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WO2025140594A1 - Lipide cationique asymétrique contenant de multiples amines tertiaires - Google Patents

Lipide cationique asymétrique contenant de multiples amines tertiaires Download PDF

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Publication number
WO2025140594A1
WO2025140594A1 PCT/CN2024/143282 CN2024143282W WO2025140594A1 WO 2025140594 A1 WO2025140594 A1 WO 2025140594A1 CN 2024143282 W CN2024143282 W CN 2024143282W WO 2025140594 A1 WO2025140594 A1 WO 2025140594A1
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group
lipid
mmol
lipids
cationic lipid
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Chinese (zh)
Inventor
林铭贵
朱琦
王爱兰
王琳琳
魏国华
翁文桂
刘超
林昇
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Xiamen Sinopeg Biotech Co Ltd
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Xiamen Sinopeg Biotech Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Definitions

  • Nucleic acid drugs are a general term for oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions, which play a role at the gene level, including but not limited to: siRNA, miRNA, antisense nucleic acid, aptamer, DNA, messenger RNA (mRNA), etc.
  • mRNA is a type of single-stranded RNA transcribed from a strand of DNA as a template, carrying genetic information and guiding protein synthesis.
  • mRNA as a drug or vaccine
  • simple production process rapid synthesis, low cost, and convenience for large-scale production
  • people have applied mRNA to the prevention and treatment of different types of diseases, especially in the field of vaccines.
  • mRNA has the disadvantages of easy degradation, instability, and difficulty in cell entry. These defects can be overcome by optimizing its own chemical structure and selecting a suitable delivery system.
  • the mRNA delivery system is one of the most critical technologies restricting the development of mRNA drugs.
  • the present invention provides a piperazine ring-branched asymmetric cationic lipid and a preparation method thereof, a lipid composition comprising the cationic lipid, a lipid pharmaceutical composition containing the lipid composition and a preparation thereof, and a liposome or lipid nanoparticle containing the lipid composition, especially an LNP-nucleic acid pharmaceutical composition containing the cationic lipid and a preparation thereof, which have the advantages of high delivery efficiency, safety and low toxicity, high biocompatibility, and reduced cationic lipid dosage, and can improve the therapeutic and/or preventive effects of the drug.
  • a cationic lipid characterized in that the structure is as shown in the general formula (1):
  • L d is -(CH 2 ) ta N ⁇ , the left end of which is connected to the piperazine ring, ta is an integer of 1-6, and a is 2;
  • a lipid composition contains a cationic lipid having a structure represented by formula (1).
  • a lipid pharmaceutical composition preparation contains the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.
  • a liposome or lipid nanoparticle contains a lipid composition, wherein the lipid composition contains a cationic lipid with a structure shown in formula (1).
  • the cationic lipid containing piperazine ring of the present invention adopts heterofunctionalized piperazine ring-containing raw materials, and the preparation process is simple, the cost is lower, and it is more environmentally friendly.
  • the LNP-mRNA composition prepared therefrom has the effects of low toxicity, high biocompatibility and high cell transfection.
  • FIG3 is a 1 H NMR spectrum of the cationic lipid E5-1 prepared in Example 5.
  • FIG. 4 is a 1 H NMR spectrum of the cationic lipid E6-1 prepared in Example 6.
  • FIG5 is a 1 H NMR spectrum of the cationic lipid E30-1 prepared in Example 30.
  • FIG6 is a high performance liquid chromatography (HPLC) test result of the cationic lipid E1-3 prepared in Example 1.3.
  • FIG. 7 is a mass spectrum (MS) of the cationic lipid E1-3 prepared in Example 1.3.
  • FIG9 is the imaging result of the LNP-mRNA pharmaceutical composition L-1-3 prepared in Example 34 after injection into mice.
  • each term has the following meaning.
  • the structure involved when the structure involved has isomers, it can be any one of the isomers unless otherwise specified.
  • a structure with cis-trans isomers it can be either a cis structure or a trans structure;
  • for a structure with E/Z isomers it can be either an E structure or a Z structure; and when optically active, it can be either levorotatory or dextrorotatory.
  • integer ranges marked in the form of intervals can represent a group consisting of all integers within the range, and the range includes two endpoints.
  • the integer range 1-6 represents a group consisting of 1, 2, 3, 4, 5, and 6.
  • the numerical ranges in the present invention including but not limited to numerical ranges represented by integers, non-integers, percentages, and fractions, all include two endpoints unless otherwise specified.
