Ionizable liposomes, their preparation and use in gene delivery

Abstract

Ionizable liposomes, their preparation and use in gene delivery. Specifically, ionizable lipid compounds of the following Formula I are provided, stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs or solvates thereof, wherein the definitions of the various groups are as described herein. Also provided are lipid nanoparticles comprising the ionizable lipid compounds of Formula (I). Compositions and related uses are also provided. The compounds of Formula (I) enable the preparation of lipid nanoparticles for the delivery of nucleic acid-based therapeutics or active agents in vivo and in vitro.

Description

Technical Field

[0001] This invention relates to ionizable liposomes, their preparation, and their application in gene delivery. Background Technology

[0002] Following small molecule drugs and antibody drugs, nucleic acid drugs have become the third major class of new drugs. Unlike other types of drugs, they are composed of nucleotide sequences. Currently, nucleic acid drugs with therapeutic efficacy include plasmids, messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), and microRNA (miRNA).

[0003] Nucleic acids and cell membranes both carry the same negative charge, making it difficult for naked nucleic acids to directly enter cells without external force. Furthermore, nucleic acids are readily degraded by nucleases in the cytoplasm, hindering gene delivery and gene therapy. External force or a vector is required for gene delivery. Commonly used external forces are physical methods including electroporation, compression, and injection. The biggest drawback of these methods is that they cannot be used for in vivo gene delivery, and large-scale in vitro gene delivery is also not feasible.

[0004] Vectors are generally classified into viral vectors and non-viral vectors. Viral vectors have extremely high transfection efficiency both in vivo and in vitro, but they have many drawbacks, such as high toxicity, strong immune responses, low gene load, poor targeting, and complex preparation processes. Non-viral vectors, on the other hand, are easy to prepare, transport, and store, and are safe, effective, and non-immunogenic, thus gaining increasing attention and application.

[0005] Liposome-based nanoparticle (LNP) technology is a non-viral vector technology that has been developed in recent years. Using this technology, Alnylam launched the world's first siRNA drug. Pfizer, Moderna, and other companies have developed mRNA COVID-19 vaccines. Liposome-based nanoparticles (LNPs) are prepared from cationic lipids and auxiliary lipids (phospholipids, cholesterol, and PEGylated lipids) using microfluidic devices. Among them, the auxiliary lipids have been commercialized, while cationic lipids directly determine the encapsulation and delivery efficiency of nucleic acid drugs, becoming a core element in the development of LNP technology.

[0006] T cells (also known as T lymphocytes) are a type of lymphocyte that plays a crucial role in the immune response. With the increasing prevalence of cell immunotherapy, especially the application of CAR-T technology, the genetic engineering of T cells has become increasingly important. However, T cell transfection typically still uses electroporation or viral vectors. Research on liposome transfection of T cells is very limited. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a delivery vector for delivering genes into cells, its preparation method, and its uses.

[0008] The first aspect of this invention provides an ionizable lipid compound, said ionizable lipid compound being a compound represented by chemical formula I, or a stereoisomer thereof, a tautomer thereof, a pharmaceutically acceptable salt thereof, a prodrug thereof, or a solvate thereof:

[0009]

[0010] In the formula:

[0011] A1 and A2 can each be independently CH or N;

[0012] B is selected from -L4-N(R9R) 10 ) or R 12 ;

[0013] L1, L2, L3, and L4 are each independently selected from optionally substituted alkylene groups and optionally substituted -[(CH2)] groups. n O] m -(CH2) o - Optionally substituted alkenyl groups and optionally substituted ynyl groups;

[0014] R1, R2, R3, R4, R9, R 10 and R 12 Each of the following can be independently H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted acyl, -NR'R", carboxyl, nitro, cyano or alkylthionyl;

[0015] R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8bEach group is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, halogen, nitro, cyano, -NR'R”, and carboxyl;

[0016] n, m, and o are each independent integers from 1 to 10; and

[0017] R' and R” are each independently selected from H or optionally substituted alkyl groups.

[0018] A second aspect of the present invention provides a lipid nanoparticle containing an ionizable lipid compound of Formula I.

[0019] A third aspect of the present invention provides a composition comprising an ionizable lipid compound of Formula I, or comprising the lipid nanoparticles thereof.

[0020] A fourth aspect of the present invention provides a method for treating or preventing a disease or symptom of a subject, the method comprising administering a pharmaceutical composition as described in any embodiment of the present invention.

[0021] The fifth aspect of the present invention provides the use of the compound of formula I according to any embodiment of the present invention in the preparation of lipid nanoparticles or pharmaceutical compositions. Attached Figure Description

[0022] Figure 1 Cell viability obtained from flow cytometry analysis.

[0023] Figure 2 The transfection efficiency of the LNP formulation of this invention on cells under conditions with or without AopE4 addition.

[0024] Figure 3 Fluorescence image obtained through cell fluorescence imaging.

[0025] Figure 4 Fluorescence imaging of mice 6 hours after intramuscular (im) and intravenous (iv) injection of lipid nanoparticles.

[0026] Figure 5 Tissue fluorescence imaging of mice 6 hours after intramuscular (im) and intravenous (iv) injection of lipid nanoparticles.

[0027] Figure 6 Fluorescence imaging of mice 24 h after intramuscular (im) and intravenous (iv) injection of lipid nanoparticles.

[0028] Figure 7 Fluorescence intensity of lipid nanoparticles in mice 6 h and 24 h after intramuscular (im) and intravenous (iv) injection.

[0029] Figure 8Results of in vitro stability experiments. Detailed Implementation

[0030] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0031] I. Terminology

[0032] Unless otherwise specified, the following terms as used herein have the following meanings:

[0033] The terms “comprising” and “including” as used herein are interpreted in an open and inclusive sense, and mean “including but not limited to” throughout the specification and claims unless the context otherwise requires.

[0034] In this invention, references to "one embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrase "in one or more embodiments" appearing in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used in the specification and claims, the singular forms “a”, “the”, and “described” include plural references unless the context clearly indicates otherwise.

[0036] The "effective amount" or "therapeutic effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect (such as an increase or inhibition of target sequence expression compared to the normal expression level detected in the absence of nucleic acid).

[0037] The term "nucleic acid" as used herein refers to polymers containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA, RNA, and their hybrids. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can be in the form of hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, small interfering RNA (siRNA), microRNA (miRNA), multivalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof.

[0038] The term "gene" as used in this article refers to a nucleic acid (such as DNA or RNA) sequence that contains a partial or full length of coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0039] The term “lipids” as used herein refers to a group of organic compounds, including but not limited to esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, including fats and oils as well as waxes; (2) “complex lipids”, including phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0040] "Steroids" are compounds that contain the following carbon skeleton:

[0041]

[0042] Unrestricted examples of steroids include cholesterol.

[0043] "Cholesterol derivative" can be any cholesterol derivative known in the art for the preparation of liposomes, with exemplary cholesterol derivatives including commonly used cholesterol, CAS: 57-88-5.

[0044] The term "polymer-conjugated lipid" as used herein refers to a molecule comprising both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycol (PEG)-modified lipid. The term "PEGylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEG-DAG (diacylglycerol), PEG-PE (phosphatidylethanolamine), PEG-S-DAG, PEG-DSPE (distearate phosphatidylethanolamine), PEG-cer (ceramide), and PEG dialkoxypropyl carbamate, etc. In a preferred embodiment of the invention, the polymer-conjugated lipid is mPEG2000-DSPE.

[0045] The term "neutral lipids" as used herein refers to lipid substances that exist as zwitterions (uncharged or neutral) at a selected pH. At physiological pH, such lipids include, but are not limited to: phosphatidylcholines such as 1,2-distearyl-sn-glycero-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate choline (POPC), 1,2-dioleoyl-sn-glycero-3-phosphate choline (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), sphingomyelin (SM), and ceramides. Neutral lipids can be synthetic or of natural origin.

[0046] The term "lipid nanoparticles" as used herein refers to particles having at least one nanometer-scale size (e.g., 1-1000 nm). Lipid nanoparticles may be included in formulations for the delivery of active agents or therapeutic agents (e.g., nucleic acids) to target sites (e.g., cells, tissues (e.g., diseased tissues such as tumor tissue), organs). In some embodiments, the lipid nanoparticles of the present invention comprise nucleic acids. Such lipid nanoparticles typically comprise one or more compounds of Formula I of the present invention, one or more auxiliary lipid molecules, one or more cholesterol or cholesterol derivatives, and / or one or more polymer-conjugated lipid molecules. The auxiliary lipid molecules may be one or more neutral lipid molecules. Active agents or therapeutic agents may be encapsulated in the lipid portions of the lipid nanoparticles or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticles, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0047] As is known in the art, the average diameter of lipid nanoparticles can be about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 3 The nanoparticles have wavelengths of approximately 5 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In some embodiments, when present in the lipid nanoparticles, the nucleic acids are resistant to degradation by nucleases in aqueous solutions. Lipid nanoparticles containing nucleic acids and methods for their preparation are known in the art, for example, see CN102712935A or related patents, the entire disclosure of which is incorporated herein by reference for all purposes.

[0048] The morphology of the lipid nanoparticles of the present invention is not particularly limited, but examples of morphologies in which the ionizable lipids of the present invention are dispersed in an aqueous solvent include monolayer liposomes, multilayer liposomes, or unspecified layered structures.

[0049] In this article, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0050] "Hydroxy" refers to the -OH group.

[0051] "Carbonyl" refers to the -C(=O)- group.

[0052] "Carboxyl group" refers to -COOH.

[0053] "Nitro" refers to -NO2.

[0054] “Cyano” refers to -CN.

[0055] "Amino" refers to -NH2.

[0056] "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group containing one to twenty-four carbon atoms, such as methyl, ethyl, propyl, butyl, tridecyl, dodecyl, tridecyl, methylpentadecanyl, hexylnonyl, etc., and the alkyl group is connected to the rest of the molecule by a single bond. In some embodiments, the alkyl group described herein has 10-24 carbon atoms, or 12-20 carbon atoms.

