Compounds, liposomes and drug carriers for drug delivery

Abstract

The present invention relates to a compound represented by formula (I) or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of a compound represented by formula (I), TIFF2026503201000033.tif3981X1, X2 and X3 are each independently an optionally substituted C1-C 15 alkylene, and R and R are each independently an optionally substituted C-C 40 Alkyl, optionally substituted C-C 40 Heteroalkyl, optionally substituted C-C 40 Alkenyl, optionally substituted C-C 40 Heteroalkenyl, optionally substituted C-C 40 Alkynyl or optionally substituted C-C 40 The present invention provides a compound comprising heteroalkynyl, wherein R3, R4, R5, and R6 are each independently H, halogen, or optionally substituted C1-C3 alkyl, and the substituents are independently selected from halogen, -OH, -SH, -NH2, -NO2, cyano, and C1-C3 alkyl, which has the advantages of low cytotoxicity, strong delivery ability, and strong immunostimulatory effect.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, specifically, the present invention relates to compounds, liposomes and drug carriers for drug delivery, more specifically, the present invention relates to compounds and uses thereof, liposomes, drug carriers, composites, pharmaceutical compositions and pharmaceutical uses thereof. [Background technology]

[0002] Gene therapy technology is a mainstream research area in modern biomedicine, and nucleic acid drugs (e.g., small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA)) can be used to prevent cancer, bacterial and viral infections, and treat genetic diseases. Because nucleic acid drugs are easily degraded and difficult to enter cells, they must be encapsulated in a carrier for delivery to target cells. Therefore, the development of safe and efficient delivery vehicles is a prerequisite for the clinical application of gene therapy.

[0003] Currently, the two most common nucleic acid delivery systems are viral vectors and non-viral vectors. While viral vectors have relatively high transfection efficiency, they suffer from issues such as safety and poor targeting. Non-viral vectors, such as liposomes, are rapidly developing and are considered ideal nucleic acid delivery systems due to their low immunogenicity, high biocompatibility, and high transfection efficiency. However, traditional lipid-based nucleic acid delivery systems suffer from issues such as low efficiency, high toxicity, and poor targeting.

[0004] Among non-viral vectors for nucleic acid delivery, LNPs have the most outstanding performance and have already been used in three approved nucleic acid drugs: Patisiran (an siRNA drug, product name ONPATTRO), BNT162b2, and mRNA-1273. LNPs typically consist of four components: ionizable lipids, neutral phospholipids, cholesterol, and polyethylene glycol-modified lipids. Ionizable lipids play a key role in LNPs, as mRNA loading, expression level, and targeting all depend on them. For example, the ionizable lipids used in Patisiran, BNT162b2, and mRNA-1273 are DLin-MC3-DMA (MC3), ALC-0315, and SM-102, respectively. The LNP delivery platform has attracted much attention in the pharmaceutical industry as a promising mRNA drug delivery vehicle and is also expanding into other research fields. However, the internal mechanism by which ionizable lipid LNPs efficiently deliver mRNA remains unclear. The mechanism by which LNPs load mRNA is by electrostatic adsorption. Even when the loading capacity (i.e., the encapsulation rate) is the same, the in vivo expression levels of LNP-mRNA are not the same. The only variable is the chemical structure of the ionizable lipid. Furthermore, the chemical structure of the ionizable lipid also affects the distribution of in vivo mRNA expression. As the application scope of mRNA drugs expands, the demands on delivery systems for mRNA drugs with different targets for different types of diseases become even higher. Therefore, targeted delivery of mRNA has become a scientific challenge that requires urgent resolution.

[0005] Therefore, there is a need to develop lipid compounds that are highly efficient, have low toxicity, and have excellent targeting properties. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent, and therefore provides a compound for drug delivery, which has advantages such as low cytotoxicity, strong delivery ability, and strong immune activation effect.

[0007] In one aspect, the present invention provides a compound of formula (I) or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of a compound of formula (I), [ka] wherein X1, X2, and X3 each independently represent an optionally substituted C1-C 15 alkylene, and R and R are each independently an optionally substituted C-C 40 Alkyl, optionally substituted C-C 40 Heteroalkyl, optionally substituted C-C 40 Alkenyl, optionally substituted C-C 40 Heteroalkenyl, optionally substituted C-C 40 Alkynyl or optionally substituted C-C 40 and R3, R4, R5, and R6 are each independently H, halogen, or optionally substituted C1-C3 alkyl, each of which is independently one or more of halogen, -OH, -SH, -NH2, -NO2, cyano, or C1-C3 alkyl. The compounds of the present invention have low cytotoxicity and high biocompatibility. They also have strong delivery capabilities and can be used as delivery carriers to deliver nucleic acid drugs to the heart, liver, spleen, lungs, and kidneys, especially the spleen, thereby effectively activating the body's immune system and increasing the level of specific antibodies in animals.

[0008] In another aspect, the present invention provides the use of the aforementioned compounds in the preparation of liposomes, drug carriers, or complexes. According to an embodiment of the present invention, the aforementioned compounds have the property of being ionizable and can be used to prepare liposomes, and the prepared liposomes can serve as drug carriers to form nucleic acid drug-liposome complexes.

[0009] In yet another aspect, the present invention provides a liposome. According to an embodiment of the present invention, the liposome comprises the compound described above. The liposome according to an embodiment of the present invention has advantages such as low cytotoxicity, high biocompatibility, and strong delivery ability, and has a strong immune activation effect.

[0010] In yet another aspect, the present invention provides a liposome, comprising the compound described above, a neutral lipid, cholesterol, and a PEG-lipid, wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), and the molar ratio of the compound, neutral lipid, cholesterol, and PEG-lipid is (40-60):(5-10):(30-50):(0.5-5).

[0011] In yet another aspect, the present invention provides a drug carrier. According to an embodiment of the present invention, the drug carrier comprises the aforementioned compound or the aforementioned liposome. According to an embodiment of the present invention, the aforementioned compound is an ionizable lipid, the drug carrier is an ionizable carrier, and the aforementioned compound or liposome can be used to load a drug and deliver the drug into cells.

[0012] In yet another aspect, the present invention provides a composite. According to an embodiment of the present invention, the composite comprises the aforementioned compound, the aforementioned liposome, or the aforementioned drug carrier, and a biologically active ingredient. As described above, the aforementioned compound is an ionizable lipid, and the drug carrier is an ionizable carrier, so that the aforementioned compound or liposome can be used to load a drug and prepare a composite containing a biologically active ingredient, and deliver the biologically active ingredient into cells of a living body for the treatment of a disease.

[0013] In yet another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned compound, the aforementioned liposome, the aforementioned drug carrier, or the aforementioned complex. As described above, the aforementioned compounds, liposomes, drug carriers, and complexes have advantages such as low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. When a bioactive component is loaded into the aforementioned compound, liposome, or drug carrier, the bioactive component can be delivered into the living body, which helps the loaded bioactive component to exert its efficacy in treating diseases.

[0014] In yet another aspect of the present invention, the present invention provides a use of the above-mentioned compound, the above-mentioned liposome, the above-mentioned drug carrier, the above-mentioned complex, or the above-mentioned pharmaceutical composition in the manufacture of a drug, wherein the drug is intended to target at least one of the heart, liver, spleen, lungs, and kidneys. As described above, the above-mentioned compounds, liposomes, drug carriers, and complexes have the advantages of low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. When a bioactive component is loaded into the above-mentioned compound, liposome, or drug carrier, the bioactive component can be delivered to the heart, liver, spleen, lungs, and kidneys, which helps the loaded bioactive component to exert its efficacy in disease treatment.

