Long-acting spleen-targeted cationic lipid compounds containing a benzene ring structure, compositions containing same and uses - Patent Application 20070122997

Abstract

To provide a long-acting spleen-targeting cationic lipid compound and a composition comprising the same.SOLUTION: Provided is a compound represented by Formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. (In the formula: G1 to G3 represent alkylene; R1 represents alkyl; R2 and R3 represent alkyl or H; M1 represents -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, or -CH(OH)-; M2 represents -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, or -CH(OH)-; R' represents alkyl or H; and X1 represents -CH- or N.)SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, specifically to a long-acting spleen-targeted cationic lipid compound containing a benzene ring structure, a composition containing the same and a use thereof. [Background technology]

[0002] Effective targeted delivery of bioactive agents, such as small molecule drugs, polypeptides, proteins, and nucleic acids, especially nucleic acids, remains a persistent medical challenge. Nucleic acid therapeutics face serious challenges due to poor cell permeability and the high susceptibility of certain nucleic acid molecules (including RNA) to degradation.

[0003] Cationic lipid-containing compositions, liposomes, and lipoplexes have been demonstrated to effectively deliver bioactive substances, such as small molecule drugs, polypeptides, proteins, and nucleic acids, to cells and / or intracellular compartments. Such compositions generally contain one or more "cationic" and / or amino (ionizable) lipids, including neutral lipids, structured lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. While these various lipid-containing nanoparticle compositions have been demonstrated, their safety, efficacy, and specificity still need to be improved. In particular, the increased complexity of lipid nanoparticles (LNPs) can complicate production and increase their toxicity, a major concern that may limit their clinical use. For example, LNP siRNA particles (e.g., patisiran) must be pre-treated with steroids and antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829.). Therefore, there is a need to develop improved cationic lipid compounds and compositions containing them that are useful for delivering therapeutic and / or prophylactic agents (e.g., nucleic acids) to cells to meet the needs of manufacturing and clinical applications. Summary of the Invention

[0004] In a first aspect of the present invention, there is provided a novel cationic lipid compound, which is a compound represented by formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof:

[0005] [ka]

[0006] however, G1 is C 1-4 is alkylene,

[0007] G2 is C 1-8 is alkylene,

[0008] G3 is C 2-8 alkylene or H,

[0009] R1 is C 6-25 Straight chain alkyl, C 6-25 Branched alkyl or C 6-25 is a substituted alkyl;

[0010] R2 is C 6-25 Straight chain alkyl, C 6-25 Branched alkyl, C 6-25 is a substituted alkyl or H,

[0011] R3 is C 1-25 Straight chain alkyl, C 1-25 Branched alkyl, C 1-25 is a substituted alkyl or H,

[0012] M1 is -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, a heteroaryl group, or H;

[0013] M2 is -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, a heteroaryl group, or H;

[0014] R' is C 1-18 Alkyl, C 2-18 alkenyl or H,

[0015] X1 is -CH- or N.

[0016] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G1 is unsubstituted C1 alkylene.

[0017] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G1 is unsubstituted C2 alkylene.

[0018] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G2 is unsubstituted C1 alkylene.

[0019] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G2 is unsubstituted C3 alkylene.

[0020] Further, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, G2 is unsubstituted C4 alkylene.

[0021] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G3 is H.

[0022] Further, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, G3 is unsubstituted C5 alkylene.

[0023] Further, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, G3 is unsubstituted C6 alkylene.

[0024] Furthermore, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, R1 is C 6-14 It is a straight chain alkyl.

[0025] Furthermore, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, R1 is C 12-25 It is a branched alkyl.

[0026] Furthermore, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, R1 is

[0027] [ka] is.

[0028] Furthermore, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, R2 is C 6-14 It is a straight chain alkyl.

[0029] Furthermore, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, R2 is C 12-25 It is a branched alkyl.

[0030] Furthermore, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, R2 is

[0031] [ka] is.

[0032] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, R2 is H.

[0033] Furthermore, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, R3 is C 4-15 It is a straight chain alkyl.

[0034] Further, in the compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, R3 is a C8 straight chain alkyl.

[0035] In the compound of the present invention represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, M1 and M2 may be the same or different.

[0036] Furthermore, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, M1 is -C(O)O-.

[0037] Furthermore, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, M 1 is —C(O)NH—.

[0038] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, M1 is -NHC(O)-. Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, M2 is H.

[0039] Further, in the compounds of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, M2 is -C(O)O-.

[0040] Furthermore, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, X1 is -CH-.

[0041] Furthermore, in the compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, X 1 is N.

[0042] In the compound represented by formula (I) of the present invention, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) may be a compound represented by formula (I-1):

[0043] [ka]

[0044] However, G3 is H,

[0045] G1, G2, R1, R3, M1, X1 are as defined herein.

[0046] Furthermore, in the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, the compound represented by formula (I) is at least one of compounds YK-901, YK-902, YK-903, YK-904, YK-905, YK-906, YK-907, YK-908, YK-909, YK-910, YK-911, and YK-912, which have the following structures:

[0047] [ka] JPEG0007813399000006.jpg151152

[0048] Furthermore, in the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) is a compound YK-908 having the following structure:

[0049] [ka]

[0050] Furthermore, in the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) is a compound YK-909 having the following structure:

[0051] [ka]

[0052] Furthermore, in the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) is a compound YK-910 having the following structure:

[0053] [ka]

[0054] Furthermore, in the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) is a compound YK-912 having the following structure:

[0055] [ka]

[0056] A second aspect of the present invention provides a composition comprising a compound of formula (I) according to any one of the first aspects, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, in a carrier comprising a cationic lipid.

[0057] In a preferred composition, the molar ratio of the cationic lipid in the carrier is 25% to 75%.

[0058] In a preferred composition, the carrier further comprises a neutral lipid.

[0059] In a preferred composition, the molar ratio of said cationic lipid to said neutral lipid is 1:1 to 15:1, preferably 4 to 5:1, and most preferably 4.9:1.

[0060] In preferred compositions, the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0061] In a preferred composition, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl- and one or more selected from sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0062] In a preferred composition, the neutral lipid is DOPE and / or DSPC.

[0063] In a preferred composition, the carrier further comprises a structured lipid.

[0064] In a preferred composition, the molar ratio of the cationic lipid to the structural lipid is from 0.6:1 to 3:1.

[0065] In preferred compositions, the structured lipid is one or more selected from cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatidine, ursolic acid, alpha-tocopherol, corticosteroids.

[0066] In a preferred composition, the structured lipid is cholesterol.

[0067] In a preferred composition, the carrier further comprises a polymer-conjugated lipid.

[0068] In a preferred composition, the molar ratio of said polymer-conjugated lipid in the carrier is 0.5% to 10%, preferably 1.5%.

[0069] In a preferred composition, the polymer-conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0070] In preferred compositions, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0071] In a preferred composition, the carrier further comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

[0072] In a preferred composition, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-55):(1-5), where the sum of the molar ratios of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 100.

[0073] In preferred compositions, the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 45:10:43.5:1.5 or 49:10:39.5:1.5.

[0074] In a preferred composition, the composition is a nanoparticle formulation, the average particle size of the nanoparticle formulation is between 10 nm and 300 nm, and the polydispersity index of the nanoparticle formulation is ≦50%.

[0075] In a preferred composition, the composition is a nanoparticle formulation, the mean particle size of the nanoparticle formulation is between 90 nm and 280 nm, and the polydispersity index of the nanoparticle formulation is ≦45%.

[0076] In preferred compositions, the cationic lipid further comprises one or more other ionizable lipid compounds.

[0077] Preferred compositions further comprise a therapeutic or prophylactic agent.

[0078] In a preferred composition, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1.

[0079] In a preferred composition, the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1.

[0080] In a preferred composition, the weight ratio of the carrier to the therapeutic or prophylactic agent is 15:1.

[0081] In preferred compositions, the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0082] In a preferred composition, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0083] In preferred compositions, the therapeutic or prophylactic agent is a nucleic acid.

[0084] In preferred compositions, the therapeutic or prophylactic agent is ribonucleic acid (RNA).

[0085] In a preferred composition, the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

[0086] In preferred compositions, the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0087] In a preferred composition, the RNA is mRNA.

[0088] In preferred compositions, the composition further comprises one or more pharmaceutically acceptable excipients or diluents.

[0089] A third aspect of the present invention provides the use of a compound represented by formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect above, or a composition as described in the second aspect above, in the manufacture of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide or protein drug.

[0090] A fourth aspect of the present invention provides the use of a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof as described in the first aspect above, or a composition as described in the second aspect above, in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

[0091] In a further use, the disease or condition is characterized by dysfunction or abnormal protein or polypeptide activity.

[0092] In a further use, 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.

[0093] In a further use, the infectious disease is selected from coronavirus, influenza or HIV virus-induced diseases, childhood pneumonia, Rift Valley fever, yellow fever, rabies, or various herpes strains.

[0094] In a further use, the subject is a mammal, preferably the mammal is a human.

[0095] In a further use, the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0096] In a further use, the composition is administered subcutaneously.

[0097] In a further use, the subject is administered a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent.

[0098] A fifth aspect of the present invention provides the use of a compound according to the first aspect or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof for increasing cell transfection efficiency and / or decreasing cytotoxicity, for example for increasing cell transfection efficiency and / or decreasing cytotoxicity in vitro.

[0099] A sixth aspect of the present invention provides use of a compound described in the first aspect or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof for improving targeting of a nucleic acid drug to an organ and / or cell and / or for improving the expression level of a nucleic acid in a target organ and / or target cell.

[0100] In a preferred embodiment of the invention, the nucleic acid drug comprises mRNA.

[0101] In a preferred embodiment of the invention, said (exogenous) nucleic acid is mRNA. [Brief explanation of the drawings]

[0102] In order to more clearly describe the technical solutions of the embodiments of the present invention, the following briefly introduces drawings of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and are not intended to limit the present invention.

[0103] [Figure 1] Figure 1 shows the results of cell transfection experiments using LNP formulations of Fluc-mRNA prepared based on YK-908, YK-909, YK-910, YK-912, SM-102, and Compound 7, where a is YK-908, b is YK-909, c is YK-910, d is YK-912, e is SM-102, and f is Compound 7.

[0104] [Figure 2] 1 shows the fluorescence absorption intensity of LNP formulations of Fluc-mRNA made from different cationic lipids.

[0105] [Figure 3] This shows the cell viability after adding LNP formulations of Fluc-mRNA made from different cationic lipids to cell culture medium and culturing for 24 hours.

[0106] [Figure 4]Mouse organ (heart, liver, spleen, lung, kidney, lymph) fluorescence micrographs of LNP formulations of Fluc-mRNA produced based on SM-102, YK-506, YK-908, YK-909, YK-910, and YK-912 are shown.

[0107] [Figure 5] Fluorescence images of LNP formulations of Cy5 fluorescent-labeled Leqvio manufactured based on YK-908, YK-909, YK-910, and YK-912, and of mouse organs (heart, liver, spleen, lungs, kidneys, and lymph) directly injected with Cy5 fluorescent-labeled Leqvio are shown. DETAILED DESCRIPTION OF THE INVENTION

[0108] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following clearly and completely describes the technical solutions of the embodiments of the present invention in combination with the drawings of the examples of the present invention. Obviously, the described embodiments are some of the embodiments of the present invention, but not all of them. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention.

[0109] The present invention may be embodied in other specific forms without departing from the essential characteristics of the invention. It is to be understood that, without contradiction, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or embodiments to obtain additional embodiments. The present invention includes the additional embodiments obtained from such combinations.

[0110] All publications and patents mentioned herein are incorporated herein by reference in their entirety. If the usage or terminology used in any publication or patent incorporated by reference conflicts with the usage or terminology used in this invention, the usage or terminology used in this invention shall control.

[0111] The chapter titles used herein are for organizational purposes only and should not be construed as limiting the categories described.

[0112] Unless otherwise defined, all technical and scientific terms used herein have their ordinary meaning within the art to which the claimed category belongs. In the event that there are multiple definitions for a term, those set forth in the specification prevail.

[0113] Unless otherwise indicated in the operating examples or otherwise, all numerical values ​​reciting quantitative properties, such as dosage, in the specification and claims should be understood as being modified in all instances by the term "about." Furthermore, any numerical range recited herein should be understood to include all subranges within that range, and any combination of the endpoints of the corresponding ranges or subranges.

[0114] As used herein, the terms "comprise," "contain," "include," or similar terms such as "comprise," mean that the elements appearing before the term include the elements listed after the term and equivalents thereof, without excluding elements not listed. The terms "comprise" or "comprise" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0115] The term "pharmaceutically acceptable" as used herein means that a compound or composition is chemically and / or toxicologically compatible with the other components that make up the formulation and / or with humans or mammals who use it to prevent or treat a disease or condition.

[0116] As used herein, the term "subject" or "patient" includes humans and mammals.

[0117] The term "treatment" as used herein refers to the administration of one or more drug substances to a subject suffering from a disease or having symptoms of said disease in order to cure, alleviate, relieve, improve or affect the disease or the symptoms of said disease. In the context of the present invention, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

[0118] The term "solvate" as used herein 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). Any solvate of a compound of formula (I) used in the treatment of a disease or condition may provide different properties (including pharmacokinetic properties), but since the compound of formula (I) is obtained when absorbed by a subject, each use of a compound of formula (I) should be understood to include the use of any solvate of the compound of formula (I).

