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WO2024192906A1 - Lipid compound for delivering therapeutic agent, preparation method therefor, and use thereof - Google Patents

Lipid compound for delivering therapeutic agent, preparation method therefor, and use thereof Download PDF

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Publication number
WO2024192906A1
WO2024192906A1 PCT/CN2023/100823 CN2023100823W WO2024192906A1 WO 2024192906 A1 WO2024192906 A1 WO 2024192906A1 CN 2023100823 W CN2023100823 W CN 2023100823W WO 2024192906 A1 WO2024192906 A1 WO 2024192906A1
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WO
WIPO (PCT)
Prior art keywords
compound
alkyl
lipid
pharmaceutically acceptable
nucleic acid
Prior art date
Application number
PCT/CN2023/100823
Other languages
French (fr)
Chinese (zh)
Inventor
王子君
桂阳
Original Assignee
尧唐(上海)生物科技有限公司
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Publication date
Application filed by 尧唐(上海)生物科技有限公司 filed Critical 尧唐(上海)生物科技有限公司
Priority to PCT/CN2024/082316 priority Critical patent/WO2024193525A1/en
Publication of WO2024192906A1 publication Critical patent/WO2024192906A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C333/02—Monothiocarbamic acids; Derivatives thereof
    • C07C333/04—Monothiocarbamic acids; Derivatives thereof having nitrogen atoms of thiocarbamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • 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
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
    • 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
    • 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
    • C07C237/12—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 atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C329/00—Thiocarbonic acids; Halides, esters or anhydrides thereof
    • C07C329/02—Monothiocarbonic acids; Derivatives thereof
    • C07C329/04—Esters of monothiocarbonic acids
    • C07C329/06—Esters of monothiocarbonic acids having sulfur atoms of thiocarbonic groups bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/04—1,3-Dioxanes; Hydrogenated 1,3-dioxanes
    • C07D319/06—1,3-Dioxanes; Hydrogenated 1,3-dioxanes not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • C07F7/1872—Preparation; Treatments not provided for in C07F7/20
    • C07F7/1892—Preparation; Treatments not provided for in C07F7/20 by reactions not provided for in C07F7/1876 - C07F7/1888

Definitions

  • the present invention relates to a lipid compound for delivering a therapeutic agent (such as a nucleic acid molecule), a lipid carrier containing the same, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation, as well as their preparation methods and related applications, and belongs to the technical field of biomedicine and gene therapy.
  • a therapeutic agent such as a nucleic acid molecule
  • a lipid carrier containing the same a nucleic acid lipid nanoparticle composition
  • a pharmaceutical preparation as well as their preparation methods and related applications, and belongs to the technical field of biomedicine and gene therapy.
  • nucleic acid drugs can be used to prevent and treat cancer, bacterial and viral infections, and diseases with genetic causes. Since nucleic acid drugs are easily degraded and difficult to enter cells, they usually need to be encapsulated with a carrier and delivered to the target cells. Therefore, the development of safe and efficient delivery carriers has become a prerequisite for the clinical application of gene therapy.
  • Lipid nanoparticles are currently a research hotspot in the field of non-viral gene carriers.
  • the FDA approved Moderna and BioNtech & Pfizer's new coronavirus vaccines for COVID-19 both of which use LNP technology to deliver mRNA drugs, thereby achieving prevention of the SARS-CoV-2 virus.
  • LNPs are usually composed of four lipid compounds, namely ionizable lipids, neutral lipids, steroids, and polymer-bound lipids, among which the choice of ionizable lipids has the greatest impact on LNPs.
  • nucleic acid therapeutic drugs still face some challenges in their effectiveness, mainly including delivery efficiency, low cell permeability, and high sensitivity to degradation of certain nucleic acid molecules including RNA. Therefore, there is still a need to develop new lipid compounds to promote the in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or preventive purposes.
  • One object of the present invention is to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g., salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, etc.), which can be used together with other lipid compounds (e.g., neutral lipids, charged lipids, steroids) to prepare lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, specifically including mRNA) to improve the delivery efficiency of nucleic acid drugs in vivo, and lipid compounds with specific structures can be selected as lipid carriers according to the organs where the nucleic acid drugs need to be enriched.
  • lipid compounds or a pharmaceutically acceptable form thereof e.g., salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, etc.
  • other lipid compounds e.g., neutral lipids, charged lipids, steroids
  • therapeutic agents e
  • Another object of the present invention is to provide a lipid carrier comprising the above compound or a pharmaceutically acceptable form thereof.
  • Another object of the present invention is to provide a nucleic acid lipid nanoparticle composition comprising the above compound or a pharmaceutically acceptable form thereof or the above lipid carrier.
  • Another object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.
  • Another object of the present invention is to provide a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.
  • Another object of the present invention is to provide a method for preventing, improving or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.
  • the present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, etc.
  • G1 is a C1-6 alkylene group
  • R 1 and R 2 are each independently C 1-6 alkyl or C 1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy is optionally further substituted by halogen, hydroxy, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxy-substituted C 1-6 alkyl;
  • R 1 , R 2 and the connected N together form a 3-8 membered heterocyclic group, or any one of R 1 and R 2 is directly connected to any carbon atom in G 1 to form a 3-8 membered heterocyclic group;
  • 0 to 3 ring Cs in the heterocyclic group are optionally further substituted by N, O or S;
  • 0 to 3 Hs in the heterocyclic group are optionally further substituted by halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxyl-substituted C 1-6 alkyl;
  • L 1 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k optional An integer from 1 to 10; j is selected from 0 or 1;
  • R 3 , R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;
  • a and b are each independently selected from integers of 0-10;
  • L 2 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k are selected from integers of 1-10; j is selected from 0 or 1;
  • R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;
  • c and d are each independently selected from integers of 0-10;
  • L 3 and L 4 are each independently selected from Alkyne group, heteroatom O, N, S or not present; l, n are integers selected from 1-10; m is selected from 0 or 1;
  • R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy, halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;
  • e, f, and g are each independently selected from integers of 0-10.
  • G1 is methylene, ethylene, propylene, butylene or pentylene.
  • R 1 and R 2 are each independently methyl, ethyl, propyl or butyl, wherein 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl; or, R 1 , R 2 and the connected N together form a 5-membered or 6-membered heterocyclic group, wherein 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and wherein 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl.
  • one of R 1 and R 2 is directly connected to any carbon atom in G 1 .
  • the other of R 1 and R 2 is methyl, ethyl, propyl or butyl, and 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl.
  • 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl.
  • G 1 , R 1 , and R 2 correspond to the groups shown in any one of Compounds 1 to 31, respectively.
  • L1 is alkynylene, O or absent; h, i, k are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; j is selected from 0 or 1;
  • At least two of R 3 , R 4 and R 5 are not hydrogen, and R 6 and R 7 are hydrogen;
  • a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • At least one of a and b or both of them are not zero.
  • L 1 , R 3 , R 4 , R 5 , R 6 , R 7 , a, and b correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.
  • L2 is alkynylene, O or absent; h is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 8 and R 9 are each independently hydrogen, and at least two of R 10 , R 11 , and R 12 are not hydrogen;
  • c and d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, at least one of c and d or both of them are not 0.
  • L 2 , R 8 , R 9 , R 10 , R 11 , R 12 , c, and d correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.
  • L 3 and L 4 are each independently selected from Alkyne, O or does not exist;
  • R 13 and R 14 are each hydrogen; at least two of R 15 , R 16 , R 17 , R 18 and R 19 are not hydrogen; preferably, at least two of R 16 , R 18 and R 19 are not hydrogen;
  • e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L3 is Alkyne, O or absent
  • R 13 and R 14 are each hydrogen; at least two of R 15 , R 16 and R 17 are not hydrogen;
  • e and f are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • L 3 , R 13 , R 14 , R 15 , R 16 , R 17 , e, and f correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.
  • the corresponding multiple R 3 s , multiple R 4 s , multiple R 6 s , multiple R 7 s , multiple R 8 s , multiple R 9 s , multiple R 10 s , multiple R 11 s , multiple R 13 s , multiple R 14 s, multiple R 15 s , and multiple R 17 s may be independently the same or different.
  • the compound is selected from one or more of Compounds 1 to 31 shown in Table 1.
  • the present invention provides an intermediate having a structure shown in Formula Ic or Formula Id:
  • the intermediates described in the present invention can be used to prepare the compounds described in the present invention or their pharmaceutically acceptable forms.
  • the present invention provides a method for preparing the compound described in ⁇ the first aspect> or a pharmaceutically acceptable form thereof, the method comprising:
  • Thiocarbamates are generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate
  • the method of preparing the compound or its pharmaceutically acceptable form it is a key intermediate that can be used to obtain secondary amines containing a long chain or its precursors through alkylation or reductive amination. Then, thiocarbamate is generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate. Finally, the ketal protecting group is removed, and the second and third fatty long chains are introduced through condensation reaction to obtain a compound of formula I or a salt thereof.
  • the present invention also provides the use of the compound or its pharmaceutically acceptable form in the preparation of liposome nanocarriers.
  • the present invention provides a lipid carrier, which comprises the compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.
  • lipid carriers have high encapsulation efficiency for nucleic acid drugs, greatly improving the delivery efficiency of nucleic acid drugs in vivo.
  • the lipid carrier comprises a first lipid compound and a second lipid compound
  • the first lipid compound comprises the compound according to the ⁇ First Aspect> or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and optionally other ionizable lipids
  • the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.
  • the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.
  • the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and a combination of other cationic or ionizable lipids.
  • the other cationic or ionizable lipid compounds include 1,2-dilinoleyloxy-N,N-dimethylaminopropane DLinDMA, 1,2-dioleyloxy-N,N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane DLin-KC2-DMA, 1,2-dioleoyl-3-trimethylammonium-propane DOT AP, 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecan-2-ol C12-200, 3 ⁇ [N-N’N’-dimethyl
  • the anionic lipid includes one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol DOPG, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine DOPS and dimyristoylphosphatidylglycerol.
  • the neutral lipid includes at least one of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine DSPC, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine DPPC, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine DOPC, dipalmitoylphosphatidylglycerol DPPG, oleoylphosphatidylcholine POPC, 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine DMPE, distearoylphosphatidylethanolamine DSPE and 1-
  • the steroid includes one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, coproposterol, and corticosteroids.
  • the polymer-bound lipids include 1,2-dimyristoyl-sn-glyceromethoxy-polyethylene glycol PEG-DMG, dimyristoylglycerol-polyethylene glycol PEG-c-DMG, polyethylene glycol-dimyristoylglycerol PEG-C14, PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, PEG modified PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O-(2
  • the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55 :15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of any one of the above compounds or its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (1-10):(0-10); illustratively, the molar ratio can be 1:1,
  • the molar ratio of the first lipid compound, anionic lipids, neutral lipids, steroids and polymer-bound lipids is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of any of the above compounds or their pharmaceutically acceptable forms such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4):(0-5).
  • the present invention provides a nucleic acid lipid nanoparticle composition, which includes the compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug or the lipid carrier according to the fifth aspect, and a nucleic acid drug.
  • a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug or the lipid carrier according to the fifth aspect, and a nucleic acid drug.
  • the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.
  • the antisense nucleic acid is an antisense oligonucleic acid.
  • the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.
  • the DNA comprises a plasmid.
  • the mRNA includes a sequence encoding an RNA-guided DNA binder, more specifically, an mRNA encoding a nuclease or a base editor.
  • the nuclease includes one or more of Cas9 (e.g., GeoCas9, CjCas9, SpCas9), CasX, CasY, Casz, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein (AGO), and homologs thereof (e.g., dCas9 and nCas9).
  • Cas9 e.g., GeoCas9, CjCas9, SpCas9
  • CasX e.g., CasX, CasY, Casz, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein (AGO)
  • AGO Argonaute protein
  • the nucleic acid drug may further include a guide RNA, specifically, the guide RNA includes a gRNA nucleic acid.
  • the nucleic acid drug includes mRNA and gRNA encoding a nuclease or a base editor.
  • the mRNA or gRNA is modified.
  • the modification is selected from pseudouracil ( ⁇ ), N1-methyl pseudouracil (N1M ⁇ ), 1-ethyl pseudouracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof. Composed of groups.
  • the mass ratio of the nucleic acid drug to any of the compounds or their pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs is 1:(3-40).
  • the mass ratio of the nucleic acid drug to the lipid carrier is 1:(3-40).
  • the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.
  • the ratio (w/w) of the mRNA to the gRNA of the nuclease or base editor is about 10: 1 to about 1: 10. In some embodiments, the ratio (w/w) is about 3: 1 to about 1: 3. In some embodiments, the ratio (w/w) is about 3: 2.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to the fifth aspect, or a nucleic acid lipid nanoparticle composition according to the sixth aspect, and a pharmaceutically acceptable excipient, carrier or diluent.
  • the present invention provides a pharmaceutical preparation comprising any one of the above-mentioned compounds or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier or diluent.
  • the particle size of the pharmaceutical preparation is 30 to 500 nm.
  • the particle size can be 30 nm, 50 nm, 100 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.
  • the encapsulation rate of the nucleic acid drug in the pharmaceutical preparation is greater than 50%.
  • the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.
  • the present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.
  • the present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.
  • the present invention also provides a method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.
  • the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal.
  • the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered, for example, via an enteral or parenteral administration route.
  • a dosage of about 0.001 mg/kg to about 10 mg/kg of the nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered to the subject.
  • the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is used to treat or prevent a disease or disorder in a subject in need thereof.
  • the disease or condition is selected from a metabolic disease, a genetic disease, cancer, a cardiovascular disease, and an infectious disease.
  • the metabolic disease includes familial hypercholesterolemia (FH)
  • the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE)
  • the infectious disease includes hepatitis B (HEPATITIS B).
  • the subject is a mammal or a human.
  • the present invention provides a series of novel structural formula I compounds, which can be used as ionizable lipids to prepare lipid carriers together with other lipid compounds, and their particle size is controllable, uniformly distributed, and has a very high encapsulation rate.
  • the compound of the present invention is an amino liposome, and the liposome compound contains both hydrophobic non-polar long chains and hydrophilic amino groups, and this amphoteric characteristic can be used to form lipid particles.
  • the group connected to the nitrogen atom in the thiocarbamate is preferably a fatty long chain
  • the C number of the fatty long chain is preferably greater than or equal to 3, further preferably greater than or equal to 4, further preferably greater than or equal to 5, and further preferably greater than or equal to 6.
  • the lipid compound synthesis method of the present invention is simple, has a high yield, can be quickly synthesized, and has low cost.
  • the compound of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs, enriching the types of ionizable lipid compounds, and is particularly important for the development and application of nucleic acid preventive and therapeutic agents.
  • FIG1 is a schematic diagram of the delivery strategy for detecting the PCSK9 gene editing efficiency in mouse liver cells in the present invention.
  • Figure 2 shows the PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells.
  • the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.
  • the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.
  • the word “may” includes both performing a certain process and not performing a certain process.
  • references to “some specific/preferred embodiments”, “other specific/preferred embodiments”, “embodiments”, etc. mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments.
  • the elements may be combined in various embodiments in any suitable manner.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention that is substantially non-toxic to an organism.
  • Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compound of the present invention with a pharmaceutically acceptable inorganic/organic acid or inorganic/organic base, and such salts are also referred to as acid addition salts or base addition salts.
  • Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.
  • common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc.
  • common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.
  • stereoisomer refers to a stable isomer that has a vertical asymmetric plane due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane polarized light. Since there are asymmetric centers and other chemical structures that may cause stereoisomerism in the compounds of the present invention, the present invention also includes these stereoisomers and mixtures thereof. Since the compounds of the present invention and their salts include asymmetric carbon atoms, they can exist in the form of a single stereoisomer, a racemate, an enantiomer, and a mixture of diastereomers.
  • these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or single stereoisomer-enriched (purity ⁇ 98%, purity ⁇ 95%, ⁇ 93%, ⁇ 90%, ⁇ 88%, ⁇ 85% or ⁇ 80%) mixtures.
  • a single stereoisomer of a compound is prepared synthetically from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column.
  • Starting compounds of particular stereochemistry are either commercially available or prepared as described herein and resolved by methods well known in the art.
  • tautomer refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved.
  • proton tautomers include (but are not limited to) interconversions through proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imino alcohol isomerization, etc. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.
  • solvate refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanolates, acetoneates, etc.
  • chelate refers to a complex having a ring structure, which is obtained by chelation of two or more ligands with the same metal ion to form a chelate ring.
  • non-covalent complex is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule.
  • complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).
  • prodrug refers to a derivative compound that can directly or indirectly provide a compound of the present invention after being applied to a patient.
  • Particularly preferred derivative compounds or prodrugs are compounds that can increase the bioavailability of the compound of the present invention when administered to a patient (e.g., more easily absorbed into the blood), or compounds that promote the delivery of the parent compound to the site of action (e.g., the lymphatic system).
  • all prodrug forms of the compounds of the present invention are within the scope of the present invention, and various prodrug forms are well known in the art.
  • substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.
  • substituent Y can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl.
  • alkyl refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no unsaturation, and connected to other fragments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl and tert-butyl, etc.
  • C 1-30 alkyl refers to a saturated monovalent straight or branched hydrocarbon group containing 1 to 30 carbon atoms.
  • alkylene refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene, etc.
  • C 1-30 alkylene refers to a saturated divalent straight or branched alkyl group containing from 1 to 30 carbon atoms.
  • cycloalkyl refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms.
  • the cycloalkyl group may include a paracyclic, bridged or spirocyclic ring system.
  • C 3-6 cycloalkyl used in the present invention refers to a cycloalkyl group having 3 to 6 carbon atoms.
  • the cycloalkyl group may be a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo[2.2.1]heptyl group, etc.
  • cycloalkylene refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including, but not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene, etc.
  • C3-30 cycloalkylene refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.
  • branched alkyl refers to an alkane radical that is attached to a parent molecule and forms at least two branched structures.
  • alkenyl refers to a monovalent straight or branched chain alkane radical consisting solely of carbon and hydrogen atoms and containing at least one Double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups.
  • alkenylene refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc.
  • alkynyl refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups.
  • C2-30 alkynyl refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.
  • alkynylene refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by two single bonds, including (but not limited to) ethynylene, etc.
  • C2-30 alkynylene refers to a divalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.
  • cycloalkenyl refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms. Cycloalkenyl may include cyclic, bridged or spirocyclic systems. For example, cyclopropenyl and cyclobutenyl, etc.
  • cycloalkenylene refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenylene and cyclobutenylene, etc.
  • C3-30 cycloalkenylene refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.
  • alkoxy refers to an -O-alkyl group.
  • branched alkenyl refers to an alkene radical attached to a parent molecule and forming at least two branched structures.
  • the term "3-8 membered heterocyclyl" used in the present invention refers to a heterocyclyl having 3 to 8 ring atoms.
  • the heterocyclyl group can be oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, or trithianyl.
  • aryl refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated ⁇ electron system.
  • C 6-10 aryl used in the present invention refers to an aryl group having 6 to 10 carbon atoms.
  • the aryl group can be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.
  • heteroaryl refers to a monocyclic or fused polycyclic aromatic group having a conjugated ⁇ electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S. If the valence bond requirements are met, the heteroaryl group can be bonded to the molecule through any of the ring atoms. The rest are connected.
  • heteroaryl refers to a heteroaryl having 5 to 10 ring atoms.
  • heteroaryl can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzo derivatives thereof, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • hydroxy refers to -OH.
  • cyano refers to -CN.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • subject includes humans and non-human animals.
  • Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits and pigs.
  • the subject is a human.
  • patient or subject are used interchangeably herein.
  • nuclease refers to an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in a nucleic acid molecule.
  • the nuclease is selected from a meganuclease, a zinc finger nuclease (ZFNs), a TAL-effector DNA binding domain-nuclease fusion protein (TALEN), and an RNA-guided nuclease or a variant thereof in which the nuclease activity has been reduced or inhibited.
  • the RNA-guided nuclease is a naturally occurring CRISPR-Cas protein or an active variant or fragment thereof.
  • CRISPR-Cas systems are classified as class I or class II systems.
  • Class II systems contain single effector nucleases and include types II, V, and VI.
  • Each category is further subdivided into types (types I, II, III, IV, V, VI), some of which are further divided into subtypes (e.g., type II-A, type II-B, type II-C, type V-A, type V-B).
  • Type II CRISPR-Cas protein refers to a CRISPR-Cas effector protein that requires a trans-activating RNA (tracrRNA) and comprises two nuclease domains (RuvC and HNH), each of which is responsible for cutting a single strand of a double-stranded DNA molecule.
  • the CRISPR-Cas protein is a naturally occurring Type V CRISPR-Cas protein or an active variant or fragment thereof.
  • V-type CRISPR-Cas protein refers to a CRISPR-Cas effector protein that cuts dsDNA and comprises a single RuvC nuclease domain or a split RuvC nuclease domain and lacks an HNH domain.
  • the CRISPR-Cas protein is a naturally occurring VI-type CRISPR-Cas protein or an active variant or fragment thereof.
  • VI-type CRISPR-Cas protein As used herein, the term "VI-type CRISPR-Cas protein”, “VI-type CRISPR-Cas effector protein” or “Cas13” refers to a CRISPR-Cas effector protein that does not require tracrRNA and comprises two HEPN domains that cut RNA.
  • gRNA refers to a nucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and guide the sequence-specific binding of the associated nuclease to the target nucleotide sequence.
  • the corresponding guide RNA is one or more RNA molecules (usually one or two) that can bind to the Cas enzyme and guide the Cas enzyme to bind to a specific target nucleotide sequence, and in which In those cases where the Cas enzyme has nickase or nuclease activity, it also cleaves the target nucleotide sequence.
  • misappropriate amount means that the amount of the added solvent or the amount of the drug can be adjusted in a wide range and has little effect on the synthesis result, and no specific limitation is made.
  • Example 1 Synthesis of 4-[7-butyl-17-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-25-ethyl-8,14,20-trioxy-19,25-diaza-9,15-dioxa-21-thiaheptacosan-19-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester
  • reaction solution was quenched with 500 ml saturated ammonium chloride solution, extracted with ethyl acetate (500 mL x 2), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate.
  • the mixture was concentrated under reduced pressure and column chromatography was performed to obtain the compound nonane-2,5-diyn-1-ol (24.00 g, yield 64.5%).
  • MS: m/z[M+H] + 137.0.
  • nickel acetate tetrahydrate 14.02 g, 79.30 mmol, 0.9 eq
  • ethanol 240 mL
  • sodium borohydride (2.95 g, 70.49 mmol, 0.8 eq) was added at 0 degrees Celsius
  • ethylenediamine 21.17 g, 352.44 mmol, 4.0 eq
  • a solution of compound 1-12 (12.00 g, 88.11 mmol, 1.0 eq) in ethanol (60 mL) was slowly added dropwise, and hydrogen was replaced three times, and the mixture was reacted at 0 degrees Celsius for 4 hours.
  • compound 1-13 (8.00 g, 57.05 mmol, 1.0 eq), 4-( ⁇ [(2-methylpropan-2-yl)oxy]carbonyl ⁇ amino)butyric acid (17.39 g, 85.58 mmol, 1.5 equiv.), dichloromethane (180 mL), 4-dimethylaminopyridine (6.97 g, 57.05 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.87 g, 114.10 mmol, 2.0 eq) were added.
  • compound 1-8 (1.09 g, 3.63 mmol, 3.0 eq), N,N-diisopropylethylamine (0.78 g, 6.05 mmol, 5.0 eq), 4-dimethylaminopyridine (0.15 g, 1.21 mmol, 1.0 eq), dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.70 g, 3.63 mmol, 3.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 2-6 (0.44 g, 1.21 mmol, 5.0 eq) was added.
  • reaction solution was stirred at room temperature for 1 hour, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 30 mL of petroleum ether, combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-(3-hydroxypropyl)-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (110.0 mg, yield 46.4%).
  • reaction solution was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give 2-butyloctanoic acid-18-butyl-8-(hydroxymethyl)-5,11,17-trioxydeca-6,10,16-trioxa-tetracosane-1-yl ester (4.50 g, yield 35.6%).
  • compound 4-1 (2.00 g, 2.98 mmol, 1.0 eq) and triethylamine (0.90 g, 8.94 mmol, 3.0 eq) were added to 30 ml of dichloromethane, and methylsulfonic anhydride (0.78 g, 4.47 mmol, 1.5 eq) was slowly added at zero degrees Celsius, and the temperature was slowly restored to room temperature and reacted for 4 hours.
  • reaction solution was diluted with water, extracted with dichloromethane 3 times, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain methanesulfonic acid-12-butyl-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-5,11-dioxy-4,10-dioxahexadecan-1-yl ester (2.00 g, yield 89.6%).
  • compound 4-2 (1.00 g, 1.34 mmol, 1.0 eq), benzylamine (287.2 mg, 2.68 mmol, 2.0 eq), potassium carbonate (555.6 mg, 4.02 mmol, 3.0 eq), potassium iodide (111.2 mg, 0.68 mmol, 0.5 eq) were added to 20 ml of acetonitrile, nitrogen was protected, heated to 90 degrees Celsius, and reacted overnight.
  • compound 4-5 (182.0 mg, 0.22 mmol, 1.0 eq), 3-(diethylamino)propane-1-thiol (48.0 mg, 1.33 mmol, 1.5 eq), 4-dimethylaminopyridine (27.0 mg, 0.22 mmol, 1.0 eq), pyridine (35.0 mg, 0.44 mmol, 2.0 eq) were added to 10 ml of acetonitrile and stirred at room temperature for 4 hours.
  • reaction solution was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 12 grams of the title compound, as well as a monosubstituted intermediate and raw material.
  • the intermediate and raw material were placed in a sealed pot again and stirred at 110 degrees Celsius for 72 hours. The above operation was repeated and combined to obtain 4,4-bis[(5Z)-octadecyl-5-en-1-yloxy]butyronitrile (26.52 g, 41.8%).
  • MS: m/z[M+H] + 357.3.
  • compound 6-2 (2.00 g, 6.22 mmol, 1.0 eq), potassium hydroxide (1.05 g, 18.66 mmol, 3.0 eq), ethanol (6 mL), and water (6 mL) were added to a jar, heated to 110 degrees Celsius, and reacted overnight.
  • Example 7 Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(4- ⁇ [(2Z)-non-2-enyl]oxy ⁇ -4-oxy-butyl)-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester
  • Example 8 Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(4- ⁇ [(5Z)-oct-5-enyl]oxy ⁇ -4-oxy-butyl)-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester
  • Example 10 Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(3- ⁇ [(10Z,12Z)-1-oxyylidene octadecane-9,12-dienyl]oxy ⁇ propyl)-5,11-dioxyylidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester
  • 1 H NMR 400 MHz, CDCl 3 ) ⁇ 5.48-5.25 (m, 4H), 4.25-3.90 (m, 10H), 3.51-3.37 (m, 3H), 2.95-2.82 (m, 1H), 2.80-2.71 (m, 1H), 2.60-2.20 (m, 10H), 2.12-1.80 (m, 8H), 1.73-1.52 (m, 14H), 1.50-1.03 (m, 56H), 0.98-0.82 (m, 15H).
  • Example 11 Synthesis of 2-butyloctanoic acid-(24Z)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiooctan-1-yl)-8-( ⁇ [(10Z,12Z)-1-oxyylidene octadec-9,12-dienyl]oxy ⁇ methyl)-18- ⁇ [(5Z)-oct-5-enyl]oxy ⁇ -5,15-dioxyylidene-10-aza-6,14,19-trioxacosaccharide-24-en-1-yl ester
  • reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound (10Z, 12Z)-octadecanoic acid-9,12-dienoic acid-4-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanyl-7-yl)-10-ethyl-2-(hydroxymethyl)-5-oxygen-4,10-diaza-6-thiadodecane-1-yl ester (1.10 g, yield 39.1%).
  • MS: m/z[M+H] + 837.6.
  • reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 2-butyloctanoic acid-(24Z)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiooctan-1-yl)-8-( ⁇ [(10Z,12Z)-1-oxyylidene octadec-9,12-dienyl]oxy ⁇ methyl)-18- ⁇ [(5Z)-oct-5-enyl]oxy ⁇ -5,15-dioxyylidene-10-aza-6,14,19-trioxacositol-24-en-1-yl ester (131.1 mg, yield 36.9%).
  • Example 12 Synthesis of (10Z,12Z)-octadecane-9,12-dienoic acid-(18Z)-4-(6-ethyl-1-oxy-6-aza-2-thiooctan-1-yl)-12- ⁇ [(5Z)-oct-5-enyl]oxy ⁇ -2-[6-(octyloxy)-3-oxy-2,7-dioxapentadecan-1-yl]-9-oxy-4-aza-8,13-dioxa-hexadecane-18-en-1-yl ester
  • Example 12 was synthesized by using the method of Example 11 except that compound 1-8 in Example 11 was replaced by compound 12-1.
  • Compound 12-1 was synthesized by using the method of Example 6 except that compound 6-1 in Example 6 was replaced by octan-1-ol. Yield: 38.8%.
  • MS: m/z[M+H] + 1247.9.
  • Example 13 Synthesis of 2-butyloctanoic acid-16-ethyl-8-( ⁇ [(10Z,12Z)-1-oxyidene octadecane-9,12-dienyl]oxy ⁇ methyl)-10-(4- ⁇ [(2Z)-non-2-enyl]oxy ⁇ -4-oxyidene butyl)-5,11-dioxyidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester
  • Example 14 Synthesis of (10Z,12Z)-octadec-9,12-dienoic acid-10-ethyl-4-(4- ⁇ [(2Z)-non-2-enyl]oxy ⁇ -4-oxydebutyl)-2-[6-(octyloxy)-3-oxyde-2,7-dioxapentadecan-1-yl]-5-oxyde-4,10-diaza-6-thiadodecane-1-yl ester
  • Lipid nanoparticles were prepared by mixing ionizable lipid or the compound of the present invention/DSPC/cholesterol/PEG-lipid at a molar ratio of 50:10:38.5:1.5.
  • Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and compound 1 to compound 14 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol, and PEG-DMG according to the above molar ratios.
  • Luciferase mRNA (L-6107, TriLink BioTechnologies, Inc.) was dissolved in 100mM enzyme-free citric acid buffer at pH 4 (mRNA concentration was 0.2mg/mL).
  • the ethanol solutions of different lipid carriers were mixed with the mRNA buffer at a ratio of 1:3 (volume/volume) (where the mass ratio of total lipids to mRNA was 40:1), and the corresponding nucleic acid lipid nanoparticles were obtained by passing through a microfluidic nanodrug manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12ml/min.
  • the obtained nucleic acid lipid nanoparticles were immediately diluted into 1 ⁇ DPBS buffer at a volume of 40 times.
  • the diluted nucleic acid lipid nanoparticle solution was passed through an ultracentrifuge tube and concentrated to the target volume. After dilution, it was used for DLS particle size measurement and encapsulation efficiency detection.
  • the particle size and polydispersity index of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscattering detection mode.
  • the encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2.
  • the cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol.
  • the LDL receptor (LDLR) is a low-density lipoprotein that binds to cholesterol that circulates back to the liver, and then breaks down cholesterol into bile acids, which are excreted from the body through the intestines.
  • PCSK9 is a protease synthesized by the liver that can bind to the LDL receptor and promote the entry of the LDL receptor into liver cells, causing the LDL receptor to be degraded by lysosomes and the number of LDL receptors to decrease. Therefore, inhibiting the activity of the enzyme PSCK9 can increase the number of LDLRs, thereby enhancing the ability to absorb and decompose cholesterol.
  • Figure 1 shows the changes in the number of LDL receptors before and after editing of specific sites in the PCSK9 gene and the resulting changes in cholesterol metabolism.
  • the strategy for gene editing and delivery of PCSK9 in mouse liver cells is shown in Figure 1.
  • the main process is as follows: mRNA and sgRNA encoding ABE8e are delivered to mouse liver cells through intravenous injection using the prepared lipid nanoparticles. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and mutations from base A to G are achieved at specific sites. The editing efficiency is calculated by sequencing.
  • the single-base editor ABE8e achieves precise base replacement from A to G without the need for a donor template and without causing DSB. Based on this, the first exon of the PCSK9 gene was selected as the mutation site for screening.
  • the mRNA encoding the single-base editing tool ABE8e and sgRNA were co-delivered to the animal body through lipid nanoparticles.
  • the mRNA encoding the base editor ABE8e was translated into protein in the cytoplasm, and entered the nucleus after forming a complex with the sgRNA.
  • the base editor ABE8e targeted the splicing donor site of the first exon of the PCSK9 gene, deaminated adenine (A) on the first exon template chain into inosine (I), and I was read and replicated as G at the DNA level, ultimately achieving the replacement of A to G, thereby destroying the splicing donor site and causing the PCSK9 gene reading frame to terminate prematurely.
  • the sequences of the first exon and the first intron of the mouse PCSK9 gene were selected as the targeting region, and the target sequence PCSK9-sgRNA for single-base editing of the PCSK9 gene was determined.
  • PCSK9-sgRNA (synthesized by Nanjing GenScript Co., Ltd.), and the sequence of the PCSK9-sgRNA is:
  • PCSK9-sgRNA 5’-CCCATACCTTGGAGCAACGG-3’ (SEQ ID NO: 1);
  • sgRNA was designed and oligonucleotides were synthesized according to the target sequence.
  • the sgRNA sequence used is shown in SEQ ID NO: 1.
  • the CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and the AAAC sequence was added to the 5' end of the downstream sequence.
  • the upstream and downstream sequences were annealed by a preset program (95°C, 5min; 95°C-85°C at -2°C/s; 85°C-25°C at -0.1°C/s; maintained at 4°C), and the annealed products were ligated to the lenti U6-sgRNA/EF1a-mCherry vector (Addgene, Plasmid, #114199) linearized with BbsI (NEB, R3539S).
  • the linearization system of lenti U6-sgRNA/EF1a-mCherry vector is as follows: 3 ⁇ g of vector; 6 ⁇ L of buffer (NEB: R0539L); 2 ⁇ L of BbsI; ddH 2 O to 60 ⁇ L, and enzyme digestion at 37°C overnight.
  • the sgRNA annealing product and the linearized vector ligation system are as follows: T4 ligase buffer (NEB: M0202L) 1 ⁇ L, linearized vector 20 ng, annealed oligo fragment (10 ⁇ M) 5 ⁇ L, T4 ligase (NEB: M0202L) 0.5 ⁇ L, ddH 2 O to make up to 10 ⁇ L, Ligation was carried out at 16°C overnight.
  • the ligated vector was transformed into E. coli DH5a competent cells (Weidi Biotechnology, DL1001).
  • the specific process is as follows: DH5 ⁇ competent cells were taken out from -80°C and quickly inserted into ice. After 5 minutes, the bacterial block was melted, and the ligation product was added and gently mixed by hand at the bottom of the centrifuge tube, and then allowed to stand in ice for 25 minutes. Heat shock was performed in a 42°C water bath for 45 seconds, and then quickly returned to ice and allowed to stand for 2 minutes. 700 ⁇ l of sterile LB medium without antibiotics was added to the centrifuge tube, and after mixing, it was revived at 37°C and 200rpm for 60 minutes.
  • the base editor ABE8e used in this experiment is a highly efficient base editor ABE8e evolved by David R. Liu's team (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR.
  • Plasmid ABE8e (Plasmid#138489) was purchased from Addgene, and the mRNA of ABE8e was expressed and purified by the laboratory for future use.
  • Lipid nanoparticles (mass ratio of total lipids to mRNA is 40:1) containing the compound of the present invention (see Table 2) encapsulating mRNA and sgRNA encoding the base editor ABE8e (ABE8e mRNA:sgRNA ratio (w/w) is 3:2) were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) via tail vein injection at a dose of 0.2 mg/kg.
  • Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered to mice of the same age and gender in a similar manner as a positive control.
  • PBS buffer was injected into the tail vein of mice of the same age and gender in a similar manner as a negative control.
  • the deep sequencing steps are as follows.
  • the designed primers are shown in Table 3.
  • the PCR program was as follows: 94°C, 2 min; 98°C 10 s, 60°C 30 s, 68°C 20 s, 34 cycles; 68°C, 5 min.
  • the ionizable lipid compound used in the present invention can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of the present invention have a better particle size distribution, a high encapsulation rate, and a delivery effect that is significantly better than that of the comparative lipid nanoparticles, and can meet the needs of in vivo delivery.

