[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2023142600A1 - 一种氨基脂质及其制备方法和应用 - Google Patents

一种氨基脂质及其制备方法和应用 Download PDF

Info

Publication number
WO2023142600A1
WO2023142600A1 PCT/CN2022/131322 CN2022131322W WO2023142600A1 WO 2023142600 A1 WO2023142600 A1 WO 2023142600A1 CN 2022131322 W CN2022131322 W CN 2022131322W WO 2023142600 A1 WO2023142600 A1 WO 2023142600A1
Authority
WO
WIPO (PCT)
Prior art keywords
lnp
alkyl
alkenyl
lipid
amino lipid
Prior art date
Application number
PCT/CN2022/131322
Other languages
English (en)
French (fr)
Inventor
查高峰
彭星华
夏恒
Original Assignee
深圳虹信生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳虹信生物科技有限公司 filed Critical 深圳虹信生物科技有限公司
Publication of WO2023142600A1 publication Critical patent/WO2023142600A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/08—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by a carboxylic acid having the esterifying carboxyl group bound to an acyclic carbon atom of an acyclic unsaturated carbon skeleton
    • 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
    • C07C229/12—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 acyclic carbon skeletons
    • 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
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • 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
    • A61K47/22—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/24—Preparation of carboxylic acid esters by reacting carboxylic acids or derivatives thereof with a carbon-to-oxygen ether bond, e.g. acetal, tetrahydrofuran
    • C07C67/26—Preparation of carboxylic acid esters by reacting carboxylic acids or derivatives thereof with a carbon-to-oxygen ether bond, e.g. acetal, tetrahydrofuran with an oxirane ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom 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
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom 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 hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • 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
    • C07D211/34—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • 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
    • C07D211/36—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 hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • 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
    • C07D211/36—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 hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/58—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/18—Ring systems of four or more rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention relates to the technical field of medicinal chemistry, in particular to an amino lipid and its preparation method and application.
  • Nucleic acid drugs have extremely broad application prospects in the prevention and treatment of cancer, infectious diseases, genetic diseases and cardiovascular diseases. However, since RNA, DNA, and siRNA are easily degraded in vivo, the bioavailability is extremely low due to direct oral or intravenous administration, so carrier delivery is required.
  • nucleic acid vectors include viral vectors and non-viral vectors.
  • Viral vectors have high transfection efficiency, but viral vectors lack targeting, and there are greater safety concerns, and the vectors have small capacity and high production costs.
  • Non-viral vectors have the advantages of high safety, easy modification of carrier molecules, suitable for mass production, etc., and have broad application prospects.
  • LNP lipid-based nanoparticle delivery system
  • LNP is generally composed of ionizable or cationic lipids, phospholipids, cholesterol and PEGylated lipids.
  • the structures are amphiphilic molecules with self-assembly properties.
  • LNP low-density polypeptide
  • the components of LNP have a definite structure, good reproducibility, and are conducive to quality control. At the same time, they have the advantages of long circulation time in vivo and good biocompatibility, which have attracted widespread attention. . After the nanoparticle enters the cell, it needs to escape from the endosome/lysosome to release RNA in the cytoplasm so that it can be expressed to produce the target protein.
  • the endosomal/lysosomal escape rate of LNP is generally low, although DLin-MC3-DMA, as the "gold standard" for the evaluation of aminolipids in vivo, is currently the most efficient aminolipid and was approved by the FDA for the first For the siRNA therapeutic drug Onpattro (patisiran), only 1%-4% of RNA escapes from endosomes/lysosomes, and endosome/lysosome escape has become a key step affecting nucleic acid delivery, so the design has a good encapsulation Amino lipids with nucleic acid ability and high endosome/lysosome escape ability to solve the problem of nucleic acid delivery have great research significance and practical needs.
  • the invention provides an amino lipid and its application.
  • a first aspect of the present invention provides:
  • a kind of amino lipid, its structure is as shown in formula (I):
  • L is C 1 -C 6 alkylene, C 1 -C 6 alkenylene, C 1 -C 6 alkynylene, C 3 -C 6 cycloalkylene, C 3 -C 6 cycloalkenylene ;
  • R 1 and R 2 are the same or different, and are each independently selected from C 1 -C 20 alkyl, C 1 -C 20 alkenyl, C 1 -C 20 alkynyl, C 1 -C 20 cycloalkyl, C 1 -C 20 cycloalkenyl, C 1 -C 20 cycloalkynyl; said C 1 -C 20 alkyl, C 1 -C 20 alkenyl, C 1 -C 20 alkynyl, C 1 -C 20 cycloalkyl , C 1 -C 20 cycloalkenyl, C 1 -C 20 cycloalkynyl can be optionally substituted by hydrogen, C1-C6 hydrocarbon group, fluorine;
  • R 3 and R 4 are the same or different from each other, and are each independently selected from H, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl; the C 1 -C 10 alkane Base, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl may be optionally substituted by C 1 -C 6 hydrocarbyl;
  • R 3 and R 4 are connected to form a 4-10 membered heterocyclic ring, wherein the multi-membered heterocyclic ring contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
  • the R 1 is selected from C 4 -C 17 alkyl, C 4 -C 17 alkenyl, C 4 -C 17 alkynyl, C 4 -C 17 cycloalkyl, C 4 -C 17 cycloalkenyl , C 4 -C 17 cycloalkynyl; said C 4 -C 17 alkyl, C 4 -C 17 alkenyl, C 4 -C 17 alkynyl, C 4 -C 17 cycloalkyl, C 4 -C 17 Cycloalkenyl, C 4 -C 17 cycloalkynyl may be optionally substituted by hydrogen, C 1 -C 6 hydrocarbyl, fluorine.
  • R is selected from E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, E19, E20, E21, E22 One of , E23, E24, E25:
  • R is selected from one of E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E15, E17, E18, E20, E21, E24.
  • the R 2 is selected from C 5 -C 19 alkyl, C 5 -C 19 alkenyl, C 5 -C 19 alkynyl, C 5 -C 19 cycloalkyl, C 5 -C 19 cycloalkenyl , C 5 -C 19 cycloalkynyl; said C 5 -C 19 alkyl, C 5 -C 19 alkenyl, C 5 -C 19 alkynyl, C 5 -C 19 cycloalkyl, C 5 -C 19 Cycloalkenyl, C 5 -C 19 cycloalkynyl may be optionally substituted by hydrogen, C 1 -C 6 hydrocarbyl, fluorine;
  • R is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22 , C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47 , C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72 , C73, C74, C75, C76, C77, C78, C79, C80, C81, C82, C83, C84
  • R is selected from C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C56, C57, C58, C60, C62, C63, C64, C66, C67, C71, C72, C74, C79, C82, C83, C102, C103, C104, C105, C106, C107, C108, C109, C110, C111, C112, C113, C114, C115, C116, C117, C118, C119, C120, C121, C122, C123, C124, C125, C126, C127, C128, C129, C130, C131, C132, C133, C134, C135, C136, C137, C138, C139, C140, C141, C142, C143, C144, C1 45.
  • C146, C147 One of C146, C147.
  • the amine-containing carboxylic acid structure of R 3 , R 4 , and L forming R 3 R 4 -NL is: And/or R 3 and R 4 are connected to form a 4-10 membered heterocyclic ring, wherein the multi-membered heterocyclic ring contains 1-6 heteroatoms, and the heteroatoms are selected from nitrogen, sulfur or oxygen.
  • the amino lipid is selected from one of the compounds shown in the following structure:
  • a second aspect of the present invention provides:
  • the preparation method of the amino lipid described in the first aspect of the present invention comprises the following steps:
  • step S2 Add R 3 R 4 NLCOOH to the reaction system in step S1, and react in the presence of a condensing agent.
  • the method comprises the steps of:
  • a third aspect of the present invention provides:
  • amino lipid or its pharmaceutically acceptable salt, prodrug or stereoisomer described in the first aspect of the present invention is used in the preparation of medicines for gene therapy, gene vaccination, antisense therapy or treatment by interfering RNA application.
  • the medicament is used for the treatment of cancer or genetic diseases.
  • the tumor includes but not limited to lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or, bladder cancer, prostate cancer, and the genetic disease includes but not limited to hemophilia disease, thalassemia or Gaucher's disease.
  • the medicament is used for the treatment of cancer, allergy, toxicity and pathogen infection.
  • the above application is the application in the preparation of medicines for nucleic acid transfer.
  • the nucleic acid is RNA, including but not limited to messenger RNA (mRNA), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small Interfering RNA (siRNA) and small nuclear RNA (snRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • siRNA small Interfering RNA
  • snRNA small nuclear RNA
  • a fourth aspect of the present invention provides:
  • a nanoparticle delivery system the raw material of which is the amino lipid described in the first aspect of the present invention.
  • the present invention has the following technical effects:
  • the shown ionizable amino lipid or its pharmaceutically available salt has mild reaction conditions in the process of constructing the amino lipid, does not require protection and deprotection, and has high atom economy. In in vitro and in vivo delivery studies, it shows excellent ability to deliver nucleic acid into cells.
  • the above-mentioned amino lipid compound has two ester bonds, the introduction of this group significantly enhances the degradation ability of the cationic polymer, while greatly reducing the cytotoxicity, it can also help the release of the delivery target such as the target drug or gene, Thereby improving delivery efficiency.
  • the preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements for instruments and equipment and simple operation.
  • test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
  • PEG2000-DMG (1-(Monomethoxypolyethylene glycol)-2,3 Dimyristoylglycerol
  • Example 1 Parallel synthesis and characterization of E7C71Ay series amino lipid compound library
  • Step I solution Transfer the Step I solution to 1.5mL 96-well plate with a pipette gun (0.1mL each, 0.01mmol), and add a DCM solution (0.1mL, 0.02mmol , 0.2M), DIPEA and EDC ⁇ HCl in DCM (0.2mL, 0.04mmol, 0.2M), DMAP in DCM (0.1mL, 0.005mmol, 0.05M) were stirred at room temperature for 6h, and no Step I material was detected by TLC. After the reaction, evaporate to dryness at room temperature to obtain 15 amino lipid compounds E7C71Ay. Mass spectrometry was performed, and the results are shown in Table 1 below.
  • Example 12 In vitro evaluation of aminolipid compounds as mRNA carriers
  • HeLa cell line HeLa cell line (ATCC)
  • Detection Use a multifunctional microplate reader to detect the fluorescence intensity. Lipofectamine 3000 (Invitrogen) was used as a positive control group according to the manufacturer's instructions.
  • Method Use an 8-channel pipette to add samples. Amounts shown are for a single well of a 96-well plate.
  • the mass ratio of aminolipid compound to luciferase mRNA (Luc mRNA) is about 10:1, and the amount of mRNA per hole is 100ng.