[0057] "Alkylene" refers to a saturated, branched, or straight-chain hydrocarbon group having 1-20 carbon atoms and having two monovalent groups at the center obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane, such as -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-. In some embodiments, the alkylene described herein has 1-10 carbon atoms, or for example, 1-4 carbon atoms.

[0058] "Alkoxy" refers to an alkyl group with an alkyl-O- terminal, that is, an alkyl group containing one to twenty-four carbon atoms with an oxygen atom attached to the end, such as methoxy, ethoxy, n-propoxy, isopropoxy, etc. Alkylene oxide refers to an alkylene group with an oxygen atom attached to one end, such as -CH2CH2-O-.

[0059] "Alkoxycarbonyl" refers to a group in which a carbonyl group is attached to the oxygen atom of an alkoxy group, such as CH3OC(=O)-, CH3CH2OC(=O)-, etc.

[0060] "alkylthionyl" refers to an alkyl-S(=O)- group.

[0061] "Alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon group containing two to twenty-four carbon atoms. It contains one or more unsaturated carbon-carbon double bonds, such as vinyl, propenyl, tridecylenyl, tetradecadienyl, octadectrienyl, etc., and is connected to the rest of the molecule by single bonds.

[0062] "Subchain alkenyl" refers to a divalent straight-chain or branched alkenyl group, typically with two to twenty-four carbon atoms, containing one or more unsaturated carbon-carbon double bonds, and connected to the rest of the molecule by two single bonds. An exemplary subchain alkenyl group could be -CHCH=CHCH-.

[0063] "Cycloalkenyl" refers to an unsaturated cyclic alkenyl group containing three to ten cyclic carbon atoms.

[0064] "Alynyl" refers to a straight-chain or branched aliphatic hydrocarbon group containing two to twenty-four carbon atoms. It contains one or more unsaturated carbon-carbon triple bonds, such as ethynyl or propynyl, and is connected to the rest of the molecule by single bonds.

[0065] "Cycloalkynyl" refers to an unsaturated cyclic alkynyl group containing three to ten cyclic carbon atoms.

[0066] "Alkenylyl" refers to a divalent straight-chain or branched alkyne group, typically with three to twenty-four carbon atoms, containing one or more unsaturated carbon-carbon triple bonds, and connected to the rest of the molecule through two single bonds. An exemplary alkenylyl group could be -CHC≡CCH-.

[0067] "Acyl" refers to an alkyl-C(O)- group, such as acetyl, propionyl, etc.

[0068] "Aryl" refers to a carbocyclic ring system group comprising hydrogen, 6 to 18 carbon atoms (preferably 6 to 14 carbon atoms), and at least one aromatic ring. Aryl groups can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, and may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from acetane, acenaphthene, phenanthrene, anthracene, azulene, benzene, fluoranthene, fluorene, asymmetric indarene, symmetric indarene, indene, indene, naphthalene, phenanthracene, heptamethrin, pyrene, and triphenylene.

[0069] "Aryloxy group" refers to -OR, where R stands for aryl.

[0070] A "carbocyclic group" refers to a stable, saturated or unsaturated non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, with the number of ring carbon atoms ranging from 3 to 18. Carbocyclic groups can include fused or bridging ring systems having 3 to 18 carbon atoms, are saturated, and are connected to the rest of the molecule via single bonds. In some embodiments, the carbocyclic group is a cycloalkyl group, with representative cycloalkyl groups including, but not limited to, those having three to fifteen carbon atoms (C3-C4). 15 cycloalkyl groups, three to ten carbon atoms (C3-C4) 10Cycloalkyl groups are cycloalkyl groups with three to eight carbon atoms (C3-C8 cycloalkyl), three to six carbon atoms (C3-C6 cycloalkyl), three to five carbon atoms (C3-C5 cycloalkyl), or three to four carbon atoms (C3-C4 cycloalkyl). Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decahydronaphthyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decahydronaphthyl, trans-decahydronaphthyl, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, as well as 7,7-dimethyl-bicyclo[2.2.1]heptyl. In a preferred embodiment, the cycloalkyl group is a 3-8 membered cycloalkyl group, such as cyclopropyl, cyclopentyl, and cyclohexyl.

[0071] A "heterocyclic group" refers to a stable 3- to 20-membered non-aromatic cyclic group consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, phosphorus, oxygen, and sulfur. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, or more ring system, and may include fused ring systems, bridged ring systems, or spirocyclic systems. The heterocyclic group can be partially or fully saturated. The heterocyclic group can be connected to the rest of the compound via a carbon atom or heteroatom and through a single bond. The heterocyclic group is preferably a stable 4- to 11-membered non-aromatic monocyclic, bicyclic, bridged, or spirocyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur; more preferably, it is a stable 4- to 8-membered non-aromatic monocyclic, bicyclic, bridged, or spirocyclic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Examples of heterocyclic groups include, but are not limited to: pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2,7-diaza-spiro[3.5]nonane-7-yl, 2-oxa-6-aza-spiro[3.3]heptane-6-yl, 2,5-diaza-bicyclo[2.2.1]heptane-2-yl, aziridine, pyranyl, tetrahydropyranyl, thiaranyl, tetrahydrofuranyl, oxazinyl, dioxocyclopentyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, quinazinyl, thiazoalkyl, isothiazyl, isoxazylalkyl, dihydroindolyl, octahydroindolyl, octahydroisoindolyl, pyrrolidinyl, pyrazolyl, phthalimide, etc.

[0072] "Heteroaryl" refers to a 5- to 16-membered conjugated cyclic group having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. The heteroaryl group can be a monocyclic, bicyclic, tricyclic, or more ring system. Preferably, the heteroaryl group is a stable 5- to 12-membered aromatic group containing 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur; more preferably, it is a stable 5- to 10-membered aromatic group containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered aromatic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups include, but are not limited to, thiophene, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indole, furanyl, pyrroleyl, triazolyl, tetrazolyl, triazinyl, indazinyl, isoindoleyl, indazole, isoindazoleyl, purine, quinolinyl, isoquinolinyl, diazonyl, naphthidyl, quinoxolinyl, pteridyl, carbazolyl, carbazolyl, phenanthridine, and phenanthridine. Linyl, acridine, phenazinyl, isothiazolyl, benzothiazolyl, benzothiophene, oxatriazolyl, cinolinyl, quinazolinyl, phenylthio, indene, o-diazaphenanthyl, isoxazolyl, phenoxazinyl, phenthiazolyl, 4,5,6,7-tetrahydrobenzo[b]thiophene, naphthopyridyl, [1,2,4]triazolo[4,3-b]pyridazine, [1,2,4]triazolo[4,3-a]pyrazine, [1,2,4]triazolo[4,3-c]pyrimidine, [1,2,4]triazolo[4,3-a]pyridine, imidazo[1,2-a]pyridine, imidazo[1,2-b]pyridazine, imidazo[1,2-a]pyrazine, etc.

[0073] In this document, when a group is "optionally substituted," it may be optionally substituted by 1 to 5 substituents, which may be selected from: alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclic groups. These aryl, heteroaryl, cycloalkyl, and heterocyclic groups may each be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro, aryl, heteroaryl, cycloalkyl, and heterocyclic groups. It should be understood that the number of substituents is influenced by the molecular structure of the compound. For example, when the substituent is aryl, heteroaryl, cycloalkyl, or heterocyclic, the number of substituents is usually 1; when the substituent is halogen, the number of halogen atoms may be 2 to 5, depending on the chain length of the substituted group or the number of ring carbon atoms.

[0074] In this document, "ionizable lipid compound" refers to a lipid compound that exists in a charged form at a specific pH value or within a specific pH range, including positively or negatively charged compounds, preferably positively charged lipid compounds (i.e., cationic lipid compounds). The specific pH value or pH range refers to the pH value or pH range of the storage or intended use environment of the lipid compound, including but not limited to physiological pH.

[0075] In this article, "pharmaceutically acceptable salts" include acid addition salts and base addition salts.

[0076] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the bioavailability and properties of the free base and form with inorganic or organic acids. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. The organic acids include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactopyric acid, gentian acid, glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, glutaric acid, 2-oxo- Glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthalic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid, etc.

[0077] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological potency and properties of the free acid. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianophenamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, heparin, choline, betaine, benzylamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0078] In this document, "pharmaceutical composition" refers to a formulation containing a compound of Formula I of the present invention and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions typically contain a therapeutically effective amount of a compound of Formula I and / or a therapeutic agent or active agent. "Therapeutically effective amount" means an amount sufficient to achieve therapeutic effect in a mammal, preferably a human, when administered. The amount of the compound of Formula I of the present invention and / or the therapeutic agent or active agent constituting a "therapeutically effective amount" will vary depending on the compound or the therapeutic agent or active agent used, the condition and its severity, the method of administration, and the age of the mammal to be treated, but can be conventionally determined by those skilled in the art based on their knowledge and the content of this disclosure.

[0079] The term "pharmaceutically acceptable carriers or excipients" as used herein includes, but is not limited to, any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by the FDA or CFDA for use in humans or domestic animals.

[0080] The term "treatment" as used herein encompasses treatment for a target disease or condition in mammals, preferably humans, and includes:

[0081] (1) To prevent the occurrence of the disease or condition in mammals, especially when such mammals are susceptible to the condition but have not yet been diagnosed with it;

[0082] (2) To suppress the disease or condition, that is, to prevent its development;

[0083] (3) To alleviate the disease or condition, that is, to cause the disease or condition to subside; or

[0084] (4) Relieve the symptoms caused by the disease or condition, that is, relieve pain without addressing the underlying disease or condition.

[0085] In this article, “disease” and “condition” may be used interchangeably or differently, because a particular disease or condition may not have a known pathogen (so the cause is not yet known), and therefore it has not been recognized as a disease but is only regarded as an undesirable condition or syndrome in which clinicians have identified more or less a specific group of symptoms.

[0086] In this article, "mammals" includes humans, as well as domesticated animals such as laboratory animals and pets (such as cats, dogs, pigs, cattle, sheep, goats, horses, and rabbits) and non-domesticated animals (such as wild animals).

