A low-toxicity, spleen-targeting cationic lipid compound containing a carbamate structure, a composition comprising the same, and use thereof

Abstract

The present invention provides a compound of Formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, compositions comprising the foregoing, and their use for delivering a therapeutic or prophylactic agent.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a cationic lipid compound, compositions comprising the same, and their uses. Background Technology

[0002] The effective targeted delivery of bioactive substances, such as small molecule drugs, peptides, proteins, and nucleic acids, especially nucleic acids, remains a persistent medical challenge. Nucleic acid therapeutics face significant challenges due to their low cell permeability and high sensitivity to degradation of certain nucleic acid molecules, including RNA.

[0003] Compositions containing cationic lipids, liposomes, and lipoplexes have been confirmed as effective transport mediators for delivering bioactive substances such as small molecule drugs, peptides, proteins, and nucleic acids into cells and / or intracellular compartments. These compositions typically contain one or more "cationic" and / or amino (ionizable) lipids, including neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, readily ionizable amine-containing lipids. Although various such lipid-containing nanoparticle compositions have been demonstrated, their safety, efficacy, and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, a major concern that could limit their clinical application. For example, LNPsiRNA particles (such as patisiran) require prior administration of steroids and antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369(2013) 819-829.). Therefore, there is a need to develop improved cationic lipid compounds, and compositions containing them, to facilitate the delivery of therapeutic and / or prophylactic agents such as nucleic acids into cells. Summary of the Invention

[0004] This invention provides a cationic lipid compound based on a tertiary carbamate, comprising its pharmaceutically acceptable salts and its stereoisomers or tautomers. This enriches the variety of cationic lipid compounds, providing more options for the efficient delivery of nucleic acid drugs, gene vaccines, small molecule drugs, peptides, or protein drugs. When formed with other lipid components to form lipid nanoparticles, it can effectively deliver mRNA or drug molecules into cells to exert their biological functions.

[0005] In a first aspect, this disclosure provides a cationic lipid compound, which is a compound of formula (I). Or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein:

[0006] G1 is C 2～10 Alkylene;

[0007] G2 is C 2～10 Alkylene;

[0008] G3 is

[0009] R1 is unsubstituted C 6～25 Straight-chain or branched alkyl groups;

[0010] R2 represents unsubstituted C. 6～25 Straight-chain or branched alkyl groups;

[0011] m and n are either 1 or 0. When m is 0, n is 1, and when m is 1, n is 0.

[0012] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is a C5, C7 or C3 alkylene.

[0013] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G2 is a C5, C3 or C7 alkylene.

[0014] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is an unsubstituted C 8～22 Straight-chain alkyl groups.

[0015] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is an unsubstituted C 10 C 11 C8, C9 or C 12 Straight-chain alkyl groups.

[0016] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is an unsubstituted C 20 C 15 C 17 Branched alkyl groups.

[0017] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is

[0018] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R2 is an unsubstituted C 8～22 Straight-chain alkyl groups.

[0019] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R2 is an unsubstituted C 10 C 11 C8, C9 or C 12 Straight-chain alkyl groups.

[0020] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R2 is an unsubstituted C 20 C 15 C 17 Branched alkyl groups.

[0021] The optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R2 is

[0022] An optional compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound of formula (I) has one of the following structures:

[0023]

[0024]

[0025]

[0026] Optional compounds of formula (I) or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, wherein said compound (I) is compound YK-803 having the following structure:

[0027]

[0028] Optional compounds of formula (I) or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, wherein said compound (I) is compound YK-810 having the following structure:

[0029]

[0030] Optional compounds of formula (I) or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, wherein said compound (I) is compound YK-813 having the following structure:

[0031]

[0032] In a second aspect, the present invention provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) as described in any one of the first aspects above, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof.

[0033] A further composition wherein the cationic lipid comprises 25% to 75% of the carrier in molar ratio.

[0034] A further composition, wherein the carrier further comprises neutral lipids.

[0035] A further composition wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1, preferably 4.5:1.

[0036] A further composition wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0037] Further compositions, wherein the neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0038] A further composition wherein the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) and / or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0039] A further composition wherein the carrier further comprises structural lipids.

[0040] A further composition wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1 to 3:1.

[0041] A further composition wherein the structural lipid is selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0042] A further composition, wherein the structural lipid is cholesterol.

[0043] A further composition wherein the carrier further comprises a polymeric conjugated lipid.

[0044] A further composition wherein the polymeric conjugated lipid accounts for 0.5% to 10% of the carrier in molar ratio, preferably 1.5%.

[0045] A further composition wherein the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0046] A further composition wherein the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0047] A further composition wherein the carrier comprises cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. The molar ratio of the cationic lipid:neutral lipid:structural lipid:polymer-conjugated lipid is (25–75):(5–25):(15–65):(0.5–10).

[0048] A further composition wherein the molar ratio of the cationic lipid: neutral lipid: structural lipid: polymer conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

[0049] A further composition wherein the molar ratio of the cationic lipid: neutral lipid: structural lipid: polymer conjugated lipid is 45:10:43.5:1.5.

[0050] A further composition, wherein the composition is a nanoparticle formulation, the average particle size of the nanoparticle formulation being 10 nm to 210 nm; and the polydispersity index (PDI) of the nanoparticle formulation being ≤50%.

[0051] A further composition wherein the average particle size of the nanoparticle formulation is 100 nm to 205 nm; and the polydispersity index (PDI) of the nanoparticle formulation is ≤30%.

[0052] A further composition wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

[0053] Further compositions may also contain a therapeutic or preventative agent.

[0054] A further composition wherein the mass ratio of the carrier to the therapeutic or preventative agent is 10:1 to 30:1.

[0055] A further composition wherein the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1 to 25:1.

[0056] A further composition wherein the mass ratio of the carrier to the therapeutic or preventative agent is 15:1.

[0057] A further composition wherein the therapeutic or preventative agent comprises one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0058] A further composition wherein the therapeutic or preventative agent is a vaccine or compound capable of evoking an immune response.

[0059] A further composition wherein the therapeutic or preventative agent is a nucleic acid.

[0060] A further composition wherein the therapeutic or preventative agent is ribonucleic acid (RNA).

[0061] A further composition wherein the therapeutic or preventative agent is deoxyribonucleic acid (DNA).

[0062] A further composition 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.

[0063] A further composition wherein the RNA is mRNA.

[0064] A further composition, wherein the composition further comprises one or more pharmaceutically acceptable excipients or diluents.

[0065] Thirdly, the present invention provides the use of the compound of formula (I) as described in any one of the first aspects above, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the composition as described in any one of the second aspects above, in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs.

[0066] Fourthly, the present invention provides the use of a compound of formula (I) as described in any of the first aspects above, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or a composition as described in any of the second aspects above, in the preparation of a medicament for treating diseases or conditions in mammals in need.

[0067] Preferred use, wherein the disease or symptom is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0068] Preferred uses, wherein the disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0069] Preferred use, wherein the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or various herpes diseases.

[0070] The preferred use is in which the drug is administered to a human.

[0071] In a preferred use, the route of administration of the drug is intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

[0072] In a preferred use, the drug is administered subcutaneously.

[0073] In a preferred use, the dosage of the drug is 0.001 to 10 mg / kg. Attached Figure Description

[0074] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit the invention.

[0075] Figure 1The results of cell transfection experiments of LNP formulations of Fluc-mRNA prepared based on YK-803, YK-810, YK-813, SM-102, P-76 and compound 13 are shown, where: a is YK-803, b is YK-810, c is YK-813, d is SM-102, e is P-76 and f is compound 13.

[0076] Figure 2 The results of cell transfection fluorescence detection of LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-801, YK-802, YK-803, YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, YK-814, SM-102, MC3, HHMA, P-76, compound 13 and Lipofectamine 3000) are shown.

[0077] Figure 3 The display shows the cell viability of LNP formulations of Fluc-mRNA prepared from different cationic lipids (YK-801, YK-802, YK-803, YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, YK-814, SM-102, MC3, HHMA, P-76, compound 13, and Lipofectamine 3000) after culturing in cell culture medium for 24 h.

[0078] Figure 4 This shows the expression of Fluc-mRNA LNP formulations prepared from different cationic lipids (SM-102, YK-803, YK-810, YK-813) in mouse liver, spleen, lung, heart, and kidney. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. Based on the described embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0080] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such further embodiments obtained through combinations.

[0081] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this disclosure, the use and terminology of this disclosure shall prevail.

[0082] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0083] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0084] Unless otherwise indicated in the working embodiments or elsewhere, all figures for quantitative qualities such as dosages set forth in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any range of figures listed in this application is intended to include all subranges within that range and any combination of the endpoints of that range or subranges.

[0085] As used herein, the words “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms “containing” or “comprising (including)” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently composed of” or “composed of”.

[0086] The term "pharmaceutically acceptable" in this application means that the compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0087] The terms “subject” or “patient” in this application include both humans and mammals.

[0088] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of such a disease, in order to cure, alleviate, reduce, improve, or affect the disease or its symptoms. In the context of this application, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0089] The term "solvent" in this application refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula I used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield a compound of formula I upon absorption into a subject, such that the use of a compound of formula I separately encompasses the use of any solvate of a compound of formula I.

[0090] The term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water.

[0091] It should be further understood that compounds of formula I or pharmaceutically acceptable salts thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this invention. For example, compounds of formula I or pharmaceutically acceptable salts thereof may exist in unsolvated forms as well as in solvated forms formed with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0092] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic addition salt of an inorganic or organic acid of the compounds disclosed herein. See, for example, SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids include, for example, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, etc. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with compounds of Formula I.

[0093] The nitrogen-containing compounds of formula (I) disclosed herein can be converted into N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Therefore, subject to the permissible valence state and structure, the compounds claimed in this application include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0094] Some of the compounds disclosed herein can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in this disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, the compounds of this disclosure can be separated by adding chiral acid resolving reagents such as chiral tartaric acid or chiral malic acid to form salts, utilizing the physicochemical properties of the products, such as differences in solubility.

[0095] This invention also includes all suitable isotopic variants of the compounds disclosed herein. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but whose atomic mass differs from that of atoms commonly or predominantly found in nature. Examples of isotopes that can be introduced into the compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively, for example… 2 H (deuterium) 3 H (tritium) 11 C 13 C 14 C 15 N、 17 O and 18 O.

[0096] The term "alkyl" in this disclosure refers to a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in this disclosure refers to a branched or straight-chain saturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. n～m This refers to groups that include carbon atoms from n to m. For example, C 2～5 Alkylenes include C2 alkylenes, C3 alkylenes, C4 alkylenes, and C5 alkylenes.

[0097] The alkyl (or alkylene) group may be unsubstituted or substituted, wherein at least one hydrogen atom is replaced by another chemical group.

[0098] "Therapeutic effective amount" is the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" is the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can determine the therapeutic and prophylactic effective amounts conventionally based on their knowledge and this disclosure.

[0099] In this application, when the name of a compound differs from its structural formula, the structural formula shall prevail.

[0100] It should be understood that the term "compound disclosed" as used in this application may, depending on the context, include: compounds of formula (I), their N-oxides, their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0101] The term cationic lipid, as used in this article, refers to lipids that carry a positive charge at a selected pH value.

[0102] Cationic liposomes readily bind to negatively charged nucleic acids, interacting with the negatively charged phosphate groups present in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery carriers.

[0103] The inventors discovered during the screening of numerous compounds that identifying suitable cationic lipid compounds that meet the following criteria is extremely difficult: a significantly different structure from representative cationic lipids in the prior art, coupled with extremely high transfection efficiency and extremely low cytotoxicity, and high and sustained expression in mice. This disclosure, through a unique design, has discovered compounds such as YK-803, YK-810, and YK-813 that, compared to cationic lipids with significantly different chemical structures in the prior art, can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression levels and duration in animals, thereby enabling rapid induction of immune responses and antibody production in mRNA vaccines. This significantly enhances preventative efficacy without altering the vaccine composition, which has significant clinical implications.

[0104] In short, this invention is based on at least the following findings:

[0105] 1. A series of cationic lipid compounds, including YK-803, YK-810 and YK-813, are designed. Some of them have significantly different chemical structures from those of representative cationic lipids in the prior art, such as SM-102, DLin-MC3-DMA (MC3) and HHMA; while others have similar chemical structures, such as compound 13.

[0106] SM-102 is a cationic lipid compound disclosed by Moderna, Inc. in WO20170409245A2 (page 29 of the specification).

[0107] DLin-MC3-DMA (MC3) is a cationic lipid compound disclosed by Alnylam Pharmaceuticals, Inc. in CN102625696B (page 6 of the specification).

[0108] HHMA is a cationic lipid compound disclosed by Suzhou Abogen Biosciences Co., Ltd. in CN112979483B (page 7 of the specification).

[0109] P-76 is a cationic lipid compound disclosed by Xiamen Sinobond Biotechnology Co., Ltd. in CN113402405B (page 46 of the specification).

[0110] Compound 13 is a cationic lipid compound disclosed by Fujifilm Corporation of Japan in CN114728016A (page 37 of the specification).

