Lipid compounds, lipid nanoparticles thereof, compositions thereof, and methods of making and uses thereof

Abstract

The present disclosure belongs to the technical field of biological medicine, and particularly relates to a 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 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 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 lipid nanoparticle drug delivery systems.

[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] 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. 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 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 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 lipid compound having a structure of Formula (I), or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof,

[0009]

[0010] wherein,

[0011] R1and R2are each independently selected from H, C 1-10 alkyl, C 3-10 cycloalkyl, and 3- to 10-membered heterocyclyl;

[0012] 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.

[0013] In some embodiments, R1and R2are each independently selected from H, C 1-6 alkyl, C 3-7 cycloalkyl, and 3- to 7-membered heterocyclyl.

[0014] In some embodiments, R1and R2are each independently selected from C 1-6 alkyl.

[0015] In some embodiments, R1and R2are each independently selected from C 1-5 alkyl.

[0016] In some embodiments, R1and R2are each independently selected from C 1-3 alkyl.

[0017] In some embodiments, R1and R2are each methyl or ethyl.

[0018] In some embodiments, R1and R2are each methyl.

[0019] In another aspect, the present disclosure provides a lipid compound having a structure of Formula (II), or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof,

[0020]

[0021] wherein,

[0022] 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.

[0023] In some embodiments, n1, n2, n3, n4, n5, n6, n7are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0024] In some embodiments, n1 is an integer from 1-8.

[0025] In some embodiments, n1 is an integer from 1-5.

[0026] In some embodiments, n1 is an integer from 3-5.

[0027] In some embodiments, n1 is 4.

[0028] In some embodiments, n2, n3, n6, n7 are each independently an integer from 1-8.

[0029] In some embodiments, n2, n3, n6, n7 are each independently an integer from 3-8.

[0030] In some embodiments, n2, n3, n6, n7 are each independently an integer from 6-8.

[0031] In some embodiments, n2, n3, n6, n7 are each 7.

[0032] In some embodiments, n4, n5 are each independently an integer from 1-8.

[0033] In some embodiments, n4, n5 are each independently an integer from 3-8.

[0034] In some embodiments, n4, n5 are each independently an integer from 5-7.

[0035] In some embodiments, n4, n5 are each 6.

[0036] In some embodiments, the cationic lipid compound is the following compound:

[0037]

[0038] In another aspect, the present disclosure provides a method of preparing a lipid compound as previously described, or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, the method comprising the step of: reacting a compound of formula (IIa) with a compound of formula (IIb) to obtain a lipid compound of the structure shown in formula (II);

[0039]

[0040] wherein n1, n2, n3, n4, n5, n6, n7 are each defined as previously described.

[0041] In another aspect, the present disclosure provides a lipid nanoparticle comprising a lipid compound as previously described, or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof, or comprising a lipid compound obtained from a method of preparation as previously described, or a pharmaceutically acceptable salt, solvate, isotopologue, tautomer, or stereoisomer thereof.

[0042] In some embodiments, the lipid nanoparticle optionally comprises a payload.

[0043] In some embodiments, the lipid nanoparticle further comprises a structural lipid, a phospholipid, and a PEGylated lipid compound.

[0044] In some embodiments, the payload is selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent.

[0045] 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.

[0046] In some embodiments, the nucleic acid is selected from one or more of an antisense oligonucleotide (ASO), an RNA, or a DNA.

[0047] In some embodiments, the RNA is selected from one or more of an interfering RNA (RNAi), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense RNA (aRNA), a messenger RNA (mRNA), a modified messenger RNA (mmRNA), a long non-coding RNA (IncRNA), a microRNA (miRNA), a small activating RNA (saRNA), a polymeric coding nucleic acid (MCNA), a polymeric coding nucleic acid (PCNA), a guide RNA (gRNA), a CRISPR RNA (crRNA), a circular RNA (circRNA), a self-replicating RNA (SrRNA), or a ribozyme, preferably one or more of a modified mRNA, a mRNA, a siRNA, a gRNA.