  • the numerical values in the present invention involving "about” or “around” generally refer to a numerical range of ⁇ 10%, which may be enlarged to ⁇ 15% in some cases, but not more than ⁇ 20%.
  • the preset numerical value is used as the base.
  • the molar percentage of steroid lipids in the total lipids in the solution containing the solvent is about 40%, which can generally be considered to include the case where the molar percentage of steroid lipids is 30%-50%.
  • “pharmaceutically acceptable salt” refers to a salt of the compound of the present invention that is approved for use in animals, and more specifically in humans, including salts formed by the compound represented by formula (1) and inorganic acids or organic acids.
  • “Pharmaceutical Salts” J.Pharm.Sci.1977,66,1-19.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid
  • organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid
  • benzoic acid salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, Pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid,
  • HCl or hydrochloric acid
  • HBr or hydrobromic acid solution
  • methanesulfonic acid sulfuric acid, tartaric acid or fumaric acid
  • sulfuric acid tartaric acid or fumaric acid
  • HBr or hydrobromic acid solution
  • methanesulfonic acid sulfuric acid, tartaric acid or fumaric acid
  • L 1 , L 2 , and L 3 are any of the following:
  • the preparation process of the present invention may involve an initial raw material PIP 0 containing a piperazine ring, wherein the PIP 0 contains the same or different functional group pairs F q and F 0 , and the structure is represented as follows Wherein, Fq is H, protected or unprotected -OH, -COOH, F0 is protected or unprotected -NH2 , and the definitions of other symbols are consistent with those described in the general formula (1).
  • IM-C can be obtained by simple esterification, amidation, alkylation, addition or substitution, etc., in a single step or multiple steps. For example, in Example 5, S5-1 With S5-2 The IM-C intermediate can be obtained by esterification and then deprotection.
  • the preparation process of the present invention may involve an intermediate PIP-C containing a piperazine ring, a primary amino group and a carbon-branched hydrophobic tail chain, the structure of which is shown as The definitions of other symbols are consistent with those in general formula (1).
  • PIP-C can be obtained by reacting the intermediate IM-C with the initial raw material PIP 0.
  • the intermediate IM-C with pip 0 The intermediate PIP-C was obtained by esterification reaction.
  • the condensing agent used in the reaction is not limited, but preferably N, N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl, EDCI), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU), and most preferably DCC.
  • a suitable catalyst such as 4-dimethylaminopyridine may be added to this reaction.
  • the oxidant used in the reaction is not particularly limited, as long as it is a compound or a combination of multiple compounds that can increase the valence of the substrate, preferably phenyliodine di(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluoric acid, sodium hypochlorite, cobalt acetate, cobalt acetate, manganese acetate, palladium acetate, copper acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzoylbenzene, 2-iodobenzoic acid, dimethyldioxycyclopropane, dimethyl sulfoxide-oxalyl chloride, dimethyl sulfoxide-acetic anhydride, DDQ, dichlorotri(triphenylpho
  • the reaction solvent can be a solvent-free solvent or an aprotic solvent.
  • the aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, dimethylformamide.
  • the base used in the reaction is an inorganic base or an organic base, preferably an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine); preferably triethylamine and pyridine.
  • an organic base such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine
  • triethylamine and pyridine preferably triethylamine and pyridine.
  • the reaction process involves the "protection” and "deprotection” processes of related groups.
  • the functional group is usually protected.
  • the target functional group is selectively reacted, so the other functional groups are protected.
  • the protecting group not only stably protects the functional group as the object, but also needs to be easily removed as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting group bonded to the specified functional group under appropriate conditions.
  • the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, etc.
  • the amino group protected by the amino protecting group is not particularly limited, and can be, for example, a primary amine, a secondary amine, a hydrazine, an amide, etc.
  • the amino group is not particularly limited, and includes, but is not limited to, a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium ion.
  • the amount of the fluorine-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the amount of fluorine-containing reagent is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will result; when the amount of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, the excess reagent or compound will cause trouble for purification and may be mixed into the subsequent steps, thereby causing side reactions.
  • the reaction solvent is preferably 0 to 30°C. When the temperature is lower than 0°C, the reaction rate is slow and the protecting group cannot be completely removed.
  • the obtained product is a mixture of an amine intermediate and excess sulfonate and halide, which can be purified by column chromatography, anion exchange resin, permeation, ultrafiltration and the like.