[0119] The term "hydrate" refers to the term "solvate" above wherein the solvent is water.

[0120] Furthermore, it should be understood that the compounds of formula (I) or pharmaceutically acceptable salts thereof can be isolated in the form of solvates, and therefore, any such solvates are all included within the scope of the present invention. For example, the compounds of formula (I) or pharmaceutically acceptable salts thereof can exist in unsolvated forms as well as solvated forms formed with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.).

[0121] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. See, for example, S.M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids include, for example, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, enanthic 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, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxybenzoic acid, ... Examples of suitable pharmaceutically acceptable salts include ethanesulfonic acid, itaconic acid, amidosulfonic 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, glucoheptonic 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 a pharmaceutically acceptable salt with the compound of Formula (I).

[0122] The nitrogen-containing compounds of formula (I) of the present invention can be converted to N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Thus, where valence and structure permit, the compounds claimed in this invention include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0123] Certain compounds of the present invention may exist in one or more stereoisomeric forms. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Accordingly, compounds claimed for protection by the present invention also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art will appreciate that one stereoisomer may have superior efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by methods such as chiral synthesis, chiral catalysis, and chiral resolution. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, chiral acid resolving agents such as chiral tartaric acid or chiral malic acid can be added to form salts with the compounds of the present invention, and the physical and chemical properties of the products, such as differences in solubility, can also be exploited.

[0124] The present invention also includes all suitable isotopic variants of the compounds of the present invention. An isotopic variant is defined as a compound in which at least one atom has been replaced with an atom having the same atomic number, but whose atomic mass is different from the atomic mass commonly or predominantly found in nature. Illustrative examples of isotopes that can be incorporated into compounds include isotopes of hydrogen, carbon, nitrogen, and oxygen, each of which can be, for example, 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N, 17 O and 18 It is O.

[0125] As used herein, the term "alkyl" includes both branched and straight-chain saturated aliphatic monovalent hydrocarbon groups having the specified number of carbon atoms. As used herein, the term "alkylene" includes both branched and straight-chain saturated aliphatic divalent hydrocarbon groups having the specified number of carbon atoms. n-m refers to a group having n to m carbon atoms. For example, C 2-5 Alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, and C5 alkylene.

[0126] An alkyl (or alkylene) can be unsubstituted, or an alkyl (or alkylene) can be substituted, where at least one hydrogen has been replaced with another chemical group. Unless otherwise specified or defined, "alkyl" (or "alkylene") in this invention includes unsubstituted and substituted alkyl (or alkylene).

[0127] A "therapeutically effective amount" is the amount of a therapeutic agent that, when administered to a patient, is capable of alleviating a disease or symptom. A "prophylactically effective amount" is the amount of a prophylactic agent that, when administered to a subject, is capable of preventing a disease or symptom. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" depends on the therapeutic / prophylactic agent, the disease state and its severity, and variables such as the age, weight, etc. of the patient / subject being treated / prevented. Those skilled in the art can routinely determine therapeutically effective amounts and prophylactically effective amounts based on their knowledge and the present invention.

[0128] In the present invention, when the compound name and structural formula do not match, the structural formula takes precedence.

[0129] The term "compounds of the invention" as used herein should be understood to include compounds represented by formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof, depending on the context.

[0130] As used herein, the term cationic lipid refers to a lipid that is positively charged at a selected pH value.

[0131] Cationic liposomes readily bind to negatively charged nucleic acids, interacting with the negatively charged phosphate groups present on nucleic acids via electrostatic forces to form lipid nanoparticles (LNPs), which are currently one of the primary delivery vehicles.

[0132] The present inventors have found that screening a large number of compounds has proved extremely difficult to find suitable cationic lipid compounds that are structurally significantly different from typical cationic lipids of the prior art, while at the same time having extremely high transfection efficiency, extremely low cytotoxicity, and high and sustained expression in mice. The present inventors have discovered several compounds, including YK-908, YK-909, YK-910, and YK-912, which are capable of delivering nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cellular toxicity, and significantly improved expression levels and duration in animals, compared to cationic lipids of the prior art that have significantly different chemical structures.

[0133] Briefly, the present invention is based on at least the following discoveries.

[0134] The cationic lipid compounds of the present invention can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins.Compared with known cationic lipid compounds, the cationic lipid compounds of the present invention show higher transfection efficiency and less cytotoxicity, and can significantly improve the expression level in the spleen of animals, thereby improving delivery efficiency and having important clinical significance.

[0135] 1. Compared with representative cationic lipids of the prior art, the designed series of cationic lipid compounds including YK-908, YK-909, YK-910, and YK-912 have significant differences in chemical structure from SM-102, MC3 (Dlin-MC3-DMA), YK-501, YK-506, and HHMA, but are similar in chemical structure to compounds 7 and I-1.

[0136] SM-102 is a cationic lipid compound disclosed by Moderna, Inc., USA, in WO20170409245A2 (page 29 of the specification).

[0137] MC3 is a cationic lipid compound disclosed by Alnylam Pharmaceuticals, Inc. in CN102625696B (page 6 of the specification).

[0138] YK-501 and YK-506 are cationic lipid compounds disclosed by Youcare Pharmaceutical group drug research institute in CN116082275B (page 2 of the specification).

[0139] HHMA is a cationic lipid compound disclosed by Suzhou Abogen Biosciences Co., Ltd. in CN112979483B (page 7 of the specification).

[0140] Compound 7 is a cationic lipid compound disclosed by Suzhou Abogen Biosciences Co., Ltd. in CN114206827A (page 45 of the specification).

[0141] I-1 is a cationic lipid compound disclosed by Acuitas Therapeutics Inc. in CN110799492A (page 19 of the specification).

[0142] The chemical structures of cationic lipids structurally similar to representative cationic lipids of the prior art are as follows:

[0143] [ka]

[0144] [ka]

[0145] [ka]

[0146] (CN116082275B, specification page 2),

[0147] [ka]

[0148] [ka]

[0149] [ka]

[0150] (CN114206827A, specification page 45)

[0151] [ka]

[0152] 2. Among the series of compounds designed in this invention, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly enhanced distribution of mRNA and small nucleic acids in the spleen of mice compared with representative and structurally similar cationic lipids of the prior art. For example, the cell transfection efficiency of YK-910 was 6.04 times that of SM-102, 186.59 times that of YK-501, 13.19 times that of YK-506, 8.53 times that of HHMA, 90.92 times that of MC3, 33.51 times that of Compound 7, 42.56 times that of I-1, and 10.24 times that of Lipofectamine 3000. The cell viability of YK-910 was 12% higher than that of SM-102, 31% higher than that of YK-501, 17% higher than that of HHMA, 20% higher than that of MC3, 21% higher than that of Compound 7, 26% higher than that of I-1, and 10.24 times higher than that of Lipofectamine 3000. 3000, the 24-hour mRNA expression level of YK-910 in animals was 6.78 times that of SM-102, 12.21 times that of YK-501, 8.28 times that of YK-506, and 11.50 times that of compound 7, and the mRNA expression level of YK-910 in the spleen was 9.49 times that of SM-102, 17.04 times that of YK-501, and 3.40 times that of YK-506. The distribution of small nucleic acids in the spleen of YK-910 was 51.19 times that of SM-102, 11.91 times that of YK-501, and 2.85 times that of YK-506. The fluorescence intensity of YK-912 in the spleen and liver was 1.25 times and 10.82 times that of SM-102, respectively, but the distribution in the kidney was 0.14 times that of SM-102, which was significantly reduced.

[0153] 3. Among a series of compounds designed in this invention with very little difference in chemical structure, LNP preparations made from YK-908, YK-909, YK-910, and YK-912 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved distribution of small nucleic acids in the spleen of mice compared to direct administration. For example, when delivering mRNA, the cell transfection efficiency of YK-910 was 345.06 times that of YK-903 and 313.25 times that of YK-906, and its cytotoxicity was 58% lower than that of YK-902. When delivering small nucleic acids, the intrasplenic fluorescence intensity of YK-908, YK-909, YK-910, and YK-912 was 100.55 times, 126.85 times, 150.67 times, and 31.80 times that of direct administration, respectively, and the intrarenal distribution volume of YK-912 was 0.09 times that of direct administration.

[0154] 4. Through unique design and screening, the present invention has discovered several compounds, such as YK-908, YK-909, YK-910 and YK-912, which, compared with other compounds with similar structures in the prior art, have significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity and significantly improved expression levels in the spleen of animals, thereby improving delivery efficiency and achieving unpredictable technical effects.

[0155] In summary, the present invention has discovered, through unique design and screening, several compounds, such as YK-908, YK-909, YK-910 and YK-912, which have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression levels in the spleen of animals, thereby improving delivery efficiency, compared with typical cationic lipids with similar structures in the prior art.

[0156] Details are as follows:

[0157] 1. Compared with representative cationic lipids of the prior art, the compounds designed in the present invention, including YK-908, YK-909, YK-910, and YK-912, have significant differences in chemical structure from SM-102, MC3, YK-501, YK-506, and HHMA, and minor differences from compounds 7 and I-1.

[0158] A comparison of representative cationic lipids from the prior art, such as SM-102, MC3, YK-501, YK-506, HHMA, compound 7, and I-1, with the designed series of compounds is provided below.

[0159] 1) SM-102, MC3, YK-501, YK-506 and HHMA have significantly different chemical structures from the compounds designed in this invention. As can be seen from the chemical structures, the compounds of the present invention incorporate a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure, linked by the amino to an ethyl tertiary amine head lipid or ester, an amide-containing linear alkyl or ester, or an amide-containing branched alkyl. However, SM-102, MC3, YK-501, YK-506, and HHMA do not incorporate the 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure. The amino head of SM-102 is a simple ethanolamine structure. The amino head of MC3 is a dimethyl tertiary amine structure without a hydroxyl group. The amino head of YK-501 and YK-506 is piperazinyl, and the amino head of HHMA is a methyl tertiary amine structure. The hydroxyl group is located at the second position of the aliphatic chain. The other groups also differ significantly.

[0160] 2) Compounds 7 and I-1 have relatively similar chemical structures to the compounds designed in the present invention, and both contain a benzene ring structure.

[0161] 2. In vitro cell transfection efficiency is significantly improved compared to typical cationic lipids and structurally similar compounds of the prior art.

[0162] 1) Among the designed compounds, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 exhibited the highest cell transfection efficiency, all of which were significantly improved compared to representative cationic lipids of the prior art (e.g., SM-102, MC3, YK-501, YK-506, HHMA, compound 7, and I-1). For example, the cell transfection efficiency of YK-910 was 6.04-fold higher than that of SM-102, 186.59-fold higher than that of YK-501, 13.19-fold higher than that of YK-506, 8.53-fold higher than that of HHMA, 90.92-fold higher than that of MC3, 33.51-fold higher than that of compound 7, 42.56-fold higher than that of I-1, and 10.24-fold higher than that of Lipofectamine 3000.

[0163] 2) The structures are similar, X1=CH, R1=C 6-25 Straight chain, branched chain alkyl, R2 is H, M1=-C(O)O-, M2 is H, G1=-CH2-, G2=C 1-4 A series of compounds, including YK-901, YK-902, YK-903, YK-904, YK-905, and YK-911, all of which have alkylene groups (G3 = -H), were compared with YK-912. The structural differences between these compounds were limited to minor variations in the individual groups (Table 1). Cell transfection results (Table 4 and Figure 2) showed that the activity of this series of compounds was significantly different. The cell transfection efficiency of YK-912 was 131.47-fold higher than that of YK-901, 18.35-fold higher than that of YK-902, 228.30-fold higher than that of YK-903, 45.65-fold higher than that of YK-904, 78.83-fold higher than that of YK-905, and 4.39-fold higher than that of YK-911, respectively, demonstrating significant improvements in transfection efficiency.

[0164] 3) The structures are similar, X1=CH, R1=C 6-25 Straight chain, branched chain alkyl, R2 is H, M1=-C(O)NH- or -NHC(O)-, M2 is H, G1=-CH2-, G2=C 1-4A series of compounds, such as YK-907, which have alkylene, G3=-H, were compared with YK-908, and the cell transfection efficiency of YK-908 was significantly improved, reaching 134.49 times that of YK-907.

[0165] 4) The structures are similar, X1=N, R1=C 10-11 Straight chain alkyl, R2=C 15 Branched alkyl or C 18 Branched alkyl, M1 = -C(O)O-, M2 = -C(O)O-, G1 = C2 alkylene, G2 = C 3-4 Alkylene, G3=C 5-6 Compared with the alkylene compound YK-906, the cell transfection efficiencies of YK-909 and YK-910 were significantly improved, being 246.15-fold and 313.25-fold higher than that of YK-906, respectively.

[0166] 3. Cytotoxicity was significantly reduced compared to representative cationic lipids and structurally similar compounds of the prior art.

