapeutically effective dose may vary depending on the specific agent selected, the route of administration, the nature of the disease being treated, the patient's age and condition, and ultimately be determined by the attending physician or clinician. For example, the therapeutic or prophylactic agent may be administered to mammals (e.g., humans) in doses ranging from approximately 0.001 mg / kg to approximately 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. Unless otherwise specified in the examples, the procedures should be carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified by the manufacturer, the reagents and equipment used may be any commercially available conventional products.

[0138] Example 1: Synthesis of cationic lipid compounds 1. Synthesis of 1.6-((4-(undecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)caproate 2-octyldecyl (YK-001) The synthesis route is as follows:

[0139] [ka]

[0140] Step 1: Synthesis of n-undecyl 4-bromobutyrate (YK-001-PM1) n-Undecanol (5.00 g, 29.02 mmol) and 4-bromobutyric acid (5.14 g, 30.78 mmol) were dissolved in methylene chloride (40 mL). To the above solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.67 g, 34.82 mmol) and 4-dimethylaminopyridine (177 mg, 1.45 mmol) were added, and the mixture was reacted at 30-35°C with stirring for 8 hours. After the reaction was complete, the reaction mixture was washed with saturated sodium carbonate and saturated brine, and dried over Na2SO4. The mixture was filtered, and the filtrate was concentrated under reduced vacuum. The residue was purified by silica gel chromatography to obtain n-undecyl 4-bromobutyric acid (6.68 g, 20.79 mmol, 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 4-((2-hydroxyethyl)amino)butyrate n-undecyl (YK-001-PM2) 4-Undecyl bromobutyrate (2.71 g, 8.43 mmol) and ethanolamine (1.40 g, 22.92 mmol) were dissolved in acetonitrile (50 mL). Potassium carbonate (3.17 g, 22.92 mmol) was added to the system, and the mixture was heated to 70°C and reacted with stirring for 2 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced vacuum to remove the solvent. The residue was purified by silica gel chromatography to obtain 4-((2-hydroxyethyl)amino)butyrate n-undecyl (1.47 g, 4.88 mmol, 57.89%). 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-bromohexanoic acid (3.05 g, 6.82 mmol, 61.50%) was obtained according to 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 6-((4-(undecyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)caproate 2-octyldecyl (YK-001) 6-2-octyldecyl bromohexanoate (200 mg, 0.45 mmol) and 4-(2-hydroxyethyl)amino)butyrate n-undecyl (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 with stirring for 20 hours. The reaction mixture was cooled to room temperature and filtered, and the filtrate was concentrated under reduced 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%). 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 (YK-002) ((5-(decyloxy)-5-oxopentyl)(2-hydroxyethyl)amino)caproate The synthesis 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-bromopentanoic acid (2.50 g, 7.78 mmol, 84.93%) was obtained according to 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 5-((2-hydroxyethyl)amino)pentanoate n-decyl (YK-002-PM2) Using 5-bromopentanoic acid n-decyl (1.92 g, 5.98 mmol) and ethanolamine (0.31 g, 5.07 mmol) as raw materials, 5-((2-hydroxyethyl)amino)pentanoic acid n-decyl (0.34 g, 1.13 mmol, 18.90%) was obtained according to 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 6-((5-(decyloxy)-5-oxopentyl)(2-hydroxyethyl)amino)caproate 2-octyldecyl (YK-002) Using 5-((2-hydroxyethyl)amino)pentanoate n-decyl (151 mg, 0.50 mmol) and 6-bromohexanoate-2-octyldecyl (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 6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)caproate 2-octyldecyl (YK-003) The synthesis 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-bromohexanoic acid (2.41 g, 7.19 mmol, 78.49%) was obtained according to 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 6-((2-hydroxyethyl)amino)caproate n-decyl (YK-003-PM2) 6-bromohexanoate 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 mixture was concentrated under reduced vacuum to remove the solvent, then diluted with ethyl acetate (80 mL) and washed with saturated brine (50 mL x 3). After concentrating the organic phase under reduced pressure, it was purified by silica gel chromatography to obtain 6-((2-hydroxyethyl)amino)caproate n-decyl (0.66 g, 2.09 mmol, 62.6%). 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 6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)caproate 2-octyldecyl (YK-003) Using 6-((2-hydroxyethyl)amino)caproate n-decyl (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 4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyrate 2-octyldecyl (YK-004) The synthesis 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 (10.52 g, 34.2 mmol, 42.0%) was obtained according to 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 4-((2-hydroxyethyl)amino)butyrate n-decyl (YK-004-PM2) Using 4-bromobutyrate n-decyl (8.00 g, 26.04 mmol) and ethanolamine (4.78 g, 78.26 mmol) as raw materials, 4-((2-hydroxyethyl)amino)butyrate n-decyl (4.40 g, 15.3 mmol, 58.8%) was obtained according to 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 4-bromobutyrate-2-heptylnonyl (YK-004-PM3) Using 2-heptyrnonanol (390 mg, 1.45 mmol) and 4-bromobutyric acid (265 mg, 1.59 mmol) as raw materials, 4-bromobutyric acid-2-heptyrnonyl (500 mg, 1.19 mmol, 82.07%) was obtained according to 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 4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyrate 2-octyldecyl (YK-004) Using 4-bromobutyrate-2-heptyrnonyl (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 5.8-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octane9-heptadecyl (YK-005) The synthesis route is as follows:

[0161] [ka]

[0162] Step 1: Synthesis of 8-bromooctanoate-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 according to the preparation method of YK-001-PM1.

[0163] Step 2: Synthesis of 8-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)octane9-heptadecyl (YK-005) Using 4-((2-hydroxyethyl)amino)butyrate n-decyl (150 mg, 0.52 mmol) and 8-bromooctanoate-9-heptadecyl (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 6.5-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)pentanoate 2-octyldecyl (YK-006) The synthesis route is as follows:

[0165] [ka]

[0166] Step 1: Synthesis of 5-bromopentanoate-2-heptylnonyl (YK-006-PM1) Using 2-heptyrnonanol (300 mg, 1.11 mmol) and 5-bromopentanoic acid (220 mg, 1.22 mmol) as raw materials, 5-bromopentanoic acid-2-heptyrnonyl (438 mg, 1.01 mmol, 91.0%) was obtained according to 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 5-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)pentanoic acid 2-octyldecyl (YK-006) Using 5-bromopentanoate-2-heptyrnonyl (200 mg, 0.46 mmol) and 4-((2-hydroxyethyl)amino)butyrate n-decyl (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 (YK-007) 7.6-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)caproate The synthesis route is as follows:

[0169] [ka]

[0170] Step 1: Synthesis of 6-bromohexanoic acid 1-undecyl (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, according to the preparation method of YK-001-PM1, 6-bromohexanoic acid 1-undecyl (2.40 g, 6.87 mmol, 59.2%) was obtained.

[0171] Step 2: Synthesis of undecyl 6-((2-hydroxyethyl)amino)hexanoate (YK-007-PM2) Using 6-bromohexanoic acid 1-undecyl (2.25 g, 6.44 mmol) and ethanolamine (1.18 g, 19.29 mmol) as raw materials, according to the preparation method of YK-001-PM2, undecyl 6-((2-hydroxyethyl)amino)hexanoate (855 mg, 2.59 mmol, forty point two percent) was obtained. C 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, according to the preparation method of YK-001, the target compound (260 mg, 0.37 mmol, forty one point one percent) was obtained. C 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 (YK-008) ((4-(nonyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate The synthesis route is as follows:

[0174] [ka]

[0175] Step 1: Synthesis of 4-bromobutyrate n-nonyl (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, 4-bromobutyric acid n-nonyl (3.66 g, 12.5 mmol, 36.0%) was obtained according to 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 4-((2-hydroxyethyl)amino)butyrate n-nonyl (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, by the preparation method of YK-001-PM2, 4-((2-hydroxyethyl)amino)butyric acid n-nonyl (1.22 g, 4.46 mmol, 53.2%) was obtained, C 15 H 31 NO3, MS(ES): m / z(M+H + ) 274.2. 1 H NMR(400 MHz, CDCl3) δ 4.06(t, J = 6.8 Hz, 2H), 3.53 - 3.47(m, 3H), 2.68(t, J = 7.0 Hz, 2H), 2.43(t, J = 8.1 Hz, 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 = 6.9 Hz, 3H).

