Ionizable cationic lipid compound and application thereof

Abstract

The invention provides an ionizable cationic lipid compound with a structure as shown in formula (I). The ionizable cationic lipid compound can be used for preparing lipid nanoparticles (LNP) for delivering therapeutic agents and / or prophylactic agents. The LNP prepared by using the ionizable cationic lipid compound of the present invention has good stability and transfection efficiency, and can efficiently and stably deliver bioactive substances (including nucleic acids such as mRNA) to target cells or organs, thereby causing high-specificity antibody response in vivo. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a cationic lipid compound and its application in the delivery of bioactive substances. Background Art

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic characteristics and pharmacological activities, and can be used for the treatment of tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. After nucleic acid drugs are injected into the human body, a highly efficient and safe drug delivery system is required to deliver them to the diseased site. This drug delivery system needs to remain for a sufficient time to accurately target the diseased site while avoiding damage to normal cells.

[0003] Current delivery systems can be divided into viral vectors and non-viral vectors. Due to their immunogenicity, tumorigenicity, and limited drug loading capacity, viral vectors have less application in nucleic acid drugs; non-viral vectors, such as polymers, liposomes or LNPs, can bind nucleic acid drugs with specific ligands to enable them to target specific cells and are widely used in current nucleic acid drugs. LNP is one of the most widely studied and applied delivery systems for nucleic acid drugs at present. The LNP delivery system can safely and effectively deliver nucleic acids, with advantages such as high nucleic acid encapsulation efficiency, effective cell transfection, strong tissue penetration, low cytotoxicity and immunogenicity, which are beneficial for drug delivery. Compared with other delivery systems, it has great advantages. Therefore, the LNP delivery system has broad development and application prospects.

[0004] In the prior art, LNP delivery systems often consist of components such as ionizable lipids (cationic lipids), steroids, neutral lipids, PEG-lipids, nucleic acid drugs, etc. For example: Patent document AU2020325221A1 discloses a composition for delivering LNPs to target cells, including (i) ionizable lipids; (ii) sterols or other structural lipids; (iii) non-cationic co-lipids or phospholipids; (iv) PEG lipids and (v) agents (such as nucleic acid molecules) encapsulated in and / or associated with the LNP. These four components enhance the delivery efficiency to target cells in a specific ratio. Patent document WO2021 / 250263A1 discloses a composition including ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids, and discloses including less than about 1 mol% of C14-PEG2000 lipid and specific percentages of other lipids. Patent CN102712935B discloses a lipid particle, which includes: cationic lipids; neutral lipids, zwitterionic lipids or anionic lipids; PEG-lipids; sterols and nucleic acids, and assembles the above components into a lipid particle with a solid core, and its solid core can achieve a higher encapsulation efficiency. Patent document WO 2021 / 055849A1 discloses a lipid with the following structure: This structure can improve its safety, effectiveness and specificity. Patent document WO2021 / 026358Al discloses a lipid nanoparticle (LNP) for delivering to target cells: including (i) ionizable lipids (ii) sterols or other structural lipids; (iii) non-cationic co-lipids or phospholipids; (iv) payload; (v) polyethylene glycol lipids. As a drug delivery system, it takes into account both safety and effectiveness. In recent years, it has been found that introducing cholesterol into ionizable lipid compounds can also be used to deliver nucleic acid drugs. Patent document US7514099B2 discloses an amino lipid compound CLinDMA of cholesterol This compound can form a four-component LNP with phospholipids, cholesterol, and PEG lipids or a five-component LNP with phospholipids, DMOBA lipids, cholesterol, and PEG lipids to deliver siRNA. Patent document CN112424214A discloses an ionizable cationic lipid compound (3) formed from cholesterol and linear olefins This compound constructs an LNP with cholesterol, DPPC, DOPE, and DMG-PEG200 to deliver nucleic acids. All of the above compounds need to be combined with 3 or even more than 4 different lipid excipients to form a nucleic acid drug delivery carrier preparation, and the construction process is relatively complex. There is an urgent need in the prior art to optimize the structures of the components in LNP, especially to optimize the structure of ionizable cationic lipids, in order to further obtain an LNP delivery system that is safe, effective, stable, has a simple construction, and can be applied to different administration routes simultaneously. Summary of the Invention

[0005] On the one hand, the present invention provides a lipid compound represented by formula (I),

[0006]

[0007] or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein,

[0008] Ar is an aryl or heteroaryl; the aryl or heteroaryl is optionally substituted with a group selected from: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl, halogen, alkoxy, alkylthio, NR4R4’, R4-C(O)-, R4-C(O)O-;

[0009] Each occurrence of G1, G2, and G3 is independently selected from a bond, an optionally substituted C1-C 20 alkylene, an optionally substituted C2-C 20 alkenylene, an optionally substituted C2-C 20 alkynylene, an optionally substituted carbocyclic moiety, an optionally substituted arylene;

[0010] Each occurrence of L1, L2, L3, L4, L5, and L6 is independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-, -NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-, -N(R a )C(=O)O-, -OC(=O)N(R a )-, -S-S-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-;

[0011] One of R1, R2, and R3 is selected from an optionally substituted C1-C 20 alkyl, an optionally substituted C2-C 20 alkenyl, an optionally substituted C2-C 20 alkynyl; wherein, the C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20One or more -CH2- in the alkynyl group may be optionally replaced by -O-, -S-, -NR a -, carbocyclyl, aryl, heteroaryl, and / or heterocyclyl substitution;

[0012] At the same time, another one of R1, R2 and R3 is selected from a steroidal group;

[0013] At the same time, the third one of R1, R2 and R3 is selected from -(R4") q -NR a R b 、-(R4”) q -nitrogen-containing heteroaryl, -(R4") q -nitrogen-containing heterocyclic group, -(R4") q wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidine group are optionally substituted by one or more groups selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, halogen, hydroxyl, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 Acyl, C1-C 20 Acyloxy, R c O-C1-C 20 alkyl;

[0014] R4 and R4' are each independently selected from H, Cl-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and / or heterocyclylalkyl;

[0015] R4" is selected from C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkyne;

[0016] R a , R b and R c are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl;

[0017] m, n and p are each independently selected from 1, 2 or 3;

[0018] q is selected from 0 or 1.

[0019] On the other hand, the present invention also provides a lipid nanoparticle, comprising the lipid compound shown by formula (I) or its stereoisomers, tautomers, pharmaceutically acceptable salts.

[0020] On the other hand, the present invention also provides a pharmaceutical composition, comprising the lipid nanoparticle described herein and a pharmaceutically acceptable carrier.

[0021] On the other hand, the present invention also provides a method for delivering a therapeutic agent and / or a prophylactic agent, comprising administering the pharmaceutical composition described herein to an individual in need thereof.

[0022] On the other hand, the present invention also provides the use of the lipid compound shown by formula (I) or its stereoisomers, tautomers, pharmaceutically acceptable salts described herein in the preparation of a delivery system for a therapeutic agent and / or a prophylactic agent.

[0023] Beneficial effects:

[0024] The three-component lipid nanoparticles prepared by using the lipid compound of the present invention or its stereoisomers, tautomers, pharmaceutically acceptable salts have a simple process, and at the same time have better stability and transfection efficiency. Using the lipid nanoparticles to deliver nucleic acids (such as mRNA), it can efficiently and stably deliver them to target cells or organs, and induce a high specific antibody response and cellular immune response in experimental animals, and have good safety. Description of the drawings

[0025] Figure 1 Showing the transfection efficiency of Luc-mRNA-LNP in HEK293T cells detected by fluorescence microscopy.

[0026] Figure 2 Showing the serum specific antibody titer after immunizing mice with the SARS-CoV-2 S protein mRNA-LNP vaccine.

[0027] Figure 3 Showing the level of specific CD8+ T cell response in the spleen after immunizing mice with the SARS-CoV-2 S protein mRNA-LNP.

[0028] Figure 4 Showing the level of specific CD4+ T cell response in the spleen after immunizing mice with the SARS-CoV-2 S protein mRNA-LNP. Detailed implementation manners

[0029] Definitions

[0030] As used in this specification, unless the context in which they are used indicates otherwise, the following words and phrases are generally intended to have the meanings set forth below.

[0031] As used herein, the term "lipid nanoparticle", or "LNP", refers to particles having a nanoscale size, such as from 1 nm to 1,000 nm, which comprise one or more types of lipid molecules.

[0032] As used herein, the term "gene drug" generally consists of a vector or delivery system containing an engineered gene construct, and its active ingredient can be DNA, RNA, genetically modified virus, bacteria or cells. By introducing exogenous genes into target cells or tissues, replacing, compensating, blocking, or correcting specific genes, it aims to achieve the purpose of treating and preventing diseases.

[0033] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids.

[0034] As used herein, the term "lipid compound" refers to a group of organic compounds, which include but are not limited to esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents. The organic solvents in the present invention include but are not limited to: benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, ether, ethyl acetate, acetone, carbon tetrachloride.

[0035] As used herein, the term "alkyl" refers to a monovalent group of a straight-chain or branched-chain saturated hydrocarbon chain having 1 to 20 carbon atoms (more typically having 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms). Illustrative examples of this term are groups such as methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, 1-nonyl, 1-decyl, etc.

[0036] As used herein, the term "alkylene" refers to a divalent group of a straight or branched chain saturated hydrocarbon chain having from 1 to 20 carbon atoms (more typically from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms). Illustrative of this term are groups such as methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like.

