Cationic lipid compound, lipid nanoparticle thereof, composition thereof, preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a cationic lipid compound, lipid nanoparticles thereof, a composition thereof, a preparation method and application thereof. The cationic lipid compound disclosed by the invention has the following advantages that the lipid nanoparticles prepared from the cationic lipid compound disclosed by the invention have good in-vivo delivery efficiency on nucleic acid.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedical technologies, and particularly relates to cationic lipid compounds, their lipid nanoparticles, their compositions, and their preparation methods and uses. Background Art

[0002] As a novel therapeutic approach, gene therapy with nucleic acid drugs has achieved breakthrough progress in multiple fields such as the treatment of infectious diseases and tumors in a short period of time. Lipid nanoparticles (LNPs) are one of the most advanced carriers for safely and efficiently delivering nucleic acid drugs (such as mRNA, siRNA, etc.) to specific target organs and protecting them from degradation. They have many advantages such as high encapsulation efficiency, good cell transfection efficiency, strong tissue penetration, low cytotoxicity and immunogenicity, and have currently been successfully applied to multiple commercial products. Taking the drugs approved by the FDA as an example, the mRNA COVID-19 vaccines developed by Moderna and Pfizer-BioNTech, as well as 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 their development, and the research and development of lipid nanoparticle technology has greatly promoted the development of nucleic acid drugs. Lipid nanoparticles are currently the most well-studied nucleic acid drug delivery systems. Lipid nanoparticles usually consist of four components: ionizable lipids / phospholipids / cholesterol / PEGylated lipids. The physical and chemical stability of the lipid components affects particle size, charge, membrane fluidity, and formulation stability, which is crucial for effective LNP drug delivery.

[0004] Cationic lipid compounds have received extensive attention as nucleic acid delivery carriers. They bind to nucleic acids through electrostatic interactions and can effectively avoid nuclease degradation 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, there is an urgent need to develop a novel cationic lipid compound, its lipid nanoparticles, its composition, and its preparation methods and uses. Summary of the Invention

[0006] The present disclosure aims to develop a novel cationic lipid compound, its lipid nanoparticles, its composition, and its preparation methods and uses.

[0007] To achieve the above technical objectives, the technical solutions adopted by the present disclosure are as follows:

[0008] On the one hand, the present disclosure provides a cationic lipid compound having the structure of formula (I') or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof,

[0009]

[0010] wherein,

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

[0012] R1 and R2 are each independently selected from H, C 1-10 alkyl, C 3-10 cycloalkyl and a 3- to 10-membered heterocyclic group;

[0013] n1, n2, n3, n4, n5, n6, n7 are each independently an integer from 1 to 10;

[0014] Preferably, Ring A is selected from

[0015] On the other hand, the present disclosure provides a cationic lipid compound having the structure of formula (I) or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof,

[0016]

[0017] wherein,

[0018] R1 and R2 are each independently selected from H, C 1-10 alkyl, C 3-10 cycloalkyl and a 3- to 10-membered heterocyclic group;

[0019] n1, n2, n3, n4, n5, n6, n7 are each independently an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0020] In some embodiments, R1 and R2 are each independently selected from H, C 1-6 alkyl, C 3-7 cycloalkyl and a 3- to 7-membered heterocyclic group.

[0021] In some embodiments, R1 and R2 are each independently selected from C 1-6 alkyl.

[0022] In some embodiments, R1 and R2 are each independently selected from C 1-5 alkyl.

[0023] In some embodiments, R1 and R2 are each independently selected from C 1-3 alkyl.

[0024] In some embodiments, both R1 and R2 are methyl or ethyl.

[0025] In some embodiments, both R1 and R2 are methyl.

[0026] On the other hand, the present disclosure provides a cationic lipid compound having the structure of formula (II) or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof,

[0027]

[0028] wherein,

[0029] n1, n2, n3, n4, n5, n6, n7 are each independently an integer from 1 to 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 and n2 are each independently an integer from 1 to 8.