[0111] The chemical structures of representative cationic lipids and structurally similar cationic lipids in the prior art are as follows:

[0112] (WO20170409245A2, page 29 of the instruction manual);

[0113] (CN102625696B, Compound of Formula I, page 6 of the specification.) (CN112979483B, page 12 of the instruction manual)

[0114] (CN113402405B, page 46 of the instruction manual);

[0115] (CN114728016A, page 37 of the instruction manual);

[0116] 2. Among the designed series of compounds, the LNP formulations prepared from YK-803, YK-810, and YK-813, compared with representative and structurally similar cationic lipids in the prior art, exhibit significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly increased mRNA expression levels and duration in mice, significantly increased expression levels in mouse spleens, and significantly reduced expression levels in livers. For example, the cell transfection efficiency of YK-810 can reach 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of compound 13, and 11.23 times that of Lipofectamine 3000; the cell viability of YK-810 is 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA, 15% higher than P-76, 26% higher than compound 13, and higher than Lipofectamine 3000. The expression level of YK-810 in mice was 60% higher than that of SM-102 after 24 hours, 26.19 times that of P-76, and 124.86 times that of compound 13. After 7 days, it was 14.93 times that of SM-102, 59.48 times that of P-76, and 215.00 times that of compound 13. The expression level of YK-810 in the spleen was 15.02 times that of SM-102, while the expression level of YK-810 in the liver was only 0.08 times that of SM-102.

[0117] 3. Among the series of compounds with very similar chemical structures designed in this application, the LNP formulations prepared from YK-803, YK-810, and YK-813, compared with other compounds, showed significantly improved cell transfection efficiency and significantly reduced cytotoxicity; the expression level and duration of mRNA in mice were also significantly increased. For example, the structures of this series of compounds differ only slightly from those of YK-803, YK-810, and YK-813 in some functional groups, but the cell transfection activity of YK-810 can reach 2020 times that of YK-814 and 1390 times that of YK-805; the cytotoxicity of YK-810 is reduced by 39% compared to YK-814; and the expression level of mRNA in mice by YK-810 can reach 200 times that of YK-802.

[0118] There is no clear correlation between the structure of cationic lipid compounds and their intracellular transfection efficiency, cytotoxicity, and the high and sustained expression of mRNA in animals in LNP formulations prepared from them. Compounds with similar structures may exhibit significant differences in transfection efficiency and / or cytotoxicity, as well as intracellular expression. Therefore, screening suitable cationic lipid compounds that simultaneously possess high cell transfection efficiency, low cytotoxicity, high and sustained mRNA expression in animals, and high expression in the spleen but low expression in the liver, is extremely difficult and requires a great deal of creative work.

[0119] 4. Through unique design and screening, this disclosure has discovered some compounds, such as YK-803, YK-810 and YK-813, which, compared with other compounds with similar structures in the prior art, can significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly increase expression level and expression time in animals, significantly increase expression level in animal spleen and significantly reduce expression level in liver, thereby improving delivery efficiency and achieving unexpected technical effects.

[0120] 5. This disclosure, through unique design and screening, has discovered compounds such as YK-803, YK-810, and YK-813 that, while ensuring high efficiency and low toxicity, can target mRNA delivery to the spleen. These compounds are not expressed in other organs, such as the lungs, heart, and kidneys, but are expressed in small amounts in the liver. The low efficiency of vaccine-induced immune responses is a reason why existing cancer therapeutic vaccines cannot achieve their maximum efficacy. The spleen is the largest secondary lymphoid organ in the body. LNP cancer vaccines targeting the spleen can effectively stimulate an immune response and significantly improve efficacy, thus having important clinical application significance in cancer treatment.

[0121] In summary, this disclosure, through unique design and screening, has identified several compounds, such as YK-803, YK-810, and YK-813. These compounds, compared to representative cationic lipids in the prior art, regardless of whether their chemical structures differ significantly (e.g., SM-102, MC3, and HHMA) or are structurally similar (e.g., P-76 and compound 13), are able to deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression levels and duration in animals. mRNA expression levels are significantly increased in the spleen and significantly decreased in the liver. These results represent unexpected technological advancements.

[0122] Specifically as follows:

[0123] 1. The compounds designed in this application have some significant differences in chemical structure compared with representative cationic lipids in the prior art, such as SM-102, MC3 and HHMA; while others have smaller differences, such as compound 13.

[0124] Compared with representative cationic lipids in the prior art, such as SM-102, MC3, HHMA, P-76, and compound 13, this series of designed compounds:

[0125] 1) Compared with SM-102, MC3, and HHMA, the compounds designed in this application have significantly different chemical structures. As can be seen from the chemical structural formulas, the series of compounds in this application introduce urethane ester -OC(O)N(G1)-, which is connected to the head of the tertiary amine structure, the long alkyl straight chain containing the ester group, or the branched tail. In contrast, SM-102, MC3, and HHMA do not have urethane ester structures. The amino head structure of SM-102 is a simple ethanolamine type, the amino head of MC3 is a dimethyl tertiary amine structure, and the amino head structure of HHMA is a methyl tertiary amine structure. The hydroxyl structure is located at the 2-position of the aliphatic chain. The series of compounds in this application have a wide variety of tertiary amine head groups.

[0126] 2) P-76 and Compound 13, like the compounds designed in this application, contain a carbamate structure. P-76 contains two carbamate groups, and the carbamates are located in the hydrophobic tail branches. Compound 13 and the series of compounds in this application have very similar structures, both containing a carbamate structure and two hydrophobic tails, and each hydrophobic tail contains an ester bond.

[0127] 2. The in vitro cell transfection efficiency is significantly improved compared to representative cationic lipids and structurally similar compounds in existing technologies.

[0128] 1) Among the designed series of compounds, the LNP formulations prepared by YK-803, YK-810, and YK-813 exhibited the highest cell transfection efficiency. Compared to representative cationic lipids in the prior art, regardless of whether the structures were significantly different (e.g., SM-102, MC3, and HHMA) or very similar (e.g., compound 13), the cell transfection efficiency was significantly improved. For example, the cell transfection efficiency of YK-810 was 5.17 times that of SM-102, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of compound 13, and 11.23 times that of Lipofectamine 3000.

[0129] 2) A series of compounds with similar structures, where the ester bonds of their carbamate structures are linked to G1 or G2 groups, including YK-801, YK-802, and YK-803, were compared. These compounds share the same carbamate structure with G1 or G2 groups linked to their ester bonds, differing only slightly in a few other structural groups. Cell transfection results showed significant differences in activity among these compounds, with YK-803 exhibiting the highest transfection efficiency. The transfection efficiency of YK-803 was 35.36 times that of YK-801 and 93.24 times that of YK-802.

[0130] 3) A series of compounds with similar structures, whose ester bonds in their carbamate structures are linked to G3 groups, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. All these compounds have ester bonds in their carbamate structures linked to G3 groups, with only slight differences in a few other structural groups. Cell transfection results showed that the activities of this series of compounds varied greatly, with YK-810 and YK-813 exhibiting the highest cell transfection efficiency. For example, the cell transfection efficiencies of YK-810 and YK-813 are 257.28 times that of YK-804 (598.49 times), 1390.52 times and 597.75 times that of YK-805 (1390.52 times and 597.75 times), and 2020.10 times and 868.39 times that of YK-814 (2020.10 times and 868.39 times), respectively.

[0131] 4) There is no direct correlation between compound structure and intracellular transfection efficiency. Compounds with very similar structures may exhibit significant differences in intracellular transfection efficiency. Therefore, screening for cationic lipids with high intracellular transfection efficiency is extremely difficult and requires a great deal of creative work.

[0132] 3. The cytotoxicity is significantly reduced compared to representative cationic lipids and structurally similar compounds in the prior art.

[0133] 1) The series of compounds designed in this application, including YK-803, YK-810, and YK-813, have chemical structures that differ significantly from those of representative cationic lipids in the prior art, such as SM-102, MC3, and HHMA; while others have similar structures, such as compound 13. LNP formulations prepared from YK-803, YK-810, and YK-813 exhibit the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids in the prior art. For example, the cell viability of YK-810 is 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA, 15% higher than P-76, 26% higher than compound 13, and 60% higher than Lipofectamine 3000.

[0134] 2) A series of compounds with similar structures, where the ester bond of the carbamate structure is linked to a G1 or G2 group, including YK-801, YK-802, and YK-803, were compared. The structural differences between these compounds were only slight variations in a few individual groups. The results showed that YK-803 exhibited the lowest cytotoxicity and significantly improved cell viability. For example, the cell viability of YK-803 was 18% higher than that of YK-801.

[0135] 3) A series of compounds with similar structures, where the ester bond of the carbamate structure is linked to the G3 group, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The results showed that YK-810 and YK-813 exhibited the lowest cytotoxicity and significantly improved cell viability. For example, the cell viability of YK-810 and YK-813 was 38% and 35% higher than that of YK-804, and 39% and 36% higher than that of YK-814, respectively.

[0136] 4) There is no direct correlation between compound structure and cytotoxicity. Even compounds with very similar structures can exhibit significant differences in cytotoxicity. Therefore, it is impossible to predict cytotoxicity based on chemical structure, making it extremely difficult to screen for cationic lipids with low cytotoxicity, requiring a great deal of creative work.

[0137] 4. The expression level and duration of mRNA in animals were significantly improved compared to representative cationic lipids and structurally similar compounds in the prior art, and it showed targeting of the spleen.

[0138] 1) The LNP formulations prepared from YK-803, YK-810, and YK-813 showed significantly increased mRNA expression levels and duration in mice compared to representative cationic lipids in the prior art. Expression levels at 6h, 24h, 48h, and 7d were significantly higher than those of representative cationic lipids in the prior art. For example, at 24 hours, YK-810 showed 5.78 times that of SM-102, 26.19 times that of P-76, and 124.86 times that of compound 13; at 48 hours, YK-810 showed 7.97 times that of SM-102, 23.17 times that of P-76, and 123.44 times that of compound 13; and at 7 days, YK-810 showed 14.93 times that of SM-102, 59.48 times that of P-76, and 215.00 times that of compound 13.

[0139] 2) Compared with the structurally similar compound YK-811, which has slight differences in allotropes, the LNP formulations prepared from YK-803, YK-810, and YK-813 significantly increased the expression level and duration of mRNA in mice. For example, YK-810 reached 130.17 times that of YK-802 at 24 hours, 200.74 times at 48 hours, and 110.86 times at 7 days.

[0140] 3) The LNP formulations prepared from YK-803, YK-810, and YK-813 showed significantly increased mRNA expression levels in mouse spleens compared to SM-102, a representative cationic lipid in the prior art. For example, the expression levels of YK-803, YK-810, and YK-813 in the spleen were 8.61-fold, 15.02-fold, and 7.58-fold, respectively, compared to SM-102. The mRNA expression in mouse spleens was consistent with the cell transfection results in Example 6. The expression levels of YK-803, YK-810, and YK-813 in the liver were very weak, at 0.02-fold, 0.08-fold, and 0.04-fold, respectively, compared to SM-102. The proportions of delivered mRNA expressed in the spleen and liver were 0.09 times higher for SM-102, 47.44 times higher for YK-803, 16.50 times higher for YK-810, and 17.25 times higher for YK-813.

[0141] The spleen is the largest secondary lymphoid organ in animals. By increasing the expression level of delivered mRNA in the spleen, mRNA vaccines can rapidly induce an immune response and produce antibodies in vivo. This can significantly improve the preventive effect without altering the vaccine composition, which has important clinical significance. It also shows good targeted efficacy for developing treatments for diseases caused by spleen damage or abnormalities, such as lymphoma and leukemia.

[0142] 4) There is no direct correlation between the compound structure of cationic lipids and the expression level and duration of mRNA in animals. Compounds with very similar structures may produce LNP formulations with significantly different mRNA expression levels in animals. Therefore, it is extremely difficult to screen cationic lipids that exhibit high and sustained mRNA expression in animals based on their chemical structure, as this cannot be predicted. This requires a great deal of creative work.

[0143] This disclosure provides a novel cationic lipid compound for delivering therapeutic or preventative agents. The cationic lipid compound of this disclosure can be used to deliver nucleic acid molecules, small molecule compounds, peptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compound of this disclosure exhibits higher transfection efficiency, lower cytotoxicity, and higher expression in animals, thus improving delivery efficiency and safety.

[0144] This disclosure provides a cationic lipid, which is a compound of formula (I).

[0145] Or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein:

[0146] G1 is C 2～10 Alkylene;

[0147] G2 is C 2～10 Alkylene;

[0148] G3 is

[0149]

[0150] R1 is unsubstituted C 6～25 Straight-chain or branched alkyl groups;

[0151] R2 represents unsubstituted C. 6～25 Straight-chain or branched alkyl groups;

[0152] m and n are 1 or 0. When m is 0, n is 1, that is, the nitrogen element in formula (I) is directly connected to G3 through a covalent bond; when m is 1, n is 0, that is, the nitrogen element in formula (I) is directly connected to G2 through a covalent bond.

[0153] In one embodiment, G1 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0154] In one embodiment, G1 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0155] In one embodiment, G1 is an unsubstituted C7 alkylene, for example, -(CH2)7-.

[0156] In one embodiment, G2 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0157] In one embodiment, G2 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0158] In one embodiment, G2 is an unsubstituted C7 alkylene, for example, -(CH2)7-.

[0159] In one implementation, G3 is

[0160] In one implementation, G3 is

[0161] In one implementation, G3 is

[0162] In one implementation, G3 is

[0163] In another implementation, G3 is...