[0177] Step 3: Synthesis of 6-((4-(nonynyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoic acid 2-octyldecyl (YK-008) Using 6-bromohexanoic acid-2-octyldecyl (200 mg, 0.45 mmol) and 4-((2-hydroxyethyl)amino)butyric acid n-nonyl (102 mg, 0.37 mmol) as raw materials, by the preparation method of YK-001, the target compound (30 mg, 0.047 mmol, 13.5%) was obtained. C 39 H 77 NO5, MS(ES): m / z(M+H + ) 640.6. 1 H NMR(400 MHz, CDCl3) δ 4.06(t, J = 6.8 Hz, 2H), 3.97(d, J = 5.8 Hz, 2H), 3.68 - 3.62(m, 2H), 2.32(dd, J = 15.2, 7.6 Hz, 4H), 1.63(dd, J = 15.1, 7.6 Hz, 6H), 1.39 - 1.17(m, 52H), 0.87(d, J = 7.0 Hz, 9H).

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

[0179] [ka]

[0180] Step 1: Synthesis of 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate 2-octyldecyl (YK-009) Using 4-(2-hydroxyethyl)amino)butyrate n-decyl (1.00 g, 3.48 mmol) and 6-bromohexanoate 2-octyldecyl (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 (YK-010) ((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate The synthesis 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-heptyrnonanol (300 mg, 1.24 mmol) as raw materials, 2-heptyrnonyl 6-bromohexanoic acid (280 mg, 0.67 mmol, 54.0%) was obtained according to 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 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate 2-heptylnonyl (YK-010) Using 4-(2-hydroxyethyl)amino)butyrate n-decyl (150 mg, 0.52 mmol) and 6-bromohexanoate 2-heptylnonyl (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 (YK-011) 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate The synthesis 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-bromohexanoic acid (240 mg, 0.61 mmol, 41.6%) was obtained according to 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 6-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate 2-hexyloctyl (YK-011) Using 4-(2-hydroxyethyl)amino)butyrate n-decyl (150 mg, 0.52 mmol) and 6-bromohexanoate 2-hexyloctyl (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. 11H NMR (400 MHz, CDCl3) δ 4.09 - 4.03 (m, 4H), 3.56 - 3.53 (m, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.22 (t, J = 7.1 Hz, 2H), 1.71 - 1.63 (m, 3H), 1.58 - 1.54 (m, 2H), 1.42 - 1.19 (m, 47H), 0.88 (t, J = 6.8 Hz, 9H).

[0189] Synthesis of nonyl 12.8 - ((2 - hydroxyethyl)(10 - ((9 - heptadecyl)oxy) - 10 - oxodecyl)amino)octanoate (Compound 23) The synthetic route is as follows.

[0190]

Chemical Structure

[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, by the preparation method of YK - 001 - PM1, n - nonyl 8 - bromooctanoate (1.85 g, 5.30 mmol, 52.0%) was obtained.

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

[0193] Step 3: Synthesis of 9 - heptadecyl 10 - bromodecanoate (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 according to the preparation method of YK-001-PM1.

[0194] Step 4: Synthesis of 8-(10-((9-heptadecyl)oxy)-10-oxodecyl)amino)octanoate nonyl (compound 23) Using 8-((2-hydroxyethyl)amino)octanoate nonyl (500 mg, 1.52 mmol) and 10-bromodecanate-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 synthesis route is as follows:

[0196] [ka]

[0197] Step 1: Synthesis of 9-heptadecyl 6-bromohexanoate (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 according to the preparation method of YK-001-PM1.

[0198] Step 2: Synthesis of 8-(6-((9-heptadecyl)oxy)-6-oxohexyl)amino)octanoate nonyl (compound 27) Using 8-((2-hydroxyethyl)amino)octanoate nonyl (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 nanolipid 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 each dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 to prepare ethanol lipid solutions. Citrate buffer (pH=4~5) was rapidly added to the ethanol lipid solutions by ethanol injection and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Qifa Experimental Agents Co., Ltd.) was diluted with citrate buffer (pH=4~5) to obtain an aqueous mRNA solution. Liposomes were prepared using fixed-volume liposome solutions and aqueous mRNA solutions so that the weight ratios of total lipids to mRNA were 4:1, 10:1, 16:1, 24:1, and 30:1, respectively. They were sonicated at 25°C for 15 minutes (ultrasonic frequency 40kHz, ultrasonic output 800W). The obtained liposomes were diluted 10-fold with PBS, and then ultrafiltration was performed using a 300 kDa ultrafiltration tube to remove ethanol. Subsequently, the mixture was fixed in PBS, and eGFP-mRNA was encapsulated in a cationic lipid YK-009 / DSPC / cholesterol / DMG-PEG2000 (molar percentage 50:10:38.5:1.5) to obtain LNP formulations.