[0048] In some embodiments, the DNA is selected from one or more of a single-stranded DNA (ssDNA), a double-stranded DNA (dsDNA), preferably one or more of a plasmid DNA (pDNA), a minicircle DNA (mcDNA), a complementary DNA (cDNA), a chloroplast DNA (cpDNA), a multicopy single-stranded DNA (msDNA), a mitochondrial DNA (mtDNA), or a ribosomal DNA (rDNA).

[0049] 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.

[0050] In another aspect, the present disclosure provides a method for preparing the lipid nanoparticle as described above, comprising: mixing a 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 cargo.

[0051] In another aspect, the present disclosure provides a pharmaceutical composition comprising a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as described above, a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof obtained by the method for preparing as described above, or a lipid nanoparticle as described above, and optionally a pharmaceutically acceptable excipient.

[0052] In another aspect, the present disclosure provides a use of a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as described above, a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof obtained by the method for preparing as described above, a lipid nanoparticle as described above, or a pharmaceutical composition as described above in the manufacture of a medicament for treating, diagnosing, or preventing a disease.

[0053] In another aspect, the present disclosure provides a use of a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as described above, a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof obtained by the method for preparing as described above, a lipid nanoparticle as described above, or a 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.

[0054] In another aspect, the present disclosure provides a use of a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as described above, a lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof obtained by the method for preparing as described above, a lipid nanoparticle as described above, or a pharmaceutical composition as described above for delivering a nucleic acid. The nucleic acid is as described above, and will not be repeated here.

[0055] 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 lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof as described above, the lipid compound obtained by the method of preparation as described above or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, the lipid nanoparticle as described above, or the pharmaceutical composition as described above.

[0056] The present disclosure has the following advantages:

[0057] (1) The lipid nanoparticle prepared from the lipid compound of the present disclosure has good in vivo delivery efficiency for nucleic acids.

[0058] (2) The lipid compound of the present disclosure is expected to provide a new strategy for nucleic acid delivery. DETAILED DESCRIPTION

[0059] Definitions and Explanations

[0060] For easier understanding of the present disclosure, certain technical and scientific terms are defined specifically below. In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the cell and tissue culture, microbiology-related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding fields. Meanwhile, for better understanding of the present disclosure, the definitions and explanations of the related terms are provided below. It should be understood that the present disclosure is not limited to the specific methods, reagents, compounds, compositions or biological systems, and changes can be made of course. It should also be understood that the terms used in the present disclosure are only for the purpose of describing the specific embodiments and are not intended to be limiting.

[0061] As used in the specification and the appended claims, "a," "an," and "the" include both singular and plural, unless the context clearly dictates otherwise.

[0062] 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 elements is not necessarily limited to only those steps or elements but can include other not expressly listed steps or elements.

[0063] In the description herein, 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 of all possible embodiments or different subsets of all possible embodiments, and can be combined with each other without conflict.

[0064] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C1to C6alkyl" is intended to include C1, C2, C3, C4, C5, C6, and sub-ranges such as C1to C2, C2to C3, C3to C4, C4to C5, C5to C6, and C1to C6. 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.

[0065] 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., C2D 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., C2D x H 5-x . 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.).

[0066] 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.

[0067] The present disclosure“compounds” also include tautomeric forms. Tautomeric forms arise from the interchange of a single bond and an adjacent double bond together with the migration of a proton. The term“tautomer” or“tautomeric forms” refers to different functional group isomers that are in dynamic equilibrium at room temperature and can rapidly interconvert. 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 tautomers in solution. In a solution where 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.

[0068] When this specification describes compounds that are susceptible to tautomerization, but only one tautomer is described, it is to be understood that all tautomers are included as part of the chemical meaning described. It is to be 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.

[0069] Of the various types of tautomerism possible, two are commonly observed. In keto-enol tautomerism, both an electron and a hydrogen atom move simultaneously.