[0164] In some preferred embodiments, the compound of formula (I) has one of the following structures:

[0165]

[0166]

[0167]

[0168]

[0169] In a more preferred embodiment, the compound of formula (I) is one of the following structures:

[0170]

[0171]

[0172] Another aspect of this disclosure provides a composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) above or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0173] In some embodiments, the composition is a nanoparticle formulation with an average size of 140 nm to 210 nm, preferably 140 nm to 205 nm; the polydispersity index of the nanoparticle formulation is ≤50%, preferably ≤30%, and more preferably ≤25%.

[0174] cationic lipids

[0175] In one embodiment of the composition / carrier disclosed herein, the cationic lipid is one or more selected from compounds of formula (I) above, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers. In some embodiments, the cationic lipid is selected from compounds of formula (I) above. For example, the cationic lipid is compound YK-801, YK-802, YK-803, YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, or YK-814. In a preferred embodiment, the cationic lipid is compound YK-810. In another preferred embodiment, the cationic lipid is compound YK-803. In another preferred embodiment, the cationic lipid is compound YK-813. In another preferred embodiment, the cationic lipid is compound YK-811. In another preferred embodiment, the cationic lipid is compound YK-809. In another preferred embodiment, the cationic lipid is compound YK-807.

[0176] In some embodiments, the cationic lipid accounts for 25% to 75% of the carrier molar ratio, for example 35%, 45%, 49%, 50%, 51%, 55%, 60%, and 65%.

[0177] This carrier can be used to deliver active ingredients, such as therapeutic or preventative agents. The active ingredient can be encapsulated within the carrier or bound to the carrier.

[0178] For example, the therapeutic or preventative agent comprises one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. The nucleic acid includes, but is 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.

[0179] neutral lipids

[0180] The carrier may contain neutral lipids. In this disclosure, neutral lipids refer to lipids that are uncharged at a selected pH or exist in a zwitterionic form and play an auxiliary role. Neutral lipids may modulate the flowability of nanoparticles to form lipid bilayer structures and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0181] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1, for example, 14:1, 13:1, 12:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5.1:1, 5:1, 4.9:1, 4.6:1, 4.5:1, 4.4:1, 4:1, 3:1, 2:1. In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 4.5:1.

[0182] For example, neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0183] The carrier component of a composition comprising cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0184] The neutral lipid moiety may be selected from the non-restricted group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-restricted group consisting of lauric acid, myristic acid, myristenoic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzanoic acid, docosapentaenoic acid, and docosahexaenoic acid. It also encompasses non-natural species including natural species with modifications and substitutions, such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of a composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components such as targeting or imaging components (e.g., dyes).

[0185] The neutral lipids that can be used in these compositions may be selected from the non-limiting group of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16Lyso PC), 1,2-dilinanoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphocholine), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0186] In some embodiments, neutral lipids include DSPC. In some embodiments, neutral lipids include DOPE. In some embodiments, neutral lipids include both DSPC and DOPE.

[0187] structural lipids

[0188] The carrier of the composition comprising cationic lipids may also include 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.

[0189] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1. In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 45:43.5.

[0190] 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 (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0191] Polymer conjugated lipids

[0192] The carrier of the composition containing cationic lipids may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages.

[0193] In some embodiments, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350–5000 Da.

[0194] For example, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0195] In one embodiment of the composition / carrier disclosed herein, the polymeric conjugated lipid is DMG-PEG2000.

[0196] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is (25–75):(5–25):(15–65):(0.5–10), for example (35–49):(4.5–15):(35–55):(1–5).

[0197] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is 45:10:43.5:1.5.

[0198] Therapeutic agents and / or preventative agents

[0199] The composition may include one or more therapeutic and / or preventive agents. In some embodiments, the mass ratio of the carrier to the therapeutic or preventive agent is 10:1 to 30:1, for example 12.5:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

[0200] In some embodiments, the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1 to 25:1, preferably 15:1.

[0201] The therapeutic or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0202] For example, the therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

[0203] The carriers disclosed herein can deliver therapeutic and / or preventive agents to mammalian cells or organs, and thus this disclosure also provides methods for treating diseases or conditions in mammals in need, including administering a composition comprising therapeutic and / or preventive agents to mammals and / or contacting mammalian cells with the composition.

[0204] Therapeutic agents and / or preventive agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic agents and / or preventive agents can be substances that, upon delivery to a cell or organ, induce a desired change in that cell or organ or other body tissue or system. Such species can be used to treat one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventive agents are small molecule drug substances that can be used to treat a specific disease, condition, or illness.Examples of pharmaceuticals that can be used in a composition include, but are not limited to, anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside). Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0205] In some implementations, the therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraquinone. Anthracindione, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, and may include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and / or novel epitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and / or compounds capable of eliciting an immune response are administered intramuscularly by a composition comprising compounds according to formulas (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, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, lactamase (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), and cyclophosphamides). Phosphoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP, cisplatin), anthracyclines (e.g. daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), and antimitotic agents (e.g. vincristine, vinblastine, paclitaxel and levothyroxine).

[0206] In other embodiments, the therapeutic and / or prophylactic agents are proteins. 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), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0207] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to 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 inducible factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribonuclease; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribonuclease, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some implementations, the RNA is mRNA.

[0208] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any polypeptide that is naturally occurring or non-naturally present or otherwise modified. The polypeptide encoded by the mRNA may have any size and may possess any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells.

[0209] In other embodiments, the therapeutic and / or preventative agent is siRNA. siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA may be chosen such that, upon administration of a composition comprising the siRNA to a subject in need, a gene associated with a specific disease, symptom, or condition is silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0210] In some implementations, the therapeutic and / or preventative agents are sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0211] In some implementations, the therapeutic and / or prophylactic agent is shRNA or its encoding vector or plasmid. shRNA can be generated within the target cell after delivery of an appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant field.

[0212] Disease or ailment

[0213] The compositions / carriers disclosed herein can deliver therapeutic or preventative agents to subjects or patients. These therapeutic or preventative agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. Therefore, the compositions disclosed herein can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs. Due to the wide variety of such therapeutic or preventative agents, the compositions disclosed herein can be used to treat or prevent a variety of diseases or conditions.

[0214] In some implementations, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0215] For example, the disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0216] In some implementations, the infectious disease is selected from diseases caused by coronaviruses, influenza viruses, or HIV viruses, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes diseases.

[0217] Other components

[0218] The composition may include one or more components other than those described in the foregoing sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0219] The composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogues).

[0220] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), and mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, and eprazinone). The composition may contain mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, streptococcal DNase α (dornasealfa), neltenexine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0221] The composition may also contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0222] In addition to these components, the composition may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients, including but not limited to one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Examples of excipients include starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0223] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0224] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0225] The compositions disclosed herein can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. For example, a sterile injectable solution can be prepared by incorporating the desired amount of the therapeutic or prophylactic agent with the various other desired ingredients described above into a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Surfactants may also be added to promote the formation of a homogeneous solution or suspension.

[0226] For example, the compositions of this disclosure can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the compositions are administered subcutaneously.

[0227] The compositions disclosed herein are administered in therapeutically effective amounts, which can vary not only with the specific reagent chosen, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, the therapeutic or prophylactic agent can be administered to mammals (e.g., humans) at doses of 0.001 to 10 mg / kg.

[0228] Example

[0229] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific embodiments of the present invention, the raw materials used are all commercially available. Unless otherwise stated, percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0230] The following abbreviations represent the following reagents:

[0231] DCM: Dichloromethane; EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP: 4-Dimethylaminopyridine; DCC: N,N'-Dicyclohexylcarbodiimide; CDI: Carbonyldiimidazole; rt: Room temperature

[0232] Example 1: Synthesis of cationic lipid compounds

[0233] 1. Synthesis of YK-801

[0234] The synthesis route is as follows:

[0235]

[0236] Step 1: Synthesis of 6-(octyloxy)-6-oxohexyl-1H-imidazolium-1-carboxylate (YK-801-PM1)

[0237] Carbonyl diimidazole (563 mg, 3.47 mmol) was dissolved in dichloromethane (10 mL). Under a nitrogen atmosphere, the solution was cooled to 0 °C in an ice-water bath. Octyl 6-hydroxyhexanoate (424 mg, 1.74 mmol) was added in portions to the system. After the addition was complete, the mixture was brought to room temperature and stirred for 3 hours. After the reaction was complete, the reaction solution was washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding YK-801-PM1 (525 mg, 1.55 mmol, 89.4%). 18 H 30 N₂O₄, MS(ES): m / z(M+H) + )339.2.

[0238] Step 2: Synthesis of (3-chloro-2-hydroxypropyl)carbamate tert-butyl ester (YK-801-PM2)

[0239] 1-Amino-3-chloro-2-propanol hydrochloride (4.0 g, 27.39 mmol) was dissolved in dioxane (180 mL) and water (60 mL), and the solution was cooled to 0 °C in an ice-water bath. 1 M sodium hydroxide solution (55 mL) was added dropwise. After the addition was complete, the pH of the system was >10. Di-tert-butyl carbonate anhydride (6.6 g, 30.24 mmol) was then added. After the addition was complete, the mixture was brought to room temperature and stirred for 5 hours. After the reaction was complete, 100 mL of water was added to the reaction solution, followed by extraction with dichloromethane (100 mL × 2). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (0-30% ethyl acetate / n-hexane) to obtain YK-801-PM2 (3.8 g, 18.12 mmol, 66.2%). 16ClNO3, MS(ES): m / z(M+H) + )210.1.

[0240] Step 3: Synthesis of tert-butyl (2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)carbamate (YK-801-PM3)

[0241] YK-801-PM2 (3.8 g, 18.12 mmol) and 2-(methylamino)ethanol (1.6 g, 21.30 mmol) were dissolved in acetonitrile (50 mL). Potassium carbonate (7.5 g, 54.27 mmol) and potassium iodide (3.0 g, 18.07 mmol) were added to the above system, and the mixture was heated to 70 °C and stirred for 5 hours. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (0-20% dichloromethane / methanol) to obtain YK-801-PM3 (3.5 g, 14.09 mmol, 77.8%). 11 H 24 N₂O₄, MS(ES): m / z(M+H) + )249.2.

[0242] Step 4: Synthesis of 1-amino-3-((2-hydroxyethyl)(methyl)amino)propane-2-ol (YK-801-PM4)

[0243] YK-801-PM3 (3.5 g, 14.09 mmol) was added to a 4M hydrogen chloride / 1,4-dioxane solution (35 mL) and stirred at room temperature for 10 hours. After the reaction was complete, the solution was concentrated, and 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate aqueous solution were added. After stirring, the mixture was separated, and then extracted with dichloromethane (500 mL × 2). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent, yielding YK-801-PM4 (1.8 g, 12.14 mmol, 86.2%). C6H 16 N₂O₂, MS(ES): m / z(M+H) + )149.1.

[0244] Step 5: Synthesis of 2-octyl dodecyl 6-((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)amino)hexanoic acid (YK-801-PM5)

[0245] The YK-801-PM4 (1.0 g, 6.75 mmol) and 2-octyl-dodecyl 6-bromohexanoate (3.2 g, 6.73 mmol) prepared above were dissolved in acetonitrile (10 mL). Potassium carbonate (2.8 g, 20.26 mmol) was added to the above system, and the mixture was heated to 70 °C and stirred for 7 hours. After the reaction was completed, 50 mL of water was added to the reaction solution, followed by extraction with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (0-20% dichloromethane / methanol) to obtain YK-801-PM5 (0.8 g, 1.47 mmol, 21.9%). 32 H 66 N₂O₄, MS(ES): m / z(M+H) + 543.5;

[0246] Step Six: Synthesis of (6-(((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)(6-(((2-octyldodecyl)oxy)-6-oxohexyl)carbamoyl)oxyhexanoate octyl ester (YK-801)

[0247] YK-801-PM5 (239 mg, 0.44 mmol), YK-801-PM1 (74 mg, 0.22 mmol), triethylamine (20 mg, 0.20 mmol), and potassium carbonate (83 mg, 0.60 mmol) were dissolved in tetrahydrofuran (5 mL), and the mixture was heated to 60 °C and stirred for 3 hours. After the reaction was complete, 10 mL of water was added to the reaction solution, followed by extraction with dichloromethane (10 mL × 2). The combined organic phases were washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (0-22% dichloromethane / methanol) to obtain YK-801 (63 mg, 0.08 mmol, 35.2%). 47 H 92 N₂O₈, MS(ES): m / z(M+H) + )813.7.

[0248] 1H NMR (CDCl3, 400MHz, 298K) δ4.40 (s, 1H), 4.33 (d, J = 4.8Hz, 1H), 4.15 (t, J = 6.7Hz ,2H),4.05(t,J=6.8Hz,2H),3.95(d,J=5.8Hz,2H),3.27(d,J=12.3Hz,2H),3.07 -2.93(m,4H),2.87(s,2H),2.57(s,3H),2.31(dd,J=10.0,4.8Hz,4H),1.90(s,1 H),1.75-1.52(m,10H),1.50-1.37(m,4H),1.26(s,44H),0.88(t,J=6.7Hz,9H).