  • lipid nanoparticle solution After incubating the lipid nanoparticle solution at room temperature for 30 minutes, add 100 ⁇ L of freshly resuspended HeLa cells (1 ⁇ 10 4 cells) to each well of a 96-well all-white microplate, and then use a pipette to transfer the lipid nanoparticle The particle solution was added to a 96-well plate (10 ⁇ L per well). Place in a 37°C incubator with 5% CO2 for incubation.
  • the substrate ONE-Glo TM Luciferase was added to the cells at 100 ⁇ L/well, and detected with a multifunctional microplate reader (Biorek Synergy H1) after 2 minutes.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • lipid nanoparticles encapsulating luciferase mRNA to the 96-well all-white ELISA plate covered with primary cells, and control the volume of mRNA lipid nanoparticles added in each well to 10 ⁇ L. They were then placed in an incubator at 37°C with 5% CO2 for 16 hours.
  • Detection of transfection efficiency 20 ⁇ L of substrate ONE-Glo TM Luciferase was added to each well of a 96-well all-white microplate plate, and detected with a multifunctional microplate reader (Biorek Synergy H1) after 1 min.
  • Table 3 shows the expression intensity of representative aminolipid compounds transfected with LucmRNA on BMDC.
  • DLin-MC3 was used as a control, and the expression intensity of many of the amino lipids was similar to that of MC3, and many of them were significantly better than the positive control.
  • Example 14 Evaluation of in vivo delivery performance of luciferase mRNA of lipid nanoparticles prepared from aminolipid compounds
  • the amino lipid compound of the present invention is mixed with neutral lipids (such as: DSPC, DOPE, cholesterol), pegylated lipids (such as: PEG2000-DMG, PEG2000-DSPE) in an optimized molar ratio and dissolved in in absolute ethanol.
  • neutral lipids such as: DSPC, DOPE, cholesterol
  • pegylated lipids such as: PEG2000-DMG, PEG2000-DSPE
  • PEG2000-DMG pegylated lipids
  • TriLink Luc-mRNA
  • the particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 20 ⁇ L of LNP solution for particle size measurement, and cycle three times for 30 s each time.
  • Determination of encapsulation efficiency determine according to the standard procedure of Quant-iT RiboGreen RNA kit.
  • In vivo delivery 9 C57BL/6 mice were randomly selected in each group, and the dosage of 0.5mg/kg mRNA was injected into the lipid nanoparticle solution by subcutaneous, intramuscular and tail vein injection respectively (each administration method 3 mice). After 12 hours, 200 ⁇ L of 10 mg/mL D-luciferin potassium salt was injected into each mouse through the tail vein, and after 10 minutes, the mouse was placed under an in vivo imaging system (IVIS-200, Xenogen), and each mouse was observed. The total fluorescence intensity of each mouse was taken and recorded. The expression intensities of Fluc mRNA delivered by representative aminolipid compounds through three administration methods are shown in Table 5-7. DLin-MC3 served as a control.
  • Table 5 Expression intensity of Luc mRNA delivered by subcutaneous administration of representative aminolipid compound LNP
  • serial number LNP number mean fluorescence intensity serial number LNP number mean fluorescence intensity 1 LNP-1 2.4E+06 10 LNP-25 2.5E+06 2 LNP-4 5.3E+06 11 LNP-29 6.3E+07
  • Table 7 Expression intensity of Luc mRNA delivered by tail vein administration of representative aminolipid compound LNP
  • Example 15 Evaluation of in vivo immune and tumor therapeutic effects of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method the amino lipid compound described in the present invention is mixed with DSPC, cholesterol, and PEG2000-DMG in a molar ratio of 50:10:38.5:1.5 and dissolved in absolute ethanol.
  • Ovalbumin mRNA Ovalbumin mRNA
  • TriLink Luc-mRNA
  • the particles were then dialyzed in 1X PBS at 4°C for 6 hours using a dialysis box (Fisher, MWCO 20,000), and filtered through a 0.22 ⁇ m microporous membrane before use.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 10:1.
  • Animal preparation 5-6 weeks old female C57BL/6 mice were selected, weighing about 18-20g, and the feeding environment was SPF-level breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • B16-OVA melanoma cells (1.5 x 105 ) were injected subcutaneously into the outer thigh of mice. Vaccination was started when the tumor size reached 50 mm 3 (approximately on the 6th or 7th day after tumor inoculation). Animals were immunized twice by intramuscular injection of LNP preparation containing 1 ⁇ g OVA-mRNA, with an interval of 7 days between the second doses. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5 ⁇ length ⁇ width. Euthanize the mice when the tumor volume reaches 1500 mm.
  • the tumor growth rate of E7C71A9 and E6C71A12 was significantly slower than that of MC3 group (as shown in Figure 3), and 60% (E7C71A9 group) and 40% (E6C71A12 group) of mice achieved complete remission, significantly better than MC3 group (as shown in Figure 4).
  • Example 20 In vitro evaluation of aminolipid compounds as mRNA carriers
  • HeLa cell line HeLa cell line (ATCC)
  • Detection Use a multifunctional microplate reader to detect the fluorescence intensity. Lipofectamine 3000 (Invitrogen) was used as a positive control group according to the manufacturer's instructions.
  • Method Use an 8-channel pipette to add samples. Amounts shown are for a single well of a 96-well plate.
  • the mass ratio of aminolipid compound to luciferase mRNA (Luc mRNA) is about 10:1, and the amount of mRNA per hole is 100ng.
  • lipid nanoparticle solution After incubating the lipid nanoparticle solution at room temperature for 30 minutes, add 100 ⁇ L of freshly resuspended HeLa cells (1 ⁇ 10 4 cells) to each well of a 96-well all-white microplate, and then use a pipette to transfer the lipid nanoparticle The particle solution was added to a 96-well plate (10 ⁇ L per well). Place in a 37°C incubator with 5% CO2 for incubation.
  • the substrate ONE-Glo TM Luciferase was added to the cells at 100 ⁇ L/well, and detected with a multi-functional microplate reader (Biorek Synergy H1) after 2 minutes.
  • Table 8 shows the transfection efficiency of some compounds on Luc-mRNA of HeLa cells.
  • Animal preparation 6-week-old female C57BL/6 mice were selected, weighing about 20 g, and the feeding environment was an SPF-grade breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • lipid nanoparticles encapsulating luciferase mRNA to the 96-well all-white ELISA plate covered with primary cells, and control the volume of mRNA lipid nanoparticles added in each well to 10 ⁇ L. They were then placed in an incubator at 37°C with 5% CO2 for 16 hours.
  • Detection of transfection efficiency 20 ⁇ L of substrate ONE-Glo TM Luciferase was added to each well of a 96-well all-white microplate plate, and detected with a multifunctional microplate reader (Biorek Synergy H1) after 1 min.
  • Table 9 shows the expression intensity of transfected LucmRNA of representative aminolipid compounds on BMDC.
  • DLin-MC3 was used as a control, and the expression intensity of many of the amino lipids was similar to that of MC3, and many of them were significantly better than the positive control.
  • Example 22 Evaluation of in vivo delivery performance of luciferase mRNA of lipid nanoparticles prepared from aminolipid compounds
  • the amino lipid compound of the present invention is mixed with neutral lipids (such as: DSPC, DOPE, cholesterol), pegylated lipids (such as: PEG2000-DMG, PEG2000-DSPE) in an optimized molar ratio and dissolved in in absolute ethanol.
  • neutral lipids such as: DSPC, DOPE, cholesterol
  • pegylated lipids such as: PEG2000-DMG, PEG2000-DSPE
  • PEG2000-DMG pegylated lipids
  • TriLink Luc-mRNA
  • the particle size and PDI of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). Take 20 ⁇ L of LNP solution for particle size measurement, and cycle three times for 30 s each time.
  • Determination of encapsulation efficiency determine according to the standard procedure of Quant-iT RiboGreen RNA kit.
  • In vivo delivery 9 C57BL/6 mice were randomly selected in each group, and the dosage of 0.5mg/kg mRNA was injected into the lipid nanoparticle solution by subcutaneous, intramuscular and tail vein injection respectively (each administration method 3 mice). After 12 hours, 200 ⁇ L of 10 mg/mL D-luciferin potassium salt was injected into each mouse through the tail vein, and after 10 minutes, the mouse was placed under an in vivo imaging system (IVIS-200, Xenogen), and each mouse was observed. The total fluorescence intensity of each mouse was taken and recorded. The expression intensities of Fluc mRNA delivered by representative aminolipid compounds through three administration methods are shown in Table 11-13. DLin-MC3 served as a control.
  • Table 11 Expression intensity of Luc mRNA delivered by subcutaneous administration of representative aminolipid compound LNP
  • serial number LNP number mean fluorescence intensity serial number LNP number mean fluorescence intensity 1 LNP-48 8.8E+06 11 LNP-70 6.2E+07 2 LNP-52 5.1E+07 12 LNP-72 8.8E+07 3 LNP-54 1.2E+08 13 LNP-73 5.6E+07 4 LNP-56 5.1E+07 14 LNP-76 6.7E+07 5 LNP-59 4.6E+07 15 LNP-79 8.1E+07 6 LNP-60 1.1E+08 16 LNP-82 9.3E+07 7 LNP-62 3.2E+07 17 LNP-83 5.5E+07 8 LNP-65 8.3E+07 18 LNP-85 7.2E+07 9 LNP-66 9.4E+07 19 LNP-87 1.0E+08 10 LNP-69 2.6E+07 20 LNP-90 8.9E+07 19 LNP-87 1.0E+08 10 LNP-69 2.6E+07 20 LNP-90 8.9E+07 19 LNP-87
  • Table 12 Expression intensity of Luc mRNA delivered by intramuscular injection of representative aminolipid compound LNP
  • serial number LNP number mean fluorescence intensity serial number LNP number mean fluorescence intensity 1 LNP-49 5.3E+07 11 LNP-72 7.8E+07 2 LNP-54 1.5E+08 12 LNP-73 6.4E+07 3 LNP-55 6.4E+07 13 LNP-77 4.5E+07 4 LNP-59 7.0E+07 14 LNP-80 4.8E+07 5 LNP-60 1.7E+08 15 LNP-81 5.9E+07 6 LNP-61 6.1E+07 16 LNP-83 6.4E+07 7 LNP-65 1.3E+08 17 LNP-84 8.3E+07 8 LNP-66 9.9E+07 18 LNP-85 6.8E+07 9 LNP-68 8.6E+07 19 LNP-88 1.0E+08 10 LNP-70 8.2E+07 20 LNP-90 9.6E+07
  • Table 13 Expression intensity of Luc mRNA delivered by tail vein administration of representative aminolipid compound LNP
  • serial number LNP number mean fluorescence intensity serial number LNP number mean fluorescence intensity 1 LNP-51 3.5E+07 11 LNP-70 5.4E+07 2 LNP-53 3.3E+07 12 LNP-72 4.9E+07 3 LNP-54 8.1E+07 13 LNP-74 2.4E+07 4 LNP-56 6.2E+07 14 LNP-78 3.8E+07 5 LNP-58 4.7E+07 15 LNP-82 2.3E+07 6 LNP-59 7.9E+07 16 LNP-83 9.9E+06 7 LNP-60 1.3E+08 17 LNP-85 1.4E+07 8 LNP-64 5.4E+07 18 LNP-87 1.0E+08 9 LNP-65 7.6E+07 19 LNP-89 8.7E+07 10 LNP-66 8.9E+07 20 LNP-91 6.3E+06
  • Example 23 In vivo immune and tumor therapeutic effect evaluation of lipid nanoparticles prepared from aminolipid compounds
  • Preparation method the amino lipid compound described in the present invention is mixed with DSPC, cholesterol and PEG2000-DMG in a molar ratio of 50:10:38.5:1.5 and dissolved in absolute ethanol.
  • the mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 8:1.
  • Animal preparation 5-6 weeks old female C57BL/6 mice were selected, weighing about 18-20g, and the feeding environment was SPF-level breeding room. Animal experiments were carried out in strict accordance with the guidelines of the national health agency and animal ethics requirements.
  • B16-OVA melanoma cells (1.5 x 105 ) were injected subcutaneously into the outer thigh of mice. Vaccination was started when the tumor size reached 50 mm 3 (approximately on the 6th or 7th day after tumor inoculation). Animals were immunized twice by intramuscular injection of LNP preparation containing 5 ⁇ g of OVA-mRNA, with an interval of 7 days between the second doses. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5 ⁇ length ⁇ width ⁇ width. Euthanize the mice when the tumor volume reaches 1500 mm.
  • the tumor growth rate of E7C115A11 was significantly slower than that of the MC3 group, 90% of the mice tumors completely regressed (as shown in Figure 5), and the survival rate reached 100%, which was significantly better than that of the MC3 group (as shown in Figure 6).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Pulmonology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