[0015] In yet another aspect of the present invention, the present invention provides a use of the above-mentioned compound, the above-mentioned liposome, the above-mentioned drug carrier, the above-mentioned complex, or the above-mentioned pharmaceutical composition in the manufacture of a drug for treating or preventing a disease. As described above, the above-mentioned compounds, liposomes, drug carriers, and complexes have advantages such as low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. When a bioactive component is loaded into the above-mentioned compound, liposome, or drug carrier, the bioactive component can be delivered into the living body, which helps the loaded bioactive component to exert its efficacy in treating a disease.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief explanation of the drawings]

[0017] The above and / or additional aspects and advantages of the present invention will become more apparent and understandable from the following detailed description of the embodiments taken in conjunction with the drawings, the description of which is set forth below. [Figure 1] 1 shows the results of cytotoxicity evaluation of different compounds in step 1 of a test example of the present invention. [Figure 2] 10 shows bioluminescence intensity images of each isolated organ of a mouse injected with different LNPs@FLuc mRNA drugs in step 2 of the test example of the present invention. [Figure 3] 1 shows the total fluorescence statistical results of each isolated organ of mice injected with different LNPs@FLuc mRNA drugs in step 2 of the test example of the present invention. [Figure 4] 1 shows the antibody titer results in the serum of mice injected with different LNPs@FLuc mRNA drugs in step 3 of the test example of the present invention. [Figure 5] 1 is a H1-NMR spectrum of compound 7 of the present invention. [Figure 6] 1 is a H1-NMR spectrum of compound 8 of the present invention. [Figure 7] 1 is a H1-NMR spectrum of compound 9 of the present invention. [Figure 8] 1 is a H1-NMR spectrum of compound 10 of the present invention. [Figure 9] 1 is a H1-NMR spectrum of compound 11 of the present invention. [Figure 10] 1 is a H1-NMR spectrum of compound 12 of the present invention. [Figure 11] 1 is a H1-NMR spectrum of compound 13 of the present invention. [Figure 12] 1 is a H1-NMR spectrum of compound 15 of the present invention. [Figure 13] 1 is a H1-NMR spectrum of compound 16 of the present invention. [Figure 14] 1 is a H1-NMR spectrum of compound 17 of the present invention. [Figure 15]1 is a H1-NMR spectrum of compound 18 of the present invention. [Figure 16] 1 is a H1-NMR spectrum of compound 19 of the present invention. [Figure 17] 1 is a H1-NMR spectrum of compound 21 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] Examples of the present invention will be described in detail below. The examples described below are illustrative and are intended only to explain the present invention, but should not be understood as limiting the present invention.

[0019] It should be noted that the terms "first" and "second" are for descriptive purposes only and should not be understood as indicating or implying the relative importance or the number of technical features indicated. Thus, a feature qualified by "first" or "second" may explicitly or implicitly include one or more of the feature. Furthermore, in the description of this invention, unless otherwise specified, "plurality" means two or more.

[0020] It should be noted that with regard to the description of structural formulas and chemical formulas in the examples or embodiments of the present invention, the present invention is intended to encompass all alternatives, modifications, and equivalent technical solutions, all of which are within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described in the present invention can be used to practice the present invention. The present invention is in no way limited to the methods and materials described in the present invention. In the event that one or more of the incorporated literature, patents, and similar materials differ or contradict this application (including, but not limited to, defined terms, term usage, described techniques, etc.), the present invention shall prevail.

[0021] Furthermore, it should be appreciated that certain features of the invention, which are apparent and described in multiple separate examples or embodiments, may also be provided in combination in a single example or embodiment. Conversely, various features of the invention, which are, for brevity, described in a single example or embodiment, may also be provided alone or in any suitable subcombination.

[0022] Unless otherwise indicated, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and unless otherwise indicated, all patent publications cited throughout the disclosure of this invention are incorporated herein by reference in their entirety.

[0023] In the present invention, the following definitions apply unless otherwise indicated. For the purposes of the present invention, chemical elements are defined according to the Periodic Table of the Elements, CAS version, and the Chemicals Handbook, 75th Edition, 1994. For general principles of organic chemistry, see also "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007. The entire content of the present invention is therefore incorporated by reference.

[0024] As used herein, the terms "comprise" or "include" are open-ended, that is to say, they include what is specified in the present invention, but do not exclude other content.

[0025] As used herein, the compounds of the present invention further include isotopically labeled compounds of the present invention, which are the same as the compounds described in the present invention except that one or more atoms have been replaced with atoms of a mass or mass number different from the mass or mass number of the common atoms in nature. Further, exemplary isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H, 3 H, 13 C. 14 C. 15 N, 16 O. 17 O. 31 P, 32 P, 36 S, 18 F and 37 It is Cl.

[0026] All compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and / or other isotopes of other atoms are included within the scope of the present invention. Isotopically labeled compounds of the present invention, e.g., 3 H and 14 Doping the compounds of the present invention with a radioactive isotope such as C can be used for drug and / or substrate tissue distribution analysis. 3 H and carbon-14 i.e. 14 C isotopes are particularly preferred. Also preferred are heavier isotopes such as deuterium, i.e. 2 Substitution with H can offer therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dose requirements, and therefore may be preferable in some cases.

[0027] The stereochemical definitions and conventions used herein essentially follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to non-enantiomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures, are contemplated as being within the scope of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S indicate the absolute configuration of the molecule about the chiral center(s) in the molecule. The prefixes d and l or (+) and (-) are symbols used to designate the rotation of plane-polarized light by a compound, where (-) or l indicates that the compound is levorotatory. A compound preceded by (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. Specific stereoisomers may be referred to as enantiomers, and mixtures of such isomers are commonly referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate; such a racemic mixture or racemate can exist when a chemical reaction or process lacks stereoselectivity or stereospecificity.

[0028] Depending on the starting materials and methods selected, the compounds of the present invention may exist in the form of one of the possible isomers or a mixture thereof, for example, as a pure optical isomer or as a mixture of isomers, such as racemic isomers and non-enantiomeric mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations, or resolved by conventional techniques. When the compound contains one double bond, the substituent can be in the E- or Z-configuration. When the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent can be in the cis- or trans-configuration.

[0029] The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to non-enantiomers, enantiomers, and atropisomers, as well as geometric (or conformational) isomers and mixtures thereof, including racemic mixtures, are all contemplated to be within the scope of the present invention.

[0030] Unless otherwise indicated, structures depicted in the present invention also represent all isomeric (e.g., enantiomeric, non-enantiomeric, atropisomer, and geometric (or conformational) isomeric) forms that encompass the structure, including, by way of example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers and enantiomeric mixtures, non-enantiomeric mixtures, and geometric (or conformational) mixtures of the compounds of the present invention are all included within the scope of the present invention.

[0031] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present invention can exist in racemic or enantiomerically enriched form, for example, in the (R)-, (S)-, or (R,S)-configuration. In some embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess for the (R)- or (S)-configuration. Where possible, substituents on atoms having unsaturated double bonds may exist in cis-(Z)- or trans-(E)-form.

[0032] Thus, as described herein, the compounds of the present invention may exist in the form of one of the possible isomers, rotamers, atropisomers, tautomers or mixtures thereof, e.g. in the form of a substantially pure geometric (cis or trans) isomer, a non-enantiomer, an optical isomer (enantiomer), a racemate or a mixture thereof.

[0033] Depending on the physicochemical differences of the components, any resulting isomeric mixtures can be separated into pure or substantially pure geometric or optical isomers, non-enantiomers, racemates, for example, by chromatography and / or fractional crystallization.

[0034] Racemates of any final products or intermediates obtained by known methods may be resolved into their optical antipodes by methods familiar to those skilled in the art, for example, by separation of the resulting non-enantiomeric salts. Racemic products may also be separated by chiral chromatography, for example, high pressure liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers may be prepared by asymmetric synthesis (e.g., Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981), Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aube, Elsevier, Oxford, UK, 2012), Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972)).

[0035] As used herein, the term "tautomer" or "tautomeric form" refers to structural isomers with different energies that are interconvertible via a low energy barrier. When tautomerism is possible (e.g., in solution), a chemical balance of tautomers can be achieved. For example, proton tautomers (prototropic tautomers) include interconversions via proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via any rearrangement of bonding electrons. A specific example of keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-keto tautomerism. A specific example of phenol-keto tautomerism is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are included within the scope of the invention.

[0036] As used herein, the term "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed between a solvent molecule and water.

[0037] As used herein, the term "pharmaceutically acceptable" refers to a substance or composition that must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith.

[0038] As used herein, the term "pharmaceutically acceptable salts" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described, for example, in SM Berge et al., "Describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19." Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochloride, hydrobromide, phosphate, sulfate, and perchlorate) formed by reaction with an amino group, and organic acid salts (such as acetate, oxalate, maleate, tartrate, citrate, succinate, and malonate) or salts obtained by other methods described in the literature, such as ion exchange methods. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxybenzoate ... Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium, and N-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecylate, valerate, and the like. +(C1-C4 alkyl)4 salts. The present invention contemplates any quaternary ammonium salts formed from compounds containing N groups. Water- or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include amine cations formed from suitable non-toxic ammonium, quaternary ammonium salts and counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-C8 sulfonates, and aromatic sulfonates.

