CN117466768B - Cationic lipid compound, preparation method and application thereof and mRNA delivery system - Google Patents
Cationic lipid compound, preparation method and application thereof and mRNA delivery system Download PDFInfo
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- CN117466768B CN117466768B CN202311819315.4A CN202311819315A CN117466768B CN 117466768 B CN117466768 B CN 117466768B CN 202311819315 A CN202311819315 A CN 202311819315A CN 117466768 B CN117466768 B CN 117466768B
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- -1 Cationic lipid compound Chemical class 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 108020004999 messenger RNA Proteins 0.000 title claims abstract 4
- 238000006243 chemical reaction Methods 0.000 claims description 67
- 239000000203 mixture Substances 0.000 claims description 52
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- 150000001875 compounds Chemical class 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- 229940125782 compound 2 Drugs 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 10
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 9
- 229940126214 compound 3 Drugs 0.000 claims description 8
- 229940125898 compound 5 Drugs 0.000 claims description 8
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 229940125904 compound 1 Drugs 0.000 claims description 7
- BKJFDZSBZWHRNH-UHFFFAOYSA-N 8-bromooctanoic acid Chemical compound OC(=O)CCCCCCCBr BKJFDZSBZWHRNH-UHFFFAOYSA-N 0.000 claims description 6
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- ZAJNGDIORYACQU-UHFFFAOYSA-N decan-2-one Chemical compound CCCCCCCCC(C)=O ZAJNGDIORYACQU-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- WTWWTKPAEZQYPW-UHFFFAOYSA-N heptadecan-9-ol Chemical compound CCCCCCCCC(O)CCCCCCCC WTWWTKPAEZQYPW-UHFFFAOYSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 5
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 3
- WUGQZFFCHPXWKQ-UHFFFAOYSA-N Propanolamine Chemical compound NCCCO WUGQZFFCHPXWKQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000012280 lithium aluminium hydride Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 235000009518 sodium iodide Nutrition 0.000 claims description 3
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims description 2
- 239000011541 reaction mixture Substances 0.000 claims description 2
- WHRNULOCNSKMGB-UHFFFAOYSA-N tetrahydrofuran thf Chemical compound C1CCOC1.C1CCOC1 WHRNULOCNSKMGB-UHFFFAOYSA-N 0.000 claims description 2
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- 108090000623 proteins and genes Proteins 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N dichloromethane Natural products ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
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- 238000001727 in vivo Methods 0.000 description 9
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
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- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 3
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- 238000011725 BALB/c mouse Methods 0.000 description 2
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- 108020004459 Small interfering RNA Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
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- 239000002253 acid Substances 0.000 description 2
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- 238000009987 spinning Methods 0.000 description 2
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 description 1
- LRFJOIPOPUJUMI-KWXKLSQISA-N 2-[2,2-bis[(9z,12z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-n,n-dimethylethanamine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCC1(CCCCCCCC\C=C/C\C=C/CCCCC)OCC(CCN(C)C)O1 LRFJOIPOPUJUMI-KWXKLSQISA-N 0.000 description 1
- WDBQJSCPCGTAFG-QHCPKHFHSA-N 4,4-difluoro-N-[(1S)-3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-pyridin-3-ylpropyl]cyclohexane-1-carboxamide Chemical compound FC1(CCC(CC1)C(=O)N[C@@H](CCN1CCC(CC1)N1C(=NN=C1C)C(C)C)C=1C=NC=CC=1)F WDBQJSCPCGTAFG-QHCPKHFHSA-N 0.000 description 1
- BWGRDBSNKQABCB-UHFFFAOYSA-N 4,4-difluoro-N-[3-[3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-thiophen-2-ylpropyl]cyclohexane-1-carboxamide Chemical compound CC(C)C1=NN=C(C)N1C1CC2CCC(C1)N2CCC(NC(=O)C1CCC(F)(F)CC1)C1=CC=CS1 BWGRDBSNKQABCB-UHFFFAOYSA-N 0.000 description 1
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- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
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- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
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- NRLNQCOGCKAESA-KWXKLSQISA-N [(6z,9z,28z,31z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCC(OC(=O)CCCN(C)C)CCCCCCCC\C=C/C\C=C/CCCCC NRLNQCOGCKAESA-KWXKLSQISA-N 0.000 description 1