涉及式（I）所示的可离子化氨基脂质或其可药用盐，该氨基脂质可用于递送核酸及小分子药物，其具备两个酯键，显著增强了可离子化氨基脂质的溶酶体逃逸能力，有助于目的药物或基因等递送目标的释放，进而提高递送效率，显示了优良的递送核酸至细胞中的能力。

Description

一种氨基脂质及其制备方法和应用 技术领域
本发明医药化学技术领域，特别涉及一种氨基脂质及其制备方法应用。
背景技术
核酸药物在癌症、传染性疾病、遗传性疾病和心血管疾病的预防与治疗方面具有极为广阔的应用前景。然而，由于RNA、DNA和siRNA等体内极易被降解，直接通过口服或静脉注射给药，生物利用度极低，因此需要载体的递送。
常用的核酸载体包括病毒载体和非病毒载体，病毒载体具有较高的转染效率，但是病毒载体缺乏靶向性，存在较大的安全顾虑，且载体容量小，生产成本较高。非病毒载体具有安全性较高，载体分子容易修饰，适合大量生产等优点，具有广阔的应用前景。其中基于脂质的纳米颗粒递送系统(Lipid Nanoparticles,LNP)的应用走在了前列，LNP一般由可离子化或阳离子脂质、膦脂、胆固醇和聚乙二醇化的脂质组成。结构上均为具有自组装性能的两亲性分子，LNP各成分结构确定，重现性好，利于质量监管，同时具有较长的体内循环时间、良好的生物相容性等优点而受到广泛关注。纳米颗粒在进入细胞后，需逃出内涵体/溶酶体，才能在细胞质中释放RNA，使其得以表达生成目的蛋白。
技术问题
目前LNP的内涵体/溶酶体逃逸率普遍较低，尽管DLin-MC3-DMA作为氨基脂质体内评价的“金标准”，是目前最高效的氨基脂质，并被FDA批准用于首个siRNA治疗性药物Onpattro(patisiran)，也仅有1％-4％的RNA逃出内涵体/溶酶体，内涵体/溶酶体逃逸已成为影响核酸递送的关键步骤，因此设计具有良好包载核酸能力，同时又具有较高的内涵体/溶酶体逃逸能力的氨基脂质，以解决核酸递送问题，具有重大的研究意义和现实需求。
技术解决方案
针对现有技术中阳离子脂质体的转染的效率低、正电荷所导致的细胞毒性等技术问题，
本发明提供了一种氨基脂质及其应用。
本发明的目的通过以下技术方案予以实现：
本发明的第一个方面，提供：
一种氨基脂质，其结构如式(I)所示：
Figure PCTCN2022131322-appb-000001
其中，L为C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚炔基、C 3-C 6亚环烷基、C 3-C 6亚环烯基；
R 1和R 2相同或不同，并且各自独立地选自C 1-C 20烷基、C 1-C 20烯基、C 1-C 20炔基、C 1-C 20环烷基、C 1-C 20环烯基、C 1-C 20环炔基；所述C 1-C 20烷基、C 1-C 20烯基、C 1-C 20炔基、C 1-C 20环烷基、C 1-C 20环烯基、C 1-C 20环炔基可任选地被氢、C1-C6烃基、氟取代；
R 3和R 4彼此相同或不同，并且各自独立地选自H，C 1-C 10烷基、C 2-C 10烯基、C 2-C 10炔基；所述C 1-C 10烷基、C 2-C 10烯基、C 2-C 10炔基可任选地被C 1-C 6烃基取代；
或R 3和R 4相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。
优选地，所述R 1选自C 4-C 17烷基、C 4-C 17烯基、C 4-C 17炔基、C 4-C 17环烷基、C 4-C 17环烯基、C 4-C 17环炔基；所述C 4-C 17烷基、C 4-C 17烯基、C 4-C 17炔基、C 4-C 17环烷基、C 4-C 17环烯基、C 4-C 17环炔基可任选地被氢、C 1-C 6烃基、氟取代。
优选地，R 1选自E1、E2、E3、E4、E5、E6、E7、E8、E9、E10、E11、E12、E13、E14、E15、E16、E17、E18、E19、E20、E21、E22、E23、E24、E25中的一种：
Figure PCTCN2022131322-appb-000002
更优选地，R 1选自E3、E4、E5、E6、E7、E8、E9、E10、E11、E12、E13、E15、E17、E18、E20、E21、E24中的一个。
优选地，所述R 2选自C 5-C 19烷基、C 5-C 19烯基、C 5-C 19炔基、C 5-C 19环烷基、C 5-C 19环烯基、C 5-C 19环炔基；所述C 5-C 19烷基、C 5-C 19烯基、C 5-C 19炔基、C 5-C 19环烷基、C 5-C 19环烯基、C 5-C 19环炔基可任选地被氢、C 1-C 6烃基、氟取代；
优选地，R 2选自C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30、C31、C32、C33、C34、C35、C36、C37、C38、C39、C40、C41、C42、C43、C44、C45、C46、C47、C48、C49、C50、C51、C52、C53、C54、C55、C56、C57、C58、C59、C60、C61、C62、C63、C64、C65、C66、C67、C68、C69、C70、C71、C72、C73、 C74、C75、C76、C77、C78、C79、C80、C81、C82、C83、C84、C85、C86、C87、C88、C89、C90、C91、C92、C93、C94、C95、C96、C97、C98、C99、C100、C101、C102、C103、C104、C105、C106、C107、C108、C109、C110、C111、C112、C113、C114、C115、C116、C117、C118、C119、C120、C121、C122、C123、C124、C125、C126、C127、C128、C129、C130、C131、C132、C133、C134、C135、C136、C137、C138、C139、C140、C141、C142、C143、C144、C145、C146、C147中的一种：
Figure PCTCN2022131322-appb-000003
Figure PCTCN2022131322-appb-000004
Figure PCTCN2022131322-appb-000005
更优选地，R 2选自C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C56、C57、C58、C60、C62、C63、C64、C66、C67、C71、C72、C74、C79、C82、C83、C102、C103、C104、C105、C106、C107、C108、C109、C110、C111、C112、C113、C114、C115、C116、C117、C118、C119、C120、C121、C122、C123、C124、C125、C126、C127、C128、C129、C130、C131、C132、C133、C134、C135、C136、C137、C138、C139、C140、C141、C142、C143、C144、C145、C146、C147中的一个。
优选地，R 3、R 4、L形成R 3R 4-N-L的含胺的羧酸结构为：
Figure PCTCN2022131322-appb-000006
和/或R 3和R 4相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。
优选地，
Figure PCTCN2022131322-appb-000007
选自：A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A11、A12、A13、A14、A15、A16、A17、A18、A19、A20、A21、A22、A23、A24、A25、A26、A27、A28、A29、A30、A31、A32、A33、A34、A35、A36、A37、A38、A39、A40中的一种：
Figure PCTCN2022131322-appb-000008
更优选地，
Figure PCTCN2022131322-appb-000009
选自A1、A7、A8、A9、A10、A11、A12、A14、A15、A16、A23、A24、A28、A33、A37中的一个。
Figure PCTCN2022131322-appb-000010
优选的，所述氨基脂质选自如下结构所示化合物的一种：
Figure PCTCN2022131322-appb-000011
Figure PCTCN2022131322-appb-000012
本发明的第二个方面，提供：
本发明第一个方面所述氨基脂质的制备方法，包括以下步骤：
S1.化合物R 2COOH与环氧类化合物在催化剂FeCl 3/Py的催化下无溶剂搅拌反应；
S2.在步骤S1反应体系中加入R 3R 4NLCOOH，并在缩合剂存在的条件下反应即得。
反应的流程如下：
Figure PCTCN2022131322-appb-000013
优选地，所述方法包括以下步骤：
(1)常温下，环氧类化合物与由R 2COOH表示的化合物在催化剂FeCl 3/Py的催化下进行第一反应，得到第一中间体；
(2)分离第一中间体，在缩合剂的作用下，加入催化量的DMAP，使所述第一中间体与R 3R 4NLCOOH的羧酸在常温下进行第二反应，得到所述式I的氨基脂质化合物。上述制备方法中使用的缩合剂为EDC·HCl、DCC等。
本发明的第三个方面，提供：
本发明第一个方面所述氨基脂质或其药物可接受的盐、前药或立体异构体在制备用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。
优选地，所述药物用于癌症或遗传疾病的治疗。
优选地，所述肿瘤包括但不限于肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌或、膀胱癌、前列腺癌，所述遗传疾病包括但不限于血友病，地中海贫血或高雪氏病中。
优选地，所述药物用于治疗癌症、过敏、毒性和病原体感染。
优选地，上述应用为在制备用于核酸转移的药物中的应用。
优选地，所述核酸为RNA，包括但不限于信使RNA(mRNA)、反义寡核苷酸、DNA、质粒、核糖体RNA(rRNA)、微RNA(miRNA)、转移RNA(tRNA)、小干扰RNA(siRNA)和小核RNA(snRNA)。
本发明的第四个方面，提供：
一种纳米颗粒递送系统，其原料本发明第一个方面所述的氨基脂质。
有益效果
与现有技术相比本发明具有以下技术效果：
本发明公开的通式(Ⅰ)
Figure PCTCN2022131322-appb-000014
所示的可离子化氨基脂质或其药物可用的盐，在构建氨基脂质的过程中反应条件温和，不需要保护和脱保护，原子经济性高。在体外、体内的递送研究中，显示了优良的递送核酸至细胞中的能力。上述氨基脂质化合物具备两个酯键，该基团的引入显著增强了阳离子聚合物的降解能力，在大幅度降低细胞毒性的同时，还能有助于目的药物或基因等递送目标的释放，进而提高递送效率。所述氨基脂质化合物的制备方法具有原料易得、反应条件温和、反应选择性好、反应产率高、仪器设备要求低和操作简单的优点。
附图说明
图1实施例5中E7C71A9的 1H-NMR图谱；
图2实施例5中E7C71A9的 13C-NMR图谱；
图3实施例15中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的肿瘤生长曲线图(分别使用E6C71A12与E7C71A9组装成的LNP)；
图4实施例15中肌肉注射OVA mRNA疫苗后的荷瘤小鼠生存曲线图(分别使用E6C71A12与E7C71A9组装成的LNP)；
图5实施例23中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的肿瘤生长曲线图(使用E7C115A11组装成的LNP)；
图6实施例23中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的肿瘤生长曲线图(使用E7C115A11组装成的LNP)。
本发明的实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
本发明所用的术语“任选地取代的”意指与原子或基团连接的一个或多个氢原子独立地未被取代，或被一个或多个例如一、二、三或四个取代基取代。当一个原子或基团被多个取代基取代时，所述多个取代基可以相同或不同。
文中的缩写：
RNA     核糖核酸
DSPC    二硬脂酰磷脂酰胆碱
DOPE    二油酰基磷脂酰乙醇胺
DOPC    二油酰磷脂酰胆碱
DOPS    二油酰基磷脂酰丝氨酸
DSPE    二硬脂酰磷脂酰乙醇胺
PEG2000-DMG    (1-(单甲氧基聚乙二醇)-2,3二肉豆寇酰基甘油
kD      千道尔顿
PBS     磷酸盐缓冲溶液。
以下实施例中如无特别说明，氨基脂质结构通式如式(Ⅰ)所示
Figure PCTCN2022131322-appb-000015
编号指代的氨基脂质结构中，E1～E25为上述定义的R 1取代基、C1～C147为上述定义的R 2取代基、A1～A40为上述定义的
Figure PCTCN2022131322-appb-000016
基团。如E7C71A1的结构式为
Figure PCTCN2022131322-appb-000017
实施例1：E7C71Ay系列氨基脂质化合物库的平行合成与表征
Figure PCTCN2022131322-appb-000018
在25mL的反应管中依次加入FeCl 3(4mg，0.005mmol)、Py(1μL，0.0025mmol)、2-己基癸酸(0.3mL,1mmol)和1,2-环氧十二烷(0.27mL,1.2mmol)，室温下搅拌反应过夜，得Step I(1mmol)，加入10mL的DCM，配成0.1M的Step I溶液。
用移液枪将Step I溶液分别转移至1.5mL的96孔板中(每个0.1mL，0.01mmol)，每孔中各加入含三级胺基团羧酸的DCM溶液(0.1mL，0.02mmol，0.2M)、DIPEA和EDC·HCl的DCM溶液(0.2mL，0.04mmol，0.2M)，DMAP的DCM溶液(0.1mL，0.005mmol，0.05M)常温搅拌6h，TLC检测无Step I原料。反应结束后，常温挥发至干，即得到15个氨基脂质化合物E7C71Ay。进行质谱检测，结果见下面的表1。
表1：E7C71Ay系列氨基脂质化合物库的MW/z值
Figure PCTCN2022131322-appb-000019
Figure PCTCN2022131322-appb-000020
实施例2：2-羟基十六烷基十二烷酸酯
Figure PCTCN2022131322-appb-000021
在25mL的反应管中依次加入FeCl 3(20mg，0.025mmol)、Py(5μL，0.0125mmol)、月桂酸(1g，5mmol)和1,2-环氧十六烷(1.7mL,6mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得2-羟基十六烷基十二烷酸酯(2.0g，90％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，6H)，1.26-1.45(m，40H)，1.47(m，2H)，1.63(m，2H)，2.02(m，1H)，2.34(t，2H)，3.82(m，1H)，3.95(m，1H)，4.13(m，1H)。ESI-MS calculated for C 28H 57O 3 +[M+H] +441.4,found 441.6。
实施例3：2-((4-(二甲氨基)丁酰基)氧基)十二烷酸十六烷酯
Figure PCTCN2022131322-appb-000022
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，4-(二甲氨基)丁酸(101mg，0.6mmol)，2-羟基十六烷十二烷酸酯(220mg，0.5mmol)和DCM(4mL)。室温下搅拌3h得到化合物E11C7A9(235mg,，85％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，6H)，1.25-1.45(m，40H)，1.58(m，4H)，1.78(m，2H)，2.23(s，6H)，2.30(m，6H)，4.01(m，1H)，4.21(m，1H)，5.08(m，1H)。 13C NMR(400MHz，CDCl 3)：δ14.03，14.08，22.59，22.64，23.35，25.11，27.39，27.43，29.23，29.27，29.29，29.45，29.53，29.59，30.80，31.65，31.85，31.89，32.16，32.39，47.39，47.69，58.86，64.49，71.53，171.87，173.43。ESI-MS calculated for C 34H 68NO 4 +[M+H] +554.5,found 554.7。