[0039] As used herein, the terms "optionally," "optional," or "optionally" generally refer to the fact that the subsequently described event or circumstance may, but does not necessarily, occur, and the description includes instances when the event or circumstance occurs and instances when the event or circumstance does not occur.

[0040] As used herein, the terms "optionally substituted" and "substituted or unsubstituted" can be used interchangeably. In general, the term "optionally," whether preceded by the term "substituted," denotes that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise specified, one optional substituent may be substituted at each substitutable position of the group. When a given structural formula contains multiple positions substitutable with one or more substituents selected from a specified group, the substituents may be substituted at each position, either identically or differently. The substituents may be, but are not limited to, F, Cl, Br, CN, OH, NH, NO, etc.

[0041] As used herein, the term "one or more" (e.g., in the definition of substituents of compounds of the general formula of the invention) refers to "one, two, three, four or five, particularly one, two, three or four, more particularly one, two or three, and even more particularly one or two."

[0042] It should also be explained that, unless otherwise specified, the expressions "each ... independently," "... each independently," and "... independently" employed in the present invention can be used interchangeably and should all be understood in a broad sense, and can indicate that specific options represented by the same symbol in different groups do not affect each other, and can also indicate that specific options represented by the same symbol in the same group do not affect each other.

[0043] As used herein, the term "halogen" refers to a fluorine, chlorine, bromine or iodine atom.

[0044] As used herein, the minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, e.g., the prefix C a -C b means that it contains "a" to "b" carbon atoms. n " refers to a linear or branched saturated / unsaturated carbon chain containing 1, 2, 3, 4, 5, ... or n carbon atoms, and is further understood as "C1-C n " includes any subranges contained therein, e.g., C1-C 40 , C2-C 40 , C1-C 24 , C3-C 24 , C1-C 11 , C4-C 10 , C4-C8, C1-C3.

[0045] As used herein, the term "C1-C 40 "Alkyl" refers to a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5, ..., or 40 carbon atoms, e.g., C2-C 40 Alkyl, C2-C 24 Alkyl, C3-C 24 Alkyl, C3-C 11 Alkyl, C4-C 10alkyl, C4-C8 alkyl, where methyl, ethyl, n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2C H3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH 2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like, wherein said alkyl groups are independently unsubstituted or substituted with one or more substituents described herein.

[0046] As used herein, the term "C2-C 40"Alkenyl" refers to a linear or branched monovalent hydrocarbon radical having 2, 3, 4, 5, ..., or 40 carbon atoms, at least one C-C position of which is sp2 double bond unsaturated, where the alkenyl radical may be independently unsubstituted or substituted with one or more substituents as described herein, and includes radicals defined as "cis," "trans," or "Z," "E," including, but not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like. For example, C2-C 40 Alkenyl, C2-C 24 Alkenyl, C3-C 24 Alkenyl, C3-C 11 Alkenyl, C4-C 10 alkenyl, C4-C8 alkenyl.

[0047] As used herein, the term "C2-C 40 "Alkynyl" refers to a linear or branched monovalent hydrocarbon radical having 2, 3, 4, 5, ..., or 40 carbon atoms, at least one C-C position of which is sp triple bond unsaturated, where the alkynyl radical may be independently unsubstituted or substituted with one or more substituents according to the invention, and examples include, but are not limited to, ethynyl (-C≡CH), propargyl (-CHC≡CH), 1-propynyl (-C≡C-CH), etc. For example, C-C 40 Alkynyl, C2-C 24 Alkynyl, C3-C 24 Alkynyl, C3-C 11 Alkynyl, C4-C 10 alkynyl, C4-C8 alkynyl.

[0048] As used herein, the term "heteroalkyl" refers to the fact that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) can be present in an alkyl to provide a heteroalkyl (e.g., an alkyl containing one or more ether, thioether, or amino linkages). The term "heteroalkenyl" refers to the fact that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) can be present in an alkenyl to provide a heteroalkenyl (e.g., an alkenyl containing one or more ether, thioether, or amino linkages). The term "heteroalkynyl" refers to the fact that heteroatoms such as oxygen, sulfur, phosphorus, and nitrogen (in the form of a tertiary amine moiety) can be present in an alkynyl to provide a heteroalkynyl (e.g., an alkynyl containing one or more ether, thioether, or amino linkages).

[0049] " in the description of the group of the present invention [ka] " is used to describe the substitution position of a group.

[0050] As used herein, "liposome" or "lipid nanoparticle (LNP)" refers to a drug delivery system in which a drug or other biologically active substance is dissolved or coated in the lipid core, or adsorbed or attached to the surface of the nanoparticle, using a biocompatible lipid material as a carrier.

[0051] As used herein, the term "pharmaceutically acceptable adjuvant" includes any solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, salts, drug stabilizers, binders, excipients, dispersing agents, lubricants, sweeteners, flavoring agents, coloring agents, or other compositions, all of which are known to those skilled in the art (e.g., as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0052] As used herein, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect may be a prophylactic effect, with respect to complete or partial prevention of a disease or its symptoms, and / or a therapeutic effect, with respect to partial or complete cure of a disease and / or side effects of a disease. As used herein, "treatment" encompasses mammalian, particularly human, disease, and includes (a) preventing the onset of a disease or condition in an individual prone to, but not diagnosed with, the disease; (b) suppressing the disease, e.g., inhibiting disease progression; or (c) alleviating disease, e.g., reducing disease-related symptoms. As used herein, "treatment" includes any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or suppress a disease in an individual, including, but not limited to, administering a drug, including a compound described herein, to an individual in need thereof.

[0053] The present invention provides compounds and uses thereof, liposomes, drug carriers, complexes, pharmaceutical compositions and pharmaceutical uses thereof, each of which is described in detail below.

[0054] compound In one aspect, the present invention provides a compound of formula (I) or a stereoisomer, tautomer, solvate or pharmaceutically acceptable salt of a compound of formula (I), [ka] wherein X1, X2, and X3 each independently represent an optionally substituted C1-C 15 alkylene, and R and R are each independently an optionally substituted C-C 40 Alkyl, optionally substituted C-C 40 Heteroalkyl, optionally substituted C-C 40 Alkenyl, optionally substituted C-C 40 Heteroalkenyl, optionally substituted C-C 40 Alkynyl or optionally substituted C-C 40 The present invention provides a compound in which R3, R4, R5, and R6 are each independently H, halogen, or optionally substituted C1-C3 alkyl, and each of the substituted groups is independently one or more selected from halogen, -OH, -SH, -NH2, -NO2, cyano, and C1-C3 alkyl. The compound of the present invention has low cytotoxicity and exhibits high biocompatibility. The compound has strong delivery ability and can be used as a delivery carrier to deliver nucleic acid drugs and the like into the animal body, for example, to the heart, liver, spleen, lungs, and kidneys, especially to the spleen, thereby effectively activating the body's immune system and increasing the level of specific antibodies in the animal body.

[0055] In some embodiments, X1 and X2 are each independently C4-C 12 It is alkylene.

[0056] In some embodiments, X3 is C2-C8 alkylene.

[0057] In some embodiments, X1 is not a C4 alkylene, X2 is a C4 alkylene, and X3 is a C2 alkylene at the same time.

[0058] In some embodiments, R3, R4, R5, and R6 are each independently H or halogen.

[0059] In some embodiments, R1 and R2 each independently have a structure represented by formula (II): [ka] In the formula, m is an integer of 1 to 10. R7 and R8 are each independently H, optionally substituted C1-C 20 Alkyl, optionally substituted C-C 20 Heteroalkyl, optionally substituted C-C 20 Alkenyl, optionally substituted C-C 20 Heteroalkenyl, optionally substituted C-C 20 Alkynyl or optionally substituted C-C 20 heteroalkynyl, wherein each substituting group is independently one or more selected from halogen, -OH, -SH, -NH2, -NO2, cyano, or C1-C3 alkyl.

[0060] In some preferred embodiments, m is 2, 3, or 4.

[0061] Illustratively, when m is 3, R1 and R2 are [ka] The structure is as follows.