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

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

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

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

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

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

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

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

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

[0041] In some embodiments, n7 is 3.

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

[0043]

[0044] On the other hand, the present disclosure provides a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, characterized in that the preparation method comprises the following steps: reacting a compound of formula (IIa) with a compound of formula (IIb) to obtain a cationic lipid compound having the structure shown in formula (II);

[0045]

[0046] wherein n1, n2, n3, n4, n5, n6, n7 are each defined as described above.

[0047] On the other hand, the present disclosure provides a lipid nanoparticle comprising the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, or comprising the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof obtained by the preparation method as described above.

[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 polyethylene glycolated lipid compound.

[0050] In some embodiments, 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), RNA or 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 (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi-coding nucleic acid (MCNA), poly-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) and 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 particle size of the lipid nanoparticles is 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] On the other hand, the present disclosure provides a method for preparing the lipid nanoparticles as described above, comprising: mixing the lipid compound or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer in the lipid nanoparticles with a structural lipid, a phospholipid, and a polyethylene glycolated lipid compound, and then mixing with the payload to obtain the lipid nanoparticles.

[0058] On the other hand, the present disclosure provides a pharmaceutical composition, which comprises the cationic lipid compound as described above or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer, the cationic lipid compound or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer obtained by the preparation method as described above, or the lipid nanoparticles as described above, and optionally a pharmaceutically acceptable excipient.

[0059] On the other hand, the present disclosure provides the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof obtained by the preparation method as described above, the lipid nanoparticle as described above, or the use of the pharmaceutical composition as described above in the preparation of a drug for treating, diagnosing or preventing a disease.

[0060] On the other hand, the present disclosure provides the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof obtained by the preparation method as described above, the lipid nanoparticle as described above, or the use of the pharmaceutical composition as described above in the preparation of a drug for delivering nucleic acid. The nucleic acid is as described above and will not be elaborated herein.

[0061] On the other hand, the present disclosure provides the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof obtained by the preparation method as described above, the lipid nanoparticle as described above, or the use of the pharmaceutical composition as described above for delivering nucleic acid. The nucleic acid is as described above and will not be elaborated herein.

[0062] On the other hand, the present disclosure provides a method for treating, diagnosing or preventing a disease, which comprises the step of administering to a patient in need the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof as described above, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof obtained by the preparation method as described above, 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 nanoparticles prepared from the cationic lipid compound of the present disclosure have good in vivo delivery efficiency for nucleic acid.

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

[0066] Definitions and Explanations

[0067] To facilitate a better understanding of the present disclosure, certain technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the terms related to cell and tissue culture, microbiology, and laboratory procedures used herein are widely used terms and conventional procedures in the respective fields. Meanwhile, to better understand the present disclosure, definitions and explanations of relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and of course, changes can be made to the above. It should also be understood that the terms used in the present disclosure are only for describing specific embodiments and are not intended to be limiting.

[0068] Unless the context clearly dictates otherwise, the words "a", "an", and "the" as used in this specification and the appended claims cover one or more.

[0069] As used herein, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that comprises a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products, or devices.

[0070] In the description herein, reference to "some embodiments", "some implementations", or "some embodiments" describes 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 numerical range is listed, it is intended to include every value and sub-range within the stated 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 of the hydrogens present in a compound, or the hydrogens in a particular group or moiety within the compound, may be replaced by deuterium or tritium. One to the maximum number of hydrogens present in the compound may be replaced by deuterium. One to the maximum number of hydrogens present in any group in a general formula compound or a specific compound may be deuterated. For example, when a certain group is described as an ethyl group, the ethyl group may be C2H5 or C2H5 in which x (1 to 5) hydrogens are replaced by deuterium, such as C2D x H 5-x 。When a certain group is described as a deuterated ethyl group, the deuterated ethyl group may be C2H5 in which x (1 to 5) hydrogens are replaced by deuterium, such as C2D x H 5-x 。The stable deuterated derivatives described in the present disclosure are preferably stable deuterated isotope derivatives in which any hydrogen atom that can be deuterated in each formula is replaced by 1 to the maximum number (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, and 1 to 7, etc.) of deuterium atoms.

