Cationic lipid compounds, lipid nanoparticles thereof, compositions thereof, and methods of making and use thereof

Abstract

The present disclosure belongs to the technical field of biological medicine, and particularly relates to a cationic lipid compound, a lipid nanoparticle thereof, a composition thereof, and a preparation method and use thereof. The present disclosure has the following advantages: the lipid nanoparticle prepared from the cationic lipid compound of the present disclosure has good in-vivo delivery efficiency for nucleic acids.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of biological medicine, and particularly relates to a cationic lipid compound, a lipid nanoparticle thereof, a composition thereof, and a preparation method and use thereof. BACKGROUND

[0002] As a new type of therapeutic method, nucleic acid drug gene therapy has made breakthrough progress in a plurality of fields such as infectious disease and tumor treatment in a short time. Lipid nanoparticle (LNP) is one of the most advanced carriers for safely and efficiently delivering nucleic acid drugs (mRNA, siRNA, etc.) to specific target organs and protecting them from degradation, and has many advantages such as high encapsulation rate, good cell transfection efficiency, strong tissue penetration, low cytotoxicity and immunogenicity, and has been successfully applied in a plurality of commercial products. Taking FDA-approved drugs as an example, the mRNA new crown vaccines developed by Moderna and Pfizer-BioNTech, and the siRNA drug Onpattro developed by Alnylam all use a lipid nanoparticle drug delivery system.

[0003] Since the development of nucleic acid drugs, the delivery system has been the main bottleneck restricting its development, and the research and development of lipid nanoparticle technology has greatly promoted the development of nucleic acid drugs. Lipid nanoparticle is the most fully researched nucleic acid drug delivery system. Lipid nanoparticle is usually composed of four components: ionizable lipid / phospholipid / cholesterol / PEGylated lipid. The physical and chemical stability of the lipid component affects the particle size, charge, membrane fluidity and formulation stability, and is crucial for effective LNP drug delivery.

[0004] Cationic lipid compounds as nucleic acid delivery carriers have attracted widespread attention, which can effectively avoid nuclease degradation by combining with nucleic acids through electrostatic interaction and deliver nucleic acid drugs to the cytoplasm. The in vivo delivery efficiency of lipid nanoparticles is of great significance in drug delivery and nucleic acid therapy. Cationic lipid compounds play a key role in lipid nanoparticles, and can significantly affect their in vivo delivery efficiency.

[0005] Therefore, it is urgent to develop a new type of cationic lipid compound, a lipid nanoparticle thereof, a composition thereof, and a preparation method and use thereof. SUMMARY

[0006] The present disclosure aims to develop a new type of cationic lipid compound, a lipid nanoparticle thereof, a composition thereof, and a preparation method and use thereof.

[0007] To achieve the above technical purpose, the technical solution adopted by the present disclosure is:

[0008] In one aspect, the present disclosure provides a cationic lipid compound having the structure of Formula (I’), or a pharmaceutically acceptable salt, solvate, isotopically enriched variant, tautomer, or stereoisomer thereof,

[0009]

[0010] wherein,

[0011] Ring A is a 5-membered heteroaryl, preferably a 5-membered nitrogen heteroaryl, preferably a diazolyl;

[0012] R1and R2are each independently selected from H, C 1-10 alkyl, C 3-10 cycloalkyl, and 3- to 10-membered heterocyclyl;

[0013] n1, n2, n3, n4, n5, n6, n7are each independently an integer from 1-10;

[0014] Preferably, Ring A is selected from

[0015] In another aspect, the present disclosure provides a cationic lipid compound having the structure of Formula (I), or a pharmaceutically acceptable salt, solvate, isotopically enriched variant, tautomer, or stereoisomer thereof,

[0016]

[0017] wherein,

[0018] R1and R2are each independently selected from H, C 1-10 alkyl, C 3-10 cycloalkyl, and 3- to 10-membered heterocyclyl;

[0019] n1, n2, n3, n4, n5, n6, n7are each independently an integer from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0020] In some embodiments, R1and R2are each independently selected from H, C 1-6 alkyl, C 3-7 cycloalkyl, and 3- to 7-membered heterocyclyl.