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Abstract

A lipid compound for delivering a therapeutic agent, a preparation method therefor, and a use thereof. A lipid compound is a compound having a structural formula I or a pharmaceutically acceptable form thereof. The lipid compound may be used in combination with other lipid components such as a neutral lipid, cholesterol, and a polymer-bound lipid to form lipid nanoparticles for delivery of therapeutic agents (e.g., nucleic acid molecules) to achieve therapeutic or prophylactic purposes (e.g., vaccination), thereby enriching the types of ionizable lipid compounds.

Description

递送治疗剂的脂质化合物及其制备方法与应用Lipid compounds for delivering therapeutic agents and preparation methods and applications thereof 技术领域Technical Field

本发明是关于一种递送治疗剂(例如核酸分子)的脂质化合物、包含其的脂质载体、核酸脂质纳米粒组合物和药物制剂，以及它们的制备方法与相关应用，属于生物医药、基因治疗技术领域。The present invention relates to a lipid compound for delivering a therapeutic agent (such as a nucleic acid molecule), a lipid carrier containing the same, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation, as well as their preparation methods and related applications, and belongs to the technical field of biomedicine and gene therapy.

背景技术Background Art

基因治疗技术是现代生物医药领域研究的热点，利用核酸药物可防治癌症，防治细菌和病毒感染，以及治疗具有遗传病因的疾病等。由于核酸药物具有易降解、难以进入细胞等特点，通常需要借助载体将其封装起来递送至靶细胞，因此，开发安全高效的递送载体就成为基因治疗在临床应用的前提。Gene therapy technology is a hot topic in modern biomedicine research. Nucleic acid drugs can be used to prevent and treat cancer, bacterial and viral infections, and diseases with genetic causes. Since nucleic acid drugs are easily degraded and difficult to enter cells, they usually need to be encapsulated with a carrier and delivered to the target cells. Therefore, the development of safe and efficient delivery carriers has become a prerequisite for the clinical application of gene therapy.

脂质纳米颗粒(Lipid nanoparticle，LNP)目前是非病毒基因载体领域的研究热点。2018年，FDA批准了LNP递送patisiran(onpattro)来治疗遗传性转甲状腺素蛋白淀粉样变性，自此运用LNP技术递送核酸药物的研究呈现迸发式地增长。特别是2020年底，FDA分别批准了Moderna和BioNtech&辉瑞针对COVID-19的新冠病毒疫苗，这两种疫苗均采用LNP技术递送mRNA药物，从而实现了对SARS-CoV-2病毒的预防。Lipid nanoparticles (LNP) are currently a research hotspot in the field of non-viral gene carriers. In 2018, the FDA approved the LNP delivery of patisiran (onpattro) to treat hereditary transthyretin amyloidosis. Since then, the research on the delivery of nucleic acid drugs using LNP technology has shown explosive growth. In particular, at the end of 2020, the FDA approved Moderna and BioNtech & Pfizer's new coronavirus vaccines for COVID-19, both of which use LNP technology to deliver mRNA drugs, thereby achieving prevention of the SARS-CoV-2 virus.

LNP通常由四种脂质化合物组成，即可电离脂质、中性脂质、类固醇和聚合物结合的脂质，其中，可电离脂质的选择对LNP的影响最大。LNPs are usually composed of four lipid compounds, namely ionizable lipids, neutral lipids, steroids, and polymer-bound lipids, among which the choice of ionizable lipids has the greatest impact on LNPs.

当前核酸治疗药物发挥作用仍面临一些挑战，主要包括递送效率、低细胞渗透性和对包括RNA在内的某些核酸分子降解的高敏感性等。因此，仍需要开发新的脂质化合物促进核酸分子的活体外或活体内递送以实现治疗和/或预防目的。Currently, nucleic acid therapeutic drugs still face some challenges in their effectiveness, mainly including delivery efficiency, low cell permeability, and high sensitivity to degradation of certain nucleic acid molecules including RNA. Therefore, there is still a need to develop new lipid compounds to promote the in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or preventive purposes.

发明内容Summary of the invention

本发明的一个目的在于提供一种脂质化合物或其药学上可接受的形式(例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物等)，该脂质化合物可与其它脂质化合物(例如，中性脂质、带电脂质、类固醇)共同制备脂质纳米颗粒，用于递送治疗剂(例如，核酸分子，具体地还包括mRNA)提升核酸药物在体内的递送效率，可根据核酸药物需要富集的器官而选用特定结构的脂质化合物作为脂质载体。One object of the present invention is to provide a lipid compound or a pharmaceutically acceptable form thereof (e.g., salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs, etc.), which can be used together with other lipid compounds (e.g., neutral lipids, charged lipids, steroids) to prepare lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acid molecules, specifically including mRNA) to improve the delivery efficiency of nucleic acid drugs in vivo, and lipid compounds with specific structures can be selected as lipid carriers according to the organs where the nucleic acid drugs need to be enriched.

本发明的另一目的还提供所述脂质化合物或其药学上可接受的形式的制备方法。Another object of the present invention is to provide a method for preparing the lipid compound or a pharmaceutically acceptable form thereof.

本发明的另一目的还提供包含上述化合物或其药学上可接受的形式的脂质载体。Another object of the present invention is to provide a lipid carrier comprising the above compound or a pharmaceutically acceptable form thereof.

本发明的另一目的还提供包含上述化合物或其药学上可接受的形式或上述脂质载体的核酸脂质纳米粒组合物。Another object of the present invention is to provide a nucleic acid lipid nanoparticle composition comprising the above compound or a pharmaceutically acceptable form thereof or the above lipid carrier.

本发明的另一目的还提供包含上述化合物或其药学上可接受的形式、或上述脂质载体、或上 述核酸脂质纳米粒组合物的药物制剂。Another object of the present invention is to provide a composition comprising the above compound or a pharmaceutically acceptable form thereof, or the above lipid carrier, or The pharmaceutical preparation of the nucleic acid lipid nanoparticle composition.

本发明的另一目的还提供上述化合物或其药学上可接受的形式或上述脂质载体或上述核酸脂质纳米粒组合物或上述药物制剂在制备核酸药物、基因疫苗、小分子药物、多肽或蛋白质药物中的用途。Another object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

本发明的另一目的还提供用于体内递送核酸药物的方法，所述方法包括向有需要的受试者施用上述核酸脂质纳米粒组合物或上述药物制剂。Another object of the present invention is to provide a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

本发明的另一目的还提供用于防止、改善或治疗有需要的受试者的疾病或状况的方法，所述方法包括对所述受试者施用核酸脂质纳米粒组合物或上述药物制剂。Another object of the present invention is to provide a method for preventing, improving or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.

<第一方面><First aspect>

本发明提供了式I化合物或其药学上可接受的形式，所述药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物等，
The present invention provides a compound of formula I or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, etc.

其中：in:

G1为C1-6亚烷基； G1 is a C1-6 alkylene group;

R1、R2各自独立地为C1-6烷基或C1-6烷氧基；所述烷基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-6烷基、C1-6烷氧基、卤素取代的C1-6烷基或羟基取代的C1-6烷基所取代；R 1 and R 2 are each independently C 1-6 alkyl or C 1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy is optionally further substituted by halogen, hydroxy, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxy-substituted C 1-6 alkyl;

任选地，R1、R2与相连的N共同形成3-8元杂环基，或者，R1、R2中任一与G1中的任一碳原子直接连接而形成3-8元杂环基；所述杂环基中有0至3个环C任选进一步被N、O或S所取代；所述杂环基中有0至3个H任选进一步被卤素、羟基、C1-6烷基、C1-6烷氧基、卤素取代的C1-6烷基或羟基取代的C1-6烷基所取代；Optionally, R 1 , R 2 and the connected N together form a 3-8 membered heterocyclic group, or any one of R 1 and R 2 is directly connected to any carbon atom in G 1 to form a 3-8 membered heterocyclic group; 0 to 3 ring Cs in the heterocyclic group are optionally further substituted by N, O or S; 0 to 3 Hs in the heterocyclic group are optionally further substituted by halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxyl-substituted C 1-6 alkyl;

L1选自 亚炔基、杂原子O、N、S或不存在；h、i、k选 自1-10的整数；j选自0或1；L 1 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k optional An integer from 1 to 10; j is selected from 0 or 1;

R3、R4、R5、R6、R7各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 3 , R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;

a、b各自独立地选自0-10的整数；a and b are each independently selected from integers of 0-10;

L2选自 亚炔基、杂原子O、N、S或不存在；h、i、k选自1-10的整数；j选自0或1；L 2 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k are selected from integers of 1-10; j is selected from 0 or 1;

R8、R9、R10、R11、R12各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;

c、d各自独立地选自0-10的整数；c and d are each independently selected from integers of 0-10;

L3、L4各自独立地选自 亚炔基、杂原子O、N、S或不存在；l、n选自1-10的整数；m选自0或1；L 3 and L 4 are each independently selected from Alkyne group, heteroatom O, N, S or not present; l, n are integers selected from 1-10; m is selected from 0 or 1;

R13、R14、R15、R16、R17、R18、R19各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy, halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl;

e、f、g各自独立地选自0-10的整数。e, f, and g are each independently selected from integers of 0-10.

根据本发明的一些具体实施方案，式I中，所述G1为亚甲基、亚乙基、亚丙基、亚丁基或亚戊基。According to some specific embodiments of the present invention, in Formula I, G1 is methylene, ethylene, propylene, butylene or pentylene.

根据本发明的一些具体实施方案，式I中，R1、R2各自独立地为甲基、乙基、丙基或丁基，所述甲基、乙基、丙基或丁基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代；或者，R1、R2与相连的N共同形成5元或6元杂环基，所述杂环基中有0或1或2个环C任选进一步被N、O或S所取代，所述杂环基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代。According to some specific embodiments of the present invention, in Formula I, R 1 and R 2 are each independently methyl, ethyl, propyl or butyl, wherein 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl; or, R 1 , R 2 and the connected N together form a 5-membered or 6-membered heterocyclic group, wherein 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and wherein 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl.

根据本发明的另一些具体实施方案，式I中，R1、R2中之一与G1中的任一碳原子直接连接而 形成5或6元杂环基，R1、R2中另一为甲基、乙基、丙基或丁基，所述甲基、乙基、丙基或丁基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代。所述杂环基中有0或1或2个环C任选进一步被N、O或S所取代，所述杂环基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代。According to other specific embodiments of the present invention, in Formula I, one of R 1 and R 2 is directly connected to any carbon atom in G 1 . To form a 5- or 6-membered heterocyclic group, the other of R 1 and R 2 is methyl, ethyl, propyl or butyl, and 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl. 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl.

根据本发明的一些具体实施方案，式I中，G1、R1、R2分别对应化合物1-化合物31中任一化合物所示的基团。According to some specific embodiments of the present invention, in Formula I, G 1 , R 1 , and R 2 correspond to the groups shown in any one of Compounds 1 to 31, respectively.

根据本发明的一些具体实施方案，式I中，According to some specific embodiments of the present invention, in Formula I,

L1为 亚炔基、O或不存在；h、i、k各自独立地选自1、2、3、4、5、6、7、8、9或10；j选自0或1； L1 is alkynylene, O or absent; h, i, k are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; j is selected from 0 or 1;

R3、R4、R5中至少二者不为氢，R6、R7为氢；At least two of R 3 , R 4 and R 5 are not hydrogen, and R 6 and R 7 are hydrogen;

a选自0、1、2、3、4、5、6、7、8、9或10；a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

b选自0、1、2、3、4、5、6、7、8、9或10；b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

优选地，a、b至少之一或均不为0。Preferably, at least one of a and b or both of them are not zero.

根据本发明的一些具体实施方案，式I中，L1、R3、R4、R5、R6、R7、a、b分别对应化合物1-化合物31中任一化合物所示的基团或取值。According to some specific embodiments of the present invention, in Formula I, L 1 , R 3 , R 4 , R 5 , R 6 , R 7 , a, and b correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.

根据本发明的一些具体实施方案，式I中，According to some specific embodiments of the present invention, in Formula I,

L2为亚炔基、O或不存在；h选自1、2、3、4、5、6、7、8、9或10； L2 is alkynylene, O or absent; h is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R8、R9各自独立地为氢，R10、R11、R12中至少二者不为氢；R 8 and R 9 are each independently hydrogen, and at least two of R 10 , R 11 , and R 12 are not hydrogen;

c、d各自独立地选自0、1、2、3、4、5、6、7、8、9或10；优选地，c、d至少之一或均不为0。c and d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, at least one of c and d or both of them are not 0.

根据本发明的一些具体实施方案，式I中，L2、R8、R9、R10、R11、R12、c、d分别对应化合物1-化合物31中任一化合物所示的基团或取值。According to some specific embodiments of the present invention, in Formula I, L 2 , R 8 , R 9 , R 10 , R 11 , R 12 , c, and d correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.

根据本发明的一些具体实施方案，式I中，According to some specific embodiments of the present invention, in Formula I,

L3、L4各自独立地选自亚炔基、O 或不存在；L 3 and L 4 are each independently selected from Alkyne, O or does not exist;

R13、R14各自为氢；R15、R16、R17、R18、R19至少二者不为氢；优选地，R16、R18、R19至少二者不为氢；R 13 and R 14 are each hydrogen; at least two of R 15 , R 16 , R 17 , R 18 and R 19 are not hydrogen; preferably, at least two of R 16 , R 18 and R 19 are not hydrogen;

e、f、g各自独立地选自0、1、2、3、4、5、6、7、8、9或10。e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

根据本发明的一些具体实施方案，式I中，According to some specific embodiments of the present invention, in Formula I,

L3为亚炔基、O或不存在； L3 is Alkyne, O or absent;

R13、R14各自为氢；R15、R16、R17至少二者不为氢；R 13 and R 14 are each hydrogen; at least two of R 15 , R 16 and R 17 are not hydrogen;

e、f各自独立地选自0、1、2、3、4、5、6、7、8、9或10。e and f are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

根据本发明的一些具体实施方案，式I中，L3、R13、R14、R15、R16、R17、e、f分别对应化合物1-化合物31中任一化合物所示的基团或取值。According to some specific embodiments of the present invention, in Formula I, L 3 , R 13 , R 14 , R 15 , R 16 , R 17 , e, and f correspond to the groups or values shown in any one of Compounds 1 to 31, respectively.