[0087] Compounds of Formula I, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers, thus yielding enantiomers, diastereomers, and other stereoisomers that can be defined, in absolute stereochemistry, as (R)- or (S)- or (D)- or (L)- (for amino acids). This invention includes all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and stepwise crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain alkene double bonds or other geometrically asymmetric centers, and unless otherwise stated, the compounds are intended to include E and Z geometric isomers. Similarly, it aims to include all tautomer forms.

[0088] II. Ionizable lipid compounds

[0089] The ionizable lipid compounds described herein have the structural formula shown in Formula I:

[0090]

[0091] In the formula, A1 and A2 are each independently CH or N; B is selected from -L4-N(R9R) 10 ) or R 12 L1, L2, L3, and L4 are each independently selected from optionally substituted alkylene groups and optionally substituted -[(CH2)] groups. n O] m -(CH2) o - Optionally substituted subalkenyl groups and optionally substituted ynyl groups; R1, R2, R3, R4, R9, R 10 and R 12 Each of the following can be independently H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted acyl, -NR'R", carboxyl, nitro, cyano, or alkyl sulfoxide; R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8bEach is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, halogen, nitro, cyano, -NR'R" and carboxyl; n, m and o are each independently integers from 1 to 10; and R' and R" are each independently selected from H or optionally substituted alkyl.

[0092] This article also includes stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs, and solvates of compounds represented by Formula I.

[0093] In Formula I, when a group is "optionally substituted", the substituent can be selected from hydroxyl, alkoxy, halogen, alkyl, haloalkyl, hydroxy-substituted alkyl, alkenyl, haloalkenyl, hydroxy-substituted alkenyl, -NR'R"", optional substituted cycloalkyl, optional substituted aryl, optional substituted heteroaryl, or optional substituted heterocyclic. The number of substituents can be 1, 2, 3, 4, 5, or 6. R' and R" are each independently selected from H and C. 1-4 Alkyl group. The alkyl group, including alkyl, alkoxy, haloalkyl, and hydroxyl-substituted alkyl groups, can be C10. 1-6 Alkyl or C 1-4 Alkyl; the alkenyl, haloalkenyl, and hydroxyl-substituted alkenyl groups may be C10-2000. 2-6 alkenyl or C 2-4 Alkenyl; the cycloalkyl group may be C3-8 cycloalkyl, optionally surrounded by 1-4 groups selected from halogens, C 1-4 The aryl group may be substituted with alkyl, hydroxyl, and -NR'R" substituents; the aryl group may be a 6-14 membered aryl group, such as phenyl and naphthyl, and the aryl group may optionally be replaced by 1-4 substituents selected from halogens, C 1-4 The heteroaryl group may be substituted with alkyl, hydroxyl, and -NR'R" substituents; the heteroaryl group may be a 5-10 member heteroaryl group, such as a 5-10 member nitrogen- and / or oxygen-containing heteroaryl group, such as pyridyl, pyrazolyl, and imidazolyl, etc., and the heteroaryl group may optionally be replaced by 1-4 substituents selected from halogens, C 1-4 The heterocyclic group is substituted with alkyl, hydroxyl, and -NR'R" substituents; the heterocyclic group can be a 4-10 member heterocyclic group, preferably a 4-10 member nitrogen- and / or oxygen-containing heterocyclic group, such as morpholino, piperidinyl, piperazine, pyrrolidinyl, etc., and the heterocyclic group may optionally be replaced by 1-4 substituents selected from halogens, C 1-4 Alkyl, hydroxyl and -NR'R" substituents.

[0094] In the preferred embodiment, in Formula I, A1 and A2 are both N.

[0095] In a preferred embodiment, in Formula I, L1, L2, L3, and L4 are each independently an optionally substituted alkylene group or an optionally substituted -[(CH2)] group. n O] m -(CH2) o- Preferably, L1, L2, L3, and L4 are each optionally selected from hydroxyl, -NR'R", C 1-4 Alkoxy and halogen are substituted with 1-5 substituents, wherein R' and R" are each independently selected from H and C. 1-4 Alkyl group. Preferably, L1, L2, L3, and L4 are each independently C1. 1-4 Alkylene or -[(CH2)] n O] m -(CH2) o -

[0096] In the preferred embodiment, in Equation I, n, m, and o are each independent integers from 1 to 4.

[0097] In some preferred embodiments, n is 2, m is 1-4, and o is 2.

[0098] In some implementations, in Equation I, L1, L2, L3, and L4 are all C 1-4 Alkylene. In some embodiments, L1, L2, and L4 are each independently -[(CH2)] n O] m -(CH2) o - Preferably, L1, L2, and L4 are the same group; L3 is C 1-4 Alkylene. Preferably, in these embodiments, n, m, and o are each independently an integer from 1 to 4; more preferably, n is 2, m is 1-4, and o is 2.

[0099] In a preferred embodiment, in Formula I, R1, R2, R3, and R4 are each independently an alkyl group optionally substituted with a hydroxyl group and / or a halogen. Preferably, R1, R2, R3, and R4 are substituted with at least one hydroxyl group. The preferred number of hydroxyl substituents is 1, 2, 3, 4, or 5, more preferably 1. Preferably, at least one hydroxyl group is located on the second carbon atom at the N-terminus of the alkyl group. Preferably, the carbon chain length of the alkyl group is 1-24 carbon atoms, more preferably 8-24 carbon atoms, more preferably 10-20 carbon atoms. In some embodiments, the carbon chain length of the alkyl group is 12-18 carbon atoms, and preferably, the alkyl group has one hydroxyl substituent on the second carbon atom at the N-terminus of the alkyl group. In some embodiments, R1, R2, R3, and R4 are each independently -CH2CH(OH)CH2R. 11 , wherein, the R 11 C that is optionally substituted with halogen 1-21 Alkyl, preferably, R 11 C 8-15 Alkyl group. In some embodiments, R 11 For unreplaced C 1-21Alkyl or perfluorinated C 1-21 Alkyl group. In a preferred embodiment, R1, R2, R3, and R4 are the same group.

[0100] In the preferred embodiment, in formula I, R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8b Each is independently selected from H and optionally substituted C 1-4 Alkyl group. The C... 1-4 The alkyl group is optionally substituted with 1 to 5 substituents selected from hydroxyl and halogen. More preferably, R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8b All are H.

[0101] In the preferred embodiment, R9, R 10 and R 12 Each is independently an alkyl group optionally substituted with a hydroxyl group and / or a halogen. In some embodiments, R9, R 10 and R 12 Each is at least substituted with a hydroxyl group, preferably at least one hydroxyl group. The preferred number of hydroxyl substituents is 1-5, more preferably 1. Preferably, at least one hydroxyl group is located on the second carbon atom at the N-terminus of the alkyl group. Preferably, the carbon chain length of the alkyl group is 1-24 carbon atoms, preferably 8-24 carbon atoms, more preferably 10-20 carbon atoms. In some embodiments, the carbon chain length of the alkyl group is 12-18 carbon atoms, and preferably, the alkyl group has one hydroxyl substituent on the second carbon atom at the N-terminus of the alkyl group. In some embodiments, R9, R... 10 and R 12 Each is independently -CH2CH(OH)CH2R 11 , wherein, the R 11 C that is optionally substituted with halogen 1-21 Alkyl, preferably, R 11 C 8-15 Alkyl group. In some embodiments, R 11 For unreplaced C 1-21 Alkyl or perfluorinated C 1-21 Alkyl groups. In a preferred embodiment, R9 and R 10 They are the same group.

[0102] In some preferred embodiments, R1, R2, R3, R4, R9 and R 10 Both are -CH2CH(OH)CH2R 11 The functional groups are preferably all the same.

[0103] In some implementations, B is R 12 R 12 C is optionally substituted with 1, 2, 3, 4 or 5 hydroxyl groups. 1-10 Alkyl group. Preferably, the second carbon atom at the N-terminus of the alkyl group is replaced by a hydroxyl group. Preferably, R 12 For unreplaced C 1-4 Alkyl groups or C groups substituted with one or two hydroxyl groups 1-4 alkyl.

[0104] In some implementations, B is R 12 R 12 C10, which is optionally substituted with 1-5 hydroxyl groups 1-10 Alkyl group, preferably, wherein the second carbon atom at the N-terminus of the alkyl group is replaced by a hydroxyl group; L1 and L2 are each independently -[(CH2)] n O] m -(CH2) o -or C 1-4 Alkylene, preferably, L1 and L2 are the same group; L3 is C 1-4 Alkylene.

[0105] In a preferred embodiment, in formula I:

[0106] Both A1 and A2 are N;

[0107] R1, R2, R3, and R4 are each independently C1 cells that are optionally substituted with hydroxyl groups. 1-24 Alkyl group; preferably, R1, R2, R3 and R4 are substituted with at least one hydroxyl group; preferably, R1, R2, R3 and R4 are each independently -CH2CH(OH)CH2R 11 , wherein, the R 11 C that is optionally substituted with halogen 1-21 Alkyl group, preferably R 11 For unreplaced C 1-21 alkyl;

[0108] R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8b All are H;

[0109] B is -L4-N(R9R) 10 ); where L1, L2, L3, and L4 are all C 1-4 Alkylenes, preferably, L1, L2, L3, and L4 are the same alkylene group; or L1, L2, and L4 are each independently -[(CH2)] n O] m -(CH2) o - Preferably, L1, L2, and L4 are the same group, and L3 is C 1-4 Alkylenes; R9 and R 10 Each is independently a C that is optionally substituted with a hydroxyl group and / or a halogen. 1-24 Alkyl groups; preferably, R9 and R 10 At least one hydroxyl group is substituted; preferably, R9 and R 10 Each is independently -CH2CH(OH)CH2R 11 , wherein, the R 11 C that is optionally substituted with halogen 1-21 Alkyl, preferably, R 11 For unreplaced C 1-21 alkyl;

[0110] Or B is R 12 Among them, R 12 C10, which is optionally substituted with 1-5 hydroxyl groups 1-10 Alkyl group, preferably, wherein the second carbon atom at the N-terminus of the alkyl group is replaced by a hydroxyl group; L1 and L2 are each independently -[(CH2)] n O] m -(CH2) o -or C 1-4 Alkylene, preferably, L1 and L2 are the same group; L3 is C 1-4 Alkylene;

[0111] n, m, and o are each an independent integer from 1 to 4.