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Classifications
-
- 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
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
- C07C209/30—Preparation of compounds containing amino groups bound to a carbon skeleton by reduction of nitrogen-to-oxygen or nitrogen-to-nitrogen bonds
- C07C209/40—Preparation of compounds containing amino groups bound to a carbon skeleton by reduction of nitrogen-to-oxygen or nitrogen-to-nitrogen bonds by reduction of hydroxylamino or oxyimino groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/04—Formation of amino groups in compounds containing carboxyl groups
- C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
- C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
- C07C237/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C249/00—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C249/04—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton of oximes
- C07C249/08—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton of oximes by reaction of hydroxylamines with carbonyl compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/58—Preparation of carboxylic acid halides
- C07C51/60—Preparation of carboxylic acid halides by conversion of carboxylic acids or their anhydrides or esters, lactones, salts into halides with the same carboxylic acid part
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to a cationic lipid compound, a preparation method and application thereof, and an mRNA delivery system, and relates to the technical field of medical biology, wherein the cationic lipid compound has the following structure:the invention is mainly used for developing the cationic lipid compound with high delivery efficiency, good spleen targeting and good biological safety.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to a cationic lipid compound, a preparation method and application thereof, and an mRNA delivery system.
Background
mRNA (messenger ribonucleic acid) is a single-stranded ribonucleic acid which is polymerized by taking one strand of a DNA (deoxyribonucleic acid) double strand as a template (template) and taking 4 kinds of ribonucleoside triphosphates (A, U, G, C) as substrates under the catalysis of RNA polymerase (RNA polymerase) through phosphodiester bonds. mRNA is capable of carrying and transmitting genetic information stored in the nuclear DNA, and plays a key role in the conversion of genetic information into functional proteins. In the cytoplasm, the immature mRNA is modified into mature mRNA through capping, tailing, intron cutting and other steps, and the mature mRNA can accurately guide the synthesis process of protein in the cytoplasm. Relatively, mRNA is easy to transfect due to its much smaller molecular weight than DNA, and there is no oncogenic risk of integration into the host DNA to initiate insertion mutations. Therefore, mRNA is taken as a preventive and therapeutic drug, and has great advantages and potential in the prevention and treatment of various diseases.
mRNA nucleic acid medicine is one kind of preventing and treating strategy for preventing and treating diseases with functional protein or subunit activating host immune system to produce corresponding humoral immunity or cell immunity reaction and for treating diseases with expressed protein or subunit possessing function of treating diseases or regulating the expression of other genes. Compared with other methods, the method has the advantages that the method can directly activate the organism on the molecular level to generate functional antibodies or cellular immune responses aiming at specific pathogens, or can purposefully repair pathogenic genes or correct the expression of abnormal genes, thereby achieving the effects of preventing and treating various diseases. The mRNA nucleic acid medicine can achieve the effect that the traditional medicine cannot replace, for example, the monoclonal antibody medicine can only act on the cell surface, but the mRNA nucleic acid medicine can not only act on the extracellular protein of the cell membrane, but also act on the intracellular protein, even act in the nucleus, and has accurate targeting. Of the 7000 diseases faced by humans, about 1/3 of the diseases are clinically almost drug-free due to problems (deletion, reduction or overexpression) of functional genes, such as Hemophilia (Hemophilia), duchenne Muscular Dystrophy (DMD), cystic fibrosis (cytosticibrosis), and severe immunodeficiency Syndrome (SCID), etc., and mRNA nucleic acid drugs are very advantageous for this monogenic disease. In the age background of popularization of personalized medicine and accurate medicine. In theory, diseases caused by gene differences or abnormal gene expression of patients can be accurately and effectively treated by using mRNA nucleic acid medicaments.