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

[0203] 2. Optimization of the ratio of cationic lipids to neutral lipids LNP preparations containing eGFP-mRNA were prepared using the method described in paragraph 1, where the molar ratios of cationic lipid YK-009 and neutral lipid DSPC were 1:1, 5:1, and 10:1, respectively. In the cell transfection experiment, when the molar ratio of the cationic lipid to the neutral lipid was 1:1 to 10:1, there was a transfection effect in all cases. Among them, when the molar ratio was 5:1, it was found that the transfection efficiency was the highest. (Figure 2)

[0204] 3. Optimization of the ratio of the polymer-conjugated lipid to the carrier (liposome) By the method in Item 1, LNP formulations encapsulating eGFP-mRNA were prepared, where 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] In the cell transfection experiment, when the molar ratio of the polymer-conjugated lipid to the carrier was in the range of 0.5% to 5%, there was a transfection effect in all cases. Also, it was shown that the transfection efficiency was the highest when the molar ratio was 1.5%, and the lowest when the molar ratio was 0.5% (Figure 3).

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

[0207] In the cell transfection experiment, when the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid was 50:10:38.5:1.5, the transfection effect was the highest. When the molar ratio was 65:8:25:2, the effect was slightly worse, but it could still be transfected (Figure 4).

[0208] Example 3: Cell transfection experiment of the LNP formulation of eGFP-mRNA Cell regeneration and subculturing: 293T cells were regenerated, cultured in a culture dish, and subculturished until the required number of cells were reached. Seeding plates: Cells in culture dishes were digested and counted. The cells were plated at 10,000 cells per well into 96-well plates and then at 150,000 cells per well into 12-well plates, after which they were cultured overnight until the cells adhered to the plate walls.

[0209] Cell transfection experiment: An LNP preparation containing 1.5 μg of eGFP-mRNA prepared in Example 2 (the cationic lipid in the carrier is YK-009) and a Lipofectamin2000 preparation containing eGFP-mRNA were added to cell culture medium in 12-well plates. After 24 hours of incubation, the cells were observed using a fluorescence microscope, and the transfection efficiency of various samples was investigated by measuring the fluorescence intensity.

[0210] Based on the experimental results, the preparation conditions for nanolipid particles (LNP formulations) were finally determined. Specifically, nanolipid particles (LNP formulations) were prepared in the following experiment under the following conditions: a carrier-to-mRNA ratio of 16:1, a molar ratio of cationic lipids to neutral lipids of 5:1, polymer-conjugated lipids accounting for 1.5% of the liposome, and a molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids of 50:10:38.5:1.5.

[0211] Example 4: Preparation of nanolipid particles (LNP formulation) (optimal ratio)

[0212] [Table 1] TIFF0007863167000027.tif243170TIFF0007863167000028.tif106170 Cationic lipids listed in Table 1, DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 were each dissolved in ethanol in a molar ratio of 50:10:38.5:1.5 to prepare ethanol lipid solutions. Citrate buffer (pH=4~5) was rapidly added to the ethanol lipid solutions by the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Qifa Experimental Agents Co., Ltd.) or Fluc-mRNA (purchased from Shanghai Qifa Experimental Agents Co., Ltd.) was diluted with citrate buffer (pH=4~5) to obtain mRNA aqueous solutions. Liposomes were prepared using a fixed volume of liposome solution and an mRNA aqueous solution to achieve a total lipid to mRNA weight ratio of 16:1. They were sonicated at 25°C for 15 minutes (ultrasonic frequency 40 kHz, ultrasonic output 800 W). The resulting liposomes were diluted 10-fold with PBS and then ultrafiltered through a 300 kDa ultrafiltration tube to remove ethanol. Subsequently, LNP preparations were obtained by fixing the volume with PBS and encapsulating eGFP-mRNA or Fluc-mRNA using the 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. A 10 μL liposome solution was taken out, diluted to 1 mL with RNase-free deionized water, and added to the sample pool. Each sample was measured three times. 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 nanolipid particles prepared in Example 4 had particle sizes ranging from 110 to 210 nm, and all were suitable for mRNA delivery. The particles prepared from compound 23 and YK-003 had the smallest particle sizes, at 114.12 nm and 119.91 nm, respectively, while the particles prepared from YK-008 and MC3 had the largest particle sizes, at 205.00 nm and 205.20 nm, respectively. The polydispersity index of all nanolipid particles ranged from 5% to 30%, with YK-004 having the smallest polydispersity index at 9.7% and YK-002 having the largest at 27.7%.

