WO2023186041A1 - 一种阳离子脂质化合物及其制备方法和应用 - Google Patents

一种阳离子脂质化合物及其制备方法和应用 Download PDF

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WO2023186041A1
WO2023186041A1 PCT/CN2023/085225 CN2023085225W WO2023186041A1 WO 2023186041 A1 WO2023186041 A1 WO 2023186041A1 CN 2023085225 W CN2023085225 W CN 2023085225W WO 2023186041 A1 WO2023186041 A1 WO 2023186041A1
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cationic lipid
compound
lipid
lipid compound
acid
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French (fr)
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章雪晴
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荣灿生物医药技术(上海)有限公司
上海交通大学
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Definitions

  • the invention relates to the field of biomedicine, in particular to a cationic lipid compound and its preparation method and application.
  • Nucleic acid vaccine also known as genetic vaccine, refers to introducing nucleic acid sequences (such as DNA, mRNA, etc.) encoding immunogenic proteins or polypeptides into the host body, and expressing the immunogenic proteins or polypeptides through host cells. , inducing host cells to generate an immune response to the immunogen to achieve the purpose of preventing and treating diseases. Among them, ensuring the smooth introduction of foreign genes is an extremely important part of the gene therapy process and gene vaccine immunity. Among the many methods of gene introduction, the method of developing suitable lipid nanoparticles (LNP) to encapsulate nucleic acids, targeting them to target cells, and delivering nucleic acids of specific genes into cells is gradually used by scientists.
  • LNP lipid nanoparticles
  • nucleic acid drugs contain a large number of phosphate groups, so they are negatively charged and have a large molecular weight.
  • various lipid compounds such as cationic lipids have been developed.
  • LNP is a type of nanoparticle that uses lipid compounds to form nanoparticles and has a double-layer or multi-layer membrane structure.
  • the outer membrane is mainly composed of PEG lipids and neutral lipids
  • the inner membrane is mainly composed of neutral lipids, with some cholesterol in the middle as structural lipids.
  • Neutral ionizable lipids, cationic lipid compounds and their wrapped nucleic acids are distributed in the nanoparticles.
  • “Lipid nanoparticles” refer to nanovesicles formed by encapsulating or associating drugs such as nucleic acids to be delivered using cationic lipid compounds.
  • Lipid nanoparticles and compositions thereof may be used for a variety of purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and in vivo to induce expression or inhibit protein of interest expression of target genes.
  • encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids
  • “Cationic lipid compound” refers to a lipid capable of being positively charged.
  • Exemplary cationic lipids include one or more positively charged amine groups.
  • Preferred cationic lipids are ionizable and can exist in either a positively charged form or a neutral form depending on the pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can influence its immune recognition in the blood, blood clearance, and tissue distribution, as well as its ability to escape inclusion bodies within cells and is critical for intracellular delivery of nucleic acids.
  • nucleic acid delivery technology In order to produce the desired therapeutic effect and/or trigger the desired immune response in biological systems, nucleic acid delivery technology still faces many challenges: first, nucleic acid molecules are easily degraded by in vivo and extracorporeal nucleases; second, nucleic acid molecules enter Cells have limited ability to interact with target organelles and regulate target gene expression or target protein expression. Lipid nanoparticles formed from cationic lipids with other lipid components (such as helplipids, cholesterol, and PEGylated lipids) and nucleic acids can be used to protect nucleic acids from degradation and promote cellular uptake of nucleic acids.
  • lipid components such as helplipids, cholesterol, and PEGylated lipids
  • the market needs an mRNA-LNP with good biocompatibility, high in vivo mRNA transfection efficiency, high stability, and high biological safety, so that it can target different diseases (such as metabolic diseases, respiratory diseases, cardiovascular and cerebrovascular diseases, tumors, etc. ), different administration methods (muscular injection, subcutaneous injection, intravenous injection, local injection, inhalation administration, transdermal administration, etc.), different application scenarios and purposes (cell therapy, gene editing, targeted technology, etc.), and Provide safe and effective delivery vector options for nucleic acid drugs or vaccines for various currently unmet clinical needs.
  • diseases such as metabolic diseases, respiratory diseases, cardiovascular and cerebrovascular diseases, tumors, etc.
  • different administration methods muscle injection, subcutaneous injection, intravenous injection, local injection, inhalation administration, transdermal administration, etc.
  • application scenarios and purposes cell therapy, gene editing, targeted technology, etc.
  • the purpose of the present invention is to provide a cationic lipid compound and its preparation method and application.
  • the mRNA-LNP prepared by the cationic lipid compound with a new structure of the present invention has good biocompatibility, In vivo mRNA transfection efficiency is high, stability is high, and biological safety is high.
  • a cationic lipid compound which is a compound with the following structure:
  • R 3 is H
  • R 4 is H
  • M 0 and M 1 are:
  • M 2 and M 3 are:
  • the structure of the present invention introduces hydroxyl groups into the head, while the overall structure is similar to a cone, with a small head and a large tail.
  • the specific meaning of the small head and large tail is: with the N atom as the center, the molecular structure of the head with the hydroxyl group occupies less space than the molecular structure of the tail ending with an alkane chain.
  • the cationic lipid compounds have achieved better biocompatibility and higher in vivo mRNA transfection efficiency.
  • At least two of M 0 , M 1 , M 2 , and M 3 are ester bonds. Introducing degradable ester bonds into the hydrophobic tail of the lipid molecule can change the lipid structure. It can improve the metabolic behavior of plasma molecules in the body, thereby improving the biosafety of mRNA-LNP.
  • the aforementioned cationic lipid compound is any compound selected from the following group:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the aforementioned cationic lipid compound is the following compound:
  • the preparation method of the aforementioned cationic lipid compound of H-1, H-3-H-16, H-18-H-20 structure includes the following steps:
  • Synthesis of the second intermediate product oxidize the double bond with m-chloroperoxybenzoic acid to generate epoxy to obtain the second intermediate product;
  • Synthesis of cationic lipid compounds A ring-opening reaction occurs between amine and epoxy under heating conditions, and the hydrophilic head and hydrophobic tail of the cation are connected to form a cationic lipid compound.
  • the preparation method of the aforementioned cationic lipid compounds with H21 and H23 structures includes the following steps:
  • Synthesis of cationic lipid compounds Dissolve the first intermediate product long-chain alkyl carboxylic acid in dichloromethane, and react with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide under activation Triethanolamine is synthesized by esterification reaction.
  • the preparation method of the aforementioned cationic lipid compounds with H22 and H24 structures includes the following steps:
  • the preparation method of the aforementioned cationic lipid compounds with H25, H26, and H27 structures includes the following steps:
  • Synthesis of the third intermediate product After dissolving the long-chain alkyl carboxylic acid in dichloromethane, it reacts with the long-chain alkyl carboxylic acid under the activation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • the preparation method of the aforementioned cationic lipid compounds with H28 and H29 structures includes the following steps:
  • Synthesis of cationic lipid compounds After dissolving the second intermediate product in dichloromethane, add 1-(3-dimethylamino It is synthesized by esterification reaction with the first intermediate product under the activation of propyl)-3-ethylcarbodiimide.
  • the preparation method of the aforementioned cationic lipid compound of H17 structure includes the following steps:
  • Synthesis of cationic lipid compounds prepared by the ring-opening reaction of the epoxy of the second intermediate product and the amine of the third intermediate product under heating conditions.
  • the preparation method of the aforementioned cationic lipid compound of H2 structure includes the following steps:
  • the first intermediate product is prepared by oxidizing long-chain alkyl alcohols to long-chain alkyl aldehydes using Dessmartin oxidant;
  • Synthesis of cationic lipid compounds prepared by reductive amination reaction between amine and aldehyde group of the second intermediate product.
  • the preparation method of the aforementioned cationic lipid compounds with H30 and H31 structures includes the following steps:
  • Synthesis of the first intermediate product oxidize the double bond with m-chloroperoxybenzoic acid to generate epoxy to obtain the first intermediate product;
  • Synthesis of cationic lipid compounds A ring-opening reaction occurs between amine and epoxy under heating conditions, and the hydrophilic head and hydrophobic tail of the cation are connected to form a cationic lipid compound.
  • a cationic lipid compound is applied to a composition containing a cationic lipid compound, its stereoisomer, its tautomer or its pharmaceutically acceptable salt.
  • the composition containing the cationic lipid compound includes: a carrier, a contained pharmaceutical reagent, and a pharmaceutical auxiliary agent.
  • the carrier is lipid nanoparticles LNP
  • the average size of the lipid nanoparticles is 30-200 nm
  • the polydispersity index of the lipid nanoparticle preparation is ⁇ 0.5.
  • the carrier includes: one or more ionizable lipid compounds.
  • the carrier also includes: a co-lipid, and the molar ratio of the cationic lipid compound to the co-lipid is 0.5:1-10:1; such a molar ratio is preferred, as long as it is
  • the compositions of cationic lipid compounds adopting the structure of the present invention are all within the protection scope of the present invention and are inspired by the present invention; the helper lipids include: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, sterols and their derivatives, One or a combination of ceramides and charged lipids.
  • the carrier also includes: structural lipid or polymer-conjugated lipid.
  • the carrier also includes: structural lipid, and the molar ratio of the cationic lipid compound to the structural lipid is 0.5:1-5:1; such a molar ratio is preferred, as long as it is
  • the compositions using the cationic lipid compound with the structure of the present invention are all within the protection scope of the present invention and are inspired by the present invention.
  • the carrier also includes: polymer-conjugated lipid, the molar ratio of the cationic lipid compound to the polymer-conjugated lipid is 20:1-250:1; the polymer-conjugated lipid
  • the substance is PEGylated lipids.
  • a cationic lipid compound contains pharmaceutical reagents including: nucleic acid molecules, small molecules One or more compounds, polypeptides or proteins; the selection and compounding of pharmaceutical reagents are not limited, as long as the cationic lipid compounds adopting the structure of the present invention are within the protection scope of the present invention, and are protected by the present invention. Inspiration.
  • pharmaceutical auxiliaries include: one or more of diluents, stabilizers, preservatives or freeze-drying protective agents; the selection and compounding of pharmaceutical auxiliaries are not limited, as long as All cationic lipid compounds adopting the structure of the present invention are within the protection scope of the present invention and are inspired by the present invention.
  • the introduction of hydroxyl groups into the head of the cationic lipid compound of the present invention increases the hydrophilicity of the compound's head and promotes the fusion of nanoparticles and cell membranes.
  • the overall structure is similar to a cone, and the head (the part containing nitrogen atoms) is small. , with a large tail (long-chain alkane in the hydrophobic part); through structural improvement, the lipid nanoparticles prepared from cationic lipid compounds have achieved better biocompatibility and higher in vivo mRNA transfection efficiency.
  • the amine in the middle of the cationic lipid compound of the present invention can be combined with nucleic acids after ionization, which is beneficial to encapsulation;
  • the present invention introduces a degradable ester bond into the hydrophobic tail of the cationic lipid compound, which can be rapidly degraded under the action of ester decomposing enzymes in the human body.
  • the introduction of the ester bond can change the metabolic behavior of lipid molecules in the human body, thereby improving the mRNA- Biosafety of LNP;
  • the lipid nanoparticles prepared from the cationic lipid compound of the present invention can form stable nanostructures with narrow size distribution, and the size varies with the structure of different lipid compounds, within the range of 30-200nm;
  • the nanostructure of the lipid nanoparticles of the present invention is stable and can be stored at low temperature for 120 days to 150 days;
  • the synthesis route of the present invention is simple and easy to implement, and the raw materials are cheap and easy to obtain, which is conducive to industrial production;
  • the mRNA-LNP prepared from the cationic lipid compound of the present invention can be used for different diseases (such as metabolic diseases, respiratory diseases, cardiovascular and cerebrovascular diseases, tumors, etc.) and different administration methods (muscular injection, subcutaneous injection, intravenous injection, topical injection, etc.) Injection, inhalation administration, transdermal administration, etc.), different application scenarios and purposes (cell therapy, gene editing, targeted technology, etc.), and various currently unmet clinical needs, provide safe and effective nucleic acid drugs or vaccines Selection of delivery vehicles.
  • diseases such as metabolic diseases, respiratory diseases, cardiovascular and cerebrovascular diseases, tumors, etc.
  • different administration methods muscle injection, subcutaneous injection, intravenous injection, topical injection, etc.
  • Injection inhalation administration, transdermal administration, etc.
  • different application scenarios and purposes cell therapy, gene editing, targeted technology, etc.
  • various currently unmet clinical needs provide safe and effective nucleic acid drugs or vaccines Selection of delivery vehicles.
  • Figure 1 is a transmission electron microscope image of lipid nanoparticles formed from cationic lipid compound H-1 in Experimental Example 3 of the present invention.
  • Nucleic acid is the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is a biological macromolecule composed of multiple nucleotide monomers; nucleic acid is composed of nucleotides, and nucleotide monomers are composed of five-carbon sugars and phosphates. base, nitrogenous base, or any modifying group. If the five-carbon sugar is ribose, the polymer formed is RNA; if the five-carbon sugar is deoxyribose, the polymer formed is DNA.
  • Nucleic acid molecules include single-stranded DNA, double-stranded DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA Component) , small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense DNA, antisense RNA, ribozyme, asymmetric interfering RNA (aiRNA ), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), gRNA, sgRNA, crRNA or tracrRNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), Morpholin antisense oligonucleotides, morpholino oligonu
  • Pharmaceutically acceptable salts refer to acid addition salts or base addition salts.
  • the acid of the acid addition salt includes but is not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acid phosphate, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartame Amino acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecane Sulfuric acid, ethane-1,2-disulfonic acid, ethane sulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, gluconic acid, dextran Sugar acid, glucuronic acid,
  • base addition salts include but are not limited to: sodium salt, potassium salt, lithium salt, ammonium salt, calcium salt, magnesium salt, iron salt, zinc salt, copper salt, manganese salt, and aluminum salt; organic bases include but are not limited to Limited to ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine Acid, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine , theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin; preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclo
  • Charged lipids refer to a type of lipid compound that exists in a positively or negatively charged form; its charge does not depend on the pH within the physiological range, such as pH 3 to 9, and is not affected by pH.
  • Charged lipids can be synthetic or naturally derived. Examples of charged lipids include, but are not limited to, DOTAP, DOTMA, 18PA.