[0167] 1) The chemical structures of the series of compounds designed in this invention, including YK-908, YK-909, YK-910, and YK-912, are significantly different from those of representative cationic lipids of the prior art, such as SM-102, MC3, and HHMA, and are slightly different from those of compounds 7 and I-1. LNP formulations made with YK-909 and YK-910 exhibited the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids of the prior art. For example, the cell viability of YK-910 was 12% higher than that of SM-102, 31% higher than that of YK-501, 17% higher than that of HHMA, 20% higher than that of MC3, 21% higher than that of compound 7, 26% higher than that of I-1, and 63% higher than that of Lipofectamine 3000. The cytotoxicity of the LNP formulations produced could not be predicted based on the structure of the cationic lipid compounds. There was a high possibility that the toxicity to transfected cells would vary greatly between compounds with very different structures and compounds with similar structures.

[0168] 2) The structures are similar, X1=CH, R1=C 6-25 Straight chain, branched chain alkyl, R2 is H, M1=-C(O)O-, M2 is H, G1=-CH2-, G2=C 1-4 A series of compounds, including YK-901, YK-902, YK-903, YK-904, YK-905, and YK-911, all of which have alkylene groups (G3 = -H), were compared with YK-912. Although the structural differences between these compounds are limited to minor differences in the individual groups, the differences in cytotoxicity between these compounds were significant. Among these, YK-912 had the highest cell viability, which was 29% higher than YK-901, 51% higher than YK-902, 40% higher than YK-903, 35% higher than YK-904, 36% higher than YK-905, and 25% higher than YK-911, thereby significantly improving cell viability.

[0169] 3) The structures are similar, X1=CH, R1=C 6-25 Straight chain, branched chain alkyl, R2 is H, M1=-C(O)NH- or -NHC(O)-, M2 is H, G1=-CH2-, G2=C 1-4 A series of compounds, such as YK-907, in which G3 is alkylene and -H, were compared with YK-908, and the cell viability of YK-908 was significantly improved, 16% higher than that of YK-907.

[0170] 4) The structure is similar, X1=N, R1=C 10-11 Straight chain alkyl, R2=C 15 Branched alkyl or C 18 Branched alkyl, M1 = -C(O)O-, M2 = -C(O)O-, G1 = C2 alkylene, G2 = C 3-4 Alkylene, G3=C 5-6 Compared with the alkylene compound YK-906, the cell transfection efficiencies of YK-909 and YK-910 were significantly improved, being 29% and 31% higher than YK-906, respectively.

[0171] 4. The protein expression and duration of mRNA in the mouse body were significantly improved compared to representative cationic lipids and structurally similar compounds of the prior art, the mRNA expression level in the animal's spleen was significantly improved compared to representative cationic lipids of the prior art, and the distribution of small nucleic acids in the animal's spleen was significantly improved compared to representative cationic lipids of the prior art and direct administration.

[0172] 1) Compared with representative cationic lipids of the prior art, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 showed the highest and most sustained mRNA expression levels in mice, with the expression levels at 6 hours, 24 hours, 48 ​​hours, and 7 days being significantly higher than those of representative cationic lipids of the prior art. For example, according to the 24-hour data, the mRNA expression levels of YK-910 in mice were 6.78 times higher than those of SM-102, 12.21 times higher than those of YK-501, 8.28 times higher than those of YK-506, and 11.50 times higher than those of Compound 7.

[0173] 2) Compared with YK-904, a compound with a similar structure but slight differences in individual groups, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 showed the highest mRNA expression intensity and longest duration in mice. For example, YK-910 reached 23.07-fold the mRNA expression intensity of YK-904 in 48 hours and 26.32-fold after 7 days.

[0174] 3) The LNP formulations prepared from YK-908, YK-909, YK-910, and YK-912 all significantly increased mRNA expression levels in mouse spleens compared with representative cationic lipids of the prior art. For example, YK-908, YK-909, YK-910, and YK-912 were highly expressed in the spleen, at 6.59-fold, 8.00-fold, 9.49-fold, and 6.86-fold higher levels than SM-102, respectively. The Fluc-mRNA-containing LNP formulations prepared from all compounds showed highly differential expression in different organs of mice. YK-908, YK-909, YK-910, and YK-912 were almost exclusively expressed in the spleen, with no expression in other organs such as the heart, lungs, and kidneys. They were expressed in small amounts or not at all in the liver. SM-102 was expressed in the liver and spleen, but not in the heart, lungs, or kidneys.

[0175] 4) The LNP formulations prepared from YK-908, YK-909, and YK-910 all significantly improved the distribution of small nucleic acid drugs in the mouse spleen compared with representative cationic lipids of the prior art. For example, the distribution of YK-908, YK-909, and YK-910 in the spleen was significantly increased, and the fluorescence intensities of YK-908, YK-909, and YK-910 in the spleen were 34.16-fold, 43.10-fold, and 51.19-fold, respectively, that of SM-102, 7.95-fold, 10.02-fold, and 11.91-fold that of YK-501, and 1.90-fold, 2.40-fold, and 2.85-fold that of YK-506, respectively.

[0176] Surprisingly, the fluorescence intensity of YK-912 in the spleen and liver was 1.25-fold and 10.82-fold higher than that of SM-102, respectively, but was significantly reduced in the kidney (0.14-fold lower than that of SM-102), demonstrating long-term small nucleic acid drug delivery. The Cy5-labeled Leqvio-containing LNP formulations produced by all compounds showed significant differences in distribution in different organs of mice. For example, YK-908, YK-909, and YK-910 were expressed in the liver, spleen, and kidney, but not in other organs such as the heart, lungs, and lymph nodes.

[0177] Compared with direct administration, the LNP formulations prepared from YK-908, YK-909, and YK-910 all significantly improved the distribution of small nucleic acid drugs in the mouse spleen. For example, the fluorescence intensity in the spleen of YK-908, YK-909, and YK-910 was 100.55-fold, 126.85-fold, and 150.67-fold higher, respectively, than that of direct administration. The distribution of YK-912 in the kidney was 0.09-fold higher than that of direct administration, further demonstrating the potential for long-term efficacy in small nucleic acid drug delivery.

[0178] 5) Compared with representative cationic lipids of the prior art, such as SM-102 and Compound 7, liposomes prepared from compounds designed by the present inventors either express a reduced amount of protein in the liver or remain in the liver and do not express the target protein. Therefore, compared with cationic lipids of existing technologies, LNP formulations prepared from compounds designed by the present inventors have reduced or no toxicity to the liver.

[0179] The present invention provides novel cationic lipid compounds for the delivery of therapeutic or prophylactic agents. The cationic lipid compounds of the present invention can be used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared with known cationic lipid compounds, the cationic lipid compounds of the present invention exhibit higher transfection efficiency and lower cytotoxicity, improving delivery efficiency and safety.

[0180] One aspect of the present invention provides novel cationic lipid compounds, which are compounds of formula (I), or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof:

[0181] [ka]

[0182] however, G1 is C 1-4alkylene, preferably C1 alkylene or C2 alkylene, more preferably unsubstituted C1 alkylene or unsubstituted C2 alkylene;

[0183] G2 is C 1-8 alkylene, preferably C 1-4 alkylene, more preferably unsubstituted C1, C3, C4 alkylene;

[0184] G3 is C 2-8 alkylene or H, preferably C 4-6 alkylene or H, more preferably unsubstituted C5, C6 alkylene or H;

[0185] R1 is C 6-25 A straight chain, branched chain alkyl or substituted alkyl, preferably unsubstituted C 8-15 Straight chain alkyl or unsubstituted C 16-24 Branched alkyl, more preferably unsubstituted C 10-15 straight chain alkyl, or

[0186] [ka] is.

[0187] R2 is C 6-25 Straight chain alkyl, C 6-25 Branched alkyl, C 6-25 substituted alkyl or H, preferably unsubstituted C 16-22 branched alkyl or H, more preferably

[0188] [ka] and

[0189] R3 is C 1-25 Straight chain alkyl, C 1-25 Branched alkyl, C 1-25 substituted alkyl or H, preferably unsubstituted C 4-15 It is a straight chain alkyl, more preferably a C8 straight chain alkyl,

[0190] M1 is —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)2—, —SS—, an aryl group, a heteroaryl group or H, preferably —C(O)O—, —C(O)NH— or —NHC(O)—;

[0191] M2 is —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)2—, —SS—, an aryl group, a heteroaryl group or H, preferably H or —C(O)O—;

[0192] R' is C 1-18 Alkyl, C 2-18 alkenyl or H, preferably H;

[0193] X1 is -CH- or N.

[0194] In one embodiment, G1 is an unsubstituted C1 alkylene, for example, -CH2-.

[0195] In one embodiment, G1 is an unsubstituted C2 alkylene, for example, -(CH2)2-.

[0196] In one embodiment, G2 is an unsubstituted C1 alkylene, for example, -CH2-.

[0197] In one embodiment, G2 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0198] In one embodiment, G2 is an unsubstituted C4 alkylene, for example, -(CH2)4-.

[0199] In one embodiment, G3 is H, for example, -H.

[0200] In one embodiment, G3 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0201] In one embodiment, G3 is an unsubstituted C6 alkylene, for example, -(CH2)6-.

[0202] In one embodiment, R is unsubstituted C 10 It is a straight chain alkyl, i.e., -(CH2)9CH3.

[0203] In one embodiment, R is unsubstituted C 11 It is a straight chain alkyl, i.e., -(CH2) 10 It is CH3.

[0204] In one embodiment, R is unsubstituted C 14 It is a straight chain alkyl, i.e., -(CH2) 13 It is CH3.

[0205] In one embodiment, R is unsubstituted C 24 For example, R1 is

[0206] [ka] is.

[0207] In one embodiment, R is unsubstituted C 15 For example, R1 is

[0208] [ka] is.

[0209] In one embodiment, R is unsubstituted C 18 For example, R1 is

[0210] [ka] is.

[0211] In one embodiment, R is unsubstituted C 22 For example, R1 is

[0212] [ka] is.

[0213] In one embodiment, R2 is H.

[0214] In two embodiments, R2 is unsubstituted C 15 For example, R2 is

[0215] [ka] is.

[0216] In two embodiments, R2 is unsubstituted C 18 For example, R2 is

[0217] [ka] is.

[0218] In three embodiments, R3 is an unsubstituted C8 straight chain alkyl, ie, -(CH2)7CH3.

[0219] In one embodiment, M1 is an ester bond, for example, M1 is -C(O)O-.

[0220] In one embodiment, M1 is an amide bond, for example, M1 is -C(O)NH- or -NHC(O)-.

[0221] In one embodiment, M2 is H.

[0222] In two embodiments, M2 is an ester bond, for example, M2 is -C(O)O-.

[0223] In one embodiment, X1 is -CH-.

[0224] In one embodiment, X 1 is N.

[0225] In the compound represented by formula (I) of the present invention, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, the compound represented by formula (I) may be a compound represented by formula (I-1):

[0226] [ka] However, G3 is H, G1, G2, R1, R3, M1, X1 are as defined herein. In an exemplary embodiment, the compound is selected from the following compounds or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof:

[0227] [Table 1] JPEG0007813399000029.jpg195166

[0228] A second aspect of the present invention provides a composition comprising a carrier containing a cationic lipid and a compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof.

[0229] In one embodiment, the composition is a nanoparticle formulation, the nanoparticle formulation having an average size of 10 nm to 300 nm, preferably 90 nm to 260 nm, and a polydispersity index of the nanoparticle formulation of ≦50%, preferably ≦40%, more preferably ≦30%.

[0230] cationic lipids

[0231] In one embodiment of the composition / carrier of the present invention, the cationic lipid is one or more selected from the group consisting of a compound represented by formula (I) above or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof. In one embodiment, the cationic lipid is a compound represented by formula (I) selected from the above. For example, the cationic lipid is compound YK-908, YK-909, YK-910, or YK-912. In one preferred embodiment, the cationic lipid is compound YK-908; in another preferred embodiment, the cationic lipid is compound YK-909; in yet another preferred embodiment, the cationic lipid is compound YK-910; and in another preferred embodiment, the cationic lipid is compound YK-912.

[0232] In another embodiment of the composition / carrier of the present invention, the cationic lipid comprises (a) one or more selected from the group consisting of the compound represented by formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, and (b) one or more other ionizable lipid compounds different from (a). The (b) cationic lipid compound may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the (b) cationic lipid compound may be SM-102 in CN102625696B, DLin-K-C2-DMA in CN102245590B, or Compound 1 in CN113039174A.

[0233] In one embodiment, the molar ratio of the cationic lipid in the carrier is 25% to 75%, for example, 30%, 40%, 50%, 55%, 60%, 65%, or 70%.

[0234] The carriers can be used to deliver active ingredients, such as therapeutic or prophylactic agents, which may be encapsulated within the carrier or combined with the carrier.

[0235] For example, the therapeutic or prophylactic agent may comprise one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. The nucleic acid may include, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNA may include, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0236] neutral lipid

[0237] The carrier may include a neutral lipid. In the present invention, a neutral lipid refers to a lipid that plays an auxiliary role and is uncharged or exists in a zwitterionic form at a selected pH value. The neutral lipid can promote lipid phase transition and adjust the fluidity of nanoparticles to form a lipid bilayer structure, thereby improving efficacy and affecting target organ specificity.

[0238] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, and 2:1. In one preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5:1 (for example, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, or a range consisting of any two of the above ratios and any intermediate ratio therebetween).