[0062] In some embodiments, R7 and R8 are each independently H, optionally substituted C4-C 18 Alkyl, optionally substituted C-C 18 Heteroalkyl, optionally substituted C-C 18 Alkenyl or optionally substituted C-C 18 heteroalkenyl, where each substituting group is independently one or more selected from halogen, -OH, -SH, -NH2, -NO2, cyano, or C1-C3 alkyl.

[0063] In some embodiments, R1 and R2 each independently have a structure represented by formula (III): [ka] In the formula, n is 1 or 2; R7 and R8 are each independently H, optionally substituted C1-C 20 Alkyl, optionally substituted C-C 20 Heteroalkyl, optionally substituted C-C 20 Alkenyl, optionally substituted C-C 20 Heteroalkenyl, optionally substituted C-C 20 Alkynyl or optionally substituted C-C 20 heteroalkynyl, wherein each substituting group is independently one or more selected from halogen, -OH, -SH, -NH2, -NO2, cyano, or C1-C3 alkyl.

[0064] In some preferred embodiments, R7 and R8 each independently represent an optionally substituted C4-C 18 Alkyl, optionally substituted C-C 18 Heteroalkyl, optionally substituted C-C 18 Alkenyl or optionally substituted C-C 18 heteroalkenyl, where each substituting group is independently one or more selected from halogen, -OH, -SH, -NH2, -NO2, cyano, or C1-C3 alkyl.

[0065] In some embodiments, R1 and R2 are each independently: [ka] The structure of the compound is as follows:

[0066] In some embodiments, the compound is [ka] [ka] The structure of the compound is as follows:

[0067] In some embodiments, the compounds of the present invention further include other salts of the compounds, which are not necessarily pharmaceutically acceptable salts, and which can be used as intermediates for preparing and / or purifying the compounds of the present invention and / or for separating the enantiomers of the compounds of the present invention.

[0068] The compounds of the present invention or their salts may be obtained in the form of their hydrates or include other solvents for their crystallization. Because the compounds of the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents (including water), the present invention is intended to encompass both solvated and unsolvated forms.

[0069] Although any structural formulas provided herein may represent the unlabeled and isotopically unlabeled forms of these compounds, the compounds of the present invention also include compounds defined herein that are labeled with various isotopes. Isotopically labeled compounds also have the structure shown in the general formulas provided herein, except that one or more atoms are replaced with atoms having a selected atomic mass or mass number. Isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example. 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 18 F, 31 P, 32 P, 36 S, 37 Cl or 125 I.

[0070] In another aspect, the present invention provides the use of the aforementioned compounds in the preparation of liposomes, drug carriers, or complexes. According to an embodiment of the present invention, the aforementioned compounds have the property of being ionizable and can be used to prepare liposomes, which can serve as drug carriers to form nucleic acid drug-liposome complexes.

[0071] In some embodiments, the compounds can be used in the form of polymers to prepare liposomes. The compounds can be covalently bonded to other substances to prepare liposomes. The compounds can be chemically reacted with other substances to prepare liposomes. According to embodiments of the present invention, the specific method for preparing liposomes using the compounds is not limited, and preparing liposomes using the compounds and containing all or part of the structure of the compounds in the liposomes is considered to be a use of the present invention.

[0072] For administration purposes, the compounds of the present invention (usually in the form of liposomes and bioactive ingredients combined) may be administered as crude chemicals or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the present invention comprise a compound of structure (I) and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compound of structure (I) is present in the composition in an amount to effectively form liposomes and deliver the bioactive ingredient, e.g., to treat the particular disease or condition involved. Concentrations and dosages can be readily determined by one of ordinary skill in the art.

[0073] Liposomes In yet another aspect, the present invention provides a liposome. According to an embodiment of the present invention, the liposome comprises the compound described above. The liposome according to an embodiment of the present invention has advantages such as low cytotoxicity, high biocompatibility, and strong delivery ability, and has a strong immune activation effect.

[0074] In some embodiments, the liposome further comprises at least one of a steroid, a neutral lipid, and a PEG-lipid.

[0075] In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DPPE). The present invention relates to a method for preparing a glycerol-based glycerol complex comprising administering to a subject ...

[0076] In some embodiments, the PEG-lipid is selected from the group consisting of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetyl, PEG-di The copolymer contains at least one selected from oleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-phosphatidylethanolamine (PEG-PE), PEG-diacylglycerol succinate (PEG-S-DAG), PEG-ceramide (PEG-cer), PEG dialkoxypropylcarbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0077] In some embodiments, the steroid comprises at least one selected from cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, and brassicasterol, preferably cholesterol.

[0078] In some embodiments, the molar ratio of the compound:neutral lipid:steroid:PEG-lipid is (20-80):(5-50):(10-60):(0.01-10).

[0079] In some embodiments, the molar ratio of the compound:neutral lipid:steroid:PEG-lipid is (40-60):(5-10):(30-50):(0.5-5).

[0080] In yet another aspect, the present invention provides liposomes. According to an embodiment of the present invention, the liposomes comprise the aforementioned compound, a neutral lipid, cholesterol, and a PEG-lipid, wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), and the molar ratio of the compound, neutral lipid, cholesterol, and PEG-lipid is (40-60):(5-10):(30-50):(0.5-5). The liposomes according to the embodiments of the present invention have advantages such as low cytotoxicity, high biocompatibility, and strong delivery ability, and have a strong immune activation effect.

[0081] Drug carriers, composites and pharmaceutical compositions In yet another aspect, the present invention provides a drug carrier. According to an embodiment of the present invention, the drug carrier comprises the aforementioned compound or the aforementioned liposome. According to an embodiment of the present invention, the aforementioned compound is an ionizable lipid, and the drug carrier is an ionizable carrier, and the aforementioned compound or liposome can be used to load a drug and deliver the drug into cells, thereby helping the loaded drug to exert its efficacy in treating related diseases.

[0082] In yet another aspect, the present invention provides a composite. According to an embodiment of the present invention, the composite comprises the aforementioned compound, the aforementioned liposome, or the aforementioned drug carrier, and a biologically active ingredient. As described above, the aforementioned compound is an ionizable lipid, and the drug carrier is an ionizable carrier. Therefore, the aforementioned compound or liposome can be used to load a drug and prepare a composite containing a biologically active ingredient, which can deliver the drug into cells of a living body and help the loaded biologically active ingredient exert its efficacy in treating related diseases.

[0083] In some embodiments, the bioactive component comprises at least one selected from a DNA molecule, an RNA molecule, a protein, a polypeptide, and a small molecule drug.

[0084] In some embodiments, the replica of the protein or polypeptide is not particularly limited and may be a peptide chain or the protein itself, or a derivative of the above substance or a complex with another substance, such as a Cas9 protein or a domain peptide thereof, a protein carrying a nuclide, or an antibody.

[0085] In some embodiments, the bioactive moiety is a nucleic acid. Illustratively, the bioactive moiety is an antisense RNA or a messenger RNA.

[0086] In some embodiments, the bioactive component may include DNA, RNA, nucleic acid-protein complexes, nucleic acid-lipid complexes, nucleic acid-nuclide complexes, etc., and the type of nucleic acid is not particularly limited. Specific examples of nucleic acids include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA (microRNA), ribozymes, antisense oligonucleotides, plasmid DNA, peptide nucleic acids, triplex-forming oligonucleotides (TFOs), and genes. Nucleic acids suitable for use as drug carriers of the present invention may be nucleic acids derived from humans, animals, plants, bacteria, viruses, etc., or may be nucleic acids prepared by chemical synthesis. Furthermore, the nucleic acids may be single-stranded, double-stranded, or triple-stranded, and their molecular weights are not particularly limited. Furthermore, in the present invention, the nucleic acids may be chemically modified, enzymatically modified, or peptide-modified. In the present invention, a single type of nucleic acid may be used, or two or more types may be used in combination. In some embodiments, the vector composition for nucleic acid delivery of the present invention is preferably a transporter mRNA or its analog.

[0087] In some embodiments, the bioactive ingredient is electronegative or hydrophobic. According to embodiments of the present invention, the drug carrier comprises an ionizable lipid, which is electropositive at a certain pH and can bind to the electronegative bioactive ingredient.

[0088] In yet another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned compound, the aforementioned liposome, the aforementioned drug carrier, or the aforementioned composite. As described above, the aforementioned compounds, liposomes, drug carriers, and composites have advantages such as low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. By loading a bioactive component into the aforementioned compound, liposome, or drug carrier, the bioactive component can be delivered into the living body, which helps the loaded bioactive component to exert its efficacy in treating diseases.