本发明中，当所述a、b、c、d、e、f的取值大于1时，所对应的多个R3、多个R4、多个R6、多个R7、多个R8、多个R9、多个R10、多个R11、多个R13、多个R14、多个R15、多个R17可各自独立地相同或不同。In the present invention, when the values of a, b, c, d, e, and f are greater than 1, the corresponding multiple R 3 s , multiple R 4 s , multiple R 6 s , multiple R 7 s , multiple R 8 s , multiple R 9 s , multiple R 10 s , multiple R 11 s , multiple R 13 s , multiple R 14 s, multiple R 15 s , and multiple R 17 s may be independently the same or different.

根据本发明的一些具体实施方案，所述化合物选自表1所示化合物1至化合物31中的一种或多种。According to some specific embodiments of the present invention, the compound is selected from one or more of Compounds 1 to 31 shown in Table 1.

表1本发明代表性化合物




Table 1 Representative compounds of the present invention




<第二方面><Second Aspect>

本发明提供了一种中间体，其具有式Ic或式Id所示结构：
The present invention provides an intermediate having a structure shown in Formula Ic or Formula Id:

其中，各取代基及e、f、g取值如前述本发明中所定义。Wherein, each substituent and the values of e, f, and g are as defined in the present invention above.

本发明所述的中间体可用于制备本发明所述的化合物或其药学上可接受的形式。The intermediates described in the present invention can be used to prepare the compounds described in the present invention or their pharmaceutically acceptable forms.

<第三方面><Third Aspect>

本发明提供了一种制备<第一方面>所述的化合物或其药学上可接受的形式的方法，该方法包括：The present invention provides a method for preparing the compound described in <the first aspect> or a pharmaceutically acceptable form thereof, the method comprising:

通过烷基化或还原胺化得到 Obtained by alkylation or reductive amination

通过三光气或羰基二咪唑或对硝基氯甲酸苯酯的作用生成硫代氨基甲酸酯 Thiocarbamates are generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate

脱去缩酮保护基，通过缩合反应引入脂肪长链，得到式I化合物或其药学上可接受的形式；Removing the ketal protecting group and introducing a long fatty chain through a condensation reaction to obtain a compound of formula I or a pharmaceutically acceptable form thereof;

其中，各取代基及e、f、g取值为前述本发明中所定义。The values of each substituent and e, f, and g are as defined in the present invention.

根据本发明的一些具体实施方案，本发明的制备所述的化合物或其药学上可接受的形式的方法中，是作为关键中间体，通过烷基化或还原胺化得到含有一条长链或其前体的二级胺然后再通过三光气或羰基二咪唑或对硝基氯甲酸苯酯的作用生成硫代氨基甲酸酯最后脱去缩酮保护基，通过缩合反应引入第二和第三条脂肪长链，得到式I或其盐形式的化合物。According to some specific embodiments of the present invention, in the method of preparing the compound or its pharmaceutically acceptable form, It is a key intermediate that can be used to obtain secondary amines containing a long chain or its precursors through alkylation or reductive amination. Then, thiocarbamate is generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate. Finally, the ketal protecting group is removed, and the second and third fatty long chains are introduced through condensation reaction to obtain a compound of formula I or a salt thereof.

<第四方面><Fourth Aspect>

本发明还提供了所述的化合物或其药学上可接受的形式在制备脂质体纳米载体中的应用。The present invention also provides the use of the compound or its pharmaceutically acceptable form in the preparation of liposome nanocarriers.

<第五方面><Fifth Aspect>

本发明提供了一种脂质载体，其包含根据<第一方面>所述的化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物。该类脂质载体对核酸药物的包封效率高，大大提升了核酸药物在体内的递送效率。 The present invention provides a lipid carrier, which comprises the compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug. Such lipid carriers have high encapsulation efficiency for nucleic acid drugs, greatly improving the delivery efficiency of nucleic acid drugs in vivo.

在一些实施方案中，脂质载体包含第一脂质化合物和第二脂质化合物，其中，第一脂质化合物包含根据<第一方面>所述的化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物以及任选的其它可电离脂质，第二脂质化合物包含阴离子脂质、中性脂质、类固醇和聚合物结合的脂质中的一种或两种以上的组合。In some embodiments, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises the compound according to the <First Aspect> or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and optionally other ionizable lipids, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

在一些实施方案中，上述第一脂质化合物为上述任一种化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物。In some embodiments, the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug.

在一些实施方案中，上述第一脂质化合物为上述任一种化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物和其它阳离子或可电离脂质的组合。In some embodiments, the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug and a combination of other cationic or ionizable lipids.

在一些实施方案中，所述其它阳离子或可电离脂质化合物包括1,2-二亚油基氧基-N,N-二甲基氨基丙烷DLinDMA、1,2-二油基氧基-N,N-二甲基氨基丙烷DODMA、DLin-MC2-MPZ、2,2-二亚油基-4-(2-二甲基氨基乙基)-[1,3]-二氧环戊烷DLin-KC2-DMA、1,2-二油酰基-3-三甲基铵-丙烷DOTAP、1,1′-(2-(4-(2-((2-(双(2-羟基十二烷基)氨基)乙基)(2-羟基十二烷基)氨基)乙基)哌嗪-1-基)乙基氮烷二基)二-十二烷-2-醇C12-200、3β[N-N’N’-二甲氨乙烷)-氨甲酰]胆固醇DC-Chol和N-[1-(2,3-二油酰氯)丙基]-N,N,N-氯化三甲胺DOTMA中的一种或两种以上的组合。In some embodiments, the other cationic or ionizable lipid compounds include 1,2-dilinoleyloxy-N,N-dimethylaminopropane DLinDMA, 1,2-dioleyloxy-N,N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane DLin-KC2-DMA, 1,2-dioleoyl-3-trimethylammonium-propane DOT AP, 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)di-dodecan-2-ol C12-200, 3β[N-N’N’-dimethylaminoethane)-carbamoyl]cholesterol DC-Chol and N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride DOTMA, or a combination of two or more thereof.

在一些实施方案中，所述阴离子脂质包括磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、磷脂酰甘油、二油酰磷脂酰甘油DOPG、1,2-二油酰-sn-甘油-3-磷脂酰丝氨酸DOPS和二豆蔻酰磷脂酰甘油中的一种或两种以上的组合。In some embodiments, the anionic lipid includes one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoylphosphatidylglycerol DOPG, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine DOPS and dimyristoylphosphatidylglycerol.

在一些实施方案中，所述中性脂质包括1,2-二油酰-sn-甘油-3-磷脂酰乙醇胺DOPE、1,2-二硬脂酰-sn-甘油-3-磷脂酰胆碱DSPC、1,2-二棕榈酰-sn-甘油-3-磷脂酰胆碱DPPC、1,2-二油酰-sn-甘油-3-磷脂酰胆碱DOPC、二棕榈酰磷酯酰甘油DPPG、油酰磷脂酰胆碱POPC、1-棕榈酰基-2-油酰基磷脂酰乙醇胺POPE、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺DPPE、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺DMPE、二硬脂酰磷脂酰乙醇胺DSPE和1-硬脂酰基-2-油酰基磷脂酰乙醇胺SOPE中的至少一种或其经阴离子或阳离子修饰基团修饰的脂质。阴离子或阳离子修饰基团不作限定。In some embodiments, the neutral lipid includes at least one of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine DSPC, 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine DPPC, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine DOPC, dipalmitoylphosphatidylglycerol DPPG, oleoylphosphatidylcholine POPC, 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine DMPE, distearoylphosphatidylethanolamine DSPE and 1-stearoyl-2-oleoylphosphatidylethanolamine SOPE, or lipids modified with anionic or cationic modifying groups. Anionic or cationic modifying groups are not limited.

在一些实施方案中，所述类固醇包括胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸、α-生育酚、粪固醇和皮质类固醇中的一种或多种。In some embodiments, the steroid includes one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, coproposterol, and corticosteroids.

在一些实施方案中，所述聚合物结合的脂质包括1,2-二肉豆蔻酰基-sn-甘油甲氧基-聚乙二醇PEG-DMG、二肉豆蔻酰甘油-聚乙二醇PEG-c-DMG、聚乙二醇-二肉豆蔻酰基甘油PEG-C14、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺PEG-c-DMA、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)]PEG-DSPE、聚乙二醇化磷脂酰乙醇胺PEG-PE、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、吐温-20、吐温-80、1,2-二棕榈基-sn-甘油-甲氧基聚乙二醇PEG-DPG、4-O-(2’，3’-二(十四烷酰氧基)丙基-1-O-(ω-甲氧基(聚乙氧基)乙基)丁二酸酯PEG-s-DMG、PEG-二烷氧基丙基PEG-DAA、mPEG2000-1,2-二-O-烷基-sn3-氨基甲酰基甘油酯PEG-c-DOMG和N-乙酰半乳糖胺 ((R)-2,3-双(十八烷氧基)丙基-1-(甲氧基聚(乙二醇)2000)丙基氨基甲酸酯))GalNAc-PEG-DSG中的一种或两种以上的组合。In some embodiments, the polymer-bound lipids include 1,2-dimyristoyl-sn-glyceromethoxy-polyethylene glycol PEG-DMG, dimyristoylglycerol-polyethylene glycol PEG-c-DMG, polyethylene glycol-dimyristoylglycerol PEG-C14, PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, PEG modified PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglyceride PEG-c-DOMG and N-acetylgalactosamine ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate))GalNAc-PEG-DSG or a combination of two or more thereof.

在一些实施方案中，在脂质载体中，第一脂质化合物、阴离子脂质、中性脂质、类固醇和聚合物结合的脂质的摩尔比为(20～65):(0～20):(5～25):(25～55):(0.3～15)；示例性地，第一脂质化合物、阴离子脂质、中性脂质、类固醇和聚合物结合的脂质的摩尔比可以为20:20:5:50:5、30:5:25:30:10、20:5:5:55:15、65:0:9.7:25:0.3等；其中，在第一脂质化合物中，上述任一种化合物或其药学上可接受的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物和其它阳离子或可电离脂质的摩尔比为(1～10):(0～10)；示例性地，该摩尔比可以为1:1、1:2、1:5、1:7.5、1:10、2:1、5:1、7.5:1、10:1等。In some embodiments, in the lipid carrier, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid is (20-65):(0-20):(5-25):(25-55):(0.3-15); illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid and the polymer-bound lipid can be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55 :15, 65:0:9.7:25:0.3, etc.; wherein, in the first lipid compound, the molar ratio of any one of the above compounds or its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (1-10):(0-10); illustratively, the molar ratio can be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

在一些实施方案中，在脂质载体中，第一脂质化合物、阴离子脂质、中性脂质、类固醇和聚合物结合的脂质的摩尔比为(20～55):(0～13):(5～25):(25～51.5):(0.5～15)；其中，在第一脂质化合物中，上述任一种化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物和其它阳离子或可电离脂质的摩尔比为(3-4):(0-5)。In some embodiments, in the lipid carrier, the molar ratio of the first lipid compound, anionic lipids, neutral lipids, steroids and polymer-bound lipids is (20-55):(0-13):(5-25):(25-51.5):(0.5-15); wherein, in the first lipid compound, the molar ratio of any of the above compounds or their pharmaceutically acceptable forms such as salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs and other cationic or ionizable lipids is (3-4):(0-5).

<第六方面><Sixth Aspect>

本发明提供了一种核酸脂质纳米粒组合物，其包括根据<第一方面>所述的化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物或根据<第五方面>所述的脂质载体，以及核酸药物。The present invention provides a nucleic acid lipid nanoparticle composition, which includes the compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug or the lipid carrier according to the fifth aspect, and a nucleic acid drug.

在一些实施方案中，所述核酸药物包括RNA、DNA、反义核酸、适配体、核酶、免疫刺激性核酸或PNA。In some embodiments, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

在一些实施方案中，所述反义核酸为反义寡核酸。In some embodiments, the antisense nucleic acid is an antisense oligonucleic acid.

在一些实施方案中，所述RNA为mRNA、rRNA、circRNA、siRNA、saRNA、tRNA、snRNA、antagomir、微RNA抑制剂、微RNA激活剂或shRNA。In some embodiments, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA.

在一些实施方案中，所述DNA包括质粒。In some embodiments, the DNA comprises a plasmid.

在一些实施方案中，所述mRNA包括编码RNA导向的DNA结合剂的序列，更具体地，包括编码核酸酶或碱基编辑器的mRNA。In some embodiments, the mRNA includes a sequence encoding an RNA-guided DNA binder, more specifically, an mRNA encoding a nuclease or a base editor.

在一些实施方案中，所述的核酸酶包括Cas9(例如，GeoCas9、CjCas9、SpCas9)、CasX、CasY、Casz、Cpf1、C2c1、C2c2、C2c3和Argonaute蛋白(AGO)，及其同源物(例如，dCas9和nCas9)中的一种或多种。In some embodiments, the nuclease includes one or more of Cas9 (e.g., GeoCas9, CjCas9, SpCas9), CasX, CasY, Casz, Cpf1, C2c1, C2c2, C2c3, and Argonaute protein (AGO), and homologs thereof (e.g., dCas9 and nCas9).

在一些实施方案中，所述核酸药物还可包括导向RNA，具体地，所述导向RNA包括gRNA核酸。In some embodiments, the nucleic acid drug may further include a guide RNA, specifically, the guide RNA includes a gRNA nucleic acid.

在一些实施方案中，所述核酸药物包括编码核酸酶或碱基编辑器的mRNA和gRNA。In some embodiments, the nucleic acid drug includes mRNA and gRNA encoding a nuclease or a base editor.

在一些实施方案中，所述mRNA或gRNA经过修饰。In some embodiments, the mRNA or gRNA is modified.

在一些实施方案中，所述修饰选自由假尿嘧啶(ψ)、N1-甲基假尿嘧啶(N1Mψ)、1-乙基假尿嘧啶、2-硫脲嘧啶(s2U)、4-硫脲嘧啶、5-甲基胞嘧啶、5-甲基尿嘧啶、5-甲氧基尿嘧啶及其任何组合 组成的组。In some embodiments, the modification is selected from pseudouracil (ψ), N1-methyl pseudouracil (N1Mψ), 1-ethyl pseudouracil, 2-thiouracil (s2U), 4-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof. Composed of groups.

在一些实施方案中，上述核酸药物与上述任一种化合物或其药学上可接受的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物的质量比为1:(3～40)。In some embodiments, the mass ratio of the nucleic acid drug to any of the compounds or their pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs is 1:(3-40).

在一些实施方案中，上述核酸药物与上述脂质载体的质量比为1:(3～40)。In some embodiments, the mass ratio of the nucleic acid drug to the lipid carrier is 1:(3-40).

示例性地，上述质量比为1:3、1:5、1:10、1:15、1:20、1:30等。Exemplarily, the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.

在一些实施方案中，所述的核酸酶或碱基编辑器的mRNA与gRNA的比率(w/w)为约10:1至约1:10。在一些实施方案中，所述的比率(w/w)为约3:1至约1:3。在一些实施方案中，所述的比率(w/w)为约3:2。In some embodiments, the ratio (w/w) of the mRNA to the gRNA of the nuclease or base editor is about 10: 1 to about 1: 10. In some embodiments, the ratio (w/w) is about 3: 1 to about 1: 3. In some embodiments, the ratio (w/w) is about 3: 2.

<第七方面><Seventh Aspect>

本发明提供了一种药物组合物，其包含根据<第一方面>所述的化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物，或根据<第五方面>所述的脂质载体，或根据<第六方面>所述的核酸脂质纳米粒组合物，以及药学上可接受的赋形剂、载体或稀释剂。The present invention provides a pharmaceutical composition comprising a compound according to the first aspect or a pharmaceutically acceptable form thereof such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or a lipid carrier according to the fifth aspect, or a nucleic acid lipid nanoparticle composition according to the sixth aspect, and a pharmaceutically acceptable excipient, carrier or diluent.

<第八方面><Eighth Aspect>

本发明提供了一种药物制剂，其包含上述任一种化合物或其药学上可接受的形式例如盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物，或上述脂质载体，或上述核酸脂质纳米粒组合物，以及药学上可接受的赋形剂、载体或稀释剂。The present invention provides a pharmaceutical preparation comprising any one of the above-mentioned compounds or a pharmaceutically acceptable form thereof, such as a salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug, or the above-mentioned lipid carrier, or the above-mentioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier or diluent.

在一些实施方案中，药物制剂的粒径为30～500nm，示例性地，粒径可以为30nm、50nm、100nm、150nm、250nm、350nm、500nm等。In some embodiments, the particle size of the pharmaceutical preparation is 30 to 500 nm. For example, the particle size can be 30 nm, 50 nm, 100 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.

在一些实施方案中，核酸药物在药物制剂中的包封率大于50％。示例性地，包封率可以为55％、60％、65％、70％、75％、79％、80％、85％、89％、90％、93％、95％等。In some embodiments, the encapsulation rate of the nucleic acid drug in the pharmaceutical preparation is greater than 50%. Exemplarily, the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

<第九方面><Ninth Aspect>

本发明还提供上述化合物或其药学上可接受的形式或上述脂质载体或上述核酸脂质纳米粒组合物或上述药物制剂在制备核酸药物、基因疫苗、小分子药物、多肽或蛋白质药物中的用途。The present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

本发明还提供用于体内递送核酸药物的方法，所述方法包括向有需要的受试者施用上述核酸脂质纳米粒组合物或上述药物制剂。The present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

本发明还提供用于防止、改善或治疗有需要的受试者的疾病或状况的方法，所述方法包括对所述受试者施用核酸脂质纳米粒组合物或上述药物制剂。The present invention also provides a method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation to the subject.

在一些实施方案中，上述核酸脂质纳米粒组合物或上述药物制剂通过以下施用途径之一施用：口服、鼻内、静脉内、腹膜内、肌肉内、关节内、病灶内、气管内、皮下以及皮内。在一些实施方案中，上述核酸脂质纳米粒组合物或上述药物制剂例如经由肠内或肠胃外施用途径施用。在一些实施方案中，将约0.001mg/kg至约10mg/kg剂量的所述核酸脂质纳米粒组合物或药物制剂施用给所述受试者。 In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation is administered, for example, via an enteral or parenteral administration route. In some embodiments, a dosage of about 0.001 mg/kg to about 10 mg/kg of the nucleic acid lipid nanoparticle composition or pharmaceutical preparation is administered to the subject.

在一些实施方案中，上述核酸脂质纳米粒组合物或上述药物制剂用于治疗或预防于治疗或预防有需要的受试者的疾病或病症。In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is used to treat or prevent a disease or disorder in a subject in need thereof.

在一些实施方案中，所述的疾病或病症选自代谢性疾病、遗传性疾病、癌症、心血管疾病和传染性疾病。In some embodiments, the disease or condition is selected from a metabolic disease, a genetic disease, cancer, a cardiovascular disease, and an infectious disease.

在一些实施方案中，所述的代谢性疾病包括家族性高胆固醇血症(FH)，所述的遗传性疾病包括转甲状腺素蛋白淀粉样变(ATTR)、原发性高草尿酸症(PH1)和遗传性血管性水肿(HAE)，所述的传染性疾病包括乙型肝炎(HEPATITIS B)。In some embodiments, the metabolic disease includes familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious disease includes hepatitis B (HEPATITIS B).

在一些实施方案中，所述的受试者为哺乳动物或人。In some embodiments, the subject is a mammal or a human.

本发明提供了一系列结构新颖的式I化合物，该化合物可作为可电离脂质，与其它脂质化合物共同制备脂质载体，其粒径可控，分布均一，具有很高的包封率。更具体地，本发明的化合物是一种氨基脂质体，脂质体化合物同时含有疏水非极性长链和亲水氨基，这种两性特征可以用于形成脂质颗粒。进一步，在本发明的化合物中，与硫代氨基甲酸酯中氮原子相连基团优选为脂肪长链，所述脂肪长链的C数优选大于等于3、进一步优选大于等于4、再进一步优选大于等于5、更进一步优选大于等于6，随着脂肪长链的增加，递送效果可显著提升，该脂肪长链的引入可增加脂质体中非极性疏水组分的比重，从而增加膜融合来增强mRNA的释放，促进mRNA递送的协同改善，得到显著增强的递送效率。The present invention provides a series of novel structural formula I compounds, which can be used as ionizable lipids to prepare lipid carriers together with other lipid compounds, and their particle size is controllable, uniformly distributed, and has a very high encapsulation rate. More specifically, the compound of the present invention is an amino liposome, and the liposome compound contains both hydrophobic non-polar long chains and hydrophilic amino groups, and this amphoteric characteristic can be used to form lipid particles. Further, in the compound of the present invention, the group connected to the nitrogen atom in the thiocarbamate is preferably a fatty long chain, and the C number of the fatty long chain is preferably greater than or equal to 3, further preferably greater than or equal to 4, further preferably greater than or equal to 5, and further preferably greater than or equal to 6. With the increase of the fatty long chain, the delivery effect can be significantly improved, and the introduction of the fatty long chain can increase the proportion of the non-polar hydrophobic component in the liposome, thereby increasing membrane fusion to enhance the release of mRNA, promote the synergistic improvement of mRNA delivery, and obtain significantly enhanced delivery efficiency.

本发明的脂质化合物合成方法简单、收率高，可以快速合成，成本低。本发明的化合物可用于递送核酸药物、基因疫苗、小分子药物、多肽或蛋白质药物，丰富了可电离脂质化合物的种类，尤其对核酸预防剂和治疗剂的发展和应用具有重要的意义。The lipid compound synthesis method of the present invention is simple, has a high yield, can be quickly synthesized, and has low cost. The compound of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs, enriching the types of ionizable lipid compounds, and is particularly important for the development and application of nucleic acid preventive and therapeutic agents.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1为本发明中小鼠肝脏细胞PCSK9基因编辑效率检测的递送策略示意图。FIG1 is a schematic diagram of the delivery strategy for detecting the PCSK9 gene editing efficiency in mouse liver cells in the present invention.

图2显示不同脂质化合物包封的碱基编辑器在小鼠肝脏细胞中的PCSK9基因编辑效率。Figure 2 shows the PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells.

具体实施方式DETAILED DESCRIPTION

为更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本领域技术人员通常理解的含义。For easier understanding of the present invention, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

本说明书中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

本说明书中，使用“基本上”或“实质上”表示与理论模型或理论数据的标准偏差在5％、优选为3％、更优选为1％范围以内。In the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

本说明书中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。In this specification, the word "may" includes both performing a certain process and not performing a certain process.

本说明书中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。 In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

本说明书中，所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如，特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中，并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外，应理解，所述要素可以任何合适的方式组合在各种实施方案中。In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

在进一步描述本发明之前，应当理解，本发明不限于本文中所述的特定实施方案；还应该理解，本文中所使用的术语仅用于描述而非限定特定实施方案。Before the present invention is further described, it is to be understood that the present invention is not limited to the particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of description only and is not intended to be limiting of the particular embodiments.

[术语定义][Definition of terms]

除非另有说明，下列术语的含义如下：Unless otherwise stated, the following terms have the following meanings:

术语“药学上可接受的盐”是指对生物体基本上无毒性的本发明的化合物的盐。药学上可接受的盐通常包括(但不限于)本发明的化合物与药学上可接受的无机/有机酸或无机/有机碱反应而形成的盐，此类盐又被称为酸加成盐或碱加成盐。常见的无机酸包括(但不限于)盐酸、氢溴酸、硫酸、磷酸等，常见的有机酸包括(但不限于)三氟乙酸、柠檬酸、马来酸、富马酸、琥珀酸、酒石酸、乳酸、丙酮酸、草酸、甲酸、乙酸、苯甲酸、甲磺酸、苯磺酸、对甲苯磺酸等，常见的无机碱包括(但不限于)氢氧化钠、氢氧化钾、氢氧化钙、氢氧化钡等，常见的有机碱包括(但不限于)二乙胺、三乙胺、乙胺丁醇等。The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is substantially non-toxic to an organism. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compound of the present invention with a pharmaceutically acceptable inorganic/organic acid or inorganic/organic base, and such salts are also referred to as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc., common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc., and common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.

术语“立体异构体”(或称“旋光异构体”)是指由于具有至少一个手性因素(包括手性中心、手性轴、手性面等)而导致具有垂直的不对称平面，从而能够使平面偏振光旋转的稳定异构体。由于本发明的化合物中存在可能导致立体异构的不对称中心以及其它化学结构，因此本发明也包括这些立体异构体及其混合物。由于本发明的化合物及其盐包括不对称碳原子，因而能够以单一立体异构体形式、外消旋物、对映异构体和非对映异构体的混合物形式存在。通常，这些化合物能够以外消旋混合物的形式制备。然而，如果需要的话，可以将这类化合物制备或分离后得到纯的立体异构体，即单一对映异构体或非对映异构体，或者单一立体异构体富集化(纯度≥98％、纯度≥95％、≥93％、≥90％、≥88％、≥85％或≥80％)的混合物。化合物的单一立体异构体是由含有所需手性中心的旋光起始原料合成制备得到的，或者是通过制备得到对映异构体产物的混合物之后再分离或拆分制备得到的，例如转化为非对映异构体的混合物之后再进行分离或重结晶、色谱处理、使用手性拆分试剂，或者在手性色谱柱上将对映异构体进行直接分离。具有特定立体化学的起始化合物既可以商购得到，也可以按照本文中描述的方法制备再通过本领域熟知的方法拆分得到。The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a vertical asymmetric plane due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane polarized light. Since there are asymmetric centers and other chemical structures that may cause stereoisomerism in the compounds of the present invention, the present invention also includes these stereoisomers and mixtures thereof. Since the compounds of the present invention and their salts include asymmetric carbon atoms, they can exist in the form of a single stereoisomer, a racemate, an enantiomer, and a mixture of diastereomers. Generally, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or single stereoisomer-enriched (purity ≥98%, purity ≥95%, ≥93%, ≥90%, ≥88%, ≥85% or ≥80%) mixtures. A single stereoisomer of a compound is prepared synthetically from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of particular stereochemistry are either commercially available or prepared as described herein and resolved by methods well known in the art.