[0112] In a preferred embodiment, in formula I: A1 and A2 are both N; R1, R2, R3, and R4 are each independently -CH2CH(OH)CH2R. 11 , wherein, the R 11 For unreplaced C 5-18 Alkyl; R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8b Both are H; B is R. 12 ;R12 C is optionally substituted with 1, 2, 3, 4 or 5 hydroxyl groups. 1-10 Alkyl, preferably C 1-4 Alkyl group, preferably, wherein the second carbon atom at the N-terminus of the alkyl group is replaced by a hydroxyl group; L1 and L2 are each independently -[(CH2)] n O] m -(CH2) o -or C 1-4 Alkylene, preferably, L1 and L2 are the same group; L3 is C 1-4 Alkylene; n is 2, m is 1-4, o is 2.

[0113] In a preferred embodiment, in formula I: A1 and A2 are both N; R1, R2, R3, and R4 are each independently -CH2CH(OH)CH2R. 11 , wherein, the R 11 For unreplaced C 5-18 Alkyl; R 5a R 5b R 6a R 6b R 7a R 7b R 8a and R 8b Both are H; B is -L4-N(R9R) 10 L1, L2, and L4 are each independently -[(CH2)] n O] m -(CH2) o -; L3 is C 1-4 Alkylenes; R9 and R 10 Each is independently -CH2CH(OH)CH2R 11 , wherein, the R 11 For unreplaced C 5-18 Alkyl; n is 2, m is 1-4, o is 2.

[0114] In a preferred embodiment, compound I has the following structure:

[0115]

[0116]

[0117] In each formula, a, b, c, d, e, and f are each independently an integer from 0 to 20, preferably an integer from 8 to 16, and more preferably an integer from 9 to 15. In some embodiments, a, b, c, d, e, and f are all the same integer from 0 to 20, preferably the same integer from 8 to 16, and more preferably the same integer from 9 to 15.

[0118] In a preferred embodiment, the compound of formula I is:

[0119]

[0120] In a preferred embodiment, the compound of formula I is:

[0121]

[0122] In a preferred embodiment, the compound of formula I is:

[0123]

[0124] III. Lipid Nanoparticles

[0125] The compound of formula I of the present invention can be used to prepare lipid nanoparticles for in vivo and in vitro delivery of nucleic acid therapeutic agents or active agents.

[0126] In addition to the compound of Formula I of the present invention, the lipid nanoparticles of the present invention may also contain one or more auxiliary lipid molecules, one or more cholesterol or cholesterol derivatives and / or one or more polymer-conjugated lipid molecules.

[0127] Preferably, the auxiliary lipid molecule is a neutral lipid molecule, preferably selected from: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In a preferred embodiment, the lipid nanoparticles contain DOPE. In some embodiments, the lipid nanoparticles contain DSPC.

[0128] Preferably, the cholesterol derivative is cholesterol (abbreviated as chole, CAS: 57-88-5).

[0129] Preferably, the polymer in the polymer-conjugated lipid molecule is polyethylene glycol. In a preferred embodiment, the polymer-conjugated lipid molecule is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-DSPE, PEG-cer, DMG-PEG, and PEG dialkoxypropyl carbamate. Preferably, the lipid nanoparticles contain PEG2000-DSPE. In some embodiments, the lipid nanoparticles contain DMG-PEG2000.

[0130] In a preferred embodiment, the liposome nanoparticles, when containing a compound of Formula I, its stereoisomers, racemates, or pharmaceutically acceptable salts, have a molar ratio of 60–5:60–5:50–5:10–1 to an accessory lipid molecule, cholesterol or a cholesterol derivative, or a polymer-conjugated lipid molecule, preferably 40–10:30–10:50–20:8–1. In some embodiments, this molar ratio is 40–30:20–10:50–40:4–1.

[0131] The evaluation particle size of the lipid nanoparticles of the present invention is typically between 30 nm and 150 nm, preferably between 40 nm and 150 nm.

[0132] In a preferred embodiment, the lipid nanoparticles of the present invention contain a therapeutic agent or active agent, preferably a nucleic acid therapeutic agent or active agent. More preferably, the nucleic acid therapeutic agent or active agent is selected from: messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and aptamers. In some embodiments, the lipid nanoparticles of the present invention can be used to express proteins encoded by mRNA. The proteins can be, for example, proteins that have therapeutic, preventive, or physiological function-enhancing effects on an organism, including but not limited to antigens, antibodies, and proteins with biological functions known in the art. In other embodiments, the lipid nanoparticles of the present invention can be used to upregulate endogenous protein expression by delivering a miRNA inhibitor targeting a specific miRNA or by regulating a target mRNA or a group of miRNAs of several mRNAs. In other embodiments, the lipid nanoparticle compositions of the present invention can be used to downregulate (e.g., silence) the protein and / or mRNA levels of a target gene. In some other embodiments, the lipid nanoparticles of the present invention can also be used to deliver mRNA and plasmids to express transgenes. In other embodiments, the lipid nanoparticles of the present invention can be used to induce pharmacological effects resulting from protein expression, such as increasing red blood cell production by delivering suitable erythropoietin mRNA, or protecting against infection by delivering mRNA encoding an antigen or antibody of interest. When the lipid nanoparticles of the present invention contain a therapeutic agent or active agent, the mass ratio of the liposome itself to the therapeutic agent or active agent, such as mRNA, can be in the range of, for example, 5 to 20:1.

[0133] The lipid nanoparticles of this invention can be prepared using conventional methods in the art. For example, compound I is mixed with an auxiliary lipid molecule, cholesterol, and polymer-conjugated lipid molecules in a specific molar ratio and dissolved in ethanol to obtain an ethanol-lipid solution. mRNA is dissolved in citrate buffer to obtain an aqueous mRNA solution. The ethanol-lipid solution and the aqueous mRNA solution are mixed at a specific volume ratio using a microfluidic device. Ultrafiltration is performed to remove the ethanol, and the solution is brought to volume using DPBS. Finally, the lipid nanoparticles are filtered through a 0.2 μm sterile filter to obtain an LNP formulation using ionizable lipids.

[0134] IV. Composition

[0135] The present invention also provides a composition comprising a compound represented by Formula I of the present invention. In some embodiments, the composition is a pharmaceutical composition comprising a compound represented by Formula I of the present invention, a therapeutic agent or active agent, and one or more auxiliary lipid molecules, one or more cholesterol or cholesterol derivatives and / or one or more polymer-conjugated lipid molecules.

[0136] Preferably, the auxiliary lipid molecule is a neutral lipid molecule, preferably selected from: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In a preferred embodiment, the lipid nanoparticles contain DOPE. In some embodiments, the lipid nanoparticles contain DSPC.

[0137] Preferably, the cholesterol derivative is cholesterol.

[0138] Preferably, the polymer in the polymer-conjugated lipid molecule is polyethylene glycol. In a preferred embodiment, the polymer-conjugated lipid molecule is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-DSPE, PEG-cer, DMG-PEG, and PEG dialkoxypropyl carbamate. Preferably, the lipid nanoparticles contain PEG2000-DSPE. In some embodiments, the lipid nanoparticles contain DMG-PEG2000.

[0139] Preferably, the therapeutic agent or active agent is a nucleic acid therapeutic agent or active agent. More preferably, the nucleic acid therapeutic agent or active agent is selected from: messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and aptamers.

[0140] Preferably, in the composition, the compound represented by Formula I, one or more auxiliary lipid molecules, one or more cholesterol or cholesterol derivatives, and one or more polymer-conjugated lipid molecules form liposome nanoparticles as described in any embodiment of this application, which encapsulate the therapeutic agent or active agent. Preferably, in the composition, the mass ratio of the liposome nanoparticles themselves to the therapeutic agent or active agent, such as mRNA, may be in the range of, for example, 5 to 20:1.

[0141] Preferably, the pharmaceutical composition contains a therapeutically or preventively effective amount of the lipid nanoparticles described in any embodiment of the present invention and a pharmaceutically acceptable carrier or excipient. Specifically, the compound of Formula I of the present invention is present in the composition in an amount that effectively forms lipid nanoparticles and delivers a therapeutically or preventively effective amount of a therapeutic agent or active agent, such as for treating a specific target disease or condition. Appropriate concentrations and dosages can be readily determined by those skilled in the art.

[0142] In some embodiments, the compositions of the present invention further comprise an apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins and their variants and fragments known to those skilled in the art, as well as apolipoprotein agonists or their analogues and fragments described below. Suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V, and ApoE, as well as their active polymorphs, isoforms, variants, and mutants, and their fragments or truncated forms. In a preferred embodiment, the apolipoprotein is ApoE4.

[0143] The pharmaceutical compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, intraperitoneal, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term "intraperitoneal" includes subcutaneous, intravenous, intramuscular, intradermal, and intrasternal injection or infusion techniques.

[0144] Appropriate pharmaceutically acceptable carriers and excipients can be selected based on the specific dosage form and route of administration. For example, as a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, sheets, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; gliding agents such as colloidal silica; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavoring; and coloring agents.

[0145] When the pharmaceutical composition is in the form of capsules, such as gelatin capsules, it may contain liquid carriers such as polyethylene glycol or oil, in addition to the materials described above.

[0146] The pharmaceutical compositions of the present invention can be prepared by methods well known in the pharmaceutical industry. For example, a pharmaceutical composition intended for injection can be prepared by combining the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a solution.

[0147] The pharmaceutical compositions of the present invention are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health, sex, and diet; the method and time of administration; the excretion rate; the combination of drugs; the severity of the specific disease or condition; and the subject of treatment.