[0239] For example, the neutral lipid may comprise one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0240] The carrier component of the composition containing cationic lipids may also contain one or more neutral lipids-phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can be formed into one or more lipid bilayers. Generally, the phospholipids may contain a phospholipid moiety and one or more fatty acid moieties.

[0241] The neutral lipid moiety can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety can be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also included are non-naturally occurring species, including naturally occurring species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, the phospholipids can be functionalized with one or more alkynes (e.g., alkenyl in which one or more double bonds are replaced with triple bonds) or cross-linked with one or more alkynes. Under appropriate reaction conditions, alkynyls can undergo copper-catalyzed cycloaddition reactions when exposed to azides. Such reactions can be used to functionalize the lipid bilayer of the composition to promote membrane permeability or cellular recognition, or to couple the composition to useful components such as targeting or imaging moieties (e.g., dyes).

[0242] Neutral lipids that can be used in such compositions include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl- The phosphatidylcholine may be selected from the non-limiting group consisting of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0243] In some embodiments, the neutral lipid comprises DSPC. In some embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0244] structured lipids

[0245] The carrier of the composition comprising a cationic lipid may further comprise one or more structured lipids, which in the present invention refer to lipids that fill the spaces between lipids and improve the stability of the nanoparticles.

[0246] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.5:1 to 5:1, preferably 0.6:1 to 3:1, for example, about 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, or 3:1.

[0247] The structured lipid can be selected from the group consisting of, but not limited to, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatidine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0248] Polymer-conjugated lipids

[0249] The carrier of the composition containing a cationic lipid may further contain one or more polymer-conjugated lipids. Polymer-conjugated lipids mainly refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, restricts lipid fusion, controls nanoparticle size, reduces nonspecific interactions with macrophages, and increases nanoparticle half-life.

[0250] In one embodiment, the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is generally 350 to 5,000 Da.

[0251] For example, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycero-methoxypolyethylene glycol-2000 (DMG-PEG2000), and methoxypoly(ethylene glycol) ditetradecylacetamide (ALC-0159).

[0252] In one embodiment of the composition / carrier of the present invention, the polymer-conjugated lipid is DMG-PEG2000.

[0253] In one embodiment of the composition / carrier of the present invention, the carrier further comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10), for example, (30-49):(7.5-15):(35-55):(1-5). The sum of the molar ratios of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 100.

[0254] In one embodiment of the composition / carrier of the present invention, the carrier further comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 40:10:48.5:1.5 or 49:10:39.5:1.5, preferably 49:10:39.5:1.5.

[0255] Therapeutic and / or preventive agents

[0256] The composition may contain one or more therapeutic and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0257] In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1, for example, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, or 20:1, and is preferably 15:1.

[0258] The therapeutic or prophylactic agent may include, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0259] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0260] Because the carriers of the present invention are capable of delivering therapeutic and / or prophylactic agents to mammalian cells or organs, the present invention also provides methods for treating a disease or condition in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and / or prophylactic agent and / or contacting mammalian cells with the composition.

[0261] Therapeutic and / or prophylactic agents include biologically active substances, also referred to as "active agents." Therapeutic and / or prophylactic agents may be substances that, after delivery to a cell or organ, cause a desired change in the corresponding cell or organ, or in other body tissues or systems. Such species can be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agents are small molecule drugs that can be used to treat specific diseases, disorders, or conditions. Examples of drugs that can be used in the compositions include antiproliferative agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside), and the like.arabinosides, anthracyclines, alkylating agents, platinum-based compounds, antimetabolites and nucleoside analogues such as methotrexate, purine and pyrimidine analogues, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline and doxepin), anticonvulsants (e.g., phenytoin), antihistamines Antibiotics (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), B)) include, but are not limited to, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives and contrast media.

[0262] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that induces an immune response, and / or other therapeutic and / or prophylactic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, and the like. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations capable of providing immunity against one or more symptoms associated with influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, tuberculosis, and may include mRNA encoding antigens and / or epitopes from infectious diseases. Vaccines may also include compounds and preparations that induce an immune response against cancer cells and may include mRNA encoding antigens, epitopes, and / or neoepitopes from tumor cells.Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and / or compounds capable of inducing an immune response are administered intramuscularly via a composition comprising a compound of Formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (III) (e.g., Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122). Other therapeutic and / or prophylactic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, racemicin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromocyclododecanoate, cyclosporine, cyclopentasiloxane, cyclohexyl 1-methylpropional ... Antibiotics include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0263] In another embodiment, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), VIR factors, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0264] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" is used in its broadest sense to include all compounds and / or substances that may be incorporated into an oligonucleotide chain or into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present invention include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), (including messenger mRNA (mRNA) and hybrids thereof), RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, and the like. In some embodiments, the therapeutic and / or prophylactic agent is RNA. RNA that can be used in the compositions and methods described herein is selected from the group consisting of, but not limited to, shortmers, antagomirs, antisense RNA, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0265] In some embodiments, the therapeutic and / or prophylactic agent is an mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA may have any secondary structure or activity, regardless of size. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell.

[0266] In other embodiments, the therapeutic and / or prophylactic agent is an siRNA. The siRNA can selectively reduce or downregulate the expression of a gene of interest. For example, the siRNA can be selected so that a gene associated with a particular disease, symptom, or condition is silenced after administration of a composition containing the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0267] In some embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, sgRNA-cas9 complexes can affect mRNA translation of cellular genes.

[0268] In some embodiments, the therapeutic and / or prophylactic agent is shRNA or a carrier or plasmid encoding it.The shRNA can be produced in target cells after appropriate constructs are delivered to the nucleus.The constructs and mechanisms associated with shRNA are well known in the art.

[0269] Disease or illness

[0270] The compositions / carriers of the present invention can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Given the wide variety of therapeutic or prophylactic agents, the compositions of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0271] In one embodiment, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0272] 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.

[0273] In one embodiment, the infectious disease is selected from coronavirus, influenza virus or HIV virus-induced disease, childhood pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes strains.

[0274] Other ingredients

[0275] The composition may include one or more ingredients other than those described in the preceding section, for example, the composition may include one or more hydrophobic small molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0276] 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, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0277] Surface modifiers include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase α), and the like. alfa, neltenexine and erdosteine) and DNA enzymes (e.g., rhDNA enzyme). Surface modifiers can be disposed (e.g., via coating, adsorption, covalent bonding, or other methods) within and / or on the surface of the nanoparticles of the composition.

[0278] The composition may also include one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl group that can undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that can effectively promote membrane permeability, cell recognition, or imaging in this manner. The surface of the composition can also be coupled to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeability are well known in the art.

[0279] In addition to such ingredients, the composition may contain any substance that can be used in pharmaceutical compositions. For example, the composition may contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, fluidization aids, liquid vehicles, adhesives, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavorings, coloring agents, etc. Excipients are, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0280] 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.

[0281] In some embodiments, a composition comprising one or more lipids according to the present invention may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (e.g., Class A or Class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0282] The compositions of the present invention can be prepared in the form of solid, semisolid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions of the present invention can be prepared by methods well known in the pharmaceutical arts. For example, a sterile injectable solution can be prepared by mixing the required amount of a therapeutic or prophylactic agent with various other necessary ingredients described above in an appropriate solvent such as sterile distilled water, followed by filtration sterilization. Furthermore, a surfactant can be added to promote the formation of a uniform solution or suspension.

[0283] For example, the compositions of the present invention may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the compositions are administered subcutaneously.

[0284] The compositions of the present invention are administered in a therapeutically effective amount, which may vary depending on the particular formulation selected, the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. For example, the therapeutic or prophylactic agent may be administered to a subject (preferably a mammal such as a human) in a dose of about 0.001 mg / kg to about 10 mg / kg.

[0285] Example

[0286] The present invention will be further described below with reference to examples. However, the present invention is not limited to the following examples. The operating conditions used in the examples can be further adjusted according to different requirements of specific applications, and operating conditions not specified are conventional conditions in the art. In certain embodiments of the present invention, all raw materials used are commercially available. Unless otherwise specified, percentages in this context are by weight percentage, and all temperatures are in degrees Celsius. The technical features related to various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0287] The following abbreviations represent the following reagents:

[0288] DCM: dichloromethane; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP: 4-dimethylaminopyridine; DIEA: N,N-diisopropylethylamine; CAN: acetonitrile; TsOH: p-toluenesulfonic acid; rt: room temperature

[0289] Example 1: Synthesis of cationic lipid compounds

[0290] 1. Synthesis of YK-901

[0291] The synthetic route is as follows:

[0292] [ka]

[0293] Step 1: Synthesis of 6-bromohexanoic acid-4-decyltetradecyl ester (YK-901-PM1)

[0294] 6-Bromohexanoic acid (280 mg, 1.44 mmol) and 4-decyltetradecan-1-ol (510 mg, 1.44 mmol) were dissolved in dichloromethane (10 mL). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (413 mg, 3.92 mmol) and 4-dimethylaminopyridine (18 mg, 0.26 mmol) were added to the resulting mixture. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, the solvent was removed under reduced pressure. The product was purified by silica gel flash chromatography (ethyl acetate / n-hexane) to give YK-901-PM1 (550 mg, 1.03 mmol, 71.8%).

[0295] Step 2: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexanoic acid-4-decyltetradecyl ester (YK-901)

[0296] YK-901-PM1 (261 mg, 0.49 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (100 mg, 0.33 mmol) were dissolved in acetonitrile (1.5 mL). Calcium carbonate (135 mg, 0.96 mmol) and potassium iodide (11 mg, 0.064 mmol) were added to the mixture and stirred at 70°C for 12 hours. After the reaction was complete, the solids were removed by filtration, the filtrate was distilled under reduced pressure, and the residue was purified by silica gel flash chromatography (methanol / dichloromethane) to obtain YK-901 (100 mg, 0.13 mmol, 40.0%). 49 H 91 NO4, MS(ES): m / z(M+H + )758.7.

[0297] 1 H NMR (400 MHz, CDCl3) δ 7.13 (s, 4H), 4.10-4.00 (m, 4H), 3.93 (d, J = 12.5 Hz, 2H), 3.04 - 2.96 (m, 2H), 2.74-2.65 (m, 2H), 2.63 - 2.54 (m, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.01 - 1.87 (m, 4H), 1.75-1.55 (m, 6H), 1.50-1.41 (m, 2H), 1.40-1.20 (m, 50H), 0.91 (t, J = 6.6 Hz, 9H).

[0298] 2. Synthesis of YK-902

[0299] The synthetic route is as follows:

[0300] [ka]

[0301] Step 1: Synthesis of 4-bromobutyric acid-4-decyltetradecyl ester (YK-902-PM1)

[0302] Using 4-bromobutyric acid (612 mg, 3.66 mmol) and 4-decyltetradecan-1-ol (1.3 g, 3.66 mmol) as raw materials, YK-902-PM1 (1.5 g, 2.98 mmol, 81.4%) was obtained according to the synthesis method of YK-901-PM1.

[0303] Step 2: Synthesis of 4-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)butyric acid 4-decyltetradecyl ester (YK-902)

[0304] YK-902-PM1 (196 mg, 0.39 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (100 mg, 0.33 mmol) were used as raw materials, and YK-902 (60 mg, 0.08 mmol, 24.9%) was obtained according to the synthesis method of YK-901. 47 H 87 NO4, MS(ES): m / z(M+H + )730.6. 1 H NMR (400 MHz, CDCl3) δ 7.12 (s, 4H), 4.13 - 4.03 (m, 2H), 3.76 (pd, J = 11.9, 6.8 Hz, 4H), 3.52 (s, 4H), 2.91 (t, J = 6.8 Hz, 2H), 2.70 - 2.54 (m, 4H), 2.50 (q, J = 7.1 Hz, 2H), 2.13 - 1.99 (m, 2H), 1.82 (dd, J = 10.2, 6.8 Hz, 2H), 1.60 (d, J = 6.8 Hz, 4H), 1.28 (d, J = 15.1 Hz, 46H), 0.91 (t, J = 6.5 Hz, 9H).

[0305] 3. Synthesis of YK-903

[0306] The synthetic route is as follows:

[0307] [ka]

[0308] Synthesis of decyl 4-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)butanoate (YK-903)

[0309] Using decyl-4-bromobutyrate (200 mg, 0.65 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (100 mg, 0.33 mmol) as raw materials, YK-903 (85 mg, 0.16 mmol, 48.2%) was obtained according to the synthesis method for YK-901. 33 H 59 NO4, MS(ES): 534.45 m / z (M+H + ). 1 H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.04-4.10 (m, 2H), 3.59-3.66 (m, 2H), 2.69 (t, J = 6.8 Hz, 2H), 2.56 (d, J = 32.0 Hz, 6H), 2.43 (t, J = 7.2 Hz, 2H), 1.86 (s, 2H), 1.68 (d, J = 17.2 Hz, 2H), 1.59 (d, J = 27.2 Hz, 4H), 1.43 - 1.15 (m, 25H), 0.88 (t, J = 6.4 Hz, 6H).

[0310] 4. Synthesis of YK-904

[0311] The synthetic route is as follows:

[0312] [ka]

[0313] Step 1: Synthesis of 7-bromoheptanoic acid-undecyl ester (YK-904-PM1)

[0314] Using 7-bromoheptanoic acid (728 mg, 3.48 mmol) and undecanol (500 mg, 2.90 mmol) as raw materials, YK-904-PM1 (900 mg, 2.48 mmol, 85.4%) was obtained according to the synthesis method of YK-901-PM1.