mRNA nucleic acid drugs have great advantages and potential in controlling gene expression and preventing and treating malignant diseases. However, there are difficulties in the development, preparation and subsequent systemic administration of such drugs. Firstly, mRNA exists in a single-chain form, so that the mRNA is extremely unstable in vitro and under physiological conditions, and is easily degraded by RNA nuclease (RNAase) in air or blood, and is also easily cleared by mononuclear macrophages in tissues and organs such as liver, spleen and the like; secondly, due to the electronegativity of mRNA, it is difficult to penetrate the cell membrane into the cell interior; again, mRNA is difficult to escape from endosomes and into the cytoplasm to function. Furthermore, uracil ribonucleoside (U) in mRNA is prone to immunogenicity, which in some cases may increase the potential toxic side effects of mRNA drugs. Finally, the susceptibility to off-target effects is also an important challenge in the preparation and administration of mRNA nucleic acid-based drugs. Therefore, the development of an intracellular delivery system of an mRNA nucleic acid drug is a key point of being capable of large-scale clinical application.
To more safely and effectively exert the therapeutic capacity of RNA, scientists use Lipid Nanoparticles (LNPs) to encapsulate and deliver RNA to specific sites in the body. This RNA delivery strategy has shown great utility in delivering double-stranded small interfering RNAs (siRNAs, 21 to 23 nucleotides in length). For example, lipid C12-200 has been widely used in the manufacture of siRNA-LNP formulations for various therapeutic applications in vivo to inhibit protein expression. The advent of synthetic ionizable lipid materials (synthetic ionizable lipids) and lipid materials (lipid-materials) has not only greatly reduced the in vivo toxicity of LNPs, but also made it possible to deliver large molecular weight RNAs (e.g., mRNA) in vivo. These amine-containing ionizable lipids or lipid molecules are positively charged and, by means of electrostatic attraction, can efficiently bind to negatively charged mRNA and self-assemble to form LNP. LNP can improve blood circulation time of RNA and increase uptake rate of cells; in the cell, RNA is released into cytoplasm through endosome escape way to express specific protein and to play a certain therapeutic role.
The LNP has the following 4 main raw material components: 1) Ionizable lipids or lipid molecules such as DLin-KC2-DMA, DLin-MC3-DMA, L319. This is the core component that enables in vivo delivery of mRNA. 2) Phospholipid molecules, a phospholipid, provide structure for the LNP bilayer and may also assist endosomal escape; 3) Cholesterol, enhancing LNP stability, promoting membrane fusion; 4) Polyethylene glycols such as DMG-PEG2000, which control and reduce the particle size of the LNP and "protect" the LNP from non-specific endocytosis of immune cells.
In vivo, changes in the raw materials and components of LNP can have profound effects on the physicochemical stability and efficacy of mRNA action of mRNA-LNP formulations. Existing LNP component protocols do not fully exploit the efficacy of mRNA-LNP formulations, requiring continuous structural adjustments to the ionizable lipid molecules and design optimization for specific RNAs. Therefore, there is a need to develop a cationic lipid compound having high delivery efficiency, good biosafety and targeting properties.
Disclosure of Invention
In view of the above, the present invention provides a cationic lipid compound, a preparation method and application thereof, and an mRNA delivery system, and the main purpose of the present invention is to develop a cationic lipid compound with high delivery efficiency, good spleen targeting and good biosafety.
In order to achieve the above purpose, the present invention mainly provides the following technical solutions:
in one aspect, embodiments of the present invention provide a cationic lipid compound, wherein the cationic lipid compound has the structure shown below:
。
preferably, the cationic lipid compound is prepared according to the following equation:
；
wherein, under the condition of room temperature, adding the compound 6, the compound 4, acetonitrile and stirring, then adding the potassium carbonate and the sodium iodide, heating to 55-65 ℃ for reaction; and (3) extracting and purifying after the reaction is finished to obtain the cationic lipid compound.
Preferably, the preparation equation of the compound 6 is as follows:
；
wherein, under the condition of room temperature, adding the compound 5, ethanol and 3-aminopropanol into a reaction vessel, and heating to 55-65 ℃ for reaction; after the reaction, ethanol was removed, followed by extraction and purification to give compound 6.