[0073] The present disclosure mentions the compounds of formula (I), and also includes their isotopic variants, tautomers, stereoisomers, mixtures of stereoisomers, solvates (solvates), or derivatives, etc.

[0074] The "compounds" in the present disclosure also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond and are accompanied by the migration of a proton. The terms "tautomer" or "tautomeric form" refer to different functional group isomers that are in dynamic equilibrium at room temperature and can rapidly interconvert. It refers to one of two or more structural isomers in which the equilibrium exists and it is easy to convert from one isomer form to another. This transformation results in the formal migration of a hydrogen atom and is accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric groups in solution. In a solution where tautomerization is possible, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH conditions. The concept of tautomers that can be interconverted by tautomerization is called tautomerism.

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

[0076] Among the various possible types of tautomerism, two are generally observed. In keto-enol tautomerism, electrons and hydrogen atoms move simultaneously.

[0077] Common tautomeric pairs are: keto - enol, amide - nitrile, lactam - lactam, amide - 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 atomic arrangements and configurations. 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). Accordingly, the present disclosure includes enantiomers, diastereomers, and mixtures thereof. The present disclosure further includes all mixtures of the above stereoisomers, regardless of the ratio, including racemates.

[0079] Depending on their structure, the compounds of the present disclosure can exist in various stable isotope 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 by 15N atoms, or those in which one or more of carbon, fluorine, chlorine, bromine, sulfur, or oxygen have been replaced by stable isotopes of their respective original 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 straight - chained (linear or branched) saturated aliphatic hydrocarbon group. When a numerical range is listed, each value and sub - ranges within the said range are specifically included. 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.

[0082] The term "C 1-6"Alkyl" refers to a straight-chain or branched-chain alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-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. More preferably, it is a lower alkyl (C 1-3 alkyl) containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, etc. The term "C 1-6 alkyl" also includes heteroalkyl, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. When substituted, the substituents are preferably one or more of the groups described in the present disclosure.

[0083] As used herein, "solvate" or "solvent complex" refers to a complex formed by a compound of the present disclosure and a solvent. They either react in the solvent or precipitate or crystallize out from the solvent. For example, a complex formed with water is called "hydrate". The solvates of the compounds represented by formula (I) of the present disclosure are within the scope of the present disclosure.

[0084] The term "pharmaceutically acceptable salt" as used in the present disclosure refers to those carboxylate salts and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic reactions, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) the zwitterionic form of the compounds of the present invention.

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

[0086] The base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and then separating the free acid. The free acid forms differ somewhat in certain physical properties, such as solubility in polar solvents, from their respective salt forms, but for the purposes of this invention, the salts are equivalent to their respective free acids.

[0087] The salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides prepared from inorganic acids, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. Representative salts include: hydrobromides, hydrochlorides, sulfates, bisulfates, nitrates, acetates, oxalates, valerates, oleates, palmitates, stearates, laurates, borates, benzoates, lactates, phosphates, toluenesulfonates, citrates, maleates, fumarates, succinates, tartrates, naphthoates, methanesulfonates, glucoheptonates, lactobionates, lauryl sulfonates, and isethionates, etc. The salts can also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkane diacids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, naphthoates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, methanesulfonates, etc. Pharmaceutically acceptable salts can include cations based on alkali metals and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also covered are salts of amino acids, such as arginates, gluconates, galacturonates, etc. (see, for example, 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 the equivalent, which is usually used to represent the molar multiple relationship of a certain substance; for example, 1.2 eq. refers to 1.2 times the molar amount.