[0315] Step 2: Synthesis of 7-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)heptanoic acid undecyl ester (YK-904)

[0316] YK-904-PM1 (182 mg, 0.50 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (100 mg, 0.33 mmol) were used as raw materials, and YK-904 (90 mg, 0.15 mmol, 46.2%) was obtained according to the synthesis method of YK-901. 37 H 67 NO4, MS(ES): m / z(M+H + )590.55. 1 H NMR (400 MHz, CDCl3) δ 7.08 (s, 4H), 4.05 - 4.02 (m, 4H), δ 3.76 - 3.70 (m, 4H), 2.72- 2.70 (m, 2H), 2.60 - 2.53 (m, 4H), 2.30 - 2.27 (m, 2H), 1.75- 1.70 (m, 2H), 1.65 - 1.60 (m, 8H), 1.30 -1.25 (m, 31H), 0.89 - 0.85(m, 6H).

[0317] 5. Synthesis of YK-905

[0318] The synthetic route is as follows:

[0319] [ka]

[0320] Step 1: Synthesis of 6-bromohexanoic acid undecyl ester (YK-905-PM1)

[0321] Using 6-bromohexanoic acid (3.00 g, 15.38 mmol) and undecanol (2.66 g, 15.44 mmol) as raw materials, YK-905-PM1 (3.20 g, 9.16 mmol, 59.6%) was obtained according to the synthesis method of YK-901-PM1.

[0322] Step 2: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexanoic acid undecyl ester (YK-905)

[0323] YK-905-PM1 (453 mg, 1.30 mmol) and 2-amino-2-(2-(4-octylphenyl)ethyl)-1,3-propanediol (200 mg, 0.65 mmol) were used as raw materials, and YK-905 (130 mg, 0.23 mmol, 34.7%) was obtained according to the synthesis method of YK-901. 36 H 65 NO4, MS(ES): m / z(M+H + )576.5. 1 H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.05 (t, J = 6.8 Hz, 2H), 3.67 (s, 3H), 2.76 - 2.44 (m, 8H), 2.31 (t, J = 6.4 Hz, 2H), 1.79 - 1.51 (m, 10H), 1.48 - 1.07 (m, 28H), 0.95 - 0.83 (m, 6H).

[0324] 6. Synthesis of YK-906

[0325] The synthetic route is as follows:

[0326] [ka]

[0327] Step 1: Synthesis of 6-((2-chloroethyl)(4-(decyloxy)-4-oxobutyl)amino)hexanoic acid 2-octyldecyl ester (YK-906-PM1)

[0328] YK-009 (600 mg, 0.92 mmol; see CN114044741B for details of the synthesis method) was dissolved in dichloromethane (6 mL), the reaction mixture was cooled to 0 °C, and dichlorosulfoxide (220 mg, 1.83 mml) was added dropwise at 0 °C. After the addition was complete, the mixture was warmed to room temperature and allowed to react at room temperature for 3 hours. The reaction mixture was then spun dry under reduced pressure to yield YK-906-PM1 (600 mg, 0.89 mmol, 97.3%).

[0329] Step 2: Synthesis of 2-octyldecyl-6-((4-(decyloxy)-4-oxobutyl)(2-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)ethyl)amino)hexanoate (YK-906)

[0330] YK-906-PM1 (600 mg, 0.89 mmol), 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (200 mg, 0.65 mmol), potassium iodide (11 mg, 0.07 mmol), and calcium carbonate (270 mg, 1.95 mmol) were added to acetonitrile (2 mL) and stirred at 80 °C for 7 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel flash chromatography (0-10% methanol / dichloromethane) to give YK-906 (200 mg, 0.21 mmol, 32.6%). 59 H 110 N2O6, MS(ES): m / z(M+H + )943.8. 1H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.06-4.02 (m, 4H), 3.94 - 3.91 (m, 4H), 2.72-2.52 (m, 4H), 2.36 - 2.27 (m, 4H), 1.93-1.58 (m, 8H), 1.64-1.26 (m, 68H), 0.88 (t, J = 6.4 Hz, 12H).

[0331] 7. Synthesis of YK-907

[0332] The synthetic route is as follows:

[0333] [ka]

[0334] Step 1: Synthesis of 6-bromohexanoyl chloride (YK-907-PM1)

[0335] 6-Bromohexanoic acid (1.0 g, 5.13 mmol) was dissolved in dichloromethane (3 mL), and oxalyl chloride (976 mg, 7.69 mmol) was added to the mixture in an ice bath. The reaction mixture was stirred overnight at room temperature, and after completion of the reaction, the solvent was removed under reduced pressure to obtain YK-907-PM1 (1.08 g, 5.06 mmol, 98.6%).

[0336] Step 2: Synthesis of 6-bromo-N-tetradecylhexanamide (YK-907-PM2)

[0337] YK-907-PM1 (1.08 g, 5.06 mmol) and tetradecylamine (1.09 g, 5.11 mmol) were dissolved in acetonitrile (8 mL). Calcium carbonate (2.12 g, 15.30 mmol) was added to the mixture and stirred at room temperature for 4 hours. After the reaction was completed, the solid was removed by suction filtration, and the filtrate was spin-dried under reduced pressure. The residue was then purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain YK-907-PM2 (650 mg, 1.66 mmol, 32.9%).

[0338] Step 3: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)-N-tetradecylhexanamide (YK-907)

[0339] YK-907-PM2 (190 mg, 0.49 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (100 mg, 0.33 mmol) were used as raw materials, and YK-907 (66 mg, 0.11 mmol, 32.4%) was obtained according to the synthesis method of YK-901. 39 H 72 N2O3, MS(ES): m / z(M+H + )617.6. 1 H NMR (400 MHz, CDCl3) δ 7.13 (s, 4H), 3.73 (s, 4H), 3.23 - 3.20 (m, 2H), 2.80 - 2.69 (m, 2H), 2.66 - 2.49 (m, 4H), 2.27 - 2.15 (m, 2H), 1.80-1.17 (m, 44H), 0.96 - 0.82 (m, 6H).

[0340] 8. Synthesis of YK-908

[0341] The synthetic route is as follows:

[0342] [ka]

[0343] Step 1: Synthesis of 6-bromohexylamine (YK-908-PM1)

[0344] tert-Butyl (6-bromohexyl)carbamate (500 mg, 1.78 mmol) was dissolved in HCl / dioxane (4 M, 2 mL) and stirred overnight at room temperature. After completion of the reaction, the solvent was removed by spinning to give YK-908-PM1 (320 mg, 1.78 mmol, 100%).

[0345] Step 2: Synthesis of 2-hexyldecanoyl chloride (YK-908-PM2)

[0346] 2-Hexyldecanoic acid (456 mg, 1.78 mmol) was dissolved in dichloromethane (2 mL). To the mixture was added oxalyl chloride (270 mg, 2.14 mmol) and N,N-dimethylformamide (0.05 mL) in an ice bath. The mixture was stirred overnight at room temperature. After completion of the reaction, the solvent was removed under reduced pressure to give YK-908-PM2 (489 mg, 1.78 mmol, 100%).

[0347] Step 3: Synthesis of N-(6-bromohexyl)-2-hexyldecanamide (YK-908-PM3)

[0348] YK-908-PM3 (60 mg, 0.14 mmol, 8.1%) was obtained using YK-908-PM1 (320 mg, 1.78 mmol) and YK-908-PM2 (489 mg, 1.78 mmol) as raw materials and following the synthesis method for YK-907-PM2.

[0349] Step 4: Synthesis of N-(6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexyl) 2-hexyldecanamide (YK-908)

[0350] YK-908-PM3 (60 mg, 0.14 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (53 mg, 0.17 mmol) were used as raw materials, and YK-908 (20 mg, 0.03 mmol, 22.1%) was obtained according to the synthesis method of YK-901. 41 H 76 N2O3, MS(ES): m / z(M+H + ) 645.6. 1H NMR (400 MHz, CDCl3) δ 7.14 (s, 4H), 4.04 (dd, J = 43.2, 12.5 Hz, 4H), 3.31 (d, J = 6.4 Hz, 2H), 3.13 (s, 2H), 2.82 - 2.65 (m, 2H), 2.63 - 2.50 (m, 2H), 2.04 (dt, J = 36.5, 25.2 Hz, 5H), 1.69 - 1.18 (m, 44H), 0.90 (dd, J = 6.3, 4.0 Hz, 9H).

[0351] 9. Synthesis of YK-909

[0352] The synthetic route is as follows:

[0353] [ka]

[0354] Step 1: Synthesis of 3-hexylnonyl 6-(2-chloroethyl)(4-(decyloxy)-4-oxobutyl)amino)hexanoate (YK-909-PM1)

[0355] Using 3-hexylnonyl 6-(4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-101, see CN116178193B for details of the synthesis method) (200 mg, 0.33 mmol) as the starting material, YK-909-PM1 (200 mg, 0.32 mmol, 96.1%) was obtained according to the synthesis method of YK-906-PM1.

[0356] Step 2: Synthesis of 3-hexylnonyl 6-((4-(decyloxy)-4-oxobutyl)(2-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)ethyl)amino)hexanoate (YK-909)

[0357] YK-909-PM1 (200 mg, 0.32 mmol) and 2-amino-2-(4-octylphenylethyl)-1,3-propanediol (100 mg, 0.33 mmol) were used as raw materials, and YK-909 (130 mg, 0.14 mmol, 45.1%) was obtained according to the synthesis method for YK-906. 56 H 104 N2O6, MS(ES): 901.8m / z(M+H + ). 1 H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.47 (s, 4H), 4.02-4.14 (m, 7H), 3.82-3.93 (m, 4H), 3.05 (d, J = 28.0 Hz, 4H), 2.62-2.71 (m, 5H), 2.53 - 2.57 (m, 2H), 2.27-2.38 (m, 4H), 2.04 (s, 3H), 1.83-1.92 (m, 4H), 1.54-1.67 (m, 11H), 1.25-1.30 (m, 38H), 0.86-0.89 (m, 14H).

[0358] 10.Synthesis of YK-910

[0359] The synthetic route is as follows:

[0360] [ka]

[0361] Step 1: Synthesis of 3-hexylnonyl-7-((2-chloroethyl)(4-oxo-4-(undecyloxy)butyl)amino)heptanoate (YK-910-PM1)

[0362] 3-Hexylnonyl 7-((2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)heptanoate (YK-108, see CN116178193B for details of the synthesis method) (300 mg, 0.47 mmol) was used as the raw material, and YK-910-PM1 (300 mg, 0.46 mmol, 96.9%) was obtained according to the synthesis method of YK-906-PM1.

[0363] Step 2: Synthesis of 3-hexylnonyl-7-((2-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)ethyl)(4-oxo-4-(undecyloxy)butyl)amino)heptanoate (YK-910)

[0364] YK-910-PM1 (300 mg, 0.46 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (106 mg, 0.34 mmol) were used as raw materials, and YK-910 (160 mg, 0.17 mmol, 50.6%) was obtained according to the synthesis method of YK-901. 58 H 108 N2O6, MS(ES): m / z(M+H + )929.8. YK-910: 1 H NMR (400 MHz, CDCl3) δ 7.08 (s, 4H), 4.09 - 4.03 (m, 4H), δ 3.83 - 3.71 (m, 4H), 2.95- 2.87 (m, 4H), 2.64 - 2.53 (m, 7H), 2.35 - 2.27 (m, 4H), 1.84- 1.80 (m, 4H), 1.59 - 1.54 (m, 9H), 1.28 -1.25 (m, 54H), 0.89 - 0.86(m, 12H).

[0365] 11.Synthesis of YK-911

[0366] The synthetic route is as follows:

[0367] [ka]

[0368] Step 1: Synthesis of 2-octyldodecyl-7-bromoheptanoate (YK-911-PM1)

[0369] Using 7-bromoheptanoic acid (255 mg, 1.22 mmol) and 2-octyldecanol (300 mg, 1.11 mmol) as raw materials, YK-911-PM1 (450 mg, 0.97 mmol, 87.8%) was obtained according to the synthesis method of YK-901-PM1.

[0370] Step 2: Synthesis of 2-octyldodecyl-7-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)heptanoate (YK-911)

[0371] Using YK-911-PM1 (260 mg, 0.56 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (174 mg, 0.57 mmol) as raw materials, YK-911 (100 mg, 0.15 mmol, 25.9%) was obtained according to the synthesis method of YK-901. 44 H 81 NO4, MS(ES): 688.6 m / z(M+H + ). 1 H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ 7.08 (s, 4H), 4.09-4.14 (m, 3H), 3.95 (d, J = 8.0 Hz, 2H), 3.71-3.79 (m, 5H), 2.75(t, J = 8.0 Hz, 2H), 2.53-2.63 (m, 4H), 2.29(t, J = 8.0 Hz, 2H), 2.04 (s, 5H), 1.76-1.80 (m, 2H), 1.65-1.71 (m, 2H), 1.53-1.64 (m, 4H), 1.37(s, 3H), 1.24-1.29(m, 34H), 0.87(t, J = 8.0 Hz, 9H).