Preferably, the preparation equation of the compound 5 is as follows:
；
adding 8-bromooctanoic acid, 9-heptadecanol and dichloromethane DCM into a reaction vessel, stirring, sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI and 4-dimethylaminopyridine DMAP, and reacting at room temperature; after the reaction, extraction and purification treatment are carried out to obtain the compound 5.
Preferably, the preparation equation of the compound 4 is as follows:
；
adding a compound 2, dichloromethane DCM and triethylamine into a reaction container at room temperature, cooling the temperature of the reaction system to below 0 ℃ by using an ice bath, dropwise adding a compound 3 into the reaction system, and reacting at room temperature; after the reaction, extraction, drying and concentration are performed to obtain a compound 4.
Preferably, the preparation equation of the compound 3 is as follows:
；
adding 8-bromooctanoic acid and dichloromethane DCM into a reaction container, dropwise adding DMF, cooling the reaction system to below 0 ℃ by ice bath, dropwise adding thionyl chloride into the reaction system, and reacting at room temperature; after the reaction was completed, dichloromethane DCM was removed to give compound 3.
Preferably, the preparation equation of the compound 2 is as follows:
；
wherein, compound 1 and tetrahydrofuran THF are added into a reaction vessel under the condition of room temperature, the reaction system is cooled to below 0 ℃ by ice bath, then lithium aluminum hydride is added into the reaction vessel to react at room temperature; after the completion of the reaction, the reaction mixture was worked up to give compound 2.
Preferably, the preparation equation of the compound 1 is as follows:
；
wherein, 2-decanone, ethanol and sodium bicarbonate are added into a reaction vessel under the condition of room temperature and stirred; then adding hydroxylamine hydrochloride into the mixture, heating the mixture to 45-55 ℃ for reaction; after the reaction, ethanol was removed, and extraction, drying and concentration were performed to obtain compound 1.
In yet another aspect, the use of the above-described cationic lipid compounds of the invention in the preparation of an mRNA delivery system.
In yet another aspect, embodiments of the present invention provide an mRNA delivery system, wherein the mRNA delivery system comprises the cationic lipid compound described above. Wherein the mRNA delivery system is an mRNA-LNP delivery system.
Compared with the prior art, the cationic lipid compound, the preparation method and application thereof and the mRNA delivery system have at least the following beneficial effects:
in one aspect, embodiments of the present invention develop a novel cationic lipid compound having the structure shown below:
；
the cationic lipid compound is used for preparing an mRNA delivery system and has the characteristics of high delivery efficiency, good spleen targeting and good biological safety.
On the other hand, the embodiment of the invention provides an mRNA delivery system (mRNA-LNP delivery system), and the mRNA delivery system comprises the cationic lipid compound, so that the LNP of the mRNA delivery system has the characteristics of high delivery efficiency, good spleen targeting and good biosafety.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a graph of in vivo transfection efficiency for XH162 lipid nanoparticle formulations of the present invention and controls; wherein, (a) graph shows distribution quantification results of luciferase in spleen after 6 hours of intravenous lipid nanoparticle preparation in tail of mice; (b) The figures are comparison graphs of spleen targeting 6 hours after intravenous lipid nanoparticle formulation at the tail of mice.
FIG. 2 is a graph of transfection efficiency in vivo from XH162 lipid nanoparticle formulations of the present invention and controls; wherein, (a) graph shows the quantitative distribution result of luciferase at the injection site after 6 hours of intramuscular injection of lipid nanoparticle preparation into mice; (b) The figure shows the quantitative results of luciferase distribution in spleen after 6 hours of intramuscular injection of lipid nanoparticle formulation into mice.
Fig. 3 is a graphical representation of the change in body weight of mice after injection of XH162 lipid nanoparticle formulation and control.
Detailed Description
In order to further describe the technical means and effects adopted for achieving the preset aim of the invention, the following detailed description refers to the specific implementation, structure, characteristics and effects according to the application of the invention with reference to the accompanying drawings and preferred embodiments. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.
The present invention aims to develop a cationic lipid compound (specifically, named XH 162) which has high delivery efficiency, good spleen targeting and good biosafety.