实施例4：2-羟基十二烷基2-己基癸酸酯
Figure PCTCN2022131322-appb-000023
在25mL的反应管中依次加入FeCl 3(20mg，0.025mmol)、Py(5μL，0.0125mmol)、2-己基癸酸(1.3g，5mmol)和1,2-环氧十二烷(1.3mL,6mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得到2-羟基十二烷基2-己基癸酸酯 (1.9g，85％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，9H)，1.26-1.45(m，36H)，1.47(m，2H)，1.63(m，2H)，2.02(m，1H)，2.34(t，2H)，3.82(m，1H),3.95(m，1H)，4.13(m，1H)。ESI-MS calculated for C 28H 56O 3 +[M+H] +441.4,found 441.5。
实施例5：2-((4-(二甲氨基)丁酰基)氧基)十二烷基2-己基癸酸酯
Figure PCTCN2022131322-appb-000024
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，4-(二甲氨基)丁酸(101mg，0.6mmol)，2-羟基十二烷基2-己基癸酸酯(220mg，0.5mmol)和DCM(4mL)。室温下搅拌3h得到化合物E7C71A9(235mg,，85％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，9H)，1.25-1.45(m，38H)，1.58(m，4H)，1.79(m，2H)，2.12-2.30(m，11H)，4.01(m，1H)，4.22(m，1H)，5.07(m，1H)(图1)。 13C NMR(400MHz，CDCl 3)：δ14.04，14.07，22.59，22.64，22.95，25.11，27.39，27.43，29.23，29.27，29.29，29.45，29.53，29.59,30.79，31.68，31.85，31.87，32.18，32.37,45.39，45.69，58.84，64.48，71.51，172.98，176.23(图2)。ESI-MS calculated for C 34H 67NO 4 +[M+H] +554.5,found 554.6。
实施例6：2-羟基癸基-十八烷-9-烯酸酯
Figure PCTCN2022131322-appb-000025
在25mL的反应管中依次加入FeCl 3(20mg，0.025mmol)、Py(5μL，0.0125mmol)、油酸(1.6mL，5mmol)和1,2-环氧癸烷(1.1mL,6mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得到2-羟基癸基-十八烷-9-烯酸酯(1.9 g，85％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，6H)，1.26-1.45(m，34H)，1.63(m，2H)，2.17(m，4H)，2.33(m，2H)，4.09-4.35(m，3H)，5.35-5.43(m，2H)。ESI-MS calculated for C 28H 55O 3 +[M+H] +439.4,found 439.6。
实施例7：1-(油酰氧基)癸烷-2-基1-甲基哌啶-4-羧酸酯
Figure PCTCN2022131322-appb-000026
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，1-甲基哌啶-4-羧酸(86mg，0.6mmol)，2-羟基癸基-十八烷-9-烯酸酯(219mg，0.5mmol)和DCM(4mL)。室温下搅拌3h得到化合物E5C82A23(226mg，80％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，6H)，1.23-1.46(m，32H)，1.49(m，2H)，1.66(m，2H)，1.73-2.03(m，4H)，2.11-2.20(m，7H)，2.33-2.51(m，7H)，4.03(m，1H)，4.24(m，1H)，5.07(m，1H)，5.43(m，2H)。 13C NMR(400MHz，CDCl 3)：δ14.04，14.06，22.59，22.64，25.01，25.26，29.23，29.25，29.27，29.29，29.45，29.53，29.59，30.80，31.65，31.85，31.90，32.16，32.39，47.39，47.69，58.86，64.49，71.53，130.57，130.63，171.97，173.73。ESI-MS calculated for C 35H 66NO 4 +[M+H] +564.5,found 564.6。
实施例8：3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯
Figure PCTCN2022131322-appb-000027
在25mL的反应管中依次加入FeCl 3(20mg，0.025mmol)、Py(5μL，0.0125mmol)、
豆蔻酸(1.1g，5mmol)和3-(全氟正己基)环氧丙烷(1.4mL,6mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯(2.7g，90％)。 1H NMR(400MHz， CDCl 3)：δ0.88(t，3H)，1.26-1.45(m，20H)，1.63(m，2H)，2.03(m，2H)，2.36(t，2H)，6.68(t，1H)。ESI-MS calculated for C 22H 32F 13O 3 +[M+H] +591.2，found 591.3。
实施例9：1-((4-(二甲氨基)丁酰基)氧基)-3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟辛基十四烷酸酯
Figure PCTCN2022131322-appb-000028
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，4-(二甲氨基)丁酸(101mg，0.6mmol)，3,3,4,4,5,5,6,6,7,7,8,8,8-十三氟-1-羟基辛基十四烷酸酯(295mg，0.5mmol)和DCM(4mL)。室温下搅拌3h得到化合物E24C9A9(263.8mg，75％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，3H)，1.23-1.60(m，20H)，1.66(m，2H)，1.88(m，2H)，2.05-2.16(m，8H)，2.36(m，4H)，3.10(t，2H)，7.46(t，1H)。 13C NMR(400MHz，CDCl 3)：δ14.07，22.69，22.95，25.06，27.39，29.25，29.41，29.57，29.59，30.79，31.85，31.87，32.18，32.37，46.39，46.69，59.84，88.37，109.05，110.09，111.89，112.37，118.49，173.67，176.23。ESI-MS calculated for C 28H 43F 13NO 4 +[M+H] +704.3，found 704.5。
实施例10：8-乙基-2-羟基癸基棕榈酸酯
Figure PCTCN2022131322-appb-000029
在25mL的反应管中依次加入FeCl 3(20mg，0.025mmol)、Py(5μL，0.0125mmol)、2-己基癸酸(1.3g，5mmol)和2-(6-乙基辛基)环氧乙烷(1.3mL,6mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得8-乙基-2-羟基癸基棕榈 酸酯(2.0g，90％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，9H)，1.26-1.45(m，37H)，1.47(m，2H)，1.63(m，2H)，2.02(m，1H)，2.34(t，2H)，3.82(m，1H)，3.95(m，1H)，4.13(m，1H)。ESI-MS calculated for C 28H 57O 3 +[M+H] +441.4,found 441.6。
实施例11：2-((4-(二甲氨基)丁酰基)氧基)-8-乙基癸基棕榈酸酯
Figure PCTCN2022131322-appb-000030
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，4-(二甲氨基)丁酸(101mg，0.6mmol)，8-乙基-2-羟基癸基棕榈酸酯(220mg，0.5mmol)和DCM(4mL)。室温下搅拌3h得到化合物E20C11A9(221mg,，80％)。 1H NMR(400MHz，CDCl 3)：δ0.88(t，9H)，1.25-1.45(m，37H)，1.58(m，4H)，1.78(m，2H)，2.23(s，6H)，2.30(m，6H)，4.01(m，1H)，4.21(m，1H)，5.08(m，1H)。 13C NMR(400MHz，CDCl 3)：δ12.01，12.08，14.09，22.59，22.64，22.95，25.11，27.39，27.43，29.23，29.25，29.31,29.44,29.56,29.59,30.80,31.64，31.83，31.87，32.21，32.33，46.48，46.89，59.45，65.36，71.70，173.48,176.73。ESI-MS calculated for C 34H 68NO 4 +[M+H] +554.5,found 554.7。
实施例12：氨基脂质化合物作为mRNA载体的体外评价
细胞系：HeLa细胞系(ATCC)
培养基：补充了10％胎牛血清的1640(Invitrogen)
筛选形式：96孔板细胞转染
检测：使用多功能酶标仪检测荧光强度。根据制造商的说明，Lipofectamine3000(Invitrogen)用作阳性对照组。
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。
1.参考实施例1中所述的路线合成得到一系列氨基脂质化合物，将其与二硬脂酰磷脂酰胆碱(DSPC)，胆固醇，PEG2000-DMG按摩尔比为50：10：38.5：1.5的比例混合溶解在无水乙醇中；Luc-mRNA(TriLink)溶解在醋酸钠缓冲液(25nM，pH＝5.0)中，使用排枪取 出上述的混合脂质溶液，加入至Luc-mRNA溶液中，充分混合，控制其配比为乙醇溶液与醋酸钠缓冲液(25nM，pH＝5.0)的比例为1:3，制得脂质纳米颗粒溶液。氨基脂质化合物与荧光素酶mRNA(Luc mRNA)的质量比约为10:1，每孔mRNA的用量为100ng。
2.脂质纳米颗粒溶液在室温下孵育30min后，于96孔全白酶标板中每孔加入100μL新鲜重悬浮的HeLa细胞(1×10 4细胞)，再用移液管将脂质纳米颗粒溶液加入到96孔板中(每孔10μL)。置于37℃含有5％CO 2的培养箱中孵育。
3.细胞初始转染后16至20小时，将底物ONE-Glo TMLuciferase以100μL/孔加入细胞中，2min后用多功能酶标仪(Biorek SynergyH1)进行检测。
4.相对转染效率如下计算：
Figure PCTCN2022131322-appb-000031
结果：部分化合物对HeLa细胞的Luc-mRNA的转染效率示于表2中。
表2：4345种化合物对HeLa细胞的mRNA相对转染效率
Figure PCTCN2022131322-appb-000032
Figure PCTCN2022131322-appb-000033
Figure PCTCN2022131322-appb-000034
Figure PCTCN2022131322-appb-000035
Figure PCTCN2022131322-appb-000036
Figure PCTCN2022131322-appb-000037
Figure PCTCN2022131322-appb-000038
Figure PCTCN2022131322-appb-000039
Figure PCTCN2022131322-appb-000040
Figure PCTCN2022131322-appb-000041
Figure PCTCN2022131322-appb-000042
实施例13：氨基脂质化合物制备的脂质纳米颗粒在BMDC原代细胞上的转染
制剂方法：同实施例7。
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
细胞获取：把C57BL/6小鼠进行脱颈臼处死，并置于75％酒精中浸泡5分钟进行消毒，解剖获取小鼠大腿及小腿胫骨，并把附着的肌肉剔除露出骨质，然后用1mL吸有PBS的注射器把胫骨中的骨髓吹出，把所得骨髓吹散后通过50μm滤网滤去杂质，向所得过滤物中加入红细胞裂解液(3～4mL)后放置5分钟后进行800g、5分钟的离心除去上清液，将所得细胞置于1640培养基(含10％胎牛血清、20ng/mL GMCSF、10ng/mL IL4)中重悬，并接种于6孔板中，接种密度为100000个细胞/毫升培养基，放置于37℃、含5％CO 2细胞培 养箱中，每2天进行半换液一次，于第七天收集悬浮细胞和松散贴壁的细胞，并接种到96孔全白酶标板接种密度为每孔20000个细胞，培养基体积为100μL。
细胞转染：向铺有原代细胞的96孔全白酶标板中加入包裹萤光素酶mRNA的脂质纳米颗粒，控制每孔中的mRNA脂质纳米颗粒加入体积为10μL。随后放置在37℃、含5％CO 2浓度的培养箱中16小时。
转染效率检测：往96孔全白酶标板中每孔加入20μL底物ONE-Glo TMLuciferase，1min后用多功能酶标仪(Biorek SynergyH1)进行检测。代表性氨基脂质化合物在BMDC上转染LucmRNA的表达强度见表3。DLin-MC3作为对照，所述的氨基脂质中有多个与MC3表达强度相似，并且还有多个显著优于阳性对照。
表3 98个氨基脂质化合物在BMDC上的转染的表达强度
Figure PCTCN2022131322-appb-000043
Figure PCTCN2022131322-appb-000044
Figure PCTCN2022131322-appb-000045
Figure PCTCN2022131322-appb-000046
Figure PCTCN2022131322-appb-000047
Figure PCTCN2022131322-appb-000048
Figure PCTCN2022131322-appb-000049
Figure PCTCN2022131322-appb-000050
Figure PCTCN2022131322-appb-000051
实施例14：氨基脂质化合物制备的脂质纳米颗粒的萤光素酶mRNA体内递送性能评价
1.脂质纳米颗粒的制备
将本发明的氨基脂质化合物与中性脂质(如：DSPC、DOPE、胆固醇)、聚乙二醇化脂质(如：PEG2000-DMG、PEG2000-DSPE)按优化后的摩尔比混合并溶解在无水乙醇中。使用微流控制备系统使所得的乙醇溶液和溶解有Luc-mRNA(TriLink)的醋酸钠缓冲液(25mM,pH＝5.0)以1:3的体积比在微流控芯片中混合以制得脂质纳米颗粒的粗溶液，然后将其用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h。使用前再通过0.22μm的微孔滤膜进行过滤。氨基脂质化合物与萤光素酶mRNA(Luc mRNA)的质量比约为10:1。
