Long-acting spleen-targeted cationic lipid compound containing benzene ring structure, composition containing same and use thereof

Abstract

The present invention relates to the field of medicine, and in particular to a long-acting spleen-targeted cationic lipid compound containing a benzene ring structure, a composition comprising the same, and uses thereof. More specifically, the present invention provides a compound represented by formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. The present invention also provides a composition comprising the aforementioned compound and their use for delivering a therapeutic agent or a preventive agent.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and specifically relates to a long-acting spleen-targeted cationic lipid compound containing a benzene ring structure, a composition containing the same and its use. Background Art

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

[0003] Confirm that compositions, liposomes and liposome complexes (lipoplex) containing cationic lipids are used as transport vehicles to effectively transport bioactive substances such as small molecule drugs, polypeptides, proteins and nucleic acids to cells and / or intracellular compartments. These compositions generally include one or more "cationic" and / or amino (ionizable) lipids, including neutral lipids, structural lipids and polymer conjugated lipids. Cationic and / or ionizable lipids include amine-containing lipids that can be easily protonated. Although multiple such nanoparticle compositions containing lipids have been demonstrated, safety, efficacy and specificity are still to be improved. It should be noted that the increase of lipid nanoparticle (Lipid Nanoparticle, LNP) complexity complicates its production and may increase its toxicity, which is a major concern that may limit its clinical application. For example, LNP siRNA particles (such as patisiran) require the use of steroids and antihistamines in advance to eliminate unnecessary immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretinamyloidosis, N Engl J Med, 369 (2013) 819-829.). Therefore, it is necessary to develop improved cationic lipid compounds that help deliver therapeutic agents and / or preventive agents (such as nucleic acids) to cells, and compositions comprising the same, to meet the needs of production and clinical applications. Summary of the invention

[0004] The first aspect of the present invention provides a novel cationic lipid compound, which is a compound represented by formula (I)

[0005] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0006] G 1 C 1-4 Alkylene;

[0007] G 2 C 1-8 Alkylene;

[0008] G 3 C 2-8 Alkylene or H;

[0009] R 1 C 6-25 Straight chain alkyl, C 6-25 Branched alkyl or C 6-25 substituted alkyl;

[0010] R 2 C 6-25 Straight chain alkyl, C 6-25 Branched alkyl, C 6-25 Substituted alkyl or H;

[0011] R 3 C 1-25 Straight chain alkyl, C 1-25 Branched alkyl, C 1-25 Substituted alkyl or H;

[0012] M 1 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 or a heteroaryl group or H; M 2 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; R' is: C 1-18 Alkyl, C 2-18 Alkenyl or H;

[0013] X 1 It is -CH- or N.

[0014] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 For unsubstituted C 1 Alkylene.

[0015] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 For unsubstituted C 2Alkylene.

[0016] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 For unsubstituted C 1 Alkylene.

[0017] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 For unsubstituted C 3 Alkylene.

[0018] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 2 For unsubstituted C 4 Alkylene.

[0019] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 3 For H.

[0020] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 3 For unsubstituted C 5 Alkylene.

[0021] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 3 For unsubstituted C 6 Alkylene.

[0022] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 C 6-14 Straight chain alkyl.

[0023] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 C 12-25 Branched chain alkyl.

[0024] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1 for:

[0025] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2C 6-14 Straight chain alkyl.

[0026] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 C 12-25 Branched chain alkyl.

[0027] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 for:

[0028] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 2 For H.

[0029] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3 C 4-15 Straight chain alkyl.

[0030] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3 C 8 Straight chain alkyl.

[0031] In the compound of formula (I) of the present invention or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, M 1 and M 2 It can be the same or different.

[0032] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein M 1 It is -C(O)O-.

[0033] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein M 1 It is -C(O)NH-.

[0034] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein M 1 It is -NHC(O)-.

[0035] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein M 2 For H.

[0036] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein M 2 It is -C(O)O-.

[0037] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 1 It is -CH-.

[0038] A further compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein X 1 is N.

[0039] Further, a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of 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 having the following structure:

[0040]

[0041]

[0042]

[0043] Further, a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-908 having the following structure:

[0044]

[0045] Further, a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-909 having the following structure:

[0046]

[0047] Further, a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-910 having the following structure:

[0048]

[0049] Further, a compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-912 having the following structure:

[0050]

[0051] The second aspect of the present invention provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises a compound of formula (I) according to any one of the aforementioned first aspects, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0052] In a preferred composition, the molar proportion of the cationic lipid in the carrier is 25%-75%.

[0053] A preferred composition is wherein the carrier further comprises a neutral lipid.

[0054] A preferred composition, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1, preferably 4-5:1, most preferably 4.9:1.

[0055] A preferred composition wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0056] A preferred composition, wherein the neutral lipid is selected from one or more of the following: 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 (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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 16.0PE), 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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0057] A preferred composition is wherein the neutral lipid is DOPE and / or DSPC.

[0058] A preferred composition is wherein the carrier further comprises a structured lipid.

[0059] A preferred composition, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1.

[0060] A preferred composition, wherein the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, α-tocopherol, corticosteroids.

[0061] A preferred composition is wherein the structured lipid is cholesterol.

[0062] A preferred composition is one in which the carrier further comprises a polymer-conjugated lipid.

[0063] In a preferred composition, the molar proportion of the polymer conjugated lipid in the carrier is 0.5%-10%, preferably 1.5%.

[0064] A preferred composition, wherein the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

[0065] A preferred composition, wherein the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0066] A preferred composition, wherein the carrier comprises a neutral lipid, a structural lipid and a polymer conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

[0067] A preferred composition, wherein 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). The sum of the molar ratios of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is 100.

[0068] A preferred composition, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 45:10:43.5:1.5 or 49:10:39.5:1.5.

[0069] A preferred composition, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm-300nm; and the polydispersity coefficient of the nanoparticle preparation is ≤50%.

[0070] A preferred composition, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 90nm-280nm; and the polydispersity coefficient of the nanoparticle preparation is ≤45%.

[0071] Preferred compositions are those wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

[0072] Preferred compositions further comprise a therapeutic or prophylactic agent.

[0073] A preferred composition, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 10:1-30:1.

[0074] A preferred composition, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5:1-20:1.

[0075] A preferred composition, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 15:1.

[0076] A preferred composition wherein the therapeutic agent or preventive agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0077] Preferred compositions are those wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0078] Preferred compositions are those wherein the therapeutic or prophylactic agent is a nucleic acid.

[0079] Preferred compositions are those wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

[0080] Preferred compositions are those wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

[0081] A preferred composition, wherein the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0082] A preferred composition is wherein said RNA is mRNA.

[0083] A preferred composition is one in which the composition further comprises one or more pharmaceutically acceptable excipients or diluents.

[0084] The third aspect of the present invention provides the use of the compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect or the composition as described in the second aspect in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0085] The fourth aspect of the present invention is the use of the compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect or the composition as described in the second aspect in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

[0086] A further use wherein the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.

[0087] A further use, wherein the disease or disorder 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.

[0088] A further use, wherein the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or various herpes.

[0089] A further use, wherein the subject is a mammal, preferably the mammal is a human.

[0090] A further use, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0091] A further use wherein the composition is administered subcutaneously.

[0092] A further use wherein a dosage of about 0.001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent is administered to the subject.

[0093] The fifth aspect of the present invention provides the use of the compound described in the first aspect or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer in improving cell transfection efficiency and / or reducing cytotoxicity, such as use in improving cell transfection efficiency and / or reducing cytotoxicity in vitro.

[0094] The sixth aspect of the present invention provides the use of the compound described in the first aspect or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer in improving the targeting of nucleic acid drugs to organs and / or cells, and / or in increasing the expression level of nucleic acids in target organs and / or target cells.

[0095] According to a preferred embodiment of the present invention, the nucleic acid drug comprises mRNA.

[0096] According to a preferred embodiment of the present invention, the (exogenous) nucleic acid is mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0098] Figure 1 The cell transfection experimental results of LNP preparations of Fluc-mRNA prepared based on YK-908, YK-909, YK-910, YK-912, SM-102 and compound 7 are shown, wherein: 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.

[0099] Figure 2 The fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared from different cationic lipids is shown.

[0100] Figure 3 The cell survival rate after 24 h of culture in cell culture medium was shown for Fluc-mRNA LNP preparations prepared with different cationic lipids.

[0101] Figure 4 Fluorescence images of mouse organs (heart, liver, spleen, lung, kidney, lymph) of LNP preparations of Fluc-mRNA prepared based on SM-102, YK-506, YK-908, YK-909, YK-910, and YK-912 are shown.

[0102] Figure 5 The fluorescence images of the LNP preparations of Cy5 fluorescently labeled Leqvio prepared based on YK-908, YK-909, YK-910, and YK-912, as well as the mouse organs (heart, liver, spleen, lung, kidney, and lymph) directly injected with Cy5 fluorescently labeled Leqvio are shown. DETAILED DESCRIPTION

[0103] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0104] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0105] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0106] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0107] Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0108] Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the present invention is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

[0109] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes (comprising)" used herein may be open, semi-closed and closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0110] The term "pharmaceutically acceptable" in the present invention means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the formulation and / or with humans or mammals for preventing or treating diseases or disorders.

[0111] The term "subject" or "patient" in the present invention includes humans and mammals.

[0112] The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, mitigate, improve or affect the disease or the symptoms of the disease. In the context of the present invention, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0113] The term "solvate" in the present invention refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield a compound of formula (I) once absorbed into a subject, such that the use of a compound of formula (I) encompasses the use of any solvate of a compound of formula (I), respectively.

[0114] The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water.

[0115] It should be further understood that the compound of formula I or its pharmaceutically acceptable salt can be isolated in the form of a solvate, and therefore any such solvate is included within the scope of the present invention. For example, the compound of formula I or its pharmaceutically acceptable salt can exist in an unsolvated form and in a solvated form formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0116] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the invention. For example, see SM Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, Pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, 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, glycerophosphoric acid, 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 shown in Formula I.

[0117] The nitrogen-containing compound of formula (I) of the present invention can be converted into N-oxide by treatment with an oxidant (e.g., meta-chloroperbenzoic acid, hydrogen peroxide, ozone). Therefore, under conditions where valence and structure permit, the compounds claimed in the present invention include not only the nitrogen-containing compound shown in the structural formula, but also its N-oxide derivatives.

[0118] Certain compounds of the present invention may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Therefore, the compounds claimed in the present invention also include racemic mixtures, single stereoisomers and optically active mixtures. It should be understood by those skilled in the art that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolution reagents such as chiral tartaric acid and 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 different solubility, can be used for separation.

[0119] The present invention also includes all suitable isotopic variations of the compounds of the present invention. An isotopic variation is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen and oxygen, for example 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 17 O and 18 O.

[0120] The term "alkyl" in the present invention refers to a branched and straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in the present invention refers to a branched and straight chain saturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. n-m It refers to a group having n to m carbon atoms. 2-5 Alkylene includes C 2 Alkylene, C 3 Alkylene, C 4 Alkylene, C 5 Alkylene.

[0121] The alkyl group (or alkylene group) may be unsubstituted, or the alkyl group (or alkylene group) may be substituted, wherein at least one hydrogen is replaced by another chemical group. Unless otherwise specified or defined, the "alkyl group" (or "alkylene group") in the present invention includes unsubstituted and substituted alkyl groups (or alkylene groups).