[0372] 12.Synthesis of YK-912

[0373] The synthetic route is as follows:

[0374] [ka]

[0375] Step 1: Synthesis of 6-bromohexanoic acid-2-octyldodecyl ester (YK-912-PM1)

[0376] Using 2-octyl-dodecanol (1.00 g, 3.35 mmol) and 6-bromohexanoic acid (0.78 g, 4.00 mmol) as starting materials, YK-912-PM1 (1.15 g, 2.42 mmol, 72.2%) was obtained according to the synthesis method of YK-901-PM1.

[0377] Step 2: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexanoic acid 2-octyldodecyl ester (YK-912)

[0378] YK-912-PM1 (302 mg, 0.63 mmol) and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (130 mg, 0.42 mmol) were used as raw materials, and YK-912 (85 mg, 0.12 mmol, 28.8%) was obtained according to the synthesis method of YK-901. 45 H 83 NO4, MS(ES): m / z(M+H + )702.6. 1H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 3.96 (d, J = 5.8 Hz, 2H), 3.66 - 3.60 (m, 4H), 2.65 - 2.53 (m, 6H), 2.32 (t, J = 7.4 Hz, 2H), 1.72 - 1.56 (m, 9H), 1.44-1.25 (m, 44H), 0.88 (t, J = 6.4 Hz, 9H).

[0379] 13. Synthesis of Compound 7

[0380] The synthetic route is as follows:

[0381] [ka]

[0382] Step 1: Synthesis of 2-(phenylamino)ethan-1-ol (compound 7-PM1)

[0383] Iodobenzene (810 mg, 3.97 mmol), ethanolamine (730 mg, 11.95 mmol), cuprous chloride (39.6 mg, 0.40 mmol), and potassium hydroxide (730 mg, 13.01 mmol) were dissolved in a 10 mL single-neck flask and stirred overnight at room temperature under nitrogen gas protection. After completion of the reaction, 10 mL of purified water was added to quench the reaction mixture. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel flash chromatography (ethyl acetate / n-hexane) to give compound 7-PM1 (430 mg, 3.13 mmol, 79.0%).

[0384] Step 2: Synthesis of bis(2-hexyldecyl)6,6'-((2-((2-hydroxyethyl)(phenyl)amino)ethyl)azadiyl)dihexanoic acid (compound 7)

[0385] Compound 7-PM1 (100 mg, 0.73 mmol) and bis(2-hexyldecyl) 6,6'-((2-chloroethyl)azetidinyl)dihexanoate (184 mg, 0.24 mmol) were used as raw materials, and compound 7 (92 mg, 0.11 mmol, 44.7%) was obtained according to the synthesis method of YK-906. 54 H 100 N2O5, MS(ES): m / z(M+H + )857.8. 1 H NMR (400 MHz, CDCl3) δ 7.27 - 7.13 (m, 2H), 6.73 - 6.43 (m, 3H), 3.93 - 3.80 (m, 4H), 3.70 - 3.58 (m, 5H), 2.68 (s, 1H), 2.42 (t, J = 7.4 Hz, 2H), 2.34 (s, 2H), 1.62 - 1.56 (m, 12H), 1.41-1.28 (m, 56H), 0.88 (t, J = 6.3 Hz, 12H).

[0386] 14.Synthesis of I-1

[0387] The synthetic route is as follows:

[0388] [ka]

[0389] Step 1: Synthesis of 2-(phenylamino)ethan-1-ol (I-1-PM1)

[0390] 5-Aminovaleric acid (2.50 g, 21.3 mmol), benzyl alcohol (6.92 g, 64.0 mmol), and p-toluenesulfonic acid monohydrate (4.45 g, 23.4 mmol) were dissolved in toluene (90 mL), and the mixture was heated under reflux overnight with stirring. After completion of the reaction, the mixture was cooled to room temperature and filtered to obtain the p-toluenesulfonic acid salt of compound I-1-PM1 (5.95 g, 15.7 mmol, 73.6%).

[0391] Step 2: Synthesis of bis(2-hexyldecyl) 6,6'-((5-(benzyloxy)-5-oxopentyl)azadiyl)dihexanoate (I-1)

[0392] Using the p-toluenesulfonate salt of I-1-PM1 (1.50 g, 3.95 mmol) and 2-hexyldecyl 6-bromohexanoate (2.65 g, 6.32 mmol) as raw materials, I-1 (797 mg, 0.90 mmol, 22.8%) was obtained according to the synthesis method of YK-906. 56 H 101 NO6, MS(ES): m / z(M+H + )884.8. 1 H NMR (400 MHz, CDCl3) δ 7.40 - 7.33 (m, 5H), 5.11 (s, 2H), 3.94 - 3.83 (m, 4H), 2.42 - 2.33 (m, 8H), 2.33 (m, 4H), 1.66 - 1.59 (m, 8H), 1.51 - 1.40 (m, 6H), 1.41-1.18 (m, 52H), 0.88 (t, J = 6.5 Hz, 12H).

[0393] 15.Synthesis of YK-501

[0394] The synthesis was carried out according to the synthesis method of YK-501 in Example 1 of CN116082275B.

[0395] 16.Synthesis of YK-506

[0396] The synthesis was carried out according to the synthesis method of YK-506 in Example 1 of CN116082275B.

[0397] Example 2: Optimization of lipid nanoparticle (LNP) manufacturing conditions

[0398] 1. Optimizing the ratio of carrier (liposome) to mRNA

[0399] The cationic lipid compounds YK-908, YK-909, YK-910, and YK-912 synthesized in Example 1 were dissolved in ethanol with DSPC (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and DMG-PEG2000 in a molar ratio of 49:10:39.5:1.5 to prepare an ethanol lipid solution. The ethanol lipid solution was quickly added to citrate buffer (pH = 4-5) using the ethanol injection method and vortexed for 30 seconds for later use. eGFP-mRNA (purchased from BIOHUB International Trade Co., Ltd.) was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared by mixing a predetermined volume of liposome solution and an mRNA solution at total lipid to mRNA weight ratios of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1, respectively. Ultrasonication was performed at 25 °C for 15 min (ultrasonic frequency 40 kHz, ultrasonic power 800 W). The resulting liposomes were diluted 10-fold with PBS and then ultrafiltered through a 300 kDa ultrafiltration tube to remove ethanol. The resulting solution was then fixed to a predetermined volume with PBS. LNP formulations encapsulating eGFP-mRNA were obtained using the cationic lipid YK-908 (or YK-909, YK-910, or YK-912) / DSPC / cholesterol / DMG-PEG2000 (molar ratio: 49:10:39.5:1.5).

[0400] The results of cell transfection experiments showed that the weight ratio of carrier to mRNA ranged from 10:1 to 30:1 and all showed excellent transfection effects. The best transfection effect was observed at 15:1, while the ratios of 5:1 and 35:1 showed poor transfection effects, indicating that these ratios cannot be used for mRNA delivery.

[0401] 2. Optimizing the ratio of cationic lipids to neutral lipids

[0402] LNP formulations encapsulating eGFP-mRNA were prepared according to the method of 1, where the molar ratios of the cationic lipid YK-908 (or YK-909, YK-910, or YK-912) to the neutral lipid DSPC were 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1, and 20:1, respectively.

[0403] Cell transfection experiments showed that cationic lipid to neutral lipid molar ratios ranging from 1:1 to 15:1 all had transfection effects, with the highest transfection efficiency being 4.9:1.

[0404] 3. Optimization of the ratio of polymer-conjugated lipids in the carrier (liposome)

[0405] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, where the cationic lipid in the carrier was YK-908 (or YK-909, YK-910, or YK-912), and the molar ratios of the polymer-conjugated lipid DMG-PEG2000 in the carrier were 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10%, and 15%, respectively.

[0406] According to the results of cell transfection experiments, all molar ratios of polymer-conjugated lipids in the carrier ranging from 0.5% to 10% had a transfection effect, with the highest transfection efficiency at 1.5% and the lowest at 10%.

[0407] 4. Optimization of the ratio of each component in the carrier (liposome)

[0408] LNP formulations encapsulating eGFP-mRNA were prepared according to method 1, where the molar ratios of the cationic lipid YK-908 (or YK-909, YK-910, or YK-912), neutral lipid DSPC, structural lipid cholesterol, and polymer-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.

[0409] Cell transfection experiments showed that the molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids were all 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. The best transfection efficiency was observed within the range of (35-49):(7.5-15):(35-55):(1-5), with the molar ratio of 49:10:39.5:1.5 showing the best transfection efficiency.

[0410] Example 3: Cell transfection experiments of LNP formulations of eGFP-mRNA

[0411] Cell resuscitation and passaging: 293T cells were resuscitated and cultured in culture dishes until the required cell number was reached.

[0412] Seeding plates: Cells in culture plates were digested and counted, and 10,000 cells per well were plated on a 96-well plate, and 150,000 cells per well on a 12-well plate, and cultured overnight until the cells attached to the wall.

[0413] Cell transfection experiment: 1.5 μg of the LNP formulation of eGFP-mRNA prepared in Example 2 (the cationic lipids in the carrier were YK-908, YK-909, YK-910, and YK-912) and the Lipofectamine 3000 formulation of eGFP-mRNA were each added to the cell culture medium in a 12-well plate. After continuous cultivation for 24 hours, the samples were observed under a fluorescence microscope, and the transfection efficiency of the samples was considered according to the fluorescence intensity.

[0414] Based on the experimental results, the final conditions for producing lipid nanoparticles (LNP formulations) were determined: the mass ratio of carrier to mRNA was 15:1, the molar ratio of cationic lipid to neutral lipid was 4.9:1, the polymer-conjugated lipid accounted for 1.5 mol% of the liposomes, and the molar ratio of cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid was 49:10:39.5:1.5. Subsequent experiments used these conditions to produce lipid nanoparticles (LNP formulations).

[0415] Example 4: Preparation of lipid nanoparticles (LNP formulation) (optimized ratio)

[0416] [Table 2] JPEG0007813399000045.jpg242166JPEG0007813399000046.jpg210166

[0417] The cationic lipids listed in Table 2 were dissolved in ethanol with DSPC (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and DMG-PEG2000 in a molar ratio of 49:10:39.5:1.5 to prepare an ethanolic lipid solution. The ethanolic lipid solution was quickly added to citrate buffer (pH = 4-5) by the ethanol injection method and vortexed for 30 s for later use. eGFP-mRNA (BIOHUB International Trade Co., Ltd.), Fluc-mRNA (BIOHUB International Trade Co., Ltd.), or Cy5 fluorescently labeled Leqvio (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared by mixing a predetermined volume of liposome solution and an mRNA aqueous solution at a total lipid to mRNA weight ratio of 15:1. Ultrasonication was performed for 15 minutes at 25°C (ultrasonic frequency 40 kHz, ultrasonic power 800 W). The resulting liposomes were diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol. The resulting solution was then fixed to a predetermined volume with PBS. LNP formulations encapsulating eGFP-mRNA or Fluc-mRNA were obtained using a cationic lipid / DSPC / cholesterol / DMG-PEG2000 (molar ratio: 49:10:39.5:1.5).

[0418] Lipofectamine 3000 transfection reagent is currently widely used for cell transfection, boasting excellent transfection performance and excellent transfection efficiency, which can improve cell activity and is suitable for difficult-to-transfect cell types. The inventors selected Lipofectamine 3000 transfection reagent to perform controls and prepared Lipofectamine 3000 formulations of eGFP-mRNA or Fluc-mRNA according to the Lipofectamine 3000 (Invitrogen Trading (Shanghai) Co., Ltd.) guidelines.

[0419] Example 5: Measurement of particle size and polydispersity index (PDI) of lipid nanoparticles

[0420] Dynamic light scattering was used to measure the polydispersity index (PDI) using a Malvern laser analyzer.

[0421] 10 μL of liposome solution was diluted to 1 mL with RNAse-free deionized water and added to the sample pool. Each sample was measured three times. Measurement conditions: 90° scattering angle, 25°C. The detection results are shown in Table 3.

[0422] [Table 3]

[0423] As can be seen in Table 3, the particle sizes of the lipid nanoparticles produced in Example 4 were 110 to 165 nm, and all of them could be used for mRNA delivery.

[0424] Here, it was found that the particles produced by YK-904 had the smallest particle size at 112 nm, and the particles produced by YK-901 had the largest particle size at 162 nm.

[0425] The polydispersity index of all lipid nanoparticles was between 8% and 18%, where YK-904 had the smallest at 8.2% and YK-901 had the largest at 17.4%.

[0426] The particle morphology produced by YK-908, YK-909, YK-910, and YK-912 was also relatively excellent, with smaller particle sizes and better uniformity compared to particles produced from other cationic lipids with similar structures in the prior art, such as SM-102, HHMA, MC3, compound 7, and I-1.

[0427] Example 6: Performance verification of in vitro LNP delivery vehicles

[0428] Cell resuscitation and passaging: The method was the same as in Example 3.

[0429] Seed plate: The method is the same as in Example 3.