[0037] As used herein, the term "alkenyl" refers to a monovalent group of a straight or branched chain unsaturated hydrocarbon chain having from 2 to 20 carbon atoms (more typically from 2 to 10 carbon atoms, from 2 to 8 carbon atoms, or from 2 to 6 carbon atoms) and having a carbon-carbon double bond (e.g., 1, 2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond can be present at any stable point along the chain. Illustrative of this term are groups such as vinyl (i.e., -CH=CH2), prop-1-enyl (i.e., -CH=CHCH3), prop-3-enyl (or allyl, i.e., -CH2CH=CH2), prop-2-enyl (i.e., -C(CH3)=CH2), butadienyl (including 1,2-butadienyl and 1,3-butadienyl), and the like.

[0038] As used herein, the term "alkenylene" refers to a divalent group of a straight or branched chain unsaturated hydrocarbon chain having from 2 to 20 carbon atoms (more typically from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms) and having a carbon-carbon double bond (e.g., 1, 2, or 3 carbon-carbon double bonds). The unsaturated carbon-carbon double bond can be present at any stable point along the chain. Illustrative of this term are groups such as vinylene, propenylene, butenylene, pentenylene, hexenylene, and the like.

[0039] As used herein, the term "alkynyl" refers to a monovalent group of a straight or branched chain unsaturated hydrocarbon chain having from 2 to 20 carbon atoms (more typically from 2 to 10 carbon atoms, from 2 to 8 carbon atoms, or from 2 to 6 carbon atoms) and having a carbon-carbon triple bond (e.g., 1, 2, or 3 carbon-carbon triple bonds). Illustrative of this term are groups such as ethynyl (i.e., -C≡CH), propargyl (i.e., -CH2C≡CH), propynyl (i.e., -C≡CCH3), and the like.

[0040] As used herein, the term "alkynylene" refers to a divalent group of a straight or branched chain unsaturated hydrocarbon chain having from 2 to 20 carbon atoms (more typically from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms) and having a carbon-carbon triple bond (e.g., 1, 2, or 3 carbon-carbon triple bonds). The unsaturated carbon-carbon triple bond can be present at any stable point along the chain. Illustrative of this term are groups such as ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.

[0041] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0042] As used herein, the term "alkoxy" refers to an "alkyl-O-" group, where the alkyl is as defined herein. Illustrative of this term are groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0043] As used herein, the term "acyl" refers to an "alkyl-C(=O)-", "alkenyl-C(=O)-", "alkynyl-C(=O)-", "aryl-C(=O)-", "heteroaryl-C(=O)-", "carbocyclic-C(=O)-", "heterocyclic-C(=O)-" group, where the alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic, heterocyclic are as defined herein. Illustrative of this term are groups such as formyl, acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, n-hexanoyl, acryloyloxy, benzoyl, cyclopropylcarbonyl, and the like.

[0044] As used herein, the term "acyloxy" refers to an "alkyl-C(=O)O-", "alkenyl-C(=O)O-", "alkynyl-C(=O)O-", "aryl-C(=O)O-", "heteroaryl-C(=O)O-", "carbocyclic-C(=O)O-", "heterocyclic-C(=O)-" group, where the alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclic, heterocyclic are as defined herein. Illustrative of this term are groups such as formyloxy, acetyloxy, propionyloxy, n-butyryloxy, isobutyryloxy, n-valeryloxy, n-hexanoyloxy, and the like.

[0045] As used herein, the term "aryl" refers to an aromatic carbocyclic group having 6 to 14 carbon atoms (more typically having 6 to 10 carbon atoms, or 6 carbon atoms) in a single ring (e.g., phenyl), multiple rings (e.g., biphenyl), or multiple fused (condensed) rings (e.g., naphthyl, fluorenyl, and anthracenyl). Illustrative of this term are groups such as phenyl, fluorenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthalene (if the point of attachment is through an aryl group), and the like.

[0046] As used herein, the term "carbocyclic" refers to a monocyclic or multiple fused (condensed) rings or bridged rings or spirocyclic monovalent saturated or partially unsaturated group having 3 to 14 carbon atoms (more typically having 3 to 8 carbon atoms, or 3 to 6 carbon atoms) as ring atoms. The carbocyclic or carbocyclic group can be saturated or partially unsaturated and can be fused to another saturated, partially unsaturated, or aromatic ring, provided that the ring atom connected to the target molecule is not an aromatic carbon. Examples of carbocyclic or carbocyclic groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclopentadiene, and the like.

[0047] As used herein, the term "heteroaryl" refers to an aromatic ring group having a monocyclic or multiple fused (condensed) ring (e.g., having 2 or 3 rings) containing 5 to 14 ring atoms in the ring (more typically having 5 to 10 ring atoms, or 5 to 6 ring atoms), wherein in addition to carbon atoms, the ring atoms further contain at least one heteroatom selected from oxygen, nitrogen, and / or sulfur. If the ring is aromatic, sulfur and nitrogen atoms may also be present in oxidized forms. The multiple fused (condensed) ring heteroaryl is formed by fusing a monocyclic heteroaryl as defined above with one or more rings selected from the following to form a multiple fused ring system: heteroaryl (to form, for example, naphthyridinyl, such as 1,8-naphthyridinyl), heterocycle (e.g., to form 1,2,3,4-tetrahydronaphthyridinyl, such as 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycle (to form, for example, 5,6,7,8-tetrahydroquinolinyl), and aryl (to form, for example, indazolyl). It should be understood that the point of attachment of the heteroaryl can be on any suitable atom of the heteroaryl, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryls include, but are not limited to: pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl, benzofuranyl, benzimidazolyl, thianthrenyl, pyrrolo[2,3-b]pyridinyl, quinazolin-4(3H)-one, triazolyl, 4,5,6,7-tetrahydro-1H-indazolyl, and 3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazolyl.

[0048] As used herein, the term "heterocyclic group" refers to a monocyclic or multiple fused (condensed), bridged or spiro cyclic monovalent or divalent saturated or partially unsaturated group having 3 to 14 ring atoms (more typically 3 to 10 ring atoms, or 3 to 6 ring atoms) within the ring, wherein in addition to carbon atoms, the ring atoms further comprise at least one nitrogen atom. Examples of heterocyclic groups include, but are not limited to, aziridine ring, azetidine ring, pyrrolidine ring, piperidine ring, azepane ring, azocane ring, tetrahydroimidazole ring, tetrahydropyrazole ring, tetrahydrooxazole ring, tetrahydroisoxazole ring, tetrahydrothiazole ring, tetrahydroisothiazole ring, piperazine ring, morpholine ring, dihydropyridyl, 4,5,6,7-tetrahydro-1H-benzo[d]imidazole, 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine, etc. The heterocyclic group in the present invention is a heterocyclic group containing a nitrogen atom in the structure, including but not limited to substituted or unsubstituted: aziridinyl, azetidinyl, β-lactamyl, pyrrolyl, piperidinylalkyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, caprolactamyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, piperazinylalkyl, indolyl, benzimidazolyl, carbazolyl, quinolinyl, isoquinolinyl, pteridinyl, acridinyl, 7H-purinyl, phenazinyl, phenothiazinyl or 1H-azepinyl.

[0049] As used herein, the term "optionally substituted" means unsubstituted or substituted by one or more groups selected from: C1-C6 alkyl, C1-C6 alkoxy, halogen, hydroxy, cyano, nitro, amino, C3-C6 cycloalkyl, oxo.

[0050] As used herein, the term "steroid" is an organic compound having a tetracyclic carbon skeleton structure as shown below.

[0051]

[0052] Steroids include naturally occurring or synthetic steroids and their analogs. Steroids or their analogs include sterols or their analogs derived from plants and / or animals. Examples of the steroids described herein include, but are not limited to, avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, chondrillasterol, epicholesterol, ergosterol, fucosterol, hexahydro-lumisterol, hydroxy cholesterol, lumisterol, saringosterol, sitostanol, stigmasterol, stigmasterol, cholanic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epi-cholesterol, demosterol, cholestanol, cholestanone, cholestenone, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl] cholesterol (DC-Chol), 24(S)-hydroxy cholesterol, 25-hydroxy cholesterol, 25(R)-27-hydroxy cholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloeucalenol, 22-ketosterol, 20-hydroxy sterol, 7-hydroxy cholesterol, 19-hydroxy cholesterol, 22-hydroxy cholesterol, 25-hydroxy cholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumisterol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, tomatine, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, etc.

[0053] As used herein, the term "therapeutically effective amount" means an amount sufficient to effect treatment, as defined below, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending on the subject being treated and the disease condition, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art.

[0054] As used herein, the term "stereoisomer" refers to a compound having the same chemical composition and connectivity, but whose atoms have different orientations in space, which orientations cannot be interchanged by rotation about a single bond. "Stereoisomers" include "diastereomers" and "enantiomers". "Diastereomers" are stereoisomers having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. A mixture of diastereomers can be separated by high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography. "Enantiomers" are two stereoisomers that are non-superimposable mirror images of each other.

[0055] As used herein, the term "tautomer" refers to the coexistence of two (or more) compounds that differ only in the position and electronic distribution of one (or more) mobile atoms, such as keto-enol tautomers.