[0122] A "therapeutically effective amount" is an amount of a therapeutic agent that ameliorates a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" varies with the therapeutic agent / prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient / subject to be treated / prevented. A person of ordinary skill in the art can routinely determine a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present invention.

[0123] In the present invention, when the name of a compound is inconsistent with its structural formula, the structural formula shall prevail.

[0124] It should be understood that the term "compounds of the present invention" used in the present invention may include, depending on the context, compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.

[0125] As used herein, the term cationic lipid refers to a lipid that has a positive charge at a selected pH value.

[0126] Cationic liposomes easily bind to negatively charged nucleic acids, that is, they interact with negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery vectors.

[0127] When screening a large number of compounds, the inventors found that it was very difficult to screen out suitable cationic lipid compounds that met the following conditions: a huge difference in structure from representative cationic lipids in the prior art, extremely high transfection efficiency and extremely low cytotoxicity, and high and sustained expression in mice. The inventors found that some compounds, such as YK-908, YK-909, YK-910 and YK-912, etc., can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression and duration in animals compared to cationic lipids with very different chemical structures in the prior art.

[0128] Briefly, the present invention is based on at least the following findings:

[0129] The cationic lipid compound 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 compound of the present invention exhibits higher transfection efficiency and less cytotoxicity, and significantly increases the expression level in the spleen of animals, thereby improving the delivery efficiency and having important clinical significance.

[0130] 1. A series of cationic lipid compounds were designed, including YK-908, YK-909, YK-910, and YK-912. Compared with the representative cationic lipids in the prior art, some of them have huge differences in chemical structure, such as SM-102, MC3 (Dlin-MC3-DMA), YK-501, YK-506, and HHMA; some of them have similar chemical structures, such as compounds 7 and I-1.

[0131] SM-102 is a cationic lipid compound disclosed by Moderna, Inc. in WO20170409245A2 (page 29 of the specification).

[0132] MC3 is a cationic lipid compound disclosed by Alnylam Pharmaceuticals, Inc. in CN102625696B (page 6 of the specification).

[0133] YK-501 and YK-506 are cationic lipid compounds disclosed by Beijing Yuekang Kechuang Pharmaceutical Technology Co., Ltd. in CN116082275B (page 2 of the specification).

[0134] HHMA is a cationic lipid compound disclosed by Suzhou Abbott Biotechnology Co., Ltd. in CN112979483B (page 7 of the specification).

[0135] Compound 7 is a cationic lipid compound disclosed by Suzhou Abbott Biotechnology Co., Ltd. in CN114206827A (page 45 of the specification).

[0136] I-1 is a cationic lipid compound disclosed by Aikangtaisheng Therapeutics in CN110799492A (page 19 of the specification).

[0137] The chemical structures of representative cationic lipids and structurally similar cationic lipids in the prior art are as follows:

[0138] (WO2017049245A2, specification page 29);

[0139]

[0140] (CN116082275B, specification page 2);

[0141] (CN112979483B, instruction manual page 12)

[0142] (CN102625696B, page 6 of the specification);

[0143]

[0144] (CN114206827A, specification page 45);

[0145] (CN110799492A, specification page 19);

[0146] 2. In this series of compounds designed by the present invention, the LNP preparations prepared from YK-908, YK-909, YK-910, and YK-912 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased distribution of mRNA and small nucleic acids in mouse spleen compared with representative and structurally similar cationic lipids in the prior art. For example, the cell transfection efficiency of YK-910 can reach 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 survival rate of YK-910 is 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 28% higher than that of Lipofectamine 3000. 3000 was 63% higher; 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; 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, while the distribution level of small nucleic acids in the spleen was 51.19 times that of SM-102, 11.91 times that of YK-501 and 2.85 times that of YK-506. Although the fluorescence intensity of YK-912 in the spleen and liver was 1.25 times and 10.82 times that of SM-102 respectively, its distribution level in the kidney was significantly reduced, which was 0.14 times that of SM-102.

[0147] 3. Among the series of compounds with small differences in chemical structure designed by the present invention, the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 have 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 is significantly improved compared with direct administration. For example, when delivering mRNA, the cell transfection efficiency of YK-910 can reach 345.06 times that of YK-903 and 313.25 times that of YK-906, and the cytotoxicity can be reduced by 58% compared with YK-902; when delivering small nucleic acids, the fluorescence intensity of YK-908, YK-909, YK-910 and YK-912 in the spleen is 100.55 times, 126.85 times, 150.67 times and 31.80 times that of direct administration, respectively, and the distribution of YK-912 in the kidney is 0.09 times that of direct administration.

[0148] 4. Through unique design and screening, the present invention has discovered some compounds, such as YK-908, YK-909, YK-910 and YK-912, which, compared with other compounds with similar structures in the prior art, can significantly improve cell transfection efficiency, significantly reduce cytotoxicity and significantly increase expression levels in the spleen of animals, thereby improving delivery efficiency and achieving unexpected technical effects.

[0149] In summary, the present invention has discovered some compounds, such as YK-908, YK-909, YK-910 and YK-912, through unique design and screening. Compared with the representative cationic lipids in the prior art and with similar structures, these compounds have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression in the spleen of animals, thereby improving delivery efficiency.

[0150] The details are as follows:

[0151] 1. The compounds designed by the present invention, including YK-908, YK-909, YK-910 and YK-912, have chemical structures that are quite different from those of representative cationic lipids in the prior art, such as SM-102, MC3, YK-501, YK-506 and HHMA; and some have relatively small differences, such as compounds 7 and I-1.

[0152] Representative cationic lipids of the prior art, such as SM-102, MC3, YK-501, YK-506, HHMA, compound 7 and I-1, are compared with this series of designed compounds:

[0153] 1) The chemical structures of SM-102, MC3, YK-501, YK-506 and HHMA are significantly different from those of the compounds designed in the present invention. As can be seen from the chemical structure, the series of compounds of the present invention introduce a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure, which is connected to the lipid or the straight-chain alkyl or the branched-chain alkyl containing ester or amide groups at the head of the ethyl tertiary amine through the amino group, while SM-102, MC3, YK-501, YK-506 and HHMA do not have a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure; the amino head structure of SM-102 is a simple ethanolamine structure, the amino head structure of MC3 is a dimethyl tertiary amine structure without a hydroxyl structure, the amino head structures of YK-501 and YK-506 are piperazine groups, the amino head structure of HHMA is a methyl tertiary amine structure, and the hydroxyl group is located at the 2-position of the fatty chain, and the other groups are also quite different.

[0154] 2) Compounds 7 and I-1 have chemical structures similar to the compounds designed in the present invention, both of which contain a benzene ring structure.

[0155] 2. The in vitro cell transfection efficiency is significantly improved compared with the representative cationic lipids and structurally similar compounds in the prior art.

[0156] 1) Among the designed series of compounds, the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 have the highest cell transfection efficiency, and compared with the representative cationic lipids in the prior art (such as SM-102, MC3, YK-501, YK-506, HHMA, compound 7 and I-1), the cell transfection efficiency is significantly improved. For example, the cell transfection efficiency of YK-910 can reach 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.

[0157] 2) Similar structures, X 1 =CH, R 1 =C 6-25 Straight chain, branched chain alkyl, R 2 H, M 1 =-C(O)O-, M 2 H, G 1 =-CH 2 -, G 2 =C 1-4 Alkylene, G 3 =-H, such as YK-901, YK-902, YK-903, YK-904, YK-905 and YK-911, were compared with YK-912. The structural differences of these compounds are only slightly different in individual groups (Table 1). Cell transfection results show (Tables 4 and Figure 2 ), the activity of this series of compounds varies greatly, among which the cell transfection efficiency of YK-912 can reach 131.47 times that of YK-901, 18.35 times that of YK-902, 228.30 times that of YK-903, 45.65 times that of YK-904, 78.83 times that of YK-905, and 4.39 times that of YK-911, respectively, and the transfection efficiency is significantly improved.

[0158] 3) Similar structures, X 1 =CH, R 1 =C 6-25 Straight chain, branched chain alkyl, R 2 H, M 1 =-C(O)NH- or -NHC(O)-, M 2 H, G 1 =-CH2 -, G 2 =C 1-4 Alkylene, G 3 =-H, such as YK-907, compared with YK-908. The cell transfection efficiency of YK-908 was significantly improved, reaching 134.49 times that of YK-907.

[0159] 4) Similar to the structure, X 1 =N、R 1 =C 10-11 Straight chain alkyl, R 2 =C 15 Branched alkyl or C 18 Branched alkyl, M 1 =-C(O)O-, M 2 =-C(O)O-, G 1 =C 2 Alkylene, G 2 =C 3-4 Alkylene, G 3 =C 5-6 Compared with the alkylene compound YK-906, the cell transfection efficiency of YK-909 and YK-910 was significantly improved, reaching 246.15 times and 313.25 times that of YK-906, respectively.

[0160] 3. The cytotoxicity is significantly lower than that of representative cationic lipids and structurally similar compounds in the prior art.

[0161] 1) The chemical structures of this series of compounds designed by the present invention, including YK-908, YK-909, YK-910, and YK-912, are greatly different from those of representative cationic lipids in the prior art, such as SM-102, MC3, and HHMA, and some are less different, such as compound 7 and I-1. The LNP preparations prepared by YK-909 and YK-910 have the lowest cytotoxicity, and the cell survival rate is significantly improved compared with the representative cationic lipids in the prior art. For example, the cell survival rate of YK-910 is 12% higher than SM-102, 31% higher than YK-501, 17% higher than HHMA, 20% higher than MC3, 21% higher than compound 7, 26% higher than I-1, and 63% higher than Lipofectamine 3000. It is impossible to infer the cytotoxicity of the LNP preparations prepared by the cationic lipid compounds based on their structures. Whether they are compounds with huge structural differences or compounds with similar structures, the toxicity to transfected cells is very likely to be very different.

[0162] 2) Similar structures, X 1 =CH, R 1 =C 6-25 Straight chain, branched chain alkyl, R2 H, M 1 =-C(O)O-, M 2 H, G 1 =-CH 2 -, G 2 =C 1-4 Alkylene, G 3 A series of compounds with =-H, such as YK-901, YK-902, YK-903, YK-904, YK-905 and YK-1411, were compared with YK-912. The structural difference between these compounds is only that the individual groups are slightly different. The cytotoxicity of this series of compounds varies greatly. Among them, YK-912 has the highest cell survival rate, which is 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, and the cell survival rate is significantly improved.

[0163] 3) Similar structures, X 1 =CH, R 1 =C 6-25 Straight chain, branched chain alkyl, R 2 H, M 1 =-C(O)NH- or -NHC(O)-, M 2 H, G 1 =-CH 2 -, G 2 =C 1-4 Alkylene, G 3 A series of compounds with =-H, such as YK-907, were compared with YK-908. The cell survival rate of YK-908 was significantly improved, which was 16% higher than that of YK-907.

[0164] 4) Similar to the structure, X 1 =N、R 1 =C 10-11 Straight chain alkyl, R 2 =C 15 Branched alkyl or C 18 Branched alkyl, M 1 =-C(O)O-, M 2 =-C(O)O-, G 1 =C 2 Alkylene, G 2 =C 3-4 Alkylene, G 3 =C 5-6 Compared with the alkylene compound YK-906, the cell transfection efficiency of YK-909 and YK-910 was significantly improved, which was 29% and 31% higher than that of YK-906, respectively.