The cationic lipid compound of the present invention has the following structure:
XH162
the synthesis method of the cationic lipid compound comprises the following steps:
1. synthesis of Compound 1
；
2-decanone (10.75 g,68.8mmol,1.0 eq), ethanol (100 mL), sodium bicarbonate (7.5 g,89.4mmol,1.3 eq) and hydroxylamine hydrochloride (5.74 g,82.5mmol,1.2 eq) were added to a 250mL three-necked flask at room temperature, stirred for 10min, warmed to 50℃and reacted for 6h; TLC monitoring, reaction completion, cooling the reaction bottle to room temperature, removing ethanol by rotation, adding water, extracting for three times by EA, combining organic phases, washing once by water, washing once by saturated saline, drying by anhydrous sodium sulfate, concentrating to obtain a crude product of the compound 1 (11.5 g, 97.6%); the crude product was used directly in the next step.
2. Synthesis of Compound 2
；
To a 100mL single flask at room temperature, crude compound 1 (11.5 g,67.1mmol,1.0 eq) and THF (40 mL) were added, the flask was cooled to below 0deg.C with an ice bath, lithium aluminum hydride (4.8 g,126.3mmol,1.88 eq) was added in portions, and the mixture was reacted at room temperature for 6h; TLC monitoring, reaction is complete, stopping reaction, extracting with 4.8mL of water, adding 15% sodium hydroxide (4.8 mL), precipitating solid, adding 14.4mL of water, and filtering; washing the filter cake twice with THF, and filtering; the filtrates are collected together, THF is removed by low temperature, the pH is adjusted to 2, methyl tertiary butyl ether is used for extracting impurities twice, the pH is adjusted to 13-14, DCM is used for extracting twice, and the organic phase is concentrated to obtain a crude product of the compound 2.
The crude compound 2 was dissolved in 100mL of THF/H 2 To O (1:7), sodium carbonate (9.5 g,89.6mmol,1.33 eq) was added, and the flask was cooled to below 0deg.C with an ice bath and added (BOC) 2 O (16.5 g,75.6mmol,1.13 eq). The reaction was carried out at room temperature for 1 hour. TLC monitoring, reaction completion, stopping reaction, turning off THF, EA extraction three times, combining the organic phases, washing once again, drying over anhydrous sodium sulfate, concentrating, column chromatography purification (PE/PE: EA=10:1, i.e. increasing polarity from PE to PE: EA with a volume ratio of 10:1) to give compound 2-1 (10 g). Compound 2-1 was dissolved in 100mL of ethanol, 6N HCl (130 mL) was added, the temperature was raised to 50℃and the reaction was carried out for 2h. TLC monitored, the reaction was complete and stopped. The ethanol was removed by spinning, the methyl tert-butyl ether was extracted twice, the pH was adjusted to 13-14, the DCM was extracted twice, the organic phase was dried over anhydrous sodium sulfate and concentrated to give Compound 2 (4.7 g, 44.5%).
3. Synthesis of Compound 3
；
To a 250mL three-necked flask at room temperature, 8-bromooctanoic acid (10 g,44.8mmol,1.0 eq), DCM (100 mL) and 2 drops of DMF were added dropwise, the flask was cooled to below 0deg.C in an ice bath, sulfoxide chloride (10.8g,90.8 mmol,2.0eq) was added dropwise, and the reaction was carried out at room temperature for 2h; TLC monitoring, reaction complete, stop reaction, spin-off DCM to give crude compound 3, which is used directly in the next step.
4. Synthesis of Compound 4
；
In a 250mL three-necked flask at room temperature, compound 2 (2 g,12.7mmol,1.0 eq), DCM (50 mL) and triethylamine (2 g,19.8mmol,1.56 eq) were added, the flask was cooled to below 0deg.C in an ice bath, crude compound 3 (3.38 g,14.0mmol,1.1 eq) was added dropwise and reacted at room temperature for 0.5h; TLC monitoring, reaction completion, adding water, extracting aqueous phase twice with DCM, combining organic phases, washing once with weak acid, drying, concentrating to give crude compound 4, which is directly used for the subsequent synthesis.