[0216] Example 6: Verification of the performance of LNP delivery carriers in vitro Cell regeneration and subculturing: The method is the same as in Example 3. Seeding plate: Same as in Example 3.

[0217] 1. Fluorescence measurement of Fluc-mRNA An LNP preparation containing 0.3 μg of Fluc-mRNA (the carrier components of the LNP preparation 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 those listed in Table 1) was added to a 96-well plate cell culture medium and cultured for 24 hours. Then, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected using an IVIS fluorescence measurement system. This experiment verified the transfection efficiency of the LNP preparation in cells. The results are shown in Table 3.

[0218] Furthermore, to compare the transfection efficiency of LNP preparations prepared from the cationic lipid YK-009 and compound 25 in cells, LNP preparations containing 0.3 μg, 0.225 μg, 0.15 μg, and 0.075 μg of Fluc-mRNA were prepared (the carrier components of the LNP preparations were cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids in a molar ratio of 50:10:38.5:1.5, where the cationic lipid was either YK-009 or compound 25). The intracellular transfection activity of the prepared LNP preparations was detected using the same method as described 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 5,479,373. YK-001, YK-002, YK-005, YK-006, and YK-008 also showed very strong fluorescence absorption, all of which were 10. 6 ~10 7 The RLU values ​​for YK-009, YK-006, YK-001, YK-005, YK-002, and YK-008 were 8.4 times, 4.1 times, 3.4 times, 2.2 times, 2.0 times, and 1.9 times, respectively, compared to compound 23. 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 ​​for YK-009 were 58 times, 39 times, 8 times, 13 times, and 42 times, respectively, compared to YK-010, YK-004, compound 23, compound 27, and MC3.

[0222] Table 4 and Figure 6 show that there was a significant difference in the fluorescence absorption of LNP preparations prepared from YK-009 and compound 25. The fluorescence absorption of LNP preparations containing 0.075 μg, 0.15 μg, 0.225 μg, and 0.3 μg of Fluc-mRNA, as well as the YK-009 preparation, was 3.7 times, 4.0 times, 7.0 times, and 4.4 times, respectively, compared to the compound 25 preparation.

[0223] Data were analyzed using GraphPad Prism software. Here, YK-009, YK-006, YK-001, YK-005, YK-002, and YK-008 showed significant differences with compound 23, and YK-009, YK-006, YK-001, YK-005, YK-002, and YK-008 showed significant differences with compound 27. All compounds except YK-004 and YK-010 showed significant differences with MC3. LNP preparations containing 0.075 μg, 0.15 μg, 0.225 μg, and 0.3 μg of Fluc-mRNA, as well as YK-009, all showed significant differences with compound 25.

[0224] From a structural standpoint, compared to YK-009, YK-001 has one more carbon atom in the L1 group but the rest of the structure is identical; YK-002 has one more carbon atom in the G1 group but the rest of the structure is identical; YK-004 has two fewer carbon atoms in the G2 group but the rest of the structure is identical; YK-006 has one fewer carbon atom in the G2 group but the rest of the structure is identical; YK-010 has one fewer carbon atom in each double strand of the L2 group but the rest of the structure is identical; and compound 23 has one more carbon atom in the G1 group. Compound 25 has four more carbon atoms, one less carbon atom in the L1 group, four more carbon atoms in the G2 group, and one less carbon atom in the single-strand L2 group, but the rest of its structure is identical. Compound 25 has two more carbon atoms in the G1 group, one more carbon atom in the L1 group, two more carbon atoms in the G2 group, and one less carbon atom in the single-strand L2 group, but the rest of its structure is identical. Compound 27 has four more carbon atoms in the G1 group, one less carbon atom in the L1 group, and one less carbon atom in the single-strand L2 group, but the rest of its structure is identical.

[0225] This indicates that there is no correspondence between the structure of a compound and its transfection efficiency within cells, and that compounds with small structural differences are likely to have very large differences in transfection efficiency. For example, compared to YK-009, YK-010, YK-004, compound 23, and compound 27 have slightly different structures, but the cell transfection efficiencies of YK-009 were 58 times, 39 times, 8 times, and 13 times higher, respectively, for these cationic lipid compounds. YK-009 and compound 25 have only slight structural differences, yet the difference in cell transfection efficiency was as much as 7 times. Therefore, compounds with similar structures do not necessarily have similar transfection efficiencies, and their transfection efficiencies can vary greatly. Selecting cationic lipid compounds with high transfection efficiency is not easy and requires various designs and a lot of creative effort.