脂质纳米颗粒的表征：
粒径的表征：所制备的脂质纳米颗粒的粒径和PDI通过Nano-ZSZEN3600(Malvern)测定。取LNP溶液20μL进行粒径测量，循环三次，每次循环30s。
包封率测定：参照Quant-iT RiboGreen RNA试剂盒标准规程进行测定。
表4：使用代表性氨基脂质化合物制备的LNP的表征数据
编号 氨基脂质编号 Z-Average(d.nm) PDI 包封率
LNP-1 E4C79A9 122.4 0.12 93.9％
LNP-2 E4C79A11 124.6 0.14 94.1％
LNP-3 E4C79A12 122.1 0.07 95.3％
LNP-4 E5C71A11 123.1 0.10 93.5％
LNP-5 E5C79A11 131.2 0.07 95.6％
LNP-6 E6C71A11 124.1 0.13 93.0％
LNP-7 E6C71A12 110.1 0.09 93.6％
LNP-8 E6C79A10 129.2 0.05 94.4％
LNP-9 E6C79A11 141.2 0.08 95.6％
LNP-10 E6C79A12 123.4 0.10 93.7％
LNP-11 E7C71A9 112.1 0.02 96.6％
LNP-12 E7C71A10 126.8 0.09 95.3％
LNP-13 E7C79A9 129.8 0.08 94.9％
LNP-14 E7C79A10 148.3 0.04 95.2％
LNP-15 E7C79A11 140.1 0.02 95.4％
LNP-16 E8C71A9 147.6 0.09 93.1％
LNP-17 E8C71A11 136.4 0.04 94.8％
LNP-18 E8C71A12 139.5 0.02 95.0％
LNP-19 E8C79A9 123.6 0.06 91.8％
LNP-20 E9C56A10 140.9 0.15 95.9％
LNP-21 E9C57A10 140.4 0.13 92.2％
LNP-22 E9C62A10 139.2 0.02 93.0％
LNP-23 E9C64A9 150.4 0.12 94.4％
LNP-24 E9C64A10 143.7 0.12 95.2％
LNP-25 E9C71A10 139.9 0.12 92.5％
LNP-26 E11C57A11 157.6 0.08 93.6％
LNP-27 E11C60A11 103.7 0.05 92.9％
LNP-28 E11C62A11 102.6 0.05 91.4％
LNP-29 E15C79A11 100.2 0.02 93.3％
LNP-30 E15C79A12 107.1 0.08 92.1％
LNP-31 E20C10A12 111.9 0.11 93.4％
LNP-32 E20C11A10 109.8 0.06 94.0％
LNP-33 E20C12A9 103.3 0.09 93.6％
LNP-34 E20C71A9 107.2 0.14 95.3％
LNP-35 E21C11A11 101.1 0.12 94.1％
LNP-36 E21C12A9 109.7 0.05 95.0％
LNP-37 E6C71A12 139.3 0.10 93.5％
LNP-38 E7C71A9 141.7 0.09 92.6％
LNP-39 DLin-MC3 139.2 0.15 94.6％
LNP-40 E6C71A12 139.8 0.05 93.5％
LNP-41 E7C71A9 141.3 0.10 94.6％
LNP-42 E6C71A12 137.3 0.08 93.6％
LNP-43 E7C71A9 140.1 0.09 93.5％
LNP-44 E6C71A12 134.7 0.13 94.6％
LNP-45 E7C71A9 138.9 0.11 94.5％
LNP-46 E6C71A12 132.3 0.04 93.4％
LNP-47 E7C71A9 124.2 0.06 94.5％
注：上表中：
LNP-1～LNP-36的脂质配方为：氨基脂质:DSPC:胆固醇:PEG2000-DMG＝50:10:38.5:1.5；
LNP-37～LNP-39的脂质配方为：氨基脂质:DOPE:胆固醇:PEG2000-DMG＝45:10:42.5:1.5；
LNP-40和LNP-41的脂质配方为：氨基脂质:DOPC:胆固醇:PEG2000-DMG＝55:5:38.5:1.5；
LNP-42和LNP-43的脂质配方为：氨基脂质:胆固醇:PEG2000-DSPE＝60:35.5:4.5；
LNP-44和LNP-45的脂质配方为：氨基脂质:DSPC:DOPC:胆固醇:PEG2000-DMG＝45:10:5:38.0:2.0；
LNP-46和LNP-47的脂质配方为：氨基脂质:DSPC:DOPE:胆固醇:PEG2000-DSPE＝50:10:5:33.5:1.5。
2.动物实验
动物准备：选取体重约20g的6周龄的雌性C57BL/6小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：每组随机选取9只C57BL/6小鼠，按0.5mg/kg mRNA的用量，分别使用皮下、肌肉和尾静脉注射三种给药方式注射脂质纳米颗粒溶液(每种给药方式3只小鼠)。12小时后，往每只小鼠体内通过尾静脉注射200μL 10mg/mL的D-萤光素钾盐，10分钟后，将小鼠放置于活体成像系统(IVIS-200,Xenogen)下，观察每只小鼠总的萤光强度并拍照记录。代表性氨基脂质化合物通过3种给药方式递送的Fluc mRNA的表达强度见表5-7。DLin-MC3作为对照。
表5：代表性氨基脂质化合物LNP皮下给药递送的Luc mRNA的表达强度
编号 LNP编号 平均荧光强度 编号 LNP编号 平均荧光强度
1 LNP-1 2.4E+06 10 LNP-25 2.5E+06
2 LNP-4 5.3E+06 11 LNP-29 6.3E+07
3 LNP-5 1.5E+06 12 LNP-32 1.8E+06
4 LNP-6 7.9E+06 13 LNP-34 5.9E+06
5 LNP-7 7.0E+07 14 LNP-35 4.8E+06
6 LNP-11 8.9E+07 15 LNP-37 5.8E+06
7 LNP-16 6.6E+06 16 LNP-38 1.4E+06
8 LNP-17 3.1E+06 17 LNP-39 4.5E+06
9 LNP-20 7.7E+06 18 LNP-43 5.7E+07
表6：代表性氨基脂质化合物LNP肌注给药递送的Luc mRNA的表达强度
编号 LNP编号 荧光强度 编号 LNP编号 荧光强度
1 LNP-2 4.3E+06 10 LNP-27 7.7E+06
2 LNP-7 5.1E+07 11 LNP-30 2.8E+06
3 LNP-8 4.5E+06 12 LNP-39 7.4E+06
4 LNP-11 7.6E+07 13 LNP-40 3.5E+06
5 LNP-12 1.9E+06 14 LNP-41 2.7E+06
6 LNP-14 8.6E+06 15 LNP-42 6.5E+06
7 LNP-15 3.1E+06 16 LNP-45 3.4E+07
8 LNP-23 9.7E+06 17 LNP-46 8.5E+06
9 LNP-26 3.3E+06 18 LNP-47 5.7E+06
表7：代表性氨基脂质化合物LNP尾静脉给药递送的Luc mRNA的表达强度
编号 LNP编号 荧光强度 编号 LNP编号 荧光强度
1 LNP-3 3.4E+06 10 LNP-22 5.7E+06
2 LNP-7 8.3E+07 11 LNP-24 6.4E+06
3 LNP-9 5.9E+06 12 LNP-28 7.9E+06
4 LNP-10 1.9E+07 13 LNP-31 5.8E+06
5 LNP-11 7.0E+07 14 LNP-33 2.6E+06
6 LNP-13 4.9E+06 15 LNP-39 4.7E+06
7 LNP-18 5.6E+06 16 LNP-42 8.4E+07
8 LNP-19 3.9E+07 17 LNP-43 7.5E+07
9 LNP-21 1.1E+07 18 LNP-44 5.3E+06
实施例15：氨基脂质化合物制备的脂质纳米颗粒的体内免疫和肿瘤治疗效果评价
制剂方法：将本发明中所述的氨基脂质化合物与DSPC，胆固醇，PEG2000-DMG按摩尔比50：10：38.5：1.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠缓冲液(50mM,pH＝4.0)中。使用微流控制备系统使所得的乙醇溶液和溶解有Luc-mRNA(TriLink)的醋酸缓冲液(25mM,pH＝5.0)以1:3的体积比在微流控芯片中混合制得脂质纳米颗粒，再使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为10:1。
动物准备：选取5-6周龄的雌性C57BL/6小鼠，体重在18-20g左右，饲养环境为SPF级的饲养室动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：将B16-OVA黑色素瘤细胞(1.5×10 5)皮下注射到小鼠大腿外侧。当肿瘤大小长到50mm 3时(约在肿瘤接种后第6天或第7天)开始接种疫苗。通过肌肉注射含有1μg OVA-mRNA的LNP制剂对动物进行两次免疫，第二针间隔7天。使用数显卡尺每周测量肿瘤生长3次，计算公式为0.5×长度×宽度。当肿瘤体积达到1500mm 3时对小鼠实施安乐死。E7C71A9和E6C71A12的肿瘤生长速度显著慢于MC3组(如图3所示)，且分别有60％(E7C71A9组)和40％(E6C71A12组)的小鼠达到了完全缓解的效果，显著优于MC3组(如图4所示)。
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。
实施例16：2-羟基十二烷基-2-庚基壬酸酯
Figure PCTCN2022131322-appb-000052
在25mL的反应管中依次加入FeCl 3(20mg，0.125mmol)、Py(5μL，0.0625mmol)、2-庚基壬酸(1.28g，5mmol)和1,2-环氧十二烷(1.84g,10mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得到2-羟基十二烷基-2-庚基壬酸酯(1.54g，70％)。 1H NMR(400MHz，CDCl 3)：δ0.85-0.89(m,9H),1.25- 1.26(m,36H),1.39-1.41(m,2H),1.58-1.62(m,4H),2.11-2.14(m,1H),4.05-4.11(m,2H),4.33-4.35(m,1H),5.37(brs,1H).ESI-MS calculated for C 28H 57O 3 +[M+H] +441.4,found 441.7。
实施例17：2-((4-(二甲氨基)丁酰基)氧基)十二烷酸十六烷酯
Figure PCTCN2022131322-appb-000053
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，4-(二甲氨基)丁酸(101mg，0.6mmol)，2-羟基十二烷基-2-庚基壬酸酯(220mg，0.5mmol)和DCM(4mL)。室温下搅拌3h，使用柱层析梯度洗脱纯化(DCM:MeOH＝100:1至100:3)得到化合物E7C114A9(222mg,，80％)。 1H NMR(400MHz，CDCl 3)：δ0.85-0.89(m,9H),1.25-1.44(m,38H),1.55-1.58(m,4H),1.75-1.83(m,2H),2.23(s,6H),2.28-2.39(m,5H),4.01-4.06(dd,J 1＝11.7Hz,J 2＝6.1Hz,1H),4.21-4.25(dd,J 1＝11.8Hz,J 2＝3.5Hz,1H),5.05-5.07(m,1H). 13C NMR(400MHz,CDCl 3):δ14.1,22.7,23.4,25.3,29.3,29.4,29.6,31.9,32.7,44.7,47.0,61.0,65.8,70.7,173.1,175.8.ESI-MS calculated for C 34H 68NO 4 +[M+H] +554.5,found 554.4。
实施例18：2-羟基十四烷基-3-己基十一酸酯
Figure PCTCN2022131322-appb-000054
在25mL的反应管中依次加入FeCl 3(20mg，0.125mmol)、Py(5μL，0.0625mmol)、3-己基十一酸(1.36g，5mmol)和1,2-环氧十四烷(2.12g,10mmol)，室温下搅拌反应过夜，使用柱层析梯度洗脱纯化(hexane：EA＝20：1至5：1)得到2-羟基十四烷基-3-己基十一酸酯(1.59g，66％)。 1H NMR(400MHz，CDCl 3)：δ0.87-0.90(m,9H),1.24-1.28(m,44H),1.39-1.41(m,2H),1.92-1.95(m,1H),2.02-2.04(m,1H),2.26-2.29(m,1H),4.09-4.14(m,2H),4.33-4.35(m,1H),5.38(brs,1H).ESI-MS calculated for C 31H 63O 3 +[M+H] +483.5,found 483.8。
实施例19：2-((3-(哌啶-1-基)丙酰基)氧基)十四烷基-3-羟基十一烷酸酯
Figure PCTCN2022131322-appb-000055
在10mL的反应管中依次加入EDC·HCl(192mg，1mmol)，DIPEA(174μL，1mmol)，DMAP(3.0mg，0.025mmol)，3-哌啶基-1-丙酸(95mg，0.6mmol)，2-羟基十四烷基-3-己基十一酸酯(242mg，0.5mmol)和DCM(4mL)。室温下搅拌3h，使用柱层析梯度洗脱纯化(DCM:MeOH＝100:1至100:3)得到化合物E9C126A24(227mg,73％)。 1H NMR(400MHz，CDCl 3)：δ0.87-0.80(m,9H),1.23-1.28(m,44H),1.38-1.39(m,2H),1.44-1.49(m,6H),1.92-1.95(m,1H),2.02-2.05(m,1H),2.27-2.29(m,1H),2.35-2.43(m,6H),3.76-3.79(m,2H),4.17-4.19(m,1H),4.42-4.44(m,1H),5.16-5.18(m,1H). 13C NMR(400MHz,CDCl 3):δ14.1,22.7,24.5,25.3,25.9,27.1,29.3,29.6,29.9,30.7,31.8,31.9,32.4,32.6,33.2,39.7,52.8,56.8,65.5,70.7,173.1.ESI-MS calculated for C 39H 76NO 4 +[M+H] +622.6,found 622.9。
实施例20：氨基脂质化合物作为mRNA载体的体外评价
细胞系：HeLa细胞系(ATCC)
培养基：补充了10％胎牛血清的1640(Invitrogen)
筛选形式：96孔板细胞转染
检测：使用多功能酶标仪检测荧光强度。根据制造商的说明，Lipofectamine3000(Invitrogen)用作阳性对照组。
方法：使用8通道移液管加样。所示的含量为96孔平板的单孔。
1.参考实施例17和实施例19中所述的路线合成得到一系列氨基脂质化合物，将其与二硬脂酰磷脂酰胆碱(DSPC)，胆固醇，PEG2000-DMG按摩尔比为47.5:10:41:1.5的比例混合溶解在无水乙醇中；Luc-mRNA(TriLink)溶解在醋酸钠缓冲液(25nM，pH＝5.0)中，使用排枪取出上述的混合脂质溶液，加入至Luc-mRNA溶液中，充分混合，控制其配比为乙醇溶液与醋酸钠缓冲液(25nM，pH＝5.0)的比例为1:3，制得脂质纳米颗粒溶液。氨基脂质化合物与荧光素酶mRNA(Luc mRNA)的质量比约为10:1，每孔mRNA的用量为100ng。