5. Synthesis of Compound 5
；
In a 1L single-necked flask under ice-bath condition, 8-bromooctanoic acid (26.1 g,117mmol,1.5 eq), 9-heptadecanol (20 g,78mmol,1.0 eq), DCM (200 mL) and stirring for 10min, EDCI (19.4 g,101.2mmol,1.3 eq) and DMAP (1.9 g,45.6mmol,0.2 eq) were added sequentially, and the reaction was carried out overnight at room temperature; TLC monitored formation of new spots (incomplete reaction), stopped the reaction, the reaction was quenched with water, extracted twice with DCM, the organic phase was washed once with weak acid water, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified directly by column chromatography (PE: ea=20:1) to give compound 5 (10 g, 27.8%).
6. Synthesis of Compound 6
；
In a 250mL three-necked flask at room temperature, compound 5 (10 g,21.7mmol,1.0 eq), ethanol (100 mL), 3-aminopropanol (16.3 g,216.7mmol,10 eq) were added, and the mixture was allowed to react overnight at 60 ℃; TLC monitored complete reaction, stopped reaction, cooled to room temperature, ethanol was removed by spinning, water was added, EA extracted twice, the organic phase dried over anhydrous sodium sulfate, concentrated, and the crude product purified directly by column chromatography (DCM: meoh=10:1) to give compound 6 (5 g, 50.7%).
7. Synthesis of Compound XH162
；
In a 100mL three-necked flask at room temperature, compound 6 (4.4 g,9.65mmol,1.0 eq), compound 4 (4.54 g,12.53mmol,1.3 eq), acetonitrile (50 mL) and potassium carbonate (2 g,14.47mmol,1.5 eq) were added and stirred for 10min, and sodium iodide (0.5 g,3.33mmol,0.3 eq) was added and the temperature was raised to 60℃for 6h; TLC monitoring, reaction completion, stopping reaction, spin-off acetonitrile, extraction three times with water, EA, collection of the organic phase, drying, concentration and purification of the crude product by column chromatography (DCM: meOH=20:1-10:1+1% Et 3 N) to give the product XH162 (2 g, 28%).
Here, the nuclear magnetic data of XH162 is as follows:
1 H NMR (400 MHz, CDCl 3 ) δ 5.40 (d,J= 8.3 Hz, 1H), 4.91 – 4.79 (m, 1H), 4.01 – 3.90 (m, 1H), 3.84 – 3.76 (m, 2H), 2.72 (d,J= 5.3 Hz, 2H), 2.56 – 2.44 (m, 4H), 2.27 (t,J= 7.5 Hz, 2H), 2.12 (t,J= 7.6 Hz, 2H), 1.77 – 1.70 (m, 2H), 1.62 (d,J= 6.7 Hz, 4H), 1.50 (d,J= 5.2 Hz, 8H), 1.31 (dd,J= 33.0, 22.1 Hz, 51H), 1.10 (d,J= 6.6 Hz, 3H), 0.94 – 0.82 (m, 9H).
the technical effect of the use of the cationic lipid compound XH162 of the present invention in an mRNA-LNP delivery system is described below by way of example.
Example 1
And (3) preparing and detecting the lipid nanoparticle preparation.
1. Preparation steps
The cationic lipid compound XH162 of the present invention is mixed with distearoyl phosphatidylcholine DSPC, cholesterol and DMG-PEG2000 at a ratio of 50:10:38.5:1.5 in ethanol to prepare an ethanol lipid solution.
N1-methyl-pseudouridine modified luciferase mRNA was diluted with 50mM citrate buffer (pH=4.0) to give an aqueous mRNA solution.
By microfluidic device 1:3 volume ratio of ethanol lipid solution and mRNA aqueous solution to prepare lipid nanoparticles, 1X PBS dialysis was performed for 18 hours to remove ethanol and complete the citrate buffer exchange procedure. Finally, the lipid nanoparticle solution was subjected to sterile filtration (0.2 μm) and ultrafiltration concentration steps to obtain a lipid nanoparticle preparation encapsulating luciferase mRNA, named: XH162 lipid nanoparticle formulation, abbreviated XH162 LNP.