[0056] As used herein, the term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a given compound and that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be acid addition salts and / or base addition salts. Acid addition salts can be prepared from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, carbonates, bisulfates, hydrogen phosphates, dihydrogen phosphates, bicarbonates, etc.; salts derived from organic acids include formates, acetates, propionates, glycolates, pyruvates, oxalates, malates, malonates, succinates, maleates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, salicylates, lactates, nicotinates, lauryl sulfates, naphthalenesulfonates, camphorsulfonates, gluconates, glucuronates, oleates, palmitates, stearates, pamoates, trifluoroacetates, etc. Base addition salts can be formed with inorganic bases or organic bases. Salts derived from inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, lithium, barium, aluminum salts, etc.; salts derived from organic bases include salts formed with various primary, secondary, and tertiary amines, such as ethylamine, diethylamine, n-propylamine, isopropylamine, diethanolamine, glucosamine, lysine, piperazine, piperidine, morpholine, tromethamine, choline, etc.

[0057] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal being treated therewith.

[0058] As used herein, the term "delivery system" refers to a formulation or composition that regulates the spatial, temporal, and dosage distribution of a bioactive ingredient in a living organism.

[0059] Compound

[0060] In some embodiments, the present invention provides a lipid compound of formula (I)

[0061]

[0062] or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein L1, L2, L3, L4, L5, L6, G1, G2, G3, R1, R2, R3, m, n, p are as defined above.

[0063] In some embodiments, the present invention provides a lipid compound of formula (I) or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein,

[0064] Ar is an aryl group; the aryl group is optionally substituted with a group selected from: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl group, halogen, alkoxy group, alkylthio group, NR4R4’, R4-C(O)-, R4-C(O)O-;

[0065] Each occurrence of G1, G2, and G3 is independently selected from a bond, an optionally substituted C1-C 20 alkylene group, an optionally substituted C2-C 20 alkenylene group, an optionally substituted C2-C 20 alkynylene group, an optionally substituted carbocyclic moiety, an optionally substituted arylene group;

[0066] Each occurrence of L1, L2, L3, L4, L5, and L6 is independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-, -NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-, -N(R a )C(=O)O-, -OC(=O)N(R a )-, -S-S-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-;

[0067] R1 is selected from an optionally substituted C1-C 20 alkyl group, an optionally substituted C2-C 20 alkenyl group, an optionally substituted C2-C 20 alkynyl group; wherein, one or more -CH2- in the C1-C 20 alkyl group, C2-C 20 alkenyl group, C2-C 20 alkynyl group may be optionally replaced by O, S, -NR a -, carbocyclic group, aryl group, heteroaryl group, and / or heterocyclic group;

[0068] R2 is selected from steroid groups;

[0069] R3 is selected from -(R4”) q -NR a R b 、-(R4”) q -a nitrogen-containing heteroaryl, -(R4”) q -a nitrogen-containing heterocyclic group, -(R4”) q -a guanidino group; wherein, the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidino group are optionally substituted with one or more groups selected from the following: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C1-C 20 alkoxy, halogen, hydroxy, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 acyl, C1-C 20 acyloxy, R c O-C1-C 20 alkyl;

[0070] R4 and R4’ are each independently selected from H, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, a carbocyclic group, an aryl group, an arylalkyl group, a heteroaryl group, a heteroarylalkyl group, a heterocyclic group and / or a heterocyclic alkyl group;

[0071] R4” is selected from C1-C 20 alkylene, C2-C 20 alkenylene, C2-C 20 alkynylene;

[0072] R a 、R b and R c are each independently selected from H, optionally substituted C1-C 20 alkyl, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclic group, optionally substituted aryl group, optionally substituted arylalkyl group, optionally substituted heteroaryl group, optionally substituted heteroarylalkyl group, optionally substituted heterocyclic group, optionally substituted heterocyclic alkyl group;

[0073] m, n and p are each independently selected from 1, 2 or 3;

[0074] q is selected from 0 or 1.

[0075] In some embodiments, the present invention provides a lipid compound of formula (I) or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein,

[0076] Ar is phenyl; the phenyl is optionally substituted with a group selected from the following: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl, halogen, alkoxy, alkylthio, NR4R4’, R4-C(O)-, R4-C(O)O-;

[0077] Each occurrence of G1, G2 and G3 is independently selected from a bond, optionally substituted C1-C 20 alkylene, optionally substituted C2-C 20 alkenylene, optionally substituted C2-C 20 alkynylene;

[0078] Each occurrence of L1, L2, L3, L4, L5 and L6 is independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-, -NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-, -N(R a )C(=O)O-, -OC(=O)N(R a )-, -S-S-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-;

[0079] R1 is selected from optionally substituted C1-C 20 alkyl, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 alkynyl; wherein, one or more -CH2- in the C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl may be optionally replaced by O, S, -NR a -, carbocyclic group, aryl, heteroaryl, and / or heterocyclic group;

[0080] R2 is selected from steroid groups;

[0081] R3 is selected from -(R4”) q -NR a R b 、-(R4”) q -nitrogen-containing heteroaryl, -(R4”) q-A nitrogen-containing heterocyclic group; wherein the nitrogen-containing heteroaryl group and the nitrogen-containing heterocyclic group are optionally substituted by one or more groups selected from the following: C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C1-C 20 alkoxy, halogen, hydroxy, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 acyl, C1-C 20 acyloxy, R c O-C1-C 20 alkyl;

[0082] R4 and R4’ are each independently selected from H, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, carbocyclic group, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic group and / or heterocyclic alkyl;

[0083] R4” is selected from C1-C 20 alkylene, C2-C 20 alkenylene, C2-C 20 alkynylene;

[0084] R a 、R b and R c are each independently selected from H, optionally substituted C1-C 20 alkyl, optionally substituted C2-C 20 alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclic group, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclic group, optionally substituted heterocyclic alkyl;

[0085] m, n and p are each independently selected from 1, 2 or 3;

[0086] q is selected from 0 or 1.

[0087] In some embodiments, the present invention provides lipid compounds represented by formula (II-1), (II-2), (II-3), (II-4), (II-5):

[0088]

[0089] or their stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein, L1, L2, L3, L4, L5, L6, G1, G2, G3, R1, R2, R3, m, n and p are as defined herein.

[0090] In some embodiments, the present invention provides lipid compounds represented by formulae (III-1), (III-2), (III-3), (III-4), (III-5):

[0091]

[0092] or their stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein, L1, L2, L3, L4, L5, L6, G1, G2, G3, R1, R2, R3, m, n, and p are as defined herein.

[0093] In some embodiments, the present invention provides lipid compounds represented by formulae (IV-1), (IV-2), (IV-3), (IV-4), (IV-5), (IV-6), (IV-7), (IV-8), (IV-9), (IV-10):

[0094]

[0095]

[0096] or their stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein, R1, R2, R3 are as defined herein, and r, s, t, and u are each independently selected from integers from 1 to 10.

[0097] In some embodiments, the steroid in the steroid group of the present invention is selected from naturally occurring steroids or their analogs; preferably, it includes phytosterols and zoosterols, or their analogs; more preferably, it is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, cholesterol, dihydrocholesterol, dihydroergosterol, marasmanol, epicholesterol, ergosterol, fucosterol, hexahydro-lumisterol, hydroxy cholesterol, lumisterol, saringosterol, sitostanol, stigmastanol, stigmasterol, cholalic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, ent-cholesterol, epi-cholesterol, demosterol, cholestanol, cholestanone, cholestenone, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl] cholesterol (DC-Chol), 24(S)-hydroxy cholesterol, 25-hydroxy cholesterol, 25(R)-27-hydroxy cholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloeucalenol, 22-ketosterol, 20-hydroxy sterol, 7-hydroxy cholesterol, 19-hydroxy cholesterol, 22-hydroxy cholesterol, 25-hydroxy cholesterol, 7-dehydrocholesterol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lumisterol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, tomatidine, ursolic acid, chenodeoxycholic acid, zymosterol, diosgenin, etc.

[0098] In some embodiments, among the lipid compounds provided by the present invention or their stereoisomers, tautomers, and pharmaceutically acceptable salts, the steroid in the steroid group is selected from cholesterol and derivatives of cholesterol.

[0099] In some embodiments, among the lipid compounds provided by the present invention or their stereoisomers, tautomers, and pharmaceutically acceptable salts, the steroid group has the following structure:

[0100]

[0101] R5 is selected from hydrogen, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C1-C 20 alkoxycarbonyl C1-C 20 alkyl-;

[0102] R6 is selected from hydrogen, halogen, cyano, hydroxy, amino, oxo, C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0103] m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0104] In some embodiments, the steroid group is selected from:

[0105]

[0106] wherein R' is C 1- C 20 alkyl.

[0107] In some embodiments, the present invention provides lipid compounds of formula (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9) or formula (V-10):

[0108]

[0109]

[0110]

[0111] or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein, R1, R3, r, s, t and u are as defined herein.

[0112] In some embodiments, the present invention provides lipid compounds of formula (VI-1), (VI-2), (VI-3), (VI-4), (VI-5), (VI-6), (VI-7), (VI-8), (VI-9) or formula (VI-10):

[0113]

[0114]

[0115]

[0116] or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof; wherein, R1, R3, r, s, t and u are as defined herein.

[0117] In some specific embodiments, R1 is selected from optionally substituted C1-C 20 alkyl; wherein, the C1-C 20 alkyl in which one or more -CH2- may optionally be replaced by O, S, -NR a -, carbocyclic group.

[0118] In some specific embodiments, R1 is selected from

[0119] In some specific embodiments,

[0120] R3 is selected from -(R4”), q -NR, a R, b -(R4”), q -a 5- or 6-membered nitrogen-containing heteroaryl, -(R4”), q -a 5- or 6-membered nitrogen-containing heterocyclic group; wherein the 5- or 6-membered nitrogen-containing heteroaryl and 5- or 6-membered nitrogen-containing heterocyclic group are optionally substituted with one or more groups selected from: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, hydroxy, mercapto, cyano, nitro, amino, C1-C6 acyl, C1-C6 acyloxy, R, c O-C1-C6 alkyl;

[0121] R4” is selected from C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene;

[0122] R, a and R, b each independently selected from H and C1-C6 alkyl;

[0123] R, c is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, phenyl-C1-C6 alkyl;

[0124] q is selected from 0 or 1.

[0125] In some specific embodiments,

[0126] R3 is selected from -R4”-NR, a R, b -R4”-a 5- or 6-membered nitrogen-containing heterocyclic group; wherein the 5- or 6-membered nitrogen-containing heterocyclic group is optionally substituted with a group selected from: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, hydroxy, mercapto, cyano, nitro, amino, C1-C6 acyl, C1-C6 acyloxy, R, c O-C1-C6 alkyl;

[0127] R4” is selected from C1-C6 alkylene;

[0128] R, a and R, b each independently selected from H, C1-C6 alkyl;

[0129] R, c is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, benzyl.

[0130] In some embodiments, the present invention provides a compound selected from the following or a pharmaceutically acceptable salt thereof.

[0131]

[0132]

[0133] Lipid nanoparticles (LNP)

[0134] In some embodiments, the present invention provides a lipid nanoparticle as a delivery vehicle for a therapeutic and / or prophylactic agent (such as nucleic acids including DNA, RNA, etc.), comprising a lipid compound described herein or a stereoisomer, tautomer thereof, and a pharmaceutically acceptable salt thereof. In some embodiments, the lipid nanoparticles described herein further comprise one or more phospholipids. In some embodiments, the lipid nanoparticles described herein further comprise one or more PEG lipids. In some embodiments, the lipid nanoparticles described herein further comprise a combination of phospholipids and PEG lipids. The lipid nanoparticles described herein can deliver a therapeutic and / or prophylactic agent to a target site of interest (such as cells, tissues, organs, etc.). Accordingly, the lipid nanoparticles described herein further comprise one or more therapeutic or prophylactic agents (such as nucleic acids, particularly therapeutic nucleic acids (TNA)).

[0135] In some embodiments, the molar ratio of the lipid compound:phospholipid:PEG lipid in the lipid nanoparticle is 30-90:10-60:0.5-20; preferably, the molar ratio of the lipid compound:phospholipid:PEG lipid is 40-80:20-50:0.5-10; more preferably, the molar ratio of the lipid compound:phospholipid:PEG-lipid is 50-70:30-40:0.5-5; most preferably, the molar ratio of the lipid compound:phospholipid:PEG-lipid is 65.7:32.8:1.5.

[0136] Phospholipid

[0137] In some embodiments, phospholipids are further included in the lipid nanoparticles described herein. Examples of phospholipids include, but are not limited to, for example, distearoyl-sn-glycero-phosphoethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), rutinoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dioleoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, hexacosyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, or mixtures thereof. It should be understood that other diacyl phosphatidylcholines and diacyl phosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0138] In some embodiments, the molar percentage of phospholipids in the total lipids of the lipid nanoparticles is from about 15% to about 65%, such as from about 20% to about 65%, from about 25% to about 65%, from about 30% to about 65%, from about 35% to about 65%, from about 40% to about 65%, from about 45% to about 65%, from about 50% to about 65%, from about 55% to about 65%, from about 60% to about 65%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%.

[0139] PEG lipid

[0140] In some embodiments, a PEG lipid is incorporated into the lipid nanoparticles described herein to inhibit aggregation of the particles, thereby enhancing the stability of the lipid nanoparticles. In some embodiments, the PEG lipid described herein is a lipid linked to one or more polyethylene glycol (PEG) chains by a covalent bond or a non-covalent bond. In some embodiments, the PEG lipid described herein is a lipid linked to one or more polyethylene glycol (PEG) chains by a covalent bond.

[0141] In some embodiments, the molecular weight of the PEG molecule suitable for the PEG lipid described herein is from about 500 to about 10,000, from about 1,000 to about 10,000, from about 1,000 to about 5,000, from about 1,000 to about 4,000, from about 1,000 to about 3,000, from about 1,000 to about 2,000, such as PEG2000, PEG2500, PEG3000, etc.

[0142] Examples of PEG lipids include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., 1-(mono-methoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramides (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (e.g., 4-0-(2’,3’-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, sodium N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, PEG-dilauroyl oxypropyl, PEG-dimyristoyl oxypropyl, PEG-dipalmitoyl oxypropyl, PEG-distearoyl oxypropyl, 1-(mono-methoxy-polyethylene glycol)-2,3-dimyristoyl glycerol-PEG (DMG-PEG), distearoyl-rac-glycerol-PEG (DSG-PEG), PEG-dilauroyl glycerol, PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol, PEG-dilauroyl glycerol amide, PEG-dimyristoyl glycerol amide, PEG-dipalmitoyl glycerol amide, PEG-distearoyl glycerol amide, (1-[8'-(cholest-5-en-3β-oxy)formylamino-3',6'-dioxaoctyl]carbamoyl-ω-methyl-poly(ethylene glycol) (PEG-cholesterol), 3,4-bis(tetradecyloxy)benzyl-ω-methyl-poly(ethylene glycol) ether (PEG-DMB), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-PEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxy (DSPE-PEG-OH).

[0143] In some embodiments, the molar percentage of PEG lipid in the total lipid of the lipid nanoparticles is from about 0.1% to about 10%, such as from about 1.0% to about 10%, from about 1.5% to about 10%, from about 2.0% to about 10%, from about 2.5% to about 10%, from about 3.0% to about 10%, from about 3.5% to about 10%, from about 4.0% to about 10%, from about 4.5% to about 10%, from about 5.0% to about 10%, from about 5.5% to about 10%, from about 6.0% to about 10%, from about 6.5% to about 10%, from about 7.0% to about 10%, from about 7.5% to about 10%, from about 8.0% to about 10%, from about 8.5% to about 10%, from about 9.0% to about 10%, from about 9.5% to about 10%, from about 1.0% to about 1.5%, from about 1.5% to about 2.0%, from about 2.0% to about 2.5%, from about 2.5% to about 3.0%, from about 3.0% to about 3.5%, from about 3.5% to about 4.0%, from about 4.0% to about 4.5%, from about 4.5% to about 5.0%, from about 5.0% to about 5.5%, from about 5.5% to about 6.0%, from about 6.0% to about 6.5%, from about 6.5% to about 7.0%, from about 7.0% to about 7.5%, from about 7.5% to about 8.0%, from about 8.0% to about 8.5%, from about 8.5% to about 9.0%, from about 9.0% to about 10%.

[0144] The particle size of the lipid nanoparticles

[0145] In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 150 nm, such as from about 45 nm to about 150 nm, from about 50 nm to about 150 nm, from about 55 nm to about 150 nm, from about 60 nm to about 150 nm, from about 65 nm to about 150 nm, from about 70 nm to about 150 nm, from about 75 nm to about 150 nm, from about 80 nm to about 150 nm, from about 85 nm to about 150 nm, from about 90 nm to about 150 nm, from about 95 nm to about 150 nm, from about 100 nm to about 150 nm, from about 105 nm to about 150 nm, from about 110 nm to about 150 nm, from about 115 nm to about 150 nm, from about 120 nm to about 150 nm, from about 125 nm to about 150 nm, from about 130 nm to about 150 nm, from about 135 nm to about 150 nm, from about 140 nm to about 150 nm, from about 145 nm to about 150 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 120 nm, such as from about 45 nm to about 120 nm, from about 50 nm to about 120 nm, from about 55 nm to about 120 nm, from about 60 nm to about 120 nm, from about 65 nm to about 120 nm, from about 70 nm to about 120 nm, from about 75 nm to about 120 nm, from about 80 nm to about 120 nm, from about 85 nm to about 120 nm, from about 90 nm to about 120 nm, from about 95 nm to about 120 nm, from about 100 nm to about 120 nm, from about 105 nm to about 120 nm, from 110 nm to about 120 nm, from 115 nm to about 120 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 110 nm, such as from about 45 nm to about 110 nm, from about 50 nm to about 110 nm, from about 55 nm to about 110 nm, from about 60 nm to about 110 nm, from about 65 nm to about 110 nm, from about 70 nm to about 110 nm, from about 75 nm to about 110 nm, from about 80 nm to about 110 nm, from about 85 nm to about 110 nm, from about 90 nm to about 110 nm, from about 95 nm to about 110 nm, from about 100 nm to about 110 nm, from about 105 nm to about 110 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 100 nm, such as from about 45 nm to about 100 nm, from about 50 nm to about 100 nm, from about 55 nm to about 100 nm, from about 60 nm to about 100 nm, from about 65 nm to about 100 nm, from about 70 nm to about 100 nm, from about 75 nm to about 100 nm, from about 80 nm to about 100 nm, from about 85 nm to about 100 nm, from about 90 nm to about 100 nm, from about 95 nm to about 100 nm.In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 90 nm, such as from about 45 nm to about 90 nm, from about 50 nm to about 90 nm, from about 55 nm to about 90 nm, from about 60 nm to about 90 nm, from about 65 nm to about 90 nm, from about 70 nm to about 90 nm, from about 75 nm to about 90 nm, from about 80 nm to about 90 nm, from about 85 nm to about 90 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 85 nm, such as from about 45 nm to about 85 nm, from about 50 nm to about 85 nm, from about 55 nm to about 85 nm, from about 60 nm to about 85 nm, from about 65 nm to about 85 nm, from about 70 nm to about 85 nm, from about 75 nm to about 85 nm, from about 80 nm to about 85 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 80 nm, such as from about 45 nm to about 80 nm, from about 50 nm to about 80 nm, from about 55 nm to about 80 nm, from about 60 nm to about 80 nm, from about 65 nm to about 80 nm, from about 70 nm to about 80 nm, from about 75 nm to about 80 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 70 nm, such as from about 45 nm to about 70 nm, from about 50 nm to about 70 nm, from about 55 nm to about 70 nm, from about 60 nm to about 70 nm, from about 65 nm to about 70 nm. In some embodiments, the particle size of the lipid nanoparticles described herein ranges from about 40 nm to about 60 nm, such as from about 45 nm to about 60 nm, from about 50 nm to about 60 nm, from about 55 nm to about 60 nm.

[0146] Lipid / nucleic acid ratio

[0147] In some embodiments, the weight ratio or molar ratio of lipid to nucleic acid in the lipid nanoparticles is from about 10:1 to about 100:1, such as from about 10:1 to about 95:1, from about 10:1 to about 90:1, from about 10:1 to about 85:1, from about 10:1 to about 80:1, from about 10:1 to about 75:1, from about 10:1 to about 70:1, from about 10:1 to about 65:1, from about 10:1 to about 60:1, from about 10:1 to about 55:1, from about 10:1 to about 50:1, from about 10:1 to about 45:1, from about 10:1 to about 40:1, from about 10:1 to about 35:1, from about 10:1 to about 30:1, from about 10:1 to about 25:1, from about 10:1 to about 20:1, from about 10:1 to about 15:1.

[0148] In some embodiments, the N / P ratio of the lipid nanoparticles (i.e., the ratio of lipid amines carrying a positive charge to nucleic acid phosphate groups carrying a negative charge) is about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or higher.

[0149] Therapeutic / Preventive Agent

[0150] In some embodiments, the lipid nanoparticles of the present invention further comprise a therapeutic agent and / or a preventive agent. In some embodiments, the therapeutic agent and / or preventive agent described herein include organic molecules, inorganic molecules, proteins, polypeptides, nucleic acids, vaccines, immunotherapeutic agents, etc. In some embodiments, the therapeutic agent and / or preventive agent described herein include nucleic acids. In some embodiments, the therapeutic agent and / or preventive agent described herein include DNA. In some embodiments, the therapeutic agent and / or preventive agent described herein include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, or viral DNA. In some embodiments, the therapeutic agent and / or preventive agent described herein include RNA. In some embodiments, the therapeutic agent and / or preventive agent described herein include small interfering RNA (siRNA). In some embodiments, the therapeutic agent and / or preventive agent described herein include messenger RNA (mRNA). In some embodiments, the therapeutic agent and / or preventive agent described herein include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme.

[0151] Pharmaceutical Compositions and Formulations

[0152] In some embodiments, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a therapeutic and / or prophylactic agent (e.g., nucleic acids including DNA, RNA, etc.) vaccine comprising the lipid nanoparticles described herein and a pharmaceutically acceptable carrier.

[0153] In some embodiments, the pharmaceutically acceptable carriers described herein include diluents, buffers, stabilizers, etc.

[0154] In some embodiments, the diluent includes ethylene glycol, glycerol, polyethylene glycol, sucrose, trehalose, or a combination thereof, etc. In some embodiments, the content of the diluent is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0155] In some embodiments, the buffer includes phosphate, citrate, imidazole, histidine, Tris, HEPES, or a combination thereof, etc. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or above 100 mM.

[0156] In some embodiments, the stabilizer includes salts, including inorganic metal salts such as sodium chloride, potassium chloride, calcium chloride, etc. In some embodiments, the concentration of the stabilizer in the pharmaceutical composition is about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, or above 200 mM.

[0157] In some embodiments, depending on the different routes of administration of the drug, the pharmaceutical composition and / or vaccine of the present invention can be prepared into oral preparations, intramuscular injection preparations, subcutaneous injection preparations, intravenous injection preparations, aerosol inhalation preparations, nasal spray inhalation preparations or dry powder inhalation preparations, ophthalmic administration preparations.

[0158] Indications

[0159] The lipid nanoparticles and / or pharmaceutical compositions provided by the present invention can be used for the prevention and / or treatment of cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal diseases, neonatal screening, nutritional diseases, parasitic diseases, skin diseases, etc.

[0160] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions provided by the present invention are mRNA vaccines and can be used for the prevention of cancer, viral infections, bacterial infections, fungal infections, etc. The viruses include, but are not limited to: norovirus, Ebola virus, coronavirus (including the novel coronavirus SARS CoV2), cytomegalovirus, dengue virus, Zika virus, Coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus, etc.

[0161] Treatment method

[0162] The present invention provides a method for in vivo delivery of the lipid nanoparticles described herein, including administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need. In some embodiments, the present invention provides a method for in vivo delivery of the lipid nanoparticles described herein, including administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by pulmonary delivery. In some embodiments, the present invention provides a method for in vivo delivery of the lipid nanoparticles described herein, including administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by intranasal delivery. In some embodiments, the present invention provides a method for in vivo delivery of the lipid nanoparticles described herein, including administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by inhalation. In some embodiments, the present invention provides a method for in vivo delivery of the lipid nanoparticles described herein, including administering the lipid nanoparticles or pharmaceutical compositions described herein to an individual in need by nebulized inhalation.

[0163] Preparation of lipid nanoparticles

[0164] Lipid nanoparticles encapsulating therapeutic and / or prophylactic agents can be prepared using a variety of methods known in the art. Typically, a solution containing a mixture of various lipids is first prepared, and this solution is mixed with a solution of the therapeutic and / or prophylactic agent before forming the lipid nanoparticles, encapsulating the therapeutic and / or prophylactic agent in the lipid nanoparticles formed from the mixture of various lipids (as described in, for example, WO2016004318, US20160038432). Alternatively, a solution containing a mixture of various lipids is first prepared and then lipid nanoparticles are formed, and then the resulting lipid nanoparticles are mixed with the therapeutic and / or prophylactic agent to encapsulate the therapeutic and / or prophylactic agent in the lipid nanoparticles formed from the mixture of various lipids (as described in, for example, WO2018089801, US20180153822). These methods can effectively encapsulate the therapeutic and / or prophylactic agent in the lipid nanoparticles, and the encapsulation efficiency is usually not less than about 80%, not less than about 85%, not less than about 90%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, not less than about 99%.

[0165] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0166] Example

[0167] Synthesis of Compound 1 in Example 1

[0168] Step 1: Synthesis of Compound A2

[0169]

[0170] Dissolve 10 g of cholesterol (A1) in dichloromethane (150 mL), add 4-bromobutyric acid (4.75 g) and DMAP (950 mg) thereto, stir the mixture until dissolved, stir in an ice bath, add EDCI (g), and then react at room temperature for 16 hours. After the reaction is completed, add 100 mL of water to the system, separate the organic phase, extract the aqueous phase with dichloromethane (50 mL × 2), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain Compound A2 (10 g, white solid), with a yield of 72%.

[0171] Step 2: Synthesis of Compound A4

[0172] Add compound A3 (30 g) and DMF (0.1 mL) to a 250 mL round-bottom flask, add 100 mL of dichloromethane to dissolve and mix, then dropwise add 49.5 mL of oxalyl chloride to the reaction flask. After the addition is complete, react at room temperature for 12 hours. After the reaction is complete, directly evaporate the solvent under reduced pressure to obtain compound A4 (32.1 g, yellow oil), with a yield of 100%.

[0173] Step 3: Synthesis of compound A5

[0174]

[0175] Add 6-bromo-1-hexanol (25.4 g) and dichloromethane (100 mL) to a 500 mL round-bottom flask. Dissolve 32.1 g of compound A4 in 100 mL of dichloromethane and slowly add it dropwise to the reaction flask. After the addition is complete, continue to react at room temperature for 12 hours. After the reaction is complete, directly evaporate the solvent under reduced pressure and separate by silica gel column chromatography to obtain compound A5 (39.5 g, pale yellow oil), with a yield of 80%.

[0176] Step 4: Synthesis of compound A7

[0177]

[0178] Add compound A6 (10 g) and DMF (100 mL) to a 250 mL round-bottom flask. Under ice bath conditions, add imidazole (6.32 g) and tert-butyldimethylchlorosilane (11.3 g) respectively, and stir at room temperature for 16 hours. After the reaction is complete, dilute and mix the reaction system with 500 mL of water, extract with ethyl acetate, combine the organic phases, wash the organic phases with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and separate by silica gel column chromatography to obtain compound A7 (14.0 g, bright yellow oil), with a yield of 77%.

[0179] Step 5: Synthesis of compound A8

[0180]

[0181] Add compound A7 (7.28 g), compound A5 (12.0 g), DMF (100 mL), potassium carbonate (19.7 g) and NaI (770 mg) to a 500 mL round-bottom flask. Stir the reaction system at 60 °C for 16 hours. After the reaction is complete, pour the reaction system into 500 mL of water, extract 3 times with ethyl acetate, combine the organic phases, wash the organic phases with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and separate by silica gel column chromatography to obtain compound A8 (11.5 g, bright yellow oil), with a yield of 38%.

[0182] Step 6: Synthesis of compound A9

[0183]

[0184] Compound A8 (7.60 g, 12.8 mmol), compound A2 (7.55 g, 14.1 mmol), potassium carbonate (5.31 g, 38.4 mmol) and sodium iodide (190 mg, 0.149 mmol) were dissolved in 75 mL of DMF and stirred at 60 °C for 16 h. 400 mL of water was added to the reaction system to quench the reaction, and the mixture was extracted with ethyl acetate (200 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound A9 (9.50 g, bright yellow oil), with a yield of 70%.

[0185] Step 7: Synthesis of compound A10

[0186]

[0187] Compound A9 (5.00 g, 4.77 mmol) was dissolved in 50 mL of tetrahydrofuran, then 1.0 M TBAF solution (5.8 mL) was added, and the mixture was stirred at room temperature for 2 h. Then 200 mL of water was added to quench the reaction, and the reaction system was extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound A10 (4.20 g, yellow oil), with a yield of 94%.

[0188] 1 H NMR (400 MHz, CDCl3): δ 6.51 (s, 2H), 6.37 (s, 1H), 5.38 (m, 1H), 5.29 (d, J = 17.2 Hz, 1H), 4.63 (m, 3H), 4.09 (t, J = 6.4 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H), 3.94 (t, J = 6.4 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 2.32 (d, J = 8.0 Hz, 3H), 2.13 - 2.08 (m, 6H), 2.05 - 1.93 (m, 3H), 1.91 - 1.74 (m, 6H), 1.71 - 1.54 (m, 8H), 1.53 - 1.39 (m, 11H), 1.37 - 1.21 (m, 24H), 1.14 - 1.09 (m, 7H), 1.02 (s, 3H), 0.96 - 0.84 (m, 13H), 0.69 (s, 3H).

[0189] Step 8: Synthesis of compound A11

[0190]

[0191] Compound A10 (1.50 g, 1.60 mmol) and pyridine (410 mg, 3.21 mmol) were dissolved in 15 mL of dichloromethane, and then thionyl chloride (290 mg, 2.41 mmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, saturated aqueous sodium bicarbonate was added to the reaction system until pH = 8. The aqueous phase was extracted twice with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound A11 (1.50 g, yellow oil), with a yield of 98%.

[0192] Step 9: Synthesis of Compound 1

[0193]

[0194] Compound A11 (1.50 g, 1.57 mmol), 1-(3-aminopropyl)imidazole (990 mg, 7.88 mmol), potassium carbonate (440 mg, 3.15 mmol), potassium iodide (50 mg, 0.105 mmol) and 15 mL of DMF were added to a reaction flask. After the mixture was dissolved, it was heated to 60 °C and reacted for 16 hours. After the reaction was completed, 20 mL of water was added to the reaction system. The aqueous phase was extracted with ethyl acetate (20 mL х 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound 1 (450 mg, yellow oil), with a yield of 27%.

[0195] 1 HNMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 7.05 (s, 1H), 6.91 (s, 1H), 6.45 (d, 2H), 6.35 (s, 1H), 5.37 (m, 1H), 4.64 (m, 1H), 4.08 (m, 4H), 3.99 (m, 2H), 3.93 (m, 2H), 3.70 (m, 2H), 2.62 (m, 2H), 2.49 (m, 2H), 2.33 (m, 3H), 2.10 (m, 2H), 2.04 - 1.93 (m, 4H), 1.90 - 1.74 (m, 6H), 1.64 (s, 5H), 1.58 - 1.53 (m, 4H), 1.44 (m, 10H), 1.30 (m, 24H), 1.13 (m, 7H), 1.02 (m, 4H), 0.96 (m, 2H), 0.92 (s, 3H), 0.88 (m, 10H), 0.68 (s, 3H).

[0196] LC-MS (ESI+): 1041

[0197] Synthesis of Compound 2 in Example 2

[0198]

[0199] Compound A10 (560 mg, 600 μmol), N,N-dimethylglycine (92.8 mg, 900 μmol), EDCI (138 mg, 720 μmol) and DMAP (88 mg, 720 μmol) were added to 10 mL of dichloromethane, and the mixture was stirred at room temperature for 15 h. The reaction system was poured into brine, and the organic phase was separated. The aqueous phase was extracted with dichloromethane three times. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound 2 (305 mg, yellow oil), with a yield of 50%. LC-MS (ESI+): 1019.6 [M+H]+

[0200] 1 H NMR (400 MHz, CDCl3) δ 6.69 (d, J = 1.4 Hz, 2H), 6.41 (t, J = 1.5 Hz, 1H), 5.28 (t, J = 6.1 Hz, 1H), 5.21 (s, 2H), 4.72 (p, J = 7.4 Hz, 1H), 4.12 (q, J = 7.5 Hz, 6H), 3.49 (s, 2H), 2.75 (s, 6H), 2.44 (t, J = 5.8 Hz, 2H), 2.27–2.06 (m, 5H), 2.01–1.82 (m, 5H), 1.77 (dq, J = 7.2, 5.4 Hz, 2H), 1.72–1.42 (m, 17H), 1.42–1.17 (m, 32H), 1.16–1.02 (m, 3H), 1.00–0.75 (m, 21H).

[0201] Synthesis of Compound 3 in Example 3

[0202]

[0203] Compound A10 (560 mg, 600 μmol), compound C1 (141.5 mg, 900 μmol), EDCI (138 mg, 720 μmol) and DMAP (88 mg, 720 μmol) were added to 10 mL of dichloromethane, and the mixture was stirred at room temperature for 15 h. The reaction system was poured into brine, and the organic phase was separated. The aqueous phase was extracted with dichloromethane three times. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound 3 (270 mg, yellow oil), with a yield of 42%. LC-MS (ESI+): 1073.7 [M+H]+

[0204] 11H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 3.0 Hz, 2H), 6.37 (t, J = 2.9 Hz, 1H), 5.27 (m, 1H), 5.20 (s, 2H), 4.60 (p, J = 14.4 Hz, 1H), 4.22–4.02 (m, 6H), 3.29 (s, 2H), 2.61–2.29 (m, 6H), 2.30–2.03 (m, 6H), 2.03–1.80 (m, 5H), 1.79–1.44 (m, 22H), 1.43–1.19 (m, 34H), 1.19–0.98 (m, 4H), 0.95–0.81 (m, 24H).

[0205] Synthesis of Compound 4 in Example 4

[0206]

[0207] Compound A10 (561 mg, 600 μmol), Compound C2 (143 mg, 900 μmol), EDCI (139 mg, 720 μmol) and DMAP (89 mg, 720 μmol) were added to 10 mL of dichloromethane and stirred at room temperature for 15 hours. The reaction system was poured into brine, and the organic phase was separated. The aqueous phase was extracted with dichloromethane three times. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain Compound 4 (238 mg, yellow oil), with a yield of 37%. LC-MS (ESI+): 1074.7 [M + H]+

[0208] 1 1H NMR (400 MHz, CDCl3) δ 6.62 (s, 2H), 6.34 (t, J = 2.9 Hz, 1H), 5.21 (tt, J = 12.5, 2.0 Hz, 1H), 5.20 (s, 2H), 4.71 (p, J = 14.8 Hz, 1H), 4.20–4.02 (m, 6H), 3.28 (s, 2H), 2.44 (td, J = 11.7, 1.2 Hz, 2H), 2.32 (s, 8H), 2.28–2.05 (m, 8H), 2.04–1.42 (m, 24H), 1.42–1.19 (m, 30H), 1.19–0.99 (m, 3H), 0.97–0.81 (m, 21H).

[0209] Synthesis of Compound 5 in Example 5

[0210] Step 1: Synthesis of Compound B2

[0211]

[0212] Compound B1 (5.00 g, 21.7 mmol) was dissolved in 50 mL of tetrahydrofuran. Sodium hydride (960 mg, 23.8 mmol) was added under an ice bath condition, and after stirring for another 30 minutes, benzyl bromide (2.84 mL, 23.8 mmol) was slowly added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, 150 mL of ice water was slowly added to the reaction system to quench the reaction. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound B2 (6.15 g, yellow oil), with a yield of 88%.

[0213] Step 2: Synthesis of compound B3

[0214]

[0215] Compound B2 (6.00 g, 18.7 mmol) was dissolved in 30 mL of methanol and placed on an ice bath. Hydrochloric acid / methanol solution (4.0 M, 70 mL) was slowly added to the reaction system, and the mixture was continuously stirred at room temperature for 6 hours. After the reaction was completed, it was directly rotary evaporated to obtain compound B3 (5.12 g, pale yellow solid), with a yield of 93%.

[0216] Step 3: Synthesis of compound B4

[0217]

[0218] Compound B3 (1.50 g, 5.11 mmol) was added to a reaction flask, and then acetonitrile (25 mL), potassium carbonate (5.89 g, 42.627 mmol), and tert-butyl 4-bromobutyrate (1.37 g, 6.13 mmol) were added. After mixing and dissolving, the mixture was stirred at room temperature for 18 hours. Then it was filtered, and the filter cake was washed with ethyl acetate. 100 mL of water was added to the filtrate, and the layers were separated. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain compound B4 (1.80 g, yellow oil), with a yield of 97%. LCMS (ESI+): 363.4 [M+H]+.