[0070] Common tautomeric pairs are: keto-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocycles, imine-enamine, and enamine-enamine.

[0071] The term“isomer” refers to different compounds that have the same molecular formula, but different arrangements and configurations of atoms. Depending on their structure, the compounds of the present disclosure can exist in different stereoisomeric forms. These forms include configurational isomers or optical conformational isomers (enantiomers and / or diastereomers, including those of atropisomers). Thus, the present disclosure includes enantiomers, diastereomers, and mixtures thereof. The present disclosure further includes all mixtures of stereoisomers described above, regardless of how the mixture came about, including racemates.

[0072] Depending on their structure, the compounds 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 original atom.

[0073] 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.

[0074] The term "alkyl" refers to a straight chain (straight or branched) saturated aliphatic hydrocarbon group. When a numerical range is listed, it is intended to include each individual value and sub-range within the range. For example, "C1-C6alkyl" includes C1, C2, C3, C4, C5, C6, 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. The term "C 1-6 alkyl" is a straight chain or branched alkyl group comprising 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 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 (C 1-3 alkyl) containing 1 to 3 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, and the like. The term "C 1-6 alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, which when substituted are preferably one or more groups recited in the present disclosure.

[0075] "Solvate" or "solvates" as mentioned herein refers to a complex of a compound of the present disclosure with a solvent. They either react in the solvent or are precipitated out of the solvent or crystallized out of the solvent. For example, a complex with water is called "hydrate". Solvates of the compounds of the present disclosure according to Formula (I) are within the scope of the present disclosure.

[0076] The term "pharmaceutically acceptable salt" as used herein denotes those carboxylic acid salts, amino acid addition salts of the compounds of the present application 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, (where possible) the zwitterion forms of the compounds of the present application.

[0077] 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 a cation are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

[0078] Base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated by contacting the salt form with a sufficient amount of an acid to produce the free acid form. The free acid and base forms can be regenerated by

[0079] The salt can be a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acid such as hydrochloric, nitric, sulfuric, hydrobromic, hydroiodic, phosphoric acid, and the like, prepared from inorganic acids. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, gluconate, lactobionate, laurylsulphate, and isethionate, and the like. The salt 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, and the like. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, mesylate, 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 encompassed (e.g., see Berge S.M. et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0080] As used herein, "PDI" (Polydispersity Index) refers to the particle size dispersion coefficient.

[0081] As used herein, "eq." (equivalent) refers to equivalent, generally used to indicate the molar multiple relationship of a certain substance; such as 1.2 eq. refers to 1.2 times the number of moles.

[0082] Examples

[0083] In order 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 clearly and completely described below in combination with the embodiments of the present disclosure. Obviously, the described embodiments are a 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.

[0084] In the specific embodiments of the present disclosure, technical means or methods, etc. not specifically described are conventional technical means or methods in the art. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained commercially. The common chemical name abbreviations of the examples and comparative examples of the present disclosure are listed in Table 1 below.

[0085] Table 1. Common chemical name abbreviations

[0086]

[0087]

[0088] Example 1: Preparation of Compound A

[0089] Synthetic route of Compound A:

[0090]

[0091] To a solution of 8-pentadecanone (1.90 g, 8.39 mmol, 1.0 eq.) in 1,2-dichloroethane (40 mL) were added 1-Boc-piperazine (1.88 g, 10.07 mmol, 1.2 eq.), acetic acid (1.51 g, 25.17 mmol, 3.0 eq.) successively, and the reaction was allowed to react at room temperature for 2 hours under nitrogen protection. Sodium cyanoborohydride (2.11 g, 33.56 mmol, 4.0 eq.) was added at 0°C, and the system was allowed to react at room temperature for 10 hours under nitrogen protection. After the reaction was completed, water (100 mL) was added to dilute the reaction solution, dichloromethane (3 x 50 mL) was used for extraction, the combined organic phase was washed with saturated aqueous sodium chloride solution (3 x 100 mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the organic solvent was removed by a rotary evaporator to obtain a crude product, which was purified by silica gel column to obtain white solid compound 2 (1.8 g).