[0148] V. Treatment methods and uses

[0149] The lipid nanoparticles and pharmaceutical compositions of the present invention can be used to deliver therapeutic agents or active agents, such as nucleic acids, in vivo and in vitro for the treatment or prevention of diseases or conditions in subjects.

[0150] Therefore, the present invention provides the use of the compound of formula I in the preparation of lipid nanoparticles or pharmaceutical compositions for treating or preventing a disease or condition of a subject, and the compound of formula I, lipid nanoparticles or pharmaceutical compositions of the present invention for treating or preventing a disease or condition of a subject.

[0151] The diseases and conditions described herein can be any of the various diseases and conditions known in the art suitable for nucleic acid therapy and prevention, and depend on the specific biological function of the nucleic acid delivered by the lipid nanoparticles. In some embodiments, the pharmaceutical composition is a vaccine, and the method includes immunizing an individual to render that individual immune to a corresponding disease or condition, including but not limited to influenza, hepatitis B, hepatitis C, and infections caused by the novel coronavirus. The diseases or conditions also include, for example, tumors, including solid tumors and hematologic malignancies, various inflammations, etc.

[0152] The routes and methods of administration are well known in the art and as described above, including but not limited to oral, local, transdermal, inhalation, intraperitoneal, sublingual, buccal, rectal, vaginal, and intranasal administration. In some embodiments, the intraperitoneal administration route includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques.

[0153] This invention also includes the use of the compound of Formula I in the delivery of therapeutic agents or active agents such as nucleic acids, or in the preparation of reagents for delivering therapeutic agents or active agents such as nucleic acids. This invention also provides a method for in vivo or in vitro delivery of therapeutic agents or active agents such as nucleic acids, the method comprising encapsulating the therapeutic agent or active agent within lipid nanoparticles made using a compound of Formula I, and delivering the therapeutic agent or active agent via the lipid nanoparticles or a composition containing the lipid nanoparticles. The nucleic acid may be as described in any embodiment herein. In some embodiments, the therapeutic agent or active agent (such as nucleic acid) is delivered to a target site (such as cells, tissues (such as diseased tissues such as tumor tissues), or organs) via the lipid nanoparticles or a composition containing the lipid nanoparticles. In some embodiments, the cells are immune cells, including but not limited to T cells, dendritic cells (DC cells), or tumor-infiltrating lymphocytes (TILs).

[0154] VI. Preparation method of compound I

[0155] The compound of formula I of this invention can be prepared using the following general synthetic routes I and II:

[0156]

[0157]

[0158] In the above general synthetic routes I and II, each R1 can be independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclic, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, optionally substituted alkoxycarbonyl, optionally substituted acyl, -NR'R"", carboxyl, nitro, cyano, or sulfoxide; wherein R' and R" are each independently selected from H or optionally substituted alkyl. Each G is independently selected from optionally substituted alkylene, optionally substituted -[(CH2)] n O] m -(CH2) o - Optionally substituted alkenyl and alkynyl groups. When synthesizing other compounds of Formula I, compounds having the corresponding groups may be used as reactants to replace the corresponding reactants in synthetic routes I and II described above.

[0159] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0160] Example

[0161] Example 1: Synthesis of the target compound

[0162] 1. Synthesis of A-C12, A-C14, A-C16, and A-C18

[0163]

[0164] Synthetic circuit

[0165]

[0166] Step 1: Synthesis of compound 3 ((2-(4-(2-(2-(((benzyloxycarbonyl)amino)ethyl)aminoethyl)piperazinylethyl)carbamate)

[0167] A mixture of 2-(piperazin-1-yl)ethyl-1-amine (1 g, 7.75 mmol, 1.0 eq), benzyl (2-bromoethyl)carbamate (7.9 g, 31.0 mmol, 4.0 eq), and K₂CO₃ (6.4 g, 46.5 mmol, 6.0 eq) was heated and stirred overnight at 80 °C in anhydrous acetonitrile (50 mL). TLC (MeOH / DCM = 1 / 10) showed complete disappearance of the starting amine. Insoluble salts or other impurities were removed by vacuum filtration, and the filter cake was washed with DCM. The organic phases were combined and concentrated by rotary evaporation to obtain the crude product. The crude product was purified using a CombiFlash column (40 g silica gel column, DCM / MeOH (containing 10% ammonia) = 100 / 1–100 / 7, v / v). The purified product fraction was evaporated to give a colorless oily compound 3 (4 g, 78.2% yield). ESI: [M+1]: 661.1.

[0168] Step 2: Synthesis of compound 4 (N1-(2-aminoethyl)-n1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethylenediamine)

[0169] (4.0 g, 6.0 mmol, 1.0 eq) of (2-(4-(2-(2-(((benzyloxycarbonyl)amino)ethyl)aminoethyl)piperazinylethyl)carbamate (4.0 g, 6.0 mmol, 1.0 eq) was dissolved in 40 mL of anhydrous methanol, and Pd / C (10%, 400 mg) was added. The reaction was carried out overnight under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered under reduced pressure. The filter cake was washed with a small amount of methanol, and the filtrate was concentrated under reduced pressure to give compound 4 (1.2 g, 76.9% yield). ESI: [M+1]: 259.1.

[0170] Step 3: Synthesis of 1,1',1",1"-((2-(4-(2-(2-(2-(2-(2-(2-(2-(2-hydroxydodecyl)aminoethyl)piperazin-1-yl)ethyl)azodimethyl)bis(2,1-diyl)bis(azotriyl)tetra(dodecyl-2-ol)

[0171] Compound 4 (100 mg, 0.387 mmol, 1.0 eq), 1,2-epoxydodecane (712 mg, 3.87 mmol, 10.0 eq), and EtOH (1 ml) were mixed thoroughly. The mixture was heated to reflux and stirred for 48 h. The reaction solution was concentrated by rotary evaporation to obtain the crude product. The crude product was purified using a CombiFlash column (20 g silica gel column, DCM / MeOH (containing 10% ammonia) = 100 / 1-100 / 7, v / v). The purified product fraction was evaporated to give the colorless oily target product A-C12 (200 mg, 37.8% yield).

[0172] The target products A-C14, A-C16, and A-C18 were synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane in step 3 above, respectively.

[0173] 2. Synthesis of B-C12, B-C14, B-C16, and B-C18

[0174]

[0175]

[0176] Synthesis steps:

[0177]

[0178] Step 1: Synthesis of compound 2 ((2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl-4-methylbenzenesulfonate)

[0179] tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate (20 g, 97.56 mmol, 1 eq), DMAP (12 g, 97.50 mmol, 1 eq), and DIEA (25 g, 193 mmol, 2 eq) were mixed in DCM (300 ml) and stirred at room temperature until all materials were dissolved. TsCl (22 g, 115.79 g, 1.2 eq) was slowly added in portions to the DCM solution at room temperature, yielding a pale yellow solution. The mixture was stirred overnight at room temperature, and TLC showed complete disappearance of the initial alcohol. The reaction solution was diluted with DCM (200 ml) and washed with water (100 ml x 3), dilute 1N hydrochloric acid (100 ml x 3), saturated NaHCO3 (100 ml x 3), and brine (40 ml x 2), respectively. The organic layers were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated by rotary evaporation to give 33 g of a pale yellow oily liquid. This was used directly in the next reaction without further purification. ESI: [M+1]: 360.1.

[0180] Step 2: Synthesis of compound 4 (tert-butyl(2-(2-(4-(11-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)-2,2-dimethyl-4-oxo-3,8-dioxo-5,11-diazatriadecan-13-yl)piperazin-1-yl)ethoxy)ethyl)carbamate)

[0181] A mixture of 2-(piperazin-1-yl)ethyl-1-amine (1 g, 7.75 mmol, 1.0 eq), 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate (11.1 g, 31.0 mmol, 4.0 eq), and K₂CO₃ (6.4 g, 46.5 mmol, 6.0 eq) was heated and stirred overnight at 80 °C in anhydrous acetonitrile (50 mL). TLC (MeOH / DCM = 1 / 10) showed complete disappearance of the starting amine. Insoluble salts or other impurities were removed by vacuum filtration, and the filter cake was washed with DCM. The organic phases were combined and concentrated by rotary evaporation to obtain a crude product. The crude product was purified using a CombiFlash column (40 g silica gel column, DCM / MeOH (containing 10% ammonia) = 100 / 1–100 / 7, v / v). The purified product fraction was evaporated to give a colorless oily compound 4 (2.6 g, 49% yield). ESI: [M+1]: 692.0.

[0182] Step 3: Synthesis of compound 5(2-(2-aminoethoxy)-N-(2-(2-aminoethoxy)ethyl)-N-(2-(4-(2-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)ethyl)ethyl-1-amine)

[0183] Compound 4 (tert-butyl(2-(2-(4-(11-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)-2,2-dimethyl-4-oxo-3,8-dioxo-5,11-diazatriadecan-13-yl)piperazin-1-yl)ethoxy)ethyl)carbamate)2,2-dimethyl-4-oxo-3,8-dioxo-5,11-diazatriadecan-13-yl)piperazin-1-yl)ethoxy)ethyl)carbamate)2,2-dioxane)2)2)2)2)2)2)2)2(2-dioxane)2)2)2)2(dioxane-3))2)2)2(dioxane-3)))2)2(dioxane-3))))))))))))))))))))))))))))""""""""""""""2—" ... The organic phases were combined and concentrated by rotary evaporation to give a pale yellow oily compound 5 (1.2 g, 82% yield). ESI: [M+1]: 391.1.

[0184] Step 4: Synthesis of B-C12(19-(2-(4-(2-(2-(di(2-hydroxydodecyl)amino)ethoxy)ethyl)piperazin-1-yl)ethyl)-13,15-di(2-hydroxydodecyl)-16,22-diox-13,19,25-triazaheptadecane-11,27-diol)

[0185] Compound 5 (100 mg, 0.256 mmol, 1.0 eq), 1,2-epoxydodecane (470 mg, 2.56 mmol, 10.0 eq), and EtOH (1 ml) were mixed thoroughly. The mixture was heated to reflux and stirred for 48 h. TLC showed that compound 5 disappeared. The reaction solution was concentrated by rotary evaporation to obtain a crude product. The crude product was purified using a CombiFlash column (20 g silica gel column, DCM / MeOH (containing 10% ammonia) = 100 / 1-100 / 7, v / v). The fraction of the purified product was evaporated to give a colorless oily product B-C12 (Target-2, 200 mg, 52.2% yield).