术语“互变异构体”(或称“互变异构形式”)是指具有不同能量的可通过低能垒互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(或称质子转移互变异构体)包括(但不限于)通过质子迁移来进行的互相转化，如酮-烯醇异构化、亚胺-烯胺异构化、酰胺-亚胺醇异构化等。除非另外指出，本发明的化合物的所有互变异构体形式都在本发明的范围之内。 The term "tautomer" (or "tautomeric form") refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include (but are not limited to) interconversions through proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-imino alcohol isomerization, etc. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

术语“溶剂化物”是指由本发明的化合物或其药学上可接受的盐与至少一种溶剂分子通过非共价分子间作用力结合而形成的物质。常见的溶剂化物包括(但不限于)水合物、乙醇合物、丙酮合物等。The term "solvate" refers to a substance formed by the combination of a compound of the present invention or a pharmaceutically acceptable salt thereof with at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanolates, acetoneates, etc.

术语“螯合物”是具有环状结构的配合物，是通过两个或多个配位体与同一金属离子形成螯合环的螯合作用而得到。The term "chelate" refers to a complex having a ring structure, which is obtained by chelation of two or more ligands with the same metal ion to form a chelate ring.

术语“非共价复合物”是通过化合物与另一分子的相互作用而形成的，其中该化合物与该分子之间不形成共价键。例如，可以通过范德华相互作用、氢键键合和静电相互作用(也称作离子键合)来发生复合。The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

术语“前体药物”是指在适用于患者后能够直接或间接地提供本发明的化合物的衍生化合物。特别优选的衍生化合物或前药是在施用于患者时可以提高本发明的化合物的生物利用度的化合物(例如，更易吸收入血)，或者促进母体化合物向作用位点(例如，淋巴系统)递送的化合物。除非另外指出，本发明的化合物的所有前药形式都在本发明的范围之内，且各种前药形式是本领域熟知的。The term "prodrug" refers to a derivative compound that can directly or indirectly provide a compound of the present invention after being applied to a patient. Particularly preferred derivative compounds or prodrugs are compounds that can increase the bioavailability of the compound of the present invention when administered to a patient (e.g., more easily absorbed into the blood), or compounds that promote the delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the present invention are within the scope of the present invention, and various prodrug forms are well known in the art.

术语“各自独立地”是指结构中存在的取值范围相同或相近的至少两个基团(或环系)可以在特定情形下具有相同或不同的含义。例如，取代基X和取代基Y各自独立地为氢、卤素、羟基、氰基、烷基或芳基，则当取代基X为氢时，取代基Y既可以为氢，也可以为卤素、羟基、氰基、烷基或芳基；同理，当取代基Y为氢时，取代基X既可以为氢，也可以为卤素、羟基、氰基、烷基或芳基。The term "independently" means that at least two groups (or ring systems) with the same or similar value ranges in the structure may have the same or different meanings in specific circumstances. For example, substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl. When substituent X is hydrogen, substituent Y can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X can be either hydrogen or halogen, hydroxyl, cyano, alkyl or aryl.

术语“包含”和“包括”以其开放、非限制性意义使用。The terms "including" and "comprising" are used in their open, non-limiting sense.

术语“烷基”是指一价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，不含有不饱和度，并且通过一个单键与其它片段连接，包括(但不限于)甲基、乙基、丙基、异丙基、丁基、仲丁基、异丁基和叔丁基等。例如，“C1-30烷基”指包含1至30个碳原子的饱和的一价直链或支链烃基。The term "alkyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no unsaturation, and connected to other fragments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl and tert-butyl, etc. For example, "C 1-30 alkyl" refers to a saturated monovalent straight or branched hydrocarbon group containing 1 to 30 carbon atoms.

术语“亚烷基”是指二价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，不含有饱和度，并且通过两个单键分别与其它片段连接，包括(但不限于)亚甲基、1,1-亚乙基和1,2-亚乙基等。例如，“C1-30亚烷基”指从包含1至30个碳原子的饱和的二价直链或支链烷基。The term "alkylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene, etc. For example, "C 1-30 alkylene" refers to a saturated divalent straight or branched alkyl group containing from 1 to 30 carbon atoms.

术语“环烷基”是指饱和的、单环或多环(例如双环、三环或四环)的非芳香族烃基，其仅由碳原子和氢原子组成。环烷基可包括并环、桥环或螺环系统。例如，本发明中所使用的术语“C3-6环烷基”是指具有3至6个碳原子的环烷基。例如，环烷基可以是环丙基、环丁基、环戊基、环己基或双环[2.2.1]庚基等。The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms. The cycloalkyl group may include a paracyclic, bridged or spirocyclic ring system. For example, the term "C 3-6 cycloalkyl" used in the present invention refers to a cycloalkyl group having 3 to 6 carbon atoms. For example, the cycloalkyl group may be a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo[2.2.1]heptyl group, etc.

术语“亚环烷基”是指通过从如上所定义的环烷基中除去氢原子而获得的二价基团，包括(但不限于)亚环丙基、亚环丁基、亚环戊基、亚环己基和亚环庚基等。例如，“C3-30亚环烷基”指从包含3至30个碳原子的环烷基中除去氢原子所获得的二价基团。The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including, but not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene, etc. For example, " C3-30 cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

术语“含支链的烷基”是指与母体分子相连并自身形成至少两个分支结构的烷烃自由基。例如 The term "branched alkyl" refers to an alkane radical that is attached to a parent molecule and forms at least two branched structures.

术语“烯基”是指一价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，含有至少一个 双键，并且通过一个单键与其它片段连接，包括(但不限于)乙烯基、丙烯基、烯丙基、异丙烯基、丁烯基和异丁烯基等基团。例如“C2-30烯基”指包含2至30个碳原子并且具有至少1个碳碳双键(>C＝C<)的一价直链或支链烃基。The term "alkenyl" refers to a monovalent straight or branched chain alkane radical consisting solely of carbon and hydrogen atoms and containing at least one Double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups. For example, " C2-30 alkenyl" refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

术语“亚烯基”是指二价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，含有至少一个双键，并且通过两个单键分别与其它片段连接，包括(但不限于)亚乙烯基等。例如，“C2-30亚烯基”指包含2至30个碳原子并且具有至少1个碳碳双键(>C＝C<)的二价直链或支链烃基。The term "alkenylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc. For example, " C2-30 alkenylene" refers to a divalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

术语“炔基”是指一价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，含有至少一个碳碳三键，并且通过一个单键与其它片段连接，包括(但不限于)乙炔基、丙炔基、丁炔基和戊炔基等基团。例如“C2-30炔基”指包含2至30个碳原子并且具有至少1个碳碳三键的一价直链或支链烃基。The term "alkynyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups. For example, " C2-30 alkynyl" refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

术语“亚炔基”是指二价的直链或支链的烷烃基团，其仅由碳原子和氢原子组成，含有至少一个碳碳三键，并且通过两个单键分别与其它片段连接，包括(但不限于)亚乙炔基等。例如，“C2-30亚炔基”指包含2至30个碳原子并且具有至少1个碳碳三键的二价直链或支链烃基。The term "alkynylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by two single bonds, including (but not limited to) ethynylene, etc. For example, " C2-30 alkynylene" refers to a divalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

术语“环烯基”是指不饱和的、单环或多环(例如双环、三环或四环)的非芳香族烃基，其仅由碳原子和氢原子组成。环烯基可包括并环、桥环或螺环系统。例如环丙烯基和环丁烯基等。The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms. Cycloalkenyl may include cyclic, bridged or spirocyclic systems. For example, cyclopropenyl and cyclobutenyl, etc.

术语“亚环烯基”是指通过从如上所定义的环烯基中除去氢原子而获得的二价基团，包括(但不限于)亚环丙烯基和亚环丁烯基等。例如，“C3-30亚环烯基”指从包含3至30个碳原子的环烯基中除去氢原子所获得的二价基团。The term "cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenylene and cyclobutenylene, etc. For example, " C3-30 cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

术语“烷氧基”是指-O-烷基。The term "alkoxy" refers to an -O-alkyl group.

术语“含支链的烯基”是与母体分子相连并自身形成至少两个分支结构的烯烃自由基。例如 The term "branched alkenyl" refers to an alkene radical attached to a parent molecule and forming at least two branched structures. For example

术语“杂环基”是指饱和或部分饱和的、单环或多环(诸如双环，例如：并环、桥环或螺环)的非芳香族基团，其环原子由碳原子以及至少一个选自N、O和S的杂原子构成，其中S原子任选地被取代以形成S(＝O)、S(＝O)2或S(＝O)(＝NRx)，Rx独立地选自H或C1-4烷基。如果满足价键要求，杂环基可以通过任意一个环原子与分子的其余部分连接。例如，本发明中所使用的术语“3-8元杂环基”是指具有3至8个环原子的杂环基。例如，杂环基可以是环氧乙烷基、氮杂环丙烷基、氮杂环丁烷基、氧杂环丁烷基、四氢呋喃基、二氧杂环戊烯基、吡咯烷基、吡咯烷酮基、咪唑烷基、吡唑烷基、四氢吡喃基、哌啶基、哌嗪基、吗啉基、硫吗啉基、二噻烷基或三噻烷基。The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as a bicyclic, for example, fused, bridged or spiro) non-aromatic group, whose ring atoms consist of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S(=O), S(=O) 2 or S(=O)(= NRx ), and Rx is independently selected from H or C1-4 alkyl. If the valence bond requirements are met, the heterocyclyl can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclyl" used in the present invention refers to a heterocyclyl having 3 to 8 ring atoms. For example, the heterocyclyl group can be oxiranyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl, or trithianyl.

术语“芳基”是指具有共轭π电子系统的单环或稠合多环的芳香族烃基。例如，本发明中所使用的术语“C6-10芳基”是指具有6至10个碳原子的芳基。例如，芳基可以是苯基、萘基、蒽基、菲基、苊基、薁基、芴基、茚基、芘基等。The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 aryl" used in the present invention refers to an aryl group having 6 to 10 carbon atoms. For example, the aryl group can be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, etc.

术语“杂芳基”是指具有共轭π电子系统的单环或稠合多环的芳香族基团，其环原子由碳原子以及至少一个选自N、O和S的杂原子构成。如果满足价键要求，杂芳基可以通过任意一个环原子与分子的 其余部分连接。例如，本发明中所使用的术语“5-10元杂芳基”是指具有5至10个环原子的杂芳基。例如，杂芳基可以是噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基及其苯并衍生物、吡咯并吡啶基、吡咯并吡嗪基、吡唑并吡啶基、咪唑并吡啶基、吡咯并嘧啶基、吡唑并嘧啶基、嘌呤基等。The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S. If the valence bond requirements are met, the heteroaryl group can be bonded to the molecule through any of the ring atoms. The rest are connected. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to a heteroaryl having 5 to 10 ring atoms. For example, heteroaryl can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzo derivatives thereof, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.

术语“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

术语“羟基”是指-OH。The term "hydroxy" refers to -OH.

术语“氰基”是指-CN。The term "cyano" refers to -CN.

术语“氨基”是指-NH2。The term "amino" refers to -NH2 .

术语“硝基”是指-NO2。The term "nitro" refers to -NO2 .

术语“氧代基”是指(＝O)。The term "oxo" refers to (=O).

术语“受试者”包括人和非人动物。非人动物包括脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类、绵羊、猫、马、牛、鸡、狗、小鼠、大鼠、山羊、兔和猪。优选地，受试者是人。除非指出时，否则术语“患者”或“受试者”在本文中可互换地使用。The term "subject" includes humans and non-human animals. Non-human animals include vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits and pigs. Preferably, the subject is a human. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

术语“核酸酶”是指是指催化核酸分子中核苷酸之间磷酸二酯键断裂的酶。在一些实施方案中，核酸酶选自大范围核酸酶、锌指核酸酶(Zinc-finger nucleases,ZFNs)、TAL-效应物DNA结合结构域-核酸酶融合蛋白(TALEN)和RNA引导的核酸酶或其在其中核酸酶活性已被降低或抑制的变体。The term "nuclease" refers to an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the nuclease is selected from a meganuclease, a zinc finger nuclease (ZFNs), a TAL-effector DNA binding domain-nuclease fusion protein (TALEN), and an RNA-guided nuclease or a variant thereof in which the nuclease activity has been reduced or inhibited.

在一些实施方案中，RNA引导的核酸酶是天然存在的CRISPR-Cas蛋白或其活性变体或片段。CRISPR-Cas系统分类为I类或II类系统。II类系统包含单一效应核酸酶，并且包括II、V和VI型。每个类别又细分为类型(I、II、III、IV、V、VI型)，其中一些类型进一步分为亚型(例如，II-A型、II-B型、II-C型、V-A型、V-B型)。In some embodiments, the RNA-guided nuclease is a naturally occurring CRISPR-Cas protein or an active variant or fragment thereof. CRISPR-Cas systems are classified as class I or class II systems. Class II systems contain single effector nucleases and include types II, V, and VI. Each category is further subdivided into types (types I, II, III, IV, V, VI), some of which are further divided into subtypes (e.g., type II-A, type II-B, type II-C, type V-A, type V-B).

术语“II型CRISPR-Cas蛋白”、“II型CRISPR-Cas效应蛋白”或“Cas9”是指需要反式激活RNA(tracrRNA)并且包含两个核酸酶结构域(RuvC和HNH)(其各自负责切割双链DNA分子的单条链)的CRISPR-Cas效应蛋白。在其他实施方案中，CRISPR-Cas蛋白是天然存在的V型CRISPR-Cas蛋白或其活性变体或片段。The term "Type II CRISPR-Cas protein", "Type II CRISPR-Cas effector protein" or "Cas9" refers to a CRISPR-Cas effector protein that requires a trans-activating RNA (tracrRNA) and comprises two nuclease domains (RuvC and HNH), each of which is responsible for cutting a single strand of a double-stranded DNA molecule. In other embodiments, the CRISPR-Cas protein is a naturally occurring Type V CRISPR-Cas protein or an active variant or fragment thereof.

如本文所用，术语“V型CRISPR-Cas蛋白”、“V型CRISPR-Cas效应蛋白”或“Cas12”是指切割dsDNA并包含单个RuvC核酸酶结构域或分裂的RuvC核酸酶结构域并且缺少HNH结构域的CRISPR-Cas效应蛋白。在其他实施方案中，CRISPR-Cas蛋白是天然存在的VI型CRISPR-Cas蛋白或其活性变体或片段。如本文所用，术语“VI型CRISPR-Cas蛋白”、“VI型CRISPR-Cas效应蛋白”或“Cas13”是指不需要tracrRNA且包含两个切割RNA的HEPN结构域的CRISPR-Cas效应蛋白。As used herein, the term "V-type CRISPR-Cas protein", "V-type CRISPR-Cas effector protein" or "Cas12" refers to a CRISPR-Cas effector protein that cuts dsDNA and comprises a single RuvC nuclease domain or a split RuvC nuclease domain and lacks an HNH domain. In other embodiments, the CRISPR-Cas protein is a naturally occurring VI-type CRISPR-Cas protein or an active variant or fragment thereof. As used herein, the term "VI-type CRISPR-Cas protein", "VI-type CRISPR-Cas effector protein" or "Cas13" refers to a CRISPR-Cas effector protein that does not require tracrRNA and comprises two HEPN domains that cut RNA.

术语“gRNA”是指与靶核苷酸序列具有足够互补性以与靶序列杂交并指导相关核酸酶与靶核苷酸序列的序列特异性结合的核苷酸序列。对于CRISPR-Cas酶，相应的引导RNA是一个或多个RNA分子(通常是一个或两个)，它们可以结合Cas酶并指导Cas酶结合特定的靶核苷酸序列，并且在其中 Cas酶具有切口酶或核酸酶活性的那些情况下还切割靶核苷酸序列。The term "gRNA" refers to a nucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and guide the sequence-specific binding of the associated nuclease to the target nucleotide sequence. For CRISPR-Cas enzymes, the corresponding guide RNA is one or more RNA molecules (usually one or two) that can bind to the Cas enzyme and guide the Cas enzyme to bind to a specific target nucleotide sequence, and in which In those cases where the Cas enzyme has nickase or nuclease activity, it also cleaves the target nucleotide sequence.

[制备方法][Preparation method]

下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

本发明中，“适量的”指的是所加入溶剂量或药品量可调范围较大，且对合成结果影响较小，可以不作具体限定。In the present invention, "appropriate amount" means that the amount of the added solvent or the amount of the drug can be adjusted in a wide range and has little effect on the synthesis result, and no specific limitation is made.

在下述的实施例中，所用溶剂和药品均为分析纯或化学纯；溶剂在使用前均经过重新蒸馏；无水溶剂均按照标准方法或文献方法进行处理。In the following examples, all solvents and chemicals used were analytically pure or chemically pure; all solvents were redistilled before use; and all anhydrous solvents were treated according to standard methods or literature methods.

实施例Example

实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

实施例1：4-[7-丁基-17-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-25-乙基-8,14,20-三氧亚基-19,25-二氮杂-9,15-二氧杂-21-硫杂二十七烷-19-基]丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯的合成
Example 1: Synthesis of 4-[7-butyl-17-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-25-ethyl-8,14,20-trioxy-19,25-diaza-9,15-dioxa-21-thiaheptacosan-19-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester

步骤1：化合物1-2的合成Step 1: Synthesis of compound 1-2

在一个1000毫升的圆底烧瓶中分别加入5-羟甲基-2,2-二甲基-1,3-二恶烷(40.00g,273.62mmol,1.0eq)，二氯甲烷(400mL)，碳酸氢钠(57.47g,684.06mmol,2.5eq),戴斯-马丁氧化剂(127.66g,300.99mmol,1.1eq)。0摄氏度下反应2小时后缓慢加入1000毫升饱和硫代硫酸钠溶液，用二氯甲烷萃取(500mL×2)，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物2,2-二甲基-1,3-二氧杂环己烷-5-甲醛(20.10g,产率50.7％)。In a 1000 ml round-bottom flask, 5-hydroxymethyl-2,2-dimethyl-1,3-dioxane (40.00 g, 273.62 mmol, 1.0 eq), dichloromethane (400 mL), sodium bicarbonate (57.47 g, 684.06 mmol, 2.5 eq), and Dess-Martin periodinane (127.66 g, 300.99 mmol, 1.1 eq) were added respectively. After reacting at 0 degrees Celsius for 2 hours, 1000 ml of saturated sodium thiosulfate solution was slowly added, and the mixture was extracted with dichloromethane (500 mL×2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound 2,2-dimethyl-1,3-dioxane-5-carboxaldehyde (20.10 g, yield 50.7%).

步骤2：化合物1-4的合成Step 2: Synthesis of Compound 1-4

在一个300毫升的三口烧瓶中依次加入3-氯-1-二乙氨基丙烷(21.00g,140.32mmol,1.0eq)，硫脲(10.89g,143.12mmol,1.0eq)和200毫升乙醇。80摄氏度下反应过夜。减压浓缩得粗品化合物3-(二乙基氨基)丙-1-硫醇，可直接用于下一步反应。In a 300 ml three-necked flask, add 3-chloro-1-diethylaminopropane (21.00 g, 140.32 mmol, 1.0 eq), thiourea (10.89 g, 143.12 mmol, 1.0 eq) and 200 ml of ethanol in sequence. React at 80 degrees Celsius overnight. Concentrate under reduced pressure to obtain a crude compound 3-(diethylamino)propane-1-thiol, which can be directly used in the next step.

步骤3：化合物1-7的合成Step 3: Synthesis of Compound 1-7

在一个250毫升的圆底烧瓶中加入5-羟基戊酸苄基酯(5.00g,24.01mmol,1.0eq)，2-丁基辛酸(7.21g,36.01mmol,1.5eq)，100毫升二氯甲烷和4-二甲氨基吡啶(2.93g,24.01mmol,1.0eq)，最后加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(9.20g,48.02mmol,2.0eq)。室温下反应4小时，加入100毫升水稀释，用二氯甲烷萃取(100mL x 2)，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物2-丁基辛酸-5-(苄基氧基)-5-氧亚基戊基酯(4.50g,产率48.0％)。In a 250 ml round-bottom flask, 5-hydroxypentanoic acid benzyl ester (5.00 g, 24.01 mmol, 1.0 eq), 2-butyloctanoic acid (7.21 g, 36.01 mmol, 1.5 eq), 100 ml of dichloromethane and 4-dimethylaminopyridine (2.93 g, 24.01 mmol, 1.0 eq) were added, and finally 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.20 g, 48.02 mmol, 2.0 eq) was added. The mixture was reacted at room temperature for 4 hours, diluted with 100 ml of water, extracted with dichloromethane (100 mL x 2), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 2-butyloctanoic acid-5-(benzyloxy)-5-oxydipentyl ester (4.50 g, yield 48.0%).

步骤4：化合物1-8的合成Step 4: Synthesis of Compound 1-8

在一个50毫升得圆底烧瓶中加入化合物1-7(2.00g,5.12mmol,1.0eq)，甲醇(10mL),四氢呋喃(10mL)，最后加入Pd/C(545.0mg,0.51mmol,0.1eq,10％纯度)。一个大气压的氢气氛围下室温反应16小时，过滤浓缩得化合物5-[(2-丁基-1-氧亚基辛基)氧基]戊酸(1.54g,产率100％)。Compound 1-7 (2.00 g, 5.12 mmol, 1.0 eq), methanol (10 mL), tetrahydrofuran (10 mL), and finally Pd/C (545.0 mg, 0.51 mmol, 0.1 eq, 10% purity) were added to a 50 ml round-bottom flask. The mixture was reacted at room temperature for 16 hours under an atmospheric pressure of hydrogen, and the mixture was filtered and concentrated to obtain compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid (1.54 g, yield 100%).

步骤5：化合物1-10的合成Step 5: Synthesis of Compound 1-10

在一个2000毫升的圆底烧瓶中依次加入己-2-炔-1-醇(65.00g,662.28mmol,1eq)，二氯甲烷(1300mL)，吡啶(52.39g,662.28mmol,1.0eq)，0摄氏度下缓慢滴加三溴化磷(215.13g,794.74mmol,1.2equiv.)，滴加完毕后升至室温反应16小时。加入500毫升水淬灭，分别用500毫升二氯甲烷萃取两次，合并有机相，饱和碳酸氢钠溶液洗涤，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物1-溴己-2-炔(24.20g,产率22.5％)。In a 2000 ml round-bottom flask, add hex-2-yn-1-ol (65.00 g, 662.28 mmol, 1 eq), dichloromethane (1300 mL), pyridine (52.39 g, 662.28 mmol, 1.0 eq), slowly add phosphorus tribromide (215.13 g, 794.74 mmol, 1.2 equiv.) at 0 degrees Celsius, and warm to room temperature to react for 16 hours after the addition is complete. Add 500 ml of water to quench, extract twice with 500 ml of dichloromethane, combine the organic phases, wash with saturated sodium bicarbonate solution, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and obtain compound 1-bromohex-2-yn (24.20 g, yield 22.5%) by column chromatography.

步骤6：化合物1-12的合成Step 6: Synthesis of Compound 1-12

在一个500毫升的三口瓶中依次加入1-溴-2-己炔(44.00g,273.22mmol,1.0eq)，N,N-二甲基甲酰胺(240mL)，炔丙醇(19.91g,355.19mmol,1.3equiv.)，碘化亚铜(67.65g,355.19mmol,1.3equiv.)，碳酸铯(115.73g,355.19mmol,1.3equiv.)，碘化钠(53.24g,355.19mmol,1.3equiv.)，室温氮气氛围下反应16小时。反应液用500毫升饱和氯化铵溶液淬灭，乙酸乙酯萃取(500mL x 2)，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥。减压浓缩，柱层析得化合物壬-2,5-二炔-1-醇(24.00g,产率 64.5％)。MS:m/z[M+H]+＝137.0。In a 500 ml three-necked flask, 1-bromo-2-hexyne (44.00 g, 273.22 mmol, 1.0 eq), N,N-dimethylformamide (240 mL), propargyl alcohol (19.91 g, 355.19 mmol, 1.3 equiv.), cuprous iodide (67.65 g, 355.19 mmol, 1.3 equiv.), cesium carbonate (115.73 g, 355.19 mmol, 1.3 equiv.), sodium iodide (53.24 g, 355.19 mmol, 1.3 equiv.) were added in sequence, and reacted at room temperature under nitrogen atmosphere for 16 hours. The reaction solution was quenched with 500 ml saturated ammonium chloride solution, extracted with ethyl acetate (500 mL x 2), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure and column chromatography was performed to obtain the compound nonane-2,5-diyn-1-ol (24.00 g, yield 64.5%). MS: m/z[M+H] + =137.0.