[0165] 4. The protein expression and duration of mRNA in mice are significantly higher than those of representative cationic lipids and compounds with similar structures in the prior art; the mRNA expression in animal spleen is significantly higher than that of representative cationic lipids in the prior art; the distribution of small nucleic acids in animal spleen is significantly higher than that of representative cationic lipids and direct administration in the prior art

[0166] 1) Compared with the representative cationic lipids in the prior art, the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 have the highest mRNA expression in mice, and the expression is sustained. The expression levels at 6h, 24h, 48h and 7d are significantly higher than those of the representative cationic lipids in the prior art. For example, the 24-hour data showed that the mRNA expression level of YK-910 in animals can reach 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.

[0167] 2) Compared with the compound YK-904, which has a similar structure but slightly different individual groups, the LNP preparations prepared from YK-908, YK-909, YK-910 and YK-912 have the highest mRNA expression intensity and the longest duration in mice. For example, YK-910 can reach 23.07 times that of YK-904 at 48 hours and 26.32 times at 7 days.

[0168] 3) The LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 have significantly increased mRNA expression in the spleen of mice compared to the representative cationic lipids in the prior art. For example, YK-908, YK-909, YK-910 and YK-912 are all expressed in large quantities in the spleen, which are 6.59 times, 8.00 times, 9.49 times and 6.86 times that of SM-102, respectively. The LNP preparations containing Fluc-mRNA prepared by all compounds have very large expression differences in different organs of mice. YK-908, YK-909, YK-910 and YK-912 are almost only expressed in the spleen, and are not expressed in other organs, such as the heart, lungs and kidneys, and are expressed in small amounts or not expressed in the liver; SM-102 is expressed in the liver and spleen, but not in the heart, lungs and kidneys.

[0169] 4) Compared with the representative cationic lipids in the prior art, the LNP preparations prepared by YK-908, YK-909 and YK-910 have significantly improved the distribution of small nucleic acid drugs in the mouse spleen. For example, the distribution of YK-908, YK-909 and YK-910 in the spleen has increased significantly, and the fluorescence intensity of YK-908, YK-909 and YK-910 in the spleen is 34.16 times, 43.10 times and 51.19 times that of SM-102, 7.95 times, 10.02 times and 11.91 times that of YK-501, and 1.90 times, 2.40 times and 2.85 times that of YK-506.

[0170] Surprisingly, although the fluorescence intensity of YK-912 in the spleen and liver was 1.25 times and 10.82 times that of SM-102, respectively, its amount in the kidney was significantly reduced, 0.14 times that of SM-102, indicating that it may have long-term effect in delivering small nucleic acid drugs. The LNP preparations containing Cy5 fluorescently labeled Leqvio prepared by all compounds have very different distributions in different organs of mice. For example, YK-908, YK-909, and YK-910 are expressed in the liver, spleen, and kidney, but not in other organs such as the heart, lungs, and lymph.

[0171] Compared with direct administration, the LNP preparations prepared by YK-908, YK-909 and YK-910 significantly improved the 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 was 100.55 times, 126.85 times and 150.67 times that of direct administration, respectively. The distribution of YK-912 in the kidney was 0.09 times that of direct administration, further showing that its delivery of small nucleic acid drugs may have long-term effects.

[0172] 5) Compared with representative cationic lipids in the prior art, such as SM-102 and compound 7, the liposomes prepared by the compounds designed by us have reduced protein expression in the liver, or do not stay in the liver and express the target protein. Therefore, compared with cationic lipids in the prior art, the LNP preparations prepared by the compounds designed by us have reduced or no toxicity to the liver.

[0173] The present invention provides a novel cationic lipid compound for delivering a therapeutic agent or a preventive agent. The cationic lipid compound 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 compound of the present invention exhibits higher transfection efficiency and less cytotoxicity, thereby improving delivery efficiency and safety.

[0174] On the one hand, the present invention provides a novel cationic lipid compound, which is a compound represented by formula (I)

[0175] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0176] G 1 C 1-4 Alkylene, preferably C 1 Alkylene or C 2 Alkylene, more preferably unsubstituted C 1 Alkylene or unsubstituted C 2 Alkylene;

[0177] G 2 C 1-8 Alkylene, preferably C 1-4 Alkylene, more preferably unsubstituted C 1 , C 3 , C 4 Alkylene;

[0178] G 3 C 2-8 Alkylene or H, preferably C 4-6 Alkylene or H, more preferably unsubstituted C 5 , C 6 Alkylene or H;

[0179] R 1 C 6-25 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

[0180]

[0181] R 2 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

[0182] R 3 C 1-25 Straight chain alkyl, C 1-25 Branched alkyl, C 1-25 Substituted alkyl or H, preferably unsubstituted C 4-15 Straight chain alkyl, more preferably C 8 Straight chain alkyl;

[0183] M 1is -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- and -NHC(O)-;

[0184] M 2 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-;

[0185] R' is C 1-18 Alkyl, C 2-18 alkenyl or H, preferably H;

[0186] X 1 It is -CH- or N.

[0187] In one embodiment, G 1 For unsubstituted C 1 Alkylene, for example, -CH 2 -.

[0188] In one embodiment, G 1 For unsubstituted C 2 Alkylene, for example, -(CH 2 ) 2 -.

[0189] In one embodiment, G 2 For unsubstituted C 1 Alkylene, for example, -CH 2 -.

[0190] In one embodiment, G 2 For unsubstituted C 3 Alkylene, for example, -(CH 2 ) 3 -.

[0191] In one embodiment, G 2 For unsubstituted C 4 Alkylene, for example, -(CH 2 ) 4 -.

[0192] In one embodiment, G 3 is H, for example, -H.

[0193] In one embodiment, G 3 For unsubstituted C 5 Alkylene, for example, -(CH 2 ) 5 -.

[0194] In one embodiment, G 3 For unsubstituted C 6 Alkylene, for example, -(CH 2 ) 6 -.

[0195] In one embodiment, R 1 For unsubstituted C 10 A straight chain alkyl group, i.e., -(CH 2 ) 9 CH 3 .

[0196] In one embodiment, R 1 For unsubstituted C 11 A straight chain alkyl group, i.e., -(CH 2 ) 10 CH 3 .

[0197] In one embodiment, R 1 For unsubstituted C 14 A straight chain alkyl group, i.e., -(CH 2 ) 13 CH 3 .

[0198] In one embodiment, R 1 For unsubstituted C 24 Branched alkyl. For example, R 1 for:

[0199] In one embodiment, R 1 For unsubstituted C 15 Branched alkyl. For example, R 1 for:

[0200] In one embodiment, R 1 For unsubstituted C 18 Branched alkyl. For example, R 1 for:

[0201] In one embodiment, R 1 For unsubstituted C 22Branched alkyl. For example, R 1 for:

[0202] In one embodiment, R 2 For H.

[0203] In one embodiment, R 2 For unsubstituted C 15 Branched alkyl. For example, R 2 for:

[0204] In one embodiment, R 2 For unsubstituted C 18 Branched alkyl. For example, R 2 for:

[0205] In one embodiment, R 3 For unsubstituted C 8 A straight chain alkyl group, i.e., -(CH 2 ) 7 CH 3 .

[0206] In one embodiment, M 1 is an ester bond. For example, M 1 Is: -C(O)O-.

[0207] In one embodiment, M 1 is an amide bond. For example, M 1 Is: -C(O)NH- or -NHC(O)-.

[0208] In one embodiment, M 2 For H.

[0209] In one embodiment, M 2 is an ester bond. For example, M 2 Is: -C(O)O-.

[0210] In one embodiment, X 1 It is -CH-.

[0211] In one embodiment, X 1 is N.

[0212] In an exemplary embodiment, the compound is selected from the following compounds or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof:

[0213]

[0214]

[0215] The second aspect of the present invention provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0216] In one embodiment, the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 10nm-300nm, preferably 90nm-260nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%, preferably ≤40%, more preferably ≤30%.

[0217] Cationic lipids

[0218] In one embodiment of the composition / carrier of the present invention, the cationic lipid is selected from one or more of the compounds of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above. For example, the cationic lipid is compound YK-908, YK-909, YK-910 or YK-912. In a preferred embodiment, the cationic lipid is compound YK-908, in a preferred embodiment, the cationic lipid is compound YK-909, in another preferred embodiment, the cationic lipid is compound YK-910, in another preferred embodiment, the cationic lipid is compound YK-912.

[0219] In another embodiment of the composition / carrier of the present invention, the cationic lipid comprises: (a) one or more selected from the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; (b) one or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound can be a commercially available cationic lipid, or a cationic lipid compound reported in the literature. For example, (b) The cationic lipid compound can be SM-102 in CN102625696B, or DLin-K-C2-DMA in CN102245590B, or Compound 1 in CN113039174A.

[0220] In one embodiment, the molar ratio of the cationic lipid to the carrier is 25%-75%, such as 30%, 40%, 50%, 55%, 60%, 65%, 70%.

[0221] The carrier can be used to deliver active ingredients such as therapeutic or prophylactic agents. The active ingredient can be encapsulated in the carrier or combined with the carrier.

[0222] For example, the therapeutic agent or preventive agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA and RNA. Suitable RNA includes, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0223] Neutral lipids

[0224] The carrier may include a neutral lipid. In the present invention, a neutral lipid refers to a lipid that is uncharged or exists in the form of a zwitterion at a selected pH value and plays an auxiliary role. The neutral lipid may adjust the fluidity of the nanoparticles to a lipid bilayer structure and improve efficiency by promoting lipid phase transition, and may also affect the specificity of the target organ.

[0225] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1: 1-15: 1, such as 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 a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 5: 1 (for example, it can be 4.5: 1, 4.6: 1, 4.7: 1, 4.8: 1, 4.9: 1, 5: 1, or it can be a range consisting of any two of the above ratios and any intermediate ratios therein).

[0226] For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

[0227] The carrier component of the composition comprising cationic lipids can include one or more neutral lipids-phospholipids, such as one or more (many) unsaturated lipids.Phospholipids can be assembled into one or more lipid bilayers.In general, phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0228] The neutral lipid part can be selected from the non-limiting group of the following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid part can be selected from the non-limiting group of the following composition: 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 encompassed are non-natural species including natural species with modification and replacement, the modification and replacement include branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., one or more double bonds are replaced by triple bond alkenyl) or cross-linked with the one or more alkynes. Under appropriate reaction conditions, alkynyl may undergo copper-catalyzed cycloaddition reactions when exposed to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cellular recognition, or to couple the composition to useful components such as targeting or imaging moieties (eg, dyes).

[0229] The neutral lipids useful in these compositions can be selected from the non-limiting group consisting of 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether). PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-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 16.0PE), 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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0230] In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0231] Structured lipids

[0232] The carrier of the composition comprising cationic lipids may further include one or more structural lipids. The structural lipids in the present invention refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0233] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.5:1-5:1, preferably 0.6:1-3:1, 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, 3:1.