  • messenger ribonucleic acid is a type of single-stranded ribonucleic acid that is transcribed from one strand of DNA as a template and carries genetic information that can guide protein synthesis.
  • the mRNA can be monocistronic or polycistronic.
  • the mRNA may also contain one or more functional nucleotide analogs. Examples of functional nucleotide analogs include: pseudouracil nucleoside, 1-methyl-pseudouracil nucleoside or 5-methylcytosine. wait.
  • the examples here are not exhaustive, and any modified mRNA or derivatives thereof can be applied to the present invention.
  • Small molecule compounds can be active ingredients in agents used for treatment or prophylaxis, such as: antineoplastic agents, anti-infectious agents, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterials, antifungals, antiparasitics Insecticides, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents, etc. are not exhaustive here.
  • Polypeptides are compounds formed by ⁇ -amino acids linked together by peptide bonds and are intermediate products of protein hydrolysis.
  • Proteins are substances with a certain spatial structure formed by twisting and folding of polypeptide chains composed of amino acids through “dehydration condensation"; proteins can be interferons, protein hormones, cytokines, chemokines or enzymes, etc.
  • the diluent is any pharmaceutically acceptable water-soluble excipient known to those skilled in the art, including: amino acids, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, other oligosaccharides, mannitol, dextran, chlorine Sodium chloride, sorbitol, polyethylene glycol, phosphate, or its derivatives, etc.
  • the stabilizer can be any pharmaceutically acceptable excipient known to those skilled in the art: Tween-80, sodium lauryl sulfate, sodium oleate, mannitol, mannose or sodium alginate, etc.
  • the preservative can be any pharmaceutically acceptable preservative known to those skilled in the art, such as thimerosal, etc.
  • the freeze-drying protective agent can be any pharmaceutically acceptable freeze-drying protective agent known to those skilled in the art, such as: grape Sugar, mannitol, sucrose, lactose, trehalose, maltose, etc.
  • DSPC English name: Distearoyl Phosphatidylcholine, 1,2-distaroyl-sn-glycero-3-phosphocholine; Chinese name: Distearoyl Phosphatidylcholine, CAS number: 816-94-4.
  • DPPC Chinese name: Dipalmitic acid phosphatidylcholine; English name: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, CAS number: 63-89-8.
  • DMPC Chinese name: Dimyristoylphosphatidylcholine; English name: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, CAS number: 18194-24-6.
  • DOPC Chinese name: 1,2-dioleoyl-sn-glycero-3-phosphocholine; English name: 1,2-dioleoyl-sn-glycero-3-phosphocholine, CAS number: 4235-95-4.
  • POPC Chinese name: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; English name: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, CAS number: 26853-31-6.
  • DOPE Chinese name: 1,2-Dioleoyl-SN-glycerol-3-phosphorylethanolamine; English name: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE, CAS number: 4004-05-1.
  • DOTAP Chinese name: (1,2-dioleoxypropyl)trimethylammonium chloride; English name: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt), CAS number: 132172-61 -3;
  • the chemical structural formula is as follows:
  • DOTMA Chinese name: N, N, N-trimethyl-2,3-bis(octadec-9-en-1-yloxy)propan-1-ammonium chloride, CAS number: 1325214-86- 5.
  • the chemical structural formula is as follows:
  • SM Chinese name: sphingomyelin (SM); English name: sphingomyelin.
  • PEG Chinese name: polyethylene glycol; English name: Polyethylene glycol.
  • 0-10 represents the number of repeating units in the compound structure, it means that the number of repeating units can optionally be 0, 1, 2, 3, 4, 5, 6, 7 ,8,9 or 10.
  • the cationic lipid compound of the present invention is a compound with the following structure:
  • R 3 is H
  • R 4 is H
  • M 0 and M 1 are:
  • M 2 and M 3 are:
  • the structure of the present invention is similar to a cone, with a small head and a large tail.
  • the cationic lipid compounds achieve better biocompatibility and higher in vivo mRNA transfer. dyeing efficiency.
  • a method for preparing a cationic lipid compound which is characterized in that it includes the following steps:
  • Synthesis of the second intermediate product oxidize the double bond with m-chloroperoxybenzoic acid to generate epoxy to obtain the second intermediate product;
  • Synthesis of cationic lipid compounds A ring-opening reaction occurs between amine and epoxy under heating conditions, and the hydrophilic head and hydrophobic tail of the cation are connected to form a cationic lipid compound.
  • the method is preferably used to prepare compounds H-1, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H-11, H-12, H-13, H-14, H-15, H-16, H-18, H-19, H-20.
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • Synthesis of cationic lipid compounds Dissolve the first intermediate product long-chain alkyl carboxylic acid in dichloromethane, and react with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide under activation Triethanolamine is synthesized by esterification reaction.
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • Synthesis of the third intermediate product After dissolving the long-chain alkyl carboxylic acid in dichloromethane, it reacts with the long-chain alkyl carboxylic acid under the activation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.
  • the method described is preferably used to prepare compounds H-25, H-26, H-27.
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • Synthesis of cationic lipid compounds prepared by the ring-opening reaction of the epoxy of the second intermediate product and the amine of the third intermediate product under heating conditions.
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • the first intermediate product is prepared by oxidizing long-chain alkyl alcohols to long-chain alkyl aldehydes using Dessmartin oxidant;
  • Synthesis of cationic lipid compounds prepared by reductive amination reaction between amine and aldehyde group of the second intermediate product.
  • the invention provides another method for preparing a cationic lipid compound, which is characterized in that it includes the following steps:
  • Synthesis of the first intermediate product oxidize the double bond with m-chloroperoxybenzoic acid to generate epoxy to obtain the first intermediate product;
  • Synthesis of cationic lipid compounds A ring-opening reaction occurs between amine and epoxy under heating conditions, and the hydrophilic head and hydrophobic tail of the cation are connected to form a cationic lipid compound.
  • the method described is preferably used to prepare compounds H-30, H-31.
  • compositions including cationic lipid compounds
  • compositions include: carriers, contained pharmaceutical reagents, and pharmaceutical auxiliaries; wherein:
  • Carriers include: one or more ionizable lipid compounds, co-lipids, structured lipids or polymer-conjugated lipids.
  • the carrier is lipid nanoparticles (LNP)
  • the average size of the lipid nanoparticles is 30-200 nm
  • the polydispersity index of the nanoparticle preparation is ⁇ 0.5.
  • any nanoparticles prepared from one or more cationic lipid compounds of the present invention are within the scope of this patent and are inspired by the present invention; for example: in addition to lipid nanoparticles, they may also be one or more Hybrid nanoparticles formed by a variety of cationic lipid compounds and polymers, such as: PLGA-PEG, PLA-PEG, PCL, etc. are not exhaustive here.
  • Helping lipids include: one or a combination of several of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), sterols and their derivatives, ceramide, and charged lipids; phosphatidylcholine is a preferred Including: DSPC, DPPC, DMPC, DOPC, POPC; phosphatidylethanolamine as one, preferably DOPE; sterol as one Preferably, it is cholesterol; as an example, the charged lipid is DOTAP, DOTMA, and 18PA; this is not an exhaustive list. As long as the composition of the cationic lipid compound using the structure of the present invention is within the protection scope of the present invention, it is protected by the present invention. Invention inspiration. This is not an exhaustive list, and the selection of lipid helpers is not limited. As long as the cationic lipid compound adopts the structure of the present invention, it is within the protection scope of the present invention and is inspired by the present invention.
  • Structural lipids include: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassisterol, tomatine, tomatine, ursolic acid, ⁇ -tocopherol or one of corticosteroids or Several kinds. This is not an exhaustive list, and the selection of structural lipids is not limited. As long as the cationic lipid compound adopts the structure of the present invention, it is within the protection scope of the present invention and is inspired by the present invention.
  • the polymer-conjugated lipid is a PEGylated lipid; as an example, the PEGylated lipid includes: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified One or more of dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol.
  • PEGylated lipid includes: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified One or more of dialkylamine, PEG-modified diacylglycerol or PEG-modified dialkylglycerol.
  • the pharmaceutical reagents contained include: one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. This is not an exhaustive list. As long as the cationic lipid compound adopts the structure of the present invention, no matter what kind of pharmaceutical reagent is selected, it can be applied in the present invention, and all are within the protection scope of the present invention and are inspired by the present invention.
  • Pharmaceutical auxiliaries include: one or more of diluents, stabilizers, preservatives or lyoprotectants. This is not an exhaustive list. As long as the cationic lipid compound adopts the structure of the present invention, no matter what kind of pharmaceutical auxiliary compound is selected, it does not deviate from the design scope of the present invention.
  • the cationic lipid compound was prepared by the preparation method of the following Examples 1-7.
  • the dichloromethane is distilled off under reduced pressure using a rotary evaporator.
  • Add 200 mL of ethyl acetate, wash 3 times with an equal volume of saturated sodium chloride solution, dry the organic phase over anhydrous sodium sulfate for 30 minutes, distill the ethyl acetate under reduced pressure with a rotary evaporator, and perform column separation and purification (silica gel column, eluent is PE :EA 3:1 (volume ratio)), and 8.2g of colorless liquid was obtained, with a yield of 81%.
  • Example 2 Using the method of Example 1, the following compounds were prepared: H-1, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H -11, H-12, H-13, H-14, H-15, H-16, H-18, H-19, H-20.
  • Synthesis of compound c Refer to the synthesis method of compound c in Example 2.
  • Synthesis of compound c Refer to the synthesis method of compound c in Example 2.
  • Synthesis of compound c Refer to the synthesis method of compound c in Example 2.
  • Synthesis of compound d Refer to the synthesis method of compound d in the synthesis process of compound H-15.
  • Compound H-17 was prepared using the method of Example 6.
  • Dissolve compound d (1.0g, 2.6mmol) in 50mL DCM, add NaHCO 3 (1.75g, 20.8mmol), stir for 5 minutes, then add compound Dess-Martin Periodinane (1.75g, 4.16mmol) Stir at room temperature for 3h. After TLC monitored that the reaction was complete, the solvent was evaporated under reduced pressure using a rotary evaporator. Add petroleum ether, wash three times with an equal volume of saturated sodium bicarbonate solution, and wash once with an equal volume of saturated brine. The organic phase is dried with anhydrous sodium sulfate for 30 minutes.
  • Compound H-2 was prepared using the method of Example 7.
  • the dichloromethane is distilled off under reduced pressure using a rotary evaporator.
  • Add 100 mL of ethyl acetate, wash with an equal volume of saturated sodium chloride solution three times, dry the organic phase over anhydrous sodium sulfate for 30 minutes, distill the ethyl acetate under reduced pressure with a rotary evaporator, and perform column separation and purification (silica gel column, eluent is PE :EA 20:1 (volume ratio)), 3.5g colorless liquid was obtained, the yield was 81%.
  • cationic lipid compounds with a hydroxyl group in the head, a small head and a large tail in the overall structure fall within the scope of the present invention.
  • the specific meaning of the small head and large tail is: with the N atom as the center, the molecular structure of the head with the hydroxyl group occupies less space than the molecular structure of the tail ending with an alkane chain.
  • Cationic lipids, DSPC or DOPE (Avito (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avito (Shanghai) Pharmaceutical Technology Co., Ltd.), and PEG-lipids were prepared in the designed prescription ratio (Lipid (cationic Lipid compound)/DOPC/Cholesterol (cholesterol)/DMG-PEG (conjugated lipid) is 40/10/50/1.7 (molar ratio)), dissolved in ethanol (Lipid concentration 20mg/mL), and fully Mix well.
  • the above cationic lipid ethanol solution and the mRNA solution are thoroughly mixed in a volume ratio of 1:5 to 1:1.
  • the obtained nanoparticles were purified by ultrafiltration and dialysis. Sterilize by filtration.
  • PDI particle size and particle size distribution index
  • mRNA-LNP mRNA-encapsulated lipid nanoparticles
  • the index reflects the uniformity of particle size and is an important indicator of particle size characterization.
  • the encapsulation efficiency of mRNA was measured using Ribogreen RNA Quantitative Assay Kit (Thermo Fisher). The results are shown in Table 1.
  • mice Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were kept under experimental conditions of 22 ⁇ 2°C and relative humidity of 45-75%, with a light/dark cycle of 12 h.
  • the mRNA encoding luciferase (luciferase mRNA) was used as a reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP is reflected by detecting the bioluminescence intensity per unit time.
  • мно-lipid formula ratio of MC3 Lipid (cationic lipid)/DSPC/Cholesterol (cholesterol)/DMG-PEG (conjugated lipid) is 50/10/38.5/1.5 (mol
  • the lipid nanoparticles prepared from (ratio)) were used as a comparison sample for positive control, and were administered via intramuscular injection at a dose of 150 ⁇ g/kg mRNA. Each group of samples had one mouse and two legs.
  • mice were intraperitoneally injected with luciferin (20 ⁇ g/mL). After 5 minutes, the mice were placed in a small animal in vivo imager to measure the fluorescence intensity. The final results were expressed as average fluorescence intensity. The mice were injected intraperitoneally. The experimental results of fluorescence intensity after administration are shown in Table 2.
  • Experimental Example 3 Use the sample prepared in Experimental Example 1 to conduct a lipid nanoparticle structure stability experiment:
  • the lipid nanoparticles of the present invention can form stable nanostructures with narrow size distribution (small PDI).
  • small PDI narrow size distribution
  • Experimental Example 4 Use the sample prepared in Experimental Example 1 and the comparative sample of Experimental Example 2 to conduct a biocompatibility verification experiment:
  • Cell viability was measured using CCK-8 (cell counting kit-8) kit.
  • A1 is the absorbance of the drug-added group
  • A0 is the absorbance of the blank group
  • A2 is the absorbance of the control group.
  • the lipid nanoparticles prepared according to the formula were stored at 4°C at low temperature.
  • Malvern was used to Zetasizer Nano ZS characterized the particle size (Size) and PDI of mRNA-LNP (mRNA-encapsulated lipid nanoparticles), and the encapsulation efficiency of mRNA was determined using Ribogreen RNA quantitative assay kit (Thermo Fisher). The measurement results are shown in Table 4.
  • the LNP lipid nanoparticle formed by the lipid molecules of the present invention can be stored at low temperature for a long time, which facilitates the transportation and storage of the product.
  • the present invention introduces hydroxyl groups into the head of the cationic lipid compound to increase the hydrophilicity of the head of the lipid compound and the fusion of nanoparticles and cell membranes.
  • the overall structure is similar to a cone, and the head is The part (the part containing nitrogen atoms) is small and the tail (the long-chain alkane of the hydrophobic part) is large.
  • the lipid nanoparticles prepared from the cationic lipid compound have achieved better biocompatibility and Higher in vivo mRNA transfection efficiency; the compound introduces a degradable ester bond at the hydrophobic tail, which can be rapidly degraded under the action of human endocrine enzymes. Compared with the long-chain alkanes of MC3, the introduction of ester bonds can change lipid molecules.
  • MC3 requires a five-step reaction, and the reaction process involves highly dangerous Grignard reagents; compared with the synthesis of MC3, the present invention
  • the route is simple and easy, and the ingredients It is cheap and easy to obtain, which is conducive to its industrial production.
  • the LNP (lipid nanoparticles) prepared by the compound of the present invention can be stored at low temperature for a long time, which facilitates the transportation and preservation of the product. Therefore, the new cationic lipid compound designed by the present invention has good application prospects.