[0021] In some embodiments, R1and R2are each independently selected from C 1-6 alkyl.

[0022] In some embodiments, R1and R2are each independently selected from C 1-5 alkyl.

[0023] In some embodiments, R1and R2are each independently selected from C 1-3 alkyl.

[0024] In some embodiments, R1and R2are each methyl or ethyl.

[0025] In some embodiments, R1and R2are each methyl.

[0026] In another aspect, the present disclosure provides a cationic lipid compound having a structure of Formula (II), or a pharmaceutically acceptable salt, solvate, isotopically enriched variant, tautomer, or stereoisomer thereof,

[0027]

[0028] wherein,

[0029] n1, n2, n3, n4, n5, n6, n7 are each independently an integer from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0030] In some embodiments, n1, n2, n3, n4, n5, n6, n7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0031] In some embodiments, n1, n2 are each independently an integer from 1-8.

[0032] In some embodiments, n1, n2 are each independently an integer from 3-8.

[0033] In some embodiments, n1, n2 are each independently an integer from 4-6.

[0034] In some embodiments, n1, n2 are each 5.

[0035] In some embodiments, n3, n4, n5, n6 are each independently an integer from 1-8.

[0036] In some embodiments, n3, n4, n5, n6 are each independently an integer from 6-8.

[0037] In some embodiments, n3, n4, n5, n6 are each 7.

[0038] In some embodiments, n7 is an integer from 1-8.

[0039] In some embodiments, n7 is an integer from 1-5.

[0040] In some embodiments, n7 is an integer from 2-4.

[0041] In some embodiments, n7 is 3.

[0042] In some embodiments, the cationic lipid compound is the following compound:

[0043]

[0044] In another aspect, the present disclosure provides a method of preparing a cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof as previously described, characterized in that the method of preparing comprises the step of reacting a compound of formula (IIa) with a compound of formula (IIb) to obtain a cationic lipid compound of the structure of formula (II):

[0045]

[0046] wherein each of n1, n2, n3, n4, n5, n6, n7 is defined as previously described.

[0047] In another aspect, the present disclosure provides a lipid nanoparticle comprising a cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof as previously described, or comprising a cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof obtained by a method of preparation as previously described.

[0048] In some embodiments, the lipid nanoparticle optionally comprises a payload.

[0049] In some embodiments, the lipid nanoparticle further comprises a structural lipid, a phospholipid, and a PEGylated lipid compound.

[0050] In some embodiments, wherein the payload is selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

[0051] In some embodiments, the therapeutic agent, prophylactic agent, or diagnostic agent is selected from one or more of a small molecule compound, a polypeptide, a protein, a nucleic acid.

[0052] In some embodiments, the nucleic acid is selected from one or more of an antisense oligonucleotide (ASO), an RNA, or a DNA.

[0053] In some embodiments, the RNA is selected from one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (IncRNA), microRNA (miRNA), small activating RNA (saRNA), multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozyme.

[0054] In some embodiments, the RNA is one or more of modified mRNA, mRNA, siRNA, gRNA.

[0055] In some embodiments, the DNA is selected from one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), preferably one or more of plasmid DNA (pDNA), minicircle DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multi-copy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA).

[0056] In some embodiments, the lipid nanoparticle has a particle size of 40-500 nm, preferably 40-250 nm, preferably 40-200 nm, more preferably 50-150 nm, preferably 70-140 nm, preferably 90-130 nm, preferably 100-120 nm.

[0057] In another aspect, the present disclosure provides a method for preparing the lipid nanoparticle as previously described, comprising: mixing the lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof in the lipid nanoparticle with a structural lipid, a phospholipid, a PEGylated lipid compound, and then mixing with a payload, to obtain the lipid nanoparticle.

[0058] In another aspect, the present disclosure provides a pharmaceutical composition comprising the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as previously described, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof obtained by the method for preparing as previously described, or the lipid nanoparticle as previously described, and optionally a pharmaceutically acceptable excipient.

[0059] In another aspect, the present disclosure provides a use of the cationic lipid compound as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the method of preparation as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the lipid nanoparticle as described above, or the pharmaceutical composition as described above in the manufacture of a medicament for treating, diagnosing, or preventing a disease.