[0234] The structured lipid can be selected from, but is not limited to, the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, alpha-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0235] Polymer-conjugated lipids

[0236] The carrier of the composition comprising cationic lipids can also include one or more polymer conjugated lipids. Polymer conjugated lipids mainly refer to lipids modified by polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNP, regulates nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

[0237] In one embodiment, the polymer conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. The molecular weight of PEG-modified PEG is generally 350-5000Da.

[0238] For example, the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0239] In one embodiment of the composition / vector of the present invention, the polymer-conjugated lipid is DMG-PEG2000.

[0240] In one embodiment of the composition / carrier of the present invention, the carrier comprises a neutral lipid, a structural lipid and a polymer conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is (25-75):(5-25):(15-65):(0.5-10), for example (30-49):(7.5-15):(35-55):(1-5). The total molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is 100.

[0241] In one embodiment of the composition / carrier of the present invention, the carrier comprises neutral lipids, structural lipids and polymer-conjugated lipids, and the molar ratio of the cationic lipids, the neutral lipids, the structural lipids and the polymer-conjugated lipids is 40:10:48.5:1.5 or 49:10:39.5:1.5, preferably 49:10:39.5:1.5.

[0242] Therapeutic and / or preventive agents

[0243] The composition may include one or more therapeutic and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1-30:1, such as 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, 25:1.

[0244] In one embodiment, the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5: 1-20: 1. For example, it can be 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, 20: 1, preferably 15: 1.

[0245] The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0246] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0247] The vectors of the present invention can deliver therapeutic agents and / or preventive agents to mammalian cells or organs. Therefore, the present invention also provides methods for treating diseases or disorders in mammals in need thereof, which methods include administering a composition including a therapeutic agent and / or preventive agent to the mammal and / or contacting mammalian cells with the composition.

[0248] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents". Therapeutic and / or prophylactic agents can be substances that, after delivery to a cell or organ, cause a desired change in the cell or organ or in other body tissues or systems. Such species can be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder or condition.Examples of drugs that can be used in the composition include, but are not limited to, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic 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

[0013] In some embodiments, the present invention includes but is not limited to: 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), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.

[0249] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracenedione, The invention relates to a radioactive ion, for example, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity for one or more conditions related to infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis, and can include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines can also include compounds and preparations that guide the immune response for cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes and / or new epitopes. Compounds that cause immune response can include vaccines, corticosteroids (e.g., dexamethasone) and other species. In some embodiments, by including compounds (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122) according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIg) or (III) intramuscular administration of a composition that can cause immune response vaccines and / or compounds.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, cyclosporine, cyclophosphamide ... The invention relates to antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol and maytansine.

[0250] In other embodiments, the therapeutic and / or prophylactic agent is a protein. The 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), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0251] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the present invention include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrid; RNAi inducing factor; RNAi factor; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamer, etc. In some embodiments, the therapeutic agent and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In certain embodiments, RNA is mRNA.

[0252] In certain embodiments, therapeutic and / or preventive agents are mRNA. mRNA can encode any polypeptide of interest, including any natural or non-natural polypeptide or otherwise modified polypeptide. The polypeptide encoded by mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in a cell.

[0253] In other embodiments, the therapeutic and / or preventive agent is siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the composition comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0254] In certain embodiments, the therapeutic and / or preventive agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect the mRNA translation of a cell gene.

[0255] In some embodiments, the therapeutic and / or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

[0256] Disease or condition

[0257] The composition / vector of the present invention can deliver a therapeutic agent or a preventive agent to a subject or patient. The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. Therefore, the composition of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. Due to the wide variety of the above-mentioned therapeutic agents or preventive agents, the composition of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0258] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0259] For example, the disease or disorder 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.

[0260] In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes.

[0261] Other components

[0262] The composition may include one or more components other than those described in the preceding section. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

[0263] The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0264] Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, , mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornasealfa, neltenexine and erdosteine) and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent can be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment or other methods).

[0265] The composition can also include one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that effectively promote membrane penetration, cell recognition or imaging in this way. 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 penetration are well known in the art.

[0266] In addition to these components, the composition can include any substance that can be used in a pharmaceutical composition. For example, the composition can include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0267] 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.

[0268] In some embodiments, a composition comprising one or more lipids described herein may also include 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.

[0269] The composition of the present invention can be made into a preparation in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The composition of the present invention can be prepared by methods well known in the pharmaceutical field. For example, a sterile injection solution can be prepared by mixing the required amount of therapeutic agent or preventive agent with the required various other ingredients mentioned above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

[0270] For example, the compositions of the invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.

[0271] The compositions of the present invention are administered in a therapeutically effective amount, which may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined by the attending physician or clinician. For example, the therapeutic or preventive agent may be administered to a subject (preferably a mammal, such as a human) at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0272] Example

[0273] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0274] The following abbreviations represent the following reagents:

[0275] 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

[0276] Example 1: Synthesis of cationic lipid compounds

[0277] 1. Synthesis of YK-901

[0278] The synthetic route is as follows:

[0279]

[0280] Step 1: Synthesis of 6-bromohexanoic acid-4-decyltetradecyl ester (YK-901-PM1)

[0281] 6-Bromohexanoic acid (280 mg, 1.44 mmol) and 4-decyltetradecane-1-ol (510 mg, 1.44 mmol) were dissolved in dichloromethane (10 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (413 mg, 3.92 mmol) and 4-dimethylaminopyridine (18 mg, 0.26 mmol) were added to the mixture in sequence. The reaction was stirred at room temperature overnight, and the solvent was removed under reduced pressure after the reaction was completed. The product was purified by silica gel flash chromatography (ethyl acetate / n-hexane) to obtain YK-901-PM1 (550 mg, 1.03 mmol, 71.8%).

[0282] Step 2: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)hexanoic acid-4-decyltetradecyl ester (YK-901)

[0283] 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), potassium carbonate (135 mg, 0.96 mmol) and potassium iodide (11 mg, 0.064 mmol) were added to the mixture, and the mixture was stirred at 70°C for 12 hours. After the reaction, the solid was filtered out, and 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 NO 4 ,MS(ES):m / z(M+H + )758.7. 1 H NMR (400 MHz, CDCl3 )δ7.13(s,4H),4.10-4.00(m,4H),3.93(d,J=12.5Hz,2H),3.04–2.96(m,2H),2.74-2.65(m,2H),2.63–2.54(m,2H),2.3 5(t,J=7.3Hz,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=6.6Hz,9H).

[0284] 2. Synthesis of YK-902

[0285] The synthetic route is as follows:

[0286]

[0287] Step 1: Synthesis of 4-decyltetradecyl 4-bromobutyrate (YK-902-PM1)

[0288] Using 4-bromobutyric acid (612 mg, 3.66 mmol) and 4-decyltetradecane-1-ol (1.3 g, 3.66 mmol) as raw materials, according to the synthesis method of YK-901-PM1, YK-902-PM1 (1.5 g, 2.98 mmol, 81.4%) was obtained.

[0289] Step 2: Synthesis of 4-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)butyric acid 4-decyltetradecyl ester (YK-902)

[0290] 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 synthetic method of YK-901. 47 H 87 NO 4 ,MS(ES):m / z(M+H + )730.6. 1 H NMR (400 MHz, CDCl 3)δ7.12(s,4H),4.13–4.03(m,2H),3.76(pd,J=11.9,6.8Hz,4H),3.52(s,4H),2.91(t,J=6.8Hz,2H),2.70–2.54(m,4H),2.50(q,J= 7.1Hz,2H),2.13–1.99(m,2H),1.82(dd,J=10.2,6.8Hz,2H),1.60(d,J=6.8Hz,4H),1.28(d,J=15.1Hz,46H),0.91(t,J=6.5Hz,9H).

[0291] 3. Synthesis of YK-903

[0292] The synthetic route is as follows:

[0293]

[0294] Synthesis of 4-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-amino)butyric acid decyl ester (YK-903)

[0295] Using decyl-4-bromobutyrate (200 mg, 0.65 mmol) and 2-amino-2-(4-octylphenylethyl) 1,3-propanediol (100 mg, 0.33 mmol) as raw materials, according to the synthetic method of YK-901, YK-903 (85 mg, 0.16 mmol, 48.2%) was obtained. 33 H 59 NO 4 ,MS(ES):534.45m / z(M+H + ). 1 H NMR (400 MHz, CDCl 3 )δ7.09(s,4H),4.04-4.10(m,2H),3.59-3.66(m,2H),2.69(t,J=6.8Hz,2H),2.56(d,J=32.0Hz,6H),2.43(t,J=7. 2Hz,2H),1.86(s,2H),1.68(d,J=17.2Hz,2H),1.59(d,J=27.2Hz,4H),1.43–1.15(m,25H),0.88(t,J=6.4Hz,6H).

[0296] 4. Synthesis of YK-904

[0297] The synthetic route is as follows:

[0298]

[0299] Step 1: Synthesis of 7-bromoheptanoic acid undecyl ester (YK-904-PM1)

[0300] Using 7-bromoheptanoic acid (728 mg, 3.48 mmol) and undecanol (500 mg, 2.90 mmol) as raw materials, according to the synthesis method of YK-901-PM1, YK-904-PM1 (900 mg, 2.48 mmol, 85.4%) was obtained.

[0301] Step 2: Synthesis of 7-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)heptanoic acid undecyl ester (YK-904)

[0302] 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 synthetic method of YK-901. 37 H 67 NO 4 ,MS(ES):m / z(M+H + )590.55. 1 H NMR (400 MHz, CDCl 3 )δ7.08(s,4H),4.05–4.02(m,4H),δ3.76–3.70(m,4H),2.72–2.70(m,2H),2.6 0–2.53(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).

[0303] 5. Synthesis of YK-905

[0304] The synthetic route is as follows:

[0305]

[0306] Step 1: Synthesis of 6-bromohexanoic acid undecyl ester (YK-905-PM1)

[0307] Using 6-bromohexanoic acid (3.00 g, 15.38 mmol) and undecanol (2.66 g, 15.44 mmol) as raw materials, according to the synthesis method of YK-901-PM1, YK-905-PM1 (3.20 g, 9.16 mmol, 59.6%) was obtained.

[0308] Step 2: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexanoic acid undecyl ester (YK-905)

[0309] 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 synthetic method of YK-901. 36 H 65 NO 4 ,MS(ES):m / z(M+H + )576.5. 1 H NMR (400 MHz, CDCl 3 )δ7.09(s,4H),4.05(t,J=6.8Hz,2H),3.67(s,3H),2.76–2.44(m,8H),2.31(t,J=6.4Hz,2H),1.79–1.51(m,10H),1.48–1.07(m,28H),0.95–0.83(m,6H).6. Synthesis of YK-906

[0310] The synthetic route is as follows:

[0311]

[0312] Step 1: Synthesis of 2-octyldecyl 6-((2-chloroethyl)(4-(decyloxy)-4-oxobutyl)amino)hexanoate (YK-906-PM1)

[0313] Dissolve YK-009 (600 mg, 0.92 mmol, see CN114044741B for the synthesis method) in dichloromethane (6 mL), cool the system to 0°C, add thionyl chloride (220 mg, 1.83 mml) dropwise at 0°C, warm to room temperature after the addition is complete, and react at room temperature for 3 hours. The system is decompressed and dried to obtain YK-906-PM1 (600 mg, 0.89 mmol, 97.3%).