步骤7：化合物1-13的合成Step 7: Synthesis of Compound 1-13

在一个500毫升的圆底烧瓶中加入四水合醋酸镍(14.02g,79.30mmol,0.9eq)，乙醇(240mL)，0摄氏度下加入硼氢化钠(2.95g,70.49mmol,0.8eq)，搅拌30分钟后加入乙二胺(21.17g,352.44mmol,4.0eq)，然后缓慢滴加溶解有化合物1-12(12.00g,88.11mmol,1.0eq)的乙醇(60mL)溶液，氢气置换三次后，0摄氏度下反应4小时。反应用饱和氯化铵淬灭，乙酸乙酯萃取(500mL x 2)，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物(2Z,5Z)-壬-2,5-二烯-1-醇(8.00g,产率64.7％)。In a 500 ml round-bottom flask, nickel acetate tetrahydrate (14.02 g, 79.30 mmol, 0.9 eq) and ethanol (240 mL) were added, sodium borohydride (2.95 g, 70.49 mmol, 0.8 eq) was added at 0 degrees Celsius, and ethylenediamine (21.17 g, 352.44 mmol, 4.0 eq) was added after stirring for 30 minutes, and then a solution of compound 1-12 (12.00 g, 88.11 mmol, 1.0 eq) in ethanol (60 mL) was slowly added dropwise, and hydrogen was replaced three times, and the mixture was reacted at 0 degrees Celsius for 4 hours. The reaction was quenched with saturated ammonium chloride, extracted with ethyl acetate (500 mL x 2), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound (2Z, 5Z)-nona-2,5-diene-1-ol (8.00 g, yield 64.7%).

步骤8：化合物1-15的合成Step 8: Synthesis of Compound 1-15

在一个250毫升的圆底烧瓶中加入化合物1-13(8.00g,57.05mmol,1.0eq)，4-({[(2-甲基丙-2-基)氧基]羰基}氨基)丁酸(17.39g,85.58mmol,1.5equiv.)，二氯甲烷(180mL)，4-二甲氨基吡啶(6.97g,57.05mmol,1.0eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(21.87g,114.10mmol,2.0eq)。室温下反应16小时，加入100毫升水稀释，然后分别用300毫升二氯甲烷萃取两次。合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物4-({[(2-甲基丙-2-基)氧基]羰基}氨基)丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(9.00g,产率48.5％)，MS:m/z[M+H]+＝326.0。In a 250 ml round-bottom flask, compound 1-13 (8.00 g, 57.05 mmol, 1.0 eq), 4-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)butyric acid (17.39 g, 85.58 mmol, 1.5 equiv.), dichloromethane (180 mL), 4-dimethylaminopyridine (6.97 g, 57.05 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.87 g, 114.10 mmol, 2.0 eq) were added. The mixture was reacted at room temperature for 16 hours, diluted with 100 ml of water, and then extracted twice with 300 ml of dichloromethane respectively. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound 4-({[(2-methylpropan-2-yl)oxy]carbonyl}amino)butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (9.00 g, yield 48.5%), MS: m/z[M+H] + =326.0.

步骤9：化合物1-16的合成Step 9: Synthesis of Compound 1-16

在一个250毫升的烧瓶中加入化合物1-15(9.00g,27.65mmol,1.0eq)，盐酸乙酸乙酯(150mL,4M)。室温下搅拌4小时，减压浓缩得化合物4-氨基丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(7.24g,产率100％)。MS:m/z[M+H]+＝226.0。Compound 1-15 (9.00 g, 27.65 mmol, 1.0 eq) and ethyl acetate hydrochloride (150 mL, 4 M) were added to a 250 ml flask. The mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure to obtain compound 4-aminobutyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (7.24 g, yield 100%). MS: m/z[M+H] + =226.0.

步骤10：化合物1-17的合成Step 10: Synthesis of Compound 1-17

在一个250毫升的烧瓶中加入化合物1-16(6.00g,26.63mmol,1.0eq)，四氢呋喃(120mL)，化合物1-2(3.45g,23.96mmol,0.9eq)，醋酸硼氢化钠(5.06g,0.9eq)，室温下反应4小时。加入100毫升水淬灭，反应液分别用200毫升二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物4-{[(2,2-二甲基-1,3-二氧杂环己-5-基)甲基]氨基}丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(4.00g,产率42.5％)。MS:m/z[M+H]+＝354.0。Compound 1-16 (6.00 g, 26.63 mmol, 1.0 eq), tetrahydrofuran (120 mL), compound 1-2 (3.45 g, 23.96 mmol, 0.9 eq), sodium acetate borohydride (5.06 g, 0.9 eq) were added to a 250 mL flask and reacted at room temperature for 4 hours. 100 mL of water was added to quench the reaction solution, and the reaction solution was extracted twice with 200 mL of dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound 4-{[(2,2-dimethyl-1,3-dioxan-5-yl)methyl]amino}butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (4.00 g, yield 42.5%). MS: m/z[M+H] + =354.0.

步骤11：化合物1-18的合成Step 11: Synthesis of Compound 1-18

在一个100毫升的圆底烧瓶中加入化合物1-17(4.00g,11.31mmol,1.0eq)，二氯甲烷(80mL)，三乙胺(5.73g,56.57mmol,5.0eq)，羰基二咪唑(7.34g,45.26mmol,4.0eq)。室温下反应16小时，加入100毫升水稀释，分别用100毫升二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物4-{[(2,2-二甲基-1,3-二氧杂环己-5-基)甲基](咪唑-1-基羰基)氨基}丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(2.00g,产率39.5％)。MS:m/z[M+H]+＝448.0。Compound 1-17 (4.00 g, 11.31 mmol, 1.0 eq), dichloromethane (80 mL), triethylamine (5.73 g, 56.57 mmol, 5.0 eq), carbonyldiimidazole (7.34 g, 45.26 mmol, 4.0 eq) were added to a 100 mL round-bottom flask. The mixture was reacted at room temperature for 16 hours, diluted with 100 mL of water, extracted twice with 100 mL of dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain compound 4-{[(2,2-dimethyl-1,3-dioxan-5-yl)methyl](imidazol-1-ylcarbonyl)amino}butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (2.00 g, yield 39.5%). MS: m/z[M+H] + =448.0.

步骤12：化合物1-19的合成 Step 12: Synthesis of Compound 1-19

在一个250毫升的圆底烧瓶中加入化合物1-18(2.00g,4.47mmol,1.0eq)，二氯甲烷(100mL)，-10摄氏度下缓慢滴加三氟甲磺酸甲酯(0.62g,3.80mmol,0.85eq)，滴加完毕后-10摄氏度下搅拌1小时，缓慢滴加化合物1-4(1.65g,11.17mmol,2.5eq)。然后-10摄氏度下反应2小时。加入100毫升水稀释，反应溶液分别用100毫升二氯甲烷萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物4-[1-(2,2-二甲基-1,3-二氧杂环己-5-基)-8-乙基-3-氧亚基-2,8-二氮杂-4-硫杂癸-2-基]丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(1.8g,产率76.47％)。MS:m/z[M+H]+＝527.0。Compound 1-18 (2.00 g, 4.47 mmol, 1.0 eq) and dichloromethane (100 mL) were added to a 250 mL round-bottom flask, and methyl trifluoromethanesulfonate (0.62 g, 3.80 mmol, 0.85 eq) was slowly added dropwise at -10 degrees Celsius. After the addition was complete, the mixture was stirred at -10 degrees Celsius for 1 hour, and compound 1-4 (1.65 g, 11.17 mmol, 2.5 eq) was slowly added dropwise. The mixture was then reacted at -10 degrees Celsius for 2 hours. 100 ml of water was added to dilute, and the reaction solution was extracted twice with 100 ml of dichloromethane respectively. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 4-[1-(2,2-dimethyl-1,3-dioxane-5-yl)-8-ethyl-3-oxygen-2,8-diaza-4-thiadecane-2-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (1.8 g, yield 76.47%). MS: m/z[M+H] + =527.0.

步骤13：化合物1-20的合成Step 13: Synthesis of Compound 1-20

在一个50毫升得烧瓶中加入化合物1-19(1.80g,3.42mmol,1.0eq)，甲醇(18mL)，对甲苯磺酸(0.29g,1.71mmol,0.5eq)。室温下搅拌2小时。减压浓缩，柱层析得黄色油状化合物4-[10-乙基-1-羟基-2-(羟基甲基)-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-4-基]丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(0.40g,产率24.0％).MS:m/z[M+H]+＝487.1。Compound 1-19 (1.80 g, 3.42 mmol, 1.0 eq), methanol (18 mL), p-toluenesulfonic acid (0.29 g, 1.71 mmol, 0.5 eq) were added to a 50 ml flask. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and subjected to column chromatography to obtain a yellow oily compound, 4-[10-ethyl-1-hydroxy-2-(hydroxymethyl)-5-oxyylidene-4,10-diaza-6-thiadodecane-4-yl]butanoic acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (0.40 g, yield 24.0%). MS: m/z[M+H] + =487.1.

步骤14：化合物1的合成Step 14: Synthesis of Compound 1

在一个50毫升的烧瓶中加入化合物1-20(275.0mg,0.56mmol,1.0eq)，化合物1-8(424.4mg,1.41mmol,2.5equiv.)，4-二甲氨基吡啶(69.0mg,0.56mmol,1.0eq)，乙腈(5mL)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(324.9mg,1.69mmol,3.0eq)。室温下反应16小时，加入50毫升水稀释，分别用100毫升乙酸乙酯萃取两次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，柱层析得化合物4-[7-丁基-17-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-25-乙基-8,14,20-三氧亚基-19,25-二氮杂-9,15-二氧杂-21-硫杂二十七烷-19-基]丁酸-(2Z,5Z)-壬-2,5-二烯-1-基酯(220.5mg,37.1％)。MS:m/z[M+H]+＝1052.2。1H NMR(300MHz,CDCl3)δ5.732-5.531(m,2H),5.500-5.246(m,2H),4.694(d,J＝6.6Hz,2H),4.083(d,J＝5.1Hz,8H),3.68-3.314(m,4H),2.932-2.884(m,4H),2.516-2.458(m,7H),2.394-2.299(m,8H),2.046-2.022(m,2H),1.731-1.717(m,2H),1.695-1.684(m,8H),1.673-1.626(m,6H),1.597-1.552(m,6H),1.467-1.307(m,24H),1.032-0.932(m,6H),0.978-0.854(m,15H)。In a 50 ml flask were added compound 1-20 (275.0 mg, 0.56 mmol, 1.0 eq), compound 1-8 (424.4 mg, 1.41 mmol, 2.5 equiv.), 4-dimethylaminopyridine (69.0 mg, 0.56 mmol, 1.0 eq), acetonitrile (5 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (324.9 mg, 1.69 mmol, 3.0 eq). The mixture was reacted at room temperature for 16 hours, diluted with 50 ml of water, extracted twice with 100 ml of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography was performed to obtain the compound 4-[7-butyl-17-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-25-ethyl-8,14,20-trioxy-19,25-diaza-9,15-dioxa-21-thiacosadecan-19-yl]butyric acid-(2Z,5Z)-nona-2,5-dien-1-yl ester (220.5 mg, 37.1%). MS: m/z[M+H] + =1052.2. 1 H NMR (300 MHz, CDCl 3 )δ5.732-5.531(m,2H),5.500-5.246(m,2H),4.694(d,J＝6.6Hz,2H),4.083(d,J＝5.1Hz,8H),3.68-3.314(m,4H),2.932-2.884(m,4H),2.516-2.458 (m,7H),2.394-2.299(m,8H ),2.046-2.022(m,2H),1.731-1.717(m,2H),1.695-1.684(m,8H),1.673-1.626(m,6H),1.597-1.552(m,6H),1.467-1.307(m,24H),1.032-0.932 (m,6H),0.978-0.854(m,15H).

实施例2：5-[(2-丁基-1-氧亚基辛基)氧基]戊酸-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-乙基-4-己基-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯的合成
Example 2: Synthesis of 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-hexyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester

步骤1：化合物2-3的合成Step 1: Synthesis of compound 2-3

在一个100毫升的圆底烧瓶中依次加入甲烷磺酸-(2,2-二甲基-1,3-二氧杂环己-5-基)甲基酯(2.00g,8.92mmol,1.0eq)，正己胺(4.51g,44.60mmol,5.0eq)，碳酸钾(1.85g,13.38mmol,1.5eq)，碘化钾(1.63g,9.81mmol,1.1eq)，乙腈(30mL)，80摄氏度下反应16个小时，减压浓缩，柱层析得化合物[(2,2-二甲基-1,3-二氧杂环己-5-基)甲基](己基)胺(1.80g,产率88.0％)。MS:m/z[M+H]+＝230.2。In a 100 ml round-bottom flask, methanesulfonic acid-(2,2-dimethyl-1,3-dioxan-5-yl)methyl ester (2.00 g, 8.92 mmol, 1.0 eq), n-hexylamine (4.51 g, 44.60 mmol, 5.0 eq), potassium carbonate (1.85 g, 13.38 mmol, 1.5 eq), potassium iodide (1.63 g, 9.81 mmol, 1.1 eq), acetonitrile (30 mL) were added in sequence, reacted at 80 degrees Celsius for 16 hours, concentrated under reduced pressure, and column chromatography was performed to obtain the compound [(2,2-dimethyl-1,3-dioxan-5-yl)methyl](hexyl)amine (1.80 g, yield 88.0%). MS: m/z[M+H] + =230.2.

步骤2：化合物2-4的合成Step 2: Synthesis of compound 2-4

在一个100毫升的圆底烧瓶中依次加入化合物2-3(1.80g,7.85mmol,1.0eq)，二氯甲烷(20mL)和三乙胺(3.97g,39.25mmol,5eq)，然后分批加入羰基二咪唑(3.82g,23.55mmol,3.0eq)，反应液在室温下搅拌3小时，减压浓缩，加入50毫升水稀释，分别用50毫升乙酸乙酯萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物N-[(2,2-二甲基-1,3-二氧杂环己-5-基)甲基]-N-己基咪唑-1-甲酰胺(2.30g,产率90.6％)。MS:m/z[M+H]+＝324.2。Compound 2-3 (1.80 g, 7.85 mmol, 1.0 eq), dichloromethane (20 mL) and triethylamine (3.97 g, 39.25 mmol, 5 eq) were added to a 100 mL round-bottom flask in sequence, and then carbonyldiimidazole (3.82 g, 23.55 mmol, 3.0 eq) was added in batches. The reaction solution was stirred at room temperature for 3 hours, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 50 mL of ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound N-[(2,2-dimethyl-1,3-dioxan-5-yl)methyl]-N-hexylimidazole-1-carboxamide (2.30 g, yield 90.6%). MS: m/z[M+H] + =324.2.

步骤3：化合物2-5的合成Step 3: Synthesis of Compound 2-5

在一个50毫升的圆底烧瓶中加入化合物2-4(1.00g,3.09mmol,1.0eq)和二氯甲烷(15mL)，冰浴下加入三氟甲磺酸甲酯(0.48g,2.94mmol,0.95eq)。反应液继续在冰浴下搅拌0.5小时后加入化合物1-4(0.68g,4.65mmol,1.5eq)和四甲基乙二胺(1.08g,9.30mmol,3.0eq)，然后在冰浴下反应2.5小时。用50毫升二氯甲烷分别萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物{[(2,2-二甲基-1,3-二氧杂环己-5-基)甲基](己基)氨基}甲烷硫代酸-S-[3-(二乙基氨基)丙基]酯(0.49g,产率39.1％)。MS:m/z[M+H]+＝403.3。Compound 2-4 (1.00 g, 3.09 mmol, 1.0 eq) and dichloromethane (15 mL) were added to a 50 mL round-bottom flask, and methyl trifluoromethanesulfonate (0.48 g, 2.94 mmol, 0.95 eq) was added under ice bath. The reaction solution was stirred under ice bath for 0.5 hours, and then compound 1-4 (0.68 g, 4.65 mmol, 1.5 eq) and tetramethylethylenediamine (1.08 g, 9.30 mmol, 3.0 eq) were added, and then reacted under ice bath for 2.5 hours. The mixture was extracted three times with 50 mL of dichloromethane, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound {[(2,2-dimethyl-1,3-dioxan-5-yl)methyl](hexyl)amino}methanethioic acid-S-[3-(diethylamino)propyl]ester (0.49 g, yield 39.1%). MS: m/z [M+H] + = 403.3.

步骤4：化合物2-6的合成Step 4: Synthesis of Compound 2-6

在一个50毫升的圆底烧瓶中加入化合物2-5(0.49g,1.21mmol,1.0eq)和10毫升甲醇，再加入对甲基苯磺酸(0.42g,2.42mmol,2.0eq)。反应液在室温下搅拌2小时后，浓缩得化合物{己基[3-羟基-2-(羟基甲基)丙基]氨基}甲烷硫代酸-S-[3-(二乙基氨基)丙基]酯(0.44g,产率100％，粗品)，无需进一步纯化，可直接用于下一步。MS:m/z[M+H]+＝363.3。Compound 2-5 (0.49 g, 1.21 mmol, 1.0 eq) and 10 ml methanol were added to a 50 ml round-bottom flask, followed by p-toluenesulfonic acid (0.42 g, 2.42 mmol, 2.0 eq). The reaction solution was stirred at room temperature for 2 hours and concentrated to obtain compound {hexyl [3-hydroxy-2- (hydroxymethyl) propyl] amino} methanethioic acid-S- [3- (diethylamino) propyl] ester (0.44 g, yield 100%, crude product), which was used directly in the next step without further purification. MS: m/z [M+H] + = 363.3.

步骤5：化合物2的合成Step 5: Synthesis of Compound 2

在一个25毫升的圆底烧瓶中依次加入化合物1-8(1.09g,3.63mmol,3.0eq)，N,N-二异丙基乙胺(0.78g,6.05mmol,5.0eq)，4-二甲氨基吡啶(0.15g,1.21mmol,1.0eq)，二氯甲烷(10mL)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.70g,3.63mmol,3.0eq)，室温下反应0.5小时后，加入化合物2-6(0.44g,1.21mmol,1.0eq)，反应液在室温下继续反应15.5小时，减压浓缩，用30毫升水稀释，分别用20毫升石油醚萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物5-[(2-丁基-1-氧亚基辛基)氧基]戊酸-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-乙基-4-己基-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯(430.0mg,产率38.2％)。MS:m/z[M+H]+＝927.7。1H NMR(300MHz,CDCl3)δ4.20-4.00(m,8H),3.49-3.10(m,4H),2.90-2.79(m,2H),2.60-2.23 (m,12H),1.80-1.52(m,17H),1.50-1.43(m,4H),1.30-1.11(m,30H),1.08-0.98(m,6H),0.97-0.80(m,15H)。In a 25 ml round-bottom flask, compound 1-8 (1.09 g, 3.63 mmol, 3.0 eq), N,N-diisopropylethylamine (0.78 g, 6.05 mmol, 5.0 eq), 4-dimethylaminopyridine (0.15 g, 1.21 mmol, 1.0 eq), dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.70 g, 3.63 mmol, 3.0 eq) were added in sequence. After reacting at room temperature for 0.5 hours, compound 2-6 (0.44 g, 1.21 mmol, 5.0 eq) was added. mol, 1.0eq), the reaction solution was continued to react at room temperature for 15.5 hours, concentrated under reduced pressure, diluted with 30 ml of water, extracted three times with 20 ml of petroleum ether respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-hexyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (430.0 mg, yield 38.2%). MS: m/z[M+H] + =927.7. 1 H NMR (300MHz, CDCl 3 ) δ4.20-4.00(m,8H),3.49-3.10(m,4H),2.90-2.79(m,2H),2.60-2.23 (m,12H),1.80-1.52(m,17H),1.50-1.43(m,4H),1.30-1.11(m,30H),1.08-0.98(m,6H),0.97-0.80(m,15H).

实施例3：5-[(2-丁基-1-氧亚基辛基)氧基]戊酸-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-乙基-4-(3-羟基丙基)-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯的合成
Example 3: Synthesis of 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-(3-hydroxypropyl)-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester

步骤1：化合物3-2的合成Step 1: Synthesis of compound 3-2

在100毫升二氯甲烷中依次加入3-氨基丙-1-醇(5.00g,66.57mmol,1.0eq)，咪唑(11.33g,166.42mmol,2.5eq)，然后分批加入叔丁基二苯基氯硅烷(27.45g,99.85mmol,1.5eq)，反应液在室温下搅拌16小时，减压浓缩，柱层析得化合物3-{[(2-甲基丙-2-基)二苯基甲硅基]氧基}丙-1-胺(19.00g,产率91.0％)。MS:m/z[M+H]+＝314.2。3-aminopropan-1-ol (5.00 g, 66.57 mmol, 1.0 eq) and imidazole (11.33 g, 166.42 mmol, 2.5 eq) were added to 100 ml of dichloromethane in sequence, and then tert-butyldiphenylsilyl chloride (27.45 g, 99.85 mmol, 1.5 eq) was added in batches. The reaction solution was stirred at room temperature for 16 hours, concentrated under reduced pressure, and column chromatography was performed to obtain compound 3-{[(2-methylpropan-2-yl)diphenylsilyl]oxy}propan-1-amine (19.00 g, yield 91.0%). MS: m/z[M+H] + =314.2.

步骤2：化合物3-3的合成Step 2: Synthesis of compound 3-3

在50毫升乙腈中依次加入化合物2-1(3.00g,13.38mmol,1.0eq)，化合物3-2(8.39g,26.76mmol,2.0eq)，碳酸钾(3.70g,26.76mmol,2.0eq)，碘化钾(2.22g,13.38mmol,1.0eq)，反应液在80摄氏度下反应16小时。减压浓缩，用100毫升水稀释，分别用50毫升乙酸乙酯萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物9-(2,2-二甲基-1,3-二氧杂环己-5-基)-2,2-二甲基-3,3-二苯基-8-氮杂-4-氧杂-3-硅杂壬烷(4.70g,产率79.5％)。MS:m/z[M+H]+＝442.3。Compound 2-1 (3.00 g, 13.38 mmol, 1.0 eq), compound 3-2 (8.39 g, 26.76 mmol, 2.0 eq), potassium carbonate (3.70 g, 26.76 mmol, 2.0 eq), potassium iodide (2.22 g, 13.38 mmol, 1.0 eq) were added to 50 ml of acetonitrile in sequence, and the reaction solution was reacted at 80 degrees Celsius for 16 hours. The mixture was concentrated under reduced pressure, diluted with 100 ml of water, extracted three times with 50 ml of ethyl acetate respectively, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 9-(2,2-dimethyl-1,3-dioxacyclohexane-5-yl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanonane (4.70 g, yield 79.5%). MS: m/z [M+H] + = 442.3.

步骤3：化合物3-4的合成Step 3: Synthesis of compound 3-4

在50毫升二氯甲烷中依次加入化合物3-3(4.70g,10.64mmol,1.0eq)，三乙胺(5.38g,53.20mmol,5.0eq)，然后分批加入羰基二咪唑(5.18g,31.92mmol,3.0eq)。反应液在25摄氏度下反应3小时后，用二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物N-[(2,2-二甲基-1,3-二氧杂环 己-5-基)甲基]-N-(2,2-二甲基-3,3-二苯基-4-氧杂-3-硅杂庚-7-基)咪唑-1-甲酰胺(4.40g,产率77.2％)。MS:m/z[M+H]+＝536.3。Compound 3-3 (4.70 g, 10.64 mmol, 1.0 eq) and triethylamine (5.38 g, 53.20 mmol, 5.0 eq) were added to 50 ml of dichloromethane in sequence, and then carbonyldiimidazole (5.18 g, 31.92 mmol, 3.0 eq) was added in batches. After the reaction solution was reacted at 25 degrees Celsius for 3 hours, it was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound N-[(2,2-dimethyl-1,3-dioxacyclopentyl)-1,3-dioxacyclopentyl] ... [0134] N-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silaheptan-7-yl)imidazole-1-carboxamide (4.40 g, yield 77.2%). MS: m/z [M+H] + = 536.3.

步骤4：化合物3-5的合成Step 4: Synthesis of Compound 3-5

在20毫升得二氯甲烷中加入化合物3-4(2.0g,3.73mmol,1.0eq)，冰浴下加入三氟甲磺酸甲酯(0.61g,3.73mmol,1.0eq)，搅拌1小时后，加入四甲基乙二胺(1.30g,11.19mmol,3.0eq)，化合物1-4(0.82g,5.59mmol,1.5eq)，反应液在冰浴下继续反应2小时后，减压浓缩，柱层析得化合物[9-(2,2-二甲基-1,3-二氧杂环己-5-基)-2,2-二甲基-3,3-二苯基-8-氮杂-4-氧杂-3-硅杂壬-8-基]甲烷硫代酸-S-[3-(二乙基氨基)丙基]酯(1.8g,产率78.5％)。MS:m/z[M+H]+＝615.4。Compound 3-4 (2.0 g, 3.73 mmol, 1.0 eq) was added to 20 ml of dichloromethane, and methyl trifluoromethanesulfonate (0.61 g, 3.73 mmol, 1.0 eq) was added under ice bath. After stirring for 1 hour, tetramethylethylenediamine (1.30 g, 11.19 mmol, 3.0 eq) and compound 1-4 (0.82 g, 5.59 mmol, 1.5 eq) were added. The reaction solution was continued to react under ice bath for 2 hours, and then concentrated under reduced pressure. Column chromatography was performed to obtain compound [9-(2,2-dimethyl-1,3-dioxane-5-yl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanonan-8-yl]methanethioic acid-S-[3-(diethylamino)propyl] ester (1.8 g, yield 78.5%). MS: m/z [M+H] + = 615.4.