[0226] 2. Measurement of cell viability An LNP preparation containing 1.5 μg of Fluc-mRNA (the carrier components of the LNP preparation 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 those listed in Table 1) was added to a 96-well plate cell culture medium and cultured for a further 24 hours. Then, 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 using a microplate reader. The results are shown in Table 4.

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

[0228] [Table 5]

[0229] [Table 6]

[0230] Table 5 and Figure 7 show that LNP formulations of Fluc-mRNA prepared with different cationic lipids exhibited very different cytotoxicities. Cell viability was highest for YK-009 at 100%, followed by YK-011 at 98%, while YK-010 was 77%, YK-003 at 84%, YK-001 at 85%, compound 23 at 87%, compound 27 at 84%, and MC3 at 88%. These were clearly lower than YK-009.

[0231] Table 6 and Figure 8 show that the LNP preparations prepared from YK-009 and compound 25 exhibited very different inhibitory activities against cells. Here, while the LNP preparations and YK-009 preparations with Fluc-mRNA content of 1.5 μg, 1.125 μg, 0.75 μg, and 0.375 μg all resulted in 100% cell viability, the inhibitory activity of compound 25 preparations against cells was up to 9% lower than that of YK-009 preparations. The data was analyzed using GraphPad Prism software. Here, YK-009, YK-011, YK-006, and YK-007 showed significant differences in cytotoxicity compared to compounds 23, 27, and MC3.

[0232] From the experimental results above, it was found that there is no correspondence between the structure of cationic lipid compounds and their cytotoxicity, and that compounds with small structural differences exhibit very large differences in cytotoxicity. For example, compared to YK-009, YK-010 has one less carbon atom in each double-strand of the L2 group, and the rest of its structure is identical, yet cell viability decreased by 23%. YK-002 has one more carbon atom in the G1 group, and the rest of its structure is identical, yet cell viability decreased by 11%. Compound 23 has four more carbon atoms in the G1 group, one less carbon atom in the L1 group, four more carbon atoms in the G2 group, and one less carbon atom in the single-strand of the L2 group, and the rest of its structure is identical, yet cell viability decreased by 13%. Compound 25 had two more carbon atoms in the G1 group, one more carbon atom in the L1 group, two more carbon atoms in the G2 group, and one less carbon atom in the single-strand L2 group, while its other structural features were identical. However, it resulted in a 9% decrease in cell viability. Compound 27 had four more carbon atoms in the G1 group, one less carbon atom in the L1 group, and one less carbon atom in the single-strand L2 group, while its other structural features were identical. However, it resulted in a 16% decrease in cell viability. YK-009 had a similar structure to YK-010, YK-002, Compound 23, Compound 27, and Compound 25, but its cytotoxicity was lower, at 23%, 11%, 13%, 16%, and 9%, respectively. Thus, it was found that cationic lipids with minute structural differences can result in significantly different cytotoxicities.

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

[0234] An LNP preparation containing 10 μg of Fluc-mRNA was intramuscularly injected into 4-6 week old, 17-19 g female BALB / C mice. Following administration, a fluorescent imaging substrate was intraperitoneally injected into the mice at predetermined time points (3, 6, 24, 48, and 72 hours). After allowing the mice to move freely for 5 minutes, the average radiation intensity (corresponding to fluorescence intensity) of proteins expressed in the mice by the mRNA carried by the LNP was detected using a small animal live imager. The detection results are shown in Table 5 and Figure 7.

[0235] To compare protein expression and duration in mice of LNP preparations prepared from the cationic lipid YK-009 and compound 25, LNP preparations containing 10 μg, 7.5 μg, 5 μg, and 2.5 μg of Fluc-mRNA were prepared (the carrier components of the LNP preparations were cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids in a molar ratio of 50:10:38.5:1.5, where the cationic lipid was either YK-009 or compound 25). The experimental method for mouse in vivo imaging was the same as described above. The results are shown in Table 8.