[0249] 2. Synthesis of YK-802

[0250] The synthesis route is as follows:

[0251]

[0252] Step 1: Synthesis of 6-(dodecyloxy)-6-oxohexyl-1H-imidazolium-1-carboxylate (YK-802-PM1)

[0253] Using carbonyl diimidazole (131 mg, 0.81 mmol) and dodecyl 6-hydroxyhexanoate (120 mg, 0.40 mmol) as starting materials, YK-802-PM1 (118 mg, 0.30 mmol, 75.0%) was obtained according to the method for synthesizing YK-801-PM1. 22 H 38 N₂O₄, MS(ES): m / z(M+H) + )395.3.

[0254] Step 2: Synthesis of (6-(((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)(6-(((2-octyldodecyl)oxy)-6-oxohexyl)carbamoyl)oxyhexanoic acid dodecyl ester (YK-802)

[0255] Using YK-801-PM5 (525 mg, 0.97 mmol) and YK-802-PM1 (39 mg, 0.10 mmol) as raw materials, YK-802 (65 mg, 0.07 mmol, 74.8%) was obtained according to the method for synthesizing YK-801. 51 H 100 N₂O₈, MS(ES): m / z(M+H) + )869.8.

[0256] 1H NMR (CDCl3, 400MHz, 298K) δ4.40 (s, 1H), 4.33 (d, J = 4.8Hz, 1H), 4.15 (t, J = 6.7Hz ,2H),4.05(t,J=6.8Hz,2H),3.95(d,J=5.8Hz,2H),3.27(d,J=12.3Hz,2H),3.07 -2.93(m,4H),2.87(s,2H),2.57(s,3H),2.31(dd,J=10.0,4.8Hz,4H),1.90(s,1 H),1.75-1.52(m,10H),1.50-1.37(m,4H),1.26(s,52H),0.88(t,J=6.7Hz,9H).

[0257] 3. Synthesis of YK-803

[0258] The synthesis route is as follows:

[0259]

[0260] Step 1: Synthesis of 6-((2-octyldodecyl)oxy)-6-oxohexyl-1H-imidazolium-1-carboxylate (YK-803-PM1)

[0261] Using carbonyl diimidazole (131 mg, 0.81 mmol) and 2-octyl dodecyl 6-hydroxyhexanoate (167 mg, 0.40 mmol) as starting materials, YK-803-PM1 (182 mg, 0.36 mmol, 89.8%) was obtained according to the method for synthesizing YK-801-PM1. 30 H 54 N₂O₄, MS(ES): m / z(M+H) + )507.4.

[0262] Step 2: Synthesis of 6-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)(((6-(((2-octyldodecyl)oxy)-6-oxohexyl)oxycarbonyl)amino)hexanoic acid-2-octyldodecyl ester (YK-803)

[0263] Using YK-801-PM5 (525 mg, 0.97 mmol) and YK-803-PM1 (45 mg, 0.09 mmol) as raw materials, YK-803 (75 mg, 0.08 mmol, 86.0%) was obtained according to the method for synthesizing YK-801. 59 H 116 N₂O₈, MS(ES): m / z(M+H) + )981.9.

[0264] 1 H NMR(CDCl3,400MHz,298K)δ4.23(t,J=5.2Hz,3H),4.15(t,J=6.8Hz,2H),3.96(d,J=5 .6Hz,4H),3.25(d,J=15.0Hz,2H),3.01(d,J=7.4Hz,2H),2.87-2.68(m,2H),2.65(d, J=5.3Hz,2H),2.40(s,3H),2.32(t,J=7.4Hz,4H),1.86(d,J=7.3Hz,2H),1.67(dt,J= 15.7,7.3Hz,8H),1.49-1.36(m,4H),1.30(d,J=28.5Hz,64H),0.88(t,J=6.7Hz,12H).

[0265] 4. Synthesis of YK-804

[0266] The synthesis route is as follows:

[0267]

[0268] Step 1: Synthesis of 8-(6-oxo-6-(undecyloxy)hexyl)aminooctanoic acid-2-octyldodecyl ester (YK-804-PM1)

[0269] Using undecyl 6-aminohexanoate (2825 mg, 9.90 mmol) and 2-octyldodecyl 8-bromooctanoate (4155 mg, 8.25 mmol) as starting materials, YK-804-PM1 (3060 mg, 4.32 mmol, 52.4%) was obtained according to the method for synthesizing YK-801-PM5. 45 H 89 NO4,MS(ES):m / z(M+H + )708.7.

[0270] Step 2: Synthesis of 8-(N-(6-oxo-6-(undecyloxy)hexyl)-1H-imidazol-1-formamido)octanoic acid-2-octyldodecyl ester (YK-804-PM2)

[0271] Using carbonyl diimidazole (286 mg, 1.76 mmol) and YK-804-PM1 (500 mg, 0.71 mmol) as raw materials, YK-804-PM2 (337 mg, 0.42 mmol, 59.2%) was obtained by following the method for synthesizing YK-801-PM1. 49 H 91 N3O5, MS(ES): m / z(M+H) + )802.7.

[0272] Step 3: Synthesis of 6-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)(((6-((2-octyldodecyl)oxy)-6-oxohexyl)oxycarbonyl)amino)hexanoic acid-2-octyldodecyl ester (YK-804)

[0273] Using YK-804-PM2 (337 mg, 0.42 mmol) and N-methyldiethanolamine (200 mg, 1.68 mmol) as raw materials, YK-804 (260 mg, 0.30 mmol, 72.5%) was obtained by following the method for synthesizing YK-801. 51 H 100 N₂O₇, MS(ES): m / z(M+H) + )853.8.

[0274] 1 H NMR (CDCl3, 400MHz, 298K) δ4.40 (s, 2H), 4.05 (t, J = 6.7Hz, 2H), 3.96 (d, J = 5.8Hz, 2H ),3.81(s,2H),3.35(dd,J=15.2,7.6Hz,4H),3.19(s,2H),3.07(d,J=25.8Hz,2H),2 .92(d,J=34.8Hz,2H),2.66(s,2H),2.30(td,J=7.3,3.6Hz,3H),2.18(s,1H),1.70- 1.56(m,10H),1.50(d,J=7.1Hz,2H),1.28(d,J=16.5Hz,54H),0.88(t,J=6.7Hz,9H).

[0275] 5. Synthesis of YK-805

[0276] The synthesis route is as follows:

[0277]

[0278] Step 1: Synthesis of bis(2-octyldodecyl)-6,6'-azadimethyldihexanoate (YK-805-PM1)

[0279] Using 2-octyldodecyl 6-aminohexanoate (436 mg, 1.06 mmol) and 2-octyldodecyl 6-bromohexanoate (504 mg, 1.06 mmol) as starting materials, YK-805-PM1 (436 mg, 0.54 mmol, 50.9%) was obtained according to the method for synthesizing YK-801-PM5. 52 H 103 NO4,MS(ES):m / z(M+H +)806.8.

[0280] Step 2: Synthesis of bis(2-octyldodecyl)-6,6'-((1H-imidazol-1-carbonyl)azadiyl)dihexanoate (YK-805-PM2)

[0281] Using carbonyl diimidazole (263 mg, 1.62 mmol) and YK-805-PM1 (436 mg, 0.54 mmol) as raw materials, YK-805-PM2 (412 mg, 0.46 mmol, 84.7%) was obtained by following the method for synthesizing YK-801-PM1. 56 H 105 N3O5, MS(ES): m / z(M+H) + )900.8.

[0282] Step 3: Synthesis of bis(2-octyldodecyl)-6,6'-((2-((2-hydroxyethyl)(methyl)amino)ethoxy)carbonyl)azadiyl)dihexanoate (YK-805)

[0283] Using YK-805-PM2 (412 mg, 0.46 mmol) and N-methyldiethanolamine (437 mg, 3.67 mmol) as raw materials, YK-805 (200 mg, 0.21 mmol, 45.7%) was obtained according to the method for synthesizing YK-801. 58 H 114 N₂O₇, MS(ES): m / z(M+H) + )951.9.

[0284] 1 H NMR (CDCl3, 400MHz, 298K) δ4.30 (s, 2H), 3.96 (d, J = 5.7Hz, 4H), 3.70 (s, 2H), 3.19 (s, 4H), 2.92 (s, 2H), 2.80 (s, 2H), 2.51(s,3H),2.30(t,J=7.5Hz,4H),1.64(dt,J=14.9,7.3Hz,6H),1.53(s,4H),1.26(s,68H),0.88(t,J=6.8Hz,12H).

[0285] 6. Synthesis of YK-806

[0286] The synthesis route is as follows:

[0287]

[0288] Step 1: Synthesis of 3-hexylnonyl 6-(((4-(decoxy)-4-oxobutyl)amino)hexanoic acid (YK-806-PM1)

[0289] Using 6-aminohexanoic acid-3-hexylnonyl ester (630 mg, 1.84 mmol) and 4-bromobutyrate-decyl ester (566 mg, 1.84 mmol) as starting materials, YK-806-PM1 (410 mg, 0.72 mmol, 39.2%) was obtained according to the method for synthesizing YK-801-PM5. 35 H 69 NO4,MS(ES):m / z(M+H + 568.5.

[0290] Step 2: Synthesis of 3-hexylnonyl 6-(N-(4-(decoxy)-4-oxobutyl)-1H-imidazol-1-carbamoyl)hexanoate (YK-806-PM2)

[0291] Using carbonyl diimidazole (234 mg, 1.44 mmol) and YK-806-PM1 (410 mg, 0.72 mmol) as raw materials, YK-806-PM2 (400 mg, 0.60 mmol, 83.9%) was obtained by following the method for synthesizing YK-801-PM1. 39 H 71 N3O5, MS(ES): m / z(M+H) + 662.5.

[0292] Step 3: Synthesis of 6-((4-(decoxy)-4-oxobutyl)(2-((2-hydroxyethyl)(methyl)amino)ethoxy)carbonyl)amino)hexanoic acid-3-hexylnonyl ester (YK-806)

[0293] Using YK-806-PM2 (300 mg, 0.45 mmol) and N-methyldiethanolamine (432 mg, 3.63 mmol) as raw materials, YK-806 (90 mg, 0.13 mmol, 28.0%) was obtained by following the method for synthesizing YK-801. 41 H 80 N₂O₇, MS(ES): m / z(M+H) + )713.6.

[0294] 1H NMR(CDCl3,400MHz,298K)δ4.57(s,2H),4.06(dd,J=12.4,6.6Hz,4H),3.38(s,2H),3.32-3.16(m,4H),2.91(s,2H),2.63(s,2H ),2.30(dd,J=17.3,7.4Hz,4H),2.18(s,3H),1.85(s,2H),1.65-1.53(m,10H),1.28(d,J=18.4Hz,35H),0.88(t,J=6.7Hz,9H).

[0295] 7. Synthesis of YK-807

[0296] The synthesis route is as follows:

[0297]

[0298] Step 1: Synthesis of 2-octyl dodecyl 6-(6-oxo-6-(undecyloxy)hexyl)amino)hexanoic acid (YK-807-PM1)

[0299] Using undecyl 6-aminohexanoate (540 mg, 1.89 mmol) and 2-octyldodecyl 6-bromohexanoate (600 mg, 1.26 mmol) as starting materials, YK-807-PM1 (398 mg, 0.59 mmol, 46.4%) was obtained according to the method for synthesizing YK-801-PM5. 43 H 85 NO4,MS(ES):m / z(M+H + 680.7.

[0300] Step 2: Synthesis of 2-octyl dodecyl 6-(N-(6-oxo-6-(undecyloxy)hexyl)-1H-imidazol-1-formamido)hexanoic acid (YK-807-PM2)

[0301] Using carbonyl diimidazole (185 mg, 1.14 mmol) and YK-807-PM1 (388 mg, 0.57 mmol) as raw materials, YK-807-PM2 (374 mg, 0.48 mmol, 84.7%) was obtained by following the method for synthesizing YK-801-PM1. 47 H 87 N3O5, MS(ES): m / z(M+H) + )774.7.

[0302] Step 3: Synthesis of 6-((2-((2-hydroxyethyl)(methyl)amino)ethoxy)carbonyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoic acid-2-octyldodecyl ester (YK-807)

[0303] Using YK-807-PM2 (374 mg, 0.48 mmol) and N-methyldiethanolamine (461 mg, 3.87 mmol) as raw materials, YK-807 (71 mg, 0.09 mmol, 17.9%) was obtained by following the method for synthesizing YK-801. 49 H 96 N₂O₇, MS(ES): m / z(M+H) + 825.7.

[0304] 1 H NMR(CDCl3,400MHz,298K)δ4.24(t,J=5.5Hz,2H),4.05(t,J=6.8Hz,2H),3.96(d,J=5.7Hz,2H),3.64(t,J=5.0Hz,2H),3.20(d,J=5.5Hz,4H),2.82 (s,2H),2.71(s,2H),2.42(s,3H),2.34-2.26(m,4H),2.18(s,1H),1.70- 1.57(m,8H),1.53(s,4H),1.28(d,J=15.8Hz,50H),0.88(t,J=6.7Hz,9H).

[0305] 8. Synthesis of YK-808

[0306] The synthesis route is as follows:

[0307]

[0308] Step 1: Synthesis of 6-(6-(nonoxy)-6-oxohexyl)amino)hexanoic acid-heptadecane-9-ester (YK-808-PM1)

[0309] Using nonyl 6-aminohexanoate (890 mg, 3.46 mmol) and heptane 9-yl-6-bromohexanoate (1.0 g, 2.31 mmol) as starting materials, YK-808-PM1 (588 mg, 0.96 mmol, 41.7%) was obtained according to the method for synthesizing YK-801-PM5. 38 H 75 NO4,MS(ES):m / z(M+H + )610.6.