步骤5：化合物3-6的合成Step 5: Synthesis of Compound 3-6

在10毫升甲醇中，加入化合物3-5(0.90g,1.46mmol,1.0eq)，对甲基苯磺酸(0.50g,2.92mmol,2.0eq)，反应液在室温下搅拌2小时后，减压浓缩，得粗品化合物[11-羟基-10-(羟基甲基)-2,2-二甲基-3,3-二苯基-8-氮杂-4-氧杂-3-硅杂十一烷-8-基]甲烷硫代酸-S-[3-(二乙基氨基)丙基]酯(0.84g,理论产率100％，粗品)。无需纯化，直接用于下一步。MS:m/z[M+H]+＝575.3。Compound 3-5 (0.90 g, 1.46 mmol, 1.0 eq) and p-toluenesulfonic acid (0.50 g, 2.92 mmol, 2.0 eq) were added to 10 ml of methanol. The reaction solution was stirred at room temperature for 2 hours and then concentrated under reduced pressure to obtain a crude compound [11-hydroxy-10-(hydroxymethyl)-2,2-dimethyl-3,3-diphenyl-8-aza-4-oxa-3-silanhexadecane-8-yl]methanethioic acid-S-[3-(diethylamino)propyl] ester (0.84 g, theoretical yield 100%, crude product). It was used directly in the next step without purification. MS: m/z[M+H] + =575.3.

步骤6：化合物3-7的合成Step 6: Synthesis of Compound 3-7

在20毫升的二氯甲烷中依次加入化合物1-8(1.32g,4.38mmol,3.0eq),N,N-二异丙基乙胺(0.57g,4.38mmol,3.0eq)，4-二甲氨基吡啶(0.36g,2.92mmol,2.0eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.84g,4.38mmol,3.0eq)，室温下反应0.5小时后，加入化合物3-6(0.84g,1.46mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物5-[(2-丁基-1-氧亚基辛基)氧基]戊酸-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-4-(2,2-二甲基-3,3-二苯基-4-氧杂-3-硅杂庚-7-基)-10-乙基-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯(1.40g,产率84.1％)。MS:m/z[M+H]+＝1139.8。Compound 1-8 (1.32 g, 4.38 mmol, 3.0 eq), N,N-diisopropylethylamine (0.57 g, 4.38 mmol, 3.0 eq), 4-dimethylaminopyridine (0.36 g, 2.92 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.84 g, 4.38 mmol, 3.0 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3-6 (0.84 g, 1.46 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-4-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanahept-7-yl)-10-ethyl-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (1.40 g, yield 84.1%). MS: m/z[M+H] + =1139.8.

步骤7：化合物3的合成Step 7: Synthesis of compound 3

在15毫升四氢呋喃中加入化合物3-7(300.0mg,0.26mmol,1.0eq)和四丁基氟化铵的四氢呋喃溶液(0.78mL,0.78mmol,1.0M,3.0eq)。反应液在室温下搅拌1小时，减压浓缩，加入50毫升水稀释，分别用30毫升石油醚萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物5-[(2-丁基-1-氧亚基辛基)氧基]戊酸-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-乙基-4-(3-羟基丙基)-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯(110.0mg,产率46.4％)。MS:m/z[M+H]+＝901.6。1H NMR(300MHz,CDCl3)δ4.25-4.00(m,8H),3.75-3.30(m,6H),2.98-2.78(m,2H),2.73-2.25(m,12H),1.80-1.50(m,16H),1.50-1.12(m,30H),1.10-0.98(m,6H),0.97-0.98(m,6H),0.97-0.75(m,12H)。Compound 3-7 (300.0 mg, 0.26 mmol, 1.0 eq) and tetrabutylammonium fluoride tetrahydrofuran solution (0.78 mL, 0.78 mmol, 1.0 M, 3.0 eq) were added to 15 mL of tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 30 mL of petroleum ether, combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-ethyl-4-(3-hydroxypropyl)-5-oxy-4,10-diaza-6-thiadodecane-1-yl ester (110.0 mg, yield 46.4%). MS: m/z[M+H] + =901.6. 1 H NMR (300MHz, CDCl 3 ) δ4.25-4.00(m,8H),3.75-3.30(m,6H),2.98-2.78(m,2H),2.73-2.25(m,12H),1.80-1.50(m,16H),1.50-1.12(m,30H),1.10- 0.98(m,6H),0.97-0.98(m,6H),0.97-0.75(m,12H).

实施例4：2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 4: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

步骤1：化合物4-1的合成Step 1: Synthesis of compound 4-1

在室温下，将5-[(2-丁基-1-氧亚基辛基)氧基]戊酸(12.46g,41.47mmol,2.1eq)、2-羟甲基丙烷-1，3-二醇(2.00g,18.85mmol,1.0eq)、4-二甲氨基吡啶(2.3g,18.85mmol,1eq)、N-(3-二甲氨基丙基)-N'-乙基碳化二亚胺盐酸盐(7.23g,37.70mmol,2.0eq)和N,N-二异丙基乙胺(7.31g,56.55mmol,3.0eq)加入到含有100毫升二氯甲烷的圆底烧瓶中，室温搅拌至反应4小时。反应液用乙酸乙酯萃取，盐水洗，无水硫酸钠干燥，过滤，浓缩，通过柱层析纯化得到2-丁基辛酸-18-丁基-8-(羟基甲基)-5,11,17-三氧亚基-6,10,16-三氧杂二十四烷-1-基酯(4.50g,产率35.6％)。At room temperature, 5-[(2-butyl-1-oxyoctyl)oxy]pentanoic acid (12.46 g, 41.47 mmol, 2.1 eq), 2-hydroxymethylpropane-1,3-diol (2.00 g, 18.85 mmol, 1.0 eq), 4-dimethylaminopyridine (2.3 g, 18.85 mmol, 1 eq), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (7.23 g, 37.70 mmol, 2.0 eq) and N,N-diisopropylethylamine (7.31 g, 56.55 mmol, 3.0 eq) were added to a round-bottom flask containing 100 ml of dichloromethane and stirred at room temperature for 4 hours. The reaction solution was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give 2-butyloctanoic acid-18-butyl-8-(hydroxymethyl)-5,11,17-trioxydeca-6,10,16-trioxa-tetracosane-1-yl ester (4.50 g, yield 35.6%).

步骤2：化合物4-2的合成Step 2: Synthesis of compound 4-2

在室温下，将化合物4-1(2.00g,2.98mmol,1.0eq)，三乙胺(0.90g,8.94mmol,3.0eq)，加入到30毫升二氯甲烷中，零摄氏度下缓慢加入甲基磺酸酐(0.78g,4.47mmol,1.5eq)，缓慢恢复至室温，反应4小时。将反应液加入水稀释，用二氯甲烷萃取3次，有机相合并，盐水洗，无水硫酸钠干燥，过滤，浓缩，通过柱层析纯化得到甲烷磺酸-12-丁基-2-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-5,11-二氧亚基-4,10-二氧杂十八烷-1-基酯(2.00g,产率89.6％)。At room temperature, compound 4-1 (2.00 g, 2.98 mmol, 1.0 eq) and triethylamine (0.90 g, 8.94 mmol, 3.0 eq) were added to 30 ml of dichloromethane, and methylsulfonic anhydride (0.78 g, 4.47 mmol, 1.5 eq) was slowly added at zero degrees Celsius, and the temperature was slowly restored to room temperature and reacted for 4 hours. The reaction solution was diluted with water, extracted with dichloromethane 3 times, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain methanesulfonic acid-12-butyl-2-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-5,11-dioxy-4,10-dioxahexadecan-1-yl ester (2.00 g, yield 89.6%).

步骤3：化合物4-3的合成Step 3: Synthesis of compound 4-3

在室温下，将化合物4-2(1.00g,1.34mmol,1.0eq)，苄胺(287.2mg,2.68mmol,2.0eq)，碳酸钾(555.6mg,4.02mmol,3.0eq),碘化钾(111.2mg,0.68mmol,0.5eq)，加到20毫升乙腈中，氮气保护，加热至90摄氏度，反应过夜。浓缩反应液，用水稀释，乙酸乙酯萃取三次，合并有机相，有机相用饱和食盐水洗，无水硫酸钠干燥，浓缩，柱层析纯化得2-丁基辛酸-4-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-7-氧亚基-1-苯基-2-氮杂-6-氧杂十一烷-11-基酯(400.0mg,产率39.4％)。MS:m/z[M+H]+＝760.6。 At room temperature, compound 4-2 (1.00 g, 1.34 mmol, 1.0 eq), benzylamine (287.2 mg, 2.68 mmol, 2.0 eq), potassium carbonate (555.6 mg, 4.02 mmol, 3.0 eq), potassium iodide (111.2 mg, 0.68 mmol, 0.5 eq) were added to 20 ml of acetonitrile, nitrogen was protected, heated to 90 degrees Celsius, and reacted overnight. The reaction solution was concentrated, diluted with water, extracted with ethyl acetate three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-4-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-7-oxy-1-phenyl-2-aza-6-oxaundecane-11-yl ester (400.0 mg, yield 39.4%). MS: m/z [M+H] + = 760.6.

步骤4：化合物4-4的合成Step 4: Synthesis of compound 4-4

将化合物4-3(400.0mg,0.52mmol,1.0eq)投入反应瓶，随后加入10毫升甲醇和钯炭(55.3mg,0.52mmol,0.1eq,10％purity)，在氢气氛围下室温搅拌过夜。反应液过滤浓缩通过柱层析纯化得到2-丁基辛酸-8-(氨基甲基)-18-丁基-5,11,17-三氧亚基-6,10,16-三氧杂二十四烷-1-基酯(246.0mg,产率70.0％)。MS:m/z[M+H]+＝670.5。Compound 4-3 (400.0 mg, 0.52 mmol, 1.0 eq) was put into a reaction flask, followed by addition of 10 ml of methanol and palladium carbon (55.3 mg, 0.52 mmol, 0.1 eq, 10% purity), and stirred overnight at room temperature under a hydrogen atmosphere. The reaction solution was filtered and concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-8-(aminomethyl)-18-butyl-5,11,17-trioxy-6,10,16-trioxa-tetracosane-1-yl ester (246.0 mg, yield 70.0%). MS: m/z[M+H] + =670.5.

步骤5：化合物4-5的合成Step 5: Synthesis of compound 4-5

在室温下，将化合物4-4(246.0mg,0.37mmol,1.0eq),4-硝基苯基氯甲酸酯(148.0mg,0.73mmol,2.0eq)和吡啶(174.0mg,2.20mmol,3.0eq)加入到反应瓶中，随后加入二氯甲烷(10mL)，氮气保护室温搅拌过夜。反应液用水洗，饱和食盐水洗，干燥，浓缩，柱层析纯化得到2-丁基辛酸-4-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-1-[(4-硝基苯基)氧基]-1,7-二氧亚基-2-氮杂-6-氧杂十一烷-11-基酯(182.0mg,59.4％)。MS:m/z[M+H]+＝835.5。At room temperature, compound 4-4 (246.0 mg, 0.37 mmol, 1.0 eq), 4-nitrophenyl chloroformate (148.0 mg, 0.73 mmol, 2.0 eq) and pyridine (174.0 mg, 2.20 mmol, 3.0 eq) were added to a reaction flask, followed by dichloromethane (10 mL), and stirred overnight at room temperature under nitrogen protection. The reaction solution was washed with water, washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-4-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-1-[(4-nitrophenyl)oxy]-1,7-dioxy-2-aza-6-oxaundecane-11-yl ester (182.0 mg, 59.4%). MS: m/z[M+H] + =835.5.

步骤6：化合物4的合成Step 6: Synthesis of compound 4

在室温下，将化合物4-5(182.0mg,0.22mmol,1.0eq)，3-(二乙氨基)丙烷-1-硫醇(48.0mg,1.33mmol,1.5eq)，4-二甲氨基吡啶(27.0mg,0.22mmol,1.0eq)，吡啶(35.0mg,0.44mmol,2.0eq)加入到10毫升乙腈中，室温搅拌4小时。反应液用乙酸乙酯洗，饱和食盐水洗，无水硫酸钠干燥，浓缩，柱层析纯化得2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯(70.0mg,产率38.0％)。MS:m/z[M+H]+＝843.6。1H NMR(300MHz,CDCl3)δ6.44(s,1H),4.40-4.20(s,1H),4.15-4.01(m,6H),3.45-3.20(m,1H),3.15-2.78(m,7H),2.45-2.03(m,8H),1.80-1.50(m,12H),1.48-1.05(m,38H),0.90-0.75(m,12H)。At room temperature, compound 4-5 (182.0 mg, 0.22 mmol, 1.0 eq), 3-(diethylamino)propane-1-thiol (48.0 mg, 1.33 mmol, 1.5 eq), 4-dimethylaminopyridine (27.0 mg, 0.22 mmol, 1.0 eq), pyridine (35.0 mg, 0.44 mmol, 2.0 eq) were added to 10 ml of acetonitrile and stirred at room temperature for 4 hours. The reaction solution was washed with ethyl acetate, saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester (70.0 mg, yield 38.0%). MS: m/z[M+H] + =843.6. 1 H NMR (300MHz, CDCl 3 ) δ6.44(s,1H),4.40-4.20(s,1H),4.15-4.01(m,6H),3.45-3.20(m,1H),3.15-2.78(m,7H),2.45-2.03(m,8H),1.80-1.50(m,1 2H),1.48-1.05(m,38H),0.90-0.75(m,12H).

实施例5：2-丁基辛酸-22-丁基-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-5,15,21-三氧亚基-10-氮杂-6,14,20-三氧杂二十八烷-1-基酯的合成
Example 5: Synthesis of 2-butyloctanoate-22-butyl-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiooctan-1-yl)-5,15,21-trioxy-10-aza-6,14,20-trioxaoctacosane-1-yl ester

步骤1：化合物5的合成Step 1: Synthesis of compound 5

在20毫升的二氯甲烷中依次加入化合物1-8(0.17g,0.56mmol,2.0eq)，N,N-二异丙基乙胺(0.11g,0.84mmol,3.0eq)，4-二甲氨基吡啶(34.0m g,0.28mmol,1.0eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.11g,0.56mmol,2.0eq)，室温下反应0.5小时后，加入化合物3(0.25g,0.28mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物2-丁基辛酸-22-丁基-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-5,15,21-三氧亚基-10-氮杂-6,14,20-三氧杂二十八烷-1-基酯(40.0mg, 产率12.2％)。MS:m/z[M+H]+＝1184.0。1H NMR(300MHz,CDCl3)δ4.25-4.03(m,12H),3.56-3.11(m,4H),2.92-2.73(m,2H),2.46-2.22(m,16H),1.89-1.50(m,20H),1.56-1.43(m,4H),1.39-1.21(m,40H),1.07-0.97(m,6H),0.99-0.80(m,18H)。Compound 1-8 (0.17 g, 0.56 mmol, 2.0 eq), N,N-diisopropylethylamine (0.11 g, 0.84 mmol, 3.0 eq), 4-dimethylaminopyridine (34.0 mg, 0.28 mmol, 1.0 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.11 g, 0.56 mmol, 2.0 eq) were added in sequence to 20 ml of dichloromethane. After reacting at room temperature for 0.5 hour, compound 3 (0.25 g, 0.28 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain the compound 2-butyloctanoic acid-22-butyl-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiooctan-1-yl)-5,15,21-trioxy-10-aza-6,14,20-trioxaoctacosane-1-yl ester (40.0 mg, Yield 12.2%). MS: m/z[M+H] + =1184.0. 1 H NMR (300MHz, CDCl 3 ) δ4.25-4.03(m,12H),3.56-3.11(m,4H),2.92-2.73(m,2H),2.46-2.22(m,16H),1.89-1.50(m,20H),1.56-1.43(m,4H),1.39- 1.21(m,40H),1.07-0.97(m,6H),0.99-0.80(m,18H).

实施例6：2-丁基辛酸-(24Z)-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-18-{[(5Z)-辛-5-烯基]氧基}-5,15-二氧亚基-10-氮杂-6,14,19-三氧杂二十七-24-烯-1-基酯的合成
Example 6: Synthesis of 2-butyloctanoic acid-(24Z)-8-(10-butyl-3,9-dioxyylidene-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiaoctan-1-yl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxyylidene-10-aza-6,14,19-trioxacosaccharide-24-en-1-yl ester

步骤1：化合物6-2的合成Step 1: Synthesis of compound 6-2

在室温下，将4,4-二甲氧基丁腈(25.00g,193.56mmol,1.0eq)、顺-5-辛烯-1-醇(49.63g,387.12mmol,2.0eq)和4-甲基苯磺酸吡啶(2.43g,9.68mmol,2.0eq)闷罐中，加热搅拌至110摄氏度，反应72小时。反应液用乙酸乙酯萃洗，盐水洗，无水硫酸钠干燥，过滤，浓缩，通过柱层析纯化得到标题化合物12克，以及单取代的中间态和原料。将中间态和原料再置于闷罐中110摄氏度下搅拌反应72小时，重复以上操作，合并得到4，4-双[(5Z)-十八烷基-5-烯-1-基氧基]丁腈(26.52g,41.8％)。MS:m/z[M+H]+＝357.3。1HNMR(400MHz-CDCl3)δ5.42-5.28(m,4H),4.57(t,J＝5.3Hz,1H),3.61(dt,J＝9.3,6.6Hz,2H),3.44(dt,J＝9.3,6.6Hz,2H),2.42(t,J＝7.3Hz,2H),2.12-1.90(m,9H),1.66-1.54(m,5H),0.97(t,J＝7.5Hz,6H)。At room temperature, 4,4-dimethoxybutyronitrile (25.00 g, 193.56 mmol, 1.0 eq), cis-5-octen-1-ol (49.63 g, 387.12 mmol, 2.0 eq) and 4-methylbenzenesulfonate pyridine (2.43 g, 9.68 mmol, 2.0 eq) were placed in a sealed pot, heated and stirred to 110 degrees Celsius, and reacted for 72 hours. The reaction solution was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 12 grams of the title compound, as well as a monosubstituted intermediate and raw material. The intermediate and raw material were placed in a sealed pot again and stirred at 110 degrees Celsius for 72 hours. The above operation was repeated and combined to obtain 4,4-bis[(5Z)-octadecyl-5-en-1-yloxy]butyronitrile (26.52 g, 41.8%). MS: m/z[M+H] + =357.3. 1 HNMR (400MHz-CDCl 3 ) δ5.42-5.28(m,4H),4.57(t,J＝5.3Hz,1H),3.61(dt,J＝9.3,6.6Hz,2H),3.44(dt,J＝9.3,6.6Hz,2H),2.42(t,J＝7.3Hz,2H),2.12-1 .90(m,9H),1.66-1.54(m,5H),0.97(t,J=7.5Hz,6H).

步骤2：化合物6-3的合成Step 2: Synthesis of compound 6-3

在室温下，将化合物6-2(2.00g,6.22mmol,1.0eq)，氢氧化钾(1.05g,18.66mmol,3.0eq)，乙醇(6mL)，水(6mL)加入到闷罐中，加热到110摄氏度，反应过夜。将反应液浓缩干，加入水稀释，用1N的HCl水溶液中和至pH＝5，用乙酸乙酯进行萃取3次，有机相合并，盐水洗，无水硫酸钠干燥，过滤，浓缩，得到4,4-双[(5Z)-十八烷基-5-烯-1-基氧基]丁酸(1.5g,产率70.8％)。1H NMR(400MHz,CDCl3)δ5.41-5.27(m,4H),4.45(t,J＝5.6Hz,1H),3.51(dt,J＝9.0,6.7Hz,2H),3.39(dt,J＝9.0,6.7Hz,2H),2.43(t,J＝7.2Hz,2H),2.12-1.98(m,8H),1.81(q,J＝7.3Hz,2H)1.65-1.49(m,4H),1.44-1.32(m,4H),0.94(t,J＝7.5Hz,6H)。 At room temperature, compound 6-2 (2.00 g, 6.22 mmol, 1.0 eq), potassium hydroxide (1.05 g, 18.66 mmol, 3.0 eq), ethanol (6 mL), and water (6 mL) were added to a jar, heated to 110 degrees Celsius, and reacted overnight. The reaction solution was concentrated to dryness, diluted with water, neutralized with 1N HCl aqueous solution to pH = 5, extracted with ethyl acetate three times, the organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4,4-bis[(5Z)-octadecyl-5-en-1-yloxy]butyric acid (1.5 g, yield 70.8%). 1 H NMR (400MHz, CDCl 3 ) δ5.41-5.27(m,4H),4.45(t,J＝5.6Hz,1H),3.51(dt,J＝9.0,6.7Hz,2H),3.39(dt,J＝9.0,6.7Hz,2H),2.43(t,J＝7.2Hz,2H),2.12-1 .98(m,8H),1.81(q,J＝7.3Hz,2H)1.65-1.49(m,4H),1.44-1.32(m,4H),0.94(t,J＝7.5Hz,6H).

步骤3：化合物6的合成Step 3: Synthesis of compound 6

在20毫升的二氯甲烷中依次加入化合物6-3(0.14g,0.42mmol,1.5eq)，N,N-二异丙基乙胺(72.0mg,0.56mmol,2.0eq)，4-二甲氨基吡啶(51.0mg,0.42mmol,1.5eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(81.0mg,0.42mmol,1.5eq)，室温下反应0.5小时后，加入化合物3(0.25g,0.28mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得2-丁基辛酸-(24Z)-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-18-{[(5Z)-辛-5-烯基]氧基}-5,15-二氧亚基-10-氮杂-6,14,19-三氧杂二十七-24-烯-1-基酯(130.0mg,产率38.3％)。MS:m/z[M+H]+＝1123.9。1H NMR(300MHz,CDCl3)δ5.47-5.25(m,4H),4.55-4.48(m,1H),4.20-4.01(m,8H),3.71-3.52(m,2H),3.51-3.28(m,5H),2.98-2.81(m,2H),2.65-2.26(m,14H),2.12-1.80(m,10H),1.81-1.50(m,20H),1.48-1.13(m,38H),1.11-0.78(m,22H)。Compound 6-3 (0.14 g, 0.42 mmol, 1.5 eq), N,N-diisopropylethylamine (72.0 mg, 0.56 mmol, 2.0 eq), 4-dimethylaminopyridine (51.0 mg, 0.42 mmol, 1.5 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (81.0 mg, 0.42 mmol, 1.5 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3 (0.25 g, 0.28 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to give 2-butyloctanoic acid-(24Z)-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-10-(6-ethyl-1-oxy-6-aza-2-thiaoctane-1-yl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxy-10-aza-6,14,19-trioxacositol-24-en-1-yl ester (130.0 mg, yield 38.3%). MS: m/z[M+H] + =1123.9. 1 H NMR (300MHz, CDCl 3 ) δ5.47-5.25(m,4H),4.55-4.48(m,1H),4.20-4.01(m,8H),3.71-3.52(m,2H),3.51-3.28(m,5H),2.98-2.81(m,2H),2.65-2.26 (m,14H),2.12-1.80(m,10H),1.81-1.50(m,20H),1.48-1.13(m,38H),1.11-0.78(m,22H).

实施例7：2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-10-(4-{[(2Z)-壬-2-烯基]氧基}-4-氧亚基丁基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 7: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(4-{[(2Z)-non-2-enyl]oxy}-4-oxy-butyl)-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

实施例7是采用实施例1的方法将实施例1中化合物1-13替换成(2Z)-壬-2-烯-1-醇合成的。产率35.1％。MS:m/z[M+H]+＝1053.8。1H NMR(400MHz,CDCl3)δ5.65(dd,J＝18.2,7.5Hz,2H),5.57–5.33(m,2H),4.59(t,J＝26.4Hz,4H),4.31–3.74(m,10H),3.42(t,J＝20.9Hz,5H),2.92(t,J＝7.0Hz,3H),2.80–2.59(m,6H),2.51–2.22(m,9H),2.09(dd,J＝14.3,7.2Hz,3H),1.97–1.82(m,4H),1.76–1.53(m,12H),1.48–1.37(m,5H),1.35–1.20(m,25H),1.13(t,J＝7.1Hz,6H),0.87(dd,J＝7.8,6.1Hz,12H)。Example 7 was synthesized by using the method of Example 1 except that compound 1-13 in Example 1 was replaced by (2Z)-non-2-en-1-ol. Yield: 35.1%. MS: m/z[M+H] + =1053.8. 1 H NMR (400MHz,CDCl 3 )δ5.65(dd,J＝18.2,7.5Hz,2H),5.57–5.33(m,2H),4.59(t,J＝26.4Hz,4H),4.31–3.74(m,10H),3.42(t,J＝20.9Hz,5H),2.92(t,J＝7.0Hz,3H),2.80–2.59(m,6H),2.51–2.2 2(m,9H),2.09(dd,J＝14.3,7.2Hz,3H),1.97–1.82(m,4H),1.76–1.53(m,12H),1.48–1.37(m,5H),1.35–1.20(m,25H),1.13(t,J＝7.1Hz,6H),0.87(dd ,J=7.8,6.1Hz,12H).