[0314] Step 2: Synthesis of 2-octyldecyl-6-((4-(decyloxy)-4-oxobutyl)(2-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)ethyl)amino)hexanoate (YK-906)

[0315] 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), potassium carbonate (270 mg, 1.95 mmol) were added to acetonitrile (2 mL) and stirred at 80°C for 7 hours. The reaction system was cooled to room temperature, filtered, and the filtrate was distilled under reduced pressure. The residue was purified by silica gel flash chromatography (0-10% methanol / dichloromethane) to obtain YK-906 (200 mg, 0.21 mmol, 32.6%). 59 H 110 N 2 O 6 ,MS(ES):m / z(M+H + )943.8. 1 HNMR (400MHz, CDCl 3 )δ7.09(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=6.4Hz,12H).

[0316] 7. Synthesis of YK-907

[0317] The synthetic route is as follows:

[0318]

[0319] Step 1: Synthesis of 6-bromohexanoyl chloride (YK-907-PM1)

[0320] Dissolve 6-bromohexanoic acid (1.0 g, 5.13 mmol) in dichloromethane (3 mL), add oxalyl chloride (976 mg, 7.69 mmol) to the mixture under ice bath, stir and react overnight at room temperature, remove the solvent under reduced pressure after the reaction is completed, and obtain YK-907-PM1 (1.08 g, 5.06 mmol, 98.6%).

[0321] Step 2: Synthesis of 6-bromo-N-tetradecylhexanamide (YK-907-PM2)

[0322] YK-907-PM1 (1.08 g, 5.06 mmol) and tetradecylamine (1.09 g, 5.11 mmol) were dissolved in acetonitrile (8 mL), potassium carbonate (2.12 g, 15.30 mmol) was added to the mixture, and the mixture was stirred at room temperature for 4 hours. After the reaction, the solid was removed by suction filtration, the filtrate was dried under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain YK-907-PM2 (650 mg, 1.66 mmol, 32.9%).

[0323] Step 3: Synthesis of 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)-N-tetradecylhexanamide (YK-907)

[0324] 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 synthetic method of YK-901. 39 H 72 N 2 O 3 ,MS(ES):m / z(M+H + )617.6. 1 H NMR (400 MHz, CDCl 3 )δ7.13(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).

[0325] 8. Synthesis of YK-908

[0326] The synthetic route is as follows:

[0327]

[0328] Step 1: Synthesis of 6-bromohexylamine (YK-908-PM1)

[0329] Dissolve tert-butyl (6-bromohexyl)carbamate (500 mg, 1.78 mmol) in HCl / dioxane (4 M, 2 mL) and stir the mixture at room temperature overnight. After the reaction is complete, remove the solvent to obtain YK-908-PM1 (320 mg, 1.78 mmol, 100%).

[0330] Step 2: Synthesis of 2-hexyldecanoyl chloride (YK-908-PM2)

[0331] Dissolve 2-hexyldecanoic acid (456 mg, 1.78 mmol) in dichloromethane (2 mL), add oxalyl chloride (270 mg, 2.14 mmol) and N,N-dimethylformamide (0.05 mL) to the mixture in an ice bath, and stir at room temperature overnight. After the reaction is complete, remove the solvent under reduced pressure to obtain YK-908-PM2 (489 mg, 1.78 mmol, 100%).

[0332] Step 3: Synthesis of N-(6-bromohexyl)-2-hexyldecylamide (YK-908-PM3)

[0333] Using YK-908-PM1 (320 mg, 1.78 mmol) and YK-908-PM2 (489 mg, 1.78 mmol) as raw materials, according to the synthesis method of YK-907-PM2, YK-908-PM3 (60 mg, 0.14 mmol, 8.1%) was obtained.

[0334] Step 4: Synthesis of N-(6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)hexyl)2-hexyldecanamide (YK-908)

[0335] 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 synthetic method of YK-901. 41 H 76 N 2 O 3 ,MS(ES):m / z(M+H + )645.6. 1 H NMR (400 MHz, CDCl 3 )δ7.14(s,4H),4.04(dd,J=43.2,12.5Hz,4H),3.31(d,J=6.4Hz,2H),3.13(s,2H),2.82–2.65(m,2H) ,2.63–2.50(m,2H),2.04(dt,J=36.5,25.2Hz,5H),1.69–1.18(m,44H),0.90(dd,J=6.3,4.0Hz,9H).

[0336] 9. Synthesis of YK-909

[0337] The synthetic route is as follows:

[0338]

[0339] Step 1: Synthesis of 3-hexylnonyl 6-(2-chloroethyl)(4-(decyloxy)-4-oxobutyl)amino)hexanoate (YK-909-PM1)

[0340] Using 3-hexylnonyl 6-(4-(decacyclyl)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoate (YK-101, for the synthesis method, see CN116178193B) (200 mg, 0.33 mmol) as raw material, according to the synthesis method of YK-906-PM1, YK-909-PM1 (200 mg, 0.32 mmol, 96.1%) was obtained.

[0341] Step 2: Synthesis of 3-hexylnonyl 6-((4-(decyloxy)-4-oxobutyl)(2-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)ethyl)amino)hexanoate (YK-909)

[0342] 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 synthetic method of YK-906. 56 H 104 N 2 O 6 ,MS(ES):901.8m / z(M+H + ). 1 H NMR (400 MHz, CDCl 3 )δ7.09(s,4H),4.47(s,4H),4.02-4.14(m,7H),3.82-3.93(m,4H),3.05(d,J=28.0Hz,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).

[0343] 10. Synthesis of YK-910

[0344] The synthetic route is as follows:

[0345]

[0346] Step 1: Synthesis of 3-hexylnonyl-7-((2-chloroethyl)(4-oxo-4-(undecyloxy)butyl)amino)heptanoate (YK-910-PM1)

[0347] Using 3-hexylnonyl 7-((2-hydroxyethyl)(4-oxo-4-(undecanyloxy)butyl)amino)heptanoate (YK-108, for the synthesis method, see CN116178193B) (300 mg, 0.47 mmol) as raw material, according to the synthesis method of YK-906-PM1, YK-910-PM1 (300 mg, 0.46 mmol, 96.9%) was obtained.

[0348] Step 2: Synthesis of 3-hexylnonyl-7-((2-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)ethyl)(4-oxo-4-(undecanyloxy)butyl)amino)heptanoate (YK-910)

[0349] 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 synthetic method of YK-901. 58 H 108 N 2 O 6 ,MS(ES):m / z(M+H + )929.8. YK-910: 1 H NMR (400 MHz, CDCl 3 )δ7.08(s,4H),4.09–4.03(m,4H),δ3.83–3.71(m,4H),2.95–2.87(m,4H),2.6 4–2.53(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).

[0350] 11. Synthesis of YK-911

[0351] The synthetic route is as follows:

[0352]

[0353] Step 1: Synthesis of 2-octyldecyl-7-bromoheptanoate (YK-911-PM1)

[0354] Using 7-bromoheptanoic acid (255 mg, 1.22 mmol) and 2-octyldecanol (300 mg, 1.11 mmol) as raw materials, according to the synthesis method of YK-901-PM1, YK-911-PM1 (450 mg, 0.97 mmol, 87.8%) was obtained.

[0355] Step 2: Synthesis of 2-octyldecyl-7-(1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butyl-2-yl)amino)heptanoate (YK-911)

[0356] YK-911-PM1 (260 mg, 0.56 mmol) and 2-amino-2-(4-octylphenylethyl) 1,3-propanediol (174 mg, 0.57 mmol) were used as raw materials and YK-911 (100 mg, 0.15 mmol, 25.9%) was obtained according to the synthetic method of YK-901. 44 H 81 NO 4 ,MS(ES):688.6m / z(M+H + ). 1 H NMR (400 MHz, CDCl 3 )1H NMR(400MHz, CDCl3)δ7.08(s,4H),4.09-4.14(m,3H),3.95(d,J=8.0Hz,2H),3.71-3.79(m,5H),2.75(t,J=8.0Hz,2H),2.53-2.63(m,4H),2.29( t,J=8.0Hz,2H),2.04(s,5H),1.76-1.80(m,2H),1.65-1.71(m,2H),1.5 3-1.64(m,4H),1.37(s,3H),1.24-1.29(m,34H),0.87(t,J=8.0Hz,9H).

[0357] 12. Synthesis of YK-912

[0358] The synthetic route is as follows:

[0359]

[0360] Step 1: Synthesis of 2-octyldodecyl 6-bromohexanoate (YK-912-PM1)

[0361] Using 2-octyl-dodecanol (1.00 g, 3.35 mmol) and 6-bromohexanoic acid (0.78 g, 4.00 mmol) as starting materials, according to the synthesis method of YK-901-PM1, YK-912-PM1 (1.15 g, 2.42 mmol, 72.2%) was obtained.

[0362] Step 2: Synthesis of 2-octyldodecyl 6-((1-hydroxy-2-(hydroxymethyl)-4-(4-octylphenyl)butan-2-yl)amino)hexanoate (YK-912)

[0363] 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 synthetic method of YK-901. 45 H 83 NO 4 ,MS(ES):m / z(M+H + )702.6. 1 H NMR (400 MHz, CDCl 3 )δ7.09(s,4H),3.96(d,J=5.8Hz,2H),3.66–3.60(m,4H),2.65–2.53(m,6H),2.3 2(t,J=7.4Hz,2H),1.72–1.56(m,9H),1.44-1.25(m,44H),0.88(t,J=6.4Hz,9H).

[0364] 13. Synthesis of Compound 7

[0365] The synthetic route is as follows:

[0366]

[0367] Step 1: Synthesis of 2-(phenylamino)ethane-1-ol (Compound 7-PM1)

[0368] 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 single-mouth bottle (10 mL), and stirred at room temperature overnight under nitrogen protection. After the reaction was completed, 10 ml of purified water was added to quench the reaction, and the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel flash chromatography (ethyl acetate / n-hexane) to obtain compound 7-PM1 (430 mg, 3.13 mmol, 79.0%).

[0369] Step 2: Synthesis of bis(2-hexyldecyl)6,6'-((2-((2-hydroxyethyl)(phenyl)amino)ethyl)azadiyl)dihexanoic acid (Compound 7)

[0370] 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 the synthesis method of YK-906 was followed to obtain compound 7 (92 mg, 0.11 mmol, 44.7%). 54 H 100 N 2 O 5 ,MS(ES):m / z(M+H + )857.8. 1 H NMR (400 MHz, CDCl 3 )δ7.27–7.13(m,2H),6.73–6.43(m,3H),3.93–3.80(m,4H),3.70–3.58(m,5H),2.68(s,1H),2.4 2(t,J=7.4Hz,2H),2.34(s,2H),1.62–1.56(m,12H),1.41-1.28(m,56H),0.88(t,J=6.3Hz,12H).

[0371] 14. Synthesis of I-1

[0372] The synthetic route is as follows:

[0373]

[0374] Step 1: Synthesis of 2-(phenylamino)ethane-1-ol (I-1-PM1)

[0375] Dissolve 5-aminopentanoic acid (2.50 g, 21.3 mmol), benzyl alcohol (6.92 g, 64.0 mmol), p-toluenesulfonic acid monohydrate (4.45 g, 23.4 mmol) in toluene (90 mL), install a water separator and a condenser, and stir under heating and reflux to react overnight. After the reaction is completed, cool to room temperature and filter to obtain p-toluenesulfonate of compound 7-PM1 (5.95 g, 15.7 mmol, 73.6%).