[0090] Examples

[0091] To make the objectives, 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 conjunction with the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[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, etc. The materials, reagents, etc. used in the examples can be obtained from commercial sources without special instructions. The following Table 1 lists the abbreviations of common chemical substance names in the examples and comparative examples of the present disclosure.

[0093] Table 1. Abbreviations of common chemical substance names

[0094] English or abbreviation Chinese DCM Dichloromethane EtOAc Ethyl acetate DMSO Dimethyl sulfoxide DMF N,N-Dimethylformamide TFA Trifluoroacetic acid MeOH Methanol <![CDATA[CDCl3]]> Chloroform-d DMAP 4-Dimethylaminopyridine EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide <![CDATA[NaBH4]]> Sodium borohydride <![CDATA[K2CO3]]> Potassium carbonate KOH Potassium hydroxide t-BuOK Potassium tert-butoxide <![CDATA[Na2SO4]]> Sodium sulfate anhydrous TBAI Tetrabutylammonium iodide TosMIC p-Toluenesulfonylmethyl isocyanide

[0095] Example 1: Preparation of Compound A

[0096] Synthesis route of Compound A:

[0097]

[0098] Dissolve Compound 1 (4.0 g, 18.35 mmol, 1.0 eq.) and 7-tridecanol (4.64 g, 22.02 mmol, 1.2 eq.) in dichloromethane in a round-bottom flask, and then sequentially add EDCI (2.92 g, 22.02 mmol, 1.2 eq.) and DMAP (0.24 g, 1.84 mmol, 0.1 eq.). React at room temperature overnight. After the reaction is completed, quench the reaction solution with water (40 mL), extract with dichloromethane (3 x 100 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution (3 x 50 mL), and dry over anhydrous sodium sulfate. Filter to collect the organic phase, concentrate under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product, and purify by silica gel column chromatography to obtain a pale yellow oily Compound 2 (2.2 g).

[0099] Dissolve Compound 2 (2.0 g, 4.76 mmol, 1.0 eq.) in trifluoroacetic acid, stir at room temperature under nitrogen protection. After the reaction is completed, quench the reaction solution with water, adjust the pH = 7 - 8 with saturated sodium bicarbonate aqueous solution at 0 °C, extract the aqueous phase with ethyl acetate (3 x 30 mL), combine the organic phases, wash with saturated sodium chloride aqueous solution (3 x 20 mL), and dry the organic phase over anhydrous sodium sulfate. Filter to collect the filtrate, concentrate under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product, and purify by silica gel column chromatography to obtain a pale yellow oily Compound 3 (1.2 g).

[0100] In a reaction flask, 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, and then potassium hydroxide (1.8 g, 30.6 mmol, 10.0 eq.) was added. Under nitrogen protection, 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 50 mL), the organic phases were combined, washed with saturated aqueous sodium chloride solution (3 x 100 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was collected by filtration and concentrated under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product, which was purified by silica gel column chromatography to obtain colorless oily compound 4 (1.0 g).

[0101] In a reaction flask, compound 4 (900 mg, 1.62 mmol, 1.0 eq.) and TosMIC (267 mg, 1.30 mmol, 0.8 eq.) were dissolved in DMF, and then TBAI (505 mg, 1.30 mmol, 0.8 eq.) and potassium carbonate (236 mg, 1.62 mmol, 1.0 eq.) were added successively. The reaction was carried out 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), the organic phases were combined, washed with saturated aqueous sodium chloride solution (3 x 100 mL), and dried over anhydrous sodium sulfate. The organic phase was collected by filtration and concentrated under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product, which was purified by silica gel column chromatography to obtain pale yellow oily compound 5 (500 mg).