实施例8：2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-10-(4-{[(5Z)-辛-5-烯基]氧基}-4-氧亚基丁基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 8: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(4-{[(5Z)-oct-5-enyl]oxy}-4-oxy-butyl)-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

实施例8是采用实施例1的方法将实施例1中化合物1-13替换成(5Z)-辛-5-烯-1-醇合成的。产率37.4％。MS:m/z[M+H]+＝1039.8。1H NMR(400MHz,CDCl3)δ5.44–5.35(m,1H),5.31(dd,J＝10.7,7.1Hz,1H),4.31–3.93(m,10H),3.60–3.25(m,5H),2.92(t,J＝7.1Hz,2H),2.79–2.56(m,6H),2.52–2.22(m,9H),2.05(td,J＝14.7,7.3Hz,4H),1.87(dd,J＝14.6,7.2Hz,4H),1.78–1.51(m,14H),1.44(dt,J＝15.0,8.0Hz,6H),1.37–1.16(m,22H),1.12(t,J＝7.1Hz,6H),1.02–0.42(m,16H)。Example 8 was synthesized by using the method of Example 1 except that compound 1-13 in Example 1 was replaced by (5Z)-oct-5-en-1-ol. Yield: 37.4%. MS: m/z[M+H] + =1039.8. 1 H NMR (400 MHz, CDCl 3 )δ5.44–5.35 (m, 1H), 5.31 (dd, J=10.7, 7.1 Hz, 1H), 4.31–3.93 (m, 10H), 3.60–3.25 (m, 5H), 2.92 (t, J=7.1 Hz, 2H), 2.79–2.56 (m, 6H), 2.52–2.22 (m, 9H), 2.05 (td, J=7.1 Hz, 2H). ＝14.7,7.3Hz,4H),1.87(dd,J＝14.6,7.2Hz,4H),1.78–1.51(m,14H),1.44(dt,J＝15.0,8.0Hz,6H),1.37–1.16(m,22H),1.12(t,J＝7.1Hz,6H),1.02–0. 42(m,16H).

实施例9：2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-10-[4-(壬-2-炔基氧基)-4-氧亚基丁基]-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 9: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-[4-(non-2-ynyloxy)-4-oxy-butyl]-5,11-dioxy-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

实施例9是采用实施例1的方法将实施例1中化合物1-13替换成壬-2-炔-1-醇合成的。产率39.5％。MS:m/z[M+H]+＝1051.8。1H NMR(400MHz,CDCl3)δ4.68(s,2H),4.08(s,8H),3.56–3.30(m,5H),2.92(t,J＝7.0Hz,2H),2.69–2.55(m,6H),2.52–2.26(m,10H),2.21(t,J＝7.1Hz,2H),1.85(dd,J＝25.8,19.0Hz,6H),1.75–1.41(m,20H),1.27(d,J＝10.7Hz,25H),1.09(t,J＝7.1Hz,6H),0.88(dd,J＝6.5,4.9Hz,14H)。Example 9 was synthesized by using the method of Example 1 except that compound 1-13 in Example 1 was replaced by non-2-yn-1-ol. Yield: 39.5%. MS: m/z [M+H] + = 1051.8. 1 H NMR (400MHz, CDCl 3 ) δ4.68 (s, 2H), 4.08 (s, 8H), 3.56–3.30 (m, 5H), 2.92 (t, J = 7.0Hz, 2H), 2.69–2.55 (m, 6H), 2.52–2.26 (m, 10H), 2.21 (t, J = 7.1Hz, 2H) ),1.85(dd,J＝25.8,19.0Hz,6H),1.75–1.41(m,20H),1.27(d,J＝10.7Hz,25H),1.09(t,J＝7.1Hz,6H),0.88(dd,J＝6.5,4.9Hz,14H).

实施例10：2-丁基辛酸-8-(10-丁基-3,9-二氧亚基-2,8-二氧杂十六烷-1-基)-16-乙基-10-(3-{[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}丙基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 10: Synthesis of 2-butyloctanoic acid-8-(10-butyl-3,9-dioxy-2,8-dioxahexadecan-1-yl)-16-ethyl-10-(3-{[(10Z,12Z)-1-oxyylidene octadecane-9,12-dienyl]oxy}propyl)-5,11-dioxyylidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

实施例10是采用实施例6的方法将实施例6中化合物6-3替换成亚油酸合成的。产率47.1％。MS:m/z[M+H]+＝1163.9。1H NMR(400MHz,CDCl3)δ5.48-5.25(m,4H),4.25-3.90(m,10H),3.51-3.37(m,3H),2.95-2.82(m,1H),2.80-2.71(m,1H),2.60-2.20(m,10H),2.12-1.80(m,8H),1.73-1.52(m,14H),1.50-1.03(m,56H),0.98-0.82(m,15H)。Example 10 was synthesized by using the method of Example 6 except that compound 6-3 in Example 6 was replaced by linoleic acid. Yield: 47.1%. MS: m/z[M+H] + =1163.9. 1 H NMR (400 MHz, CDCl 3 )δ5.48-5.25 (m, 4H), 4.25-3.90 (m, 10H), 3.51-3.37 (m, 3H), 2.95-2.82 (m, 1H), 2.80-2.71 (m, 1H), 2.60-2.20 (m, 10H), 2.12-1.80 (m, 8H), 1.73-1.52 (m, 14H), 1.50-1.03 (m, 56H), 0.98-0.82 (m, 15H).

实施例11：2-丁基辛酸-(24Z)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-8-({[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}甲基)-18-{[(5Z)-辛-5-烯基]氧基}-5,15-二氧亚基-10-氮杂-6,14,19-三氧杂二十七-24-烯-1-基酯的合成
Example 11: Synthesis of 2-butyloctanoic acid-(24Z)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiooctan-1-yl)-8-({[(10Z,12Z)-1-oxyylidene octadec-9,12-dienyl]oxy}methyl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxyylidene-10-aza-6,14,19-trioxacosaccharide-24-en-1-yl ester

步骤1：化合物11-2的合成Step 1: Synthesis of compound 11-2

在20毫升的二氯甲烷中依次加入化合物11-1(0.94g,3.36mmol,1.0eq),N,N-二异丙基乙胺(1.30g,10.08mmol,3.0eq)，4-二甲氨基吡啶(0.08g,0.67mmol,0.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.29g,6.72mmol,2.0eq)，室温下反应0.5小时后，加入化合物3-6(1.93g,3.36mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物(10Z,12Z)-十八-9,12-二烯酸-4-(2,2-二甲基-3,3-二苯基-4-氧杂-3-硅杂庚-7-基)-10-乙基-2-(羟基甲基)-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯(1.10g,产率39.1％)。MS:m/z[M+H]+＝837.6。Compound 11-1 (0.94 g, 3.36 mmol, 1.0 eq), N,N-diisopropylethylamine (1.30 g, 10.08 mmol, 3.0 eq), 4-dimethylaminopyridine (0.08 g, 0.67 mmol, 0.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.29 g, 6.72 mmol, 2.0 eq) were added to 20 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 3-6 (1.93 g, 3.36 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound (10Z, 12Z)-octadecanoic acid-9,12-dienoic acid-4-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanyl-7-yl)-10-ethyl-2-(hydroxymethyl)-5-oxygen-4,10-diaza-6-thiadodecane-1-yl ester (1.10 g, yield 39.1%). MS: m/z[M+H] + =837.6.

步骤2：化合物11-3的合成Step 2: Synthesis of compound 11-3

在10毫升的二氯甲烷中依次加入化合物1-8(0.27g,0.90mmol,1.5eq),N,N-二异丙基乙胺(0.23g,1.80mmol,3.0eq)，4-二甲氨基吡啶(0.02g,0.67mmol,0.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.23g,1.20mmol,2.0eq)，室温下反应0.5小时后，加入化合物11-2(500.0mg,0.60mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物2-丁基辛酸-10-(2,2-二甲基-3,3-二苯基-4-氧杂-3-硅杂庚-7-基)-16-乙基-8-({[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}甲基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯(590.0mg,产率88.2％)。MS:m/z[M+H]+＝1119.8。Compound 1-8 (0.27 g, 0.90 mmol, 1.5 eq), N,N-diisopropylethylamine (0.23 g, 1.80 mmol, 3.0 eq), 4-dimethylaminopyridine (0.02 g, 0.67 mmol, 0.2 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.23 g, 1.20 mmol, 2.0 eq) were added to 10 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 11-2 (500.0 mg, 0.60 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 2-butyloctanoic acid-10-(2,2-dimethyl-3,3-diphenyl-4-oxa-3-silanylhept-7-yl)-16-ethyl-8-({[(10Z,12Z)-1-oxyylidene octadecane-9,12-dienyl]oxy}methyl)-5,11-dioxyylidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester (590.0 mg, yield 88.2%). MS: m/z[M+H] + =1119.8.

步骤3：化合物11-4的合成Step 3: Synthesis of compound 11-4

在5毫升四氢呋喃中加入化合物11-3(590.0mg,0.53mmol,1.0eq)和四丁基氟化铵的四氢呋喃溶液(1.59mL,1.59mmol,1.0M,3.0eq)。反应液在室温下搅拌1小时，减压浓缩，加入50毫升水稀释，分别用30毫升石油醚萃取三次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物2-丁基辛酸-16-乙基-10-(3-羟基丙基)-8-({[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}甲基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯(260.0mg,产率58.8％)。MS:m/z[M+H]+＝881.7。Compound 11-3 (590.0 mg, 0.53 mmol, 1.0 eq) and tetrabutylammonium fluoride tetrahydrofuran solution (1.59 mL, 1.59 mmol, 1.0 M, 3.0 eq) were added to 5 mL of tetrahydrofuran. The reaction solution was stirred at room temperature for 1 hour, concentrated under reduced pressure, diluted with 50 mL of water, extracted three times with 30 mL of petroleum ether, combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography to obtain the compound 2-butyloctanoic acid-16-ethyl-10-(3-hydroxypropyl)-8-({[(10Z,12Z)-1-oxyylidene octadecane-9,12-dienyl]oxy}methyl)-5,11-dioxyylidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester (260.0 mg, yield 58.8%). MS: m/z [M+H] + = 881.7.

步骤4：化合物11的合成Step 4: Synthesis of compound 11

在10毫升的二氯甲烷中依次加入化合物6-3(0.15g,0.45mmol,1.5eq),N,N-二异丙基乙胺(0.12g,0.90mmol,3.0eq)，4-二甲氨基吡啶(0.01g,0.06mmol,0.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.12g,0.90mmol,2.0eq)，室温下反应0.5小时后，加入化合物11-4(260.0mg,0.30mmol,1.0eq)。反应液在室温下继续反应15.5小时后，二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，柱层析得化合物2-丁基辛酸-(24Z)-10-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-8-({[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}甲基)-18-{[(5Z)-辛-5-烯基]氧基}-5,15-二氧亚基-10-氮杂-6,14,19-三氧杂二十七-24-烯-1-基酯(131.1mg,产率36.9％)。MS:m/z[M+H]+＝1203.9。1H NMR(400MHz,CDCl3)δ5.61–5.19(m,8H),4.51(s,2H),4.09(s,8H),3.79–3.52(m,3H),3.50–3.23(m,6H),2.93(t,J＝7.1Hz,3H),2.75(dd,J＝33.7,27.6Hz,8H),2.37(ddd,J＝29.7,15.2,7.5Hz,8H),2.05(dt,J＝9.6,4.7Hz,10H),2.01–1.78(m,8H),1.63(ddd,J＝21.8,10.7,5.0Hz,12H),1.39(ddd,J＝50.8,28.4,13.6Hz,30H),1.11(s, 6H),1.01–0.82(m,14H)。Compound 6-3 (0.15 g, 0.45 mmol, 1.5 eq), N,N-diisopropylethylamine (0.12 g, 0.90 mmol, 3.0 eq), 4-dimethylaminopyridine (0.01 g, 0.06 mmol, 0.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.12 g, 0.90 mmol, 2.0 eq) were added to 10 ml of dichloromethane in sequence. After reacting at room temperature for 0.5 hour, compound 11-4 (260.0 mg, 0.30 mmol, 1.0 eq) was added. The reaction solution was reacted at room temperature for 15.5 hours, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to obtain the compound 2-butyloctanoic acid-(24Z)-10-(6-ethyl-1-oxyylidene-6-aza-2-thiooctan-1-yl)-8-({[(10Z,12Z)-1-oxyylidene octadec-9,12-dienyl]oxy}methyl)-18-{[(5Z)-oct-5-enyl]oxy}-5,15-dioxyylidene-10-aza-6,14,19-trioxacositol-24-en-1-yl ester (131.1 mg, yield 36.9%). MS: m/z[M+H] + =1203.9. 1 H NMR (400MHz, CDCl 3 )δ5.61–5.19(m,8H),4.51(s,2H),4.09(s,8H),3.79–3.52(m,3H),3.50–3.23(m,6H),2.93(t,J＝7.1Hz,3H),2.75(dd,J＝33.7,27.6Hz,8H),2.37(ddd,J ＝29.7,15.2,7.5Hz,8H),2.05(dt,J＝9.6,4.7Hz,10H),2.01–1.78(m,8H),1.63(ddd,J＝21.8,10.7,5.0Hz,12H),1.39(ddd,J＝50.8,28.4,13.6Hz,30H), 1.11(s, 6H), 1.01–0.82 (m, 14H).

实施例12：(10Z,12Z)-十八-9,12-二烯酸-(18Z)-4-(6-乙基-1-氧亚基-6-氮杂-2-硫杂辛-1-基)-12-{[(5Z)-辛-5-烯基]氧基}-2-[6-(辛基氧基)-3-氧亚基-2,7-二氧杂十五烷-1-基]-9-氧亚基-4-氮杂-8,13-二氧杂二十一-18-烯-1-基酯的合成
Example 12: Synthesis of (10Z,12Z)-octadecane-9,12-dienoic acid-(18Z)-4-(6-ethyl-1-oxy-6-aza-2-thiooctan-1-yl)-12-{[(5Z)-oct-5-enyl]oxy}-2-[6-(octyloxy)-3-oxy-2,7-dioxapentadecan-1-yl]-9-oxy-4-aza-8,13-dioxa-hexadecane-18-en-1-yl ester

化合物12-1：4,4-双(辛基氧基)丁酸
Compound 12-1: 4,4-Bis(octyloxy)butyric acid

实施例12是采用实施例11的方法将实施例11中化合物1-8替换成化合物12-1合成的。化合物12-1是采用实施例6的方法将实施例6中化合物6-1替换成辛-1-醇合成的。产率38.8％。MS:m/z[M+H]+＝1247.9。1H NMR(400MHz,CDCl3)δ5.36(dt,J＝18.4,8.2Hz,8H),4.51(d,J＝4.8Hz,2H),4.09(s,6H),3.71–3.53(m,4H),3.52–3.24(m,8H),3.07–2.57(m,10H),2.55–2.23(m,8H),2.20–1.78(m,20H),1.58(td,J＝14.4,7.2Hz,10H),1.49–1.21(m,38H),1.15(s,6H),1.02–0.84(m,14H)。Example 12 was synthesized by using the method of Example 11 except that compound 1-8 in Example 11 was replaced by compound 12-1. Compound 12-1 was synthesized by using the method of Example 6 except that compound 6-1 in Example 6 was replaced by octan-1-ol. Yield: 38.8%. MS: m/z[M+H] + =1247.9. 1 H NMR (400MHz, CDCl 3 ) δ5.36 (dt, J＝18.4, 8.2Hz, 8H), 4.51 (d, J＝4.8Hz, 2H), 4.09 (s, 6H), 3.71–3.53 (m, 4H), 3.52–3.24 (m, 8H), 3.07–2.57 (m, 10H), 2. 55–2.23(m,8H),2.20–1.78(m,20H),1.58(td,J＝14.4,7.2Hz,10H),1.49–1.21(m,38H),1.15(s,6H),1.02–0.84(m,14H).

实施例13：2-丁基辛酸-16-乙基-8-({[(10Z,12Z)-1-氧亚基十八-9,12-二烯基]氧基}甲基)-10-(4-{[(2Z)-壬-2-烯基]氧基}-4-氧亚基丁基)-5,11-二氧亚基-10,16-二氮杂-6-氧杂-12-硫杂十八烷-1-基酯的合成
Example 13: Synthesis of 2-butyloctanoic acid-16-ethyl-8-({[(10Z,12Z)-1-oxyidene octadecane-9,12-dienyl]oxy}methyl)-10-(4-{[(2Z)-non-2-enyl]oxy}-4-oxyidene butyl)-5,11-dioxyidene-10,16-diaza-6-oxa-12-thiaoctadecane-1-yl ester

实施例13是采用实施例1的方法合成的。产率55.2％。MS:m/z[M+H]+＝1033.8。1H NMR(400MHz,CDCl3)δ5.68–5.30(m,8H),4.22–4.01(m,6H),3.55–3.28(m,2H),3.25–3.20(m,2H),3.10–2.55(m,8H),2.31–2.23(m,4H),2.20–1.53(m,26H),1.45–1.18(m,34H),1.15–1.05(m,6H),1.02–0.84(m,12H)。Example 13 was synthesized by the method of Example 1. Yield: 55.2%. MS: m/z [M+H] + = 1033.8. 1 H NMR (400 MHz, CDCl 3 ) δ 5.68–5.30 (m, 8H), 4.22–4.01 (m, 6H), 3.55–3.28 (m, 2H), 3.25–3.20 (m, 2H), 3.10–2.55 (m, 8H), 2.31–2.23 (m, 4H), 2.20–1.53 (m, 26H), 1.45–1.18 (m, 34H), 1.15–1.05 (m, 6H), 1.02–0.84 (m, 12H).

实施例14：(10Z,12Z)-十八-9,12-二烯酸-10-乙基-4-(4-{[(2Z)-壬-2-烯基]氧基}-4-氧亚基丁基)-2-[6-(辛基氧基)-3-氧亚基-2,7-二氧杂十五烷-1-基]-5-氧亚基-4,10-二氮杂-6-硫杂十二烷-1-基酯的合成
Example 14: Synthesis of (10Z,12Z)-octadec-9,12-dienoic acid-10-ethyl-4-(4-{[(2Z)-non-2-enyl]oxy}-4-oxydebutyl)-2-[6-(octyloxy)-3-oxyde-2,7-dioxapentadecan-1-yl]-5-oxyde-4,10-diaza-6-thiadodecane-1-yl ester

实施例14是采用实施例1的方法合成的。产率39.3％。MS:m/z[M+H]+＝1077.8。1H NMR(400MHz,CDCl3)δ5.62–5.38(m,8H),4.37–4.11(m,7H),3.63–3.15(m,4H),3.13–2.42(m,12H),2.33–2.12(m,6H),2.11–1.53(m,27H),1.45–1.12(m,40H),0.98–0.75(m,12H)。Example 14 was synthesized by the method of Example 1. Yield: 39.3%. MS: m/z [M+H] + = 1077.8. 1 H NMR (400 MHz, CDCl 3 ) δ 5.62-5.38 (m, 8H), 4.37-4.11 (m, 7H), 3.63-3.15 (m, 4H), 3.13-2.42 (m, 12H), 2.33-2.12 (m, 6H), 2.11-1.53 (m, 27H), 1.45-1.12 (m, 40H), 0.98-0.75 (m, 12H).

实施例15、脂质纳米颗粒的制备和表征Example 15. Preparation and characterization of lipid nanoparticles

1、可电离脂质或本发明的化合物/DSPC/胆固醇/PEG-脂质以50:10:38.5:1.5的摩尔比制备脂质纳米颗粒。1. Lipid nanoparticles were prepared by mixing ionizable lipid or the compound of the present invention/DSPC/cholesterol/PEG-lipid at a molar ratio of 50:10:38.5:1.5.

将二亚油基甲基-4-二甲基氨基丁酸酯(DLin-MC3-DMA，通常缩写为MC3)以及本发明的化合物1-化合物14分别与DSPC、胆固醇、PEG-DMG按照上述摩尔比溶于无水乙醇中。将荧光素酶mRNA(L-6107,TriLink BioTechnologies,Inc.)溶于100mM pH为4的无酶柠檬酸缓冲液中(mRNA浓度为0.2mg/mL)。使不同脂质载体的乙醇溶液与mRNA的缓冲液以1:3(体积/体积)混合(其中总脂质和mRNA的质量比为40:1)，以12ml/min的流速通过微流体纳米药物制造系统(NanoAssemblr Ignite，加拿大)获得对应的核酸脂质纳米颗粒。获得的核酸脂质纳米颗粒立即以40倍体积稀释到1×DPBS缓冲液中。稀释后的核酸脂质纳米颗粒溶液通过超速离心管，浓缩达到目的体积。稀释后用于DLS粒径测量和包封效率检测。Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated as MC3) and compound 1 to compound 14 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol, and PEG-DMG according to the above molar ratios. Luciferase mRNA (L-6107, TriLink BioTechnologies, Inc.) was dissolved in 100mM enzyme-free citric acid buffer at pH 4 (mRNA concentration was 0.2mg/mL). The ethanol solutions of different lipid carriers were mixed with the mRNA buffer at a ratio of 1:3 (volume/volume) (where the mass ratio of total lipids to mRNA was 40:1), and the corresponding nucleic acid lipid nanoparticles were obtained by passing through a microfluidic nanodrug manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12ml/min. The obtained nucleic acid lipid nanoparticles were immediately diluted into 1×DPBS buffer at a volume of 40 times. The diluted nucleic acid lipid nanoparticle solution was passed through an ultracentrifuge tube and concentrated to the target volume. After dilution, it was used for DLS particle size measurement and encapsulation efficiency detection.

2、使用Malvern Zetasizer Nano ZS(Malvern UK)，以173°反向散射检测模式通过动态光散射测定脂质纳米颗粒的粒径及多分散指数。2. The particle size and polydispersity index of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscattering detection mode.

根据制造商的说明，使用Quant-it Ribogreen RNA定量测定试剂盒(ThermoFisher Scientific,UK)确定脂质纳米颗粒的包封效率，测试结果见表2。The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2.

表2包含各脂质化合物脂质纳米粒子的表征
Table 2 Characterization of lipid nanoparticles containing various lipid compounds

实施例16、动物实验Example 16: Animal Experiment

靶向PCSK9的碱基编辑器ABE8e的mRNA、sgRNA递送实验mRNA and sgRNA delivery experiment of base editor ABE8e targeting PCSK9

血液中的胆固醇主要由肝脏合成，肝脏也是分解多余胆固醇的主要器官，在肝脏表面，存在一 种低密度脂蛋白LDL受体(LDLR)，LDL受体能够与循环回到肝脏的胆固醇结合，进而将胆固醇分解为胆汁酸，经过肠道排泄到体外；PCSK9是一种肝脏合成的蛋白酶，能够与LDL受体结合，促进LDL受体进入肝细胞，导致LDL受体被溶酶体降解，LDL受体数量减少；因而抑制PSCK9这种酶的活性，就能够增加LDLR的数量，从而加强对胆固醇的摄取和分解能力。基础和临床研究表明，PCSK9基因是一个治疗高血脂症、动脉粥样硬化症的有效靶点。图1示出了对PCSK9基因特定位点编辑前后导致的LDL受体的数量变化及最终导致的胆固醇代谢的改变。The cholesterol in the blood is mainly synthesized by the liver, which is also the main organ for breaking down excess cholesterol. The LDL receptor (LDLR) is a low-density lipoprotein that binds to cholesterol that circulates back to the liver, and then breaks down cholesterol into bile acids, which are excreted from the body through the intestines. PCSK9 is a protease synthesized by the liver that can bind to the LDL receptor and promote the entry of the LDL receptor into liver cells, causing the LDL receptor to be degraded by lysosomes and the number of LDL receptors to decrease. Therefore, inhibiting the activity of the enzyme PSCK9 can increase the number of LDLRs, thereby enhancing the ability to absorb and decompose cholesterol. Basic and clinical studies have shown that the PCSK9 gene is an effective target for the treatment of hyperlipidemia and atherosclerosis. Figure 1 shows the changes in the number of LDL receptors before and after editing of specific sites in the PCSK9 gene and the resulting changes in cholesterol metabolism.

小鼠肝脏细胞PCSK9基因编辑递送的策略如图1所示，主要流程如下：通过静脉注射，利用制备出的脂质纳米颗粒将编码ABE8e的mRNA、sgRNA靶向递送至小鼠肝细胞，在ABE8e、sgRNA的作用下，在PCSK9基因引入突变，在特定位点实现碱基A到G的突变，通过测序计算出编辑效率。The strategy for gene editing and delivery of PCSK9 in mouse liver cells is shown in Figure 1. The main process is as follows: mRNA and sgRNA encoding ABE8e are delivered to mouse liver cells through intravenous injection using the prepared lipid nanoparticles. Under the action of ABE8e and sgRNA, mutations are introduced into the PCSK9 gene, and mutations from base A to G are achieved at specific sites. The editing efficiency is calculated by sequencing.