[0430] 1. Fluorescent detection of Fluc-mRNA (transfection efficiency)

[0431] An LNP formulation containing 3 μg of Fluc-mRNA (the LNP formulation carrier components were cationic lipid, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 49:10:39.5:1.5, where the cationic lipid was one listed in Table 1) was added to the cell culture medium in a 96-well plate and cultured for 24 hours. The corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence intensity of each well was detected using an IVIS fluorescence detection system. The chemical structures of the designed compounds and representative cationic lipids from the prior art are listed in Table 2. The results of the intracellular transfection efficiency of LNP formulations produced using a series of cationic lipid compounds designed according to the present invention and prior art cationic lipids, including SM-102, HHMA, MC3, compound 7, and I-1, are shown in Table 4.

[0432] Table 4 listed the fluorescence detection results of Fluc-mRNA-containing LNP formulations made from different cationic lipids.

[0433] [Table 4]

[0434] Analysis of the experimental results:

[0435] (1) The compounds designed in the present invention, including YK-908, YK-909, YK-910, and YK-912, have significant differences in chemical structure compared with representative cationic lipids of the prior art, such as SM-102, MC3, YK-501, YK-506, and HHMA, and minor differences between compounds 7 and I-1.

[0436] Compared with SM-102, MC3, YK-501, YK-506 and HHMA, the chemical structures of the compounds designed in this invention were shown to be significantly different. As can be seen from the chemical structures, the compounds of the present invention incorporate a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure, linked by an amino group to an ethyl tertiary amine head group or ester, an amide-containing linear alkyl group or ester, or an amide-containing branched alkyl group. However, SM-102, MC3, YK-501, YK-506, and HHMA do not incorporate a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure. The amino head group of SM-102 is a simple ethanolamine structure. The amino head group of MC3 is a dimethyl tertiary amine structure without a hydroxyl group. The amino head group of YK-501 and YK-506 is piperazinyl, and the amino head group of HHMA is a methyl tertiary amine, with the hydroxyl group at the second position of the aliphatic chain. The other groups also differ significantly.

[0437] Compound 7, I-1, and the compound designed in the present invention all have a benzene ring structure, and it was shown that there is almost no difference in their chemical structures.

[0438] (2) Among the designed compounds, the LNP formulations prepared from YK-908, YK-909, YK-910, and YK-912 all showed significantly improved cell transfection efficiencies compared to representative cationic lipids of the prior art. For example, the cell transfection efficiency of YK-910 ("YK-910 cell transfection efficiency" refers to the cell transfection efficiency of LNP formulations prepared using YK-910, and other similar expressions have similar meanings) was 6.04-fold higher than that of SM-102, 186.59-fold higher than that of YK-501, 13.19-fold higher than that of YK-506, 8.53-fold higher than that of HHMA, 90.92-fold higher than that of MC3, 33.51-fold higher than that of compound 7, 42.56-fold higher than that of I-1, and 10.24-fold higher than that of Lipofectamine 3000.

[0439] SM-102, HHMA, and MC3 are representative cationic lipids in the prior art, and have relatively excellent transfection performance.

[0440] As shown in Table 4, the Fluc-mRNA-containing LNP formulations prepared from YK-908, YK-909, YK-910, and YK-912 exhibited the strongest fluorescence absorption, with RLU values ​​of 5,070,083, 6,780,306, 8,628,595, and 5,708,988, respectively (Figures 1 and 2).

[0441] The cell transfection efficiency of YK-908 was 3.55 times that of SM-102, 109.64 times that of YK-501, 7.75 times that of YK-506, 5.01 times that of HHMA, 53.42 times that of MC3, 19.69 times that of Compound 7, 25.01 times that of I-1, and 6.02 times that of Lipofectamine 3000, thereby significantly improving the transfection efficiency.

[0442] The cell transfection efficiency of YK-909 was 4.75 times higher than that of SM-102, 146.62 times higher than that of YK-501, 10.36 times higher than that of YK-506, 6.71 times higher than that of HHMA, 71.44 times higher than that of MC3, 26.33 times higher than that of Compound 7, 33.44 times higher than that of I-1, and 8.05 times higher than that of Lipofectamine 3000, thereby significantly improving the transfection efficiency.

[0443] The cell transfection efficiency of YK-910 was 6.04 times that of SM-102, 186.59 times that of YK-501, 13.19 times that of YK-506, 8.53 times that of HHMA, 90.92 times that of MC3, 33.51 times that of compound 7, 42.56 times that of I-1, and 10.24 times that of Lipofectamine 3000, thereby significantly improving the transfection efficiency.

[0444] The cell transfection efficiency of YK-912 was 4.00 times higher than that of SM-102, 123.46 times higher than that of YK-501, 8.73 times higher than that of YK-506, 5.65 times higher than that of HHMA, 60.16 times higher than that of MC3, 22.17 times higher than that of Compound 7, 28.16 times higher than that of I-1, and 6.77 times higher than that of Lipofectamine 3000, thereby significantly improving the transfection efficiency.

[0445] The cell transfection efficiency of YK-902 was 0.22-fold that of SM-102, 6.73-fold that of YK-501, 0.48-fold that of YK-506, 0.31-fold that of HHMA, 3.28-fold that of MC3, 1.21-fold that of compound 7, and 1.53-fold that of I-1.

[0446] The cell transfection efficiency of LNP formulations made from cationic lipid compounds cannot be predicted based on their structure; there may be large differences in cell transfection efficiency between compounds with very large structural differences and compounds with similar structures.

[0447] (3) Among the compounds with M1 = -C(O)O-, the LNP formulation prepared from YK-912 showed significantly improved cell transfection efficiency compared with other compounds, for example, YK-912 was 228-fold higher than YK-903 and 131-fold higher than YK-901.

[0448] A series of compounds with similar structures, including YK-901, YK-902, YK-903, YK-904, YK-905, YK-911, and YK-912, in which M1 is -C(O)O-, were compared. The structural differences between these compounds were limited to slight variations in the individual groups (Table 2). Cell transfection results showed a significant difference in activity between these compounds. The LNP formulation prepared from YK-912 had the highest cell transfection efficiency, significantly improved compared to the other compounds. YK-912's transfection efficiency was 131.47-fold higher than YK-901, 18.35-fold higher than YK-902, 228.30-fold higher than YK-903, 45.65-fold higher than YK-904, 78.83-fold higher than YK-905, and 4.39-fold higher than YK-911, respectively.

[0449] (4) Among the compounds in which M1 is an amide bond (-C(O)NH- or -NHC(O)-), the LNP preparation prepared from YK-908 has significantly improved cell transfection efficiency compared to YK-907, reaching 134-fold that of YK-907.

[0450] We compared YK-907 and YK-908, compounds with similar structures where M1 is -C(O)NH- or -NHC(O)-, and found that the LNP formulation prepared from YK-908 had significantly improved cell transfection efficiency compared to YK-907, reaching 134.49-fold.

[0451] (5) Among the series of compounds in which X1 is N, the LNP formulations prepared from YK-909 and YK-910 showed significantly improved cell transfection efficiencies compared with YK-906, reaching 246-fold and 313-fold, respectively, of that of YK-906.

[0452] A series of compounds with similar structures, where X1 is N, including YK-906, YK-909, and YK-910, were compared. The results showed that the cell transfection efficiencies of LNP formulations made from YK-909 and YK-910 were significantly improved compared to YK-906, reaching 246.15-fold and 313.25-fold higher than that of YK-906, respectively.

[0453] As can be seen from the above, it is not possible to predict the cell transfection activity of LNP formulations made from cationic lipid compounds based on their structure, and even a series of cationic lipid compounds with very similar structures are likely to have very large differences in cell transfection efficiency.

[0454] 2. Measuring Cell Viability

[0455] An LNP formulation containing 5 μg of Fluc-mRNA (the components of the LNP formulation carrier were cationic lipid, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 49:10:39.5:1.5, where the cationic lipid was the cationic lipid listed in Table 1) was added to the cell culture medium in a 96-well plate and cultured for 24 hours. After that, 10 μL of CCK-8 solution was added to each well, and the culture plate was cultured in an incubator for 1 hour. After that, the absorbance at 450 nm was measured using a microplate reader, and the cell viability results are shown in Table 5.

[0456] [Table 5]

[0457] Analysis of the experimental results:

[0458] (1) The chemical structures of the series of compounds designed in the present invention, including YK-908, YK-909, YK-910, and YK-912, are significantly different from those of representative cationic lipids of the prior art, such as SM-102, MC3, YK-501, YK-506, and HHMA, and are slightly different from those of compounds 7 and I-1.

[0459] Experimental results showed that LNP formulations made from YK-909 and YK-910 exhibited the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids of the prior art. For example, the cell viability of YK-910 ("YK-910 cell viability" refers to the cell viability resulting from an LNP formulation prepared using YK-910, and other similar expressions have similar meanings) was 12% higher than that of SM-102, 31% higher than that of YK-501, 17% higher than that of HHMA, 20% higher than that of MC3, 21% higher than that of Compound 7, 26% higher than that of I-1, and 63% higher than that of Lipofectamine 3000 (Figure 3).

[0460] (2) Comparing a series of compounds with similar structures, including YK-901, YK-902, YK-903, YK-904, YK-905, YK-911, and YK-912, in which M1 is —C(O)O—, the structural differences among these compounds were only slight variations in the individual groups.

[0461] Experimental results showed that the difference in cytotoxicity among this series of compounds was very large, with YK-912 showing the highest cell viability, which was 29% higher than YK-901, 51% higher than YK-902, 40% higher than YK-903, 35% higher than YK-904, 36% higher than YK-905, and 25% higher than YK-911, respectively, significantly improving cell viability.

[0462] (3) We compared the structurally similar compounds YK-907 and YK-908, in which M1 is an amide bond (-C(O)NH- or -NHC(O)-). The results showed that YK-908 significantly improved cell viability, 16% higher than that of YK-907.

[0463] (4) A series of compounds with similar structures, including YK-906, YK-909, and YK-910, where X1 is N, were compared. The results showed that the cell viability of YK-909 and YK-910 was significantly improved compared to YK-906, being 29% and 31% higher than that of YK-906, respectively.

[0464] This indicates that the cytotoxicity of LNP formulations made from cationic lipid compounds cannot be predicted based on the structure of the cationic lipid compound, and that compounds with very large structural differences, as well as compounds with similar structures, are likely to have very large differences in toxicity to transfected cells.

[0465] Example 7: Performance verification of in vivo cationic lipid (LNP) delivery carriers

[0466] This example examined the in vivo expression and duration of mRNA delivered by the designed cationic lipid in mice. In vivo experiments further demonstrated that, compared with typical cationic lipids of the prior art, the LNP delivery vehicle of the present invention (1) can effectively deliver mRNA into the animal body and achieve efficient and sustained expression, (2) can achieve high expression of delivered mRNA in the spleen but low expression in the liver, and (3) can achieve high expression of delivered small nucleic acid drugs in the spleen.

[0467] 1. Expression of delivered mRNA in mice

[0468] An LNP formulation containing 10 μg of Fluc-mRNA was injected into female BALB / C mice (4-6 weeks old, weighing 17-19 g) via the tail vein. At specific time points after administration (6 hours, 24 hours, 48 ​​hours, and 7 days), the mice were intraperitoneally injected with a fluorescent imaging substrate. After allowing the mice to move freely for 5 minutes, the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA encapsulated in the LNPs was detected in the mice using an IVIS Spectrum small animal live imager. The results of in vivo imaging of the mice are shown in Table 6.

[0469] [Table 6]

[0470] Analysis of the experimental results:

[0471] (1) The LNP formulations prepared from YK-908, YK-909, YK-910, and YK-912 delivered mRNA that was highly and continuously expressed in the mouse body. Compared with representative cationic lipids of the prior art, such as SM-102, YK-501, YK-506, and Compound 7, the mRNA expression levels were significantly improved at 6 hours, 24 hours, 48 ​​hours, and 7 days. For example, the mRNA expression level delivered by the LNP formulation prepared from YK-910 at 24 hours was 6.78-fold higher than that of SM-102, 12.21-fold higher than that of YK-501, 8.28-fold higher than that of YK-506, and 11.50-fold higher than that of Compound 7; and the mRNA expression level delivered by the LNP formulation prepared from YK-910 at 7 days was 9.24-fold higher than that of SM-102, 15.15-fold higher than that of YK-501, 2.49-fold higher than that of YK-506, and 10.03-fold higher than that of Compound 7.

[0472] (2) Compared with the compound YK-904, which has a similar structure but slight differences in individual groups, the LNP formulations made from YK-908, YK-909, YK-910, and YK-912 significantly improved both the intensity and duration of mRNA expression in mice. For example, the mRNA expression levels delivered by the LNP formulation made from YK-910 were 12.03-fold, 18.49-fold, 23.07-fold, and 26.32-fold higher than those of YK-904 at 6 hours, 24 hours, 48 ​​hours, and 7 days, respectively.

[0473] As can be seen from the above, even when cationic lipid compounds have very small structural differences, there is a high possibility that the mRNA expression in the animal body of the LNP preparation produced from them will be significantly different. Based on the structure of the cationic lipid compound, it is not possible to predict whether mRNA will be highly and persistently expressed in the animal body, and selecting a cationic lipid compound having mRNA that is highly and persistently expressed is extremely difficult and requires a great deal of creative effort.

[0474] 2. Expression of delivered mRNA in different organs of mice

[0475] Liposomes prepared from the cationic lipid compounds of the present invention either reduced the amount of protein expressed in the liver or remained in the liver and did not express the target protein, compared with representative cationic lipids of the prior art, such as SM-102, YK-501, YK-506, and Compound 7. Therefore, compared with cationic lipids of existing technologies, LNP formulations prepared from the designed compounds of the present invention were less toxic or non-toxic to the liver.