  • the anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin, preferably an ion exchange resin with a tertiary amine or quaternary ammonium salt having a skeleton of dextran, agarose, polypropionate, polystyrene, polystyrene and the like.
  • the solvent for permeation and ultrafiltration is not limited, generally water or an organic solvent can be used, wherein the organic solvent is not particularly limited, as long as the product can be dissolved therein, preferably dichloromethane, chloroform and the like.
  • Case (7) also contains phospholipids, steroid lipids and PEGylated lipids;
  • the composition contains three types of lipids, namely, neutral lipid, steroid lipid and PEGylated lipid.
  • the phospholipids in the lipid composition are preferably 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl
  • the steroid lipid in the lipid composition is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, ⁇ -tocopherol and a combination thereof.
  • the PEGylated lipid in the lipid composition is preferably polyethylene glycol-1,2 dimyristin (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-distearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-
  • the present invention comprises 20-80% of the cationic lipid represented by formula (1), 5-16% of the phospholipid, 25-55% of the steroid lipid and 0.5-10% of the pegylated lipid, wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.
  • any of the aforementioned lipid compositions is preferred, wherein the molar percentage of cationic lipids in the total lipids in the solution containing a solvent is 30-65%; more preferably, about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%.
  • the molar percentage of phospholipids in the total lipids in the solution containing the solvent is about 7.5-16%; more preferably, it is about any one of 8%, 9%, 10%, 11%, 12%, and 16%.
  • the molar percentage of steroid lipids in the total lipids in the solution containing the solvent is 35-50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
  • the molar percentage of PEGylated lipids in the total lipids in the solution containing the solvent is preferably 0.5-5%; preferably 1-3%; more preferably about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%.
  • the lipid composition can be prepared by the following methods, including but not limited to ethanol injection method, microfluidics method, T-tube mixing method, membrane extrusion method, preferably ethanol injection method and microfluidics method.
  • lipid pharmaceutical composition comprising any of the lipid compositions described above and a drug, wherein the lipid composition comprises any of the cationic lipids described above whose structure is represented by the general formula (1), and the drug is selected from any of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.
  • the lipid pharmaceutical composition is preferably used as a drug selected from any one of the following drugs: anti-tumor agents, antiviral agents, antifungal agents and vaccines.
  • the drugs in the lipid pharmaceutical composition include but are not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, razithromycin, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, dimerol, labetalol, clonidine, hydralazine, imipramine, ami
  • the N/P ratio of the lipid composition to the nucleic acid is preferably (0.1-100):1, more preferably (0.2-30):1, and most preferably (0.5-20):1.
  • the equilibration time is 0.1 to 12 h, preferably 0.2 to 6 h, more preferably 0.5 to 3 h; preferably, the recombination time is 0.1 to 12 h, preferably 0.2 to 5 h, more preferably 0.5 to 2 h.
  • lipid nanoparticles can be prepared by the following methods, including but not limited to microemulsion method, double emulsion method, high shear homogenization ultrasound method, thin film hydration extrusion method, and microfluidics method.
  • the liposomes are dried in a freeze dryer to form liposome powder.
  • the ratio of liposomes: phosphate buffer dissolved with a cryoprotectant can be 1 mg: (0.1-100) mL, preferably 1 mg: (0.3-50) mL, and more preferably 1 mg: (0.5-5) mL.
  • each lipid component in an organic solvent to obtain a lipid composition dissolved in an organic phase;
  • the organic phase is preferably ethanol;
  • the preparation process is as follows:
  • Step a Dissolve tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (S1-1, 0.69 g, 3.0 mmol) in isopropanol, add sufficient anhydrous potassium carbonate under stirring, and stir at room temperature until the reaction solution is alkaline, add 2-dodecyl acrylate (S1-2, 2.16 g, 9.0 mmol) to the reaction solution, and place the reaction solution in a reflux device (90° C.) and continue to stir and react for 36 hours. After the reaction is completed, concentrate the reaction solution to obtain a crude product.
  • S1-1 tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate
  • 2-dodecyl acrylate S1-2, 2.16 g, 9.0 mmol
  • Step b Under nitrogen protection, N, N'-dicyclohexylcarbodiimide (DCC, 2.27 g, 11.0 mmol) was added to a round-bottom flask containing 2-hexyldecanoic acid (S1-4, 1.28 g, 5.0 mmol), 5-hexene-1-ol (S1-5, 0.6 g, 6.0 mmol) dissolved in dichloromethane (50 mL) under nitrogen protection, and the mixture was reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain S1-6 (1.39 g).
  • DCC N'-dicyclohexylcarbodiimide
  • Step c S1-6 (0.68 g, 2.0 mmol) was dissolved in 20 mL of dichloromethane, and m-chloroperbenzoic acid (m-CPBA, 0.52 g, 3.0 mmol) was added under ice bath conditions. After stirring for 15 min, the ice bath was removed and the reaction was stirred overnight. After the reaction was completed, an excess of saturated sodium bisulfite solution was added and the dichloromethane was removed by concentration. The residue was washed with ethyl acetate (20 mL), saturated sodium bicarbonate solution (20 mL) 3 times, and saturated sodium chloride solution (20 mL) once. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and purified by column chromatography to obtain compound S1-7 (0.53 g).
  • m-CPBA m-chloroperbenzoic acid
  • Step c S1-3 (0.49 g, 0.8 mmol) and DIPEA (0.08 g, 0.6 mmol) were added to a methanol solution of S1-11 (0.46 g, 1.2 mmol) in sequence, and the reaction solution was placed in a reflux device (90° C.) and stirred for 24 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E1-2 (0.58 g).
  • the preparation process is as follows:
  • Step a Under nitrogen protection, DCC (3.17 g, 15.4 mmol) was added to a round-bottom flask containing 6-bromohexanoic acid (S1-12, 1.37 g, 7.0 mmol), undecanol (S1-13, 1.44 g, 8.4 mmol) and DMAP (0.21 g, 1.8 mmol) dissolved in dichloromethane (30 mL) and the mixture was reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain S1-14 (1.98 g).
  • Step b Under nitrogen protection, compound S1-1 (0.46 g, 2.0 mmol) was dissolved in acetonitrile (20 mL), and S1-14 (1.75 g, 5.0 mmol) and DIPEA (0.52 g, 4.0 mmol) were added in sequence under slow stirring, and the mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and then extracted with 0.6 M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and then the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated, and purified by column chromatography to obtain compound S1-15 (1.05 g).
  • Step c S1-15 (0.53 g, 0.8 mmol) and DIPEA (0.08 g, 0.6 mmol) were added to a methanol solution of S1-7 (0.43 g, 1.2 mmol) in sequence, and the reaction solution was placed in a reflux device (90° C.) and stirred for 24 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E1-3 (0.59 g).
  • the preparation process is as follows:
  • Step a At room temperature, 4,4-diethoxybutyronitrile (S2-1, 1.88 g, 12.0 mmol) and 2-octen-1-ol (S2-2, 4.61 g, 36.0 mmol) were added to a round-bottom flask containing pyridinium p-toluenesulfonate (PPTS, 0.15 g, 0.6 mmol), and the mixture was reacted at 105 ° C for 20 h. After the reaction was completed, the reaction solution was cooled to room temperature. The compound S2-3 (1.62 g) was obtained by column chromatography separation and purification.
  • PPTS pyridinium p-toluenesulfonate
  • KOH potassium hydroxide
  • the preparation process is as follows:
  • Step c Under nitrogen protection, compound S1-3 (0.61 g, 1.0 mmol) was dissolved in acetonitrile (20 mL), and S3-4 (0.72 g, 1.3 mmol) and DIPEA (0.17 g, 1.3 mmol) were added in sequence under slow stirring, and the mixture was stirred at room temperature for about 20 h.
  • Step b S4-2 (2.80 g, 10.0 mmol) was dissolved in a mixed solution of dichloromethane (30 mL) and acetonitrile (30 mL), and then ruthenium chloride (RuCl 3 , 0.37 g, 1.8 mmol) was added, the mixture was cooled to 10°C, and an aqueous solution of sodium periodate (0.22 g, 1.0 mmol) was added dropwise, and the mixture was stirred at 10°C for 20 h.
  • ruthenium chloride RuCl 3 , 0.37 g, 1.8 mmol
  • Step d Under nitrogen protection, DCC (1.85 g, 9.0 mmol) was added to a round-bottom flask containing 2-nonene-1-ol (S4-5, 0.71 g, 5.0 mmol), S4-4 (0.86 g, 2.0 mmol) and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (30 mL), and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration and the filtrate was concentrated. The residue was dissolved in tetrahydrofuran (10 mL), and 10 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove the TBS protection.
  • Step e S4-7 (0.75 g, 4.0 mmol) was dissolved in isopropanol, and a sufficient amount of anhydrous potassium carbonate was added under stirring, and stirred at room temperature until the reaction solution was alkaline, S1-2 (2.88 g, 12.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux device (90° C.) and continued to stir and react for 36 hours. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography to obtain compound S4-8 (1.95 g).
  • Step b Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S4-8 (0.67 g, 1.0 mmol), S4-10 (0.68 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E4-2 (1.02 g).
  • the preparation process is as follows:
  • Step a Under nitrogen protection, add DCC (1.85 g, 9.0 mmol) to a round-bottom flask containing S4-4 (0.86 g, 2.0 mmol), 3-decyne-1-ol (S6-1, 0.72 g, 5.0 mmol) and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (30 mL) and react at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration and concentrate the filtrate. Dissolve the residue in tetrahydrofuran (10 mL), add 10 mL of TBAF tetrahydrofuran solution (1 M), react overnight, and remove TBS protection.
  • Step b Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S4-8 (0.67 g, 1.0 mmol), S6-2 (0.71 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E6-1 (1.03 g).
  • Example 7 Cationic lipid (E7-1)
  • Step a 2-(4-(2-aminoethyl)piperazine-1-yl)ethanol (S7-1, 0.52 g, 3.0 mmol) was dissolved in isopropanol, and a sufficient amount of anhydrous potassium carbonate was added under stirring, and stirred at room temperature until the reaction solution was alkaline, S1-2 (2.16 g, 9.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux device (90° C.) and continued to stir and react for 36 h. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography to obtain compound S7-2 (1.46 g).
  • Step b Under ice bath conditions, S7-3 (0.60 g, 5.0 mmol) was dissolved in dichloromethane solution, and then DCC (1.13 g, 5.5 mmol) and DMAP (0.31 g, 2.5 mmol) were added. After the mixture was stirred for 10 minutes, tert-butyl alcohol (t-BuOH, 0.66 g, 7.5 mmol) was slowly added, and the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, washed three times with ether, and washed once with saturated brine. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain a crude product of S7-4, which was purified by column chromatography to obtain S7-4 (0.80 g, 90.8%).
  • Step c Under nitrogen protection, add DCC (1.85 g, 9.0 mmol) to a round-bottom flask containing S7-4 (0.35 g, 2.0 mmol), S5-1 (1.40 g, 5.0 mmol) and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (30 mL), and react at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, and concentrate the filtrate to obtain a crude product.
  • Step d Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S7-5 (0.65 g, 1.0 mmol), S7-2 (0.78 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E7-1 (1.07 g).
  • the preparation process is as follows:
  • Step a Add (9Z, 12Z)-9,12-octadecadien-1-ol (S8-1, 2.93 g, 11.0 mmol) and triethylamine (2.59 g, 31.4 mmol) to dichloromethane (30 mL) at 0°C, then dropwise add a dichloromethane (15 mL) solution containing acryloyl chloride (S8-2, 1.50 g, 16.5 mmol) to the reaction system, and stir the reaction solution at 20°C for 2 h. After the reaction is completed, filter to remove the precipitate, wash the filtrate with water and 5% hydrochloric acid in turn, and dry the organic phase with magnesium sulfate. Filter, concentrate the filtrate, and then purify the residue by column chromatography to obtain the target compound (9Z, 12Z)-9,12-diene octadecane acrylate (S8-3, 2.65 g).
  • Step c S8-4 (0.77 g, 1.0 mmol) and DIPEA (0.10 g, 0.8 mmol) were added to a methanol solution of S1-11 (0.57 g, 1.5 mmol) in sequence, and the reaction solution was placed in a reflux device (90° C.) and stirred for 24 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E8-1 (0.84 g).
  • the preparation process is as follows:
  • Example 10 Cationic lipid (E10-1)
  • the preparation process is as follows:
  • the preparation process is as follows:
  • Step a S4-7 (0.56 g, 3.0 mmol) was dissolved in isopropanol, and a sufficient amount of anhydrous potassium carbonate was added under stirring, and stirred at room temperature until the reaction solution was alkaline, S8-3 (2.89 g, 9.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux device (90° C.) and continued to stir and react for 36 h. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography to obtain compound S11-1 (1.87 g).
  • Example 12 Cationic lipid (E12-1)
  • the preparation process is as follows:
  • the preparation process is as follows:
  • the preparation process is as follows:
  • Step a S7-1 (0.52 g, 3.0 mmol) was dissolved in isopropanol, and a sufficient amount of anhydrous potassium carbonate was added under stirring, and stirred at room temperature until the reaction solution was alkaline, S8-3 (2.89 g, 9.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux device (90° C.) and continued to stir and react for 36 hours. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography to obtain compound S14-1 (1.83 g).
  • Step b Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S7-5 (0.65 g, 1.0 mmol), S14-1 (0.98 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E14-1 (1.18 g).
  • Step b S15-2 (0.61 g, 1.0 mmol) and DIPEA (0.10 g, 0.8 mmol) were added to a methanol solution of S1-7 (0.53 g, 1.5 mmol) in sequence, and the reaction solution was placed in a reflux device (90° C.) and stirred for 24 h. After the reaction, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E15-1 (0.70 g).
  • reaction solution was concentrated and dissolved in dichloromethane, and then extracted with 0.6 M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and then the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated, and the cationic lipid E16-1 (0.85 g) was obtained by column chromatography purification.
  • Example 17 Cationic lipid (E17-1)
  • Step a S4-7 (0.75 g, 4.0 mmol) was dissolved in isopropanol, and sufficient anhydrous potassium carbonate was added under stirring, and stirred at room temperature until the reaction solution was alkaline, S15-1 (2.87 g, 12.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux device (90° C.) and continued to stir and react for 36 hours. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was purified by column chromatography to obtain compound S18-1 (1.94 g).
  • Step c S19-3 (0.70 g, 1.0 mmol) and DIPEA (0.10 g, 0.8 mmol) were added to a methanol solution of S1-11 (0.57 g, 1.5 mmol) in sequence, and the reaction solution was placed in a reflux device (90° C.) and stirred for 24 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E19-1 (0.78 g).
  • Example 20 Cationic lipid (E20-1)
  • reaction solution was concentrated and dissolved in dichloromethane, and then extracted with 0.6 M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and then the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated, and the cationic lipid E21-1 (0.97 g) was obtained by column chromatography purification.
  • the preparation process is as follows:
  • Step b Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S22-1 (0.76 g, 1.0 mmol), S4-6 (0.68 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E22-1 (1.09 g).
  • Example 23 Cationic lipid (E23-1)
  • the preparation process is as follows:
  • Step a S1-3 (2.58 g, 15.0 mmol) was dissolved in dichloromethane (80 mL), triethylamine (5 mL) and N,N'-carbonyldiimidazole (CDI, 2.43 g, 15.0 mmol) were added in sequence, and the mixture was reacted at 50 °C for 1.5 h. Then 5-amino-1-butanol (S23-1, 2.32 g, 22.5 mmol) was added, and the reaction was continued at 50 °C for 16 h.
  • CDI N,N'-carbonyldiimidazole
  • reaction solution was cooled to room temperature, washed with 5% citric acid (40 mL*2) and saturated brine (40 mL) in sequence, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain a crude product.
  • Dichloromethane 40 mL was added to the crude product, stirred for 10 min, filtered, and the filter cake was washed with a small amount of dichloromethane. After concentration, the product S23-2 (2.96 g) was obtained.
  • Step b S23-2 (2.41 g, 8.0 mmol) was dissolved in dichloromethane (100 mL), triphenylphosphine (PPh 3 , 3.14 g, 12.0 mmol) was added, and carbon tetrabromide (CBr 4 , 3.97 g, 12.0 mmol) was added in batches under ice bath, and the mixture was reacted under ice bath for 20 min. 10 mL of methanol was added to quench the reaction, and the crude product was directly concentrated, and the crude product was purified by column chromatography to obtain product S23-3 (1.71 g).
  • PPh 3 triphenylphosphine
  • CBr 4 carbon tetrabromide
  • Step c Under nitrogen protection, compound S4-7 (0.28 g, 1.5 mmol) was dissolved in acetonitrile (20 mL), and S23-3 (1.37 g, 3.8 mmol) and DIPEA (0.39 g, 3.0 mmol) were added in sequence under slow stirring, and the mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and then extracted with 0.6 M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and then the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated, and purified by column chromatography to obtain compound S23-4 (0.95 g).