[0092] A solution of compound 2 (1.8 g, 4.54 mmol, 1.0 eq.) in hydrogen chloride ethyl acetate (4M, 10 mL) was stirred at room temperature for 2 hours. After the reaction was completed, white solid compound 3 (1.4 g) was obtained by concentration under reduced pressure. Without purification, it was directly used in the next step reaction.

[0093] Tert-butyl 8-bromooctanoate (2.0 g, 7.16 mmol, 1.0 eq.) and TosMIC (699 mg, 3.58 mmol, 0.5 eq.) were dissolved in DMSO (20 mL), then sodium hydride (344 mg, 8.59 mmol, 1.2 eq., 60%) and tetrabutylammonium iodide (266 mg, 0.72 mmol, 0.1 eq.) were added at 0 °C, after the addition, the system was heated to 60 °C under nitrogen protection, and the reaction was carried out for 2 hours. After the reaction was completed, water (100 mL) was added to dilute the reaction solution, dichloromethane (3 x 80 mL) was extracted, the organic phase was combined and washed with saturated sodium chloride aqueous solution (3 x 150 mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the organic solvent was removed by rotary evaporator to obtain the crude product, which was purified by silica gel column to obtain compound 5 (2.8 g) as colorless oil.

[0094] Compound 5 (2.8 g, 4.73 mmol, 1.0 eq.) was dissolved in concentrated hydrochloric acid solution (3 mL) and dichloromethane (10 mL) solution, and stirred at room temperature for 2 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution (30 mL) was added to quench the reaction, dichloromethane (3 x 30 mL) was extracted, the organic phase was combined and washed with saturated sodium chloride aqueous solution (3 x 50 mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the organic solvent was removed by rotary evaporator to obtain the crude product, which was purified by silica gel column to obtain compound 6 (862 mg) as colorless oil.

[0095] Compound 6 (862 mg, 2.02 mmol, 1.0 eq.) was dissolved in a mixture of trifluoroacetic acid (5 mL) and dichloromethane (4 mL), and stirred at room temperature for 2 hours. After the reaction was completed, water was added to quench the reaction, dichloromethane (3 x 15 mL) was extracted, the organic phase was combined and washed with saturated sodium chloride aqueous solution (2 x 30 mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and concentrated under reduced pressure to obtain compound 7 (640 mg) as white solid. Without separation and purification, it was directly used in the next reaction.

[0096] To a solution of compound 7 (640 mg, 2.04 mmol, 1.0 eq.) in dichloromethane (6 mL) were added 9-heptadecanol (523 mg, 2.04 mmol, 1.0 eq.), EDCI (587 mg, 3.06 mmol, 1.5 eq.) and DMAP (50 mg, 0.41 mmol, 0.2 eq.) successively. The reaction was stirred at room temperature for 16 hours under nitrogen atmosphere. After the reaction was completed, the reaction was quenched by water, extracted with dichloromethane (3 x 30 mL), and the combined organic phase was washed with saturated aqueous sodium chloride solution (2 x 50 mL). The organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration and concentrated under reduced pressure to obtain a crude product. The crude product was purified by high performance liquid chromatography (column: Xselect CSH F-Phenyl OBD column 30*150 mm, 5 μm; A phase: water (0.1% formic acid), B phase: acetonitrile; flow rate: 60 mL / min; gradient: 35% B to 60% B, 12 min; 7.0 min) to obtain compound 8 (380 mg) as a white solid.

[0097] A solution of compound 8 (300 mg, 0.54 mmol, 1.0 eq.) in pyridine (4 mL) was stirred at room temperature for 10 minutes under nitrogen atmosphere. To the above solution were added 1-propylphosphonic anhydride (50 wt.% in ethyl acetate) (1.72 g, 2.70 mmol, 5.0 eq.), and stirred at room temperature for 10 minutes. To the reaction mixture were added compound 3 (200 mg, 0.54 mmol, 1.0 eq.), and heated to 60°C for 3 hours. After the reaction was completed, the reaction was quenched by water, extracted with dichloromethane (3 x 30 mL), and the combined organic phase was washed with saturated aqueous sodium chloride solution (2 x 50 mL). The organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to obtain compound 9 (360 mg) as a yellow oil.

[0098] To a solution of compound 9 (360 mg, 0.43 mmol, 1.0 eq.) in methanol (18 mL) was added sodium borohydride (49 mg, 1.29 mmol, 3.0 eq.), and stirred at room temperature for 2 hours. After the reaction was completed, the reaction was diluted with water (10 mL), extracted with dichloromethane (3 x 20 mL), and the combined organic phase was washed with saturated aqueous sodium chloride solution (2 x 50 mL). The organic phase was dried over anhydrous sodium sulfate. The filtrate was collected by filtration, and the organic solvent was removed using a rotary evaporator to obtain compound 10 (280 mg) as a yellow oil. The compound was used in the next reaction without isolation and purification.

[0099] To a solution of compound 10 (270 mg, 0.32 mmol, 1.0 eq.) in dichloromethane (6 mL) were added 5-(dimethylamino)pentanoic acid (55 mg, 0.38 mmol, 1.2 eq.), EDCI (73 mg, 0.38 mmol, 1.2 eq.), DIEA (103 mg, 0.80 mmol, 2.5 eq.) and DMAP (10 mg, 0.08 mmol, 0.25 eq.) successively, and the mixture was stirred at room temperature for 16 h under nitrogen. After the reaction was completed, 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: UniHybrid8-200 C8 OBD Column 30*150 mm, 8 pm; A phase: water (100 mmol / L ammonium bicarbonate) / acetonitrile = 6:4, B phase: isopropanol / acetonitrile = 9:1; flow rate: 60 mL / min; gradient: 50% B to 70% B, 12 min; 9.5 min) to obtain compound A (110.8 mg) in the form of yellow oil.

[0100] 1H NMR (300 MHz, CDCl3) δ: 4.88-4.83 (m, 2H), 3.56 (t, J = 4.8 Hz, 2H), 3.40 (t, J = 4.8 Hz, 2H), 2.49-2.44 (m, 4H), 2.35-2.27 (m, 10H), 2.24 (s, 6H), 1.72-1.23 (m, 79H), 0.92-0.85 (m, 12H); ESI-MS m / z: 960.90 [M+H]+.

[0101] Example 2: Nanoparticle preparation

[0102] 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.

[0103] Table 2. Names and structural formulas of lipid nanoparticle assembly materials

[0104]

[0105] 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.

[0106] The preparation method of the 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.

[0107] Experimental Example 1: Physical property characterization of lipid nanoparticles