[0310] Step 2: Synthesis of heptadecanoyl-9-ester 6-(N-(6-(nonoxy)-6-oxohexyl)-1H-imidazol-1-carbamate)hexanoate (YK-808-PM2)

[0311] Using carbonyl diimidazole (313 mg, 1.93 mmol) and YK-808-PM1 (588 mg, 0.96 mmol) as raw materials, YK-808-PM2 (569 mg, 0.81 mmol, 84.2%) was obtained by following the method for synthesizing YK-801-PM1. 42 H 77 N3O5, MS(ES): m / z(M+H) + )704.6.

[0312] Step 3: Synthesis of heptadecano-9-yl-6-(((2-((2-hydroxyethyl)(methyl)amino)ethoxy)carbonyl)(6-(nonoxy)-6-oxohexyl)amino)hexanoate (YK-808)

[0313] Using YK-808-PM2 (469 mg, 0.67 mmol) and N-methyldiethanolamine (636 mg, 5.34 mmol) as raw materials, YK-808 (135 mg, 0.18 mmol, 26.7%) was obtained according to the method for synthesizing YK-801. 44 H 86 N₂O₇, MS(ES): m / z(M+H) + )755.7.

[0314] 1 H NMR(CDCl3,400MHz,298K)δ4.85(t,J=6.3Hz,1H),4.40(s,2H),4.05(t,J=6.7Hz,2H),3.81(s,2H),3.20(d,J=6.5Hz,4H),3.10(s,2H),2.96 (s,2H),2.65(d,J=9.4Hz,3H),2.29(dd,J=12.9,7.4Hz,4H),1.63(dt,J=15.4,7.6Hz,8H),1.51(s,8H),1.26(s,38H),0.88(t,J=6.8Hz,9H).

[0315] 9. Synthesis of YK-809

[0316] The synthesis route is as follows:

[0317]

[0318] Step 1: Synthesis of 6-(6-oxo-6-(undecyloxy)hexyl)amino)hexanoic acid-heptadecane-9-ester (YK-809-PM1)

[0319] Using undecyl 6-aminohexanoate (2240 ​​mg, 7.85 mmol) and heptadecanoate-9-bromohexanoate (3403 mg, 7.85 mmol) as starting materials, YK-809-PM1 (1740 mg, 2.73 mmol, 34.7%) was obtained according to the method for synthesizing YK-801-PM5. 40 H 79 NO4,MS(ES):m / z(M+H + 638.6.

[0320] Step 2: Synthesis of 6-(N-(6-oxo-6-(undecyloxy)hexyl)-1H-imidazol-1-formamido)hexanoic acid-heptadecane-9-ester (YK-809-PM2)

[0321] Using carbonyl diimidazole (864 mg, 5.33 mmol) and YK-809-PM1 (1700 mg, 2.66 mmol) as raw materials, YK-809-PM2 (1600 mg, 2.19 mmol, 82.0%) was obtained by following the method for synthesizing YK-801-PM1. 44 H 81 N3O5, MS(ES): m / z(M+H) + )732.6.

[0322] Step 3: Synthesis of heptadecan-9-yl-6-(((2-(bis(2-hydroxyethyl)amino)ethoxy)carbonyl)(6-oxo-6-(undecyloxy)hexyl)amino)hexanoate (YK-809)

[0323] Using YK-809-PM2 (600 mg, 0.82 mmol) and triethanolamine (978 mg, 6.56 mmol) as raw materials, YK-809 (110 mg, 0.14 mmol, 16.5%) was obtained according to the method for synthesizing YK-801. 47 H 92 N₂O₈, MS(ES): m / z(M+H) + )813.7.

[0324] 1H NMR (CDCl3, 400MHz, 298K) δ4.85 (s, 1H), 4.25 (s, 2H), 4.05 (t, J = 6.7Hz, 2H), 3.64 (s, 4H), 3.19 (d, J = 5.9Hz, 4H), 2.92 (dd, J = 18.7, 10.9Hz, 2H ),2.79(s,4H),2.31(dd,J=13.7,6.9Hz,4H),1.63(dd,J=15.3,7.7Hz,8H),1.57-1.45(m,8H),1.28(d,J=16.7Hz,42H),0.88(t,J=6.7Hz,9H).

[0325] 10. Synthesis of YK-810

[0326] The synthesis route is as follows:

[0327]

[0328] Step 1: Synthesis of 2-octyl dodecyl 6-(4-(decoxy)-4-oxobutyl)amino)hexanoic acid (YK-810-PM1)

[0329] Using decyl 4-bromobutyrate (922 mg, 3.00 mmol) and 2-octyl dodecyl 6-aminohexanoate (1235 mg, 3.00 mmol) as starting materials, YK-810-PM1 (1116 mg, 1.75 mmol, 58.3%) was obtained according to the method for synthesizing YK-801-PM5. 40 H 79 NO4,MS(ES):m / z(M+H + 638.6.

[0330] Step 2: Synthesis of 2-octyl dodecyl hexanoate (YK-810-PM2)

[0331] Using carbonyl diimidazole (851 mg, 5.25 mmol) and YK-810-PM1 (1116 mg, 1.75 mmol) as starting materials, YK-810-PM2 (1180 mg, 1.61 mmol, 92.1%) was obtained by following the method for synthesizing YK-801-PM1. 44 H 81 N3O5, MS(ES): m / z(M+H) + )732.6.

[0332] Step 3: Synthesis of 6-(((2-(bis(2-hydroxyethyl)amino)ethoxy)carbonyl)(4-(decoxy)-4-oxobutyl)amino)hexanoic acid-2-octyl dodecyl ester (YK-810)

[0333] Using YK-810-PM2 (590 mg, 0.81 mmol) and triethanolamine (962 mg, 6.45 mmol) as raw materials, YK-810 (131 mg, 0.16 mmol, 20.0%) was obtained by following the method for synthesizing YK-801. 47 H 92 N₂O₈, MS(ES): m / z(M+H) + )813.7.

[0334] 1 H NMR (CDCl3, 400MHz, 298K) δ4.35 (s, 2H), 4.06 (t, J = 6.6Hz, 2H), 3.96 (d, J = 5.8Hz, 2H), 3.76 (s, 4H), 3.24 (dd, J = 15.7, 7.9Hz, 4H ),3.06(s,2H),2.96(s,4H),2.31(t,J=7.0Hz,4H),1.85(s,3H),1.69-1.49(m,6H),1.33-1.25(m,48H),0.88(t,J=6.7Hz,9H).

[0335] 11. Synthesis of YK-811

[0336] The synthesis route is as follows:

[0337]

[0338] Step 1: Synthesis of decanoic acid 6-((6-((3-hexylnonyl)oxy)-6-oxohexyl)amino)decanoic acid ester (YK-811-PM1)

[0339] Using decyl 6-aminohexanoate (1000 mg, 3.68 mmol) and 3-hexylnonyl 6-bromohexanoate (1000 mg, 2.47 mmol) as starting materials, YK-811-PM1 (534 mg, 0.90 mmol, 36.3%) was obtained according to the method for synthesizing YK-801-PM5. 37 H 73 NO4,MS(ES):m / z(M+H + 596.6.

[0340] Step 2: Synthesis of decanoic acid 6-(N-(6-((3-hexylnonyl)oxy)-6-oxohexyl)-1H-imidazol-1-carbamate)decanoate (YK-811-PM2)

[0341] Using carbonyl diimidazole (290 mg, 1.79 mmol) and YK-811-PM1 (534 mg, 0.90 mmol) as raw materials, YK-811-PM2 (549 mg, 0.80 mmol, 88.8%) was obtained by following the method for synthesizing YK-801-PM1. 41 H 75 N3O5, MS(ES): m / z(M+H) + 690.6.

[0342] Step 3: Synthesis of 6-(((2-(bis(2-hydroxyethyl)amino)ethoxy)carbonyl)(6-(((3-hexylnonyl)oxy)-6-oxohexyl)amino)decanoic acid ester (YK-811)

[0343] Using YK-811-PM2 (499 mg, 0.72 mmol) and triethanolamine (862 mg, 5.78 mmol) as raw materials, YK-811 (70 mg, 0.09 mmol, 12.6%) was obtained according to the method for synthesizing YK-801. 44 H 86 N₂O₈, MS(ES): m / z(M+H) + )771.6.

[0344] 1 H NMR(CDCl3,400MHz,298K)δ4.36(s,2H),4.06(dd,J=15.6,7.6Hz,4H),3.77(d,J=5.9Hz,4H),3.20(d,J=6.8Hz,4H),3.09 (s,2H),2.98(s,4H),2.30(s,4H),1.61(ddd,J=22.4,14.6,7.2Hz,14H),1.28(d,J=18.4Hz,37H),0.88(t,J=6.7Hz,9H).

[0345] 12. Synthesis of YK-812

[0346] The synthesis route is as follows:

[0347]

[0348] Step 1: Synthesis of 6-(4-oxo-4-(undecyloxy)butyl)amino)hexanoic acid-heptadecane-9-ester (YK-812-PM1)

[0349] Using undecyl 4-bromobutyrate (964 mg, 3.00 mmol) and heptadecanoyl 6-aminohexanoate (1110 mg, 3.00 mmol) as starting materials, YK-812-PM1 (1630 mg, 2.67 mmol, 89.0%) was obtained according to the method for synthesizing YK-801-PM5. 38 H 75 NO4,MS(ES):m / z(M+H + )610.6.

[0350] Step 2: Synthesis of heptadecano-9-yl ester of 6-(N-(4-oxo-4-(undecyloxy)butyl)-1H-imidazol-1-carbamoyl)hexanoate (YK-812-PM2)

[0351] Using carbonyl diimidazole (486 mg, 3.00 mmol) and YK-812-PM1 (610 mg, 1.00 mmol) as raw materials, YK-812-PM2 (319 mg, 0.45 mmol, 45.3%) was obtained by following the method for synthesizing YK-801-PM1. 42 H 77 N3O5, MS(ES): m / z(M+H) + )704.6.

[0352] Step 3: Synthesis of heptadecan-9-yl-1-hydroxy-3,6-bis(2-hydroxyethyl)-10-oxo-11-(4-oxo-4-(undecyloxy)butyl)-9-oxo-3,6,11-triaza-heptadecan-17-ester (YK-812)

[0353] Using YK-812-PM2 (319 mg, 0.45 mmol) and ethylenediaminetetraethanol (643 mg, 2.72 mmol) as raw materials, YK-812 (68 mg, 0.08 mmol, 17.2%) was obtained according to the method for synthesizing YK-801. 49 H 97 N3O9, MS(ES): m / z(M+H) + )872.7.

[0354] 1 H NMR (CDCl3, 400MHz, 298K) δ4.84 (s, 1H), 4.10-3.99 (m, 4H), 3.36 (s, 6H), 3.22 (s, 6H), 2.30 (dd, J = 1 7.7,7.0Hz,6H),1.83(s,4H),1.62(s,6H),1.51(s,12H),1.36-1.21(m,40H),0.88(t,J=6.7Hz,9H).

[0355] 13. Synthesis of YK-813

[0356] The synthesis route is as follows:

[0357]

[0358] Step 1: Synthesis of 8-(6-oxo-6-(undecyloxy)hexyl)aminooctanoic acid-2-octyldodecyl ester (YK-813-PM1)

[0359] Using undecyl 6-aminohexanoate (1865 mg, 6.53 mmol) and 2-octyldodecyl 8-bromooctanoate (3290 mg, 6.53 mmol) as starting materials, YK-813-PM1 (1820 mg, 2.57 mmol, 39.4%) was obtained according to the method for synthesizing YK-801-PM5. 45 H 89 NO4,MS(ES):m / z(M+H + )708.7.

[0360] Step 2: Synthesis of 8-(N-(6-oxo-6-(undecyloxy)hexyl)-1H-imidazol-1-formamido)octanoic acid-2-octyldodecyl ester (YK-813-PM2)

[0361] Using carbonyl diimidazole (830 mg, 5.12 mmol) and YK-813-PM1 (1820 mg, 2.57 mmol) as raw materials, YK-813-PM2 (2000 mg, 2.49 mmol, 96.9%) was obtained by following the method for synthesizing YK-801-PM1. 49 H 91 N3O5, MS(ES): m / z(M+H) + )802.7.

[0362] Step 3: Synthesis of 8-((2-(bis(2-hydroxyethyl)amino)ethoxy)carbonyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid-2-octyldodecyl ester (YK-813)

[0363] Using YK-813-PM2 (500 mg, 0.62 mmol) and triethanolamine (930 mg, 6.23 mmol) as raw materials, YK-813 (60 mg, 0.07 mmol, 10.9%) was obtained by following the method for synthesizing YK-801. 52 H 102 N₂O₈, MS(ES): m / z(M+H) + )883.8.

[0364] 1 H NMR (CDCl3, 400MHz, 298K) δ4.46 (s, 2H), 4.05 (t, J = 6.6Hz, 2H), 3.96 (d, J = 5.8Hz, 2H), 3.89 (s, 4H ),3.20(s,6H),2.30(s,4H),1.70-1.45(m,17H),1.28(d,J=16.3Hz,54H),0.88(t,J=6.8Hz,9H).