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Abstract

公开了一种阳离子脂质化合物及其制备方法及其应用，阳离子脂质化合物在头部引入羟基，同时整体结构类似于锥形，头部小，尾部大；通过结构改进的配合，使得阳离子脂质化合物制备得到的脂质纳米粒均具有更好的生物相容性以及更高的体内mRNA转染效率，具有意想不到的技术效果；公开的阳离子脂质化合物的合成路线简单易行，原料廉价易得，有利于工业化生产；通过阳离子脂质化合物制成的脂质纳米粒具有稳定的纳米结构，可在低温储存较长时间，延长药品保质期的同时降低药品的运输条件。

Description

一种阳离子脂质化合物及其制备方法和应用 技术领域
本发明涉及生物医药领域，特别是一种阳离子脂质化合物及其制备方法和应用。
背景技术
基因治疗(gene therapy)是通过将外源基因导入靶细胞，以纠正或补偿缺陷、异常基因引起的疾病，达到治疗目的的一种治疗方法。核酸疫苗(nucleic acid vaccine)，也称基因疫苗(genetic vaccine)，是指将含编码免疫原蛋白或多肽的核酸序列(如DNA、mRNA等)导入宿主体内，通过宿主细胞表达免疫原蛋白或多肽，诱导宿主细胞产生对该免疫原的免疫应答，以达到预防和治疗疾病的目的。其中，确保外源基因的顺利导入是基因治疗过程和基因疫苗免疫极为重要的一环。在众多基因导入的方法中，开发合适的脂质纳米粒(Lipid Nanoparticle，LNP)包裹核酸，使其靶向至目标细胞，并将特定基因的核酸递送至细胞内的方法逐渐为科学家所使用。
核酸药物与普通化学药物的一个明显区别是核酸带有数量庞大的磷酸根，因而呈负电，且分子量大。为了使其能够被脂质纳米粒更好地包裹，人们开发了阳离子脂质等多种脂质化合物。
LNP是使用脂质化合物形成纳米粒的一种，具有双层或多层膜结构。外层膜主要是PEG脂质，也有中性脂质，内层膜主要是中性脂质，中间有些胆固醇作为结构脂质。纳米粒中分布有中性可离子脂质、阳离子脂质化合物及其包裹着的核酸。“脂质纳米粒”指使用阳离子脂质化合物等将需要递送的核酸等药物封装或缔合后形成的纳米囊泡体。脂质纳米粒和其组合物可以用于各种目的，包括在体外和体内将封装的或缔合的(例如，复合的)诸如核酸等治疗剂递送至细胞，从而诱导目标蛋白质的表达或者抑制靶基因的表达。
“阳离子脂质化合物”指能够带正电的脂质。示例性的阳离子脂质包括一种或多种带有正电荷的胺基团。优选的阳离子脂质是可电离的，可以根据pH值以带正电的形式或中性形式存在。阳离子脂质的电离影响脂质纳米粒在不同pH条件下的表面电荷。这种电荷状态可以影响其在血液中的免疫识别、血液清除和组织分布，以及其在细胞内的内含体逃逸的能力，对于核酸的细胞内递送是至关重要的。
为了在生物系统中产生所期望的治疗效果和/或引发所期望的免疫应答，核酸递送技术还存在着诸多挑战：第一，核酸分子易被体、外核酸酶降解；第二，核酸分子进入细胞、与目标细胞器相互作用、调节目标基因表达或目标蛋白表达的能力有限。由阳离子脂质与其他脂质组分(如助脂质、胆固醇和PEG化的脂质)和核酸形成的脂质纳米粒可以用于保护核酸不被降解并促进核酸的细胞摄取。市场需要一种生物相容性好、体内mRNA转染效率高、稳定性高、生物安全性高的mRNA-LNP，从而能够针对不同疾病(如代谢疾病、呼吸道疾病、心脑血管疾病、肿瘤等)、不同给药方式(肌肉注射、皮下注射、静脉注射、局部注射、吸入给药、经皮给药等)、不同的应用场景和目的(细胞治疗、基因编辑、靶向技术等)、及各种目前尚未满足的临床需求等，提供安全有效的核酸药物或疫苗的递送载体的选择。
发明内容
为解决现有技术的不足，本发明的目的在于提供一种阳离子脂质化合物及其制备方法和应用，通过本发明全新结构的阳离子脂质化合物制备得到的mRNA-LNP的生物相容性好、体内mRNA转染效率高、稳定性高、生物安全性高。
为了实现上述目标，本发明采用如下的技术方案：
一种阳离子脂质化合物，为如下结构的化合物：
R1为
R2为
R3为H，
R4为H，
R5为
R6为
R7为
R8为
R9为
R10为
R11为
M0、M1为：
M2、M3为：
本发明的结构在头部引入羟基，同时整体结构类似于锥形，头小，尾部大。头部小，尾部大的具体意思为：以N原子为中心，带羟基的头部的分子结构空间占位比以烷烃链为结尾的尾部的分子结构空间占位小。通过结构改进的配合；使得阳离子脂质化合物均取得了更好的生物相容性以及更高的体内mRNA转染效率。
前述的一种阳离子脂质化合物，作为一种优选，M0、M1、M2、M3至少有两个为酯键，在脂质分子的疏水尾部引入可降解的酯键，可以改变脂质分子在体内的代谢行为，进而提高mRNA-LNP的生物安全性。
前述的一种阳离子脂质化合物，作为一种实施例，为选自下组的任一化合物：