[0060] In another aspect, the present disclosure provides a use of the cationic lipid compound as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the method of preparation as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the lipid nanoparticle as described above, or the pharmaceutical composition as described above in the manufacture of a medicament for delivering a nucleic acid. The nucleic acid is as described above, and will not be repeated here.

[0061] In another aspect, the present disclosure provides a use of the cationic lipid compound as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the method of preparation as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the lipid nanoparticle as described above, or the pharmaceutical composition as described above in the manufacture of a medicament for delivering a nucleic acid. The nucleic acid is as described above, and will not be repeated here.

[0062] In another aspect, the present disclosure provides a method of treating, diagnosing, or preventing a disease, comprising the step of administering to a patient in need thereof the cationic lipid compound as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the method of preparation as described above, or a pharmaceutically acceptable salt, solvate, isotopically-labeled variant, tautomer, or stereoisomer thereof, the lipid nanoparticle as described above, or the pharmaceutical composition as described above.

[0063] The present disclosure has the following advantages:

[0064] (1) The lipid nanoparticle prepared from the cationic lipid compound of the present disclosure has good in vivo delivery efficiency for nucleic acids.

[0065] (2) The cationic lipid compound of the present disclosure is expected to provide a new strategy for nucleic acid delivery. DETAILED DESCRIPTION

[0066] Definitions and Descriptions

[0067] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. In the present disclosure, the scientific and technical terms used herein have the meanings that are commonly understood by one of ordinary skill in the art unless indicated otherwise. Also, the terms and procedures employed in cell and tissue culture, microbiology and molecular biology are those typically used by persons skilled in the art and are described and explained throughout the present disclosure. Also, the nomenclature used in connection with, and the laboratory procedures and techniques of, cellular and molecular biology, which are described in the present disclosure, are those conventionally used by persons skilled in the art. Consistent with the present disclosure, the following terms are provided below. It should be understood that this disclosure is not limited to the particular methodology, reagents, compounds, compositions, or biological systems, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0068] As used in the specification and the appended claims, "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of steps or

[0070] In the description of the present disclosure, reference is made to "some embodiments," "some implementations," or "some aspects," which describe a subset of all possible embodiments, but it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments, and can be combined with each other without conflict.

[0071] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0072] In any embodiment, any or all hydrogens present in a compound, or in a particular group or moiety within a compound, can be replaced by deuterium or tritium. One to the maximum number of hydrogens present in a compound of the general formula or in any group in a specific compound can be replaced by deuterium. For example, when a group is described as ethyl, the ethyl group can be C2H5or C2H5in which x (1 to 5) hydrogens are replaced by deuterium, e.g., C2D5 x H 5-x When a group is described as deuterated ethyl, the deuterated ethyl group can be C2H5in which x (1 to 5) hydrogens are replaced by deuterium, e.g., C2D5 x H 5-x The stable deuterated derivatives described in the present disclosure are preferably stable deuterated isotopic derivatives obtained by replacing any hydrogen atom in the general formula that can be deuterated with 1 to the maximum number of deuterium atoms (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.).

[0073] The present disclosure refers to a compound of Formula (I), and also includes isotopic variants, tautomers, stereoisomers, mixtures of stereoisomers, solvates (solvates) or derivatives thereof, and the like.

[0074] The present disclosure “compound” also includes tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond together with the migration of a proton. The term “tautomer” or “tautomer form” refers to different functional group isomers that are in dynamic equilibrium at room temperature and can quickly interconvert. It refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This transformation results in the formal migration of a hydrogen atom and is accompanied by the conversion of an adjacent conjugated double bond. Tautomers exist as a mixture of tautomeric forms in solution. In a solution in which tautomerization can occur, a chemical equilibrium of tautomers will be reached. The exact proportions of tautomers depend on several factors, including temperature, solvent, and pH conditions. The concept of tautomers that can interconvert by tautomerization is known as tautomerism.

[0075] When the present specification describes a compound that is susceptible to tautomerization, but only one of the tautomers is described, it is understood that all tautomers are included as part of the chemical meaning described. It is understood that when a compound has tautomeric forms, all tautomeric forms are intended to be included, and the naming of the compound does not exclude any tautomeric form.