[0476] 5 μg of an LNP formulation containing Fluc-mRNA was subcutaneously injected into female BALB / C mice (4-6 weeks old, weighing 17-19 g). Six hours after administration, the mice received an intraperitoneal injection of a fluorescent imaging substrate. After allowing the mice to move freely for 5 minutes, the mean radioactivity (equivalent to fluorescence intensity) of the proteins expressed by the LNP-loaded mRNA was detected in vivo using an IVIS Spectrum small animal live imager. After sampling was completed, the cervical vertebrae were cut, and the mice were dissected to precisely separate the mouse internal organs, including the heart, liver, spleen, lungs, kidneys, and lymph nodes. The mean radioactivity (equivalent to fluorescence intensity) of the proteins expressed by the LNP-loaded mRNA in each organ was detected using an IVIS Spectrum small animal live imager. The results of in vivo mouse imaging are shown in Table 7.

[0477] [Table 7]

[0478] Analysis of the experimental results:

[0479] Compared with representative cationic lipids of the prior art, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 significantly improved mRNA expression levels in mouse spleens. For example, the expression levels of YK-908, YK-909, YK-910, and YK-912 in the spleen were 6.59-fold, 8.00-fold, 9.49-fold, and 6.86-fold higher than those of SM-102, 11.83-fold, 14.37-fold, 17.04-fold, and 12.33-fold higher than those of YK-501, and 2.36-fold, 2.87-fold, 3.40-fold, and 2.46-fold higher than those of YK-506, respectively.

[0480] The liver expression levels of YK-908, YK-909, YK-910, and YK-912 were significantly reduced compared to SM-102, with the liver expression levels of SM-102 being 17.12-fold, 2.67-fold, and 2.13-fold higher than those of YK-908, YK-909, and YK-910, respectively, while YK-912 was not expressed in the liver. Therefore, compared to cationic lipids of existing technology, LNP formulations made from the designed compounds of the present invention had reduced or no liver toxicity.

[0481] Furthermore, the Fluc-mRNA-containing LNP formulations produced by all compounds showed very large differences in expression in different organs of mice. YK-908, YK-909, YK-910, and YK-912 were expressed almost exclusively in the spleen, but not in other organs such as the heart, lungs, or kidneys. They were expressed in small amounts or not at all in the liver. SM-102 was expressed in the liver and spleen, but not in the heart, lungs, or kidneys (Figure 4).

[0482] 3. Expression of delivered small nucleic acid drugs in different organs of mice

[0483] Small nucleic acid drugs delivered by delivery vehicles made from the cationic lipids YK-908, YK-909, YK-910, and YK-912 designed in this invention were significantly concentrated in the spleen of mice, with significantly reduced renal excretion. Compared with conventional cationic lipids, delivery of Cy5-conjugated small nucleic acid drugs (Cy5 fluorescently labeled Leqvio) resulted in significantly increased fluorescence in the mouse liver and spleen, and significantly reduced fluorescence in the kidney. In vivo experiments further demonstrated that small nucleic acid drugs delivered by the LNP delivery vehicles of this invention were similarly effectively delivered into the animal body, resulting in efficient and sustained therapeutic effects in the spleen.

[0484] LNP formulations containing 5 μg of Cy5-labeled Leqvio were injected via the tail vein into female BALB / C mice (4-6 weeks old, weighing 17-19 g). At a specific time point (4 hours) after administration, the mean radioactivity (equivalent to fluorescence intensity) of Cy5-labeled Leqvio in the LNPs was detected using an IVIS Spectrum small animal live imager. After sampling, the cervical vertebrae were cut, and the mice were dissected to precisely separate the mouse's internal organs, including the heart, liver, spleen, lungs, kidneys, and lymph nodes. The mean radioactivity (equivalent to fluorescence intensity) of Cy5-labeled Leqvio in the LNPs was detected using the IVIS Spectrum small animal live imager. The mouse organ imaging detection results are shown in Table 8.

[0485] [Table 8]

[0486] Analysis of the experimental results:

[0487] Compared with conventional cationic lipids, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 all showed significantly improved distribution of small nucleic acid drugs in the mouse spleen. For example, the fluorescence intensity of YK-908, YK-909, and YK-910 in the spleen ("YK-908 fluorescence intensity" refers to the fluorescence intensity resulting from Cy5 fluorescent-labeled Leqvio delivered by LNP formulations made from YK-908, and other similar expressions have similar meanings) was 34.16-fold, 43.10-fold, and 51.19-fold higher than that of SM-102, and 7.95-fold, 10.02-fold, and 11.91-fold higher than that of YK-501, respectively (Figure 5).

[0488] Surprisingly, the distribution of YK-912 in the kidney was 0.14-fold that of SM-102, which was significantly reduced, indicating its long-term efficacy for small nucleic acid drug delivery.

[0489] (2) Compared with direct administration, the LNP formulations prepared from YK-908, YK-909, YK-910, and YK-912 all significantly improved the distribution of small nucleic acid drugs in the mouse spleen, and the fluorescence intensity in the spleen was 100.55-fold, 126.85-fold, 150.67-fold, and 31.86-fold higher, respectively, than that of direct administration.

[0490] Furthermore, the amount of YK-912 distributed in the kidney was significantly reduced compared to direct administration, being only 0.09-fold lower, demonstrating its long-term efficacy for small nucleic acid drug delivery.

[0491] In conclusion, the series of cationic lipid compounds designed in this invention, including YK-908, YK-909, YK-910, and YK-912, significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly increased the expression levels of mRNA and small nucleic acids in mouse spleen, and significantly reduced renal excretion when YK-912 delivered small nucleic acids.

[0492] 1. The chemical structures of a series of compounds designed in the present invention, including YK-908, YK-909, YK-910, and YK-912, are significantly different from those of representative cationic lipids of the prior art, such as SM-102, MC3, YK-501, YK-506, and HHMA, and are slightly different from those of compounds 7 and I-1.

[0493] 2. In the designed series of compounds, LNP formulations made from YK-908, YK-909, YK-910, and YK-912 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved mRNA and small nucleic acid expression levels in mouse spleen, and significantly reduced renal excretion when YK-912 delivered small nucleic acids, compared with representative and structurally similar cationic lipids in the prior art.

[0494] For example, the cell transfection efficiency of YK-910 was 6.04 times that of SM-102, 186.59 times that of YK-501, 13.19 times that of YK-506, 8.53 times that of HHMA, 90.92 times that of MC3, 33.51 times that of Compound 7, 42.56 times that of I-1, and 10.24 times that of Lipofectamine 3000. The cell viability of YK-910 was 12% higher than that of SM-102, 31% higher than that of YK-501, 17% higher than that of HHMA, 20% higher than that of MC3, 21% higher than that of Compound 7, 26% higher than that of I-1, and 10.24 times higher than that of Lipofectamine 3000. 3000, the 24-hour mRNA expression level of YK-910 in animals was 6.78 times that of SM-102, 12.21 times that of YK-501, 8.28 times that of YK-506, and 11.50 times that of Compound 7, and the mRNA expression level of YK-910 in the spleen was 9.49 times that of SM-102, 17.04 times that of YK-501, and 3.40 times that of YK-506. The distribution of small nucleic acids in the spleen of YK-910 was 51.19 times that of SM-102, 11.91 times that of YK-501, and 2.85 times that of YK-506. The fluorescence intensity of YK-912 in the spleen and liver was 1.25 times and 10.82 times that of SM-102, respectively, but the distribution in the kidney was 0.14 times that of SM-102, which was significantly reduced.

[0495] 3. Among a series of compounds designed in the present invention with very little difference in chemical structure, the LNP formulations made from YK-908, YK-909, YK-910, and YK-912 showed significantly improved cell transfection efficiency and significantly reduced cytotoxicity compared with other compounds, and the distribution of small nucleic acids in the spleen of mice was significantly improved compared with that of direct administration.

[0496] For example, when delivering mRNA, the cell transfection efficiency of YK-910 was 345.06-fold higher than that of YK-903 and 313.25-fold higher than that of YK-906, and its cytotoxicity was 58% lower than that of YK-902. When delivering small nucleic acids, the intrasplenic fluorescence intensity of YK-908, YK-909, YK-910, and YK-912 was 100.55-fold, 126.85-fold, 150.67-fold, and 31.80-fold higher, respectively, than that of direct administration, and the intrarenal distribution volume of YK-912 was 0.09-fold lower than that of direct administration.

[0497] 4. Through unique design and screening, the present invention has discovered several compounds, such as YK-908, YK-909, YK-910 and YK-912, which, compared with other compounds with similar structures in the prior art, have significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity and significantly improved expression levels in the spleen of animals, thereby improving delivery efficiency and achieving unpredictable technical effects.

[0498] Although the present invention has been described in detail above, this is for the purpose of enabling those skilled in the art to understand and practice the content of the present invention, and does not limit the protection scope of the present invention, and all equivalent changes or modifications within the spirit and essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt or stereoisomer. 【Chemistry 1】 (however, G １ is C １－４ is alkylene, G ２ is C １－８ is alkylene, G ３ is C ２－８ is alkylene, R １ is C ６－２５ Straight chain alkyl, or C ６－２５ is a branched alkyl; R ２ is C ６－２５ Straight chain alkyl, C ６－２５ branched alkyl, or H; R ３ is C １－２５ Straight chain alkyl, C １－２５ branched alkyl, or H; M １ is —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, or —CH(OH)—; M ２ is —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, or —CH(OH)—; R' is C １－１８ alkyl, or H; X １ is —CH— or N.

2. G １ is unsubstituted C １ Alkylene or unsubstituted C ２ is alkylene, Or, G ２ is unsubstituted C １ Alkylene, unsubstituted C ３ Alkylene or unsubstituted C ４ is alkylene, Or, G ３ is unsubstituted C ５ Alkylene or unsubstituted C ６ is alkylene, Or, R １ is C ６－１４ Straight chain alkyl or C １２－２５ is a branched alkyl; Or, R ２ is C ６－１４ Straight chain alkyl, C １２－２５ is branched alkyl or H, Or, M １ is —C(O)O—, —C(O)NH—, or —NHC(O)—, Or, M ２ is —C(O)O—; 2. The compound of claim 1, a pharmaceutically acceptable salt or stereoisomer.

3. R １ teeth, 【Chemistry 2】 and Or, R ２ teeth, 【Transformation 3】 That is, 2. The compound of claim 1, a pharmaceutically acceptable salt or stereoisomer.

4. R ３ is C ８ 2. The compound of claim 1, a pharmaceutically acceptable salt or stereoisomer thereof, which is a straight chain alkyl.

5. A compound, pharmaceutically acceptable salt, or stereoisomer which is at least one of compounds YK-901, YK-902, YK-903, YK-904, YK-905, YK-906, YK-907, YK-908, YK-909, YK-910, YK-911, and YK-912, having the following structures: 【Chemistry 4】 【change】

6. A composition comprising a carrier comprising a cationic lipid comprising a compound, pharmaceutically acceptable salt or stereoisomer of any one of claims 1 to 5.

7. The composition of claim 6 , wherein the carrier further comprises a neutral lipid.

8. 8. The composition of claim 7, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

9. The neutral lipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl -sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,9. The composition of claim 8, wherein the phosphatidyl group is one or more selected from 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and mixtures thereof.

10. The composition of claim 6 , wherein the carrier further comprises a structured lipid.

11. the structured lipids are selected from one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatidine, ursolic acid, α-tocopherol, and corticosteroids; The composition of claim 10.

12. The composition of claim 6 , wherein the carrier further comprises a polymer-conjugated lipid.

13. 13. The composition of claim 12, wherein the polymer-conjugated lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

14. 14. The composition of claim 13, wherein the polymer-conjugated lipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000, dimyristoylglycero-3-methoxypolyethylene glycol 2000, and methoxypolyethylene glycol ditetradecylacetamide.

15. 7. The composition of claim 6, wherein the carrier further comprises a neutral lipid, a structured lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

16. 16. The composition of claim 15, wherein the molar ratio of the cationic lipid, the neutral lipid, the structured lipid, and the polymer-conjugated lipid is 45:10:43.5:1.5 or 49:10:39.5:1.

5.

17. the composition is a nanoparticle formulation, the nanoparticle formulation has an average particle size of 10 nm to 300 nm, and the nanoparticle formulation has a polydispersity index of ≦50%; or the cationic lipid further comprises one or more other ionizable lipid compounds; Or, the composition further comprises one or more of a pharmaceutically acceptable excipient or diluent. The composition of claim 6.

18. The composition of claim 6 further comprising a therapeutic or prophylactic agent.

19. 19. The composition of claim 18, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:

1.

20. the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; Alternatively, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

19. The composition of claim 18.

21. The composition of claim 18 , wherein the therapeutic or prophylactic agent is a ribonucleic acid and / or a deoxyribonucleic acid.

22. 22. The composition of claim 21, wherein the ribonucleic acid is selected from the group consisting of small interfering RNA, asymmetric interfering RNA, microRNA, dicer substrate RNA, small hairpin RNA, messenger RNA, and mixtures thereof.

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