[0376] Step 2: Synthesis of bis(2-hexyldecyl) 6,6'-((5-(benzyloxy)-5-oxopentyl)azadiyl) dihexanoate (I-1)

[0377] Using the p-toluenesulfonate of I-1-PM1 (1.50 g, 3.95 mmol) and 2-hexyldecyl 6-bromohexanoic acid (2.65 g, 6.32 mmol) as raw materials, according to the synthesis method of YK-906, I-1 (797 mg, 0.90 mmol, 22.8%) was obtained. 56 H 101 NO6 ,MS(ES):m / z(M+H + )884.8. 1 H NMR (400 MHz, CDCl 3 )δ7.40–7.33(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=6.5Hz,12H).

[0378] 15. Synthesis of YK-501

[0379] The synthesis was carried out according to the synthesis method of YK-501 in Example 1 of CN116082275B.

[0380] 16. Synthesis of YK-506

[0381] The synthesis was carried out according to the synthesis method of YK-506 in Example 1 of CN116082275B.

[0382] Example 2: Optimization of preparation conditions of nanolipid particles (LNP preparation)

[0383] 1. Optimization of the ratio of carrier (liposome) to mRNA

[0384] The cationic lipid compounds YK-908, YK-909, YK-910 and YK-912 synthesized in Example 1 were dissolved in ethanol at a molar ratio of 49:10:39.5:1.5 with DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG2000 to prepare an ethanol lipid solution. The ethanol lipid solution was quickly added to a citrate buffer (pH = 4-5) by an ethanol injection method and vortexed for 30 seconds for standby use. eGFP-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in a citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. A certain volume of liposome solution and mRNA aqueous solution were prepared with a total lipid to mRNA weight ratio of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1 to prepare liposomes. Ultrasound at 25°C for 15min (ultrasonic frequency 40kHz, ultrasonic power 800W). The obtained liposomes were diluted to 10 times the volume with PBS, and then ultrafiltered to remove ethanol with a 300KDa ultrafiltration tube. The volume was then fixed to a certain volume with PBS to obtain an LNP preparation encapsulating eGFP-mRNA using cationic lipid YK-908 (or YK-909, YK-910, YK-912) / DSPC / cholesterol / DMG-PEG2000 (molar ratio of 49:10:39.5:1.5).

[0385] The results of cell transfection experiments showed that the weight ratio of vector to mRNA in the range of 10:1-30:1 had good transfection effects, with the best transfection effect at 15:1. The transfection effects at ratios of 5:1 and 35:1 were poor, and these ratios should not be used to transport mRNA.

[0386] 2. Optimization of the ratio of cationic lipids to neutral lipids

[0387] The LNP preparation encapsulating eGFP-mRNA was prepared according to the method in 1, wherein the molar ratios of cationic lipid YK-908 (or YK-909, YK-910, YK-912) and neutral lipid DSPC were 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

[0388] It can be seen from the cell transfection experiment that the molar ratio of cationic lipid to neutral lipid is 1:1-15:1, and all have transfection effect, among which the highest transfection efficiency is 4.9:1.

[0389] 3. Optimization of the ratio of polymer-conjugated lipids to carrier (liposome)

[0390] According to the method in 1, LNP preparations encapsulating eGFP-mRNA were prepared, wherein the cationic lipid in the carrier was YK-908 (or YK-909, YK-910, YK-912), and the molar ratio of the polymer conjugated lipid DMG-PEG2000 to the carrier was 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15%, respectively.

[0391] The results of cell transfection experiments showed that the polymer conjugated lipid had a transfection effect when the molar ratio of the carrier was in the range of 0.5%-10%, the transfection efficiency was the highest when it was 1.5%, and the lowest when it was 10%.

[0392] 4. Optimization of the ratio of each component in the carrier (liposome)

[0393] The LNP preparation encapsulating eGFP-mRNA was prepared according to the method in 1, wherein the molar ratios of cationic lipid YK-908 (or YK-909, YK-910, 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.

[0394] Cell transfection experiments show that cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids can be transfected at molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5, and have good transfection effects within the range of (35-49):(7.5-15):(35-55):(1-5), with the best transfection effect achieved at a molar ratio of 49:10:39.5:1.5.

[0395] Example 3: Cell transfection experiment of LNP preparation of eGFP-mRNA

[0396] Cell recovery and passaging: Recover 293T cells and culture them in culture dishes to the required cell number.

[0397] Seeding: Digest and count the cells in the culture dish, seed 10,000 cells per well in a 96-well plate, or 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere.

[0398] Cell transfection experiment: LNP preparations containing 1.5 μg of eGFP-mRNA prepared in Example 2 (the cationic lipids in the carrier are YK-908, YK-909, YK-910, and YK-912) and Lipofectamine 3000 preparations of eGFP-mRNA were added to the cell culture medium of a 12-well plate, and cultured for 24 hours. The transfection efficiency of the samples was investigated by fluorescence intensity under a fluorescence microscope.

[0399] Based on the experimental results, the preparation conditions of nanolipid particles (LNP preparations) were finally determined: the ratio of carrier to mRNA was 15:1; the molar ratio of cationic lipids to neutral lipids was 4.9:1; the polymer conjugated lipids accounted for 1.5% of the liposomes; the molar ratio of cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids was 49:10:39.5:1.5. Subsequent experiments prepared nanolipid particles (LNP preparations) according to these conditions.

[0400] Example 4: Preparation of Nanolipid Particles (LNP Preparation) (Optimal Ratio)

[0401] Table 2 Cationic lipid structures

[0402]

[0403]

[0404]

[0405] The cationic lipids listed in Table 2 were dissolved in ethanol with DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) and DMG-PEG2000 at a molar ratio of 49:10:39.5:1.5 to prepare an ethanol lipid solution, and the ethanol lipid solution was quickly added to a citrate buffer (pH = 4-5) by an ethanol injection method, and vortexed for 30 seconds for use. eGFP-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (purchased from Shanghai Qifa Experimental Reagent Co., Ltd.) or Cy5 fluorescently labeled Leqvio (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.) was diluted in a citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. A certain volume of liposome solution and mRNA aqueous solution were mixed to prepare liposomes at a weight ratio of total lipid to mRNA of 15:1. Ultrasound was performed at 25°C for 15 min (ultrasonic frequency 40 kHz, ultrasonic power 800 W). The obtained liposomes were diluted to 10 times the volume with PBS, and then ultrafiltered to remove ethanol with a 300KDa ultrafiltration tube. Then, the volume was fixed to a certain volume with PBS to obtain an LNP preparation encapsulating eGFP-mRNA or Fluc-mRNA using cationic lipid / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 49:10:39.5:1.5).

[0406] Lipofectamine 3000 transfection reagent is currently widely used for cell transfection. It has very good transfection performance and excellent transfection efficiency, can improve cell activity, and is suitable for difficult-to-transfect cell types. We used Lipofectamine 3000 transfection reagent as a control and prepared Lipofectamine 3000 preparations of eGFP-mRNA or Fluc-mRNA according to the method in the instruction manual of Lipofectamine 3000 (Invitrogen (Shanghai) Trading Co., Ltd.).

[0407] Example 5: Determination of particle size and polydispersity index (PDI) of nanolipid particles

[0408] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer using dynamic light scattering.

[0409] Take 10 μL of liposome solution, dilute to 1 mL with RNase-free deionized water, add to the sample pool, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25°C. The test results are shown in Table 3:

[0410] Table 3 Particle size and polydispersity index (PDI) of nanolipid particles

[0411]

[0412]

[0413] As can be seen from Table 3, the particle size of the nanolipid particles prepared in Example 4 is between 110-165 nm, and can be used to deliver mRNA:

[0414] Among them, the particle size prepared by YK-904 is the smallest, which is 112nm; the particle size prepared by YK-901 is the largest, which is 162nm.

[0415] The polydispersity coefficients of all nanolipid particles were between 8% and 18%, with the smallest being 8.2% for YK-904 and the largest being 17.4% for YK-901.

[0416] The particle morphology prepared by YK-908, YK-909, YK-910, and YK-912 is also at a good level, with smaller particle size and better uniformity than particles prepared by other cationic lipids with similar structures in the prior art, such as SM-102, HHMA, MC3, compound 7, and I-1.

[0417] Example 6: In vitro validation of the performance of LNP delivery vehicles

[0418] Cell recovery and passage: The method is the same as in Example 3.

[0419] Seed board: The method is the same as Example 3.

[0420] 1. Fluorescence detection of Fluc-mRNA (transfection efficiency)

[0421] The LNP preparation containing 0.3 μg Fluc-mRNA (the LNP preparation carrier components are cationic lipids, DSPC, cholesterol and DMG-PEG2000, the molar ratio is 49:10:39.5:1.5, wherein the cationic lipids are cationic lipids listed in Table 1) was added to the cell culture medium of the 96-well plate, and the culture was continued for 24 hours. The corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence detection system. The chemical structures of the designed compounds and representative cationic lipids in the prior art are shown in Table 2. The transfection efficiency of the LNP preparations prepared by a series of cationic lipid compounds designed by the present invention and cationic lipids in the prior art, including SM-102, HHMA, MC3, compound 7 and I-1, is shown in Table 4.

[0422] Table 4 lists the fluorescence detection results of LNP preparations containing Fluc-mRNA prepared by different cationic lipids.

[0423] Table 4 Fluorescence detection results of Fluc-mRNA

[0424]

[0425] Experimental results analysis:

[0426] (1) The compounds designed in the present invention, including YK-908, YK-909, YK-910 and YK-912, have relatively large differences in chemical structure compared with the representative cationic lipids in the prior art, such as SM-102, MC3, YK-501, YK-506 and HHMA; some have relatively small differences, such as compounds 7 and I-1.

[0427] Compared with SM-102, MC3, YK-501, YK-506 and HHMA, the chemical structures of the compounds designed in the present invention are significantly different. As can be seen from the chemical structure, the series of compounds of the present invention introduce a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure, which is connected to the lipid or the straight-chain alkyl or the branched-chain alkyl containing ester or amide groups at the head of the ethyl tertiary amine through the amino group, while SM-102, MC3, YK-501, YK-506 and HHMA do not have a 2-amino-2-[2-(4-(alkyl)phenyl)ethyl]-1,3-propanediol structure; the amino head structure of SM-102 is a simple ethanolamine structure, the amino head structure of MC3 is a dimethyl tertiary amine structure without a hydroxyl structure, the amino head structures of YK-501 and YK-506 are piperazine groups, the amino head structure of HHMA is a methyl tertiary amine structure, and the hydroxyl group is located at the 2-position of the fatty chain, and the other groups are also quite different.

[0428] Compounds 7 and I-1, like the compound designed by the present invention, all have a benzene ring structure, and their chemical structures are slightly different.

[0429] (2) Among the designed series of compounds, the cell transfection efficiency of LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 was significantly improved compared with the representative cationic lipids in 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.

[0430] SM-102, HHMA and MC3 are representative cationic lipids in the prior art and have good transfection performance.

[0431] As shown in Table 4, the LNP preparations containing Fluc-mRNA prepared by YK-908, YK-909, YK-910 and YK-912 have the strongest fluorescence absorption, and the RLU values ​​are 5070083, 6780306, 8628595 and 5708988, respectively. Figure 1 and Figure 2 )

[0432] The cell transfection efficiency of YK-908 can reach 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 Lipofectamine3000, and the transfection efficiency is significantly improved.