[0076] Of the various types of tautomerism possible, two are commonly observed. In keto-enol tautomerism, both an electron and a hydrogen atom move simultaneously.

[0077] Common tautomeric pairs are: keto-enol, amide-nitrile, lactam-lactim, amidine-imidic acid tautomerism in heterocycles, imine-enamine, and enamine-enamine.

[0078] The term "isomer" refers to different compounds that have the same molecular formula, but different arrangements and configurations of atoms. Depending on its structure, a compound of the present disclosure can exist in different stereoisomeric forms. These forms include configurational isomers or optical conformational isomers (enantiomeric and / or diastereomeric, including those of atropisomers). Thus, the present disclosure includes enantiomers, diastereomers, and mixtures thereof. The present disclosure further includes all mixtures of the above stereoisomers, whether racemic or otherwise.

[0079] Depending on its structure, a compound of the present disclosure can exist in various stable isotopic forms. These forms include those in which one or more hydrogen atoms have been replaced by deuterium atoms, those in which one or more nitrogen atoms have been replaced by15N atoms, or those in which one or more carbon, fluorine, chlorine, bromine, sulfur, or oxygen atoms have been replaced by stable isotopes of the respective atoms.

[0080] Some compounds and salts according to the present disclosure can exist in different crystalline forms (polymorphs), which are within the scope of the present disclosure.

[0081] The term "alkyl" refers to a chain (straight-chained or branched) saturated aliphatic hydrocarbon group. When a numerical range is listed, it is intended to include each value and sub-range within the range. For example, "C1-C6alkyl" includes C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6alkyl. 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.

[0082] The term "C 1-6"Alkyl" is a straight-chain or branched chain alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof, and the like. More preferred is lower alkyl containing 1 to 3 carbon atoms (C1-C3alkyl). 1-3 "Alkyl" is a straight-chain or branched chain alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof, and the like. More preferred is lower alkyl containing 1 to 3 carbon atoms (C1-C3alkyl). 1-6 "Alkyl" is a straight-chain or branched chain alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof, and the like. More preferred is lower alkyl containing 1 to 3 carbon atoms (C1-C3alkyl).

[0083] "Solvate" or "solvate forms" mentioned in the present disclosure refers to a compound of the present disclosure forming a coordinate covalent bond with a solvent. They are either reacted in a solvent or precipitated out of a solvent or crystallized out of a solvent. For example, a coordinate covalent bond with water is called "hydrate". Solvates of the compounds of the present disclosure represented by Formula (I) are within the scope of the present disclosure.

[0084] The term "pharmaceutically acceptable salt" as used herein denotes those carboxylate salts, amino acid addition salts of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without an undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, including the zwitterionic forms, where possible, of the compounds of the present disclosure.

[0085] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals, hydrides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

[0086] Base addition salts of acidic compounds can be prepared by conventional means by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid can be regenerated by contacting the salt form with an acid and isolating the free acid. The free acid form differs from the salt form somewhat in certain physical properties, such as solubility in polar solvents, but otherwise the salt and free acid forms are equivalent for the purposes of the present invention.

[0087] Salts can be prepared from inorganic acids such as sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include the following: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate, and isethionate, and the like. Salts can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkyldioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthalene-2- carboxylate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Pharmaceutically acceptable salts can include cations based on alkali and alkaline earth metals, for example, sodium, lithium, potassium, calcium, magnesium, and aluminum, as well as ammonium, quaternary ammonium, and amine cations such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids can also be included, such as arginate, gluconate, galacturonate, and the like (see, e.g., Berge S.M. et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0088] As used herein, "PDI" (Polydispersity Index) refers to the particle size dispersion coefficient.

[0089] As used herein, "eq." (equivalent) refers to equivalent weight, generally used to express the molar relationship of a certain substance; for example, 1.2 eq. refers to 1.2 times the molar amount.

[0090] Examples

[0091] To make the purposes, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below in conjunction with the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited to this.