[0089] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable adjuvant.

[0090] The pharmaceutical compositions of the present invention can be administered in any acceptable dosage form. The pharmaceutical compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols, and existing methods for producing these dosage forms are known or will be apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques. The pharmaceutical compositions of the present invention are formulated so that the bioactive ingredients contained therein are bioavailable after administration of the composition to a patient. The composition administered to a subject or patient may be in the form of one or more dosage units, and, for example, a tablet may be a single dosage unit, and a container of the aerosol form of the compound of the present invention may contain multiple dosage units.

[0091] Purpose In yet another aspect of the present invention, the present invention provides a use of the above-mentioned compound, the above-mentioned liposome, the above-mentioned drug carrier, the above-mentioned complex, or the above-mentioned pharmaceutical composition in the manufacture of a drug, wherein the drug is intended to target at least one of the heart, liver, spleen, lungs, and kidneys. As described above, the above-mentioned compounds, liposomes, drug carriers, and complexes have the advantages of low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. By loading a biologically active ingredient into the above-mentioned compound, liposome, or drug carrier, the biologically active ingredient can be delivered to the heart, liver, spleen, lungs, and kidneys, which helps the loaded biologically active ingredient to exert its efficacy in treating diseases.

[0092] In some embodiments, the drug is for targeting the spleen.

[0093] As used herein, the term "targeting" refers to the ability to deliver a bioactive ingredient loaded in a liposome to a predetermined target, allowing the bioactive ingredient to be abundantly present at the predetermined target, and the targeting is non-specific. For example, "targeting to the spleen" refers to allowing the bioactive ingredient to be abundantly present in the spleen, but does not exclude the bioactive ingredient from being present in other parts of the body.

[0094] In yet another aspect of the present invention, the present invention provides a use of the above-mentioned compound, the above-mentioned liposome, the above-mentioned drug carrier, the above-mentioned complex, or the above-mentioned pharmaceutical composition in the manufacture of a drug, wherein the drug is for treating or preventing a disease. As described above, the above-mentioned compound, liposome, drug carrier, and complex have advantages such as low cytotoxicity, high biocompatibility, strong delivery ability, and strong immune activation effect. When a bioactive component is loaded into the above-mentioned compound, liposome, or drug carrier, the bioactive component can be delivered into the living body, which helps the loaded bioactive component to exert its efficacy in treating a disease.

[0095] In some embodiments, the disease comprises one selected from a tumor or tumor-related disease, a virus-induced related disease.

[0096] In some embodiments, the tumor comprises melanoma, brain cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colon cancer, lung cancer, kidney cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, skin cancer, neuroblastoma, glioma, sarcoma, bone cancer, uterine cancer, endometrial cancer, head and neck cancer, multiple myeloma, B-cell lymphoma, polycythemia vera, leukemia, thyroid tumor, bladder cancer, or gallbladder cancer.

[0097] In some embodiments, the virus-induced associated disease includes, but is not limited to, an associated disease induced by a novel coronavirus.

[0098] In some embodiments, the drug is for delivering a bioactive ingredient into a living body.

[0099] In some embodiments, the medicament is for delivering a bioactive ingredient to at least one of the heart, liver, spleen, lungs, and kidneys.

[0100] In some embodiments, the drug is for delivering bioactive ingredients to the spleen.The inventors have found through experiments that, compared with traditional liposomes or drug carriers, the compounds, liposomes or drug carriers of the present invention can deliver bioactive ingredients to the spleen and improve the content or expression of bioactive ingredients in the spleen.Therefore, the drug of the present invention has a strong therapeutic effect on spleen-related diseases.

[0101] method In another aspect, the present invention provides a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the above-mentioned compound or the above-mentioned pharmaceutical composition. The method of the present invention can effectively treat or prevent a disease, in particular a tumor or a tumor-related disease.

[0102] The effective amount of the compound or pharmaceutical composition according to the present invention may vary depending on the administration pattern, the severity of the disease being treated, etc. A preferred effective amount can be selected by one skilled in the art based on various factors (e.g., through clinical trials), including, but not limited to, the bioavailability, metabolism, pharmacokinetic parameters such as half-life of the bioactive component, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. For example, depending on the exigencies of the therapeutic situation, multiple individual doses may be administered daily, or the dose may be proportionally reduced.

[0103] The conjugate or pharmaceutical composition of the present invention may be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (e.g., injection and infusion solutions) or lyophilized powders. A typical drug is an injection solution. The conjugate or pharmaceutical composition described above may be administered by intravenous infusion or injection, intramuscular injection, or subcutaneous injection.

[0104] According to an embodiment of the present invention, the route of administration of the method is intramuscular injection or intravenous injection.

[0105] The present invention will be explained in conjunction with the following examples. Those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they should be in accordance with the techniques or conditions or product instructions described in the literature in the field. Unless the manufacturer is specified, the reagents or equipment used are all commercially available general products.

[0106] Example 1 Preparation of Compound 7 Synthetic route and characterization of compound 7 [ka] Synthesis of compound 2a Compound 1a (2.0 g, 8.3 mmol) and CDI (2.0 g, 12.3 mmol, i.e., N,N'-carbonyldiimidazole) were added to a solution of DCM (85.0 mL, i.e., dichloromethane) at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that 1a was completely consumed. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. The crude product of compound 2a was used directly in the next step without purification.

[0107] Synthesis of compound 4a Compound 2a (2.5 g, 7.5 mmol) and compound 3a (1.8 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2a had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4a was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0108] Synthesis of compound 5a Compound 4a (2.1 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol, i.e., triphenylphosphine), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4a had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5a was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0109] Synthesis of compound 7 Compound 5a (3.3 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5a was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 7 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 5.

[0110] Example 2 Preparation of Compounds 8 to 21 Synthetic route and characterization of compound 8 Compounds 2a, 4a and 5a were synthesized in this order according to the method described in Example 1. [ka]

[0111] Synthesis of compound 8 Compound 5a (3.3 g, 6.6 mmol), compound 6b, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5a was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 8 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 6.

[0112] Synthetic route and characterization of compound 9 Compounds 2a, 4a and 5a were synthesized in this order according to the method described in Example 1. [ka]

[0113] Synthesis of compound 9 Compound 5a (3.3 g, 6.6 mmol), 6c, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5a was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 9 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 7.

[0114] Synthetic route and characterization of compound 10 [ka] Compounds 2a, 4a and 5a were synthesized in this order according to the method described in Example 1.

[0115] Synthesis of compound 10 Compound 5a (3.3 g, 6.6 mmol), 6d, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5a was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 10 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 8.

[0116] Synthetic route and characterization of compound 11 [ka] Compound 2a was synthesized according to the method described in Example 1.

[0117] Synthesis of compound 4b Compound 2a (2.5 g, 7.5 mmol) and compound 3b (1.5 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2a had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4b was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0118] Synthesis of compound 5b Compound 4b (2.0 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4b had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5b was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0119] Synthesis of compound 11 Compound 5b (3.2 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5b was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 11 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 9.

[0120] Synthetic route and characterization of compound 12 [ka] Compound 2a was synthesized according to the method described in Example 1.

[0121] Synthesis of compound 4c Compound 2a (2.5 g, 7.5 mmol) and compound 3c (2.4 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. LCMS showed that compound 3c had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4c was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0122] Synthesis of compound 5c Compound 4c (2.3 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4c had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5c was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0123] Synthesis of compound 11 Compound 5c (3.5 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5c was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 12 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 10.

[0124] Synthetic route and characterization of compound 13 [ka] Compound 2a was synthesized according to the method described in Example 1.

[0125] Synthesis of compound 4d Compound 2a (2.5 g, 7.5 mmol) and compound 3d (3.0 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. LCMS showed that compound 3c had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4d was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0126] Synthesis of compound 5d Compound 4d (2.6 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4d had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5d was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0127] Synthesis of compound 11 Compound 5d (3.8 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5d was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 13 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 11.

[0128] Synthetic route and characterization of compound 15 [ka] Compound 5d was synthesized according to the method described for Compound 13.

[0129] Synthesis of compound 14 Compound 5d (3.6 g, 6.6 mmol), compound 6b (0.6 g, 6.6 mmol), and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5d was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 14 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1).

[0130] Compound 5a was synthesized in turn according to the method described for compound 13.