[0433] The cell transfection efficiency of YK-909 can reach 4.75 times that of SM-102, 146.62 times that of YK-501, 10.36 times that of YK-506, 6.71 times that of HHMA, 71.44 times that of MC3, 26.33 times that of compound 7, 33.44 times that of I-1 and 8.05 times that of Lipofectamine3000, and the transfection efficiency is significantly improved.

[0434] The cell transfection efficiency of YK-910 can reach 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 Lipofectamine3000, and the transfection efficiency is significantly improved.

[0435] The cell transfection efficiency of YK-912 can reach 4.00 times that of SM-102, 123.46 times that of YK-501, 8.73 times that of YK-506, 5.65 times that of HHMA, 60.16 times that of MC3, 22.17 times that of compound 7, 28.16 times that of I-1 and 6.77 times that of Lipofectamine3000, and the transfection efficiency is significantly improved.

[0436] The cell transfection efficiency of YK-902 is 0.22 times that of SM-102, 6.73 times that of YK-501, 0.48 times that of YK-506, 0.31 times that of HHMA, 3.28 times that of MC3, 1.21 times that of compound 7, and 1.53 times that of I-1.

[0437] It can be seen that the cell transfection efficiency of the LNP preparation prepared from the cationic lipid compound cannot be inferred based on the structure of the compound. Whether the compounds have huge structural differences or similar structures, the cell transfection efficiency is likely to be very different.

[0438] (3) In M 1 Among a series of compounds with -C(O)O-, the LNP preparation prepared by YK-912 has significantly improved cell transfection efficiency compared with other compounds. For example, YK-912 can reach 228 times that of YK-903 and 131 times that of YK-901.

[0439] The structure is similar to M 1 A series of compounds with -C(O)O-, including YK-901, YK-902, YK-903, YK-904, YK-905, YK-911 and YK-912, were compared. The structural differences of these compounds were only slightly different in individual groups (Table 2). The cell transfection results showed that the activity of this series of compounds was very different, among which the LNP preparation prepared by YK-912 had the highest cell transfection efficiency, which was significantly improved compared with other compounds. YK-912 could reach 131.47 times that of YK-901, 18.35 times that of YK-902, 228.30 times that of YK-903, 45.65 times that of YK-904, 78.83 times that of YK-905 and 4.39 times that of YK-911, and the transfection efficiency was significantly improved.

[0440] (4) In M 1 Among the compounds with an amide bond (-C(O)NH- or -NHC(O)-), the LNP preparation prepared by YK-908 has a significantly improved cell transfection efficiency compared to YK-907, which can be up to 134 times that of YK-907.

[0441] The structure is similar to M 1 The compounds YK-907 and YK-908, which are -C(O)NH- or -NHC(O)-, were compared. It can be seen that the cell transfection efficiency of the LNP preparation prepared by YK-908 was significantly improved compared with that of YK-907, which can reach 134.49 times that of YK-907.

[0442] (5) In X 1 Among a series of compounds with N as N, the LNP preparations prepared by YK-909 and YK-910 have significantly improved cell transfection efficiency compared to YK-906, reaching 246 times and 313 times that of YK-906, respectively.

[0443] The structure is similar to X 1 A series of compounds containing N, including YK-906, YK-909 and YK-910, were compared. The results showed that the cell transfection efficiency of LNP preparations prepared by YK-909 and YK-910 was significantly improved compared with that of YK-906, reaching 246.15 times and 313.25 times that of YK-906, respectively.

[0444] It can be seen from this that the cell transfection activity of LNP preparations prepared from cationic lipid compounds cannot be inferred based on their structure. Even if a series of cationic lipid compounds are very similar in structure, the cell transfection efficiency is likely to be very different.

[0445] 2. Cell Viability Determination

[0446] A LNP preparation containing 1.5 μg Fluc-mRNA (the LNP preparation carrier components are cationic lipids, DSPC, cholesterol and DMG-PEG2000, with a molar ratio of 49:10:39.5:1.5, wherein the cationic lipids are the cationic lipids listed in Table 1) was added to the cell culture medium of a 96-well plate, and cultured for 24 hours. Then, 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in an incubator for 1 hour. The absorbance at 450 nm was measured by a microplate reader, and the cell viability results are shown in Table 5.

[0447] Table 5 Cell viability

[0448] Serial number Cationic lipids Cell survival rate (%) 1 YK-901 58 2 YK-902 36 3 YK-903 47 4 YK-904 52 5 YK-905 51 6 YK-906 63 7 YK-907 73 8 YK-908 89 9 YK-909 92 10 YK-910 94 11 YK-911 62 12 YK-912 87 13 SM-102 82 14 YK-501 63 15 YK-506 90 16 HHMA 77 17 MC3 74 18 Compound 7 73 19 I-1 68 20 Lipofectamine 3000 31

[0449] Experimental results analysis:

[0450] (1) The chemical structures of the series of compounds designed by the present invention, including YK-908, YK-909, YK-910, and YK-912, are significantly different from those of the representative cationic lipids in the prior art, such as SM-102, MC3, YK-501, YK-506, and HHMA, while the differences are relatively small, such as compounds 7 and I-1.

[0451] The experimental results show that the LNP preparations prepared by YK-909 and YK-910 have the lowest cytotoxicity and significantly improve the cell survival rate compared with the representative cationic lipids in the prior art. For example, the cell survival rate of YK-910 is 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 )

[0452] (2) Structures similar to M 1 A series of compounds containing -C(O)O-, including YK-901, YK-902, YK-903, YK-904, YK-905, YK-1411 and YK-912, were compared. The structural differences among these compounds are only slight differences in individual groups.

[0453] The experimental results show that the cytotoxicity of this series of compounds varies greatly, among which YK-912 has the highest cell survival rate, which is 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, and the cell survival rate is significantly improved.

[0454] (3) Structures similar to M 1 Compounds YK-907 and YK-908, which are amide bonds (-C(O)NH- or -NHC(O)-), were compared. The results showed that the cell survival rate of YK-908 was significantly improved, which was 16% higher than that of YK-907.

[0455] (4) Similar structures, X 1 A series of compounds containing N, including YK-906, YK-909 and YK-910, were compared. The results showed that the cell survival rates of YK-909 and YK-910 were significantly higher than those of YK-906, which were increased by 29% and 31% respectively.

[0456] It can be seen from this that the cytotoxicity of LNP preparations prepared from cationic lipid compounds cannot be inferred based on their structure. Whether the compounds have huge structural differences or are structurally similar, their toxicity to transfected cells is very likely to be very different.

[0457] Example 7: In vivo validation of the performance of cationic lipid (LNP) delivery vehicles

[0458] This example verifies the expression and duration of the mRNA delivered by the designed cationic lipid in mice. In vivo experiments further demonstrate that the LNP delivery vector of the present invention, compared with the representative cationic lipids in the prior art: (1) can effectively deliver mRNA into animals and express it efficiently and continuously; (2) the delivered mRNA can be highly expressed in the spleen and lowly expressed in the liver; (3) the delivered small nucleic acid drug is highly expressed in the spleen.

[0459] 1. Expression of delivered mRNA in mice

[0460] The LNP preparation containing 10 μg Fluc-mRNA was injected into female BALB / C mice of 4-6 weeks old and weighing 17-19 g via the tail vein, and the mice were intraperitoneally injected with fluorescent imaging substrate at specific time points (6h, 24h, 48h and 7d) after administration. The mice moved freely for 5 minutes, and then the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by LNP in the mouse was detected by IVIS Spectrum small animal in vivo imaging. The average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by LNP in the mouse was detected by IVIS Spectrum small animal in vivo imaging, and the results of mouse in vivo imaging detection are shown in Table 6.

[0461] Table 6 Mouse in vivo imaging experimental data

[0462]

[0463] Experimental results analysis:

[0464] (1) The delivered mRNA of the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 was highly expressed and continuously expressed in mice, and the mRNA expression levels were significantly increased at 6h, 24h, 48h and 7d compared with the representative cationic lipids in the prior art, such as SM-102, YK-501, YK-506 and compound 7. For example, YK-910 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 at 24h, and was 9.24 times that of SM-102, 15.15 times that of YK-501, 2.49 times that of YK-506 and 10.03 times that of compound 7 at 7d.

[0465] (2) Compared with the compound YK-904, which has a similar structure but slightly different individual groups, the LNP preparations prepared from YK-908, YK-909, YK-910 and YK-912 significantly increased the mRNA expression intensity and duration in mice. For example, YK-910 was 12.03 times, 18.49 times, 23.07 times and 26.32 times that of YK-904 at 6h, 24h, 48h and 7d, respectively.

[0466] It can be seen from this that even cationic lipid compounds with small structural differences are very likely to have very different mRNA expression in LNP preparations prepared from them. It is impossible to predict whether mRNA is highly expressed and continuously expressed in animals based on the chemical structure of cationic lipids. It is very difficult to screen cationic lipid compounds with extremely high and continuous mRNA expression, which requires a lot of creative work.

[0467] 2. Expression of delivered mRNA in different organs of mice

[0468] The liposomes prepared from the cationic lipid compounds of the present invention have a reduced amount of protein expressed in the liver, or do not stay in the liver and express the target protein, compared with representative cationic lipids in the prior art, such as SM-102, YK-501, YK-506 and compound 7. Therefore, compared with the cationic lipids in the prior art, the LNP preparations prepared from the compounds designed in the present invention have reduced or no toxicity to the liver.

[0469] The LNP preparation containing 5 μg Fluc-mRNA was subcutaneously injected into female BALB / C mice aged 4-6 weeks and weighing 17-19 g, and the mice were intraperitoneally injected with fluorescent imaging substrates at a specific time point (6 h) after administration. The mice were allowed to move freely for 5 min, and then the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by LNP in the mouse was detected by IVIS Spectrum small animal in vivo imaging. After sampling, the mice were killed by cervical dissection, and the mice were dissected to accurately separate the internal organs of the mice: heart, liver, spleen, lung, kidney and lymph. The average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by LNP in various organs of the mouse was detected by IVIS Spectrum small animal in vivo imaging, and the results of mouse in vivo imaging detection are shown in Table 7.

[0470] Table 7 Experimental data of organ imaging 6 hours after administration of Fluc-mRNA to mice

[0471]

[0472] Experimental results analysis:

[0473] Compared with the representative cationic lipids in the prior art, the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 all showed significantly higher mRNA expression in the mouse spleen. For example, YK-908, YK-909, YK-910 and YK-912 were all expressed in large quantities in the spleen, which were 6.59 times, 8.00 times, 9.49 times and 6.86 times that of SM-102, 11.83 times, 14.37 times, 17.04 times and 12.33 times that of YK-501, and 2.36 times, 2.87 times, 3.40 times and 2.46 times that of YK-506.

[0474] The expression levels of YK-908, YK-909, YK-910 and YK-912 in the liver were significantly lower than those of SM-102, and the expression levels of SM-102 in the liver were 17.12 times, 2.67 times and 2.13 times of those of YK-908, YK-909 and YK-910, respectively, while YK-912 was not expressed in the liver. Therefore, compared with the cationic lipids in the prior art, the LNP preparations prepared by the compounds designed in the present invention have reduced or no toxicity to the liver.