[0092] In the specific embodiments of the present disclosure, technical means or methods not specifically described are conventional technical means or methods in the art. The materials, reagents and the like used in the embodiments, unless otherwise specified, can be obtained from commercial channels. The common chemical substance name abbreviations in the following Table 1 are listed.

[0093] Table 1. Common chemical substance name abbreviations

[0094] English or abbreviation Chinese DCM dichloromethane EtOAc ethyl acetate DMSO dimethyl sulfoxide DMF N,N-dimethylformamide TFA trifluoroacetic acid MeOH methanol CDCl3 deuterated chloroform DMAP 4-dimethylaminopyridine EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide NaBH4 sodium borohydride K2CO3 potassium carbonate KOH potassium hydroxide t-BuOK potassium tert-butoxide Na2SO4 anhydrous sodium sulfate TBAI tert-butylammonium iodide TosMIC p-toluenesulfonylmethyl isocyanide

[0095] Example 1: Preparation of compound A

[0096] Synthetic route of compound A:

[0097]

[0098] In a round-bottom flask, compound 1 (4.0 g, 18.35 mmol, 1.0 eq.) and 7-tridecanol (4.64 g, 22.02 mmol, 1.2 eq.) were dissolved in dichloromethane, and then EDCI (2.92 g, 22.02 mmol, 1.2 eq.) and DMAP (0.24 g, 1.84 mmol, 0.1 eq.) were added in sequence. The reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was quenched with water (40 mL), extracted with dichloromethane (3 x 100 mL), and the organic phases were combined and washed with saturated aqueous sodium chloride solution (3 x 50 mL) and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 2 (2.2 g) as a light yellow oil.

[0099] Compound 2 (2.0 g, 4.76 mmol, 1.0 eq.) was dissolved in trifluoroacetic acid, and stirred at room temperature under nitrogen protection. After the reaction was completed, the reaction solution was quenched with water, and the pH was adjusted to 7-8 at 0°C with saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the organic phases were combined and washed with saturated aqueous sodium chloride solution (3 x 20 mL) and dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography to obtain compound 3 (1.2 g) as a light yellow oil.

[0100] Compound 3 (1.0 g, 3.06 mmol, 1.0 eq.) and 1,7-dibromoheptane (3.3 g, 12.24 mmol, 4.0 eq.) were dissolved in DMSO in a reaction flask, and then potassium hydroxide (1.8 g, 30.6 mmol, 10.0 eq.) was added. The reaction was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was quenched with water (50 mL), extracted with ethyl acetate (3 x 50 mL), and the organic phases were combined and washed with saturated aqueous sodium chloride solution (3 x 100 mL). The organic phase was dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. Purification by silica gel column chromatography gave compound 4 (1.0 g) as a colorless oil.

[0101] Compound 4 (900 mg, 1.62 mmol, 1.0 eq.) and TosMIC (267 mg, 1.30 mmol, 0.8 eq.) were dissolved in DMF in a reaction flask, and then TBAI (505 mg, 1.30 mmol, 0.8 eq.) and potassium carbonate (236 mg, 1.62 mmol, 1.0 eq.) were sequentially added. The reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was quenched with water (50 mL), extracted with ethyl acetate (3 x 80 mL), and the organic phases were combined and washed with saturated aqueous sodium chloride solution (3 x 100 mL). The organic phase was dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. Purification by silica gel column chromatography gave compound 5 (500 mg) as a light yellow oil.

[0102] Compound 5 (500 mg, 0.86 mmol, 1.3 eq.) and potassium tert-butoxide (116 mg, 0.98 mmol, 1.5 eq.) were dissolved in DMF, and then compound 6 (450 mg, 0.65 mmol, 1.0 eq.) was added at 0°C. The reaction was stirred at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was quenched with water (30 mL), and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined, washed with saturated aqueous sodium chloride solution (3 x 80 mL), and the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product 7. The obtained crude product was dissolved in dichloromethane (15 mL), and concentrated hydrochloric acid (5 mL) was added and stirred. After the reaction was completed, the reaction solution was quenched with water, and the pH was adjusted to 7-8 with saturated aqueous sodium bicarbonate solution at 0°C. The organic phase was extracted with dichloromethane (3 x 20 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. Purification by silica gel column chromatography gave compound 8 (350 mg) as a light yellow oil.