[0219] Step 4: Synthesis of compound B5

[0220]

[0221] Compound B4 (1.80 g, 4.96 mmol) was dissolved in 10 mL of dichloromethane. Under ice-bath conditions, a hydrochloric acid / 1,4-dioxane mixed solution (4.0 M, 20 mL) was slowly added to the reaction system. After warming to room temperature, the mixture was stirred for 2 hours. After the reaction was completed, the solvent was directly removed by rotary evaporation to obtain Compound B5 (1.80 g, yellow oil), with a yield of 95%.

[0222] Step 5: Synthesis of Compound 5

[0223]

[0224] Compound A10 (2.00 g, 2.14 mmol), Compound B5 (810 mg, 2.14 mmol), DIEA (420 mg, 3.21 mmol), and DMAP (30 mg, 0.214 mmol) were mixed and dissolved in 20 mL of dichloromethane. DCC (1.33 g, 6.43 mmol) was added under ice-bath conditions. After the reaction system was warmed to room temperature, the mixture was stirred for 18 hours. After the reaction was completed, 20 mL of water was added, and the organic phase was separated. The aqueous phase was extracted with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain Compound 5 (1.90 g, yellow oil), with a yield of 72%.

[0225] 1 H NMR (400 MHz, CDCl3): δ 7.34 - 7.27 (m, 5H), 6.46 (s, 2H), 6.38 (s, 1H), 5.37 (m, 1H), 5.02 (s, 2H), 4.64 - 4.62 (m, 1H), 4.54 (s, 2H), 4.09 (t, J = 6.4 Hz, 4H), 3.98 (t, J = 6.0 Hz, 2H), 3.92 (t, J = 6.4 Hz, 2H), 3.59 (t, J = 6.0 Hz, 2H), 3.50 - 3.48 (m, 2H), 2.62 (t, J = 6.0 Hz, 2H), 2.49 (d, J = 6.4 Hz, 2H), 2.40 - 2.31 (m, 6H), 2.13 - 2.05 (m, 2H), 1.97 - 1.90 (m, 4H), 1.84 - 1.78 (m, 6H), 1.72 - 1.67 (m, 5H), 1.61 - 1.58 (m, 5H), 1.50 - 1.42 (m, 8H), 1.37 - 1.34 (m, 4H), 1.25 (m, 20H), 1.18 - 1.08 (m, 10H), 1.02 (s, 3H), 0.96 (m, 1H), 0.92 (d, J = 6.4 Hz, 3H), 0.89 - 0.86 (m, 12H), 0.68 (s, 3H).

[0226] Synthesis of Compound 6 in Example 6

[0227]

[0228] Potassium carbonate (0.47 mmol) and 5 mL of dichloromethane were added to a reaction flask. Chlorosulfonyl isocyanate (CSI) (0.32 mmol) was added, and the mixture was stirred evenly. Then 250 mg of Compound 5 (0.21 mmol) was added. The reaction was heated to 30 °C for 20 hours, then cooled to 0 °C. 4 mL of methanol and sodium hydroxide (1 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was removed by rotary evaporation. 10 mL of water was added, and after neutralization with 1 N hydrochloric acid, the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography to obtain Compound 6 (166 mg, yellow oil), with a yield of 70%. LC-MS (ESI+): 1131.8 [M+H]+.

[0229] 1 1H NMR (400 MHz, CDCl3): δ 6.45 (s, 2H), 6.33 (s, 1H), 5.39 (m, 1H), 4.64 - 4.62 (m, 1H), 4.54 (s, 2H), 4.09 (t, J = 6.2 Hz, 4H), 3.98 (m, 2H), 3.92 (m, 2H), 3.59 (t, J = 6.4 Hz, 2H), 3.50 - 3.48 (m, 2H), 2.61 (t, J = 5.8 Hz, 2H), 2.48 (d, J = 6.0 Hz, 2H), 2.40 - 2.31 (m, 6H), 2.13 - 2.05 (m, 2H), 1.97 - 1.90 (m, 4H), 1.84 - 1.78 (m, 6H), 1.72 - 1.40 (m, 18H), 1.39 - 1.05 (m, 34H), 1.02 (s, 3H), 0.96 (m, 1H), 0.92 (d, J = 6.4 Hz, 3H), 0.94 - 0.84 (m, 12H), 0.68 (s, 3H).

[0230] Example 7: Preparation and Characterization of mRNA-LNP

[0231] 1. Materials and Instruments

[0232] Table 1 Main Experimental Consumables

[0233]

[0234] Table 2 Main Experimental Equipment

[0235]

[0236] Table 3 Other Main Reagents

[0237]

[0238] 2. Experimental Scheme

[0239] Preparation of mRNA-LNP

[0240] Mix each lipid component in an ethanol solution according to a molar ratio of 65.7:32.8:1.5. Firefly luciferase mRNA (Luc-mRNA) is dissolved in 25 mM sodium acetate buffer at pH 4.0 to a final concentration of 135 ng / μL. The aqueous phase and the ethanol phase are mixed through a microfluidic device with a mixing flow rate of 9 mL / min for the aqueous phase and 3 mL / min for the ethanol phase. The prepared mRNA-LNP is dialyzed and ultrafiltered and concentrated into a buffer solution of 20 mM Tris, 25 mM sodium acetate, 87% by mass sucrose, and pH 7.5. After sterile filtration, the experimental sample is obtained.

[0241] Characterization of mRNA-LNP

[0242] Dilute the prepared mRNA-LNP experimental sample 50-fold with buffer (final concentration 2 - 100 ng / μL), and use a Malvern particle size potentiometer to detect the average particle size, PDI, and ζ potential of the nanoparticles; among them, the average particle size and PDI use a ZEN0040 type DLS sample cell with a sample loading volume of 200 μL; the detection of ζ potential uses a DTS1070 potential cell with a sample loading volume of 800 μL. The detection of mRNA content and encapsulation efficiency uses Quant-iT TM RiboGreen RNA Detection Kit, and TE buffer is used to detect the content C of free mRNA 游离 , and 2% Triton buffer is used to detect the total mRNA content C 总 , and the encapsulation efficiency is calculated according to the formula EE=(1 - C 游离 / C 总 )×100%. The experimental results are shown in Table 4.

[0243] Table 4 Physicochemical Parameter Characterization of Luc-mRNA-LNP

[0244]

[0245] *Note: For the comparative lipid compounds, the synthesis of each compound was carried out according to the method provided in the corresponding patent example, and the structural characterization showed that the compound structure was correctly synthesized.

[0246]

[0247] The results showed that the mRNA-LNP formed by the cationic lipid compound of the present invention, phospholipid (DOPE), PEG lipid (DMG-PEG), and mRNA had good physicochemical parameters. The average particle size was in the range of about 60 - 102 nm, the PDI was less than 0.15, with a good polydispersity coefficient, the zeta potential was between -5 mV and 5 mV, and the encapsulation efficiency of LNP for mRNA was greater than 90%, and the encapsulation efficiency was significantly better than that of the control group LNP.

[0248] Example 8 In vitro cell transfection activity of mRNA-LNP

[0249] The transfection activity of mRNA-LNP against HEK293T cells was evaluated by detecting the expression level of firefly luciferase Luc using a fluorescence microscope. The HEK293T cell solution at 6.5×10 5 cells / mL was inoculated into a 96-well cell culture plate at a volume of 200 μL / well. After 24 hours, 100 ng of Luc-mRNA-LNP was transfected into each well, and then the cell culture plate was placed in a cell culture incubator at 37 °C and 5% CO2. The negative control group was transfected with an equal volume of PBS. After 24 hours, after the culture was completed, bioluminescence detection was carried out with reference to the instruction manual of the Fire-Lumi TM luciferase detection kit, and the results were as Figure 1 shown. The results showed that the three-component LNP composition formed by using the cationic lipid compound of the present invention could achieve high expression of Luc-mRNA in cells, and the expression level was significantly better than that of the control group.

[0250] Example 9 Animal immunization test of mRNA-LNP

[0251] We used the SARS-CoV-2 spike protein mRNA to evaluate the immune activity of mRNA-LNP in mice. The LNP formulation used was an ionizable cationic lipid compound: DOPE: DMG-PEG2000 = 65.7:32.8:1.5 molar ratio for mRNA encapsulation. The specific formulation is shown in Table 4. Female BALB / c mice at 6 - 8 weeks of age were randomly grouped at 5 mice / group and immunized by intramuscular injection in the hind leg. Immunization was carried out on day 0 and day 14 respectively, and the immunization dose was 5 μg mRNA-LNP / mouse. Blood was collected on day 28 and serum was separated. The specific antibody titer against the SARS-CoV-2 virus spike protein antigen was detected by ELISA (enzyme-linked immunosorbent assay), and the intracellular cytokine staining (ICS) was used to detect the immune response levels of specific CD8+ T and CD4+ T cells in the spleen. The antibody titer, CD8+ T cell immunity, and CD4+ T cell immune response results are respectively as Figure 2 , Figure 3 and Figure 4As shown, after the three-component LNP constructed with the lipid compound provided by the present invention delivers the COVID-19 mRNA vaccine, the antibody titers, CD8+ T cell, and CD4+ T cell immune responses induced in mice are significantly better than those of the three-component LNP composed of the control compound. The results indicate that the immunogenicity of the mRNA vaccine composition formed by the lipid nanoparticles provided by the present invention is higher than that of the control group.