[0475] In addition, the expression of Fluc-mRNA-containing LNP preparations prepared by all compounds in different organs of mice was very different. YK-908, YK-909, YK-910 and YK-912 were almost only expressed in the spleen, but not in other organs such as heart, lung and kidney, and slightly or not expressed in the liver; SM-102 was expressed in the liver and spleen, but not in the heart, lung and kidney ( Figure 4 ).

[0476] 3. Expression of delivered small nucleic acid drugs in different organs of mice

[0477] The small nucleic acid drugs delivered by the delivery vectors prepared by the cationic lipids YK-908, YK-909, YK-910 and YK-912 designed by the present invention are significantly enriched in the spleen of mice, and renal excretion is significantly reduced. Compared with the cationic lipids of the prior art, the delivery of small nucleic acid drugs conjugated with Cy5 (Leqvio labeled with Cy5 fluorescence) significantly increased the fluorescence in the liver and spleen of mice, and significantly reduced the fluorescence in the kidneys. In vivo experiments further demonstrated that our LNP delivery vectors are also effective in delivering small nucleic acid drugs to animals, and produce efficient and long-lasting therapeutic effects in the spleen.

[0478] The LNP preparation containing 5 μg of Cy5 fluorescently labeled Leqvio was injected into female BALB / C mice of 4-6 weeks of age and weighing 17-19g via the tail vein, and the average radiation intensity (corresponding to the fluorescence intensity) of the Cy5 fluorescently labeled Leqvio carried by the LNP in the mouse body was detected by IVIS Spectrum small animal in vivo imager at a specific time point (4h) after administration. After the sampling was completed, the mice were killed by cervical dissection, and the internal organs of the mice were accurately separated: heart, liver, spleen, lung, kidney and lymph. The average radiation intensity (corresponding to the fluorescence intensity) of the Cy5 fluorescently labeled Leqvio carried by the LNP in each organ of the mouse was detected by IVIS Spectrum small animal in vivo imager, and the results of the mouse organ imaging test are shown in Table 8.

[0479] Table 8 Experimental data of organ imaging 4 hours after administration of Cy5 fluorescently labeled Leqvio to mice

[0480]

[0481] Experimental results analysis:

[0482] (1) Compared with the representative cationic lipids in the prior art, the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912 significantly improved the 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 was 34.16 times, 43.10 times and 51.19 times that of SM-102, and 7.95 times, 10.02 times and 11.91 times that of YK-501 ( Figure 5 )

[0483] Surprisingly, the renal distribution of YK-912 was significantly lower, 0.14 times that of SM-102, indicating its potential long-term efficacy in delivering small nucleic acid drugs.

[0484] (2) Compared with direct administration, the distribution of small nucleic acid drugs in the mouse spleen was significantly improved in the LNP preparations prepared by YK-908, YK-909, YK-910 and YK-912, and the fluorescence intensity in the spleen was 100.55 times, 126.85 times, 150.67 times and 31.86 times that of direct administration, respectively.

[0485] Moreover, the amount of YK-912 distributed in the kidneys was significantly lower than that of direct administration, which was only 0.09 times that of direct administration, indicating that it may have long-term effectiveness in delivering small nucleic acid drugs.

[0486] In summary, the present invention designs a series of cationic lipid compounds, for example, YK-908, YK-909, YK-910, and YK-912, which significantly improve cell transfection efficiency, significantly reduce cytotoxicity, significantly increase the expression levels of mRNA and small nucleic acids in mouse spleen, and significantly reduce renal excretion when YK-912 delivers small nucleic acids.

[0487] 1. A series of designed compounds, including YK-908, YK-909, YK-910, and YK-912, have chemical structures that are very different from those of representative cationic lipids in the prior art. Some of them, such as SM-102, MC3, YK-501, YK-506, and HHMA, have chemical structures that are slightly different, such as compounds 7 and I-1.

[0488] 2. Among this series of designed compounds, the LNP preparations prepared from YK-908, YK-909, YK-910, and YK-912 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased mRNA and small nucleic acid expression in mouse spleen compared with representative and structurally similar cationic lipids in the prior art. The renal excretion of YK-912 when delivering small nucleic acids is significantly reduced.

[0489] For example, the cell transfection efficiency of YK-910 can reach 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 survival rate of YK-910 is 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 28% higher than that of Lipofectamine 3000. 3000 was 63% higher; the 24-hour expression level of YK-910 mRNA in the animal body 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; 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, while the distribution level of small nucleic acids in the spleen 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 its distribution in the kidney was significantly reduced, which was 0.14 times that of SM-102.

[0490] 3. Among the series of compounds with small differences in chemical structures designed by the present invention, the LNP preparations prepared from YK-908, YK-909, YK-910 and YK-912 have significantly improved cell transfection efficiency and significantly reduced cytotoxicity compared with other compounds, and the distribution amount of small nucleic acids in the mouse spleen is significantly increased compared with direct administration.

[0491] For example, when delivering mRNA, the transfection efficiency of YK-910 cells can reach 345.06 times that of YK-903 and 313.25 times that of YK-906, and the cytotoxicity can be reduced by 58% compared with YK-902; when delivering small nucleic acids, the fluorescence intensities of YK-908, YK-909, YK-910 and YK-912 in the spleen are 100.55 times, 126.85 times, 150.67 times and 31.80 times that of direct administration, respectively, and the distribution amount of YK-912 in the kidney is 0.09 times that of direct administration.

[0492] 4. Through unique design and screening, the present invention has discovered some compounds, such as YK-908, YK-909, YK-910 and YK-910, which, compared with other compounds with similar structures in the prior art, can significantly improve cell transfection efficiency, significantly reduce cytotoxicity and significantly increase expression levels in the spleen of animals, thereby improving delivery efficiency and achieving unexpected technical effects.

[0493] The above is a detailed description of the present invention, the purpose of which is to enable those familiar with the technology in this field to understand the content of the present invention and implement it, and it is not intended to limit the scope of protection of the present invention. All equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound represented by formula (I) , or a pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1-4 Alkylene; G2 is C 1-8 Alkylene; G3 is C 2-8 Alkylene; R1 is C 6-25 Straight chain alkyl or C 6-25 Branched chain alkyl; R2 is C 6-25 Straight chain alkyl, C 6-25 Branched alkyl or H; R3 is C 1-25 Straight chain alkyl, C 1-25 Branched alkyl or H; M1 is -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)- or -CH(OH)-; M2 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; X1 is -CH- or N.

2. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is unsubstituted C1 alkylene or unsubstituted C2 alkylene.

3. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 is unsubstituted C1 alkylene, unsubstituted C3 alkylene or unsubstituted C4 alkylene.

4. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein G3 is unsubstituted C5 alkylene or unsubstituted C6 alkylene.

5. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is C 6-14 Straight chain alkyl or C 12-25 Branched chain alkyl.

6. The compound according to claim 1 or 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is: , , or .

7. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is C 6-14 Straight chain alkyl, C 12-25 Branched alkyl or H.

8. The compound according to claim 1 or 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is: or .

9. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3 is C 4-15 Straight chain alkyl.

10. The compound according to claim 9 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3 is a C8 straight chain alkyl group.

11. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein M1 is -C(O)O-, -C(O)NH- or -NHC(O)-.

12. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein M2 is -C(O)O-.

13. A compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound 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 structure: ， ， ， ， ， ， ， ， ， ， ， 。 14. The compound according to claim 13 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is at least one of compounds YK-908, YK-909, YK-910 and YK-912 having the following structure: ； ； ； 。 15. A composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt or stereoisomer thereof.

16. The composition of claim 15, wherein the molar ratio of the cationic lipid in the carrier is 25%-75%.

17. The composition of claim 15, wherein the carrier further comprises a neutral lipid.

18. The composition of claim 17, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:

1.

19. The composition of claim 18, wherein the molar ratio of the cationic lipid to the neutral lipid is 4.9:

1.

20. The composition of claim 17, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

21. The composition of claim 20, wherein the neutral lipid is selected from one or more of the following: 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-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2- Oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero oleyl-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, 1,2-dioleoyl-sn-glycero-3-phospho-rac -(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and mixtures thereof.

22. The composition of claim 21, wherein the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

23. The composition of claim 15, wherein the carrier further comprises a structured lipid.

24. The composition of claim 23, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:

1.

25. The composition of claim 23, wherein the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol and corticosteroids.

26. The composition of claim 25, wherein the structured lipid is cholesterol.

27. The composition of claim 15, wherein the carrier further comprises a polymer-conjugated lipid.

28. The composition of claim 27, wherein the molar ratio of the polymer-conjugated lipid in the carrier is 0.5%-10%.

29. The composition of claim 28, wherein the molar ratio of the polymer-conjugated lipid in the carrier is 1.5%.

30. The composition of claim 27, wherein the polymer-conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

31. The composition of claim 30, wherein the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecyl acetamide.

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

33. A composition according to claim 32, wherein 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).

34. The composition of claim 33, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 45:10:43.5:1.5 or 49:10:39.5:1.

5.

35. The composition according to claim 15, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm-300nm; and the polydispersity coefficient of the nanoparticle preparation is ≤50%.

36. The composition of claim 35, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 90nm-280nm; and the polydispersity coefficient of the nanoparticle preparation is ≤45%.

37. The composition of claim 15, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

38. The composition of claim 15, further comprising a therapeutic or prophylactic agent.

39. The composition of claim 38, wherein the mass ratio of the carrier to the therapeutic or preventive agent is 10:1-30:

1.

40. The composition of claim 39, wherein the mass ratio of the carrier to the therapeutic or preventive agent is 12.5:1-20:

1.

41. The composition of claim 40, wherein the mass ratio of the carrier to the therapeutic or preventive agent is 15:

1.

42. The composition of claim 38, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

43. The composition of claim 38, wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

44. The composition of claim 38, wherein the therapeutic or prophylactic agent is a nucleic acid.

45. The composition of claim 44, wherein the therapeutic or preventive agent is ribonucleic acid and / or deoxyribonucleic acid.

46. ​​The composition of claim 45, wherein the RNA is selected from the group consisting of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA, and mixtures thereof.

47. The composition of claim 46, wherein the RNA is messenger RNA.

48. The composition of claim 15, wherein the composition further comprises one or more of a pharmaceutically acceptable excipient or diluent.

49. Use of the compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 to 14 or the composition according to any one of claims 15 to 48 in the preparation of a nucleic acid drug or a gene vaccine drug.

50. Use of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1 to 14 or a composition according to any one of claims 15 to 48 in the preparation of a medicament for treating a disease or condition in a subject in need thereof, wherein the disease or condition is a metabolic disease.

51. The use according to claim 50, wherein the subject is a mammal.

52. The use according to claim 51, wherein the subject is a human.

53. The use according to any one of claims 49-52, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

54. The use according to claim 53, wherein the composition is administered subcutaneously.

55. The use of claim 50, wherein a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent is administered to the subject.

56. Use of the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt or stereoisomer thereof for improving cell transfection efficiency and / or reducing cytotoxicity in vitro.

57. Use of the compound or pharmaceutically acceptable salt or stereoisomer thereof as described in any one of claims 1 to 14 in improving the targeting of nucleic acid drugs to organs and / or cells, and / or in increasing the expression of nucleic acids in target organs and / or target cells, wherein the use is non-therapeutic.

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