[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. Under nitrogen protection, the reaction was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was quenched with water (30 mL), 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), the organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was collected by filtration and concentrated under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product 7. The obtained crude product was redissolved in dichloromethane (15 mL), and concentrated hydrochloric acid (5 mL) was added and stirred for reaction. 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, extracted with dichloromethane (3 x 20 mL), the organic phases were combined, and dried over anhydrous sodium sulfate. The organic phase was collected by filtration and concentrated under reduced pressure using a rotary evaporator to remove the organic solvent to obtain a crude product, which was purified by silica gel column chromatography to obtain pale yellow oily compound 8 (350 mg).

[0103] In a reaction flask, compound 8 (350 mg, 0.40 mmol, 1.0 eq.) was dissolved in methanol (6 mL), and then sodium borohydride (48 mg, 1.20 mmol, 3.0 eq.) was added at 0 °C. Under nitrogen protection, the reaction was stirred for 2 hours. After the reaction was completed, it was quenched with aqueous hydrochloric acid solution (1 N), and the aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic phases were washed with saturated aqueous sodium chloride 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 concentration under reduced pressure using a rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography to obtain a pale yellow oily compound 9 (220 mg).

[0104] In a reaction flask, compound 9 (220 mg, 0.25 mmol, 1.0 eq.) and 4-dimethylaminobutyric acid (39 mg, 0.30 mmol, 1.2 eq.) were dissolved in dichloromethane, and then EDCI (73 mg, 0.38 mmol, 1.5 eq.) and DMAP (31 mg, 0.25 mmol, 1.0 eq.) were added successively. Under nitrogen protection, the reaction was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was quenched with water, extracted with ethyl acetate (3 x 20 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The organic phase was collected by filtration, and the organic solvent was removed by concentration under reduced pressure using a rotary evaporator 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; phase A: water (100 mmol / L ammonium bicarbonate) / acetonitrile = 6:4, phase B: isopropanol / acetonitrile = 9:1; flow rate: 60 mL / min; gradient: 50% B to 70% B, 12 min; 9.5 min) to obtain a pale yellow oily compound A (90.2 mg).

[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: Preparation of nanoparticles

[0107] Materials for lipid nanoparticle assembly include: (1) Cationic lipid compounds: such as the cationic lipids designed and synthesized in the present invention or DLin-MC3-DMA (purchased from AVT) as a control group; (2) Structural lipids: such as Cholesterol (purchased from Sigma-Aldrich); (3) Phospholipids: such as DSPC, which is 1,2-distearoyl-sn-glycero-3-phosphocholine (Distearoylphosphatidylcholine, purchased from AVT); (4) PEGylated lipid compounds: such as DMG-PEG2000, which is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (purchased from AVT); (5) Active ingredients of nucleic acid fragments: such as Luciferase mRNA, siRNA, CRISPR Cas 9 mRNA, etc. (commercially purchased or self-made). The names and structural formulas of the lipid nanoparticle assembly materials are shown in Table 2 in detail.

[0108] Table 2. Names and Structural Formulas of Lipid Nanoparticle Assembly Materials

[0109]

[0110]

[0111] Preparation method of lipid nanoparticles: (1) Dissolve and mix cationic lipid compounds, cholesterol, phospholipids, and PEGylated lipids in ethanol in sequence according to (mole percentage) 50%, 38.5%, 10%, and 1.5%; (2) Dissolve the active ingredient of mRNA in 25 mM sodium acetate solution (pH = 4.5); (3) Use an automated high-throughput microfluidic system to mix the organic phase dissolved with the lipid mixture and the aqueous phase dissolved with the mRNA component at a flow rate ratio in the range of 1:1 to 1:4, and the mixing speed is between 10 mL / min and 18 mL / min; (4) Dilute the prepared lipid nanoparticles (N / P ratio is 6) with phosphate buffered saline solution, and ultrafilter the nanoparticle solution with an ultrafiltration tube with a molecular weight cut-off of 30 kDa (purchased from Millipore) to the original preparation volume; (5) Filter and sterilize the obtained nanoparticles through a 0.2 μm sterile filter membrane and store them at low temperature in a sealed glass bottle.