具体实验设计如下：The specific experimental design is as follows:

1、选择合适的突变位点及编辑设计1. Select appropriate mutation sites and editing design

单碱基编辑器ABE8e在不需要供体模板及不造成DSB的条件下实现A到G的碱基精准替换，基于此，选择PCSK9基因的第一外显子作为筛选突变位点。通过脂质纳米颗粒将编码单碱基编辑工具ABE8e的mRNA和sgRNA共同递送到动物体内，编码碱基编辑器ABE8e的mRNA在细胞质中被翻译成蛋白质，与sgRNA形成复合体后进入细胞核，在sgRNA指导下，碱基编辑器ABE8e靶向PCSK9基因第一外显子剪接供体位点，将第一外显子模板链上的腺嘌呤(A)脱氨变成肌苷(I)，I在DNA水平会被当成G进行读码与复制，最终实现A到G的替换，从而破坏剪接供体位点使得PCSK9基因阅读框提前终止。The single-base editor ABE8e achieves precise base replacement from A to G without the need for a donor template and without causing DSB. Based on this, the first exon of the PCSK9 gene was selected as the mutation site for screening. The mRNA encoding the single-base editing tool ABE8e and sgRNA were co-delivered to the animal body through lipid nanoparticles. The mRNA encoding the base editor ABE8e was translated into protein in the cytoplasm, and entered the nucleus after forming a complex with the sgRNA. Under the guidance of the sgRNA, the base editor ABE8e targeted the splicing donor site of the first exon of the PCSK9 gene, deaminated adenine (A) on the first exon template chain into inosine (I), and I was read and replicated as G at the DNA level, ultimately achieving the replacement of A to G, thereby destroying the splicing donor site and causing the PCSK9 gene reading frame to terminate prematurely.

2、碱基编辑器ABE8e的mRNA、sgRNA制备2. Preparation of mRNA and sgRNA for base editor ABE8e

选定小鼠PCSK9基因(NCBI Gene ID:100102)第一外显子、第一内含子的序列作为靶向区域，确定PCSK9基因单碱基编辑的靶点序列PCSK9-sgRNA。The sequences of the first exon and the first intron of the mouse PCSK9 gene (NCBI Gene ID: 100102) were selected as the targeting region, and the target sequence PCSK9-sgRNA for single-base editing of the PCSK9 gene was determined.

通过分析PCSK9基因的跨第一外显子与内含子的序列，设计出靶向区域的sgRNA：PCSK9-sgRNA(由南京金斯瑞公司合成)，所述PCSK9-sgRNA序列为：By analyzing the sequence across the first exon and intron of the PCSK9 gene, an sgRNA targeting the region was designed: PCSK9-sgRNA (synthesized by Nanjing GenScript Co., Ltd.), and the sequence of the PCSK9-sgRNA is:

PCSK9-sgRNA：5’-CCCATACCTTGGAGCAACGG-3’(SEQ ID NO:1)；PCSK9-sgRNA: 5’-CCCATACCTTGGAGCAACGG-3’ (SEQ ID NO: 1);

根据目标序列设计sgRNA并合成寡核苷酸(oligos)，所用到的sgRNA序列如SEQ ID NO:1所示。在每个sgRNA的上游序列5’端加CACC序列，下游序列的5’端加AAAC序列。经合成后，上、下游序列通过预设程序(95℃，5min；95℃-85℃以-2℃/s；85℃-25℃以-0.1℃/s；保持在4℃)进行退火，将退火产物连接到经过BbsI(NEB，R3539S)线性化的lenti U6-sgRNA/EF1a-mCherry载体(Addgene，Plasmid，#114199)。sgRNA was designed and oligonucleotides were synthesized according to the target sequence. The sgRNA sequence used is shown in SEQ ID NO: 1. The CACC sequence was added to the 5' end of the upstream sequence of each sgRNA, and the AAAC sequence was added to the 5' end of the downstream sequence. After synthesis, the upstream and downstream sequences were annealed by a preset program (95°C, 5min; 95°C-85°C at -2°C/s; 85°C-25°C at -0.1°C/s; maintained at 4°C), and the annealed products were ligated to the lenti U6-sgRNA/EF1a-mCherry vector (Addgene, Plasmid, #114199) linearized with BbsI (NEB, R3539S).

其中，sgRNA质粒构建中所使用的体系如下：Among them, the system used in the construction of sgRNA plasmid is as follows:

lenti U6-sgRNA/EF1a-mCherry载体的线性化体系如下：载体3μg；缓冲液(NEB：R0539L)6μL；BbsI 2μL；ddH2O补齐到60μL，37℃酶切过夜。The linearization system of lenti U6-sgRNA/EF1a-mCherry vector is as follows: 3 μg of vector; 6 μL of buffer (NEB: R0539L); 2 μL of BbsI; ddH 2 O to 60 μL, and enzyme digestion at 37°C overnight.

sgRNA退火产物与线性化载体连接体系如下：T4连接酶缓冲液(NEB：M0202L)1μL，线性化载体20ng，经退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH2O补齐到10μL， 16℃连接过夜。The sgRNA annealing product and the linearized vector ligation system are as follows: T4 ligase buffer (NEB: M0202L) 1 μL, linearized vector 20 ng, annealed oligo fragment (10 μM) 5 μL, T4 ligase (NEB: M0202L) 0.5 μL, ddH 2 O to make up to 10 μL, Ligation was carried out at 16°C overnight.

将连接的载体转化大肠杆菌DH5a感受态细胞(唯地生物，DL1001)。具体流程如下：DH5α感受态细胞从-80℃拿出，迅速插入冰中，5分钟后待菌块融化，加入连接产物并用手拨打离心管底轻轻混匀，冰中静置25分钟。42℃水浴热激45秒，迅速放回冰中并静置2分钟。向离心管中加入700μl不含抗生素的无菌LB培养基，混匀后37℃，200rpm复苏60分钟。5000rpm离心一分钟收菌，留取100μl左右上清轻轻吹打重悬菌块并涂布到Amp抗生素的LB培养基上。将平板倒置放于37℃培养箱过夜培养。挑取单菌落，经过测序确认后对阳性克隆摇菌并提取质粒(TIANGEN：DP120-01)后测定浓度，-20℃冰箱中保存备用。The ligated vector was transformed into E. coli DH5a competent cells (Weidi Biotechnology, DL1001). The specific process is as follows: DH5α competent cells were taken out from -80℃ and quickly inserted into ice. After 5 minutes, the bacterial block was melted, and the ligation product was added and gently mixed by hand at the bottom of the centrifuge tube, and then allowed to stand in ice for 25 minutes. Heat shock was performed in a 42℃ water bath for 45 seconds, and then quickly returned to ice and allowed to stand for 2 minutes. 700μl of sterile LB medium without antibiotics was added to the centrifuge tube, and after mixing, it was revived at 37℃ and 200rpm for 60 minutes. Centrifuge at 5000rpm for one minute to collect the bacteria, and about 100μl of supernatant was retained. The bacterial block was gently blown and resuspended and coated on the LB medium with Amp antibiotics. The plate was inverted and placed in a 37℃ incubator for overnight culture. Single colonies were picked, and after sequencing confirmation, the positive clones were shaken and the plasmid (TIANGEN: DP120-01) was extracted and the concentration was determined, and stored in a -20℃ refrigerator for later use.

本实验所采用的碱基编辑器ABE8e为David R.Liu团队所进化出的高效碱基编辑器ABE8e(Richter MF,Zhao KT,Eton E,Lapinaite A,Newby GA,Thuronyi BW,Wilson C,Koblan LW,Zeng J,Bauer DE,Doudna JA,Liu DR.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat Biotechnol.2020Jul；38(7):883-891.doi:10.1038/s41587-020-0453-z.Epub 2020 Mar 16.Erratum in:Nat Biotechnol.2020May 20；PMID:32433547；PMCID:PMC7357821)。从Addgene上购买获得质粒ABE8e(Plasmid#138489)，由实验室表达、纯化ABE8e的mRNA备用。The base editor ABE8e used in this experiment is a highly efficient base editor ABE8e evolved by David R. Liu's team (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor or with improved Cas domain compatibility and activity.Nat Biotechnol.2020Jul；38(7):883-891.doi:10.1038/s41587-020-0453-z.Epub 2020 Mar 16.Erratum in:Nat Biotechnol.2020May 20；PMID:32433547；PMCID:PMC7357821). Plasmid ABE8e (Plasmid#138489) was purchased from Addgene, and the mRNA of ABE8e was expressed and purified by the laboratory for future use.

3、动物研究3. Animal research

将包封编码碱基编辑器ABE8e的mRNA和sgRNA(ABE8e mRNA：sgRNA比率(w/w)为3:2)的、包含本发明的化合物(参见表2)的脂质纳米颗粒(总脂质和mRNA的质量比为40:1)，以0.2mg/kg的剂量通过尾静脉注射经全身施用于6-7周龄的C57BL/6雌性小鼠(购自江苏集萃药康公司)。Lipid nanoparticles (mass ratio of total lipids to mRNA is 40:1) containing the compound of the present invention (see Table 2) encapsulating mRNA and sgRNA encoding the base editor ABE8e (ABE8e mRNA:sgRNA ratio (w/w) is 3:2) were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) via tail vein injection at a dose of 0.2 mg/kg.

将包封碱基编辑器ABE8e的mRNA和sgRNA的、包含二亚油基甲基-4-二甲基氨基丁酸酯(DLin-MC3-DMA，缩写为MC3)的脂质纳米颗粒以类似方式施用于周龄和性别相当组的小鼠作为阳性对照。另外，还将PBS缓冲液以类似方式尾部静脉注射周龄和性别相当组的小鼠作为阴性对照。Lipid nanoparticles containing dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, abbreviated as MC3) encapsulating the mRNA and sgRNA of the base editor ABE8e were administered to mice of the same age and gender in a similar manner as a positive control. In addition, PBS buffer was injected into the tail vein of mice of the same age and gender in a similar manner as a negative control.

4、编辑效率检测4. Editing efficiency detection

小鼠给药一周后进行编辑效率检测，将小鼠处死后取肝脏组织，裂解后提取基因组，通过深度测序进行效率分析。深度测序步骤如下。设计引物见表3。One week after administration, the editing efficiency of the mice was tested. The mice were killed and liver tissue was taken. The genome was extracted after lysis and the efficiency was analyzed by deep sequencing. The deep sequencing steps are as follows. The designed primers are shown in Table 3.

表3
Table 3

PCR程序如下：94℃，2min；98℃ 10s，60℃ 30s，68℃ 20s，34个循环；68℃，5min。The PCR program was as follows: 94°C, 2 min; 98°C 10 s, 60°C 30 s, 68°C 20 s, 34 cycles; 68°C, 5 min.

PCR结束后通过凝胶电泳验证，选择大小合适且单一的条带，确定扩增产物正确，并将获得的PCR产物，送至南京金斯瑞公司测序。将深度测序结果通过crispresso软件分析读数，进行特异性位点分析，实现编辑效率计算，计算结果参见表4，各个脂质纳米颗粒所对应的编辑效率可参见图2。 After PCR, gel electrophoresis was performed to verify that the amplified product was correct by selecting a single band of appropriate size, and the obtained PCR product was sent to Nanjing GenScript for sequencing. The deep sequencing results were analyzed by Crispresso software for reading, specific site analysis, and editing efficiency calculation. The calculation results are shown in Table 4, and the editing efficiency corresponding to each lipid nanoparticle can be seen in Figure 2.

表4
Table 4

如表4中所示出的，本发明所采用的可电离脂质化合物能够有效递送核酸分子、小分子化合物等药物；且通过对比，本发明的脂质纳米颗粒粒径分布较好，包封率高，且递送效果可明显好于对比脂质纳米颗粒，能够满足体内递送的需求。As shown in Table 4, the ionizable lipid compound used in the present invention can effectively deliver drugs such as nucleic acid molecules and small molecule compounds; and by comparison, the lipid nanoparticles of the present invention have a better particle size distribution, a high encapsulation rate, and a delivery effect that is significantly better than that of the comparative lipid nanoparticles, and can meet the needs of in vivo delivery.

以上示例性实施方式所呈现的描述仅用以说明本发明的技术方案，并不想要成为毫无遗漏的，也不想要把本发明限制为所描述的精确形式。显然，本领域的普通技术人员根据上述教导做出很多改变和变化都是可能的。选择示例性实施方式并进行描述是为了解释本发明的特定原理及其实际应用，从而使得本领域的其它技术人员便于理解、实现并利用本发明的各种示例性实施方式及其各种选择形式和修改形式。本发明的保护范围意在由请求保护范围及其等效形式所限定。 The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be limited by the scope of protection requested and its equivalent form.

Claims (19)

一种具有结构式I的化合物或其药学上可接受的形式，
A compound of formula I or a pharmaceutically acceptable form thereof,
其中：in: G1为C1-6亚烷基； G1 is a C1-6 alkylene group; R1、R2各自独立地为C1-6烷基或C1-6烷氧基；所述烷基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-6烷基、C1-6烷氧基、卤素取代的C1-6烷基或羟基取代的C1-6烷基所取代；R 1 and R 2 are each independently C 1-6 alkyl or C 1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy is optionally further substituted by halogen, hydroxy, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxy-substituted C 1-6 alkyl; 任选地，R1、R2与相连的N共同形成3-8元杂环基，或者，R1、R2中任一与G1中的任一碳原子直接连接而形成3-8元杂环基；所述杂环基中有0至3个环C任选进一步被N、O或S所取代；所述杂环基中有0至3个H任选进一步被卤素、羟基、C1-6烷基、C1-6烷氧基、卤素取代的C1-6烷基或羟基取代的C1-6烷基所取代；Optionally, R 1 , R 2 and the connected N together form a 3-8 membered heterocyclic group, or any one of R 1 and R 2 is directly connected to any carbon atom in G 1 to form a 3-8 membered heterocyclic group; 0 to 3 ring Cs in the heterocyclic group are optionally further substituted by N, O or S; 0 to 3 Hs in the heterocyclic group are optionally further substituted by halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxyl-substituted C 1-6 alkyl; L1选自 亚炔基、杂原子O、N、S或不存在；h、i、k选自1-10的整数；j选自0或1；L 1 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k are selected from integers of 1-10; j is selected from 0 or 1; R3、R4、R5、R6、R7各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 3 , R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl; a、b各自独立地选自0-10的整数；a and b are each independently selected from integers of 0-10; L2选自 亚炔基、杂原子O、N、S或不存在；h、i、k选自1-10的整数；j选自0或1；L 2 is selected from Alkyne group, heteroatom O, N, S or not present; h, i, k are selected from integers of 1-10; j is selected from 0 or 1; R8、R9、R10、R11、R12各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 8 , R 9 , R 10 , R 11 , and R 12 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy , halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl; c、d各自独立地选自0-10的整数；c and d are each independently selected from integers of 0-10; L3、L4各自独立地选自 亚炔基、杂原子O、N、S或不存在；l、n选自1-10的整数；m选自0或1；L 3 and L 4 are each independently selected from Alkyne group, heteroatom O, N, S or not present; l, n are integers selected from 1-10; m is selected from 0 or 1; R13、R14、R15、R16、R17、R18、R19各自独立地为氢、C1-12烷基、C1-12烯基、C1-12炔基或C1-12烷氧基；所述烷基、烯基、炔基或烷氧基中有0至3个H任选进一步被卤素、羟基、C1-12烷基、C1-12烯基、C1-12炔基、C1-12烷氧基、卤素取代的C1-12烷基或羟基取代的C1-12烷基所取代；R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; wherein 0 to 3 H in the alkyl, alkenyl, alkynyl, or alkoxy is optionally further substituted by halogen, hydroxy, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy, halogen-substituted C 1-12 alkyl, or hydroxy-substituted C 1-12 alkyl; e、f、g各自独立地选自0-10的整数。e, f, and g are each independently selected from integers of 0-10. 根据权利要求1所述的化合物或其药学上可接受的形式，其中：The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: G1为亚甲基、亚乙基、亚丙基、亚丁基或亚戊基。 G1 is methylene, ethylene, propylene, butylene or pentylene. 根据权利要求1或2所述的化合物或其药学上可接受的形式，其中：The compound according to claim 1 or 2, or a pharmaceutically acceptable form thereof, wherein: R1、R2各自独立地为甲基、乙基、丙基或丁基，所述甲基、乙基、丙基或丁基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代；或者，R1、R2与相连的N共同形成5元或6元杂环基，所述杂环基中有0或1或2个环C任选进一步被N、O或S所取代，所述杂环基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代；R 1 and R 2 are each independently methyl, ethyl, propyl or butyl, wherein 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl; or, R 1 , R 2 and the connected N together form a 5-membered or 6-membered heterocyclic group, wherein 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and wherein 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl; 或者，R1、R2中之一与G1中的任一碳原子直接连接而形成5或6元杂环基，R1、R2中另一为甲基、乙基、丙基或丁基，所述甲基、乙基、丙基或丁基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代；所述杂环基中有0或1或2个环C任选进一步被N、O或S所取代，所述杂环基中有0或1或2个H任选进一步被卤素、羟基或C1-3烷基所取代。Alternatively, one of R 1 and R 2 is directly connected to any carbon atom in G 1 to form a 5- or 6-membered heterocyclic group, and the other of R 1 and R 2 is methyl, ethyl, propyl or butyl, and 0, 1 or 2 H in the methyl, ethyl, propyl or butyl group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl; 0, 1 or 2 ring C in the heterocyclic group are optionally further substituted by N, O or S, and 0, 1 or 2 H in the heterocyclic group are optionally further substituted by halogen, hydroxyl or C 1-3 alkyl. 根据权利要求1至3任一项所述的化合物或其药学上可接受的形式，其中：The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable form thereof, wherein: L1为亚炔基、O或不存在；h、i、k各自独立地选自1、2、3、4、5、6、7、8、9或10；j选自0或1； L1 is alkynylene, O or absent; h, i, k are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; j is selected from 0 or 1; R3、R4、R5中至少二者不为氢，R6、R7为氢； At least two of R 3 , R 4 and R 5 are not hydrogen, and R 6 and R 7 are hydrogen; a选自0、1、2、3、4、5、6、7、8、9或10；a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; b选自0、1、2、3、4、5、6、7、8、9或10；b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 优选地，a、b至少之一或均不为0。Preferably, at least one of a and b or both of them are not zero. 根据权利要求1至4任一项所述的化合物或其药学上可接受的形式，其中：The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable form thereof, wherein: L2为亚炔基、O或不存在；h选自1、2、3、4、5、6、7、8、9或10； L2 is alkynylene, O or absent; h is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R8、R9各自独立地为氢，R10、R11、R12中至少二者不为氢；R 8 and R 9 are each independently hydrogen, and at least two of R 10 , R 11 , and R 12 are not hydrogen; c、d各自独立地选自0、1、2、3、4、5、6、7、8、9或10；优选地，c、d至少之一或均不为0。c and d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, at least one of c and d or both of them are not 0. 根据权利要求1至5任一项所述的化合物或其药学上可接受的形式，其中：The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable form thereof, wherein: L3、L4各自独立地选自亚炔基、O或不存在；L 3 and L 4 are each independently selected from Alkyne, O or absent; R13、R14各自为氢；R15、R16、R17、R18、R19至少二者不为氢；R 13 and R 14 are each hydrogen; at least two of R 15 , R 16 , R 17 , R 18 and R 19 are not hydrogen; e、f、g各自独立地选自0、1、2、3、4、5、6、7、8、9或10。e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 根据权利要求1-6任一项所述的化合物或其药学上可接受的形式，其中，所述化合物选自化合物1至化合物31中的一种或多种；The compound or pharmaceutically acceptable form thereof according to any one of claims 1 to 6, wherein the compound is selected from one or more of Compound 1 to Compound 31; 优选地，所述药学上可接受的形式选自药学上可接受的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价复合物或前体药物。Preferably, the pharmaceutically acceptable form is selected from a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug. 一种中间体，其具有式Ic或式Id所示结构：

An intermediate having a structure shown in Formula Ic or Formula Id:

其中，各取代基及e、f、g取值如权利要求1-7任一项所定义。Wherein, each substituent and the values of e, f, and g are as defined in any one of claims 1-7. 一种制备权利要求1-7任一项所述的化合物或其药学上可接受的形式的方法，该方法包括：通过烷基化或还原胺化得到 A method for preparing a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, the method comprising: Obtained by alkylation or reductive amination 通过三光气或羰基二咪唑或对硝基氯甲酸苯酯的作用生成硫代氨基甲酸酯 Thiocarbamates are generated by the action of triphosgene, carbonyldiimidazole or p-nitrophenyl chloroformate 脱去缩酮保护基，通过缩合反应引入脂肪长链，得到式I化合物或其药学上可接受的形式；Removing the ketal protecting group and introducing a long fatty chain through a condensation reaction to obtain a compound of formula I or a pharmaceutically acceptable form thereof; 其中，各取代基及e、f、g取值如权利要求1-7任一项所定义。Wherein, each substituent and the values of e, f, and g are as defined in any one of claims 1-7. 权利要求1-7任一项所述的化合物或其药学上可接受的形式在制备脂质体纳米载体中的应用。Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof in the preparation of liposome nanocarriers. 一种脂质载体，其包含根据权利要求1-7中任一项所述的化合物或其药学上可接受的形式； A lipid carrier comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof; 优选地，所述脂质载体包含第一脂质化合物和第二脂质化合物，其中，所述第一脂质化合物包含根据权利要求1-7中任一项所述的化合物或其药学上可接受的形式以及任选的其它脂质化合物，所述第二脂质化合物包含阴离子脂质、中性脂质、类固醇和聚合物结合的脂质中的一种或两种以上的组合。Preferably, the lipid carrier comprises a first lipid compound and a second lipid compound, wherein the first lipid compound comprises a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof and optionally other lipid compounds, and the second lipid compound comprises one or a combination of two or more of anionic lipids, neutral lipids, steroids and polymer-bound lipids. 一种核酸脂质纳米粒组合物，其包括根据权利要求1-7中任一项所述的化合物或其药学上可接受的形式或根据权利要求11所述的脂质载体，以及核酸药物。A nucleic acid lipid nanoparticle composition, comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 11, and a nucleic acid drug. 根据权利要求12所述的核酸脂质纳米粒组合物，所述核酸药物包括RNA、DNA、反义核酸、适配体、核酶、免疫刺激性核酸或PNA；优选地，所述反义核酸为反义寡核酸；优选地，所述RNA为mRNA、rRNA、circRNA、siRNA、saRNA、tRNA、snRNA、antagomir、微RNA抑制剂、微RNA激活剂或shRNA；优选地，所述DNA包括质粒。According to the nucleic acid lipid nanoparticle composition of claim 12, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA; preferably, the antisense nucleic acid is an antisense oligonucleic acid; preferably, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator or shRNA; preferably, the DNA comprises a plasmid. 一种药物制剂，其包含根据权利要求1-7中任一项所述的化合物或其药学上可接受的形式或根据权利要求11所述的脂质载体或根据权利要求12或13所述的核酸脂质纳米粒组合物、以及药学上可接受的赋形剂、载体或稀释剂。A pharmaceutical preparation comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 11, or a nucleic acid lipid nanoparticle composition according to claim 12 or 13, and a pharmaceutically acceptable excipient, carrier or diluent. 根据权利要求1-7中任一项所述的化合物或其药学上可接受的形式或根据权利要求11所述的脂质载体或根据权利要求12或13所述的核酸脂质纳米粒组合物或根据权利要求14所述的药物制剂在制备核酸药物、基因疫苗、小分子药物、多肽或蛋白质药物中的用途。Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or the lipid carrier according to claim 11, or the nucleic acid lipid nanoparticle composition according to claim 12 or 13, or the pharmaceutical preparation according to claim 14 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. 根据权利要求15所述的用途，所述核酸脂质纳米粒组合物或上述药物制剂用于治疗或预防于治疗或预防有需要的受试者的疾病或病症。According to the use of claim 15, the nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation is used to treat or prevent a disease or condition in a subject in need thereof. 根据权利要求16所述的用途，所述的受试者为哺乳动物或人。The use according to claim 16, wherein the subject is a mammal or a human. 根据权利要求16或17所述的用途，所述的疾病或病症选自代谢性疾病、遗传性疾病、癌症、心血管疾病和传染性疾病；优选地，所述的代谢性疾病包括家族性高胆固醇血症(FH)，所述的遗传性疾病包括转甲状腺素蛋白淀粉样变(ATTR)、原发性高草尿酸症(PH1)和遗传性血管性水肿(HAE)，所述的传染性疾病包括乙型肝炎(HEPATITIS B)。According to the use according to claim 16 or 17, the disease or condition is selected from metabolic diseases, hereditary diseases, cancer, cardiovascular diseases and infectious diseases; preferably, the metabolic diseases include familial hypercholesterolemia (FH), the hereditary diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluria (PH1) and hereditary angioedema (HAE), and the infectious diseases include hepatitis B (HEPATITIS B). 一种用于防止、改善或治疗有需要的受试者的疾病或状况的方法，所述方法包括对所述受试者施用权利要求12或13所述的核酸脂质纳米粒组合物或权利要求14所述的药物制剂。 A method for preventing, ameliorating or treating a disease or condition in a subject in need thereof, the method comprising administering the nucleic acid lipid nanoparticle composition of claim 12 or 13 or the pharmaceutical preparation of claim 14 to the subject.
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