[0252] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lipid compound represented by formula (I): or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein, Ar is aryl or heteroaryl; the aryl or heteroaryl is optionally substituted by a group selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, carbocyclyl, aryl, halogen, alkoxy, alkylthio, NR4R4', R4-C(O)-, R4-C(O)O-; G1, G2 and G3 are each independently selected from a bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 alkynylene, optionally substituted carbocyclylene, optionally substituted arylene; L1, L2, L3, L4, L5 and L6 are each independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-、-NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-、-N(R a )C(=O)O-、-OC(=O)N(R a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-; One of R1, R2 and R3 is selected from an optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl; wherein the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 One or more -CH2- in the alkynyl group may be optionally replaced by -O-, -S-, -NR a -, carbocyclyl, aryl, heteroaryl, and / or heterocyclyl substitution; At the same time, another one of R1, R2 and R3 is selected from a steroidal group; At the same time, the third one of R1, R2 and R3 is selected from -(R4") q -NR a R b 、-(R4”) q -nitrogen-containing heteroaryl, -(R4") q -nitrogen-containing heterocyclic group, -(R4") q wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidine group are optionally substituted by one or more groups selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, halogen, hydroxyl, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 Acyl, C1-C 20 Acyloxy, R c O-C1-C 20 alkyl; R4 and R4' are each independently selected from H, Cl-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and / or heterocyclylalkyl; R4" is selected from C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkyne; R a , R b and R c are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1.

2. The compound according to claim 1 or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein Ar is an aryl group; the aryl group is optionally substituted by a group selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, carbocyclyl, aryl, halogen, alkoxy, alkylthio, NR4R4', R4-C(O)-, R4-C(O)O-; G1, G2 and G3 are each independently selected from a bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 alkynylene, optionally substituted carbocyclylene, optionally substituted arylene; L1, L2, L3, L4, L5 and L6 are each independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-、-NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-、-N(R a )C(=O)O-、-OC(=O)N(R a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-; R1 is selected from optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl; wherein C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 One or more -CH2- in the alkynyl group may be optionally replaced by O, S, -NR a -, carbocyclyl, aryl, heteroaryl, and / or heterocyclyl substitution; R2 is selected from a steroid group; R3 is selected from -(R4″) q -NR a R b 、-(R4”) q -nitrogen-containing heteroaryl, -(R4") q -nitrogen-containing heterocyclic group, -(R4") q wherein the nitrogen-containing heteroaryl, nitrogen-containing heterocyclic group and guanidine group are optionally substituted by one or more groups selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, halogen, hydroxyl, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 Acyl, C1-C 20 Acyloxy, R c O-C1-C 20 alkyl; R4 and R4' are each independently selected from H, Cl-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and / or heterocyclylalkyl; R4" is selected from C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkyne; R a , R b and R c are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1.

3. The compound according to claim 2 or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein Ar is phenyl; the phenyl group is optionally substituted by a group selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, carbocyclyl, aryl, halogen, alkoxy, alkylthio, NR4R4', R4-C(O)-, R4-C(O)O-; G1, G2 and G3 are each independently selected from a bond, an optionally substituted C1-C 20 Alkylene, optionally substituted C2-C 20 Alkenylene, optionally substituted C2-C 20 Alkyne; L1, L2, L3, L4, L5 and L6 are each independently selected from a bond, -O-, -S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)N(R a )-、-NR a C(=O)-, -S(=O)O-, -OS(=O)O-, -S(=O)2O-, -OS(=O)2O-, -C(=O)S-, -C(=S)S-, -OP(=O)(OR a )O-、-N(R a )C(=O)O-、-OC(=O)N(R a )-, -SS-, -OC(=O)S-, -SC(=O)O-, -N(R a )C(=O)N(R b )-; R1 is selected from optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 Alkynyl; wherein C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 One or more -CH2- in the alkynyl group may be optionally replaced by O, S, -NR a -, carbocyclyl, aryl, heteroaryl, and / or heterocyclyl substitution; R2 is selected from a steroid group; R3 is selected from -(R4″) q -NR a R b 、-(R4”) q -nitrogen-containing heteroaryl, -(R4") q - nitrogen-containing heterocyclic group; wherein the nitrogen-containing heteroaryl group and the nitrogen-containing heterocyclic group are optionally substituted by one or more groups selected from the following: C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxy, halogen, hydroxyl, mercapto, cyano, nitro, amino, carboxyl, C1-C 20 Acyl, C1-C 20 Acyloxy, R c O-C1-C 20 alkyl; R4 and R4' are each independently selected from H, Cl-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 alkynyl, carbocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl and / or heterocyclylalkyl; R4" is selected from C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkyne; R a , R b and R c are each independently selected from H, optionally substituted C1-C 20 Alkyl, optionally substituted C2-C 20 Alkenyl, optionally substituted C2-C 20 alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl; m, n and p are each independently selected from 1, 2 or 3; q is selected from 0 or 1.

4. A compound according to any one of the preceding claims, selected from or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; wherein L1, L2, L3, L4, L5, L6, G1, G2, G3, R1, R2, R3, m, n and p are as defined in any one of claims 1 to 3.

5. A compound according to any one of the preceding claims, selected from or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; in, L1, L2, L3, L4, L5, L6, G1, G2, G3, R1, R2, R3, m, n and p are as defined in any one of claims 1-3.

6. A compound according to any one of the preceding claims, selected from or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; in, R1, R2 and R3 are as defined in any one of claims 1 to 3, and r, s, t and u are each independently selected from an integer of 1-10.

7. A compound according to any one of the preceding claims, or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; in, The steroidal compound in the steroidal compound group is selected from naturally occurring steroidal compounds or their analogs; preferably, it includes plant sterols and animal sterols, or their analogs; more preferably, it is selected from cholesterol and its derivatives.

8. A compound according to any one of the preceding claims, or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof; in, The steroidal compound group has the following structure: R5 is selected from hydrogen, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C1-C 20 Alkoxycarbonyl C1-C 20 alkyl-; R6 is selected from hydrogen, halogen, cyano, hydroxyl, amino, oxo, C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the steroid group is selected from: Where R' is C 1-20 alkyl.

9. A compound according to any one of the preceding claims, selected from: or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof, wherein R1 and R3 are as defined in any one of claims 1 to 3, and r, s, t and u are each independently selected from an integer of 1 to 10.

10. A compound according to any one of the preceding claims, selected from: or a stereoisomer, a tautomer, and a pharmaceutically acceptable salt thereof, wherein R1 and R3 are as defined in any one of claims 1 to 3, and r, s, t and u are each independently selected from an integer of 1 to 10.

11. A compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; wherein R1 is selected from optionally substituted C1-C 20 Alkyl; wherein C1-C 20 One or more -CH2- in the alkyl group may be optionally replaced by O, S, -NR a -, carbocyclic group substitution.

12. A compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; wherein R1 is selected from 13. A compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; wherein R3 is selected from -(R4″) q -NR a R b 、-(R4”) q -5 or 6-membered nitrogen-containing heteroaryl, -(R4") q -5 or 6-membered nitrogen-containing heterocyclic group; wherein, The 5- or 6-membered nitrogen-containing heteroaryl and the 5- or 6-membered nitrogen-containing heterocyclic group are optionally substituted by one or more groups selected from the following: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, hydroxyl, thiol, cyano, nitro, amino, C1-C6 acyl, C1-C6 acyloxy, R c O-C1-C6 alkyl; R4" is selected from C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene; R a and R b Each independently selected from H and C1-C6 alkyl; R c Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, phenyl-C1-C6 alkyl; q is selected from 0 or 1.

14. A compound according to any one of the preceding claims, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; wherein R3 is selected from -R4"-NR a R b , -R4"-5 or 6-membered nitrogen-containing heterocyclic group; wherein, The 5- or 6-membered nitrogen-containing heterocyclic group is optionally substituted by a group selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, hydroxy, thiol, cyano, nitro, amino, C1-C6 acyl, C1-C6 acyloxy, R c O-C1-C6 alkyl; R4" is selected from C1-C6 alkylene; R a and R b Each independently selected from H, C1-C6 alkyl; R c Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, benzyl.

15. The compound according to claim 1, selected from: or its stereoisomers, tautomers, and pharmaceutically acceptable salts.

16. A lipid nanoparticle comprising the lipid compound according to any one of claims 1 to 15 or its stereoisomers, tautomers, and pharmaceutically acceptable salts thereof.

17. The lipid nanoparticle of claim 16, further comprising phospholipids and / or polyethylene glycol lipids.

18. The lipid nanoparticle of claim 17, further comprising a therapeutic and / or preventive agent.

19. The lipid nanoparticle according to claim 18, wherein the therapeutic and / or preventive agent comprises one or more nucleic acids, such as DNA, RNA, etc.

20. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 16-19 and a pharmaceutically acceptable carrier.

21. A method for treating and / or preventing a disease, comprising administering a therapeutically effective amount of the lipid nanoparticle according to any one of claims 16-19 or the pharmaceutical composition according to claim 20 to an individual in need thereof.

22. Use of a compound according to any one of claims 1 to 15 and / or a lipid nanoparticle according to claims 16 to 19 in the preparation of a therapeutic and / or preventive agent delivery system.

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