[0112] The preparation methods of lipid nanoparticles include microfluidic mixing systems, but are not limited to this method, and also include T-type mixers and ethanol injection methods, etc.

[0113] Experimental Example 1: Characterization of Physical Properties of Lipid Nanoparticles

[0114] The particle size and particle size dispersity 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 encapsulation degree of the lipid nanoparticles for RNA was characterized by the encapsulation efficiency (Encapsulation Efficiency %), which reflects the binding degree between the lipid nanoparticles and the RNA fragments. This coefficient was measured by the method of Quant-it TM RiboGreen RNAAssay (purchased from Invitrogen). The lipid nanoparticle sample was diluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH = 7.5). A portion of the sample solution was taken and 0.5% Triton X-100 was added, and the mixture was allowed to stand at 37 °C for 30 minutes. After adding the reaction solution, the fluorescence value was immediately read on a Varioskan LUX multimode microplate reader (purchased from Thermofisher) at an absorption wavelength of 485 nm and an emission wavelength of 528 nm to obtain the encapsulation rate value.

[0115] Experimental Example 2: Animal Experiment

[0116] The delivery effect and safety of the nanoparticles encapsulating luciferase mRNA (Trilink, L-7202) in mice were evaluated. The test mice were SPF-grade C57BL / 6 mice, female, 6 - 8 weeks old, weighing 18 - 22 g, purchased from Beijing Speyford Biotechnology Co., Ltd. All animals were adaptively fed for more than 7 days before the experiment. During the experiment, they had free access to food and water, with a 12 / 12 h light / dark cycle, an indoor temperature of 20 - 26 °C, and a humidity of 40 - 70%. The mice were randomly grouped. The above-prepared lipid nanoparticles encapsulating luciferase mRNA were injected into the mice by intravenous administration at a single dose of 0.5 mg / kg mRNA. Six hours after administration, in vivo bioluminescence detection of the mice was performed using a small animal in vivo imaging system (IVIS LUMINA III, purchased from PerkinElmer). The specific operation steps for the detection were as follows: A D-luciferin solution with a concentration of 15 mg / mL was prepared with physiological saline, and the substrate was administered to each mouse by intraperitoneal injection. Ten minutes after administering the substrate, the mice were placed in an anesthesia chamber and anesthetized with isoflurane at a concentration of 2.5%. The anesthetized mice were placed in the IVIS for fluorescence imaging, and data collection and data analysis were performed on the sites where the fluorescence was concentrated.

[0117] The in vivo delivery efficiency of the lipid nanoparticle carrier is represented by the average fluorescence intensity and total number of photons of different animals within the same test group, as shown in Table 3. The higher the values of the fluorescence intensity and total number of photons, the higher the in vivo delivery efficiency of the lipid nanoparticles for the mRNA fragment. The lipid nanoparticles containing the cationic lipid compound of the present disclosure have good in vivo delivery efficiency.

[0118] Table 3

[0119]

[0120] The foregoing description of specific exemplary embodiments of the present disclosure is for purposes of illustration and exemplification. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it is obvious that many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present disclosure, as well as various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. A cationic lipid compound having the structure of formula (II) or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof wherein n1, n2, n3, n4, n5, n6, n7 are each independently an integer from 1 to 10.

2. The cationic lipid compound according to claim 1, or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, characterized in that, n1, n2, n3, n4, n5, n6, n7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, n1 and n2 are each independently an integer from 1 to 8; preferably, n1 and n2 are each independently an integer from 3 to 8; preferably, n1 and n2 are each independently an integer from 4 to 6; more preferably, n1 and n2 are both 5; Preferably, n3, n4, n5, n6 are each independently an integer from 1 to 8; preferably, n3, n4, n5, n6 are each independently an integer from 6 to 8; Preferably, n7 is an integer from 1 to 8; preferably, n7 is an integer from 1 to 5; preferably, n7 is an integer from 2 to 4.