[0365] 14. Synthesis of YK-814

[0366] The synthesis route is as follows:

[0367]

[0368] Step 1: Synthesis of 6-(4-(decoxy)-4-oxobutyl)amino)hexanoic acid-heptadecane-9-ester (YK-814-PM1)

[0369] Using decyl 4-bromobutyrate (550 mg, 1.79 mmol) and heptadecanoyl 6-aminohexanoate (1000 mg, 2.68 mmol) as starting materials, YK-814-PM1 (330 mg, 0.55 mmol, 30.9%) was obtained according to the method for synthesizing YK-801-PM5. 37 H 73 NO4,MS(ES):m / z(M+H + 596.6.

[0370] Step 2: Synthesis of heptadecanoyl-9-ester 6-(N-(4-(decoxy)-4-oxobutyl)-1H-imidazol-1-carbamoyl)heptadecanoate (YK-814-PM2)

[0371] Using carbonyl diimidazole (180 mg, 1.11 mmol) and YK-814-PM1 (330 mg, 0.55 mmol) as raw materials, YK-814-PM2 (378 mg, 0.55 mmol, 98.9%) was obtained by following the method for synthesizing YK-801-PM1. 41 H 75 N3O5, MS(ES): m / z(M+H) + 690.6.

[0372] Step 3: Synthesis of 6-((4-(decoxy)-4-oxobutyl)(3-(4-(3-hydroxypropyl)piperazin-1-yl)propoxy)carbonyl)amino)heptadecanoate-9-ester (YK-814)

[0373] Using YK-814-PM2 (240 mg, 0.35 mmol) and 3,3'-(piperazine-1,4-diyl)bis(propane-1-ol) (830 mg, 4.11 mmol) as starting materials, YK-814 (53 mg, 0.06 mmol, 18.5%) was obtained by following the method for synthesizing YK-801. 48 H 93 N3O7, MS(ES): m / z(M+H) + )824.7.

[0374] 1 H NMR (CDCl3, 400MHz, 298K) δ4.90-4.80 (m, 1H), 4.12 (s, 2H), 4.05 (t, J = 6.9Hz, 2H), 3.85 (s, 2H), 3.20 (s, 4H), 2.95 (s, 4H) ),2.28(t,J=7.5Hz,8H),2.00(s,2H),1.83(s,4H),1.58(dd,J=36.3,13.8Hz,16H),1.26(s,38H),0.88(t,J=6.7Hz,9H).

[0375] 15. Synthesis of P-76

[0376] The synthesis route is as follows:

[0377]

[0378] Step 1: Synthesis of di-tert-butyl(((2-(2-hydroxyethoxy)ethyl)azadiyl)bis(hexane-6,1-diyl))dicarbamate (P-76-PM1)

[0379] Diethylene glycolamine (1051 mg, 10.00 mmol) and tert-butyl (6-bromohexyl)carbamate (5604 mg, 20.00 mmol) were dissolved in acetonitrile (50 mL). Potassium carbonate (4146 mg, 30.00 mmol) was added to the above system, and the mixture was heated to 70 °C and stirred for 5 hours. After the reaction was complete, the reaction mixture was filtered, the filtrate was concentrated under vacuum, and the residue was purified by silica gel chromatography (0-24% methanol / dichloromethane) to give P-76-PM1 (2720 mg, 5.40 mmol, 54.0%). 26 H 53 N3O6, MS(ES): m / z(M+H) + )504.4.

[0380] Step Two: N 1 -(6-aminohexyl)-N 1Synthesis of 1,6-(2-((tert-butyldiphenylsilyl)oxy)ethoxy)ethyl)hexane-1,6-diamine (P-76-PM2)

[0381] P-76-PM1 (2720 mg, 5.40 mmol), tert-butyldiphenylchlorosilane (1520 mg, 5.53 mmol), and imidazole (1021 mg, 15.00 mmol) were dissolved in N,N-dimethylformamide (50 mL) and stirred at room temperature for 5 h. After the reaction was complete, ethyl acetate (100 mL) and water (100 mL) were added. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was dissolved in 50 mL of dichloromethane, and trifluoroacetic acid (30 mL) was slowly added. The mixture was stirred at room temperature for 10 h. After the reaction was complete, the mixture was concentrated, and 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate aqueous solution were added. After stirring, the mixture was separated, and then extracted with dichloromethane (50 mL × 2). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (0-30% methanol / dichloromethane) to give P-76-PM2 (2568 mg, 4.74 mmol, 87.8%). 32 H 53 N3O2Si, MS(ES): m / z(M+H) + )542.4.

[0382] Step 3: Synthesis of 2-hexyldecyl(4-nitrophenyl) carbonate (P-76-PM3)

[0383] 4-Nitrophenyl chloroformate (806 mg, 4.00 mmol) was dissolved in tetrahydrofuran (10 mL), and the system was kept at a temperature below 5 °C in an ice-water bath. 2-Hexyldecyl-1-ol (980 mg, 4.04 mmol) was added to the solution, and the mixture was brought to room temperature and stirred for 1 hour. Then, ethyl acetate (20 mL) and water (10 mL) were added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-20% ethyl acetate / n-hexane) to give P-76-PM3 (1378 mg, 3.38 mmol, 84.5%).

[0384] Step 4: Synthesis of bis(2-hexyldecyl)(((2-(2-hydroxyethoxy)ethyl)azadiyl)bis(hexane-6,1-diyl))dicarbamate (P-76)

[0385] Compounds P-76-PM3 (1378 mg, 3.38 mmol), P-76-PM2 (2492 mg, 4.60 mmol), and triethylamine (0.5 mL) were dissolved in tetrahydrofuran (15 mL), and the mixture was heated to 50 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature, and then 1 M tetrabutylammonium bromide aqueous solution (10 mL) was added. The mixture was stirred at room temperature for 2 hours, followed by the addition of ethyl acetate (20 mL) and water (10 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-20% methanol / dichloromethane) to give P-76 (1368 mg, 1.63 mmol, 48.2%). 50 H 101 N3O6, MS(ES): m / z(M+H) + )840.8.

[0386] 1 H NMR (CDCl3, 400MHz, 298K) δ4.90-4.80 (m, 4H), 4.12 (s, 2H), 4.05 (t, J = 6.9Hz, 4H), 3.85 (s, 4H) ),3.20(s,4H),2.95(s,2H),1.83(s,2H),1.58(s,4H),1.26(s,60H),0.88(t,J=6.7Hz,12H).

[0387] 16. Synthesis of Compound 13

[0388] The synthesis route is as follows:

[0389]

[0390] Step 1: Synthesis of 2-((2-(dimethylamino)ethyl)(methyl)amino)ethane-1-ol (compound 13-PM1)

[0391] N 1 N 1 N 2 Trimethylethane-1,2-diamine (1022 mg, 10.00 mmol) and bromoethanol (625 mg, 5.00 mmol) were dissolved in acetonitrile (20 mL). Potassium carbonate (2073 mg, 15.00 mmol) was added to the above system, and the mixture was heated to 70 °C and stirred for 5 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-24% methanol / dichloromethane) to give compound 13-PM1 (590 mg, 4.03 mmol, 80.7%). C7H 18 N₂O, MS(ES): m / z(M+H) +)147.1.

[0392] Step 2: Synthesis of 2-((2-(dimethylamino)ethyl)(methyl)amino)ethyl(4-nitrophenyl)carbonate (compound 13-PM2)

[0393] 4-Nitrophenyl chloroformate (806 mg, 4.00 mmol) was dissolved in tetrahydrofuran (10 mL), and the system was kept at a temperature below 5 °C using an ice-water bath. Compound 13-PM1 (590 mg, 4.03 mmol) was added to the solution, and the mixture was brought to room temperature and stirred for 1 hour. Then, ethyl acetate (20 mL) and water (10 mL) were added, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-50% ethyl acetate / n-hexane) to give compound 13-PM2 (1050 mg, 3.37 mmol, 84.3%). 14 H 21 N3O5, MS(ES): m / z(M+H) + )312.2.

[0394] Step 3: Synthesis of dinonyl 8,8'-azadiyldioctanoate (compound 13-PM3)

[0395] Using 8-bromooctanoic acid diester (349 mg, 1.00 mmol) and 8-aminooctanoic acid nonyl ester (571 mg, 2.00 mmol) as starting materials, compound 13-PM3 (510 mg, 0.92 mmol, 92.1%) was obtained according to the method for synthesizing YK-801-PM5. 34 H 67 NO4,MS(ES):m / z(M+H + 554.5.

[0396] Step 4: Synthesis of nonyl-2,5-dimethyl-10-(8-(nonoxy)-8-oxooctyl)-9-oxo-8-oxo-2,5,10-triazaoctadecane-18-ester (compound 13)

[0397] Compound 13-PM2 (286 mg, 0.92 mmol), compound 13-PM3 (510 mg, 0.92 mmol), and triethylamine (0.5 mL) were dissolved in tetrahydrofuran (5 mL), and the mixture was heated to 50 °C and stirred for 5 hours. The reaction mixture was cooled to room temperature, and then ethyl acetate (10 mL) and water (5 mL) were added. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (0-20% methanol / dichloromethane) to give compound 13 (288 mg, 0.40 mmol, 43.1%).42 H 83 N3O6, MS(ES): m / z(M+H) + )726.6.

[0398] 1 H NMR(CDCl3,400MHz,298K)δ4.20-4.01(6H,m),3.24-3.09(4H,m),2.71-2.51(4H,m),2.44-2.38( 2H,m),2.31(3H,s),2.26(6H,s),1.79-1.43(12H,m),1.37-1.23(40H,m),0.88(6H,t,J=6.0Hz).

[0399] Example 2: Optimization of preparation conditions for lipid nanoparticles (LNP formulation)

[0400] 1. Optimization of the ratio of vector (liposome) to mRNA

[0401] The cationic lipid compounds YK-803, YK-810, and YK-813 synthesized in Example 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 to prepare ethanol lipid solutions. The ethanol lipid solutions were rapidly added to citrate buffer (pH = 4–5) using the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH = 4–5) to obtain an aqueous mRNA solution. A certain volume of liposome solution and the aqueous mRNA solution were mixed to prepare liposomes at total lipid to mRNA weight ratios of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1, respectively. The liposomes were sonicated at 25°C for 15 min (ultrasound frequency 40 kHz, ultrasound power 800 W). The obtained liposomes were diluted 10 times with PBS and then ultrafiltered to remove ethanol using a 300 kDa ultrafiltration tube. The solution was then brought to a final volume with PBS to obtain an LNP formulation containing eGFP-mRNA encapsulated with cationic lipids YK-810 / DSPC / cholesterol / DMG-PEG2000 (molar ratio 49:10:39.5:1.5).

[0402] Cell transfection experiments showed that a vector-to-mRNA weight ratio of 10:1 to 30:1 resulted in good transfection effects, with 15:1 being the most effective. Ratios of 5:1 and 35:1 showed poor transfection effects and should not be used to transport mRNA.

[0403] 2. Optimization of the ratio of cationic lipids to neutral lipids

[0404] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, wherein the molar ratios of cationic lipid YK-810 to neutral lipid DSPC were 1:1, 3:1, 3.5:1, 4:1, 4.5:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

[0405] Cell transfection experiments showed that transfection was effective at molar ratios of cationic lipids to neutral lipids ranging from 1:1 to 15:1, with the highest transfection efficiency achieved at 4.5:1.

[0406] 3. Optimization of the ratio of polymer-conjugated lipids to carriers (liposomes)

[0407] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1. The cationic lipid in the carrier was YK-810 (or YK-803, YK-813), and the molar ratio of polymeric conjugated lipid DMG-PEG2000 in the carrier was 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10%, and 15%, respectively.

[0408] Cell transfection experiments showed that transfection was effective when the molar ratio of polymer conjugated lipids to carrier was in the range of 0.5% to 10%, with the highest transfection efficiency at 1.5% and the lowest at 10%.

[0409] 4. Optimization of the proportions of components in the carrier (liposome)

[0410] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, wherein the molar ratios of cationic lipid YK-810 (or YK-803, YK-813), neutral lipid DSPC, structural lipid cholesterol, and polymeric conjugated lipid DMG-PEG2000 were 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.

[0411] Cell transfection experiments showed that transfection was achieved with molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5. Good transfection effects were observed within the range of (35–49):(7.5–15):(35–55):(1–5), with the best transfection effect observed at a molar ratio of 45:10:43.5:1.5.

[0412] Example 3: Cell transfection experiment with LNP formulation of eGFP-mRNA

[0413] Cell resuscitation and passage: 293T cells were resuscitated and passaged in culture dishes to the required number of cells.

[0414] Seeding: Digest and count the cells in the culture dish, seed 10,000 cells per well into a 96-well plate or 150,000 cells per well into a 12-well plate, and culture overnight until the cells adhere.

[0415] Cell transfection experiment: LNP formulation containing 1.5 μg of eGFP-mRNA prepared in Example 2 (the cationic lipid in the vector is YK-803, YK-810 or YK-813) and Lipofectamine 3000 formulation containing eGFP-mRNA were added to the cell culture medium of 12-well plates. After culturing for 24 h, the transfection efficiency of the samples was examined by fluorescence microscopy based on the fluorescence intensity.