[0131] Synthesis of compound 15 Compound 14 (3.6 g, 6.6 mmol), compound 5a (4.0 g, 8.0 mmol), and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 14 was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 15 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 12.

[0132] Synthetic route and characterization of compound 16 [ka] Synthesis of compound 2b Compound 1b (1.8 g, 8.3 mmol) and CDI (2.0 g, 12.3 mmol) were added to a solution of DCM (85.0 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that 1b was completely consumed. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. The crude product of compound 2b was used directly in the next step without purification.

[0133] Synthesis of compound 4e Compound 2b (2.3 g, 7.5 mmol) and compound 3a (1.8 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2b had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4e was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0134] Synthesis of compound 5e Compound 4e (2.0 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4e had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5e was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0135] Synthesis of compound 16 Compound 5e (3.1 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5e was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 16 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 13.

[0136] Synthetic route and characterization of compound 17 [ka] Synthesis of compound 2c Compound 1c (2.2 g, 8.3 mmol) and CDI (2.0 g, 12.3 mmol) were added to a solution of DCM (85.0 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that 1b was completely consumed. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. The crude product of compound 2c was used directly in the next step without purification.

[0137] Synthesis of compound 4f Compound 2c (2.7 g, 7.5 mmol) and compound 3a (1.8 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2c had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4f was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0138] Synthesis of compound 5f Compound 4f (2.3 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4f had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5f was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0139] Synthesis of compound 16 Compound 5f (3.4 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5f was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 17 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 14.

[0140] Synthetic route and characterization of compound 18 [ka] Synthesis of compound 2d Compound 1d (2.2 g, 8.3 mmol) and CDI (2.0 g, 12.3 mmol) were added to a solution of DCM (85.0 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that 1b was completely consumed. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. The crude product of compound 2d was used directly in the next step without purification.

[0141] Synthesis of compound 4f Compound 2d (2.1 g, 7.5 mmol) and compound 3a (1.8 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2d had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4g was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0142] Synthesis of compound 5f Compound 4g (1.8 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4g had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5g was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0143] Synthesis of compound 16 Compound 5g (2.9 g, 6.6 mmol), 6a, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5g was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 18 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 15.

[0144] Synthetic route and characterization of compound 19 [ka] Synthesis of compound 2e Compound 1e (2.2 g, 8.3 mmol) and CDI (2.0 g, 12.3 mmol) were added to a solution of DCM (85.0 mL) at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that 1e was completely consumed. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. The crude product of compound 2e was used directly in the next step without purification.

[0145] Synthesis of compound 4h Compound 2e (2.7 g, 7.5 mmol) and compound 3a (1.8 g, 15 mmol) were added to a DCM (75.0 mL) solution at room temperature. The reaction mixture was stirred overnight at room temperature. TLC showed that compound 2h had reacted completely. The reaction mixture was added to water and extracted with DCM. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 4h was purified by column chromatography (PE:EA = 7:1 to 1:1).

[0146] Synthesis of compound 5f Compound 4h (2.1 g, 5.5 mmol), I2 (2.2 g, 8.7 mmol), PPh3 (2.3 g, 8.8 mmol), and imidazole (0.6 g, 6.6 mmol) were added to a DCM (20.0 mL) solution at room temperature. The reaction mixture was stirred at 60 °C for 3 h. LCMS showed that compound 4h had reacted completely. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 5h was purified by column chromatography (PE:EA = 9:1 to 7:1).

[0147] Synthesis of compound 16 Compound 5h (3.3 g, 6.6 mmol), 6b, and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5g was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 19 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 16.

[0148] Synthetic route and characterization of compound 21 [ka] Compound 5a was synthesized according to the method described in Example 1.

[0149] Synthesis of compound 20 Compound 5a (3.3 g, 6.6 mmol), compound 6b (0.6 g, 6.6 mmol), and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 5a was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 20 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1).

[0150] Compound 5g was synthesized according to the method described for Compound 18.

[0151] Synthesis of compound 21 Compound 20 (3.0 g, 6.6 mmol), compound 5g (3.5 g, 8.0 mmol), and K2CO3 (1.5 g, 9.3 mmol) were dissolved in DCM (10.0 mL) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. TLC showed that compound 20 was completely consumed. The reaction mixture was added to water and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, and then concentrated. Compound 21 was purified by column chromatography (DCM:MeOH = 150:0 to 20:1). The H1-NMR spectrum is shown in Figure 17.

[0152] Example 3: Preparation of LNPs@mRNA Furthermore, the inventors constructed an mRNA delivery system LNPs@mRNA (LNPs loaded with mRNA) based on the compounds obtained in Examples 1 and 2 (Compounds 7 to 13, Compounds 15 to 19, and Compound 21) and using commercially available MC3, SM-102, and ALC-0315 as control lipids, and examined its pharmaceutical properties, such as particle size, potential, encapsulation rate, mRNA integrity, and TEM, to evaluate its formulation suitability. Hereinafter, the compounds of the present invention, MC3, SM-102, and ALC-0315, are collectively referred to as ionizable lipids.

[0153] The method for producing LNPs@mRNA is as follows. (1) Solution preparation Ionizable lipids, DOPE (or DSPC), cholesterol (Chol), and DMG-PEG2000 were dissolved in absolute ethanol to obtain an ionizable lipid solution, where the concentration of the ionizable lipid in the ionizable lipid solution was 10 mg / mL. The molar ratio of the ionizable lipids, DSPC, cholesterol (Chol), and DMG-PEG2000 was 50:10:38.5:1.5. The mRNA was diluted to an appropriate concentration with 10 mM PBS buffer (prepared with RNase-free water) at pH 6.0 for use. Typically, the mRNA is selected to encode a viral antigen protein (in the present experimental example, an mRNA sequence encoding the novel coronavirus Delta S protein was selected, and LNPs@mRNA formulations were prepared and their immunostimulatory efficacy was further tested), a tumor antigen, or another therapeutic protein or polypeptide, or a gene editing tool such as Cas9 protein.

[0154] (2) LNP preparation The ionizable lipid solution obtained in step (1) was mixed with the mRNA solution to obtain an LNP preformulation. The mass ratio of ionizable lipid to mRNA was controlled at 8:1. The mixing process was performed using a microfluidic device, and the microfluidic process parameters were a volume ratio of ethanol phase to aqueous phase of 1:4 and a flow rate of 9 mL / min.

[0155] (3) Ultrafiltration The LNP preformulation was diluted 25-fold with PBS buffer and then ultrafiltered to the initial volume in an ultrafiltration cup to obtain the final LNP formulation. The ethanol in the preformulation was removed during the ultrafiltration process, yielding different LNPs@mRNA drugs, designated Compounds 7–13, 15–19, 21, MC3, SM-102, and ALC-0315, respectively. The ultrafiltration process parameters were a 100 kDa filter membrane, 0.2 MPa air pressure, and a rotation speed of 100–200 rpm.

[0156] Examination of pharmaceutical properties of LNPs@mRNA Particle size and potential measurement An appropriate amount of LNPs@mRNA formulation was diluted 10 times with purified water, and its pharmaceutical properties such as particle size, particle size distribution, and zeta potential were detected using a Malvern Nano Zetasizer (n = 3, i.e., three measurements per formulation).

[0157] Characterization by transmission electron microscopy The LNPs@mRNA was diluted with purified water to a nanoparticle mass concentration of 2 mg / mL, carefully dropped onto a copper mesh designed for TEM, and left to stand for 2 minutes before removing excess liquid by suction using filter paper. After negative staining with 2% phosphotungstic acid for 3 minutes, the excess staining solution was removed by suction, the sample was dried using an ear tube, and then photographs were taken.

[0158] Encapsulation rate detection The encapsulation rate was detected with the Quant-iT™ RiboGreen™ kit.

[0159] mRNA integrity detection To further examine the mRNA protective effect of the LNPs of the present invention, the integrity of the mRNA in the formulation was characterized and compared to a sample of the mRNA stock solution that underwent the formulation process without the addition of ionizable lipids.

[0160] The LNPs prepared with different ionizable lipids obtained in this invention had similar particle sizes, with average particle sizes of approximately 100 nm, Di(90) values ​​within 500 nm, and PDI values ​​below 0.3, indicating uniform LNP particle size distribution. The potential of the formulations was approximately 5 mV.