[0103] In a reaction flask, compound 8 (350 mg, 0.40 mmol, 1.0 eq.) was dissolved in methanol (6 mL), then sodium borohydride (48 mg, 1.20 mmol, 3.0 eq.) was added at 0 °C. The reaction was stirred for 2 hours under nitrogen protection. After the reaction was completed, the reaction was quenched by adding hydrochloric acid aqueous solution (1 N), and the aqueous phase was extracted with ethyl acetate (3 x 20 mL), and the combined organic phase was washed with saturated sodium chloride aqueous solution (3 x 50 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product, which was purified by silica gel column to obtain compound 9 (220 mg) as a light yellow oil.

[0104] In a reaction flask, compound 9 (220 mg, 0.25 mmol, 1.0 eq.) and 4-dimethylamino butyric acid (39 mg, 0.30 mmol, 1.2 eq.) were dissolved in dichloromethane, then EDCI (73 mg, 0.38 mmol, 1.5 eq.) and DMAP (31 mg, 0.25 mmol, 1.0 eq.) were added in turn. The reaction was stirred at room temperature for 12 hours under nitrogen protection. After the reaction was completed, the reaction was quenched by adding water, extracted with ethyl acetate (3 x 20 mL), and the combined organic phase was dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain a crude product, which was purified by high performance liquid chromatography (column: UniHybrid 8-200C8 OBD Column, 30 x 150 mm, 8 μm; A phase: water (100 mmol / L ammonium bicarbonate) / acetonitrile = 6:4, B phase: isopropyl alcohol / acetonitrile = 9:1; flow rate: 60 mL / min; gradient: 50% B to 70% B, 12 min; 9.5 min) to obtain compound A (90.2 mg) as a light yellow oil.

[0105] 1 H NMR (400 MHz, CDCl3) δ: 7.88 (s, 1H), 7.86 (s, 1H), 5.07-5.01 (m, 1H), 4.89-4.82 (m, 2H), 4.11 (t, J = 7.2 Hz, 2H), 2.40-2.24 (m, 12H), 1.88-1.76 (m, 4H), 1.62-1.24 (m, 70H), 0.93-0.87 (m, 12H); ESI-MS m / z: 917.80 [M+H] + .

[0106] Example 2: Nanoparticle preparation

[0107] Materials for lipid nanoparticle assembly: (1) Cationic lipid compound: cationic lipid designed and synthesized according to the present application or DLin-MC3-DMA (purchased from AVT) as a control group; (2) Structural lipid: Cholesterol (purchased from Sigma-Aldrich); (3) Phospholipid: DSPC as 1,2-distearoyl-SN-glycero-3-phosphocholine (purchased from AVT); (4) PEGylated lipid compound: DMG-PEG2000 as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (purchased from AVT); (5) Nucleic acid fragment active ingredient: LuciferasemRNA, siRNA, CRISPR Cas 9mRNA, etc. (commercially available or self-made). The names and structural formulas of the lipid nanoparticle assembly materials are shown in Table 2.

[0108] Table 2. Names and structural formulas of lipid nanoparticle assembly materials

[0109]

[0110]

[0111] Method for preparing lipid nanoparticles: (1) Dissolve and mix cationic lipid compound, cholesterol, phospholipid and PEGylated lipid in ethanol in the order of 50%, 38.5%, 10% and 1.5% (molar percentage), respectively; (2) Dissolve mRNA active ingredient in 25mM sodium acetate solution (pH = 4.5); (3) Use an automated high-throughput microfluidic system to mix the organic phase containing the lipid mixture and the aqueous phase containing the mRNA component at a flow rate ratio ranging from 1:1 to 1:4, and the mixing speed is 10mL / min to 18mL / min; (4) Dilute the prepared lipid nanoparticles (N / P ratio of 6) with phosphate buffered saline solution, and use an ultrafiltration tube with a molecular weight cutoff of 30kDa (purchased from Millipore) to ultrafiltrate the nanoparticle solution to the original preparation volume; (5) The obtained nanoparticles are filtered through a 0.2μm sterile filter membrane, and then stored in a sealed glass bottle at low temperature.