[0108] The particle size and polydispersity index (PDI) of the prepared lipid nanoparticles were measured using Zetasizer Pro (purchased from Malvern Instruments Ltd) and DynaPro NanoStar (purchased from Wyatt). The encapsulation efficiency of the lipid nanoparticles for RNA was measured by the method of RiboGreen RNA Assay (purchased from Invitrogen). The 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 with 0.5% Triton X-100 and incubated at 37°C for 30 minutes. The fluorescence value was read immediately after adding the reaction solution in the Varioskan LUX multifunctional microplate reader (purchased from Thermofisher) at an absorption light wavelength of 485 nm and an emission light wavelength of 528 nm to obtain the encapsulation efficiency value. TM The particle size and polydispersity index (PDI) of the prepared lipid nanoparticles were measured using Zetasizer Pro (purchased from Malvern Instruments Ltd) and DynaPro NanoStar (purchased from Wyatt). The encapsulation efficiency of the lipid nanoparticles for RNA was measured by the method of RiboGreen RNA Assay (purchased from Invitrogen). The 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 with 0.5% Triton X-100 and incubated at 37°C for 30 minutes. The fluorescence value was read immediately after adding the reaction solution in the Varioskan LUX multifunctional microplate reader (purchased from Thermofisher) at an absorption light wavelength of 485 nm and an emission light wavelength of 528 nm to obtain the encapsulation efficiency value. The particle size and polydispersity index (PDI) of the prepared lipid nanoparticles were measured using Zetasizer Pro (purchased from Malvern Instruments Ltd) and DynaPro NanoStar (purchased from Wyatt). The encapsulation efficiency of the lipid nanoparticles for RNA was measured by the method of RiboGreen RNA Assay (purchased from Invitrogen). The 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 with 0.5% Triton X-100 and incubated at 37°C for 30 minutes. The fluorescence value was read immediately after adding the reaction solution in the Varioskan LUX multifunctional microplate reader (purchased from Thermofisher) at an absorption light wavelength of 485 nm and an emission light wavelength of 528 nm to obtain the encapsulation efficiency value.

[0109] Experimental Example 2: Animal Experiment

[0110] The delivery effect and safety of the nanoparticles encapsulating luciferase mRNA (Trilink, L-7202) in mice were evaluated. The test mice were SPF C57BL / 6 mice, female, 6-8 weeks old, weighing 18-22 g, purchased from Beijing Sbielof Biotechnology Co., Ltd. All animals were adaptively fed for more than 7 days before the test, and free access to food and water during the test, 12 / 12h light and dark alternation, indoor temperature 20-26℃, humidity 40-70%. The mice were randomly divided into groups. The above prepared lipid nanoparticles encapsulating luciferase mRNA were injected into the mice at a single dose of 0.5 mg / kg mRNA by intravenous administration, and the mice were detected by small animal living imaging system (IVIS LUMINA III, purchased from PerkinElmer) for living bioluminescence at 6 hours after administration. The specific operation steps of the detection are as follows: D-luciferin solution with a concentration of 15 mg / mL was prepared with normal saline, and each mouse was given the substrate by intraperitoneal injection. After 10 minutes of substrate administration, the mice were placed in a narcotizing box for anesthesia with isoflurane with a concentration of 2.5%. The anesthetized mice were placed in IVIS for fluorescence imaging and data collection and analysis of the fluorescence concentrated distribution sites.

[0111] 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.

[0112] Table 3

[0113]

[0114] 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 lipid compound having the structure of formula (II) or a pharmaceutically acceptable salt thereof, (II) in, n1 is an integer between 3 and 5; n2, n3, n6, and n7 are each an independent integer between 6 and 8; n4 and n5 are each an independent integer between 5 and 7.

2. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, n1 is 4.

3. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, n2, n3, n6, and n7 are all 7.

4. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Both n4 and n5 are 6.

5. The lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The lipid compound is one of the following compounds: 。 6. The method for preparing the lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: reacting compound (IIa) with compound (IIb) to obtain a lipid compound with the structure shown in formula (II); Wherein, n1, n2, n3, n4, n5, n6, and n7 are as defined in any one of claims 1-5.

7. Lipid nanoparticles, comprising any one of the lipid compounds of claims 1-5 or pharmaceutically acceptable salts thereof, or comprising lipid compounds of claims 6 or pharmaceutically acceptable salts thereof obtained by the preparation method described in claim 6.

8. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles optionally include a load; the load is selected from one or more therapeutic agents, preventive agents, or diagnostic agents.

9. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles also include structural lipids, phospholipids, and polyethylene glycol-modified lipid compounds.

10. The lipid nanoparticles according to claim 8, characterized in that, The therapeutic agent, preventive agent, or diagnostic agent is selected from one or more small molecule compounds, peptides, proteins, and nucleic acids.

11. The lipid nanoparticles according to claim 10, characterized in that, The nucleic acid is selected from one or more of antisense oligonucleotides, RNA, or DNA.