[0161] The results of the encapsulation rate detection showed that the encapsulation rates of the LNPs@mRNA prepared with different ionizable lipids obtained in Examples 1 and 2 of the present invention and the commercially available different ionizable lipids were both 80% or higher, indicating that the ionizable lipids provided by the present invention have high protection for mRNA in different formulation methods.

[0162] The integrity detection results showed that the mRNA stock solution without ionizable lipids had a significant decrease in integrity after undergoing the formulation process compared to the initial mRNA stock solution, while the LNPs@mRNA had no obvious decrease in integrity compared to the initial mRNA stock solution.

[0163] The above test results show that the LNPs@mRNA prepared with the ionizable lipids of the present invention have excellent nanoformulation properties, with an encapsulation rate of over 80% and high protection for mRNA.

[0164] Test Example: Verification of the effects of a series of compounds of the present invention 1. Cytotoxicity evaluation of the compounds of the present invention CHL cells were seeded into a 96-well culture plate at 4,000 cells per well and cultured overnight in an incubator at 37°C and 5% CO2. The medium was then discarded, and 100 μL of medium containing different drug concentrations of ALC-0315, SM-102, and the compounds obtained in Examples 1 and 2 (see Figure 1 for specific concentrations) was added, followed by 48 hours of culture. Then, 10 μL of CCK8 solution was added to each well, and the cells were incubated for 4 hours in a cell incubator. After incubation, the absorbance value at 450 nm of each well was measured using a microplate reader, and cell activity was calculated. IC 50 The values ​​were calculated using GraphPad Prism software (version 8.01).

[0165] The results of the cytotoxicity experiment are shown in Figure 1 and Table 1. Under the experimental conditions, the ionizable lipid of the present invention exhibited lower cytotoxicity than the ionizable lipid components in commercially available mRNA vaccine products, indicating higher biocompatibility. Advantageously, the ionizable lipid of the present invention exhibited lower cytotoxicity than the ionizable lipid components in commercially available mRNA vaccine products, such as ALC-0315 and SM-102 (IC 50 IC values ​​of compound 8 were 29.9 and 55.9, respectively. 50 The value was 166.5. [Table 1]

[0166] 2. In vivo expression and distribution of the LNPs@mRNA of the present invention Furthermore, the in vivo mRNA delivery ability of LNPs@mRNA prepared with the compounds of the present invention was examined. The inventors marked mRNA using firefly luciferase as a reporter gene, i.e., Fluc mRNA, and then prepared LNPs@Fluc mRNA using different ionizable lipids (compound 8, MC3, ALC-0315, or SM-102) according to the method of Example 3. These LNPs@Fluc mRNA formulations were designated LNP@Fluc mRNA, MC3@Fluc mRNA, ALC-0315@Fluc mRNA, and SM-102@Fluc mRNA, respectively. The in vivo mRNA expression ability and expression distribution of the LNPs@mRNA formulation systems prepared with the compounds of the present invention were examined via two administration routes: intravenous injection (step 2.1 in this example) and intramuscular injection (step 2.2 in this example).

[0167] 2.1 In vivo expression testing via intravenous route The following experiments were performed on the above-prepared formulation LNP@Fluc mRNA (for example, when the ionizable lipid is selected as compound 8, the prepared LNPs@mRNA is abbreviated as compound 8, and the same applies hereinafter), and the positive control formulations MC3@Fluc mRNA, ALC-0315@Fluc mRNA, and SM-102@Fluc mRNA, hereinafter referred to as "formulation." The mRNA concentration of each formulation was adjusted to 0.05 mg / mL with PBS, and the osmolality of each formulation was adjusted to isotonicity. 200 μL of the formulation was injected via the tail vein of each NIH mouse (i.e., 10 μg of FLuc mRNA per mouse, 3 mice per group). PBS served as a negative control (CTL). After administration, mice were maintained on a normal diet. 6 h after administration, 200 μL of substrate solution (15 mg / mL, luciferin potassium salt) was intraperitoneally injected. Timing began after substrate injection, and mice were euthanized 10 min later. The heart, liver, spleen, lungs, and kidneys were immediately dissected, and the bioluminescence intensity of each isolated organ was detected using an IVIS device. The exposure time was 60 s. After imaging, the total fluorescence (total flux) of each organ was calculated. The results are shown in Figure 2(iv) and Figure 3(iv).

[0168] The results of the in vivo expression test via intravenous injection showed that the ionizable lipid of compound 8 had stronger mRNA expression ability than the three positive control materials in terms of the total in vivo luminescence intensity via intravenous injection. It was also found that the LNPs@mRNA produced with the lipid of the present invention had a certain expression level in the spleen, which is useful for the research and development of intravenous tumor vaccines.

[0169] 2.2 In vivo expression testing via intramuscular injection route The following experiments were carried out on the above-prepared formulation LNP@Fluc mRNA (compound 8), hereinafter abbreviated as formulation, and the positive control formulations MC3@Fluc mRNA, ALC-0315@Fluc mRNA, and SM-102@Fluc mRNA. The mRNA concentration of each formulation was adjusted to 0.1 mg / mL with PBS, and the osmolality of the formulation was adjusted to isotonicity. 200 μL of the solution was injected intramuscularly into the hind limbs of BALB / c mice (20 μg of FLuc mRNA per mouse, 3 mice per group). PBS served as a negative control. After administration, mice were maintained on a normal diet. 8 h after administration, 200 μL of substrate solution (15 mg / mL, luciferin potassium salt) was intraperitoneally injected (3 mg per mouse). The timing began 10 min after substrate injection, and the mice were placed in a gas anesthesia system. After complete anesthesia, the bioluminescence intensity of the entire body was detected using an IVIS device. The exposure time was 60 s. After imaging, the total fluorescence (total flux) of each organ was calculated. The results are shown in Figures 2(im) and 3(im).

[0170] The experimental results of in vivo expression testing via intramuscular injection showed that the ionizable lipid of compound 8 had stronger mRNA expression ability than the three positive controls in terms of the total in vivo luminescence intensity via intramuscular injection. It was also discovered that the LNPs@mRNA produced with the lipid of the present invention had a certain expression level in the spleen, which is useful for the research and development of intramuscularly administered tumor vaccines.

[0171] 3. Immune activation efficacy of LNPs@Delta S-2P mRNA Following the method of Example 3, LNPs@Delta S-2P mRNA was prepared using Compound 8 and SM-102 as ionizable lipids, respectively. These are abbreviated as Compound 8 and SM-102, respectively. The commercially available lipid SM-102 was used as a positive control. In this example, Delta S-2P mRNA encodes the Delta S-2P protein shown in the amino acid sequence of SEQ ID NO: 1. Delta S-2P is a protein with the K986P and V987P mutations introduced into the full-length S protein of the Delta mutant strain B.1.617.2, and its amino acid sequence is as follows:

[0172] Male Balb / c mice aged 6-8 weeks were divided into two groups and immunized with the compound 8 and SM-102 drugs prepared above. Each group had three dose gradients, with three mice per gradient. PBS was used as a negative control (CTL) (see Table 2 for details). Two doses were administered intramuscularly, with a 14-day interval between each dose. Serum was collected 14 and 28 days after the first dose, and antibody titers against the RBD of the wild-type Delta virus S protein were detected in the serum by ELISA. The experimental results are shown in Figure 4 and Table 2. The geometric mean titers (GMTs) of the compounds were calculated.

[0173] The results showed that the mRNA vaccine prepared with compound 8 could effectively activate the host's immune system and generate specific antibodies, with a binding antibody titer of 10 6 Furthermore, compared with the positive control, the immune activation effect of the mRNA vaccine produced with compound 8 was significantly superior, with a GMT increase of more than 2-fold, reaching a maximum of 14-fold. [Table 2]

[0174] Furthermore, the immunostimulatory efficacy (antibody titer) of LNPs@Delta S-2P mRNA produced with each compound of the present invention was examined. Male Balb / c mice aged 6 to 8 weeks were immunized intramuscularly twice, with a dose of 1 μg per injection, and an interval of 14 days between the two injections. Serum was collected 28 days after the first injection, and antibody titers against the RBD of the S protein of wild-type Delta virus strains were detected in the serum by ELISA. Commercially available lipid SM-102 was used as a positive control. The geometric mean titers (GMTs) of each compound were calculated, and the experimental results are shown in Table 3. The compounds obtained in Examples 1 and 2 were used as lipids to prepare LNPs@Delta S-2P mRNA, abbreviated as Compounds 7 to 13, Compounds 15 to 19, and Compound 20, respectively. See Example 3 for specific preparation methods. Delta S-2P mRNA encodes the Delta S-2P protein with the amino acid sequence set forth in SEQ ID NO: 1.