[0112] The method for preparing lipid nanoparticles includes a microfluidic mixing system, but is not limited to this method, and also includes a T-type mixer and an ethanol injection method, etc.

[0113] Experimental Example 1: Physical property characterization of lipid nanoparticles

[0114] The particle size and particle size distribution index (PDI) of the prepared lipid nanoparticles were measured using a Zetasizer Pro (purchased from Malvern Instruments Ltd) and a DynaPro NanoStar (purchased from Wyatt) dynamic light scattering instrument. The degree of RNA encapsulation by the lipid nanoparticles was characterized by the encapsulation efficiency (%), which reflects the degree of binding between the lipid nanoparticles and the RNA fragments. This coefficient was obtained from Quant-it... TM The RiboGreen RNAAssay (purchased from Invitrogen) method was used for measurement. Lipid nanoparticle samples were diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and a portion of the sample solution was added to 0.5% Triton X-100 and incubated at 37°C for 30 minutes. [The remaining text appears to be incomplete and requires further context.] Immediately after the reaction, the fluorescence values ​​were read using a Varioskan LUX multi-functional microplate reader (purchased from Thermofisher) at an absorption wavelength of 485 nm and an emission wavelength of 528 nm to obtain the encapsulation rate.

[0115] Experiment Example 2: Animal Experiment

[0116] The delivery efficiency and safety of nanoparticles loaded with luciferase mRNA (Trilink, L-7202) in mice were evaluated. SPF-grade female C57BL / 6 mice, 6-8 weeks old and weighing 18-22g, were purchased from Beijing Spefol Biotechnology Co., Ltd. All animals underwent acclimatization for at least 7 days prior to the experiment, with free access to food and water, 12 / 12-hour light / dark cycles, an indoor temperature of 20-26℃, and a humidity of 40-70%. Mice were randomly assigned to groups. The prepared lipid nanoparticles loaded with luciferase mRNA were administered intravenously to mice at a single dose of 0.5 mg / kg mRNA. Six hours after administration, in vivo bioluminescence detection was performed using a small animal in vivo imaging system (IVIS LUMINA III, purchased from PerkinElmer). The specific procedure was as follows: a 15 mg / mL D-luciferin solution was prepared with physiological saline, and the substrate was administered to each mouse via intraperitoneal injection. Ten minutes after administration of the substrate, mice were anesthetized in an anesthesia box with 2.5% isoflurane. Anesthetized mice were then placed in an IVIS (In Vitro Irradiation System) for fluorescence imaging, and data were collected and analyzed from areas of concentrated fluorescence distribution.

[0117] The in vivo delivery efficiency of the lipid nanoparticle carriers was expressed as the average of the fluorescence intensity and total photon number of different animals within the same test group, as shown in Table 3. The higher the numerical value of the fluorescence intensity and total photon number, the higher the in vivo delivery efficiency of the lipid nanoparticle for the mRNA fragment. The lipid nanoparticle containing the cationic lipid compound of the present disclosure has good in vivo delivery efficiency.

[0118] Table 3

[0119]

[0120] The foregoing description of specific exemplary embodiments of the present disclosure is intended to be illustrative only and is not intended to limit the present disclosure to the precise forms disclosed. Many modifications and variations are possible in light of the above teachings without departing from the intended scope of the present disclosure. It is, therefore, to be understood that it is intended to cover all such modifications and variations as fall within the scope of the claimed disclosure. It is intended, for example, that the scope of the present disclosure extend to uses of the disclosed cationic lipid compounds in the preparation of a medicament for the treatment of a disease or condition.

Claims

1. A cationic lipid compound having a structure of formula (II) or a pharmaceutically acceptable salt thereof: wherein, (I) n1, n2 are each independently an integer from 4 to 6; n3, n4, n5, n6 are each independently an integer from 6 to 8; n7 is an integer from 2 to 4. n1, n2 are each 5.

2. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, n3, n4, n5, n6 are each 7.

3. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, n7 is 3.

4. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The cationic lipid compound is the following compound:

5. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following step: reacting a compound of formula (IIa) with a compound of formula (IIb) to obtain a cationic lipid compound having a structure of formula (II). 。 6. A method of preparing the cationic lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized by, wherein n1, n2, n3, n4, n5, n6, n7 are as defined in any one of claims 1-5.

7. A lipid nanoparticle comprising the cationic lipid compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, or comprising the cationic lipid compound obtained by the preparation method of claim 6 or a pharmaceutically acceptable salt thereof. The lipid nanoparticle optionally comprises a payload; the payload is selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

8. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle further comprises a structural lipid, a phospholipid, and a PEGylated lipid compound.

9. The lipid nanoparticle of claim 7, wherein, The therapeutic agent, prophylactic agent, or diagnostic agent is selected from one or more of a small molecule compound, a polypeptide, a protein, a nucleic acid.

10. The lipid nanoparticle of claim 8, wherein, The nucleic acid is selected from one or more of an antisense oligonucleotide, an RNA, or a DNA.

11. The lipid nanoparticle of claim 10, wherein, The RNA is selected from one or more of an interfering RNA, a small interfering RNA, a short hairpin RNA, an antisense RNA, a messenger RNA, a modified messenger RNA, a long non-coding RNA, a microRNA, a polymeric coding nucleic acid, a guide RNA, a CRISPR RNA, or a circular RNA.

12. The lipid nanoparticle of claim 11, wherein, The RNA is selected from one or more of a modified mRNA, an mRNA, an siRNA, a gRNA.

13. The lipid nanoparticle of claim 11, wherein, The DNA is selected from one or more of a single-stranded DNA, a double-stranded DNA.

14. The lipid nanoparticle of claim 11, wherein, The DNA is selected from one or more of a plasmid DNA, a minicircle DNA, a complementary DNA, a chloroplast DNA, a multicopy single-stranded DNA, a mitochondrial DNA, or a ribosomal DNA.

15. The lipid nanoparticle of claim 11, wherein, The lipid nanoparticle has a particle size of 40-500 nm.

16. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 40-250 nm.

17. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 40-200 nm.

18. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 50-150 nm.

19. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 70-140 nm.

20. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 90-130 nm.

21. The lipid nanoparticle of claim 7, wherein, The lipid nanoparticle has a particle size of 100-120 nm.

22. The lipid nanoparticle of claim 7, wherein, The lipid compound or a pharmaceutically acceptable salt thereof in the lipid nanoparticle is mixed with the structural lipid, the phospholipid, the PEGylated lipid compound, and then mixed with the payload.

23. The method of preparing a lipid nanoparticle of any one of claims 7-22, comprising: ​ 24. A pharmaceutical composition comprising the cationic lipid compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, the cationic lipid compound obtained by the production method of claim 6 or a pharmaceutically acceptable salt thereof, or the lipid nanoparticle of any one of claims 7 to 22, and optionally a pharmaceutically acceptable excipient.

25. Use of the cationic lipid compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, the cationic lipid compound obtained by the production method of claim 6 or a pharmaceutically acceptable salt thereof, the lipid nanoparticle of any one of claims 7 to 22, or the pharmaceutical composition of claim 24 for the manufacture of a medicament for the delivery of a nucleic acid.

26. Use according to claim 25, characterized in that, The nucleic acid is selected from one or more of an antisense oligonucleotide, RNA, or DNA.

27. Use according to claim 26, characterized in that, The RNA is selected from one or more of an interfering RNA, small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long non-coding RNA, microRNA, polymeric coding nucleic acid, guide RNA, CRISPR RNA, or circular RNA.

28. The use according to claim 26, characterized in that The RNA is selected from one or more of a modified mRNA, mRNA, siRNA, gRNA.

29. The use according to claim 26, characterized in that, The DNA is selected from one or more of single-stranded DNA, double-stranded DNA.

30. The use of claim 26, wherein, The DNA is selected from one or more of plasmid DNA, minicircle DNA, complementary DNA, chloroplast DNA, multicopy single-stranded DNA, mitochondrial DNA, or ribosomal DNA.

31. The use of claim 26, wherein, The nucleic acid is selected from one or more of an ASO, mRNA, modified mRNA, siRNA, gRNA, mcDNA, pDNA.

Images