  • Step d Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S23-4 (0.75 g, 1.0 mmol), S4-10 (0.68 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E23-1 (1.07 g).
  • Example 24 Cationic lipid (E24-1)
  • the preparation process is as follows:
  • the preparation process is as follows:
  • Example 26 Cationic lipid (E26-1)
  • the preparation process is as follows:
  • Step b Under nitrogen protection, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing S7-5 (0.65 g, 1.0 mmol), S26-1 (0.89 g, 1.2 mmol) and DMAP (0.03 g, 0.3 mmol) dissolved in dichloromethane (20 mL) and reacted at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E26-1 (1.13 g).
  • the preparation process is as follows:
  • Step a S1-1 (0.46 g, 2.0 mmol) was dissolved in dichloromethane solution (20 mL) and cooled to 0°C in an ice bath. (9Z, 12Z)-octadecane-9,12-dienal (S27-1, 1.58 g, 6.0 mmol) was added under vigorous stirring, and then sodium triacetoxyborohydride (NaBH(OAc) 3 , 1.27 g, 6.0 mmol) was added in three portions within 10 minutes. The ice bath was removed, and the reaction solution was stirred at room temperature for 2 h.
  • Example 28 Cationic lipid (E28-1)
  • reaction solution was concentrated and dissolved in dichloromethane, and then extracted with 0.6 M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and then the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the filtrate was concentrated, and the cationic lipid E29-1 (0.92 g) was obtained by column chromatography purification.
  • the preparation process is as follows:
  • Step b Under nitrogen protection, add DCC (1.36 g, 6.6 mmol) to a round-bottom flask containing S4-6 (2.03 g, 3.6 mmol), S30-1 (0.86 g, 3.0 mmol) and DMAP (0.09 g, 0.8 mmol) dissolved in dichloromethane (30 mL) and react at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, and concentrate the filtrate to obtain a crude product.
  • the above LNP-mRNA was added to a culture medium containing 10% fetal bovine serum (FBS), stirred at 37°C, and the particle size change of LNP-mRNA was measured by sampling at regular intervals.
  • FBS fetal bovine serum
  • the serum stability of the nucleic acid pharmaceutical preparation was analyzed by testing its particle size change. The experimental results showed that within 7 days, the particle size change of the experimental group was 0-14%, and the particle size change of the experimental group was 2-8%.
  • DMEM high-glucose complete medium containing 10% FBS was prepared, and the LNP-mRNA lipid pharmaceutical composition (L-1-1 to L-33 and L-CT1 to L-CT2) of the sample group was prepared into 0.1, 0.15, 0.2, 0.25, and 0.3 ⁇ g/100 ⁇ L working solution using complete medium, respectively, and stored for later use.
  • 293T cells in the logarithmic growth phase were taken and inoculated into a 96-well plate at 7 ⁇ 10 3 /well and 100 ⁇ L/well. Six replicate wells were set for both the control group and the experimental group.
  • Relative activity % (absorbance value of sample group - background absorbance value)/(absorbance value of control group - background absorbance value) ⁇ 100%; wherein the background absorbance value is the absorbance when only CCK-8 reagent and culture medium are added.
  • the experimental results show that the LNP-mRNA pharmaceutical composition prepared by the cationic lipid of the present invention does not produce obvious cytotoxicity under 5 concentration gradients, and the cell survival rate is greater than 95%.
  • the results of experimental group L-1-3 are shown in Figure 8.
  • Luciferase bioluminescence was used for testing.
  • the LNP-mRNA composition preparation was dissolved in the culture medium to prepare the required dose, and HeLa cells were used as a cell model.
  • the cell suspension was inoculated into a 96-well plate with a black-rimmed transparent bottom at a seeding density of 6000 cells/well at 100 ⁇ L/well. After inoculation, the cells were incubated in a cell culture incubator for 24 hours, and then administered at a dose of 0.2 ⁇ g mRNA per well.
  • the blank control group was added with a corresponding dose of free Fluc-mRNA.
  • the LNP-mRNA pharmaceutical compositions prepared by the present invention have excellent in vitro transfection effects, that is, the LNPs in the experimental groups are all effective nucleic acid delivery vectors, which may be because the cationic lipids of the present application contain three ionizable tertiary amine structures, and the transfection efficiency of the LNPs prepared therefrom is better than that of the L-CT1 and L-CT2 groups prepared by the nitrogen-containing heterocyclic cationic lipids in the prior art; from the comparison of the experimental groups L-1-1, L-1-2, L-1-3 and L-1-4, it can be seen that the carbon chain length between the tertiary amine and the ester bond of the linker affects the ionizability of the tertiary amine.