[0186] The target products B-C14 (Target-1), B-C16 and B-C18 were synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane and 1,2-epoxyoctadecane in step 3, respectively.

[0187] 3. Synthesis of C-C12, C-C14, C-C16, and C-C18

[0188]

[0189]

[0190] The synthesis steps are as follows:

[0191]

[0192] C-C12 was synthesized using the same method as compound B-C12, except that tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate was used instead of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate. C-C14, C-C16, and C-C18 were prepared by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane, respectively.

[0193] 4. Synthesis of D-C12, D-C14, D-C16, and D-C18

[0194]

[0195] The synthesis steps are as follows:

[0196]

[0197] The method for synthesizing D-C12 using compound B-C12 differs in that tert-butyl(2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate is used instead of tert-butyl(2-(2-hydroxyethoxy)ethyl)carbamate. D-C14, D-C16, and D-C18 are synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane, respectively.

[0198] 5. Synthesis of E-C12, E-C14, E-C16, and E-C18

[0199]

[0200]

[0201] Synthesis route:

[0202]

[0203] E-C12 was synthesized using the same method as A-C12, except that 2-(piperazin-1-yl)ethyl-1-amine was replaced with 4-methyl-1-piperazinethylamine. E-C14, E-C16, and E-C18 were synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane, respectively.

[0204] 6. Synthesis of F-C12, F-C14, F-C16, and F-C18

[0205]

[0206] Synthesis route:

[0207]

[0208] F-C12 was synthesized using the B-C12 method, except that 2-(piperazin-1-yl)ethyl-1-amine was replaced with 4-methyl-1-piperazinethylamine. F-C14, F-C16, and F-C18 were synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane, respectively.

[0209] 7. Synthesis of G-C12, G-C14, G-C16, and G-C18

[0210]

[0211] Synthesis route:

[0212]

[0213] G-C12 was synthesized using the C-C12 synthesis method, except that 2-(piperazin-1-yl)ethyl-1-amine was replaced with 4-methyl-1-piperazinethylamine. 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane were used to replace 1,2-epoxydodecane and G-C14, G-C16, and G-C18, respectively.

[0214] 8. Synthesis of H-C12, H-C14, H-C16, and H-C18

[0215]

[0216] Synthesis route:

[0217]

[0218] H-C12 was synthesized using the same method as D-C12, except that 2-(piperazin-1-yl)ethyl-1-amine was replaced with 4-methyl-1-piperazinethylamine. H-C14, H-C16, and H-C18 were synthesized by replacing 1,2-epoxytetradecane, 1,2-epoxyhexadecane, and 1,2-epoxyoctadecane, respectively.

[0219] 9. Synthesis of I-C12, I-C14, I-C16, I-C18, J-C12, J-C14, J-C16, J-C18, K-C12, K-C14, K-C16, K-C18, L-C12, L-C14, L-C16, L-C18

[0220]

[0221]

[0222]

[0223] Intermediate compound 4 is a common starting material for the synthesis of I, J, K, and L, and its synthetic route is shown below:

[0224]

[0225] Synthesis of Compound 3

[0226] N-(2-bromoethyl)phthalimide (8.78 g, 34.56 mmol, 0.9 eq) and anhydrous potassium carbonate (12.53 g) were added to a 500 mL round-bottom flask and protected under nitrogen. Separately, compound 1 (5 g, 38.41 mmol, 1 eq) was dissolved in 50 mL of anhydrous acetonitrile (10 m / v) under nitrogen protection and added to the round-bottom flask using a syringe, followed by stirring. The mixture was heated and stirred overnight at 80 °C. The reaction was monitored by TLC (TLC conditions: DCM∶MeOH = 10∶1). After the reaction was complete as indicated by UV, the reaction mixture was filtered through a sintered glass funnel. The filter cake was washed three times with 50 mL of acetonitrile, and all filtrates were collected. 10 g of silica gel was added to the filtrate, and the mixture was rotary evaporated until the silica gel was dry. The crude product was purified using a CombiFlash column (40 g silica gel column, DCM / MeOH (containing 10% ammonia) = 100 / 1-100 / 7, v / v). The target product compound 3 was obtained (8.22 g, yield 70.56%). ESI: [M+1]: 304.2

[0227] Synthesis of Compound 4

[0228] Compound 3 (8.2 g, 27.03 mmol, 1 eq) was added to a round-bottom flask containing 160 mL of ethanol and stirred. Then, 26.05 mL of 35% concentrated ammonia was added, and the mixture was refluxed overnight at 90 °C. A white suspension was obtained the next day. After confirming the disappearance of the starting material by TLC monitoring (TLC conditions: DCM∶MeOH(NH3)=10∶1, UV and ninhydrin colorimetric comparison), the mixture was cooled to room temperature. 10 g of diatomaceous earth was placed in a sintered funnel, and the reaction mixture was filtered, collecting the filtrate. 200 mL of acetonitrile was added to the filtrate, yielding a large amount of white precipitate. The white precipitate was filtered through diatomaceous earth, and the filtrate was collected. The filtrate was concentrated and dried to obtain the colorless oily target product compound 4 (4.3 g, 92% yield), ESI: [M+1]: 174.1.

[0229] Compounds of series I, J, K, and L were synthesized from compounds of series A, B, C, and D, respectively. The specific synthetic methods are not described here.

[0230] The MS and HNMR spectra of the synthesized compounds are shown in the table below:

[0231]

[0232]

[0233] Example 2: Preparation and Detection of Lipid Nanoparticles (LNP Formulation)

[0234] The ionizable lipid compound from Example 1 was dissolved in ethanol at a molar ratio of 35:16:46.5:2.5 with DOPE, cholesterol, and DSPE-PEG2000 (all purchased from Avitol (Shanghai) Pharmaceutical Technology Co., Ltd.) to prepare an ethanol lipid solution. The enhanced green fluorescent protein (EGFP) mRNA (Shanghai Jiliang Pharmaceutical Engineering Co., Ltd., JN-4201) was then diluted in 20 mM citrate buffer (pH = 6.1) to obtain an aqueous mRNA solution. The solution was then processed using a microfluidic device (INano, Myanna (Shanghai) Instrument Technology Co., Ltd.). TM L) Liposomes were prepared by mixing ethanol lipid solution and mRNA aqueous solution at a volume ratio of 1:3, with a total lipid to mRNA weight ratio of approximately 10:1. The solution was replaced by ultrafiltration to remove ethanol, and the volume was adjusted using DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain an LNP formulation encapsulating eGFP mRNA with ionizable lipid / DOPE / cholesterol / DSPE-PEG2000 (35 / 16 / 46.5 / 2.5 mol%), with an mRNA concentration of 0.002 mg / mL–0.5 mg / mL.

[0235] The size and polydispersity index (PDI) of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode. The test results are shown in Table 1.

[0236] Example 3: In vitro cell transfection experiment using lipid nanoparticles (LNP formulation)

[0237] This invention uses peripheral blood mononuclear cells (PBMCs) (Shanghai Aoneng Biotechnology Co., Ltd.), human peripheral blood leukemia T cells Jurkat (ATCC), tumor-infiltrating lymphocytes TILs (isolated from excised tumor tissue of liver cancer patients, preparation method see CN114763530A), mouse bone marrow-derived dendritic cells DC2.4 (Chinese Academy of Sciences Cell Bank), and Chinese hamster ovary cells CHO (ATCC) as validation cell lines for liposome transfection experiments. PBMCs, Jurkat, and TILs are grown in suspension, while DC2.4 and CHO cells are grown adherently. All cell culture procedures must strictly adhere to the prescribed procedures, using sterile reagents and performing aseptic operations under a sterile workbench. The main operational steps involved are as follows:

[0238] 1) Cell thawing: Remove the preserved cell cryopreservation solution from the -80℃ freezer and immediately place it in a 37℃ water bath with rapid and continuous shaking until completely thawed within one minute. Add fresh culture medium to a 15mL centrifuge tube beforehand, and then add the cell suspension to the centrifuge tube in a biosafety cabinet. Place a centrifuge tube of the same mass symmetrically in a centrifuge and centrifuge at 1200rpm for 5 minutes. After centrifugation, remove the supernatant, and then slowly add an appropriate amount of culture medium along the tube wall. After the cells are gently dispersed by pipetting, transfer the cell suspension to a prepared culture flask. Observe the state and distribution of cells in the early stages of thawing under a microscope. Incubate the culture flask in a 5% CO2 incubator at 37℃ and record the thawing time.

[0239] 2) Cell Passage: Under a microscope, if the cell density reaches 80%-90% of the field of view, the cells can be passaged. Adherent cells require trypsin digestion. To avoid residual serum in the culture medium reducing trypsin activity, wash once with DPBS solution after removing the original culture medium. After aspirating the DPBS solution, add trypsin to degrade proteins at the cell junctions, thus separating the cells and facilitating further culture. Gently shake to mix well and place in an incubator for 2 minutes of digestion. After digestion, immediately add an appropriate amount of culture medium to the original culture dish to stop digestion. Finally, carefully blow off the cells with a pipette and agitate until evenly dispersed. Then, seed and passage according to the experimental requirements at a certain ratio. Suspension cells do not require trypsin digestion.

[0240] The culture media and specific experimental procedures used for each cell line are as follows:

[0241] PBMC cell transfection

[0242] 1) Antibody coating: Pre-coat 6-well plates. Take CD3 and CD28 antibodies from a -20℃ freezer and thaw them for later use. Add the two antibodies to DPBS solution at a concentration of 5 μg / mL, mix well, and then add 1 mL / well to each well of the 6-well plate. Incubate at 37℃ for 4 hours before use.