2.脂质纳米颗粒溶液在室温下孵育30min后，于96孔全白酶标板中每孔加入100μL新鲜重悬浮的HeLa细胞(1×10 4细胞)，再用移液管将脂质纳米颗粒溶液加入到96孔板中(每孔10μL)。置于37℃含有5％CO 2的培养箱中孵育。
3.细胞初始转染后16至20小时，将底物ONE-Glo TMLuciferase以100μL/孔加入细胞中， 2min后用多功能酶标仪(Biorek SynergyH1)进行检测。
4.相对转染效率如下计算：
Figure PCTCN2022131322-appb-000056
结果：部分化合物对HeLa细胞的Luc-mRNA的转染效率示于表8中。
表8：462种化合物对HeLa细胞的mRNA相对转染效率
  A9 A11 A12 A16 A23 A24
E3C116 1.2 3.2 0.9 0.7 0.7 0.8
E5C104 1.5 1.3 0.7 0.8 0.9 0.9
E5C106 0.8 0.9 0.6 0.5 0.4 0.6
E5C109 1.1 0.5 0.4 0.3 0.5 0.4
E5C111 0.8 1.2 0.7 0.9 0.5 0.3
E5C114 1.4 2.1 0.5 0.4 0.4 0.6
E5C115 1.2 6.1 0.9 0.7 0.5 0.5
E5C116 1.2 3.0 0.8 0.6 0.6 0.5
E5C119 1.1 0.8 0.6 0.4 0.9 0.2
E5C123 0.7 0.8 0.6 0.3 0.2 0.5
E5C126 0.4 0.5 0.1 0.1 0.4 0.2
E5C128 1.2 1.5 0.8 0.9 0.8 0.4
E5C131 0.3 0.1 0.4 0.2 0.4 0.5
E5C135 0.9 0.4 0.41 0.3 0.6 0.6
E7C104 1.3 0.9 0.7 0.5 0.6 0.1
E7C106 1.1 0.3 0.6 0.5 0.6 0.3
E7C109 0.5 1.1 0.5 0.8 0.7 0.2
E7C111 0.1 0.2 0.9 0.7 0.7 0.2
E7C114 4.6 5.9 0.9 0.7 0.5 0.4
E7C115 1.6 8.3 1.0 0.9 0.3 0.4
E7C116 1.1 5.0 0.6 0.8 0.3 0.5
E7C119 0.7 0.8 1.0 0.7 0.9 0.2
E7C123 1.3 0.9 0.7 0.4 0.3 0.2
E7C126 0.6 1.1 0.4 0.2 0.5 0.1
E7C128 1.00 1.3 0.8 0.7 0.4 0.6
E7C131 0.2 0.4 0.2 0.1 0.2 0.2
E7C135 0.8 0.8 0.7 0.5 0.4 0.2
E8C104 1.6 0.9 0.2 0.3 0.5 0.3
E8C106 0.9 0.4 0.5 0.3 0.6 0.5
E8C109 1.6 0.3 0.6 0.8 0.6 0.7
E8C111 1.3 0.7 0.3 0.2 0.4 0.9
E8C114 2.9 4.8 0.4 0.3 0.5 0.3
E8C115 1.7 6.2 0.9 0.9 0.7 0.6
E8C116 1.2 3.1 0.7 0.8 0.2 0.5
E8C119 0.9 0.5 0.5 0.3 0.7 0.2
E8C123 0.8 2.0 0.6 0.4 0.3 0.4
E8C126 0.3 0.9 0.8 0.7 0.6 0.6
E8C128 2.1 1.0 0.9 0.6 0.4 0.4
E8C131 0.8 0.8 0.5 0.2 0.3 0.3
E8C135 0.9 0.5 0.6 0.5 0.5 0.2
E9C104 1.5 1.8 0.6 0.6 0.8 0.4
E9C106 0.7 0.5 0.5 0.5 0.6 0.5
E9C109 1.8 1.6 0.8 0.5 0.6 0.5
E9C111 0.9 1.3 0.6 0.7 0.4 0.3
E9C114 3.6 4.9 0.9 0.9 0.8 0.6
E9C115 6.0 7.5 1.0 0.8 0.7 0.8
E9C116 1.1 4.7 0.4 1.0 0.3 0.3
E9C119 0.9 0.9 0.8 0.5 0.4 0.6
E9C123 0.4 2.4 0.6 0.3 0.5 0.3
E9C126 0.6 0.4 0.3 0.4 0.3 0.3
E9C128 1.8 1.6 0.8 0.5 0.4 0.2
E9C131 0.6 0.5 0.4 0.6 0.5 0.4
E9C135 0.7 0.7 0.8 0.6 0.3 0.2
E10C104 0.5 0.4 0.6 0.5 0.3 0.5
E10C106 1.6 0.6 0.5 0.5 0.4 0.4
E10C109 0.7 1.1 0.9 0.7 0.7 0.8
E10C111 1.1 0.5 0.7 0.4 0.7 0.5
E10C114 1.2 2.5 0.9 0.8 0.7 0.2
E10C115 1.2 2.7 0.7 0.6 0.6 0.8
E10C119 0.3 0.9 0.8 0.5 0.3 0.4
E10C123 0.6 0.7 0.4 0.7 0.4 0.2
E10C126 0.4 0.8 0.6 0.6 0.9 0.5
E10C128 1.1 1.4 0.2 0.3 0.2 0.4
E10C131 0.5 0.5 0.6 0.5 0.8 0.1
E10C135 0.9 0.6 0.7 0.5 0.7 0.6
E11C104 0.7 0.9 0.6 0.8 0.5 0.5
E11C106 0.8 0.7 0.8 0.5 0.4 0.3
E11C109 1.3 1.4 0.3 0.8 0.5 0.4
E11C111 0.5 0.6 0.5 0.6 0.8 0.6
E11C114 1.6 1.9 0.9 0.3 0.6 0.5
E11C115 1.3 1.8 0.6 0.7 0.5 0.6
E11C119 0.8 0.8 0.7 0.5 0.5 0.4
E11C123 0.9 0.8 0.9 0.8 0.6 0.5
E11C126 1.5 0.9 0.8 0.5 0.6 0.3
E11C128 1.1 1.2 0.9 0.8 0.3 0.7
E11C131 0.2 0.7 0.4 0.5 0.3 0.3
E11C135 0.4 0.3 0.6 0.6 0.5 0.2
实施例21：氨基脂质化合物制备的脂质纳米颗粒在BMDC原代细胞上的转染
制备方法：同实施例17。
动物准备：选取6周龄的雌性C57BL/6小鼠，体重在20g左右，饲养环境为SPF级的饲养室，动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
细胞获取：把C57BL/6小鼠进行脱颈臼处死，并置于75％酒精中浸泡5分钟进行消毒，解剖获取小鼠大腿及小腿胫骨，并把附着的肌肉剔除露出骨质，然后用1mL吸有PBS的注射器把胫骨中的骨髓吹出，把所得骨髓吹散后通过50μm滤网滤去杂质，向所得过滤物中加入红细胞裂解液(3～4mL)后放置5分钟后进行800g、5分钟的离心除去上清液，将所得细胞置于1640培养基(含10％胎牛血清、20ng/mL GMCSF、10ng/mL IL4)中重悬，并接种于6孔板中，接种密度为100000个细胞/毫升培养基，放置于37℃、含5％CO 2细胞培养箱中，每2天进行半换液一次，于第七天收集悬浮细胞和松散贴壁的细胞，并接种到96孔全白酶标板接种密度为每孔20000个细胞，培养基体积为100μL。
细胞转染：向铺有原代细胞的96孔全白酶标板中加入包裹萤光素酶mRNA的脂质纳米颗粒，控制每孔中的mRNA脂质纳米颗粒加入体积为10μL。随后放置在37℃、含5％CO 2浓度的培养箱中16小时。
转染效率检测：往96孔全白酶标板中每孔加入20μL底物ONE-Glo TMLuciferase，1min后用多功能酶标仪(Biorek SynergyH1)进行检测。代表性氨基脂质化合物在BMDC上转染LucmRNA的表达强度见表9。DLin-MC3作为对照，所述的氨基脂质中有多个与MC3表达强度相似，并且还有多个显著优于阳性对照。
表9：65个氨基脂质化合物在BMDC上的转染的表达强度
Figure PCTCN2022131322-appb-000057
Figure PCTCN2022131322-appb-000058
Figure PCTCN2022131322-appb-000059
Figure PCTCN2022131322-appb-000060
Figure PCTCN2022131322-appb-000061
Figure PCTCN2022131322-appb-000062
Figure PCTCN2022131322-appb-000063
实施例22：氨基脂质化合物制备的脂质纳米颗粒的萤光素酶mRNA体内递送性能评价
1.脂质纳米颗粒的制备
将本发明的氨基脂质化合物与中性脂质(如：DSPC、DOPE、胆固醇)、聚乙二醇化脂质(如：PEG2000-DMG、PEG2000-DSPE)按优化后的摩尔比混合并溶解在无水乙醇中。使用微流控制备系统使所得的乙醇溶液和溶解有Luc-mRNA(TriLink)的醋酸钠缓冲液(25mM,pH＝5.0)以1:3的体积比在微流控芯片中混合以制得脂质纳米颗粒的粗溶液，然后将其用透析盒(Fisher,MWCO 20,000)在1X PBS、控温4℃下透析6h。使用前再通过0.22μm的微孔滤膜进行过滤。氨基脂质化合物与萤光素酶mRNA(Luc mRNA)的质量比约为10:1。
脂质纳米颗粒的表征：
粒径的表征：所制备的脂质纳米颗粒的粒径和PDI通过Nano-ZSZEN3600(Malvern)测定。取LNP溶液20μL进行粒径测量，循环三次，每次循环30s。
包封率测定：参照Quant-iT RiboGreen RNA试剂盒标准规程进行测定。
表10：使用代表性氨基脂质化合物制备的LNP的表征数据
Figure PCTCN2022131322-appb-000064
Figure PCTCN2022131322-appb-000065
注：上表中
LNP-48～LNP-86的脂质配方为：氨基脂质：DSPC：胆固醇：PEG2000-DMG＝47.5:10:41:1.5；
LNP-87的脂质配方为：氨基脂质：DOPE：胆固醇：PEG2000-DMG＝45:10:43.5:1.5；LNP-88的脂质配方为：氨基脂质：DSPC:：DOPS:胆固醇：PEG2000-DMG:DOPS＝50:10:5:38.5:1.5；
LNP-89的脂质配方为：氨基脂质：DSPC：DOPC：胆固醇：PEG2000-DMG＝45:10:5:38.0:2.0；
LNP-90的脂质配方为：氨基脂质：DSPC：DOPE：胆固醇：PEG2000-DSPE＝50:10:5:33.5:1.5.
LNP-91的脂质配方为：氨基脂质：胆固醇：PEG2000-DSPE＝60:35.5:4.5；
2.动物实验
动物准备：选取体重约20g的6周龄的雌性C57BL/6小鼠，于SPF级的饲养室中饲养。动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：每组随机选取9只C57BL/6小鼠，按0.5mg/kg mRNA的用量，分别使用皮下、肌肉和尾静脉注射三种给药方式注射脂质纳米颗粒溶液(每种给药方式3只小鼠)。12 小时后，往每只小鼠体内通过尾静脉注射200μL 10mg/mL的D-萤光素钾盐，10分钟后，将小鼠放置于活体成像系统(IVIS-200,Xenogen)下，观察每只小鼠总的萤光强度并拍照记录。代表性氨基脂质化合物通过3种给药方式递送的Fluc mRNA的表达强度见表11-13。DLin-MC3作为对照。
表11：代表性氨基脂质化合物LNP皮下给药递送的Luc mRNA的表达强度
编号 LNP编号 平均荧光强度 编号 LNP编号 平均荧光强度
1 LNP-48 8.8E+06 11 LNP-70 6.2E+07
2 LNP-52 5.1E+07 12 LNP-72 8.8E+07
3 LNP-54 1.2E+08 13 LNP-73 5.6E+07
4 LNP-56 5.1E+07 14 LNP-76 6.7E+07
5 LNP-59 4.6E+07 15 LNP-79 8.1E+07
6 LNP-60 1.1E+08 16 LNP-82 9.3E+07
7 LNP-62 3.2E+07 17 LNP-83 5.5E+07
8 LNP-65 8.3E+07 18 LNP-85 7.2E+07
9 LNP-66 9.4E+07 19 LNP-87 1.0E+08
10 LNP-69 2.6E+07 20 LNP-90 8.9E+07
表12：代表性氨基脂质化合物LNP肌注给药递送的Luc mRNA的表达强度
编号 LNP编号 平均荧光强度 编号 LNP编号 平均荧光强度
1 LNP-49 5.3E+07 11 LNP-72 7.8E+07
2 LNP-54 1.5E+08 12 LNP-73 6.4E+07
3 LNP-55 6.4E+07 13 LNP-77 4.5E+07
4 LNP-59 7.0E+07 14 LNP-80 4.8E+07
5 LNP-60 1.7E+08 15 LNP-81 5.9E+07
6 LNP-61 6.1E+07 16 LNP-83 6.4E+07
7 LNP-65 1.3E+08 17 LNP-84 8.3E+07
8 LNP-66 9.9E+07 18 LNP-85 6.8E+07
9 LNP-68 8.6E+07 19 LNP-88 1.0E+08
10 LNP-70 8.2E+07 20 LNP-90 9.6E+07
表13：代表性氨基脂质化合物LNP尾静脉给药递送的Luc mRNA的表达强度
编号 LNP编号 平均荧光强度 编号 LNP编号 平均荧光强度
1 LNP-51 3.5E+07 11 LNP-70 5.4E+07
2 LNP-53 3.3E+07 12 LNP-72 4.9E+07
3 LNP-54 8.1E+07 13 LNP-74 2.4E+07
4 LNP-56 6.2E+07 14 LNP-78 3.8E+07
5 LNP-58 4.7E+07 15 LNP-82 2.3E+07
6 LNP-59 7.9E+07 16 LNP-83 9.9E+06
7 LNP-60 1.3E+08 17 LNP-85 1.4E+07
8 LNP-64 5.4E+07 18 LNP-87 1.0E+08
9 LNP-65 7.6E+07 19 LNP-89 8.7E+07
10 LNP-66 8.9E+07 20 LNP-91 6.3E+06
实施例23：氨基脂质化合物制备的脂质纳米颗粒的体内免疫和肿瘤治疗效果评价
制剂方法：将本发明中所述的氨基脂质化合物与DSPC，胆固醇，PEG2000-DMG的摩尔比为50：10：38.5：1.5的比例混合溶解在无水乙醇中。卵清蛋白mRNA(OVA mRNA)溶解在醋酸钠溶液(50mM,pH＝4.0)中。使用两个微量注射泵，控制其配比为乙醇溶液与醋酸溶液(50mM,pH＝4.0)的比例为1：3，在微流道芯片中制得脂质纳米颗粒的粗溶液，再使用透析盒(Fisher，MWCO 20,000)在1X PBS、控温4℃下透析6h，使用前用0.22μm的微孔滤膜过滤。氨基脂质化合物与卵清蛋白mRNA(OVA mRNA)的质量比约为8:1。
动物准备：选取5-6周龄的雌性C57BL/6小鼠，体重在18-20g左右，饲养环境为SPF级的饲养室动物试验严格按照国家健康机构的指南以及动物伦理要求进行。
体内递送：将B16-OVA黑色素瘤细胞(1.5×10 5)皮下注射到小鼠大腿外侧。当肿瘤大小长到50mm 3时(约在肿瘤接种后第6天或第7天)开始接种疫苗。通过肌肉注射含有5μg OVA-mRNA的LNP制剂对动物进行两次免疫，第二针间隔7天。使用数显卡尺每周测量肿瘤生长3次，计算公式为0.5×长度×宽度×宽度。当肿瘤体积达到1500mm 3时对小鼠实施安乐死。E7C115A11的肿瘤生长速度显著慢于MC3组，90％的小鼠肿瘤完全消退(如图5所示)，生存率达到100％，显著优于MC3组(如图6所示)。
以上所述仅为本发明专利的较佳实施例而已，并不用以限制本发明专利，凡在本发明专利的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明专利的保护范围之内。