[0236] [Table 7]

[0237] [Table 8]

[0238] Table 7 and Figure 9 show that LNP preparations of Fluc-mRNA prepared from different cationic lipids exhibited significantly different expression levels in mice. From 3h to 72h, the expression levels of YK-003 and YK-009 were both the highest, indicating that the LNP preparations prepared from them were highly and sustainably expressed in vivo. At 3h, the average radiation intensities for YK-003 and YK-009 were 1,500,820 and 1,234,280, respectively, while YK-004 and YK-010 were the lowest at 69,640 and 60,100, respectively. The mRNAs possessed by these four molecules showed a difference of more than 20-fold in Fluc-mRNA expression between the highest and lowest expression levels in the animal body. At 72 hours, the average radiation intensities of YK-003 and YK-009 were 39538 and 29435, respectively, while the average radiation intensities of YK-004 and YK-010 were the lowest at 2066 and 810.2. The mRNA expression of these four compounds differed by more than 50-fold between the highest and lowest expression levels in the animal body. The values ​​of compounds 23 and 27 were 554600 and 632450, respectively, at 3 hours, but decreased rapidly between 6 and 48 hours, reaching only 4801 and 5512 at 72 hours. This indicates that the mRNA contained in the LNP preparations prepared with these compounds was rapidly degraded or metabolized in the mouse body. The mRNA expression of MC3 was very low at all time points, indicating that its mRNA was low in vivo and its expression was not sustained.

[0239] Table 8 and Figure 10 show that in mice, there was a significant difference in the expression of LNP preparations prepared from YK-009 and compound 25. In LNP preparations with Fluc-mRNA content of 2.5 μg, 5.0 μg, 7.5 μg, and 10 μg, YK-009 expression was 1.6 to 2.1 times higher at 3 hours than compound 25, 1.4 to 1.7 times higher at 6 hours, 5.2 to 5.5 times higher at 24 hours, 6.5 to 7.1 times higher at 48 hours, and 6.1 to 6.8 times higher at 72 hours. In LNP preparations with different Fluc-mRNA content, all YK-009 preparations were highly expressed for a long period in the body, while compound 25 preparations maintained higher expression at 3 and 6 hours, but decreased rapidly from 6 to 48 hours.

[0240] The data was analyzed using GraphPad Prism software. Here, in terms of high and sustained expression of mRNA possessed by LNPs in animal bodies, YK-003 and YK-009 showed significant differences compared to compound 23, YK-003 and YK-009 showed significant differences compared to compound 25, YK-003 and YK-009 showed significant differences compared to compound 27, and YK-003 and YK-009 showed significant differences compared to MC3.

[0241] From the experimental results above, it was found that there is no correspondence between the structure of cationic lipid compounds and the high and sustained expression of mRNA carried by LNPs in the animal body. Cationic lipid compounds with small structural differences show significant differences in mRNA expression promotion in mice. For example, compared to YK-009, YK-004 has two fewer carbon atoms in the G2 group but the rest of its structure is completely identical; YK-010 has one fewer carbon atom in each double strand of the L2 group but the rest of its structure is completely identical; compound 23 has four more carbon atoms in the G1 group, one fewer carbon atom in the L1 group, four more carbon atoms in the G2 group, and one fewer carbon atom in the single strand of the L2 group but the rest of its structure is completely identical; and compound 27 has four more carbon atoms in the G1 group, one fewer carbon atom in the L1 group, and one fewer carbon atom in the single strand of the L2 group but the rest of its structure is completely identical. However, at 3 hours, the mRNA levels of these four strains differed by more than 20 times between their highest and lowest expression levels in the animal body. At 72 hours, the mRNA levels of these four strains differed by more than 50 times between their highest and lowest expression levels in the animal body.

[0242] Compared to YK-009, compound 25 has two more carbon atoms in the G1 group, one more carbon atom in the L1 group, two more carbon atoms in the G2 group, and one less carbon atom in the single strand of the L2 group, while all other structures are identical. However, LNP preparations prepared from YK-009 and compound 25, with Fluc-mRNA content of 2.5 μg, 5.0 μg, 7.5 μg, and 10 μg respectively, showed that at 3 hours, YK-009 was up to 2.1 times that of compound 25; at 6 hours, YK-009 was up to 1.7 times that of compound 25; at 24 hours, YK-009 was up to 5.5 times that of compound 25; at 48 hours, YK-009 was up to 7.1 times that of compound 25; and at 72 hours, YK-009 was up to 7.2 times that of compound 25.