[0175] The results showed that the mRNA vaccines prepared using the lipid materials (compounds 7 to 13, compounds 15 to 19, and compound 20) prepared in Examples 1 and 2 could effectively activate the immune system and generate specific antibodies, with a binding antibody titer of 10 6 Furthermore, compared to the positive control, the immune activation effect of the mRNA vaccines produced with each lipid material of the present invention was clearly superior, with a GMT increase rate of more than two times. [Table 3]

[0176] In the description herein, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, exemplary references to the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined as appropriate in any one or more embodiments or examples. Furthermore, unless inconsistent, those skilled in the art may associate and combine different embodiments or examples and features of different embodiments or examples described herein.

[0177] Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and do not limit the present invention, and that those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A compound represented by formula (I), or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of a compound represented by formula (I), 【Chemistry 1】 In the formula, X １ , X ２ and X ３ Each of these is independently and arbitrarily substituted C １ -C １５ It is alkylene, R １ and R ２ are each independently optionally substituted C1-C40 alkyl, optionally substituted C1-C4 ０ heteroalkyl, optionally substituted C ２ -C ４０ alkenyl, optionally substituted C ２ -C ４０ heteroalkenyl, optionally substituted C ２ -C ４０ alkynyl or optionally substituted C ２ -C ２４ heteroalkynyl, R ３ , R ４ , R ５ and R ６ Each of these is independently H, halogen, or optionally substituted C. １ -C ３ It is alkyl, The groups to be substituted are, independently, halogen, -OH, -SH, and -NH. ２ , -NO ２ , cyano or C １ -C ３ A compound that is one or more selected from alkyl groups.

2. X １ and X ２ Each is independent of C ４ -C １２ It is alkylene, Optionally, X ３ is C ２ -C ８ It is alkylene, Optionally, X simultaneously １ C ４ It is alkylene, X ２ C ４ It is alkylene, X ３ C ２ The compound according to claim 1, characterized in that it is not alkylene.

3. R ３ , R ４ , R ５ and R ６ The compound according to claim 1, characterized in that each of them is independently H or a halogen.

4. R １ and R ２ Each of them independently has the structure shown by formula (II), 【Chemistry 2】 In the formula, m is any integer between 1 and 10. R ７ and R ８ These are H and C, which are substituted independently of each other. １ -C ２０ Alkyl, optionally substituted C １ -C ２０ Heteroalkyl, optionally substituted C ２ -C ２０ Alkenyl, optionally substituted C ２ -C ２０ Heteroalkenyl, optionally substituted C ２ -C ２０ Alkynyl or optionally substituted C ２ -C ２０ It is a heteroalkynyl, and the substituted groups are, independently, halogen, -OH, -SH, and -NH. ２ , -NO ２ , cyano or C １ -C ３ The compound according to claim 1, characterized by being one or more selected from alkyl groups.

5. m is 2, 3, or 4. Optionally, R ７ and R ８ These are H and C, which are substituted independently of each other. ４ -C １８ Alkyl, optionally substituted C ４ -C １８ Heteroalkyl, optionally substituted C ４ -C １８ Alkenyl or optionally substituted C ４ -C １８ It is a heteroalkenyl, and the substituted groups are, independently, halogen, -OH, -SH, and -NH. ２ , -NO ２ , cyano or C １ -C ３ The compound according to claim 4, characterized by being one or more selected from alkyl groups.

6. R １ and R ２ Each of them independently has the structure shown by formula (III), 【Transformation 3】 In the formula, n is either 1 or 2. R ７ and R ８ are each independently H, optionally substituted C １ -C ２０ alkyl, optionally substituted C １ -C ２０ heteroalkyl, optionally substituted C ２ -C ２０ alkenyl, optionally substituted C ２ -C ２０ heteroalkenyl, optionally substituted C ２ -C ２０ alkynyl or optionally substituted C ２ -C ２０ heteroalkynyl, and the substituting groups are each independently one or more selected from halogen, -OH, -SH, -NH ２ , -NO ２ , cyano or C １ -C ３ alkyl, Optionally, R ７ and R ８ Each of these is independently and arbitrarily substituted C ４ -C １８ Alkyl, optionally substituted C ４ -C １８ Heteroalkyl, optionally substituted C ４ -C １８ Alkenyl or optionally substituted C ４ -C １８ It is a heteroalkenyl, and the substituted groups are, independently, halogen, -OH, -SH, and -NH. ２ , -NO ２ , cyano or C １ -C ３ The compound according to claim 4, characterized by being one or more selected from alkyl groups.

7. R １ and R ２ Each is independent of the others. 【Chemistry 4】 The compound according to claim 6, characterized by having at least one of the structures of the following.

8. The aforementioned compound, 【Chemistry 5-1】 【Chemistry 5-2】 The compound according to claim 1, characterized by having at least one of the structures of the following.

9. Uses of the compound according to claim 1 in the production of liposomes, drug carriers, or complexes.

10. A liposome characterized by containing the compound described in claim 1.

11. The liposomes described above are It further comprises at least one of a steroid, a neutral lipid, and a PEG-lipid. Optionally, the neutral lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine (DM It comprises at least one selected from PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM), Optionally, the PEG-lipids are 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetrail, PEG-dioleil, It comprises at least one selected from PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-phosphatidylethanolamine (PEG-PE), PEG-diacylglycerol succinate (PEG-S-DAG), PEG-ceramide (PEG-cer), PEG-dialkoxypropyl carbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), Preferably, the steroid comprises at least one selected from cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, and brassicasterol, and is preferably cholesterol, characterized in that the liposome is as described in claim 10.

12. The molar ratio of the compound:neutral lipid:steroid:PEG-lipid is (20-80):(5-50):(10-60):(0.01-10). Preferably, the liposome according to claim 11 is characterized in that the molar ratio of the compound:neutral lipid:steroid:PEG-lipid is (40-60):(5-10):(30-50):(0.5-5).

13. A liposome comprising the compound described in claim 1, a neutral lipid, cholesterol, and a PEG-lipid, wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) and / or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), the PEG-lipid is 1,2-dimiristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), and the molar ratio of the compound, the neutral lipid, the cholesterol, and the PEG-lipid is (40-60):(5-10):(30-50):(0.5-5).

14. The compound according to claim 1, or Liposomes characterized by containing the compound described in claim 1 A drug carrier characterized by containing the following:

15. The compound according to claim 1, A liposome characterized by containing the compound described in claim 1, or A drug carrier characterized by containing the compound described in claim 1, or a liposome characterized by containing the compound described in claim 1, Bioactive ingredients and, A composite material characterized by containing the following:

16. The said biologically active component comprises at least one selected from DNA molecules, RNA molecules, proteins, polypeptides, and small molecule drugs. Optionally, the bioactive component is electronegative or hydrophobic. Preferably, the biologically active component is nucleic acid. The composite according to claim 15, more preferably characterized in that the biologically active components are antisense RNA and messenger RNA.

17. The compound according to claim 1, A liposome characterized by containing the compound described in claim 1, A drug carrier characterized by containing the compound described in claim 1, or a liposome characterized by containing the compound described in claim 1, or A compound comprising: the compound described in claim 1; a liposome characterized by containing the compound described in claim 1; or a drug carrier characterized by containing the compound described in claim 1 or a liposome characterized by containing the compound described in claim 1; and a bioactive component; A pharmaceutically acceptable adjuvant, A pharmaceutical composition characterized by containing the following:

18. An application in the manufacture of a drug for a compound according to any one of claims 1 to 8, a liposome according to any one of claims 10 to 13, a drug carrier according to claim 14, a complex according to claim 15 or 16, or a pharmaceutical composition according to claim 17, wherein the drug is for targeting at least one of the heart, liver, spleen, lungs, and kidneys. Preferably, the drug is used to target the spleen.

19. Uses in the manufacture of a drug, wherein the drug is for treating or preventing a disease, the compound according to any one of claims 1 to 8, the liposome according to any one of claims 10 to 13, the drug carrier according to claim 14, the composite according to any one of claims 15 or 16, or the pharmaceutical composition according to claim 17. The use may optionally include any disease selected from tumors or tumor-related diseases, or virus-induced related diseases.

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