3. The cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof according to claim 1 or 2, characterized in that, The cationic lipid compound is the following compound:

4. A method for preparing the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof according to any one of claims 1-3, characterized in that, The preparation method comprises the following steps: reacting a compound of formula (IIa) with a compound of formula (IIb) to obtain a cationic lipid compound having the structure shown in formula (II); wherein, n1, n2, n3, n4, n5, n6, n7 are as defined in any one of claims 1 - 3.

5. A lipid nanoparticle comprising the cationic lipid compound according to any one of claims 1 - 3 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, or comprising the cationic lipid compound obtained by the preparation method according to claim 4 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof; Preferably, the lipid nanoparticle optionally comprises a payload; Preferably, the lipid nanoparticle further comprises a structural lipid, a phospholipid and a polyethylene glycolated lipid compound; Preferably, the payload is selected from one or more of a therapeutic agent, a prophylactic agent or a diagnostic agent; Preferably, 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; Preferably, the nucleic acid is selected from one or more of an antisense oligonucleotide (ASO), RNA or DNA; Preferably, 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 (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi - coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self - replicating RNA (SrRNA) or ribozyme, preferably one or more of modified mRNA, mRNA, siRNA, gRNA; Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), preferably one or more of plasmid DNA (pDNA), minicircle DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multiple-copy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); Preferably, the lipid nanoparticles have 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.

6. The method for preparing the lipid nanoparticles according to claim 5, comprising: The lipid compound in the lipid nanoparticles or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof is mixed with a structural lipid, a phospholipid, and a polyethylene glycolated lipid compound, and then mixed with the payload to obtain the product.

7. A pharmaceutical composition, which comprises the cationic lipid compound according to any one of claims 1 - 3 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the preparation method according to claim 4 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, or the lipid nanoparticles according to claim 5, and optionally a pharmaceutically acceptable excipient.

8. Use of the cationic lipid compound according to any one of claims 1 - 3 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, the cationic lipid compound obtained by the preparation method according to claim 4 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, the lipid nanoparticles according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of a drug for delivering nucleic acid; Preferably, the nucleic acid is selected from one or more of antisense oligonucleotides (ASO), RNA, or DNA; Preferably, 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 (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi-coding nucleic acid (MCNA), poly-coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozyme, preferably one or more of modified mRNA, mRNA, siRNA, gRNA; Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), preferably one or more of plasmid DNA (pDNA), minicircle DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multiple-copy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); More preferably, the nucleic acid is selected from one or more of ASO, mRNA, modified mRNA, siRNA, gRNA, mcDNA, pDNA.

9. Use of the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer, or stereoisomer thereof according to any one of claims 1-3, the cationic lipid compound or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer, or stereoisomer thereof obtained by the preparation method according to claim 4, the lipid nanoparticle according to claim 5, or the pharmaceutical composition according to claim 7 for delivering nucleic acids; Preferably, the nucleic acid is selected from one or more of antisense oligonucleotides (ASO), RNA, or DNA; Preferably, 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 (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi-polycoding nucleic acid (MCNA), poly-coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), circular RNA (circRNA), self-replicating RNA (SrRNA), or ribozyme, preferably one or more of modified mRNA, mRNA, siRNA, gRNA; Preferably, the DNA is selected from one or more of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), preferably one or more of plasmid DNA (pDNA), minicircle DNA (mcDNA), complementary DNA (cDNA), chloroplast DNA (cpDNA), multiple-copy single-stranded DNA (msDNA), mitochondrial DNA (mtDNA), or ribosomal DNA (rDNA); More preferably, the nucleic acid is selected from one or more of ASO, mRNA, modified mRNA, siRNA, gRNA, mcDNA, pDNA.

10. Use of the cationic lipid compound according to any one of claims 1-3 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, the cationic lipid compound obtained by the preparation method according to claim 4 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, the lipid nanoparticle according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating, diagnosing or preventing a disease.

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