12. The lipid nanoparticles according to claim 11, characterized in that, The RNA is selected from one or more of the following: interfering RNA, small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long noncoding RNA, microRNA, small activating RNA, polynucleotide, polymeric nucleotide, guide RNA, CRISPR RNA, circular RNA, self-replicating RNA, or ribozyme.

13. The lipid nanoparticles according to claim 11, characterized in that, The RNA is selected from one or more of modified mRNA, mRNA, siRNA, and gRNA.

14. The lipid nanoparticles according to claim 11, characterized in that, The DNA is selected from one or more of single-stranded DNA and double-stranded DNA.

15. The lipid nanoparticles according to claim 11, characterized in that, The DNA is selected from one or more of plasmid DNA, microcircular DNA, complementary DNA, chloroplast DNA, multicopy single-stranded DNA, mitochondrial DNA, or ribosomal DNA.

16. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 40-500 nm.

17. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 40-250 nm.

18. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 40-200 nm.

19. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 50-150 nm.

20. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 70-140 nm.

21. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 90-130 nm.

22. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have a particle size of 100-120 nm.

23. The method for preparing lipid nanoparticles according to claim 8, comprising: The lipid nanoparticles are then mixed with a loading agent to obtain the final product.

24. A pharmaceutical composition comprising a lipid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, a lipid compound or a pharmaceutically acceptable salt thereof obtained by the preparation method of claim 6, or lipid nanoparticles as described in any one of claims 7-22, and optionally pharmaceutically acceptable excipients.

25. Use of the lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, the lipid compound or a pharmaceutically acceptable salt thereof obtained by the preparation method of claim 6, the lipid nanoparticles of any one of claims 7-22, or the pharmaceutical composition of claim 24 in the preparation of a medicament for delivering nucleic acids.

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

27. The use according to claim 26, characterized in that, The RNA is selected from one or more of the following: interfering RNA, small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long noncoding RNA, microRNA, small activating RNA, polynucleotide, polymeric nucleotide, guide RNA, CRISPR RNA, circular RNA, self-replicating RNA, or ribozyme.

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

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

30. The use according to claim 26, characterized in that, The DNA is selected from one or more of plasmid DNA, microcircular DNA, complementary DNA, chloroplast DNA, multicopy single-stranded DNA, mitochondrial DNA, or ribosomal DNA.

31. The use according to claim 25, characterized in that, The nucleic acid is selected from one or more of ASO, mRNA, modified mRNA, siRNA, gRNA, mcDNA, and pDNA.

32. Use of the lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, the lipid compound or a pharmaceutically acceptable salt thereof obtained by the preparation method of claim 6, the lipid nanoparticles of any one of claims 7-22, or the pharmaceutical composition of claim 24 for the delivery of nucleic acids.

33. The use according to claim 32, characterized in that, The nucleic acid is selected from one or more of antisense oligonucleotides, RNA, or DNA.

34. The use according to claim 33, characterized in that, The RNA is selected from one or more of the following: interfering RNA, small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long noncoding RNA, microRNA, small activating RNA, polynucleotide, polymeric nucleotide, guide RNA, CRISPR RNA, circular RNA, self-replicating RNA, or ribozyme.

35. The use according to claim 33, characterized in that, The RNA is selected from one or more of modified mRNA, mRNA, siRNA, and gRNA.

36. The use according to claim 33, characterized in that, The DNA is selected from one or more of single-stranded DNA and double-stranded DNA.

37. The use according to claim 33, characterized in that, The DNA is selected from one or more of plasmid DNA, microcircular DNA, complementary DNA, chloroplast DNA, multicopy single-stranded DNA, mitochondrial DNA, or ribosomal DNA.

38. The use according to claim 32, characterized in that, The nucleic acid is selected from one or more of ASO, mRNA, modified mRNA, siRNA, gRNA, mcDNA, and pDNA.

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