In addition, lipid nanoparticle formulations of compound SM102 (SM 102 is a conventional compound), compound XH106, and compound XH160 were prepared as controls, respectively named: SM102 lipid nanoparticle formulation (abbreviated as SM102 LNP), XH106 lipid nanoparticle formulation (abbreviated as XH106 LNP), XH160 lipid nanoparticle formulation (abbreviated as XH160 LNP).
SM102 was a cationic lipid used in the Moderna covd-19 vaccine, used as a control.
In addition, the structural formula of compound XH160 is as follows:
；
the structural formula of compound XH106 is as follows:
；
the preparation methods of the compound XH106 and the compound XH160 are basically the same as those of the cationic lipid compound XH162 of the present invention.
2. Detection step
Lipid nanoparticle particle size, polydispersity index (PDI) and potential (Zeta) were determined using a Litesizer ™ (Anton Paar, austria). Wherein, the particle size and the potential were measured in 0.1% PBS. Lipid nanoparticle formulation encapsulation efficiency (EE%) was measured by the RiboGreen method. The test results are shown in table 1:
as can be seen from table 1: the cationic lipid compound XH162 of the present invention can form lipid nanoparticles as cationic lipid.
Example 2
The lipid nanoparticle formulation prepared in example 1 was subjected to in vivo transfection experiments.
BALB/c mice were randomly divided into eight groups of 3. Each group was injected with SM102 lipid nanoparticle formulation or XH106 lipid nanoparticle formulation or XH160 lipid nanoparticle formulation or XH162 lipid nanoparticle formulation by single tail vein (0.3 mg/kg dose) or muscle (0.15 mg/kg dose). Specifically, a first set of single tail veins (0.3 mg/kg dose) were injected with the XH162 lipid nanoparticle formulation. A second set of single tail veins (0.3 mg/kg dose) were injected with XH106 lipid nanoparticle formulations. A third set of single tail veins (0.3 mg/kg dose) were injected with XH160 lipid nanoparticle formulations. A fourth set of single tail vein (0.3 mg/kg dose) was injected with SM102 lipid nanoparticle formulation. A fifth group of single muscles (0.15 mg/kg dose) was injected with the XH162 lipid nanoparticle formulation. A sixth group of single muscles (0.15 mg/kg dose) was injected with XH106 lipid nanoparticle formulation. A seventh group of single muscles (0.15 mg/kg dose) was injected with XH160 lipid nanoparticle formulation. An eighth group of single muscles (0.15 mg/kg dose) was injected with SM102 lipid nanoparticle formulation. After 6h of injection, the luciferase substrate was injected intraperitoneally, and after 10min, organs such as liver, spleen, lung, kidney and the like were dissected and taken, and fluorescence signals were observed and quantified using a small animal living body imaging system.
Results of tail vein injection referring to fig. 1, the expression of the XH162 lipid nanoparticle formulation of the present invention was significantly higher at the spleen site than that of the SM102 lipid nanoparticle formulation, the XH106 lipid nanoparticle formulation, the XH160 lipid nanoparticle formulation (refer to fig. 1 (a)). Meanwhile, the XH162 lipid nanoparticle formulation of the present invention has excellent spleen targeting (see (b) in fig. 1).
Intramuscular injection results are shown in figure 2. Referring to fig. 2 (a), the expression of the XH162 lipid nanoparticle formulation of the present invention at the injection site is equivalent to the SM102 lipid nanoparticle formulation, significantly higher than the XH106 lipid nanoparticle formulation, XH160 lipid nanoparticle formulation. Referring to fig. 2 (b), the expression of the XH162 lipid nanoparticle formulation of the present invention was significantly higher at the spleen site than that of the SM102 lipid nanoparticle formulation, the XH106 lipid nanoparticle formulation, and the XH160 lipid nanoparticle formulation.
Example 3
Lipid nanoparticle formulations are biosafety.