[0416] Based on the experimental results, the preparation conditions for the lipid nanoparticles (LNP formulation) were finally determined as follows: the ratio of carrier to mRNA was 15:1; the molar ratio of cationic lipids to neutral lipids was 4.5:1; the proportion of polymer-conjugated lipids in liposomes was 1.5%; and the molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids was 45:10:43.5:1.5. Subsequent experiments were conducted to prepare lipid nanoparticles (LNP formulation) under these conditions.

[0417] Example 4: Preparation of lipid nanoparticles (LNP formulation) (optimal ratio)

[0418] Table 1. Structure of cationic lipids

[0419]

[0420]

[0421]

[0422]

[0423] The cationic lipids listed in Table 1 were dissolved in ethanol at a molar ratio of 45:10:43.5:1.5 with DSPC (Avitol (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avitol (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000, respectively, to prepare ethanol lipid solutions. The ethanol lipid solutions were rapidly added to citrate buffer (pH = 4–5) using the ethanol injection method and vortexed for 30 seconds. eGFP-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH = 4–5) to obtain an aqueous mRNA solution. A certain volume of liposome solution and the aqueous mRNA solution were mixed to prepare liposomes at a total lipid to mRNA weight ratio of 15:1. The liposomes were sonicated at 25℃ for 15 min (ultrasound frequency 40 kHz, sonic power 800 W). The obtained liposomes were diluted 10 times with PBS and then ultrafiltered in a 300 kDa ultrafiltration tube to remove ethanol. The volume was then adjusted to a certain level using PBS to obtain an LNP formulation in which eGFP-mRNA or Fluc-mRNA was encapsulated using cationic lipids / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 45:10:43.5:1.5).

[0424] Lipofectamine 3000 transfection reagent is widely used for cell transfection due to its excellent transfection performance and efficiency, as well as its ability to improve cell viability. It is also suitable for difficult-to-transfect cell types. We used Lipofectamine 3000 transfection reagent as a control and prepared eGFP-mRNA or Fluc-mRNA formulations according to the method described in the Lipofectamine 3000 (Invictus (Shanghai) Trading Co., Ltd.) instructions.

[0425] Example 5: Determination of particle size and polydispersity index (PDI) of nanolipid particles

[0426] Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering.

[0427] Take 10 μL of liposome solution, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and measure each sample three times. The measurement conditions are: 90° scattering angle, 25℃. The detection results are shown in Table 2.

[0428] Table 2. Particle size and polydispersity index (PDI) of nanolipid particles.

[0429] name Particle size (nm) PDI (%) YK-801 165 11.6 YK-802 158 17.9 YK-803 143 14.7 YK-804 200 24.2 YK-805 170 15.6 YK-806 164 11.5 YK-807 158 15.9 YK-808 169 17.2 YK-809 155 12.6 YK-810 168 14.9 YK-811 145 14.7 YK-812 205 23.8 YK-813 170 5.6 YK-814 148 13.9 SM-102 188 15.2 MC3 175 14.3 HHMA 163 19.2 P-76 159 18.9 Compound 13 197 17.1

[0430] As shown in Table 2, the nanolipid particles prepared in Example 4 have a particle size between 140 and 210 nm, and all of them can be used to deliver mRNA.

[0431] Among them, the particles prepared by YK-803 have the smallest particle size, at 143 nm; while the particles prepared by YK-812 have the largest particle size, at 205 nm.

[0432] The polydispersity index of all nanolipid particles ranged from 5% to 25%, with the smallest being YK-813 at 5.6% and the largest being YK-804 at 24.2%.

[0433] The particle morphology prepared by YK-803, YK-810 and YK-813 is also at a good level.

[0434] Example 6: In vitro validation of the performance of the LNP delivery vector

[0435] Cell resuscitation and passage: The method is the same as in Example 3.

[0436] Seed plate: The method is the same as in Example 3.

[0437] 1. Fluorescence detection of Fluc-mRNA (transfection efficiency)

[0438] An LNP formulation containing 0.3 μg Fluc-mRNA (the LNP formulation carrier components were cationic lipids, DSPC, cholesterol, and DMG-PEG2000, with a molar ratio of 45:10:43.5:1.5, where the cationic lipids were those listed in Table 1) was added to the cell culture medium of a 96-well plate. After culturing for 24 h, the appropriate reagents were added according to the Gaussian Luciferase Assay Kit instructions, and the fluorescence expression intensity of each well was detected by an IVIS fluorescence detection system. The chemical structures of the designed compounds and representative cationic lipids in the prior art are shown in Table 1. The transfection efficiency of the LNP formulation prepared from a series of cationic lipid compounds designed in this application and existing cationic lipids, including SM-102, MC3, HHMA, P-76, compound 13, and Lipofectamine 3000, in cells is shown in Table 3.

[0439] Table 3 lists the fluorescence detection results of LNP formulations containing Fluc-mRNA prepared from different cationic lipids.

[0440] Table 3 Fluorescence detection results of Fluc-mRNA

[0441]

[0442]

[0443] Analysis of experimental results:

[0444] (1) The series of cationic lipid compounds designed in this application, including YK-803, YK-810 and YK-813, have chemical structures that are very different from those of representative cationic lipids in the prior art, such as SM-102, MC3 and HHMA; and chemical structures that are similar, such as compound 13.

[0445] Compared with existing representative cationic lipids SM-102, MC3, and HHMA, the compounds designed in this application have significantly different chemical structures. As can be seen from the chemical structural formulas, the series of compounds in this application creatively introduces a carbamate ester – OC(O)N(G1)- – which is connected to the head of a tertiary amine structure, or to the tail of a long alkyl chain containing an ester group, or a branched chain. SM-102, MC3, and HHMA lack a carbamate ester structure. The amino head structure of SM-102 is a simple ethanolamine type, the amino head structure of MC3 is a dimethyl tertiary amine structure, and the amino head structure of HHMA is a methyl tertiary amine structure. The hydroxyl group is located at the 2-position of the aliphatic chain. The series of compounds in this application have a diverse range of tertiary amine head groups.

[0446] Compared to existing cationic lipids containing urethane structures, such as P-76 and Compound 13, the compounds designed in this application have similar chemical structures, all containing urethane structures. P-76 contains two urethane groups, while Compound 13 and the series of compounds in this application each have one urethane group. Furthermore, P76, Compound 13, and the compounds in this application all have two hydrophobic tails, and the hydrophobic tail group of Compound 13 also contains an ester bond.

[0447] (2) Among the designed series of compounds, the LNP formulations prepared by YK-803, YK-810, and YK-813 showed the highest cell transfection efficiency. Compared with representative cationic lipids in the prior art, regardless of whether the structures were significantly different (e.g., SM-102, MC3, and HHMA) or had relatively small structural differences (e.g., compound 13), the cell transfection efficiency was significantly improved. For example, the cell transfection efficiency of YK-810 was 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of compound 13, and 11.23 times that of Lipofectamine 3000.

[0448] As shown in Table 3, the LNP formulations containing Fluc-mRNA prepared from YK-803, YK-810, and YK-813 exhibited the strongest fluorescence absorption, with RLU values ​​of 5879074, 8789452, and 3778372, respectively. Figure 1 and Figure 2 )

[0449] YK-803 is 3.46 times that of SM-102, 34.77 times that of MC3, 5.33 times that of HHMA, 35.15 times that of P-76, 126.77 times that of compound 13, and 7.51 times that of Lipofectamine 3000.

[0450] YK-810 is 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of compound 13, and 11.23 times that of Lipofectamine 3000.

[0451] YK-813 is 2.22 times that of SM-102, 22.35 times that of MC3, 3.43 times that of HHMA, 22.59 times that of P-76, 81.47 times that of compound 13, and 4.83 times that of Lipofectamine 3000.

[0452] Data analysis using GraphPad Prism software showed that any of YK-803, YK-810, and YK-813 exhibited significant differences compared to SM-102, MC3, HHMA, P-76, compound 13, and Lipofectamine 3000, indicating a significant improvement in transfection efficiency.

[0453] The cell transfection efficiency of LNP preparations made from cationic lipid compounds cannot be inferred from their structures. Whether the compounds have huge structural differences or similar structures, their cell transfection efficiency is very likely to vary greatly.

[0454] (3) Compared with a series of compounds with similar structures and ester bonds in the carbamate structure linked to G1 or G2 groups, YK-803 has the highest cell transfection efficiency. The cell transfection efficiency of YK-803 can reach 35.36 times that of YK-801 and 93.24 times that of YK-802.

[0455] A series of compounds with similar structures, whose ester bonds in their carbamate structures are linked to G1 or G2 groups, including YK-801, YK-802, and YK-803, were compared. These compounds have ester bonds in their carbamate structures linked to G1 or G2 groups, with only minor differences in a few other structural groups.

[0456] Cell transfection results showed that the activities of this series of compounds varied greatly, with YK-803 exhibiting the highest cell transfection efficiency. The cell transfection efficiency of YK-803 was 35.36 times that of YK-801 and 93.24 times that of YK-802.

[0457] Data analysis using GraphPad Prism software showed that YK-803 was significantly different from YK-801 and YK-802, with a significantly improved transfection efficiency.

[0458] (4) Compared with a series of compounds with similar structures and ester bonds in the carbamate structure linked to G3 groups, YK-810 and YK-813 have the highest cell transfection efficiency. For example, the cell transfection efficiency of YK-810 and YK-813 can reach 1390.52 times and 597.75 times that of YK-805, and 2020.10 times and 868.39 times that of YK-814, respectively.

[0459] A series of compounds with similar structures and ester bonds in their carbamate structures linked to G3 groups, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. In all these compounds, the ester bonds in their carbamate structures are linked to G3 groups, with only slight differences in a few other structural groups.

[0460] Cell transfection results showed that the activities of this series of compounds varied greatly, with YK-810 and YK-813 showing the highest cell transfection efficiency.

[0461] The cell transfection efficiencies of YK-810 were 598.49 times that of YK-804, 1390.52 times that of YK-805, 21.26 times that of YK-806, 9.94 times that of YK-807, 244.42 times that of YK-808, 26.30 times that of YK-809, 138.95 times that of YK-811, 80.64 times that of YK-812, and 2020.10 times that of YK-814.

[0462] The cell transfection efficiencies of YK-813 were 257.28 times that of YK-804, 597.75 times that of YK-805, 9.14 times that of YK-806, 4.27 times that of YK-807, 105.07 times that of YK-808, 11.31 times that of YK-809, 59.73 times that of YK-811, 34.67 times that of YK-812, and 868.39 times that of YK-814.

[0463] Data analysis using GraphPad Prism software showed that YK-810 and YK-813 were significantly different from YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-811, YK-812, YK-814, and YK-802, indicating a significant improvement in transfection efficiency.

[0464] The cell transfection efficiency of LNP preparations made from cationic lipid compounds cannot be inferred from their structures. Even a series of compounds with very similar structures may have very different cell transfection efficiencies.

[0465] 2. Cell viability assay

[0466] An LNP formulation containing 1.5 μg Fluc-mRNA (the LNP formulation carrier components were cationic lipids, DSPC, cholesterol, and DMG-PEG2000, with a molar ratio of 45:10:43.5:1.5, where the cationic lipids were those listed in Table 1) was added to the cell culture medium of a 96-well plate. After culturing for 24 h, 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in an incubator for 1 h, and the absorbance at 450 nm was measured using a microplate reader. The cell viability results are shown in Table 4.

[0467] Table 4 Cell viability

[0468]

[0469]

[0470] Analysis of experimental results:

[0471] (1) This application designs a series of cationic lipid compounds, including YK-803, YK-810, and YK-813, which differ significantly in chemical structure from representative cationic lipids in the prior art, such as SM-102, MC3, and HHMA; while others have similar chemical structures, such as compound 13. LNP formulations prepared from YK-803, YK-810, and YK-813 exhibit the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids in the prior art. For example, the cell viability of YK-810 is 8% higher than SM-102, 12% higher than MC3, 23% higher than HHMA, 15% higher than P-76, 26% higher than compound 13, and 60% higher than Lipofectamine 3000. Figure 3 )

[0472] (2) A series of compounds with similar structures, where the ester bonds of the carbamate structure are linked to G1 or G2 groups, including YK-801, YK-802, and YK-803, were compared. The structural differences between these compounds were only slight variations in a few individual groups. The results showed that YK-803 exhibited the lowest cytotoxicity and significantly improved cell viability. For example, the cell viability of YK-803 was 18% higher than that of YK-801.

[0473] (3) A series of compounds with similar structures, whose ester bonds of carbamate structures are linked to G3 groups, including YK-804, YK-805, YK-806, YK-807, YK-808, YK-809, YK-810, YK-811, YK-812, YK-813, and YK-814, were compared. The results showed that YK-810 and YK-813 had the lowest cytotoxicity and significantly improved cell survival. For example, the cell survival rates of YK-810 and YK-813 were 38% and 35% higher than those of YK-804, and 39% and 36% higher than those of YK-814, respectively.

[0474] The cytotoxicity of LNP preparations made from cationic lipid compounds cannot be inferred from their structures. Compounds with vastly different or similar structures are likely to have very different toxicities to transfected cells.

[0475] Example 7: In vivo validation of the performance of cationic lipid (LNP) delivery carriers

[0476] 1. mRNA expression in mice

[0477] The protein expression and duration of the designed cationic lipid-delivered mRNA in mice were verified. In vivo experiments further demonstrated that the LNP delivery vector prepared from the cationic lipid of this application can efficiently deliver mRNA into animals and achieve efficient and sustained expression.