前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，作为一种实施例，为如下的化合物：
前述的一种阳离子脂质化合物，为如下的化合物：
前述的H-1、H-3-H-16，H-18-H-20结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与端烯长链烷基醇发生酯化反应合成，得到第一中间产物；
第二中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧，得到第二中间产物；
阳离子脂质化合物的合成：加热条件下由胺与环氧发生开环反应，将阳离子的亲水头部与疏水尾部连接生成阳离子脂质化合物。
前述的H21、H23结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成，得到第一中间产物；
阳离子脂质化合物的合成：将第一中间产物长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与三乙醇胺发生酯化反应合成。
前述的H22、H24结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与三乙醇胺发生酯化反应合成；
第二中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成，得到第二中间产物；
阳离子脂质化合物的合成：将第二中间产物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与第一中间产物发生酯化反应合成。
前述的H25、H26、H27结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：碱性条件下长链烷基胺与二硫化碳反应后，在4-二甲氨基吡啶以及二碳酸二叔丁酯的催化下，生成相应的异硫氰酸酯；
第二中间产物的合成：在溶剂中，异硫氰酸酯与胺发生亲核取代反应，生成相应的硫脲；
第三中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成；阳离子脂质化合物的合成：将第三中间产物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与第二中间产物发生酯化反应合成。
前述的H28、H29结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：碱性条件下，胺与三光气反应生成异氰酸酯，异氰酸酯与胺反应生成相应的含脲结构的第一中间产物；
第二中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成；
阳离子脂质化合物的合成：将第二中间产物溶于二氯甲烷后，在1-(3-二甲基氨基 丙基)-3-乙基碳二亚胺活化下与第一中间产物发生酯化反应合成。
前述的H17结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：亚油醇与溴代酰氯化合物在碱性条件下发生酯化反应得到溴代的长链烷烃；
第二中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧；
第三中间产物的合成：碱性条件下由第一中间产物溴代的长链烷烃与胺发生亲核取代反应制得；
阳离子脂质化合物的合成：加热条件下由第二中间产物的环氧和第三中间产物的胺发生开环反应制得。
前述的H2结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸化合物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与二元醇发生酯化反应合成；
第二中间产物的合成：将第一中间产物使用戴斯马丁氧化剂将长链烷基醇氧化成长链烷基醛制得；
阳离子脂质化合物的合成：使用胺和第二中间产物的醛基发生还原胺化反应制得。
前述的H30、H31结构的阳离子脂质化合物的制备方法，包括如下步骤：
第一中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧，得到第一中间产物；
第二中间产物的合成：将长链环氧烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与端烯长链烷基醇发生酯化反应合成，得到第二中间产物；
阳离子脂质化合物的合成：加热条件下由胺与环氧发生开环反应，将阳离子的亲水头部与疏水尾部连接生成阳离子脂质化合物。
前述的一种阳离子脂质化合物的应用，应用于包含阳离子脂质化合物的组合物、其立体异构体、其互变异构体或其在药学上可接受的盐。
前述的一种阳离子脂质化合物的应用，包含阳离子脂质化合物的组合物包括：载体，所载的药物试剂，药物辅助剂。
前述的一种阳离子脂质化合物的应用，载体为脂质纳米粒LNP，脂质纳米粒的平均尺寸为30-200nm，脂质纳米粒的制剂的多分散指数≤0.5。
前述的一种阳离子脂质化合物的应用，载体包括：一种或多种可电离的脂质化合物。
前述的一种阳离子脂质化合物的应用，载体还包括：助脂质，阳离子脂质化合物与助脂质的摩尔比为0.5∶1-10∶1；这样的摩尔比是一种优选，只要是采用本发明结构的阳离子脂质化合物的组合物均在本发明的保护范围内，均受本发明启示；助脂质包括：磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、甾醇及其衍生物、神经酰胺、带电脂质中的一种或几种的组合。
前述的一种阳离子脂质化合物的应用，载体还包括：结构脂质或聚合物缀合脂质。
前述的一种阳离子脂质化合物的应用，载体还包括：结构脂质，阳离子脂质化合物与结构脂质的摩尔比为0.5∶1-5∶1；这样的摩尔比是一种优选，只要是采用本发明结构的阳离子脂质化合物的组合物均在本发明的保护范围内，均受本发明启示。
前述的一种阳离子脂质化合物的应用，载体还包括：聚合物缀合脂质，阳离子脂质化合物与聚合物缀合脂质的摩尔比为20∶1-250∶1；聚合物缀合脂质为聚乙二醇化脂质。
前述的一种阳离子脂质化合物的应用，所载的药物试剂包括：核酸分子，小分子 化合物，多肽或蛋白质中的一种或多种；药物试剂的选择和复配不受限制，只要是采用了本发明结构的阳离子脂质化合物均在本发明的保护范围内，且均受本发明的启示。
前述的一种阳离子脂质化合物的应用，药物辅助剂包括：稀释剂，稳定剂，防腐剂或冻干保护剂中的一种或多种；药物辅助剂的选择和复配不受限制，只要是采用了本发明结构的阳离子脂质化合物均在本发明的保护范围内，且均受本发明的启示。
本发明的有益之处在于：
本发明的阳离子脂质化合物在头部引入羟基增加了化合物头部的亲水性，以及促进了纳米粒与细胞膜的融合，同时整体结构类似于锥形，头部(含氮原子的部分)小，尾部(疏水部分的长链烷烃)大；通过结构改进的配合，使得阳离子脂质化合物制备得到的脂质纳米粒均取得了更好的生物相容性以及更高的体内mRNA转染效率，具有意想不到的技术效果；
本发明的阳离子脂质化合物中间的胺电离后可以与核酸结合，利于包封；
本发明在阳离子脂质化合物的疏水尾部引入可降解的酯键，可在人体内酯分解酶的作用下迅速降解，酯键的引入可以改变脂质分子在人体内的代谢行为，进而提高mRNA-LNP的生物安全性；
本发明阳离子脂质化合物制备得到的脂质纳米粒都能形成稳定的纳米结构，尺寸分布较窄，尺寸随不同的脂质化合物结构的不同而有所变化，在30-200nm范围内；
本发明的脂质纳米粒的纳米结构稳定，可在低温储存120天-150天；
本发明相比现有技术普遍使用的阳离子脂质化合物(如MC3)的合成，合成路线简单易行，原料廉价易得，有利于工业化生产；
由本发明的阳离子脂质化合物制备得到的mRNA-LNP，能够针对不同疾病(如代谢疾病、呼吸道疾病、心脑血管疾病、肿瘤等)、不同给药方式(肌肉注射、皮下注射、静脉注射、局部注射、吸入给药、经皮给药等)、不同的应用场景和目的(细胞治疗、基因编辑、靶向技术等)、及各种目前尚未满足的临床需求，提供安全有效的核酸药物或疫苗的递送载体的选择。
附图说明
图1是本发明实验实施例3阳离子脂质化合物H-1形成的脂质纳米粒的透射电镜图。
术语、英文缩写解释说明：
核酸是脱氧核糖核酸(DNA)和核糖核酸(RNA)的总称，是由多个核苷酸单体组成的生物大分子；核酸由核苷酸组成，核苷酸单体由五碳糖、磷酸基、含氮碱基、或任何修饰基团组成。如果五碳糖是核糖，则形成的聚合物是RNA；如果五碳糖是脱氧核糖，则形成的聚合物是DNA。
核酸分子包括单链DNA、双链DNA、短异构体、mRNA、tRNA、rRNA、长链非编码RNA(lncRNA)、微小非编码RNA(miRNA和siRNA)、端粒酶RNA(Telomerase RNA Component)、小分子RNA(snRNA和scRNA)、环状RNA(circRNA)、合成miRNA(miRNA mimics、miRNA agomir、miRNA antagomir)、反义DNA、反义RNA、核酶(ribozyme)、不对称干扰RNA(aiRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)、gRNA、sgRNA、crRNA或tracrRNA、锁核酸(LNA)、肽核酸(PNA)、吗啉反义寡核苷酸、吗啉代寡核苷酸或生物定制寡核苷 酸等。这里的举例也并非穷举，只要是由核苷酸单体聚合成的都可以应用于本发明。
药物可用的盐是指酸加成盐或碱加成盐。
其中酸加成盐的酸包括但不限于：盐酸、氢溴酸、硫酸、硝酸、磷酸、酸式磷酸盐、乙酸、2，2-二氯乙酸、己二酸、海藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸，柠檬酸、环酰胺酸、十二烷基硫酸、乙烷-1，2-二磺酸、乙烷磺酸、2-羟基乙磺酸、甲酸、富马酸、半乳糖酸、龙胆酸、葡庚酸、葡糖酸、葡聚糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、粘酸、萘-1，5二甲酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、棕榈酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸、季铵酸以及十一碳烯酸。
其中碱加成盐举例包括但不限于：钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐，锌盐、铜盐、锰盐、以及铝盐；有机碱包括但不限于氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、脱醇、2-二甲基氨基乙醇、2-二乙基氨基乙醇、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、肼苯胺、胆碱、甜菜碱、苯那敏(benethamine)、苄星青霉素(benzathine)、乙二胺、葡糖胺、甲基葡糖胺、可可碱、三乙醇胺、嘌呤、哌嗪、哌啶、N-乙基哌啶、以及聚胺树脂；优选地，有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。
带电脂质是指一类脂质化合物以带正电荷或带负电荷的形式存在；其所带电荷不依赖于生理学范围内的pH，例如pH3～9，不受pH的影响。带电脂质可以是合成的或天然来源的。带电脂质的实例包括但不限于DOTAP、DOTMA、18PA。
mRNA，信使RNA，中文译名：信使核糖核酸，是由DNA的一条链作为模板转录而来的、携带遗传信息能指导蛋白质合成的一类单链核糖核酸。mRNA可以是单顺反子mRNA也可以是多顺反子mRNA。mRNA也可以包含一种或多种功能性核苷酸类似物，功能性核苷酸类似物举例包括：假尿嘧啶核苷、1-甲基-假尿嘧啶核苷或5-甲基胞嘧啶等。这里的举例也并非穷举，任何修饰的mRNA或其衍生物都可以应用于本发明。
小分子化合物可以是用于治疗或预防的试剂中的有效成分，例如：抗肿瘤药、抗感染药、局部麻醉药、抗抑郁药、抗惊厥药、抗生素/抗菌剂、抗真菌药、抗寄生虫药、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼剂、麻醉剂、或成像剂等，这里并非穷举。
多肽是α-氨基酸以肽键连接在一起而形成的化合物，是蛋白质水解的中间产物。
蛋白质是由氨基酸以“脱水缩合”的方式组成的多肽链经过盘曲折叠形成的具有一定空间结构的物质；蛋白质可以是干扰素、蛋白质激素、细胞因子、趋化因子或者酶类等。
稀释剂是本领域技术人员可知的任意可以药用的水溶性辅料，包括：氨基酸、单糖、二糖、三糖、四糖、五糖、其它寡聚糖、甘露醇、右旋糖苷、氯化钠、山梨醇、聚乙二醇、磷酸盐，或其衍生物等。
稳定剂可以是本领域技术人员可知的任意可以药用的辅料：吐温-80，十二烷基硫酸钠，油酸钠，甘露醇，甘露糖或海藻酸钠等。
防腐剂可以是本领域技术人员可知的任意可以药用的防腐剂，比如：硫柳汞等。
冻干保护剂可以是领域技术人员可知的任意可以药用的冻干保护剂，比如：葡萄 糖、甘露醇、蔗糖、乳糖，海藻糖，麦芽糖等。
DSPC：英文名称：Distearoyl Phosphatidylcholine，1，2-distearoyl-sn-glycero-3-phosphocholine；中文名称：二硬脂酰基卵磷脂，CAS号：816-94-4。
DPPC：中文名称：二棕榈酸磷脂酰胆碱；英文名称：1，2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE，CAS号：63-89-8。
DMPC：中文名称：二肉豆蔻酰磷脂酰胆碱；英文名称：1，2-Dimyristoyl-sn-glycero-3-phosphocholine，CAS号：18194-24-6。
DOPC：中文名称：1，2-二油酰基-sn-甘油-3-磷酸胆碱；英文名称：1，2-dioleoyl-sn-glycero-3-phosphocholine，CAS号：4235-95-4。
POPC：中文名称：2-油酰-1-棕榈锡甘油-3-磷酸胆碱；英文名称：2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine，CAS号：26853-31-6。
DOPE：中文名称：1，2-二油酰-SN-甘油-3-磷酰乙醇胺；英文名称：1，2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE，CAS号：4004-05-1。
DOTAP：中文名称：(1，2-二油氧基丙基)三甲基氯化铵，；英文名称：1，2-dioleoyl-3-trimethylammonium-propane(chloride salt)，CAS号：132172-61-3；化学结构式如下所示：
DOTMA：中文名称：N，N，N-三甲基-2，3-双(十八碳-9-烯-1-基氧基)丙-1-铵氯化物，CAS号：1325214-86-5，化学结构式如下所示：
18PA：CAS号：108392-02-5，化学结构式如下所示：
SM：中文名称：鞘磷脂(SM)；英文名称：sphingomyelin。
PEG：中文名称：聚乙二醇；英文名称：Polyethylene glycol。
在本发明中，当以“0-10”表示化合物结构中的重复单元个数时，指该重复单元的个数可任选地为0、1、2、3、4、5、6、7、8、9或10。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
阳离子脂质化合物
本发明的阳离子脂质化合物，为如下结构的化合物：
R1为
R2为
R3为H，
R4为H，
R5为
R6为
R7为
R8为
R9为
R10为
R11为
M0、M1为：
M2、M3为：
本发明的结构类似于锥形，头部小，尾部大，通过这样的结构和在头部引入羟基配合，使得阳离子脂质化合物均取得了更好的生物相容性以及更高的体内mRNA转染效率。
阳离子脂质化合物的制备
本发明中，提供了根据一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与端烯长链烷基醇发生酯化反应合成，得到第一中间产物；
第二中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧，得到第二中间产物；
阳离子脂质化合物的合成：加热条件下由胺与环氧发生开环反应，将阳离子的亲水头部与疏水尾部连接生成阳离子脂质化合物。
所述的方法优选地用于制备化合物H-1、H-3、H-4、H-5、H-6、H-7、H-8、H-9、H-10、H-11、H-12、H-13、H-14、H-15、H-16、H-18、H-19、H-20。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成，得到第一中间产物；
阳离子脂质化合物的合成：将第一中间产物长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与三乙醇胺发生酯化反应合成。
所述的方法优选地用于制备化合物H-21或H-23。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与三乙醇胺发生酯化反应合成；
第二中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成，得到第二中间产物；
阳离子脂质化合物的合成：将第二中间产物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与第一中间产物发生酯化反应合成。
所述的方法优选地用于制备化合物H-22、H-24。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：碱性条件下长链烷基胺与二硫化碳反应后，在4-二甲氨基吡啶以及二碳酸二叔丁酯的催化下，生成相应的异硫氰酸酯；
第二中间产物的合成：在溶剂中，异硫氰酸酯与胺发生亲核取代反应，生成相应的硫脲；
第三中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成；阳离子脂质化合物的合成：将第三中间产物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与第二中间产物发生酯化反应合成。
所述的方法优选地用于制备化合物H-25、H-26、H-27。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：碱性条件下，胺与三光气反应生成异氰酸酯，异氰酸酯与胺反应生成相应的含脲结构的第一中间产物；
第二中间产物的合成：将长链烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与长链烷基醇化合物发生酯化反应合成；
阳离子脂质化合物的合成：将第二中间产物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与第一中间产物发生酯化反应合成。
所述的方法优选地用于制备化合物H-28、H-29。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：亚油醇与溴代酰氯化合物在碱性条件下发生酯化反应得到溴代的长链烷烃；
第二中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧；
第三中间产物的合成：碱性条件下由第一中间产物溴代的长链烷烃与胺发生亲核取代反应制得；
阳离子脂质化合物的合成：加热条件下由第二中间产物的环氧和第三中间产物的胺发生开环反应制得。
所述的方法优选地用于制备化合物H-17。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：将长链烷基羧酸化合物溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与二元醇发生酯化反应合成；
第二中间产物的合成：将第一中间产物使用戴斯马丁氧化剂将长链烷基醇氧化成长链烷基醛制得；
阳离子脂质化合物的合成：使用胺和第二中间产物的醛基发生还原胺化反应制得。
所述的方法优选地用于制备化合物H-2。
本发明提供了另一种阳离子脂质化合物的制备方法，其特征在于，包括如下步骤：
第一中间产物的合成：由间氯过氧苯甲酸将双键氧化生成环氧，得到第一中间产物；
第二中间产物的合成：将长链环氧烷基羧酸溶于二氯甲烷后，在1-(3-二甲基氨基丙基)-3-乙基碳二亚胺活化下与端烯长链烷基醇发生酯化反应合成，得到第二中间产物；
阳离子脂质化合物的合成：加热条件下由胺与环氧发生开环反应，将阳离子的亲水头部与疏水尾部连接生成阳离子脂质化合物。
所述的方法优选地用于制备化合物H-30、H-31。
包括阳离子脂质化合物的药物组合物
作为一种应用，以上化合物可以用于制备医药用途的组合物，组合物包括：载体，所载的药物试剂，药物辅助剂；其中：
载体包括：一种或多种可电离的脂质化合物，助脂质，结构脂质或聚合物缀合脂质。作为一种实施例，载体为脂质纳米粒(LNP)，脂质纳米粒的平均尺寸为30-200nm，纳米粒制剂的多分散指数≤0.5。需要说明的是：一种或多种本发明阳离子脂质化合物制备成的任何纳米粒都在本专利范围内，均受本发明的启示；比如：除了脂质纳米粒还可能是一种或多种阳离子脂质化合物与高分子形成的杂化纳米粒，比如：PLGA-PEG，PLA-PEG，PCL等这里不再穷举。
助脂质包括：磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂(SM)、甾醇及其衍生物、神经酰胺、带电脂质中的一种或几种的组合；磷脂酰胆碱作为一种优选包括：DSPC，DPPC，DMPC，DOPC，POPC；磷脂酰乙醇胺作为一种优选为DOPE；甾醇作为一种 优选为胆固醇；带电脂质作为一种实施例为DOTAP、DOTMA、18PA；这里并非穷举，只要是采用本发明结构的阳离子脂质化合物的组合物均在本发明的保护范围内，均受本发明启示。这里并非穷举，助脂质的选择不受限制，只要是采用本发明结构的阳离子脂质化合物均在本发明的保护范围内，均受本发明启示。
结构脂质包括：胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、番茄碱、熊果酸、α-生育酚或皮质类固醇中的一种或几种。这里并非穷举，结构脂质的选择不受限制，只要是采用本发明结构的阳离子脂质化合物均在本发明的保护范围内，均受本发明启示。
聚合物缀合脂质为聚乙二醇化脂质；作为一种实施例，聚乙二醇化脂质包括：PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油或PEG修饰的二烷基甘油中的一种或多种。这里并非穷举，聚合物缀合脂质的选择不受限制，只要是采用本发明结构的阳离子脂质化合物均在本发明的保护范围内，均受本发明启示。
所载的药物试剂包括：核酸分子，小分子化合物，多肽或蛋白质中的一种或多种。这里并非穷举，只要是采用本发明结构的阳离子脂质化合物，无论选用何种药物试剂均可以应用于本发明，均在本发明的保护范围内，均受本发明启示。
药物辅助剂包括：稀释剂，稳定剂，防腐剂或冻干保护剂中的一种或多种。这里并非穷举，只要是采用本发明结构的阳离子脂质化合物，无论选用何种药物辅助剂复配均不脱离本发明的设计范围。
通过以下实施例1-7的制备方法制备阳离子脂质化合物。
实施例1：
化合物c的合成：将2-己基癸酸(化合物b，9.22g，36mmol)溶于100mL二氯甲烷(DCM)中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，9.2g，36mmol)，4-二甲氨基吡啶(DMAP，1.46g，12mmol)以及N，N-二异丙基乙胺(DIPEA，7.74g，60mmol)，搅拌10min后，加入5-己烯-1-醇(化合物a，3g，30mmol)，室温搅拌过夜。TLC监测反应完全后，旋蒸仪减压蒸馏除去二氯甲烷。加入200mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝3∶1(体积比))，得8.2g无色液体，收率81％。
化合物d的合成：将化合物c(8g，24mmol)溶于100mL DCM中，冰浴条件下加入间氯过氧苯甲酸(m-CPBA，7.2g，36mmol，质量分数85％)，搅拌15min后，撤去冰浴，搅拌过夜。TLC监测反应完全后，加入过量的饱和亚硫酸氢钠溶液(10mL)以 消耗未反应完的间氯过氧苯甲酸，旋蒸仪减压蒸馏除去二氯甲烷。加入200mL乙酸乙酯，200mL饱和碳酸氢钠溶液洗涤3次，200mL饱和氯化钠溶液洗涤1次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶1(体积比))，得6g无色液体，收率72％。
化合物H-15的合成：将二甘醇胺(化合物e，0.6g，6mmol)溶于无水甲醇中，加入化合物d(6g，0.17mmol)，室温搅拌10min后，加热回流反应，反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。柱分离纯化(硅胶柱，洗脱液为DCM∶MeOH＝200∶1(体积比))，得3.9g无色液体，收率85％。MS m/z(ESI)：814.72[M+H]+
采用实施例1的方法，制备得到如下各个化合物：H-1，H-3，H-4，H-5，H-6，H-7，H-8，H-9，H-10，H-11，H-12，H-13，H-14，H-15，H-16，H-18，H-19，H-20。


实施例2：
化合物c的合成：将辛二酸(化合物b，1.52g，8.71mmol)溶于50mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，3.34g，17.4mmol)，4-二甲氨基吡啶(DMAP，0.3g，2.4mmol)以及N，N-二异丙基乙胺(DIPEA，1.5g，11.6mmol)，搅拌10min后，加入6-十一烷醇(化合物a，1g，5.8mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝20∶1(体积比))，得1.2g无色液体，收率63％。
化合物H-21的合成：将化合物c(0.88g，2.68mmol)溶于20mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，0.64g，3.35mmol)，4-二甲氨基吡啶(DMAP，0.065g，0.54mmol)以及N，N-二异丙基乙胺(DIPEA，0.35g，2.68mmol)，搅拌10min后，加入三乙醇胺(0.2g，1.34mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入50mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶1(体积比))，得0.4g无色液体，收率39％。MS m/z(ESI)：770.71[M+H]+
采用实施例2的方法，制备得到化合物H-21，H-23：
实施例3
化合物b的合成：将十四烷酸(化合物a，1.57g，6.48mmol)溶于70mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，1.86g，9.72mmol)，4-二甲氨基吡啶(DMAP，0.32g，2.6mmol)以及N，N-二异丙基乙胺(DIPEA，1.67g，12.3mmol)，搅拌10min后，加入三乙醇胺(2.9g，19.44mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入120mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶5(体积比))，得1.8g无色液体，收率74％。
化合物c的合成：参考实施例2中化合物c的合成方法。
化合物H-22的合成：将化合物c(0.88g，2.68mmol)溶于30mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，0.51g，2.68mmol)，4-二甲氨基吡啶(DMAP，0.13g，1.07mmol)以及N，N-二异丙基乙胺(DIPEA，0.52g，4.02mmol)，搅拌10min后，加入化合物b(1g，2.68mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入70mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶1(体积比))，得1.0g无色液体，收率55％。MS m/z(ESI)：684.57[M+H]+
采用实施例3的方法，制备得到化合物H-22，H-24。
实施例4
化合物a的合成：将油胺(2g，7.48mmol)溶于50mL THF中，加入三乙胺(1.13g，11.21mmol)，冰浴条件下滴加CS2(0.74g，9.72mmol)，室温搅拌反应12h。之后，4-二甲氨基吡啶(DMAP，0.27g，2.24mmol)，冰浴条件下加入二碳酸二叔丁酯((Boc)2O，2.12g，9.72mmol)室温搅拌反应3h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗3次，无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝100∶1(体积比))，得1.78g浅黄色液体，收率77％。
化合物b的合成：将化合物a(0.5g，1.62mmol)溶于10mL DMF中，加化合物d(0.24g，1.62mmol)，室温搅拌反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去大部分溶剂。加入70mL乙酸乙酯，等体积饱和氯化钠溶液洗3次，无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶1(体积比))，得0.5g无色液体，收率68％。
化合物c的合成：参考实施例2中化合物c的合成方法。
化合物H-25的合成：将化合物c(0.36g，1.09mmol)溶于20mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，0.32g，1.64mmol)，4-二甲氨基吡啶(DMAP，0.05g，0.44mmol)以及N，N-二异丙基乙胺(DIPEA，0.28g，2.81mmol)，搅拌10min后，加入化合物b(0.5g，1.09mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入50mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝5∶1(体积比))，得0.5g无色液体，收率60％。MS m/z(ESI)：768.62[M+H]+。
采用实施例4的方法，制备得到化合物H-25，H-26，H-27。

实施例5
化合物b的合成：将油胺(1g，3.74mmol)溶于50mL DCM，加入三乙胺(1.13g，11.21mmol)，冰浴条件下滴加三光气(0.44g，1.5mmol)，室温搅拌反应3h。TLC监测油胺反应完全后，旋蒸仪减压蒸馏除去溶剂。加入30mL DMF，然后加入化合物a(1.11g，7.48mmol)，室温搅拌反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去大部分溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗3次，无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶1(体积比))，得1.0g浅黄色液体，收率61％。
化合物c的合成：参考实施例2中化合物c的合成方法。
化合物H-28的合成：将化合物c(0.5g，1.52mmol)溶于20mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，0.44g，2.28mmol)，4-二甲氨基吡啶(DMAP，0.07g，0.61mmol)以及N，N-二异丙基乙胺(DIPEA，0.39g，3.04mmol)，搅拌10min后，加入化合物b(0.67g，1.52mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝5∶1(体积比))，得0.4g无色液体，收率35％。MS m/z(ESI)：752.64[M+H]+。
采用实施例5的方法，制备得到化合物H-28，H-29。
实施例6
化合物b的合成：将亚油醇(1.25g，4.68mmol)溶于50mL DCM中，加入三乙胺(0.62g，6.09mmol)，冰浴搅拌10min后，滴加6-溴己酰氯(化合物a，1g，4.68mmol)，逐渐恢复室温，室温搅拌反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗3次，等体积饱和氯化钠溶液洗3次，无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝200∶1(体积比))，得1.8g无色液体，收率87％。
化合物d的合成：参考化合物H-15合成过程中的化合物d的合成方法。
化合物e的合成：将b(1.5g，3.38mmol)溶于50mL无水乙醇中，加入三乙胺(0.44g，4.4mmol)，加化合物c(1.07g，10.25mmol)，50℃加热搅拌反应24h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗3次，无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为DCM∶MeOH＝20∶1(体积比))，得1.1g无色液体，收率70％。
化合物H-17的合成：将化合物e(0.4g，0.85mmol)溶于无10mL水甲醇中，加入化合物d(0.3g，0.85mmol)，搅拌10min后，加热回流反应，反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝5∶1(体积比))，得0.4g无色液体，收率57％。MS m/z(ESI)：822.72[M+H]+
采用实施例6的方法，制备得到化合物H-17。
实施例7
化合物c的合成：将化合物b(1.6g，5.64mmol)溶于50mL二氯甲烷中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，1.3g，6.77mmol)，4-二甲氨基吡啶(DMAP，0.28g，2.26mmol)以及N，N-二异丙基乙胺(DIPEA，1.09g，8.46mmol)，搅拌10min后，加入己二醇(化合物a，1g，8.46mmol)室温搅拌12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝50∶1(体积比))，得1.5g无色液体，收率69％。
将化合物d(1.0g，2.6mmol)溶于50mL DCM中，加入NaHCO3(1.75g，20.8mmol)，搅拌5min后，加入化合物戴斯-马丁氧化剂(Dess-Martin Periodinane，1.75g，4.16mmol)室温搅拌3h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入石油醚，等体积饱和碳酸氢钠溶液洗3次，等体积饱和食盐水洗1次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝10∶1(体积比))，得0.5g无色液体，收率50％。
化合物H-2的合成：将化合物d(0.3g，0.78mmol)溶于10mL DCM中，加入化合物e(0.08g，0.78mmol)，搅拌10min后，加三乙酰氧基硼氢化钠(0.22g，1.02mmol)室温搅拌过夜。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。加入50mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相使用无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝5∶1(体积比))，得0.3g无色液体，收率46％。MS m/z(ESI)：838.79[M+H]+
采用实施例7的方法，制备得到化合物H-2。
实施例8：
化合物b的合成：将化合物a(5g，29mmol)溶于100mL DCM中，冰浴条件下加入间氯过氧苯甲酸(m-CPBA，8.9g，44mmol，质量分数85％)，搅拌15min后，撤去冰浴，搅拌过夜。TLC监测反应完全后，加入过量的饱和亚硫酸氢钠溶液(10mL)以消耗未反应完的间氯过氧苯甲酸，旋蒸仪减压蒸馏出去二氯己烷。加入200mL乙酸乙酯，200mL饱和碳酸氢钠溶液洗涤3次，200mL饱和氯化钠溶液洗涤1次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏出去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝1∶2(体积比))，得4.5g无色液体，收率82％。
化合物d的合成：将化合物b(3.0g，16mmol)溶于50mL二氯甲烷(DCM)中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC，4.01g，21mmol)，4-二甲氨基吡啶(DMAP，0.79g，6mmol)以及N，N-二异丙基乙胺(DIPEA，3.12g，24mmol)，搅拌10min后，加入己-5-烯-2-醇(化合物c，1.94g，19mmol)，室温搅拌过夜。TLC监测反应完全后，旋蒸仪减压蒸馏除去二氯甲烷。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE∶EA＝20∶1(体积比))，得3.5g无色液体，收率81％。
化合物H-30的合成：将二甘醇胺(化合物e，0.65g，6mmol)溶于无水甲醇中，加入化合物d(5g，0.19mmol)，室温搅拌10min后，加热回流反应，反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。柱分离纯化(硅胶柱，洗脱液为DCM∶MeOH＝100∶1(体积比))，得3.0g无色液体，收率75％。MS m/z(ESI)：642.47[M+H]+。
采用实施例8的方法，制备得到化合物H-30、H-31；

需要说明的是：以上各个化合物并非穷举，只要是采用本发明的合成思路，头部带羟基，整体结构头部小，尾部大的阳离子脂质化合物均落在本发明的保护范围内。头部小，尾部大的具体意思为：以N原子为中心，带羟基的头部的分子结构空间占位比以烷烃链为结尾的尾部的分子结构空间占位小。
实验实施例1：脂质纳米粒的制备和检测：
H-1到H-29的化合物制备mRNA-LNP用于以下实验：
将阳离子脂质，DSPC或DOPE(艾维拓(上海)医药科技有限公司)，胆固醇(艾维拓(上海)医药科技有限公司)，以及PEG-脂质以设计的处方配比(Lipid(阳离子脂质化合物)/DOPC/Cholesterol(胆固醇)/DMG-PEG(缀合脂质)为40/10/50/1.7(摩尔比))，溶于乙醇中(Lipid的浓度20mg/mL)，并充分混匀。脂质纳米粒(LNP)与mRNA的质量比为10∶1到30∶1。使用柠檬酸盐或醋酸钠缓冲液(pH＝3或5)将mRNA稀释至0.2mg/mL。将上述阳离子脂质乙醇溶液与mRNA溶液以体积比为1∶5到1∶1的比例充分混匀。所获纳米粒通过超滤和透析的手段纯化。过滤除菌。使用Malvern Zetasizer Nano ZS，以173反相散射检测模式通过动态光散射表征mRNA-LNP(包载mRNA的脂质纳米粒)的粒径和粒径分布指数(particle dispersion index，PDI)，粒径分布指数体现了粒子粒径均一程度，是粒径表征的一个重要指标。mRNA的包封率均使用Ribogreen RNA定量测定试剂盒(Thermo Fisher)测定，结果见表1。
表1



实验实施例2 采用实验1制得的样品进行脂质纳米粒动物试验：
雄性ICR小鼠(6-8week，上海杰思捷实验动物有限公司)饲养在22±2℃以及相对湿度为45-75％的实验条件下，光照/黑暗周期为12h。使用编码荧光素酶的mRNA(luciferase mRNA)作为报道基因。荧光素酶催化荧光素产生生物荧光，通过检测单位时间内生物荧光强度，反映LNP的转染效率。以荧光素酶mRNA(购自ApexBio Technology)为例，将上述获得的mRNA-LNP样品1-29，将现有的商业可得的化合物MC3：作为对比样品，用MC3公知最优PEG-脂质处方配比(Lipid(阳离子脂质)/DSPC/Cholesterol(胆固醇)/DMG-PEG(缀合脂质)为50/10/38.5/1.5(摩尔比))制得的脂质纳米粒作为对比样品进行阳性对照，以150μg/kg mRNA的剂量，通过肌肉注射给药，每组样品一只小鼠，两条腿。取特定的时间点，于小鼠腹腔注射荧光素(20μg/mL)，5分钟后，将小鼠置于小动物活体成像仪测定荧光强度，最后的结果以平均荧光强度表示，小鼠腹腔注射给药后荧光强度实验结果如表2所示。
表2



结果显示，相较于现有的商业可得的阳离子脂质化合物MC3，本发明的阳离子脂质化合物均取得了显著更好的生物相容性以及显著更高的体内mRNA转染效率。
实验实施例3：采用实验实施例1制得的样品进行脂质纳米粒结构稳定性实验：
透射电镜样品的制备及表征(以样品1为例)。在铜网上滴上10μL制备好的样品15，放置10min后吸干样品并晾干。醋酸双氧铀染色5min，滤纸吸干染液后干燥过夜，利用透射电子显微镜(TEM)观察其形貌。
[根据细则91更正 26.06.2023]
如图1所示，本发明的脂质纳米粒都能形成稳定的纳米结构，尺寸分布较窄(PDI小)，尺寸随不同的脂质纳米粒的结构有所变化，在30-200nm范围内。
实验实施例4：采用实验实施例1制得的样品和实验实施例2的对比样品进行生物相容性验证实验：
使用CCK-8(cell counting kit-8)试剂盒测定细胞活力。将处于指数增长期的Hep3B细胞(100μL，细胞密度为2×104个/ml)悬浮液加入96孔板中，于细胞培养箱中孵育24h，然后从每个孔中除去细胞培养液，并添加100μL含mRNA 20μg/mL的LNP的新制细胞培养液，和细胞共孵育4h。随后，除去细胞上清液，加入新鲜细胞培养液，继续孵育20h。然后，除去上清液，加入含CCK-8工作溶液(10μL/mL)的新鲜的细胞培养液100μL，孵育2h，空白孔的设置：加含CCK-8工作溶液的细胞培养液。使用多功能微孔板检测仪检测每孔于450nm处的吸光度(检测过程中孔板中不能出现气泡)，将未经LNP处理的细胞作为对照组，将其细胞活力设为100％。
细胞活力(％)＝[A1-A0]/[A2-A0]×100。
A1为加药组吸光度，A0为空白组吸光度，A2为对照组吸光度。实验结果如表3所不。
表3

实验结果表明在限定的LNP浓度内，大多数的细胞活力不小于95％，未见明显细胞毒性。
实验实施例5：脂质纳米粒低温储存效果实验：
以样品1为例，将按照配方制作的脂质纳米粒置于4℃条件下低温保存，取不同时间点(0天、6天、10天、15天、30天、45天)，使用Malvern Zetasizer Nano ZS表征mRNA-LNP(包载mRNA的脂质纳米粒)的粒径(Size)和PDI，mRNA的包封率均使用Ribogreen RNA定量测定试剂盒(Thermo Fisher)测定。测定结果见表4所示。
表4
由表4可知：本发明的脂质分子形成的LNP(脂质纳米粒)可在低温储存较长时间，方便产品的运输和保存。
综上所述，本发明在结构上，在阳离子脂质化合物的头部引入羟基，增加脂质化合物头部的亲水性，以及纳米粒与细胞膜的融合，同时整体结构类似于锥形，头部(含氮原子的部分)小，尾部(疏水部分的长链烷烃)大，通过结构改进的配合；使得阳离子脂质化合物制备得到的脂质纳米粒均取得了更好的生物相容性以及更高的体内mRNA转染效率；化合物在疏水尾部引入可降解的酯键，可在人体内酯分解酶的作用下迅速降解，相对于MC3的长链烷烃，酯键的引入可以改变脂质分子在体内的代谢行为，进而提高mRNA-LNP的生物安全性；在合成上，MC3需要五步反应制得，且反应过程中涉及到危险性较高的格氏试剂；本发明相比较MC3的合成路线简单易行，原料 廉价易得，有利于其工业化生产。本发明化合物制备得到的LNP(脂质纳米粒)可在低温储存较长时间，方便产品的运输和保存，所以本发明设计的新阳离子脂质化合物具有良好的应用前景。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。

Claims (18)

  1. 一种阳离子脂质化合物，其特征在于，为如下结构的化合物：
    所述R1为
    所述R2为
    所述R3和R4各自独立地为-OH，且M0、M1各自独立地为：
    或R3和R4各自独立地为H，且M0、M1各自独立地为：且M0、M1中至少一个选自
    所述R5为：
    所述R6为：
    所述R7为：
    所述R8为：
    所述R9为：
    所述R10为：
    所述R11为：
    M2、M3为：
    或M0、M1、M2、M3各自独立地为
  2. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，所述M0、M1、M2、M3至少有两个为酯键。
  3. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：

  4. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：
  5. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：
  6. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：
  7. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：
  8. 根据权利要求1所述的一种阳离子脂质化合物，其特征在于，为如下的化合物：
  9. 根据权利要求1-8任意一项所述的一种阳离子脂质化合物的应用，其特征在于，应用于包含阳离子脂质化合物的组合物、其立体异构体、其互变异构体或其在药学上可接受的盐。
  10. 根据权利要求9所述的一种阳离子脂质化合物的应用，其特征在于，所述包含阳离子脂质化合物的组合物包括：载体，所载的药物试剂，药物辅助剂。
  11. 根据权利要求10所述的一种阳离子脂质化合物的应用，其特征在于，所述载体为脂质纳米粒LNP，所述脂质纳米粒的平均尺寸为30-200nm，所述脂质纳米粒的制剂的多分散指数≤0.5。
  12. 根据权利要求11所述的一种阳离子脂质化合物的应用，其特征在于，所述载体包括：一种或多种可电离的脂质化合物。
  13. 根据权利要求12所述的一种阳离子脂质化合物的应用，其特征在于，所述载体还包括：助脂质；所述阳离子脂质化合物与助脂质的摩尔比为0.5∶1-10∶1；所述助脂质包括：磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、甾醇及其衍生物、神经酰胺、带电脂质中的一种或几种的组合。
  14. 权利要求12或13所述的一种阳离子脂质化合物的应用包含阳离子脂质化合物的组合物，其特征在于，所述载体还包括：结构脂质或聚合物缀合脂质。
  15. 根据权利要求14所述的一种阳离子脂质化合物的应用，其特征在于，所述载体还包括：结构脂质；所述阳离子脂质化合物与结构脂质的摩尔比为0.5∶1-5∶1。
  16. 根据权利要求14所述的一种阳离子脂质化合物的应用，其特征在于，所述载体还包括：聚合物缀合脂质；所述阳离子脂质化合物与聚合物缀合脂质的摩尔比为20∶1-250∶1；所述聚合物缀合脂质为聚乙二醇化脂质。
  17. 根据权利要求10所述的一种阳离子脂质化合物的应用，其特征在于，所述所载的药物试剂包括：核酸分子，小分子化合物，多肽或蛋白质中的一种或多种。
  18. 根据权利要求10所述的一种阳离子脂质化合物的应用，其特征在于，所述药物辅助剂包括：稀释剂，稳定剂，防腐剂或冻干保护剂中的一种或多种。
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