BALB/c mice were randomly divided into three groups of 5 mice each. The first group was intramuscular injected with PBS, the second group was intramuscular injected with SM102 lipid nanoparticle formulation (dose 5 ug/dose), the third group was intramuscular injected with XH162 lipid nanoparticle formulation (dose 5 ug/dose), and the dosing was repeated on days 7 and 14 after the first dosing. The mice were scored for body weight changes before, 3 days after the first dose, 7 days, 10 days, and 14 days, respectively (see fig. 3 for recorded results).
As can be seen from fig. 3: similar to the control SM102 lipid nanoparticle formulation, the multiple intramuscular injections of the XH162 lipid nanoparticle formulation, all the weight changes of the mice were within a safe range, thus demonstrating: the XH162 lipid nanoparticle formulation has good biosafety.
In summary, the ionizable lipid compound (cationic lipid compound) developed by the present invention has the technical effects of high delivery efficiency, good spleen targeting and good biosafety in vivo mRNA delivery.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention in any way, but any simple modification, equivalent variation and modification made to the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. A cationic lipid compound, characterized in that the cationic lipid compound has the structure shown below:
。
2. the method for preparing a cationic lipid compound according to claim 1, wherein the cationic lipid compound is prepared by the following formula:
；
wherein, under the condition of room temperature, adding the compound 6, the compound 4, acetonitrile and stirring, then adding the potassium carbonate and the sodium iodide, heating to 55-65 ℃ for reaction; and (3) extracting and purifying after the reaction is finished to obtain the cationic lipid compound.
3. The method for producing a cationic lipid compound according to claim 2, wherein the formula for producing the compound 6 is as follows:
；
wherein, under the condition of room temperature, adding the compound 5, ethanol and 3-aminopropanol into a reaction vessel, and heating to 55-65 ℃ for reaction; after the reaction, ethanol was removed, followed by extraction and purification to give compound 6.
4. A method for preparing a cationic lipid compound according to claim 3, wherein the preparation equation of the compound 5 is as follows:
；
adding 8-bromooctanoic acid, 9-heptadecanol and dichloromethane DCM into a reaction vessel, stirring, sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI and 4-dimethylaminopyridine DMAP, and reacting at room temperature; after the reaction, extraction and purification treatment are carried out to obtain the compound 5.
5. The method for producing a cationic lipid compound according to claim 2, wherein the formula for producing the compound 4 is as follows:
；
adding a compound 2, dichloromethane DCM and triethylamine into a reaction container at room temperature, cooling the temperature of the reaction system to below 0 ℃ by using an ice bath, dropwise adding a compound 3 into the reaction system, and reacting at room temperature; after the reaction, extraction, drying and concentration are performed to obtain a compound 4.
6. The method for producing a cationic lipid compound according to claim 5, wherein the formula for producing the compound 3 is as follows:
；
adding 8-bromooctanoic acid and dichloromethane DCM into a reaction container, dropwise adding DMF, cooling the reaction system to below 0 ℃ by ice bath, dropwise adding thionyl chloride into the reaction system, and reacting at room temperature; after the reaction was completed, dichloromethane DCM was removed to give compound 3.
7. The method for producing a cationic lipid compound according to claim 5, wherein the formula for producing the compound 2 is as follows:
；
wherein, compound 1 and tetrahydrofuran THF are added into a reaction vessel under the condition of room temperature, the reaction system is cooled to below 0 ℃ by ice bath, then lithium aluminum hydride is added into the reaction vessel to react at room temperature; after the completion of the reaction, the reaction mixture was worked up to give compound 2.
8. The method for producing a cationic lipid compound according to claim 7, wherein the formula for producing the compound 1 is as follows:
；
wherein, 2-decanone, ethanol and sodium bicarbonate are added into a reaction vessel under the condition of room temperature and stirred; then adding hydroxylamine hydrochloride into the mixture, heating the mixture to 45-55 ℃ for reaction; after the reaction, ethanol was removed, and extraction, drying and concentration were performed to obtain compound 1.
9. Use of the cationic lipid compound of claim 1 in the preparation of an mRNA delivery system.
10. An mRNA delivery system comprising the cationic lipid compound of claim 1.
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