[0243] conclusion Experiments conducted to verify the performance of LNP delivery carriers in vitro and in vivo revealed no clear correspondence between the structure of cationic lipid compounds and intracellular transfection efficiency, cellular toxicity, and high and sustained expression in animal bodies. Compounds with small structural differences may exhibit very large differences in transfection efficiency and / or cellular toxicity and high intracellular expression. For example, compounds YK-009 and YK-010 in this application show a difference of nearly 60 times in cellular transfection efficiency and a difference of more than 25% in toxicity to transfected cells. Furthermore, compounds YK-003 and YK-010 show a difference of nearly 50 times in expression and sustained expression in mice. Therefore, it is difficult to select an appropriate cationic lipid compound that has high transfection efficiency and cellular toxicity, as well as high and sustained expression in mice.

[0244] In this application, through a unique design and extensive screening, we discovered several compounds, such as YK-009, YK-003, YK-006, YK-008, and YK-011. Compared to other compounds in the prior art, these compounds can deliver nucleic acids with high cell transfection efficiency, low toxicity or non-toxicity to cells, and high and sustained expression in animal bodies, yielding unexpected technological benefits.

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

Claims

1. A compound, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein the compound has one of the following structures. 【Chemistry 1】

2. A composition comprising a carrier containing a cationic lipid that includes the compound described in claim 1, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer.

3. The composition according to claim 2, wherein the molar ratio of the cationic lipid to the carrier is 30% to 70%.

4. (1) Under conditions in which the carrier further contains neutral lipids, (2) The carrier further contains structured lipids, (3) Conditions in which the carrier further contains polymer-conjugated lipids The composition according to claim 2, which satisfies any one of the following conditions.

5. (1) The condition that the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1, (2) The condition that the molar ratio of the cationic lipid to the structured lipid is 1:1 to 5:1, (3) The condition that the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 5%, The composition according to claim 4, which satisfies any one of the following conditions.

6. (1) The neutral lipid contains one or more of the following: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and their derivatives. (2) The structured lipid is one or more selected from the group consisting of cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, 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 4, which satisfies any one of the following conditions.

7. (1) The neutral lipids 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 Dieter 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-difytanoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1,The condition is that it is one or more selected from the group consisting of 2-dimiristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof. (2) The condition that 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), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159). The composition according to claim 4, which satisfies any one of the following conditions.

8. The composition according to claim 7, wherein the neutral lipid is DOPE and / or DSPC.

9. (1) The carrier comprises 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 (25-65):(5-25):(25-45):(0.5-5), (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 agent or a prophylactic agent, (4) The composition further comprises one or more pharmaceutically acceptable excipients or diluents. The composition according to claim 2, which satisfies any one of the following conditions.

10. (1) The condition that 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 condition that the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1 The composition according to claim 9, which satisfies any one of the following conditions.

11. (1) The cationic lipid further comprises one or more other ionizable lipid compounds, (2) Under the condition that the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1 to 25:1, (3) Conditions that the therapeutic or prophylactic agent contains one or more of the following: nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The composition according to claim 9, which satisfies any one of the following conditions.

12. (1) Under the condition that 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 compound capable of eliciting an immune response, (3) Conditions under which the therapeutic agent or prophylactic agent is nucleic acid The composition according to claim 11, which satisfies any one of the following conditions.

13. The composition according to claim 12, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA), or the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

14. The composition according to claim 13, 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.

15. The composition according to claim 14, wherein the RNA is mRNA.

16. Use of the compound described in claim 1, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the composition described in any one of claims 2 to 15, in the manufacture of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide drugs, or protein drugs.

17. Use of the compound described in claim 1 or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the composition described in any one of claims 2 to 15, in the manufacture of a pharmaceutical product for treating a disease or condition of a mammal as required.

18. The use according to claim 17, wherein the disease or condition is characterized by dysfunction, abnormal protein or abnormal polypeptide activity.

19. The use according to claim 17, wherein the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative disorders, genetic disorders, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases.

20. The use according to claim 19, wherein the infectious disease is selected from the group consisting of coronavirus, influenza virus or HIV virus-related diseases, childhood pneumonia, Rift Valley fever, yellow fever, rabies, and multiple types of herpes.

21. (1) The condition that the mammal is a human, (2) Under conditions in which the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation, (3) Conditions under which the therapeutic or prophylactic agent is administered to the mammal at a dose of approximately 0.001 mg / kg to approximately 10 mg / kg A use according to any one of claims 17 to 20, which satisfies any one of the following conditions.

22. The use according to claim 21, wherein the composition is administered subcutaneously.

Images