Claims (20)

  1. 一种氨基脂质，其特征在于，其结构如式(I)所示：
    Figure PCTCN2022131322-appb-100001
    其中，L为C 1-C 6亚烷基、C 1-C 6亚烯基、C 1-C 6亚炔基、C 3-C 6亚环烷基、C 3-C 6亚环烯基；
    R 1和R 2相同或不同，并且各自独立地选自C 1-C 20烷基、C 1-C 20烯基、C 1-C 20炔基、C 1-C 20环烷基、C 1-C 20环烯基、C 1-C 20环炔基；所述C 1-C 20烷基、C 1-C 20烯基、C 1-C 20炔基、C 1-C 20环烷基、C 1-C 20环烯基、C 1-C 20环炔基可任选地被氢、C1-C6烃基、氟取代；
    R 3和R 4彼此相同或不同，并且各自独立地选自H，C 1-C 10烷基、C 2-C 10烯基、C 2-C 10炔基；
    所述C 1-C 10烷基、C 2-C 10烯基、C 2-C 10炔基可任选地被C 1-C 6烃基取代；
    或R 3和R 4相连接形成4～10元杂环，其中，多元杂环包含1～6个杂原子，所述杂原子选自氮、硫或氧。
  2. 根据权利要求1所述氨基脂质，其特征在于，所述R 1选自C 4-C 17烷基、C 4-C 17烯基、C 4-C 17炔基、C 4-C 17环烷基、C 4-C 17环烯基、C 4-C 17环炔基；所述C 4-C 17烷基、C 4-C 17烯基、C 4-C 17炔基、C 4-C 17环烷基、C 4-C 17环烯基、C 4-C 17环炔基可任选地被氢、C 1-C 6烃基、氟取代；和/或
    所述R 2选自C 5-C 19烷基、C 5-C 19烯基、C 5-C 19炔基、C 5-C 19环烷基、C 5-C 19环烯基、C 5-C 19环炔基；所述C 5-C 19烷基、C 5-C 19烯基、C 5-C 19炔基、C 5-C 19环烷基、C 5-C 19环烯基、C 5-C 19环炔基可任选地被氢、C 1-C 6烃基、氟取代；和/或
    R 3、R 4、L形成R 3R 4-N-L的含胺的羧酸结构为
    Figure PCTCN2022131322-appb-100002
    和/或
    R 3和R 4相连接形成4～6元杂环，其中，多元杂环包含1～2个杂原子，所述杂原子选自氮或氧。
  3. 根据权利要求2所述氨基脂质，其特征在于，所述R 1选自C 4-C 17烷基、C 4-C 17烯基；所述C 4-C 17烷基、C 4-C 17烯基可任选地被氢、C 1-C 6烃基、氟取代；和/或所述R 2选自C 5-C 19烷基、C 5-C 19烯基、C 5-C 19环烷基；所述C 5-C 19烷基、C 5-C 19烯基、C 5-C 19环烷基、可任选地被氢、C 1-C 6烃基、氟取代；
  4. 根据权利要求3所述氨基脂质，其特征在于，其结构如式(II)所示：
    Figure PCTCN2022131322-appb-100003
    m＝3-16
    n＝0,1,2,3,4,5,6,7,8,9,10；
  5. o,p相同或不同，且o,p＝3,4,5,6,7,8根据权利要求3所述氨基脂质，其特征在于，所述R 1选自C 4-C 17烷基；所述C 4-C 17烷基可任选地被氢、C 1-C 6烃基、氟取代；和/或所述R 2选自C 5-C 19烯基、C 5-C 19环烷基；所述C 5-C 19烯基、C 5-C 19环烷基、可任选地被氢、C 1-C 6烃基、氟取代；
  6. 根据权利要求3所述氨基脂质，其特征在于，所述R 1选自C 4-C 17烯基；所述C 4-C 17烯基可任选地被氢、C 1-C 6烃基、氟取代；和/或所述R 2选自C 5-C 19烷基、C 5-C 19环烷基；所述C 5-C 19烷基、C 5-C 19环烷基、可任选地被氢、C 1-C 6烃基、氟取代；
  7. 根据权利要求2所述的氨基脂质，其特征在于，所述R 1选自E1、E2、E3、E4、E5、E6、E7、E8、E9、E10、E11、E12、E13、E14、E15、E16、E17、E18、E19、E20、E21、E22、E23、E24、E25中的一种：
    Figure PCTCN2022131322-appb-100004
    Figure PCTCN2022131322-appb-100005
    和/或
    所述R 2选自C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25、C26、C27、C28、C29、C30、C31、C32、C33、C34、C35、C36、C37、C38、C39、C40、C41、C42、C43、C44、C45、C46、C47、C48、C49、C50、C51、C52、C53、C54、C55、C56、C57、C58、C59、C60、C61、C62、C63、C64、C65、C66、C67、C68、C69、C70、C71、C72、C73、C74、C75、C76、C77、C78、C79、C80、C81、C82、C83、C84、C85、C86、C87、C88、C89、C90、C91、C92、C93、C94、C95、C96、C97、C98、C99、C100、C101、C102、C103、C104、C105、C106、C107、C108、C109、C110、C111、C112、C113、C114、C115、C116、C117、 C118、C119、C120、C121、C122、C123、C124、C125、C126、C127、C128、C129、C130、C131、C132、C133、C134、C135、C136、C137、C138、C139、C140、C141、C142、C143、C144、C145、C146、C147中的一种：
    Figure PCTCN2022131322-appb-100006
    Figure PCTCN2022131322-appb-100007
    Figure PCTCN2022131322-appb-100008
    和/或
    Figure PCTCN2022131322-appb-100009
    选自：A1、A2、A3、A4、A5、A6、A7、A8、A9、A10、A11、A12、A13、A14、A15、A16、A17、A18、A19、A20、A21、A22、A23、A24、A25、A26、A27、A28、A29、A30、A31、A32、A33、A34、A35、A36、A37、A38、A39、A40中的一种：
    Figure PCTCN2022131322-appb-100010
  8. 根据权利要求3所述的氨基脂质，其特征在于，所述R 1选自E3、E4、E5、E6、E7、E8、 E9、E10、E11、E12、E13、E15、E17、E18、E20、E21、E24中的一个；和/或
    所述R 2选自C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C56、C57、C58、C60、C62、C63、C64、C66、C67、C71、C72、C74、C79、C82、C83、C102、C103、C104、C105、C106、C107、C108、C109、C110、C111、C112、C113、C114、C115、C116、C117、C118、C119、C120、C121、C122、C123、C124、C125、C126、C127、C128、C129、C130、C131、C132、C133、C134、C135、C136、C137、C138、C139、C140、C141、C142、C143、C144、C145、C146、C147
    中的一个；和/或
    Figure PCTCN2022131322-appb-100011
    选自A1、A7、A8、A9、A10、A11、A12、A14、A15、A16、A23、A24、A28、A33、A37中的一个。
    Figure PCTCN2022131322-appb-100012
  9. 根据权利要求1所述的氨基脂质，其特征在于，选自如下结构所示化合物的一种：
    Figure PCTCN2022131322-appb-100013
    Figure PCTCN2022131322-appb-100014
  10. 权利要求1～9任一项所述氨基脂质的制备方法，其特征在于，包括以下步骤：
    S1.化合物R 2COOH与环氧类化合物在催化剂FeCl 3/Py的催化下无溶剂搅拌反应；
    其中，所述环氧类化合物的结构为：
    Figure PCTCN2022131322-appb-100015
    S2.在步骤S1反应体系中加入R 3R 4NLCOOH，并在缩合剂存在的条件下反应即得。
  11. 权利要求1至9任一项所述氨基脂质或其药学上可接受的盐在制备用于基因治疗、基因疫苗接种、反义治疗或通过干扰RNA的治疗的药物中的应用。
  12. 根据权利要求11所述应用，其特征在于，所述药物用于治疗癌症、遗传疾病过敏、毒性或病原体感染疾病。
  13. 根据权利要求11所述应用，其特征在于，所述癌症为肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌、膀胱癌或前列腺癌。
  14. 一种核酸药物给药系统，其特征在于，其原料包括权利要求1至9任一项所述的氨基脂质。
  15. 根据权利要求1-9中任一项所述的氨基脂质与其它脂质形成组合物，其中所述组合物与核酸药物形成药物制剂，所述核酸药物包括DNA和RNA。
  16. 权利要求15所述的药物制剂包含30-50％氨基脂质、40-50％结构脂质、5-20％辅助脂质及0.5-5％聚乙二醇化脂质(PEG)，上述四组分摩尔含量合计为100％；氨基脂质与核酸的质量比为1：1-50：1。
  17. 权利要求16所述的结构脂质包括胆固醇及其衍生物，优先选择为胆固醇。
  18. 权利要求16所述的辅助脂质包括DSPC、DSPE、DOPE、DOPC、DOPS，优选DSPC、DOPE，优先选择为DSPC。
  19. 权利要求16所述的聚乙二醇化脂质包括PEG-DMG、PEG-DSPE，优先选择PEG-DMG。
  20. 权利要求16所述的药物制剂，其给药方式包括雾化给药、静脉注射、皮下注射、肌肉注射、眼部给药，优选静脉注射、皮下注射、肌肉注射。
PCT/CN2022/131322 2022-01-25 2022-11-11 一种氨基脂质及其制备方法和应用 WO2023142600A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210082815.3A CN114105799B (zh) 2022-01-25 2022-01-25 一种氨基脂质及其制备方法和应用
CN202210082815.3 2022-01-25