[0243] 2) PBMC thawing: Take the frozen PBMC cells out of the -80℃ freezer and immediately put them into a 37℃ water bath to thaw them quickly. After thawing, add them to the preheated culture medium, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the PBMCs in an appropriate amount of AIM-V culture medium containing 500 U / ml IL-2.

[0244] 3) Remove the DPBS coated with antibody using a pipette, transfer the cell suspension to a pre-coated antibody-containing six-well plate (4 mL per well), and incubate at 37°C in a 5% CO2 incubator for 3-5 days before proceeding with subsequent experiments.

[0245] 4) As required by the experiment, PBMC cells were seeded into 24-well plates 30 minutes in advance for transfection. After cell counting, the cell number and cell viability were recorded, with 5 × 10⁶ cells seeded per well. 5 Collect cell suspension, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Calculate the required volume of serum-free Opti-MEM medium to be added at 300 μL per well, gently pipette to mix, and then seed into a 24-well plate for subsequent transfection experiments. ApoE4 can be added at 0.5 μg / well as needed.

[0246] 5) Dilute the LNP preparation prepared in Example 2 with Opti-MEM medium to a mRNA concentration of 1ug / ml.

[0247] 6) Add 200 μL of the diluted mixture evenly to the top of each well, containing 200 ng of mRNA per well. Incubate at 37°C in a 5% CO2 incubator for activation.

[0248] 7) Cell viability, EGFP positivity rate, and average fluorescence intensity data were obtained by cell fluorescence imaging and flow cytometry after 24h and 48h.

[0249] Jurkat cell transfection

[0250] 1) Jurkat cell resuscitation: Take the frozen Jurkat cells out of the -80℃ freezer and immediately place them in a 37℃ water bath to thaw them quickly. After thawing, add them to preheated culture medium, centrifuge at 1200 rpm for 5 min, discard the supernatant, add an appropriate amount of RPMI-1640 medium containing 10% FBS to resuspend the resuscitated Jurkat cells, and place them in a 37℃, 5% CO2 incubator for activation culture.

[0251] 2) As required by the experiment, Jurkat cells were seeded into 24-well plates 30 minutes in advance for transfection. Cell counts and cell viability were recorded, with 5 × 10⁶ cells seeded per well. 5 Collect cell suspension, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add 300 μL of serum-free Opti-MEM medium per well, gently pipette to mix, and then plate into a 24-well plate for subsequent transfection experiments. ApoE4 (0.5 μg / well) can be added as needed.

[0252] The subsequent steps are the same as the transfection protocol for PBMC cells.

[0253] TIL cell transfection

[0254] 1) TIL cell resuscitation: Take the frozen TIL cells out of the -80℃ freezer and immediately put them into a 37℃ water bath to thaw them quickly. After thawing, add them to the preheated culture medium, centrifuge at 1200 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the resuscitated TILs, and place them in a 37℃, 5% CO2 incubator for activation culture.

[0255] 2) As required by the experiment, TIL cells were seeded into 24-well plates 30 minutes in advance for transfection. Cell counts and cell viability were recorded, with 5 × 10⁶ cells seeded per well. 5 Collect cell suspension, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add 300 μL of serum-free Opti-MEM medium per well, gently pipette to mix, and then plate into a 24-well plate for subsequent transfection experiments. ApoE4 (0.5 μg / well) can be added as needed.

[0256] The subsequent steps are the same as the transfection protocol for PBMC cells.

[0257] DC2.4 cell transfection

[0258] 1) DC2.4 cell resuscitation: Take the frozen DC2.4 cells out of the -80℃ freezer and immediately put them into a 37℃ water bath to thaw them quickly. After thawing, add them to preheated culture medium, centrifuge at 1200 rpm for 5 min, discard the supernatant, add an appropriate amount of DMEM culture medium containing 10% FBS to resuspend the resuscitated DC2.4 cells, and place them in a 37℃, 5% CO2 incubator for activation culture.

[0259] 2) As required by the experiment, DC2.4 cells were seeded into 24-well plates one day in advance for transfection. Cell counts were performed to record cell number and viability, with 2 × 10⁶ cells seeded per well. 5 Take cell suspension from each cell, add a certain volume of DMEM medium containing 10% FBS, mix well, and add 1 mL to each well for subsequent transfection experiments.

[0260] 3) After 24 hours, gently aspirate the culture medium with a pipette 30 minutes in advance, add 1 mL of DPBS to each well to wash and then aspirate. Add 300 μL of serum-free Opti-MEM culture medium to each well for subsequent transfection experiments.

[0261] The subsequent steps are the same as the transfection protocol for PBMC cells.

[0262] CHO cell transfection

[0263] 1) CHO cell resuscitation: Take out the frozen CHO cells from the -80℃ freezer and immediately put them into a 37℃ water bath to thaw the cells quickly. After thawing, add them to the preheated culture medium, centrifuge at 1200 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the resuscitated CHO cells, and place them in a 37℃, 5% CO2 incubator for activation culture.

[0264] 2) As required by the experiment, CHO cells were seeded into 24-well plates one day in advance for transfection. Cell counts were performed to record cell number and viability, with 2 × 10⁶ cells seeded per well. 5 Take cell suspension from each cell, add a certain volume of culture medium and mix well. Add 1 mL to each well for subsequent transfection experiments.

[0265] 3) After 24 hours, gently aspirate the culture medium with a pipette 30 minutes in advance, add 1 mL of DPBS to each well to wash and aspirate, and then add 300 μL of serum-free Opti-MEM culture medium to each well for subsequent transfection experiments.

[0266] The subsequent steps are the same as the transfection protocol for PBMC cells.

[0267] Experimental results

[0268] Cell viability and EGFP positivity were obtained by flow cytometry analysis. Table 1 shows the characteristics of the lipid nanoparticles prepared in Example 2 and the expression level of EGFP mRNA on DC2.4, normalized to the sample with the highest fluorescence intensity.

[0269] Table 1

[0270] Sample number Particle size PDI Encapsulation rate Cell viability Positive rate fluorescence intensity A-C12 91.6 0.173 89.80％ 87.1％ 73.3％ 0.84 A-C14 97.1 0.107 80.31％ 95.4 92.3％ 0.30 A-C16 90.8 0.183 90.30％ 90.0％ 66.3％ 0.80 A-C18 96.9 0.144 90.56％ 93.0％ 61.9％ 0.87 B-C12 102.9 0.147 89.93％ 85.0％ 83.0％ 0.20 B-C14 90.5 0.133 95.21％ 95.0％ 90.2％ 1.00 B-C16 101.7 0.071 85.25％ 90.2％ 65.7％ 0.49 B-C18 108.3 0.118 94.79％ 89.6％ 68.6％ 0.27 C-C12 92.5 0.070 95.24％ 94.0％ 64.4％ 0.71 C-C14 92.2 0.171 92.82％ 94.6 93.6％ 0.75 C-C16 94.1 0.108 90.26％ 97.6％ 85.2％ 0.87 C-C18 107.6 0.134 81.92％ 89.6％ 86.1％ 0.74 D-C12 90.5 0.073 91.80％ 90.8％ 65.6％ 0.07 D-C14 97.2 0.168 92.36％ 91.3 96.7％ 0.95 D-C16 91.6 0.117 86.56％ 91.1％ 71.7％ 0.82 D-C18 103.0 0.105 88.54％ 81.5％ 64.4％ 0.45 E-C12 96.9 0.133 95.02％ 84.8％ 56.4％ 0.69 E-C14 85.2 0.119 85.62％ ＞99.0 84.3％ 0.45 E-C16 76.9 0.107 91.51％ 94.4％ 59.9％ 0.71 E-C18 84.5 0.136 92.86％ 98.5％ 65.2％ 0.14 F-C12 77.7 0.133 92.31％ 87.1％ 63.9％ 0.28 F-C14 82.4 0.131 95.21％ 93.2 73.5％ 0.33 F-C16 91.9 0.099 92.39％ 96.8％ 60.3％ 0.17 F-C18 77.2 0.081 80.61％ 93.5％ 61.5％ 0.67 G-C12 89.3 0.172 89.95％ 92.9％ 83.1％ 0.29 G-C14 86.4 0.180 84.02％ ＞99.0％ 67.5 0.30 G-C16 78.2 0.102 85.40％ 94.5％ 79.6％ 0.54

[0271] G-C18 99.8 0.195 80.79％ 95.2％ 63.9％ 0.63 H-C12 95.9 0.136 94.13％ 86.0％ 74.3％ 0.78 H-C14 94.8 0.099 91.67％ 91.7％ 66.8％ 0.52 H-C16 95.5 0.162 86.56％ ＞99.0％ 59.0％ 0.77 H-C18 73.8 0.143 80.61％ 97.4％ 81.9％ 0.38 I-C12 83.8 0.114 88.38％ 98.5％ 63.9％ 0.05 I-C14 120.3 0.180 87.59％ 97.0％ 62.7％ 0.05 I-C16 96.3 0.147 91.60％ 81.3％ 68.7％ 0.06 I-C18 72.3 0.109 83.06％ 86.0％ 66.4％ 0.04 J-C12 99.7 0.105 92.88％ 81.8％ 62.6％ 0.04 J-C14 126.1 0.121 83.58％ ＞99.0％ 59.1％ 0.06 J-C16 104.1 0.077 81.08％ 85.2％ 62.0％ 0.04 J-C18 93.7 0.163 88.56％ 83.6％ 60.9％ 0.04 K-C12 75.9 0.075 92.83％ ＞99.0％ 62.8％ 0.07 K-C14 122.7 0.155 87.00％ 82.3％ 59.8％ 0.03 K-C16 76.0 0.185 95.74％ 93.4％ 60.2％ 0.04 K-C18 124.2 0.134 92.77％ ＞99.0％ 74.7％ 0.05 L-C12 88.4 0.181 93.38％ 84.9％ 58.1％ 0.07 L-C14 101.5 0.126 91.54％ 99.0％ 60.0％ 0.04 L-C16 111.5 0.085 92.34％ 97.2％ 64.8％ 0.06 L-C18 124.0 0.182 91.70％ 98.6％ 59.7％ 0.02

[0272] Figure 1 This shows the cell viability obtained by flow cytometry analysis after in vitro transfection of cells with an LNP formulation prepared using B-C14. Figure 1 As can be seen, PBMC, TIL, Jurkat, DC2.4, and CHO cells exhibited high viability, indicating that the formulation is non-cytotoxic. The addition of ApoE4 during transfection did not affect cell viability.