  • the longer the carbon chain length the higher the transfection efficiency. This may be because the stronger the ionizability, the more nucleic acids are bound, and the higher the transfection rate.
  • the carbon chain length between the non-piperazine ring tertiary amine and the ester bond of the cationic lipids E1-1 and E1-2 in L-1-1 and L-1-2 is C2
  • the carbon chain length between the non-piperazine ring tertiary amine and the ester bond of the cationic lipids E1-3 and E1-4 in L-1-3 and L-1-4 is C5, so L-1-3 and L-1-4 show better encapsulation rate and transfection rate; in addition, although the L-27, L-28, L-29, L-32 and L-33 groups showed higher encapsulation rates, their transfection rates were not optimal.
  • E27-1, E28-1, E29-1, E32-1 and E33-1 contain fewer degradable groups, which hinders the endosomal escape of mRNA and leads to a lower transfection rate than L-1-3; from the comparison between L-6 and L-8, it can be seen that E8-1 containing hydroxyl groups shows better encapsulation rate and transfection rate than E6-1 without hydroxyl groups. This may be because hydroxyl groups can interact with phosphate groups on nucleic acids through hydrogen bonds, thereby improving delivery efficiency.
  • the lipid nanoparticle L-1-3 was delivered to female BALB/c mice of 6-8 weeks of age by tail vein injection at a dosage of 10 ⁇ g/ only, and the mouse in vivo fluorescence imaging was performed respectively after administration for 6, 12 and 24 hours. After the last time point imaging, the mice were euthanized, and the major organs heart, liver, spleen, lung, and kidney (from left to right in the figure) were imaged. 10-15min before imaging, 0.2mL of D-luciferin sodium (15mg/mL) was injected intraperitoneally. Experimental results (Fig.
  • the lipid nucleic acid pharmaceutical composition prepared by the cationic lipid of the present invention can realize efficient nucleic acid drug delivery in vivo, and the LNP-mRNA pharmaceutical composition delivered into the body is mainly distributed in the liver and spleen.
  • the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions.
  • the present invention provides specific embodiments, it should be understood that the present invention can be further improved.
  • the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

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Abstract

La présente invention se rapporte au domaine de l'apport de médicaments. L'invention concerne un lipide cationique asymétrique contenant de multiples amines tertiaires, dont la structure est représentée par la formule générale (1), la définition de chaque symbole étant cohérente avec celle de la description. Dans la présente invention, un groupe dégradable est introduit dans une position appropriée du lipide cationique et la présence du groupe dégradable permet à une composition de médicament LNP, préparée à partir du lipide cationique, d'être dégradable dans des endosomes à un moment approprié et d'avoir une faible cytotoxicité, ce qui permet de résoudre le problème selon lequel, dans l'état de la technique, des compositions de médicament LNP préparées à partir de lipides non dégradables s'accumulent dans des endosomes et acidifient l'environnement endosomal, ce qui entrave l'échappement endosomal de médicaments (tels que des ARNm) et fait que les médicaments apportés dans les cellules ne parviennent pas à exercer suffisamment les effets thérapeutiques. Le lipide cationique de la présente invention utilise une petite molécule contenant de la pipérazine hétérofonctionnalisée en tant que matière première, implique un procédé de préparation simple et un coût inférieur, et est plus écologique et respectueux de l'environnement. Une composition d'ARNm-LNP préparée à partir du lipide cationique présente les avantages d'une faible toxicité, d'une biocompatibilité élevée, d'une transfection cellulaire élevée, etc.
PCT/CN2024/143282 2023-12-29 2024-12-27 Lipide cationique asymétrique contenant de multiples amines tertiaires Pending WO2025140594A1 (fr)

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CN116947785A (zh) * 2022-06-20 2023-10-27 成都威斯津生物医药科技有限公司 用于核酸递送的可电离脂质及其复合物
WO2024144009A1 (fr) * 2022-12-29 2024-07-04 이화여자대학교 산학협력단 Formulation de nanoparticules lipidiques comprenant des lipides ionisés à structure ramifiée et son utilisation
CN117550985A (zh) * 2023-11-08 2024-02-13 深圳厚存纳米药业有限公司 一种脂质化合物及其用途

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