[0478] LNP formulation containing 10 μg Fluc-mRNA was injected intravenously into female BALB / c mice aged 4–6 weeks and weighing 17–19 g. At specific time points after administration (6 h, 24 h, 48 h, and 7 d), mice were intraperitoneally injected with a fluorescence imaging substrate. Mice were allowed free movement for 5 min, and the mean radiometric intensity (corresponding to fluorescence expression intensity) of the protein expressed by the LNP-carrying mRNA in mice was detected using an IVIS Spectrum small animal in vivo imaging system. The mean radiometric intensity (corresponding to fluorescence expression intensity) of the protein expressed by the LNP-carrying mRNA in mice, as detected by the IVIS Spectrum small animal in vivo imaging system, is shown in Table 5.

[0479] Table 5. Mouse in vivo imaging experimental data

[0480]

[0481] Analysis of experimental results:

[0482] (1) The LNP formulations prepared from YK-803, YK-810, and YK-813 showed significantly increased and sustained mRNA expression levels in mice compared to representative cationic lipids in the prior art. For example, the mRNA expression levels of the LNP formulation prepared from YK-810 in animals reached 5.78 times that of SM-102, 26.19 times that of P-76, and 124.86 times that of compound 13 at 24 h; 7.97 times that of SM-102, 23.17 times that of P-76, and 123.44 times that of compound 13 at 48 h; and 14.93 times that of SM-102, 59.48 times that of P-76, and 215.00 times that of compound 13 at 7 days. The mRNA expression in mice was consistent with the cell transfection activity.

[0483] (2) Compared with compound YK-802, which has a similar structure but slightly different individual groups, the LNP formulations prepared from YK-803, YK-810, and YK-813 showed significantly increased mRNA expression intensity and duration in mice. For example, the mRNA expression level of the LNP formulation prepared from YK-810 in animals reached 130.17 times that of YK-802 at 24h, 200.74 times at 48h, and 110.86 times at 7d. The mRNA expression in mice was consistent with the cell transfection activity.

[0484] 2. Distribution of liposomes in mice

[0485] Furthermore, it was further verified that the delivery vectors prepared by the cationic lipids designed in this application, such as YK-803, YK-810 and YK-813, can be enriched in the mouse spleen. The delivered mRNA significantly increased the protein expression level in the mouse spleen compared with the cationic lipids of the prior art, such as SM-102, while the expression in the liver was very weak.

[0486] The specific experimental procedure is as follows:

[0487] LNP formulation containing 10 μg Fluc-mRNA was injected intravenously into 4-6 week old female BALB / c mice weighing 17-19 g. At a specific time point (6 h) after administration, the mice were intraperitoneally injected with a fluorescent imaging substrate. The mice were allowed free movement for 5 min, and then the mean radiation intensity (corresponding to fluorescence expression intensity) of the proteins expressed by the LNP-carrying mRNA in the mice was detected using an IVIS Spectrum small animal in vivo imaging system. After sampling, the mice were euthanized using carbon dioxide, and their internal organs—heart, liver, spleen, lung, and kidney—were precisely isolated. The mean radiation intensity (corresponding to fluorescence expression intensity) of the proteins expressed by the LNP-carrying mRNA in each mouse organ was detected using an IVIS Spectrum small animal in vivo imaging system. The results of the mouse in vivo imaging are shown in Table 6.

[0488] Table 6. Organ imaging data of mice at specific time points (6 h) after drug administration.

[0489]

[0490] Analysis of experimental results:

[0491] The LNP formulations prepared from YK-803, YK-810, and YK-813 showed significantly increased mRNA expression levels in mouse spleen compared to SM-102, a representative cationic lipid in the prior art. For example, the expression levels of YK-803, YK-810, and YK-813 in the spleen were 8.61-fold, 15.02-fold, and 7.58-fold, respectively, compared to SM-102. The mRNA expression in the mouse spleen was consistent with the cell transfection results in Example 6. The expression of YK-803, YK-810, and YK-813 in the liver was very weak, at 0.02-fold, 0.08-fold, and 0.04-fold, respectively, compared to SM-102. The proportions of delivered mRNA expressed in the spleen and liver were 0.09 times higher for SM-102, 47.44 times higher for YK-803, 16.50 times higher for YK-810, and 17.25 times higher for YK-813.

[0492] The spleen is the largest secondary lymphoid organ in animals. By increasing the expression level of delivered mRNA in the spleen, mRNA vaccines can rapidly induce an immune response and produce antibodies in vivo. This can significantly improve the preventive effect without altering the vaccine composition, which has important clinical significance. It also shows good targeted efficacy for developing treatments for diseases caused by spleen damage or abnormalities, such as lymphoma and leukemia.

[0493] Furthermore, the LNP formulations containing Fluc-mRNA prepared from all compounds showed highly variable expression in different mouse organs. YK-803, YK-810, and YK-813 were expressed almost exclusively in the spleen, with minimal expression in the liver, and no expression in other organs such as the heart, lungs, and kidneys. SM-102 was expressed in the liver and spleen, but not in the heart, lungs, or kidneys. Figure 4 ).

[0494] In summary, this application designed a series of cationic lipid compounds, such as YK-803, YK-810 and YK-813, which significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased mRNA expression levels and duration in mice.

[0495] 1. A series of compounds were designed, including YK-803, YK-810 and YK-813, which are representative cationic lipids in the prior art. Some have huge differences in chemical structure, such as SM-102, MC3 and HHMA; others have similar chemical structures, such as compound 13.

[0496] 2. Among the designed series of compounds, the LNP formulations prepared by YK-803, YK-810 and YK-813 significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased mRNA expression levels and duration in mice compared with representative cationic lipids in the prior art (regardless of whether they have huge structural differences, such as SM-102, MC3 and HHMA, or similar structures, such as P-76 and compound 13). For example, the cell transfection efficiency of YK-810 can reach 5.17 times that of SM-102, 51.99 times that of MC3, 7.97 times that of HHMA, 52.56 times that of P-76, 189.53 times that of compound 13, and 11.23 times that of Lipofectamine 3000; the cell viability of YK-810 is 8% higher than that of SM-102, 12% higher than that of MC3, 23% higher than that of HHMA, 15% higher than that of P-76, 26% higher than that of compound 13, and higher than that of Lipofectamine 3000. The expression level of YK-810 in animals was 60% higher than that of SM-102 within 24 hours, 26.19 times that of P-76, and 124.86 times that of compound 13. Within 7 days, it was 14.93 times that of SM-102, 59.48 times that of P-76, and 215.00 times that of compound 13. The expression level of YK-810 in the spleen was 15.02 times that of SM-102, while the expression level of YK-810 in the liver was only 0.08 times that of SM-102.

[0497] 3. Among the series of compounds with very similar chemical structures designed in this application, the LNP formulations prepared from YK-803, YK-810, and YK-813, compared with other compounds, showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased mRNA expression levels in mice. For example, the cell transfection efficiency of YK-810 could reach 2020 times that of YK-814, its cytotoxicity could be reduced by 38% compared to YK-804, and its mRNA expression level in mice could be 200 times that of YK-802.

[0498] 4. This disclosure, through unique design and screening, has discovered compounds such as YK-803, YK-810, and YK-813 that, compared to other compounds with similar structures in the prior art, significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly increase expression levels and duration in animals, significantly increase expression levels in the spleen while significantly decreasing expression levels in the liver, thereby improving delivery efficiency and achieving unexpected technical effects. It enables rapid induction of immune responses and antibody production in vivo using mRNA vaccines. It significantly enhances preventative efficacy without altering the vaccine composition, which has important clinical significance.

[0499] 5. This disclosure, through unique design and screening, has discovered compounds such as YK-803, YK-810, and YK-813 that, while ensuring high efficiency and low toxicity, can target mRNA delivery to the spleen. These compounds are not expressed in other organs, such as the lungs, heart, and kidneys, but are expressed in small amounts in the liver. The low efficiency of vaccine-induced immune responses is a reason why existing cancer therapeutic vaccines cannot achieve their maximum efficacy. The spleen is the largest secondary lymphoid organ in the body. LNP cancer vaccines targeting the spleen can effectively stimulate an immune response and significantly improve efficacy, thus having important clinical application significance in cancer treatment.

[0500] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be covered within the scope of protection of the invention.

Claims

1. A compound of formula (I) Or its pharmaceutically acceptable salt, wherein, G1 is C 2～10 Alkylene; G2 is C 2～10 Alkylene; G3 is m and n are either 1 or 0. When m is 0, n is 1, and when m is 1, n is 0. Wherein G1 is a C5, C7, or C3 alkylene; Wherein G2 is a C5, C3, or C7 alkylene; Where R1 is Where R2 is unsubstituted C 10 C 11 C8, C9 or C 12 Straight-chain alkyl, or: Branched alkyl groups.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has one of the following structures:

3. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) is YK-803, which has the following structure:

4. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) is YK-810, which has the following structure:

5. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, The compound of formula (I) is YK-813, which has the following structure:

6. A composition comprising a carrier comprising a cationic lipid comprising a compound of formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. The composition according to claim 6, wherein the cationic lipid accounts for 25% to 75% of the molar ratio of the carrier.

8. The composition of claim 7, wherein the carrier further comprises neutral lipids.

9. The composition according to claim 8, wherein, The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

10. The composition according to claim 8, wherein, The molar ratio of the cationic lipid to the neutral lipid is 4.5:

1.

11. The composition according to claim 10, wherein, The neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

12. The composition according to claim 11, wherein, The neutral lipid is selected from one or more of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether) 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

13. The composition according to claim 11, wherein the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) and / or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

14. The composition according to claim 13, wherein, The carrier also contains structural lipids.

15. The composition according to claim 14, wherein, The molar ratio of the cationic lipid to the structural lipid is 0.6:1 to 3:

1.

16. The composition according to claim 15, wherein, The structural lipids are selected from one or more of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

17. The composition of claim 16, wherein the structural lipid is cholesterol.

18. The composition according to claim 6, wherein, The carrier also contains polymeric conjugated lipids.

19. The composition according to claim 18, wherein, The polymer-conjugated lipid accounts for 0.5% to 10% of the carrier molar ratio.

20. The composition according to claim 18, wherein, The polymer conjugated lipid accounts for 1.5% of the carrier molar ratio.

21. The composition according to claim 18, wherein, The polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

22. The composition according to claim 21, wherein, The polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

23. The composition according to claim 6, wherein the carrier comprises cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids; wherein the molar ratio of cationic lipids:neutral lipids:structural lipids:polymer-conjugated lipids is (25-75):(5-25):(15-65):(0.5-10).

24. The composition according to claim 23, wherein the molar ratio of the cationic lipid: neutral lipid: structural lipid: polymer conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

25. The composition according to claim 24, wherein the molar ratio of the cationic lipid: neutral lipid: structural lipid: polymer conjugated lipid is 45:10:43.5:1.

5.

26. The composition according to claim 6, wherein, The composition is a nanoparticle formulation with an average particle size of 10 nm to 210 nm and a polydispersity index (PDI) of ≤50%.

27. The composition according to claim 26, wherein, The average particle size of the nanoparticle formulation is 100 nm to 205 nm; the polydispersity index (PDI) of the nanoparticle formulation is ≤30%.

28. The composition according to claim 6, wherein, The cationic lipids also include one or more other ionizable lipid compounds.

29. The composition according to claim 6, wherein, It also includes therapeutic or preventative agents.

30. The composition according to claim 29, wherein, The mass ratio of the carrier to the therapeutic or preventive agent is 10:1 to 30:

1.

31. The composition according to claim 30, wherein, The mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1 to 25:

1.

32. The composition according to claim 31, wherein, The mass ratio of the carrier to the therapeutic or preventative agent is 15:

1.

33. The composition according to claim 29, wherein, The therapeutic or preventative agent includes one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

34. The composition according to claim 29, wherein, The therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

35. The composition according to claim 29, wherein, The therapeutic or preventative agent is a nucleic acid.

36. The composition according to claim 35, wherein, The therapeutic or preventative agent is ribonucleic acid (RNA).

37. The composition according to claim 35, wherein, The therapeutic or preventative agent is deoxyribonucleic acid (DNA).

38. The composition according to claim 36, wherein, The ribonucleic acid (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.

39. The composition according to claim 38, wherein, The RNA in question is mRNA.

40. The composition according to claim 6, wherein, The composition also includes one or more pharmaceutically acceptable excipients or diluents.

41. Use of a compound of formula (I) as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, or a composition as described in any one of claims 6-40, in the preparation of a nucleic acid drug or a gene vaccine.

42. Use of a compound of formula (I) as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, or a composition as described in any one of claims 6-40, in the preparation of a nucleic acid medicament for treating diseases or conditions in animals in need of lactation.

43. The use according to claim 42, wherein, The disease or condition is characterized by dysfunction or abnormal protein or polypeptide activity.

44. The use according to claim 42 or 43, wherein, The disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

45. The use according to claim 44, wherein, The infectious diseases mentioned are selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or various herpes diseases.

46. ​​The use according to claim 42, wherein, The drug is administered to humans.

47. The use according to claim 42, wherein, The drug can be administered via intravenous, intramuscular, intradermal, subcutaneous, intranasal, or inhalation.

48. The use according to claim 47, wherein, The drug is administered subcutaneously.

49. The use according to claim 42, wherein, The dosage of the drug is 0.001–10 mg / kg.

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