[0273] Table 2 shows the expression level of EGFP mRNA on PBMCs after ApoE4 was added to the LNP formulation prepared in Example 2 and the expression level was normalized based on the sample with the highest fluorescence intensity.

[0274] Table 2

[0275]

[0276]

[0277] Figure 2 and 3 This shows the transfection efficiency of the LNP formulation prepared with B-C14 under conditions with and without AopE4. Under conditions without AopE4, this LNP formulation showed high transfection efficiency on Jurkat, DC2.4, and CHO cells, but very low transfection efficiency on PBMC and TIL T cells. Figure 2 Adding ApoE4 improved the transfection efficiency of both PBMCs and TILs, with the transfection efficiency of PBMCs reaching over 75%. Figure 3 ).

[0278] Example 4: In vitro stability experiment of lipid nanoparticles (LNP formulation)

[0279] The formulation from Example 2 was stored at 4°C–8°C. Samples were taken every week to test the transfection performance on DC2.4, the size of the nanoparticles, and the polydispersity index (PDI). The entire process lasted 16 weeks. The results for LNPs prepared using B-C14 are as follows: Figure 8 As shown, after 16 weeks of storage at 4-8℃, the particle size, PDI, and cell transfection efficiency remained at their initial values. Other LNP formulations also maintained their initial values, PDI, and cell transfection efficiency, after 16 weeks of storage at 4-8℃.

[0280] Example 5: In vivo transfection experiment of lipid nanoparticles (LNP formulation)

[0281] Fluc-mRNA was prepared according to the method in Example 2. LNP lipid nanoparticles containing FLuc mRNA (5 moU) L-72020.1 / 1 / 5 mg, MC3 (CAS No.: 1224606-06-7, Xiamen Sinobond Biotechnology Co., Ltd.), and C12-200 (CAS No.: 1220890-25-4, synthesized according to literature PNAS, 2010, Vol(107), 5, 1864-1869) were used. Specific parameters are shown in Table 3. DSPC was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd. DMG-PEG2000 was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd. Six mice (Finoc Biotechnology (Shanghai) Co., Ltd.) were randomly selected and administered the lipid nanoparticles at a dose of 0.5 mg / kg via intravenous and intramuscular injection (3 mice per group). DPBS and Fluc-mRNA were used as controls. Six hours later, each mouse was injected with 200 μL of 10 mg / ml D-fluorescein potassium salt via the tail vein. Ten minutes later, the mice were placed under an in vivo imaging system to observe and photograph the total fluorescence intensity of each mouse. Dissected tissues (heart, liver, spleen, lung, kidney, brain, pancreas, and muscle) were placed in petri dishes for luminescence photography. Twenty-four hours later, the mice were again placed under an in vivo imaging system to observe and photograph the total fluorescence intensity of each mouse.

[0282] Table 3: Preparation parameters of each LNP

[0283]

[0284] Note: Target-1 is compound B-C14.

[0285] When administered intramuscularly, some lipid nanoparticles remain at the injection site, with the remainder primarily deposited in the liver. When administered intravenously, all lipid nanoparticles are transferred to the chest and abdomen, mainly remaining in the liver, followed by the spleen. Figure 4-7 As shown, compared to MC3 and C12-200, the CX-A sample exhibited higher fluorescence intensity. In particular, CX-A maintained a high fluorescence intensity even after 24 hours, significantly higher than the other groups. Other LNP formulations also maintained high fluorescence intensity.

[0286] Example 6: Safety Experiment of Ionizable Lipid Compounds

[0287] The genotoxicity of ionizable lipid compounds was tested according to the "Technical Guidelines for Drug Genotoxicity Research" in the "SFDA (State Food and Drug Administration) Guidelines". The bacterial reverse mutation assay, mammalian erythrocyte micronucleus assay, and in vitro mammalian cell chromosome aberration assay for the LNP formulation prepared in Example 2 were all negative.

[0288] The toxicity of empty LNPs was tested according to the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs". The results showed that none of the LNP formulations prepared in Example 2 caused acute toxic reactions.

[0289] According to the Biological Examination Method of the General Rules in the 2020 edition of the Pharmacopoeia of the People's Republic of China (Volume IV), the hemolytic activity of empty LNP was tested using the 1148 Hemolysis and Aggregation Test. The results showed that none of the LNP preparations obtained in Example 2 exhibited hemolysis or aggregation.

Claims

1. An ionizable lipid compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the following structure: 、 ； In each expression, a, b, c, d, e, and f are each an independent integer from 0 to 20.

2. The ionizable lipid compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, a, b, c, d, e, and f are each independent integers from 8 to 16.

3. The ionizable lipid compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, a, b, c, d, e, and f are all the same integer from 0 to 20.

4. The ionizable lipid compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, a, b, c, d, e, and f are the same integer from 8 to 16.

5. The ionizable lipid compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, The compound is selected from , , , , , , and .

6. A lipid nanoparticle comprising any one of claims 1-5, wherein the ionizable lipid compound or a pharmaceutically acceptable salt thereof is present.

7. The lipid nanoparticles as described in claim 6, characterized in that, The lipid nanoparticles also contain: one or more auxiliary lipid molecules, cholesterol and / or one or more polymer-conjugated lipid molecules.

8. The lipid nanoparticles as described in claim 7, characterized in that: The accessory lipid molecule is a neutral lipid molecule; and / or In the polymer-conjugated lipid molecules, the polymer is polyethylene glycol.

9. The lipid nanoparticles as described in claim 8, characterized in that, The accessory lipid molecules are selected from: DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

10. The lipid nanoparticles as described in claim 8 or 9, characterized in that, The auxiliary lipid molecule is DOPE or DSPC.

11. The lipid nanoparticles as described in claim 8, characterized in that, The polymer-conjugated lipid molecules are selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-DSPE, PEG-cer, DMG-PEG and PEG dialkoxypropyl carbamate.

12. The lipid nanoparticles as described in claim 8 or 11, characterized in that, The polymer-conjugated lipid molecules are PEG2000-DSPE or DMG-PEG2000.

13. The lipid nanoparticles according to any one of claims 6-9 and 11, characterized in that, In the lipid nanoparticles, the molar ratio of the ionizable lipid compound or its pharmaceutically acceptable salt to the auxiliary lipid molecule, cholesterol, or polymer-conjugated lipid molecule is 60~5:60~5:50~5:10~1.

14. The lipid nanoparticles according to claim 13, characterized in that, The molar ratio of the ionizable lipid compound or its pharmaceutically acceptable salt to the auxiliary lipid molecule, cholesterol, or polymer-conjugated lipid molecule is 40~10:30~10:50~20:8~1.

15. The lipid nanoparticles according to any one of claims 6-9 and 11, characterized in that, The lipid nanoparticles are used to express proteins encoded by mRNA, wherein the proteins are proteins that have therapeutic, preventive, or ameliorative effects on the physiological functions of an organism, including antigens and antibodies; or The lipid nanoparticles are used to upregulate endogenous protein expression by delivering miRNA inhibitors that target specific miRNAs or by regulating a group of miRNAs that target one or more mRNAs; or The lipid nanoparticles are used to downregulate the protein and / or mRNA levels of target genes; or The lipid nanoparticles are used to deliver mRNA and plasmids to express transgenes; or The lipid nanoparticles are used to induce pharmacological effects resulting from protein expression.

16. The lipid nanoparticles according to any one of claims 6-9 and 11, characterized in that, The lipid nanoparticles also contain surfactants.

17. The lipid nanoparticles according to claim 16, characterized in that, The active agent is a nucleic acid-based active agent.

18. The lipid nanoparticles according to claim 17, characterized in that, The nucleic acid active agents are selected from: messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and aptamers.

19. A composition comprising any one of claims 1-5 of an ionizable lipid compound or a pharmaceutically acceptable salt thereof.

20. The composition of claim 19, characterized in that, The composition is a pharmaceutical composition containing any one of claims 1-5, an ionizable lipid compound or a pharmaceutically acceptable salt thereof, an active agent, and one or more auxiliary lipid molecules, cholesterol, and / or one or more polymer-conjugated lipid molecules.

21. The composition according to claim 20, characterized in that, The accessory lipid molecule is a neutral lipid molecule; and / or In the polymer-conjugated lipid molecules, the polymer is polyethylene glycol; and / or The active agent is a nucleic acid-based active agent.

22. The composition according to claim 21, characterized in that, The accessory lipid molecules are selected from: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

23. The composition of claim 21, characterized in that, The auxiliary lipid molecule is DOPE.

24. The composition according to claim 21, characterized in that, The polymer-conjugated lipid molecules are selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-DSPE, PEG-cer, and PEG-dialkoxypropylcarbamate.

25. The composition according to claim 21, characterized in that, The polymer-conjugated lipid molecule is PEG2000-DSPE.

26. The composition of claim 21, characterized in that, Nucleic acid active agents are selected from: messenger RNA (mRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and aptamers.

27. The composition according to any one of claims 19-26, characterized in that, The composition also contains apolipoproteins.

28. The composition of claim 27, characterized in that, The apolipoproteins are selected from: ApoA-I, ApoA-II, ApoA-IV, ApoA-V and ApoE.

29. The composition of claim 28, characterized in that, The apolipoprotein is ApoE4.

30. The use of any one of the ionizable lipid compounds of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of lipid nanoparticles for the treatment or prevention of a disease.

31. Use of any one of the ionizable lipid compounds of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating or preventing a disease of the subject.

32. Use of any one of the ionizable lipid compounds of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a reagent for delivering an active agent.

33. The application as described in claim 32, characterized in that, The active agent is a nucleic acid molecule.

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