Publications (1)

Publication Number Publication Date
WO2023142600A1 true WO2023142600A1 (zh) 2023-08-03

Family

ID=80360824

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/131322 WO2023142600A1 (zh) 2022-01-25 2022-11-11 一种氨基脂质及其制备方法和应用

Country Status (4)

Country Link
US (1) US20230348360A1 (zh)
EP (1) EP4215188A1 (zh)
CN (1) CN114105799B (zh)
WO (1) WO2023142600A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114105799B (zh) * 2022-01-25 2022-05-10 中山大学附属第七医院(深圳) 一种氨基脂质及其制备方法和应用
CN114874146B (zh) * 2022-03-25 2023-09-12 圣诺生物医药技术(苏州)有限公司 组氨酸脂质、组氨酸脂质纳米颗粒及其制备方法和应用
WO2024149331A1 (zh) * 2023-01-11 2024-07-18 深圳深信生物科技有限公司 一种氨基脂质化合物、其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160311759A1 (en) * 2013-12-19 2016-10-27 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
CN114105799A (zh) * 2022-01-25 2022-03-01 中山大学附属第七医院(深圳) 一种氨基脂质及其制备方法和应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7514574B2 (en) * 2003-08-07 2009-04-07 Ooi Wong Biodegradable penetration enhancers with multiple hydrophilic moieties
US20110065769A1 (en) * 2009-05-01 2011-03-17 Ooi Wong Transdermal delivery of ionizable drugs
CN104119227A (zh) * 2013-04-27 2014-10-29 复旦大学 一种羧酸与环氧乙烷化合物酯化反应的方法
EP3083579B1 (en) 2013-12-19 2022-01-26 Novartis AG Lipids and lipid compositions for the delivery of active agents
KR20220154740A (ko) * 2020-03-17 2022-11-22 제네반트 사이언시즈 게엠베하 간 성상 세포로 치료제의 지질 나노입자 전달을 위한 양이온성 지질
CN113264842B (zh) * 2021-07-21 2022-03-01 苏州科锐迈德生物医药科技有限公司 一种脂质化合物及包含其的脂质载体、核酸脂质纳米粒组合物和药物制剂

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160311759A1 (en) * 2013-12-19 2016-10-27 Luis Brito Lipids and Lipid Compositions for the Delivery of Active Agents
CN114105799A (zh) * 2022-01-25 2022-03-01 中山大学附属第七医院(深圳) 一种氨基脂质及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Registry CAS; 29 July 2016 (2016-07-29), XP093081953, retrieved from STN Database accession no. 1962944-97-3 *

Also Published As

Publication number Publication date
US20230348360A1 (en) 2023-11-02
CN114105799B (zh) 2022-05-10
CN114105799A (zh) 2022-03-01
EP4215188A1 (en) 2023-07-26

Similar Documents

Publication Publication Date Title
WO2023142600A1 (zh) 一种氨基脂质及其制备方法和应用
CN112472822B (zh) 一类细胞内质网靶向纳米载药系统的构建与应用
CA2251999A1 (fr) Composes glycerolipidiques utiles pour le transfert d'une substance active dans une cellule cible
CA3215389C (en) Ionizable lipids and compositions for nucleic acid delivery
WO2023029928A1 (zh) 一种氨基脂质及其应用
WO2023142167A1 (zh) 一种阳离子脂质类似物、其组合物及应用
CN116199646B (zh) 一种基于Tris的可电离脂质及其制备方法与应用
CA2292837A1 (fr) Nouvelle classe d'agents transfectants cationiques des acides nucleiques
CN113214171B (zh) 两亲性树形分子、合成及其作为药物递送系统的应用
CN114874107A (zh) 一种氨基脂质及其制备方法和应用
WO2024198497A1 (zh) 氨基脂质、脂质纳米颗粒及其应用
JPWO2005054486A1 (ja) 遺伝子導入試薬調製法
CN114874106B (zh) 一种氨基脂质及其制备方法和应用
CN116284006B (zh) 可电离脂质化合物、包含其的脂质载体及应用
WO2024131717A1 (zh) 一种阳离子脂质材料的制备及应用
US20240325521A1 (en) Ionizable lipid compound for nucleic acid delivery and lnp composition thereof
CN115872893B (zh) 阳离子脂质化合物及其制备方法和应用
CN112717140B (zh) 一种含HP1γ的胍基化聚氨基胺类聚合物基因载体复合物的制备及其应用
JPH09263579A (ja) ピペリジン誘導体及びそれを構成成分とする薬物担体
CN118084699A (zh) 一种可电离脂质化合物及其制备方法与医学上的应用
CN118125932A (zh) 一种可电离脂质分子及其纳米颗粒组合物在核酸递送中的应用
JP5433866B2 (ja) 組織透過を改善するための薬剤
CN117534585A (zh) 一种新型可电离阳离子脂质化合物及其制备方法与应用
CN118271388A (zh) 一种类固醇-阳离子脂质化合物及其应用
CN116947680A (zh) 一种阳离子脂质化合物及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22923394

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE