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WO2024198497A1 - 氨基脂质、脂质纳米颗粒及其应用 - Google Patents

氨基脂质、脂质纳米颗粒及其应用 Download PDF

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Publication number
WO2024198497A1
WO2024198497A1 PCT/CN2023/137365 CN2023137365W WO2024198497A1 WO 2024198497 A1 WO2024198497 A1 WO 2024198497A1 CN 2023137365 W CN2023137365 W CN 2023137365W WO 2024198497 A1 WO2024198497 A1 WO 2024198497A1
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lipid
group
amino
cancer
alkyl
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PCT/CN2023/137365
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English (en)
French (fr)
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方菀茵
秦皓勇
徐岩
吴启新
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深圳虹信生物科技有限公司
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Publication of WO2024198497A1 publication Critical patent/WO2024198497A1/zh

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    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • 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
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    • 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
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    • 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
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    • 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
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    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • 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
    • C07C237/10—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 nitrogen atoms not being part of nitro or nitroso groups
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    • 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
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    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/12—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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Definitions

  • the invention relates to the technical field of biochemistry, and in particular to an amino lipid, lipid nanoparticles and applications thereof.
  • LNPs Lipid nanoparticles
  • ionizable lipids which can self-assemble with mRNA into virus-sized particles and release mRNA from endosomes into the cytoplasm
  • polymer-bound lipids which can increase the half-life of LNPs in the blood
  • steroids which can increase the stability of nanoparticles
  • neutral phospholipids which help form the lipid bilayer structure.
  • LNP can protect mRNA from being decomposed by RNases and from being recognized by TLRs, thus avoiding overactivation of the innate immune system.
  • the selection of ionizable lipids has the greatest impact on LNP, which can promote cell uptake and help drug molecules escape from endosomes, and at the same time affect the encapsulation rate of nucleic acid drugs, the delivery efficiency of nucleic acid drugs in the body, and cytotoxicity.
  • LNP lipoprotein
  • ionizable lipids and formulations mainly focuses on ionizable lipids and formulations.
  • the present invention provides an amino lipid having a structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof:
  • M 1 , M 2 , M 3 , and M 4 are the same or different from each other and are each independently selected from C 1 -C 24 alkylene, C 3 -C 24 cycloalkylene, Alkyl, C 2 -C 24 alkenylene or C 3 -C 24 cycloalkenylene;
  • R 1 and R 2 are the same as or different from each other, and are each independently selected from H, C 1 -C 24 alkyl, C 3 -C 24 cycloalkyl, C 2 -C 24 alkenyl or C 3 -C 24 cycloalkenyl;
  • R, R′, and R′′ are the same as or different from each other, and are each independently selected from H, a C 1 -C 10 alkyl group, a C 3 -C 10 cycloalkyl group, a C 3 -C 10 alkenyl group or a C 3 -C 10 cycloalkenyl group, a C 1 -C 10 alkyl group terminally connected to a tertiary amine group, a C 3 -C 10 cycloalkyl group terminally connected to a tertiary amine group, a C 3 -C 10 alkenyl group terminally connected to a tertiary amine group, or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or substituted by one or more C 1 -C 4 alkyl groups, C 2 -C 4 alkenyl groups, C 3 -C 8 cycloalkyl groups, or C 3
  • M5 is independently selected from a single bond, C1 - C16 alkylene, C2 - C16 alkenylene, C3 - C8 cycloalkylene or C3 - C8 cycloalkenylene;
  • the present invention provides a method for preparing the amino lipids.
  • the synthesis is mainly divided into three steps: the first step is a ring-opening reaction; the second step is a condensation reaction; and the third step is a substitution reaction.
  • the preparation method of the amino lipid comprises the following steps:
  • intermediate 1' and a carboxylic acid compound undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2'TBSO-M 1 -L 2 -M 2 -leaving group;
  • the present invention provides a lipid nanoparticle comprising any one of the above-mentioned amino lipids.
  • the lipid nanoparticles further include steroids, neutral lipids and/or polymer-bound lipids;
  • the polymer-bound lipid has the chemical formula P-Y-L, wherein P is the hydrophilic polymer portion, Y is an optional linker, and L is the lipid portion.
  • the present invention provides a pharmaceutical composition comprising the above-mentioned lipid nanoparticles and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.
  • the present invention also provides a method for using the amino lipid or lipid nanoparticle or pharmaceutical composition to perform gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.
  • the present invention also provides the use of the amino lipids or lipid nanoparticles in the preparation of drugs for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.
  • the present invention also provides the use of the amino lipids or lipid nanoparticles in drugs for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.
  • the present invention also provides a method for delivering a drug to a subject, comprising: administering the drug formulated in the above-mentioned lipid nanoparticles to the subject.
  • the amino lipids of the present invention are combined with steroids, neutral lipids and polymers to obtain LNPs through self-assembly.
  • the LNPs can further increase the translation expression level of the load-nucleic acid in cells, improve the effect of the nucleic acid-LNP preparation, and provide a basis for the theoretical treatment of personalized nucleic acid-LNP preparations.
  • Fig. 1 is the 1H-NMR spectrum of E12LA6B6O3 in Example 2;
  • Figure 2 is a tumor size monitoring curve of tumor-bearing mice after intramuscular injection of OVA mRNA vaccine in Example 39;
  • Figure 3 is a survival curve of tumor-bearing mice after intramuscular injection of OVA mRNA vaccine in Example 39.
  • neutral when applied to a compound such as a lipid or steroid, or a group or moiety, means that it is neither a cationic nor anionic, e.g. a compound having no ionizable functional groups under physiological conditions, such as hydrocarbons; or that it is both cationic and anionic under typical physiological conditions, i.e. a zwitterionic, such as the typical natural phosphatidylcholine.
  • lipid refers to a group of organic compounds that are derivatives of fatty acids (such as esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three categories: (1) “simple lipids”, including fats, oils, and waxes; (2) “complex lipids”, including phospholipids and glycolipids; and (3) “derivatized lipids”, such as steroids. With respect to glycolipids, in certain embodiments, LNPs comprise glycolipids (e.g., monosialoganglioside GM1).
  • poly refers to a plurality of atoms or groups having respective properties in a compound.
  • a polycationic compound is also a cationic compound and may be referred to as a cationic compound.
  • nucleic acid refers to any compound comprising or consisting of DNA or RNA.
  • the term may be applied to oligonucleotides or oligonucleotides.
  • the immune system protects an organism from infection. If a pathogen breaches an organism's physical barriers and enters the organism, the innate immune system provides an immediate but non-specific response. If a pathogen evades this innate response, vertebrates have a second layer of protection, the adaptive immune system. Here, the immune system adjusts its response during infection to improve its ability to recognize the pathogen. This improved response is then retained in the form of immunological memory after the pathogen has been eliminated and allows the adaptive immune system to mount a faster and stronger attack each time the pathogen is encountered. Accordingly, the immune system consists of the innate and adaptive immune systems. Each of these two parts consists of so-called humoral and cellular components.
  • the adaptive immune system consists of highly specialized systemic cells and processes that eliminate or prevent pathogenic growth.
  • the adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (generate immunity) and to mount a stronger attack each time the pathogen is encountered.
  • the system is highly adaptive due to somatic hypermutation (a process by which somatic cell mutations increase in frequency) and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to produce a large number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte.
  • the immune network theory is a theory of how the adaptive immune system works, based on the variable nature of receptors for T cells, B cells, and molecules produced by T cells and B cells that have variable regions. Interactions between regions.
  • vaccine is generally understood to mean a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.
  • antigen generally refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, such as by the formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • artificial mRNA can generally be understood as an mRNA molecule that is not naturally occurring.
  • an artificial mRNA molecule can be understood as a non-natural mRNA molecule.
  • Such an mRNA molecule may be non-natural due to its single sequence (which is not naturally occurring) and/or due to other modifications, such as structural modifications of non-naturally occurring nucleotides.
  • artificial mRNA molecules can be designed and/or generated by genetic engineering methods to correspond to the desired artificial nucleotide sequence (heterologous sequence).
  • an artificial sequence is generally a non-naturally occurring sequence, i.e., it differs from the wild-type sequence by at least one nucleotide.
  • wild type can be understood as a sequence that exists in nature.
  • pharmaceutically acceptable salt refers to a form of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.
  • Compound of the present invention may have multiple crystal forms, i.e. different lattice arrangements of the same elements of the compound.
  • Polymorphs usually have different X-ray diffraction spectra, infrared spectra, fusing points, density, hardness, crystal forms, optical and electrical properties, stability and solubility. Different factors such as recrystallization solvents, crystallization rate and storage temperature may obtain a recrystallization product taking a single crystal form as the leading one. It is understandable that amino lipids of the present invention include all such crystal forms.
  • the compounds of the present invention may have chiral centers and/or axial chirality, and therefore appear in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers, and cis-trans isomers.
  • Each chiral center or axial chirality will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures as well as pure or partially pure compounds are included within the scope of the present invention.
  • the "isomers" defined in the present invention include all such isomeric forms of the compounds.
  • the compounds of the present invention may also exist in a variety of hydrates or solvates, and the solvates contain stoichiometric or non-stoichiometric solvents and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water or other solvents such as ethanol. Hydrates are formed when the solvent is water, or solvates are formed when the solvent is other solvents such as ethanol.
  • prodrug also known as prodrug, drug precursor, prodrug, etc.
  • prodrug refers to a compound that is inactive or less active in vitro after chemical structure modification of the drug, and releases active drugs through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect.
  • carrier prodrug There are two major categories of prodrugs: one is carrier prodrug, referred to as carrier prodrug; the other is bioprodrug.
  • Carrier prodrug refers to an active compound that is covalently bonded to its transport carrier. The carrier is removed by simple hydrolysis in the body, and the active compound exerts its pharmacological effect.
  • Carrier prodrugs are generally more effective than parent compounds. The activity is usually weak or inactive.
  • Bioprodrugs are different from carrier prodrugs.
  • the active substance does not need to be temporarily combined with the carrier, but exerts its effect through the change of its own molecular structure.
  • Bioprodrugs themselves are inactive, and the active ones are their metabolites in the organism.
  • the present invention unexpectedly discovered that the use of the novel amino lipids and/or lipid nanoparticles provided by the present invention can effectively solve the shortcomings of LNP in the prior art, such as low encapsulation efficiency, low endosomal escape rate, low expression level, and low safety, thereby promoting the development of LNP in the field of mRNA drugs and vaccines.
  • the amino lipid is preferably cationizable, that is, when the pH is lowered below the pKa of the lipid's ionizable group, the amino lipid will be protonated and, when positively charged, the lipid will be able to bind to negatively charged nucleic acids.
  • the present invention provides an amino lipid having a structure shown in general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof:
  • M 1 , M 2 , M 3 , and M 4 are the same as or different from each other, and are each independently selected from a C 1 -C 24 alkylene group, a C 3 -C 24 cycloalkylene group, a C 2 -C 24 alkenylene group, or a C 3 -C 24 cycloalkenylene group;
  • R 1 and R 2 are the same as or different from each other, and are each independently selected from H, C 1 -C 24 alkyl, C 3 -C 24 cycloalkyl, C 2 -C 24 alkenyl or C 3 -C 24 cycloalkenyl;
  • R, R', and R" are the same as or different from each other and are each independently selected from H, a C1 - C10 alkyl group (preferably a C1 - C6 alkyl group, more preferably a C1 - C4 alkyl group), a C3 - C10 cycloalkyl group, a C3 - C10 alkenyl group, a C3 - C10 cycloalkenyl group, a C1 - C10 alkyl group terminally connected to a tertiary amine group, a C3 - C10 cycloalkyl group terminally connected to a tertiary amine group, a C3 - C10 alkenyl group terminally connected to a tertiary amine group, or a cyclic alkyl group containing at least one heteroatom, the cyclic alkyl group being unsubstituted or substituted by one or more C1 - C4 alkyl groups, C2
  • M5 is selected from a single bond, a C1 - C16 alkylene group, a C2 - C16 alkenylene group, a C3 - C8 cycloalkylene group or a C3 - C8 cycloalkenylene group.
  • the alkyl, alkenyl, alkylene, alkenylene containing multiple carbon atoms mentioned in the present invention may be straight-chain or branched.
  • the alkyl, alkenyl, alkylene, alkenylene, and cycloalkyl, cycloalkenyl, cycloalkylene, cycloalkenylene mentioned in the present invention may be unsubstituted or substituted, and the substituent may be any suitable substituent, i.e., any straight-chain or branched alkyl, aryl, heteroalkyl, heteroaromatic structure, which may optionally contain other functional groups, such as ester groups or amide groups.
  • G is selected from H, OR, NR′R′′ or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N; wherein R, R′, R′′ are the same as or different from each other and are each independently selected from H, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 alkenyl or C 3 -C 8 cycloalkenyl.
  • G is selected from H, OH or NR′R′′, wherein R′ and R′′ are the same or different from each other and are each independently selected from H or C 1 -C 4 alkyl; that is, NR′R′′ can be NH 2 , NHCH 3 , NHC 2 H 5 , NHC 3 H 7 , NHC 4 H 9 , N(CH 3 ) 2 , CH 3 -NC 2 H 5 , CH 3 -NC 3 H 7 , CH 3 -NC 4 H 9 , C 2 H 5 -NC 2 H 5 , C 2 H 5 -NC 3 H 7 , C 2 H 5 -NC 4 H 9 , C 3 H 7 -NC 3 H 7 , C 3 H 7 -NC 4 H 9 or C 4 H 9 -NC 4 H 9 .
  • G is selected from substituted or unsubstituted oxa-5-membered cycloalkyl, aza-5-membered cycloalkyl, aza-6-membered cycloalkyl, diaza-6-membered cycloalkyl or aza-oxa-6-membered cycloalkyl.
  • the oxa-5-membered cycloalkyl is Aza five-membered cycloalkyl is
  • the aza six-membered cycloalkyl group is The diaza six-membered cycloalkyl group is Azaoxa six-membered cycloalkyl group is The * position is connected to M 5 .
  • the substituent position is a nitrogen atom that is not connected to M 5 .
  • R, R', R" are the same or different from each other and are independently selected from H, C1 - C6 alkyl, C3 - C10 cycloalkyl, C1-C10 alkyl with -N( C1-6 alkyl) 2 connected to the terminal, or C1 - C10 alkyl with a 4-6-membered cyclic alkyl containing 1-2 ring nitrogen atoms connected to the terminal.
  • 10 alkyl, The C 3 -C 10 cycloalkyl group and the 4-6-membered cyclic alkyl group are optionally substituted with a C 1 -C 6 alkyl group.
  • M 5 is selected from a single bond, C 2 -C 16 alkylene, C 2 -C 16 alkenylene, C 4 -C 8 cycloalkylene, or C 3 -C 8 cycloalkenylene.
  • M5 is selected from a single bond, C2 - C16 alkylene or C4 - C6 cycloalkylene. More preferably, M5 is selected from a single bond, C2 - C6 alkylene or C4 - C6 cycloalkylene; or selected from C3 - C5 alkylene.
  • M 5 and G are connected to form One selected from A1 to A38:
  • M 5 and G are connected to form One selected from A39 to A52:
  • L 1 , L 2 , L 3 , and L 4 are the same as or different from each other, and are independently selected from —C( ⁇ O)O—, —OC( ⁇ O)—, —C( ⁇ O)NR—, or —NRC( ⁇ O)—.
  • R is independently selected from H or C 1 -C 10 alkyl.
  • R is H.
  • M1 , M2 , M3 , and M4 are the same as or different from each other, and M1 and M4 are each independently selected from a branched C4 - C22 alkylene group, a branched C4- C22 cycloalkylene group, a branched C4 - C22 alkenylene group, or a branched C4 - C22 cycloalkenylene group, and M2 and M3 are each independently selected from a C4 - C22 alkylene group, a C4 - C22 cycloalkylene group, a C4 - C22 alkenylene group, or a C4 - C22 cycloalkenylene group.
  • M 1 , M 2 , M 3 , and M 4 are the same as or different from each other, and M 1 and M 4 are each independently selected from a branched C 4 -C 22 alkylene group or a branched C 4 -C 22 alkenylene group, preferably a branched C 6 -C 16 alkylene group; M2 and M3 are each independently selected from C4 - C22 alkylene or C4 - C22 alkenylene, preferably C3 - C8 alkylene.
  • M 2 and M 3 are the same and are C 4 -C 22 alkylene.
  • M 1 is the same as M 4 and is a branched C 4 -C 22 alkylene group.
  • R 1 and R 2 are the same as or different from each other, and are each independently selected from C 4 -C 22 alkyl or C 4 -C 22 alkenyl, preferably C 5 -C 12 alkyl.
  • M 1 , M 2 , M 3 , and M 4 are the same or different from each other, and M 1 and M 4 are each independently selected from a branched C 4 -C 22 alkylene group or a branched C 4 -C 22 alkenylene group; M 2 and M 3 are each independently selected from a C 4 -C 22 alkylene group or a C 4 -C 22 alkenylene group; R 1 and R 2 are the same or different from each other, and are each independently selected from a C 4 -C 22 alkylene group or a C 4 -C 22 alkenyl group.
  • the structure of general formula (I) is selected from one of the following structures:
  • the structure of general formula (I) is selected from one of the following structures:
  • the present invention provides a method for preparing the above-mentioned amino lipid, comprising the following steps:
  • intermediate 1' and a carboxylic acid compound undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2'TBSO-M 1 -L 2 -M 2 -leaving group;
  • the carboxylic acid in step S2' is a HL 2 -M 2 -leaving group.
  • the carboxylic acid in step S3' is R 1 -L 1 -H.
  • the leaving group is a halogen
  • each intermediate and target product prepared are purified by column chromatography.
  • the preparation method comprises the following steps:
  • Ring-opening reaction mix the carboxylic acid compound (1.0 eq), the epoxy compound raw material (0.6-3.0 eq), ferric chloride (0.5-10 mol%), and pyridine (0.2-20 mol%), and stir at room temperature overnight. After the reaction, purify by column chromatography to obtain intermediate 1 with a yield of 56%-99.0%.
  • the preparation method comprises the following steps:
  • Condensation reaction 2 The intermediate 2' (1.0 eq) was deprotected from TBS with ammonium fluoride (5.0-20.0 eq), and mixed with carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM, and stirred at room temperature overnight, and purified by column chromatography to obtain intermediate 2;
  • the present invention provides a lipid nanoparticle, comprising any one of the above amino lipids.
  • the lipid nanoparticles further comprise a steroid, a neutral lipid and/or a polymer-bound lipid.
  • a “steroid” is an organic compound that has four rings arranged in a specific molecular configuration. It contains the following carbon skeleton:
  • Steroids and neutral steroids include naturally occurring steroids and their analogs (such as the amphiphilic lipid cholesterol hemisuccinate (CHEMS), composed of succinate esterified to the ⁇ -hydroxyl group of cholesterol as a cholesterol derivative).
  • Neutral steroids can be steroids that do not have ionizable atoms or groups under physiological conditions, or can be zwitterionic steroids.
  • the neutral steroid does not contain an atom or group that is ionizable under physiological conditions.
  • the steroid or steroid analog is cholesterol.
  • the terms "steroid” and "neutral steroid" are used interchangeably herein.
  • neutral lipids of the present invention, also referred to as “helper lipids”, are preferably phospholipids or neutral phospholipids.
  • neutral phospholipids are amphiphilic compounds consisting of molecules that typically have two hydrophobic fatty acid “tails” and a hydrophilic "head” containing a phosphate group.
  • the phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine.
  • Phospholipids exist in large quantities in nature.
  • phospholipids” or “neutral phospholipids” include both natural phospholipids and synthetic phospholipids.
  • polymer-bound lipid refers to a molecule that comprises both a lipid portion and a polymer portion.
  • the polymer-bound lipid is a pegylated lipid or a PEG-lipid.
  • pegylated lipid or “PEG-lipid” refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include PEG-DMG and the like.
  • the polymer-bound lipid has a chemical formula of P-Y-L, wherein P is a hydrophilic polymer portion, Y is an optional linker, and L is a lipid portion.
  • the hydrophilic polymer portion P can be polyethylene glycol PEG.
  • the average molecular weight of the PEG portion is between 1 kDa and 3 kDa, such as between 1.5 kDa and 2.5 kDa, between 1.7 kDa and 2.3 kDa, between 1.8 kDa and 2.2 kDa, between 1.9 kDa and 2.1 kDa, or 2 kDa.
  • PEG can be the PEG commonly referred to as "PEG 2000”.
  • the hydrophilic polymer portion P in the polymer-bound lipid can also be a substantially hydrophilic polymer different from the above-mentioned hydrophilic polymer portion, that is, the hydrophilic polymer portion P in the polymer-bound lipid can be based on poly(propylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl) methacrylamide, hydroxyethyl starch (HESylation) process (according to PMID 24681396), PASylation (PASylation) method (i.e., proline-alanine-serine), XTEN method known in the art (i.e., PEG-based peptides), polysarcosin or poly(vinyl acetate).
  • PASylation PASylation
  • the optional linker Y can be any useful spacer structure, for example selected from those commonly found useful in PEGylated lipids, such as, but not limited to, succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate linkers or combinations thereof.
  • the lipid moiety L may be derived from a phospholipid, a sphingolipid or a ceramide.
  • the term "derived from a phospholipid or a ceramide” includes free radicals of phospholipids and ceramides. Examples are free radicals containing phosphatidylethanolamine or phosphatidylglycerol. Part of the polymer is bound to lipids.
  • the polymer-bound lipid is a pegylated lipid.
  • the pegylated lipid includes, but is not limited to, the following pegylated lipids: pegylated diacylglycerol lipids (PEG-DAG); pegylated ceramide lipids (PEG-Cer); pegylated phosphatidylethanolamine lipids (PEG-PE); pegylated succinic diacylglycerol lipids (PEG-S-DAG); pegylated dialkoxypropylcarbamate lipids; 1,2-dimyristyl-rac-glycerol-3-methoxypolyethylene glycol ("PEG-DMG” or "DMG-PEG”).
  • PEG-DAG pegylated diacylglycerol lipids
  • PEG-Cer pegylated ceramide lipids
  • PEG-PE pegylated phosphatidylethanol
  • the polymer-bound lipid is DMG-PEG2000.
  • DMG-PEG 2000 is considered to be a mixture of 1,2-DMG PEG 2000 and 1,3-DMG PEG 2000 in a ratio of about 97:3.
  • the molar ratio of amino lipid, steroid, neutral lipid and polymer-bound lipid is 30-70: 30-65: 0-30: 0.2-5, and more preferably 30-60: 35-60: 0-20: 0.3-3.
  • the amino lipid is the aforementioned preferred amino lipid
  • the steroid is cholesterol
  • the neutral lipid is a phospholipid
  • the polymer-bound lipid is a pegylated lipid
  • the molar ratio of the amino lipid, cholesterol, phospholipid and pegylated lipid is 40-50:40-45:10-15:0.5-2.
  • the PEGylated lipid is DMG-PEG2000.
  • the lipid nanoparticles of the present invention are not limited to any particular morphology, and should be interpreted as including any morphology produced when an amino lipid and optionally one or more other lipids are combined, such as in an aqueous environment and/or in the presence of a nucleic acid compound.
  • liposomes, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles.
  • the lipid nanoparticle of the present invention can be combined with at least one pharmaceutically acceptable carrier or excipient to obtain a pharmaceutical composition.
  • the composition can be a dry composition, such as a powder or granule, or a solid unit, such as a lyophilized form or a tablet.
  • the composition can be in liquid form, and each excipient can be added independently in a dissolved or dispersed (such as suspended or emulsified) form.
  • the composition is formulated into a sterile solid composition, such as a powder or lyophilized form, for reorganization with an aqueous liquid carrier.
  • this preparation is also preferred.
  • nanoparticle is a submicron particle of any structure or morphology.
  • Submicron particles may also be referred to as colloids or colloids.
  • Submicron particles may also be referred to as colloids or colloids.
  • Nanoparticles may be classified with respect to the material on which they are based, as well as the structure or morphology, for example, as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polymers, hybrids or hybrid complexes, to mention only a few possible nomenclatures for specific types of nanoparticles.
  • LNPs Lipoprotein nanoparticles
  • lipids typically comprising at least one of two Affinity, membrane-forming lipids and optional other lipids, further optionally comprising a load material such as a nucleic acid compound.
  • lipid nanoparticle or “LNP” includes any subtype and morphology of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes.
  • lipid nanoparticles include any type of nanoparticles formed or co-formed by lipids.
  • lipid nanoparticles can be co-formed by a combination of lipids comprising at least one amphipathic, vesicle-forming lipid.
  • Liposomes and lipoplexes are examples of lipid nanoparticles.
  • the lipid nanoparticles further comprise a biologically active component.
  • a biologically active component refers to any compound or material that has biological activity and, because of that activity, is useful in the prevention, management, amelioration, treatment or therapy of a disease or condition in a subject, such as an animal, particularly a human subject.
  • the biologically active component is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA and replicon RNA; or any combination thereof.
  • the biologically active component is mRNA or an mRNA compound.
  • the biologically active component is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetric interfering RNA
  • miRNA micro RNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • the mRNA comprises one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal and/or a 5' cap structure.
  • the encapsulation rate of the bioactive component is at least 50-90%. More preferably, the encapsulation rate of the bioactive component is at least 60-80%.
  • the weight/weight ratio of the lipid component to the bioactive component is about 10:1 to about 60:1. More preferably, the weight/weight ratio of the lipid component to the bioactive component is about 20:1.
  • the N:P ratio in the lipid nanoparticles is about 2:1 to about 30:1. Further preferably, the N:P ratio is about 5.67:1.
  • the average size of the lipid nanoparticles is about 70 nm to about 100 nm.
  • the polydispersity index of the lipid nanoparticles is about 0.10 to about 0.20.
  • the lipid nanoparticles have a zeta potential of about -10 mV to about +20 mV.
  • the bioactive component is complexed or combined with one or more lipids (e.g., amino lipids and/or neutral lipids) to form liposomes, lipid nanoparticles (LNPs), lipoplexes and/or nanoliposomes.
  • lipids e.g., amino lipids and/or neutral lipids
  • LNPs lipid nanoparticles
  • lipoplexes and/or nanoliposomes e.g., amino lipids and/or neutral lipids
  • the term “complexed” or “combined” means that the bioactive component is substantially stable with one or more lipids.
  • the proteins are combined into larger complexes or assemblies that are not covalently bound.
  • the present invention provides a method for treating or preventing infectious diseases, cancer, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases by using the amino lipids or lipid nanoparticles or pharmaceutical compositions.
  • the present invention provides the use of the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions in the preparation of drugs for treating or preventing infectious diseases, cancer, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases.
  • the infectious disease includes viral, bacterial or protozoan infectious diseases.
  • the virus includes but is not limited to SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), bunyavirus, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extra-intestinal pathogenic Escherichia coli, Lassa virus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, vac
  • the cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.
  • the present invention also provides a method for using the amino lipid or lipid nanoparticle or pharmaceutical composition to perform gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.
  • the present invention also provides the use of the above amino lipids or lipid nanoparticles in the preparation of drugs for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.
  • the present invention also provides a method for delivering a drug to a subject, comprising: administering the drug formulated in the above-mentioned lipid nanoparticles to the subject.
  • the mode of administration is determined according to the dosage form of the drug.
  • the dosage form of the drug is related to the excipient and/or the carrier.
  • the general route of systemic administration includes: transdermal, oral, parenteral, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injection and/or intranasal administration.
  • the route of local administration generally includes intradermal, transdermal, subcutaneous or intramuscular injection, or intralesional, intracranial, intrapulmonary, intracardiac, intratumoral and sublingual injection.
  • the preferred route of administration is intramuscular injection and intradermal injection.
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.
  • E8LA12 (328.54 g/mol, 1.2 g, 3.65 mmol), 6-bromohexanoic acid (195.06 g/mol, 855 mg, 4.38 mmol), EDCI (191.70 g/mol, 2.8 g, 14.6 mmol), DMAP (45 mg, 0.365 mmol), DIPEA (2.83 g, 21.9 mmol) and DCM (10 mL) were added in sequence and the reaction was stirred at room temperature overnight. The reaction was completed. Afterwards, the product was separated and purified by column chromatography to obtain 1.65 g of the intermediate E8LA12B6 (colorless oily liquid) with a yield of 89%.
  • E8LA12B6 (505.58 g/mol, 1.65 g, 3.26 mmol), potassium carbonate (138.21 g/mol, 902 mg, 6.52 mmol), sodium iodide (149.89 g/mol, 489 mg, 3.26 mmol), aminopropanol (75.11 g/mol, 113 mg, 1.5 mmol) and acetonitrile (10 mL) were added sequentially into a 25 mL reaction tube, and the mixture was stirred at 75° C. to react overnight. After the reaction was completed, 0.94 g of E8LA12B6O3 (colorless oily liquid) was obtained by column chromatography separation and purification, with a yield of 68%.
  • E12LA6 (300.48 g/mol, 901 mg, 3.0 mmol), 6-bromohexanoic acid (195.06 g/mol, 703 mg, 3.6 mmol), EDCI (191.70 g/mol, 2.3 g, 12.0 mmol), DMAP (37 mg, 0.3 mmol) and DIPEA (2.33 g, 18 mmol) and DCM (10 mL) were added sequentially into a 25 mL reaction tube, and the mixture was stirred at room temperature overnight. After the reaction was completed, 1.08 g of the intermediate E12LA6B6 (colorless oily liquid) was obtained by column chromatography separation and purification, with a yield of 76%.
  • E12LA6B6 (477.52 g/mol, 956 mg, 2.0 mmol), potassium carbonate (138.21 g/mol, 221 mg, 1.6 mmol), sodium iodide (149.89 g/mol, 120 mg, 0.8 mmol), aminopropanol (75.11 g/mol, 60 mg, 0.8 mmol) and acetonitrile (5 mL) were added sequentially into a 25 mL reaction tube, and the mixture was stirred at 75° C. to react overnight. After the reaction was completed, 458 mg of E12LA6B6O3 (colorless oily liquid) was obtained by column chromatography separation and purification, with a yield of 66%.
  • Example 1 The lauric acid in Example 1 is replaced by caprylic acid, and the amino lipid E8LA8B6O3 can be prepared according to the same method, and its structural formula is as follows:
  • Example 1 The lauric acid in Example 1 is replaced by capric acid, and the amino lipid E8LA10B6O3 can be prepared according to the same method, and its structural formula is as follows:
  • the amino lipid E10LA6B6O3 can be prepared by replacing 1,2-epoxydodecane in Example 2 with 1,2-epoxydecane in the same manner, and its structural formula is as follows:
  • the 1,2-epoxyoctane in Example 1 is replaced by 1,2-epoxydecane, and the lauric acid is replaced by n-octanoic acid.
  • the amino lipid E10LA8B6O3 can be prepared according to the same method, and its structural formula is as follows:
  • the 1,2-epoxyoctane in Example 1 is replaced by 1,2-epoxydecane, and the lauric acid is replaced by capric acid.
  • the amino lipid E10LA10B6O3 can be prepared according to the same method, and its structural formula is as follows:
  • the amino lipid E12LA8B6O3 can be prepared by replacing the n-hexanoic acid in Example 2 with n-octanoic acid in the same manner, and the structural formula thereof is as follows:
  • E12LA12B6O3-2 (1.2 g, 3.65 mmol, 1.0 eq), 6-bromohexanoic acid (973 mg, 4.38 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.8 g, 14.6 mmol, 4.0 eq), 4-dimethylaminopyridine (45 mg, 0.365 mmol, 0.1 eq) and N,N-diisopropylethylamine (2.83 g, 21.9 mmol, 6.0 eq) and 20 mL of dichloromethane were added in sequence, and the reaction was stirred at room temperature overnight.
  • E12LA12B6O3-3 (1.65 g, 3.36 mmol, 2.2 eq), potassium carbonate (464 mg, 3.36 mmol, 2.2 eq), sodium iodide (150 mg, 1.0 mmol, 0.67 eq), 3-aminopropanol (113 mg, 1.5 mmol), and 10 mL of acetonitrile were added in sequence.
  • the reaction was stirred at 70-80°C overnight. After the reaction was completed, 0.94 g E12LA12B6O3 (0.94 g, 68%) was obtained by column chromatography.
  • the amino lipid E8CA5B6O3 can be prepared by replacing 1,2-epoxydodecane in Example 9 with 1,2-epoxyoctane and lauric acid with 2-pentyl-heptanoic acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O9 can be prepared by replacing the 3-aminopropanol in Example 2 with n-pentylamine in the same manner, and its structural formula is as follows:
  • the amino lipid E12LA6B6O10 can be prepared by replacing the 3-aminopropanol in Example 2 with trans-p-aminocyclohexanol in the same manner, and its structural formula is as follows:
  • the amino lipid E12LA6B6O12 can be prepared by replacing the 3-aminopropanol in Example 2 with 3-dimethylaminopropylamine in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O13 can be prepared by replacing the 3-aminopropanol in Example 2 with 1-(2-aminoethyl)piperidine in the same manner, and the structural formula thereof is as follows:
  • Example 2 The 3-aminopropanol in Example 2 is replaced by N-(2-aminoethyl)-4-hydroxypiperidine, and the amino lipid E12LA6B6O15 can be prepared according to the same method, and its structural formula is as follows:
  • the amino lipid E12LA12B6O30 can be prepared by replacing the 3-aminopropanol in Example 2 with ⁇ -alanine in the same manner, and its structural formula is as follows:
  • the amino lipid E12LA12B6O31 can be prepared by replacing the 3-aminopropanol in Example 2 with 4-aminobutyronitrile in the same manner, and its structural formula is as follows:
  • Imidazole (6.54 g, 96 mmol, 1.2 eq) was added to a 250 mL round-bottom flask, and the flask was filled with nitrogen by evacuation.
  • Tert-butyldimethylsilyl chloride (12.06 g, 80 mmol, 1.0 eq) was dissolved in dichloromethane (60 mL) and added dropwise to the mixture. The resulting mixture was stirred at room temperature for 4 hours.
  • K3LA6B6O3-5 (12.79 g, 25.2 mmol, 1.0 eq), ammonium fluoride (9.33 g, 252 mmol, 10.0 eq), methanol (75 mL), refluxed at 65°C with stirring for 3 hours.
  • the amino lipid K4LA6B6O3 can be prepared by replacing the 1,3-propylene glycol in Example 18 with 1,4-butylene glycol in the same manner, and the structural formula thereof is as follows:
  • the amino lipid K5LA6B6O3 can be prepared by replacing the 1,3-propylene glycol in Example 18 with 1,5-pentanediol in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O3A3 can be prepared by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 4-(4-methyl-1-piperazinyl)butyric acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O3A4 can be prepared by replacing the 3-(4-phenyl-piperazine-1-yl)-propionic acid in Example 21 with 3-(dimethylamino)propionic acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O3A5 can be prepared by replacing the 3-(4-phenyl-piperazine-1-yl)-propionic acid in Example 21 with 5-(dimethylamino)pentanoic acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O3A3 can be prepared by replacing the 3-(4-phenyl-piperazine-1-yl)-propionic acid in Example 21 with 7-(dimethylamino)heptanoic acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E8LA6B6O3A6 can be prepared by replacing E12LA6B6O3 in Example 21 with E8LA6B6O3 in the same manner, and its structural formula is as follows:
  • the amino lipid E12LA6B6O3A7 can be prepared by replacing the 3-(4-phenyl-piperazine-1-yl)-propionic acid in Example 21 with 1-methylpiperidine-4-carboxylic acid in the same manner, and the structural formula thereof is as follows:
  • the amino lipid E12LA6B6O3A8 can be prepared by replacing the 3-(4-phenyl-piperazine-1-yl)-propionic acid in Example 21 with 1-piperidine propionic acid in the same manner, and the structural formula thereof is as follows:
  • E12LA12B6O30A9-1 was synthesized.
  • the amino lipid E12LA12B6O30A10 can be prepared by replacing the 3-dimethylaminopropanol in Example 29 with 3-dimethylaminopropylamine in the same manner, and the structural formula thereof is as follows:
  • E12LA6B6O3A11-1 was synthesized.
  • 1,2-Epoxydodecane (3.69 g, 20 mmol, 1.0 eq), 20 mL of ammonia water (25% aqueous solution by mass), 10 mL of ethanol, and 10 mL of water were stirred at 60°C for 16 hours.
  • the reaction solution was spin-dried, 3 mL of ethanol and 30 mL of n-heptane were added, and the mixture was slurried at 60°C for 4 hours, filtered, and the filter cake was dried at 50°C for 10 hours to obtain compound E12N1LA6B6O3-2 (3.46 g, 86%), ESI-MS C 12 H 28 NO + [M+H] + calculated value 202.2, found value 202.2.
  • the amino lipid E12N1LA8B6O3 can be prepared by replacing the caproic acid in Example 32 with caprylic acid in the same manner, and its structural formula is as follows:
  • E12S1LA6B6O3-2 (1.2 g, 3.6 mmol, 1.0 eq), triethylamine (0.36 g, 3.6 mmol, 1.0 eq), methanesulfonyl chloride (0.49 g, 4.32 mmol, 1.2 eq), and 10 mL of dichloromethane were added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was washed once with water and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated.
  • amino lipid compound of the present invention or DLin-MC3 or SM-102 (both purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.)
  • phospholipid lipid DSPC
  • cholesterol phospholipid lipid
  • DMG-PEG2000 pegylated lipid
  • the mass ratio of the amino lipid compound to luciferase mRNA (Luc mRNA) is about 40:1.
  • the particle size and PDI (polydispersity index) of the prepared lipid nanoparticles were measured by Nano-ZSZEN3600 (Malvern). 20 ⁇ L of lipid nanoparticle (LNP) solution was taken for particle size measurement, and the cycle was repeated three times, with each cycle lasting 30 seconds.
  • LNP lipid nanoparticle
  • the encapsulation efficiency was determined according to the standard procedure of the Quant-iT RiboGreen RNA kit.
  • the encapsulation efficiency of the lipid nanoparticles provided by the present invention is higher than that of DLin-MC3, and the encapsulation efficiency of most of the lipid nanoparticles is higher than that of SM-102.
  • C57BL/6 mice were killed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection.
  • the thigh and tibia of the mice were dissected and the attached muscles were removed to expose the bone.
  • the bone marrow in the tibia was blown out with a 1 mL syringe filled with PBS.
  • the obtained bone marrow was blown out and filtered through a 50 ⁇ m filter to remove impurities.
  • Red blood cell lysis buffer (3-4 mL) was added to the obtained filtrate and placed for 5 minutes. After that, the supernatant was removed by centrifugation at 800 g for 5 minutes.
  • the obtained cells were resuspended in 1640 culture medium (containing 10% fetal bovine serum, 20 ng/mL GMCSF, 10 ng/mL IL4) and inoculated in a 6-well plate at a seeding density of 100,000 cells/mL culture medium.
  • the plate was placed at 37°C with 5% CO. 2 In the cell culture incubator, half of the medium was changed every 2 days. On the seventh day, the suspended cells and loosely attached cells were collected and inoculated into a 96-well all-white ELISA plate with a seeding density of 20,000 cells per well and a culture medium volume of 100 ⁇ L.
  • lipid nanoparticles encapsulating luciferase mRNA to a 96-well all-white ELISA plate containing primary cells, and control the volume of mRNA lipid nanoparticles added to each well to be 10 ⁇ L. Then place in an incubator at 37°C and 5% CO 2 for 12 hours.
  • the expression intensity of the amino lipids provided by the present invention is significantly better than that of DLin-MC3, and the expression intensity of some of the amino lipids is better than that of SM-102.
  • In vivo delivery Five C57BL/6 mice were randomly selected from each group and injected with lipid nanoparticle solution at a dose of 0.5 mg/kg mRNA by intramuscular injection. After 12 hours, 200 ⁇ L 10 mg/mL D-luciferin potassium salt was injected into the peritoneal cavity of each mouse. After 5 minutes, the mice were placed under the in vivo imaging system (IVIS-200, Xenogen) to observe the total fluorescence intensity of each mouse and take photos. The expression intensity of Luc mRNA delivered by LNP intramuscular injection composed of representative amino lipid compounds is shown in Table 3, with DLin-MC3 and SM-102 as controls.
  • Example 39 Evaluation of the in vivo immune and tumor therapeutic effects of lipid nanoparticles prepared from amino lipid compounds
  • the amino lipid compound of the present invention is mixed with DSPC, cholesterol, and DMG-PEG2000 at a molar ratio of 47.5:10:41:1.5 and dissolved in anhydrous ethanol.
  • the mass ratio of the amino lipid compound to ovalbumin mRNA (OVA mRNA) is about 40:1.
  • Animal preparation 5-6 week old female C57BL/6 mice weighing about 18-20 g were selected and kept in an SPF-grade feeding room. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.
  • B16-OVA melanoma cells (1.5 ⁇ 10 5 ) were injected subcutaneously into the outer thigh of mice. Vaccination was initiated when the tumor size grew to 50 mm 3 (approximately on the 6th or 7th day after tumor inoculation). Animals were immunized twice by intramuscular injection of LNP formulation containing 1 ⁇ g OVA-mRNA, with the second injection 7 days apart. Tumor growth was measured 3 times a week using a digital caliper, and the calculation formula was 0.5 ⁇ length ⁇ width ⁇ width. Mice were euthanized when the tumor volume reached 1500 mm 3.
  • the tumor growth rate of the E12LA6B6O3 group and the E10LA6B6O3 group was significantly slower than that of the MC3 group (as shown in Figure 2), and 100% (E12LA6B6O3 group) and 80% (E10LA6B6O3 group) of mice achieved complete remission, which was significantly better than the MC3 group (as shown in Figure 3).
  • Preparation of lipid nanoparticles Keep the ionizable lipid (E12LA6B6O3) content at 42.5%, the pegylated lipid DMG-PEG2000 content at 1.5%, adjust the phospholipid lipid ratio to 15, 12.5, 10, 7.5, 5.0, 2.5, 0, increase or decrease the adjusted phospholipid lipid molar ratio to the cholesterol ratio, and verify its effect on the quality control evaluation of lipid nanoparticles and the in vivo delivery performance of luciferase mRNA.
  • Each group is numbered B1-B7, and the specific ratio is shown in Table 4.
  • ALC-0315 and SM-102 are used as positive controls.
  • E12LA6B6O3, phospholipid lipid (DSPC), cholesterol, and pegylated lipid DMG-PEG2000 were mixed and dissolved in anhydrous ethanol according to the molar ratio of Table 4.
  • the base molar ratio of the amino lipid compound to the luciferase mRNA (Luc mRNA) was about 6.5:1.
  • the quality control of lipid nanoparticles was tested on a Malvern particle size analyzer and an ELISA reader, and SM-102 and ALC-0315 (both purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were added as control groups.
  • Animal preparation Six-week-old female Balb/C mice weighing approximately 20 g were selected and raised in an SPF-grade breeding room. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.
  • In vivo delivery Three Balb/C mice were randomly selected from each group, and lipid nanoparticle solution was injected by tail vein injection at a dosage of 0.1 mg/kg mRNA. After 6 hours, 150 ⁇ L of 15 mg/mL D-luciferin potassium salt was injected into the peritoneal cavity of each mouse. After 10 minutes, the mice were placed under the in vivo imaging system (IVIS Spectrum), and the total fluorescence intensity of each mouse was observed and photographed. The expression intensity of Luc mRNA delivered by tail vein injection of LNP with different DSPC content is shown in Table 5.
  • Table 5 LNP quality control data and expression intensity of different phospholipid lipid content

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Abstract

本发明提供一种氨基脂质、脂质纳米颗粒及其应用，所述氨基脂质具有通式(I)所示的结构，或其异构体、药学上可接受的盐、前药或溶剂合物。本发明还提供一种包含所述氨基脂质的脂质纳米颗粒。本发明通过采用具有通式(I)所示结构的氨基脂质作为可离子化脂质化合物，其与类固醇、中性脂质和聚合物结合的脂质通过自组装得到LNP，该LNP能进一步提高负载物-核酸在细胞内的翻译表达水平，提高核酸-LNP制剂的作用，给予个性化核酸-LNP制剂理论治疗提供了基础。

Description

氨基脂质、脂质纳米颗粒及其应用 技术领域
本发明涉及生物化学技术领域，尤其涉及一种氨基脂质、脂质纳米颗粒及其应用。
背景技术
脂质纳米颗粒(LNP)是一种新型的核酸生物分子递送技术，LNP通常由四种成分组成：(1)可离子化脂质，可与mRNA自组装成病毒大小的颗粒，并可将mRNA从内涵体释放到细胞质；(2)聚合物结合的脂质，可提高LNP在血液中半衰期；(3)类固醇，可增加纳米颗粒稳定性；(4)中性磷脂，有助于脂质双层结构的形成。
LNP既可以保护mRNA不被RNA酶分解，又可保护mRNA分子不被TLRs识别，避免先天免疫系统的过度激活的作用。其中可离子化脂质的选择对LNP的影响最大，可离子化脂质既起到促进细胞摄取，也可帮助药物分子从内涵体逃逸，同时又影响核酸药物的包封率、核酸药物在体内的递送效率以及细胞毒性等。
目前，已有Moderna和BioNtech&辉瑞针对COVID-19新冠病毒的疫苗，这两种疫苗均是采用LNP技术递送mRNA药物，从而实现了对COVID-19新冠病毒的预防，展现了LNP在mRNA药物与疫苗领域的巨大应用潜力，但目前LNP仍然具有包封率不高、内涵体逃逸率低、表达水平低、安全性不高等缺点，LNP递送系统的发展方向主要集中在可离子化脂质和配方上。
鉴于此，开发新型可离子化脂质化合物对核酸药物的发展具有重要的意义。
发明内容
第一方面，本发明提供一种氨基脂质，具有通式(I)所示的结构，或其异构体、药学上可接受的盐、前药或溶剂合物：
其中，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、NR′R″或至少含有一个杂原子的环状烷基结构；所述环状烷基结构中可被取代的碳原子或杂原子未被取代或被一个或多个羟基、C1-C4烷基、C2-C4烯基、C3-C8环烷基或C3-C8环烯基取代；
M1、M2、M3、M4彼此相同或不同，并且各自独立地选自C1-C24亚烷基、C3-C24亚环 烷基、C2-C24亚烯基或C3-C24亚环烯基；
R1、R2彼此相同或不同，并且各自独立地选自H、C1-C24烷基、C3-C24环烷基、C2-C24烯基或C3-C24环烯基；
L1、L2、L3、L4彼此相同或不同，并且独立地选自-C(＝O)O-、-OC(＝O)-、-C(＝O)S-、-SC(＝O)-、-C(＝O)NR-、-NRC(＝O)-、-S(＝O)-、-OS(＝O)2-、-S(＝O)2O-、-O-、-S-或-S-S-；
R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C10烷基、C3-C10环烷基、C3-C10烯基或C3-C10环烯基、末端连接叔胺基的C1-C10烷基、末端连接叔胺基的C3-C10环烷基、末端连接叔胺基的C3-C10烯基、或至少含有一个杂原子的环状烷基，所述环状烷基未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷基、C3-C8环烯基取代；
M5独立地选自单键、C1-C16亚烷基、C2-C16亚烯基、C3-C8亚环烷基或C3-C8亚环烯基；
第二方面，本发明提供上述氨基脂质的制备方法。主要分三步反应合成：第一步反应为开环反应；第二步为缩合反应；第三步为取代反应。
在本发明的一些实施方式中，所述氨基脂质的制备方法包括以下步骤：
S1：环氧化合物和羧酸发生开环反应，制备中间体1 R1-L1-M1-OH；
S2：中间体1和羧酸化合物原料在缩合剂下发生缩合反应，制备中间体2 R1-L1-M1-L2-M2-离去基团；
S3：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物；
或包括以下步骤：
S1’：二醇经TBS保护后，发生氧化反应，氧化产物与格氏试剂发生加成反应，制备中间体1’HO-M1-OTBS；
S2’：中间体1’和羧酸化合物在缩合剂下发生缩合反应，制备中间体2’TBSO-M1-L2-M2-离去基团；
S3’：中间体2’TBSO-M1-L2-M2-离去基团脱去TBS保护，并与羧酸化合物在缩合剂下发生缩合反应，生成中间体2 R1-L1-M1-L2-M2-离去基团；
S4’：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物。
第三方面，本发明提供一种脂质纳米颗粒，包括上述任一氨基脂质。
根据本发明提供的脂质纳米颗粒，所述脂质纳米颗粒还包括类固醇、中性脂质和/或聚合物结合的脂质；
所述聚合物结合的脂质其化学式为P-Y-L，其中P是亲水性聚合物部分，Y是任选的连接物，L是脂质部分。
第四方面，本发明提供一种药物组合物，包括上述脂质纳米颗粒和药学上可接受的载体。
第五方面，本发明还提供利用上述氨基脂质或脂质纳米颗粒或药物组合物治疗或预防传染病、癌症、遗传疾病、过敏、毒性、自身免疫性疾病的方法。
本发明还提供利用上述氨基脂质或脂质纳米颗粒或药物组合物进行基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的方法。
本发明还提供上述氨基脂质或脂质纳米颗粒在制备用于治疗或预防传染病、癌症、遗传疾病、过敏、毒性、自身免疫性疾病药物中的应用。
本发明还提供上述氨基脂质或脂质纳米颗粒在用于基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的药物中的应用。
第六方面，本发明还提供一种向受试者递送药剂的方法，包括：向受试者施用配制在上述脂质纳米颗粒中的药剂。
本发明的氨基脂质与类固醇、中性脂质和聚合物结合的脂质通过自组装得到LNP，该LNP能进一步提高负载物-核酸在细胞内的翻译表达水平，提高核酸-LNP制剂的作用，给予个性化核酸-LNP制剂理论治疗提供了基础。
附图说明
图1是实施例2中E12LA6B6O3的1H-NMR图谱；
图2是实施例39中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的肿瘤大小监测曲线；
图3是实施例39中肌肉注射OVA mRNA疫苗后的荷瘤小鼠的生存曲线图。
具体实施方式
定义
为了清晰易读，提供了以下科学背景信息和定义。本文提及或由此公开的任何技术特征可以是本发明的每个实施方式的一部分，也可以在本发明每个实施方式上读取。在本发明的上下文中可提供其他定义和解释。
除非另有定义，或除非特定上下文另有要求，本文中使用的所有技术术语的含义与相关技术领域的技术人员通常理解的含义相同。
除非上下文另有指示或要求，词语“包括”、“包含”和“含有”以及类似表达应以开放包容性意义在本说明书和权利要求书中解释为“包括但不限于”。
表述“一个实施方式”、“实施方式”、“一个特定实施方式”等意味着在本发明的至少一个实施方式中存在特定的特点、性质或特征，或如与相应表述结合所述的特点、性质或特征的特定组或组合。在整个描述中不同地方出现的这些表述不一定指同一实施方式。此外，可以在一个或多个实施方式中以任何合适的方式组合特定特点、性质或特征。
除非上下文另有明确规定，单数形式“一”、“一个”和“所述”应理解为包括复数含义。
当表述“中性”应用于化合物如脂质或类固醇、或基团或部分时，表示它既不是阳离子也不是阴离子，例如在生理条件下不具有可电离官能团的化合物，例如烃；或者表示它在典型的生理条件下既是阳离子的又是阴离子的，即两性离子，如典型的天然磷脂酰胆碱。
如本文中所用，“脂质”是指一组有机化合物，其是脂肪酸的衍生物(如酯)，通常以不溶于水但溶于许多有机溶剂为特征。脂质通常分为至少三类：(1)“简单脂质”，包括脂肪、油和蜡；(2)“复合脂质”，包括磷脂和糖脂；和(3)“衍生脂质”，如类固醇。关于糖脂，在某些实施方式中，LNP包含糖脂(例如单唾液酸神经节苷脂GM1)。
在上下文中，前缀“聚”指化合物中具有各自性质的多个原子或基团。然而，前缀的缺失不应被解释为排除复数。例如，聚阳离子化合物也是阳离子化合物，并且可被称为阳离子化合物。
术语“核酸”是指包含DNA或RNA或由其组成的任何化合物。该术语可用于寡核苷酸或寡核苷酸。
免疫系统：免疫系统可以保护生物体免受感染。如果病原体突破生物体的物理屏障并进入该生物体，则先天性免疫系统会提供即时但非特异性的应答。如果病原体逃避这种先天应答，脊椎动物则拥有第二层保护，即适应性免疫系统。此处，免疫系统在感染期间调整其应答，以改进其对病原体的识别能力。然后，这种改进的应答在病原体被消灭后以免疫记忆的形式保留下来，并允许适应性免疫系统在每次遇到这种病原体时开展更快、更强的攻击。据此，免疫系统包括先天性免疫系统和适应性免疫系统。这两部分中的每一部分都包含所谓的体液和细胞成分。
适应性免疫系统：适应性免疫系统由高度特化的系统性细胞和过程组成，可消除或防止致病性生长。适应性免疫应答为脊椎动物免疫系统提供识别和记忆特定病原体的能力(产生免疫)，并在每次遇到病原体时开展更强的攻击。由于体细胞超变(体细胞突变频率增加的过程)和V(D)J重组(抗原受体基因片段的不可逆基因重组)，该系统具有高度适应性。这种机制允许少量基因产生大量不同的抗原受体，然后在每个单独的淋巴细胞上唯一表达。由于基因重排导致每个细胞的DNA发生不可逆转的变化，因此该细胞的所有子代(后代)于是将继承编码相同受体特异性的基因，包括记忆B细胞和记忆T细胞，它们是长寿命特异性免疫的关键。免疫网络理论是一种关于适应性免疫系统如何工作的理论，它基于T细胞、B细胞以及由T细胞和B细胞产生的具有可变区的分子的受体的可变 区之间的相互作用。
术语“疫苗”通常被理解为提供至少一种抗原或抗原功能的预防性或治疗性材料。抗原或抗原功能可刺激身体的适应性免疫系统，以提供适应性免疫应答。
术语“抗原”通常指可被免疫系统识别的物质，优选由适应性免疫系统识别，并且能够触发抗原特异性免疫应答，例如通过形成抗体和/或抗原特异性T细胞作为适应性免疫应答的一部分。
术语“人工mRNA”(序列)通常可以理解为并非天然存在的mRNA分子。换言之，人工mRNA分子可以理解为非天然mRNA分子。这种mRNA分子可能是非天然的，由于它的单个序列(不是天然存在的)和/或由于其他修饰，例如非天然存在的核苷酸的结构修饰。通常，可以通过基因工程方法设计和/或生成人工mRNA分子，以对应于所需的人工核苷酸序列(异源序列)。在上下文中，人工序列通常是非天然存在的序列，即它与野生型序列至少有一个核苷酸不同。术语“野生型”可以理解为自然界中存在的序列。
术语“药学上可接受的盐”指一种化合物的存在形式，该形式不会引起对给药有机体的重要的刺激，且不会使化合物的生物活性和性质消失。
本发明所述化合物可能存在多种结晶型，即化合物的相同元素组成的不同晶格排列。多晶型通常有不同的X-射线衍射光谱、红外光谱、熔点、密度、硬度、晶型、光和电的性质、稳定性和溶解性。不同的因素如重结晶溶剂，结晶速率和贮存温度可能得到以单一晶型为主导的重结晶产物。可以理解的是，本发明所述的氨基脂质包括所有这种晶型。
本发明所述化合物可能存在手性中心和/或轴手性，并因此以消旋体、外消旋混合物、单一对映体、非对映异构体化合物和单一非对映体的形式、和顺反异构体的形式出现。每个手性中心或轴手性将独立地产生两个旋光异构体，并且所有可能的旋光异构体和非对映体混合物以及纯或部分纯的化合物包括在本发明的范围之内。本发明所限定的“各异构体”包括所述化合物的所有这种异构形式。
本发明所述的化合物还可能存在多种水合物或溶剂合物，溶剂合物含有化学计量或非化学计量的溶剂，且是在与药学上可接受溶剂如水或者乙醇等其他溶剂，结晶化过程中选择性形成的。当溶剂是水时形成水合物，或当溶剂是乙醇等其他溶剂时形成溶剂合物。
术语“前药”也称前体药物、药物前体、前驱药物等，是指药物经过化学结构修饰后得到的在体外无活性或活性较小、在体内经酶或非酶的转化释放出活性药物而发挥药效的化合物。前药有两大类：一类是载体前体药物，简称载体前药；另一类是生物前体药物。载体前体药物是指具有活性的化合物与其运输作用的载体通过共价键结合，在体内通过简单的水解作用卸掉载体，由活性化合物发挥药理作用。载体前体药物与母体化合物相比往 往活性微弱或无活性。对于载体的结构，多是亲脂性，要求对生物体无害，且能及时释放活性化合物。生物前体药物不同于载体前体药物，活性物质不用与载体暂时性结合，而是通过自身分子结构的改变来发挥作用。生物前体药物本身没有活性，有活性的是其在生物体内的代谢物。
本发明经过研究意外发现，本发明提供的新型氨基脂质和/或脂质纳米颗粒的使用，可以有效解决现有技术存在的LNP具有包封率不高、内涵体逃逸率低、表达水平低、安全性不高等缺点，从而促进LNP在mRNA药物与疫苗领域的发展。
氨基脂质
氨基脂质优选可阳离子化，即当pH值降低到脂质可电离基团的pKa以下时，氨基脂质会质子化，当带正电荷时，脂质便能与带负电荷的核酸结合。
在一个方面，本发明提供一种氨基脂质，具有通式(I)所示的结构，或其异构体、药学上可接受的盐、前药或溶剂合物：
其中，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、NR′R″或至少含有一个杂原子(优选地，所述杂原子为N或O，优选为1或2个选自N或O的杂原子)的环状烷基结构(优选为3-10元环烷基，更优选为4-6元环烷基)；所述环状烷基结构中可被取代的碳原子或杂原子未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷基或C3-C8环烯基取代；
M1、M2、M3、M4彼此相同或不同，并且各自独立地选自C1-C24亚烷基、C3-C24亚环烷基、C2-C24亚烯基或C3-C24亚环烯基；
R1、R2彼此相同或不同，并且各自独立地选自H、C1-C24烷基、C3-C24环烷基、C2-C24烯基或C3-C24环烯基；
L1、L2、L3、L4彼此相同或不同，并且独立地选自-C(＝O)O-、-OC(＝O)-、-C(＝O)S-、-SC(＝O)-、-C(＝O)NR-、-NRC(＝O)-、-S(＝O)-、-OS(＝O)2-、-S(＝O)2O-、-O-、-S-或-S-S-；
R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C10烷基(优选为C1-C6烷基，更优选为C1-C4烷基)、C3-C10环烷基、C3-C10烯基、C3-C10环烯基、末端连接叔胺基的C1-C10烷基、末端连接叔胺基的C3-C10环烷基、末端连接叔胺基的C3-C10烯基、或至少含有一个杂原子的环状烷基，所述环状烷基未被取代或被一个或多个C1-C4烷基、C2-C4烯基、 C3-C8环烷基、C3-C8环烯基取代；优选地为末端连接有-N(C1-6烷基)2的C1-C10烷基或末端连接有含有1-2个环氮原子的3-8元环状烷基(优选地为4-6元环状烷基)的C1-C10烷基，所述C3-C10环烷基和3-8元环状烷基(优选地为4-6元环状烷基)任选地被C1-C6烷基取代。
M5选自单键、C1-C16亚烷基、C2-C16亚烯基、C3-C8亚环烷基或C3-C8亚环烯基。
需要说明的是，若未明确不允许，本发明提及的含多个碳原子的烷基、烯基、亚烷基、亚烯基可以为直链，也可以为支链。另外，本发明提及的烷基、烯基、亚烷基、亚烯基，以及环烷基、环烯基、亚环烷基、亚环烯基，可以为未取代的或取代的，取代基可以是任何合适的取代基，即任何直链或支链烷基、芳基、杂烷基、杂芳香结构，其可以任选地包含其他官能团，例如酯基或酰胺基。
在一些优选实施方式中，G选自H、OR、NR′R″或至少含有一个杂原子的环状烷基结构；其中杂原子为O或N；其中R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C8烷基、C3-C8环烷基、C3-C8烯基或C3-C8环烯基。
在进一步优选的实施方式中，G选自H、OH或NR′R″、其中R′、R″彼此相同或不同，并且各自独立地选自H或C1-C4烷基；即NR′R″可以为NH2、NHCH3、NHC2H5、NHC3H7、NHC4H9、N(CH3)2、CH3-N-C2H5、CH3-N-C3H7、CH3-N-C4H9、C2H5-N-C2H5、C2H5-N-C3H7、C2H5-N-C4H9、C3H7-N-C3H7、C3H7-N-C4H9或C4H9-N-C4H9。
或者，G选自取代或未被取代的氧杂五元环烷基、氮杂五元环烷基、氮杂六元环烷基、双氮杂六元环烷基或氮杂氧杂六元环烷基。优选地，氧杂五元环烷基为氮杂五元环烷基为氮杂六元环烷基为双氮杂六元环烷基为氮杂氧杂六元环烷基为其中*号位置与M5相连。
在一些实施方式中，当G为取代的双氮杂六元环烷基时，取代基位置为不与M5相连的氮原子。
在一些优选的实施方式中，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、NR′R″或含有1或2个环N原子的环状烷基结构(优选为3-10元环烷基，更优选为4-6元环烷基)；所述环状烷基结构中可被取代的碳原子或杂原子未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷基或C3-C8环烯基取代。R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C6烷基、C3-C10环烷基、末端连接有-N(C1-6烷基)2的C1-C10烷基或末端连接有含有1-2个环氮原子的4-6元环状烷基的C1-C10烷基，所述 C3-C10环烷基和4-6元环状烷基任选地被C1-C6烷基取代。
在一些实施方式中，M5选自单键、C2-C16亚烷基、C2-C16亚烯基、C4-C8亚环烷基或C3-C8亚环烯基。
进一步优选地，M5选自单键、C2-C16亚烷基或C4-C6亚环烷基。更优选地，M5选自单键、C2-C6亚烷基或C4-C6亚环烷基；或选自C3-C5亚烷基.
在优选的实施方式中，通式(I)中，M5和G相连形成选自A1～A38中的一种：
进一步优选地，所述选自A1-A18、A22-A24、A28-A38中的一种。
更优选地，所述选自A15、A16、A17、A23、A29、A30、A33、A37、A38、A42中的一种。
在本发明的一些实施方式中，通式(I)中，M5和G相连形成的选自A39～A52中的一种：
在本发明的一些实施方式中，L1、L2、L3、L4彼此相同或不同，并且独立地选自-C(＝O)O-、-OC(＝O)-、-C(＝O)NR-或-NRC(＝O)-。
其中，当L1、L2、L3、L4独立地选自-C(＝O)NR-或-NRC(＝O)-时，其中R独立地选自H或C1-C10烷基。优选地，R均为H。
在本发明的优选实施方式中，L1与L4相同，且为-C(＝O)O-或-OC(＝O)-。
在本发明的一些实施方式中，M1、M2、M3、M4彼此相同或不同，并且M1和M4各自独立地选自含有支链的C4-C22亚烷基、含有支链的C4-C22亚环烷基、含有支链的C4-C22亚烯基或含有支链的C4-C22亚环烯基，M2和M3各自独立地选自C4-C22亚烷基、C4-C22亚环烷基、C4-C22亚烯基或C4-C22亚环烯基。
进一步优选地，M1、M2、M3、M4彼此相同或不同，并且M1和M4各自独立地选自含有支链的C4-C22亚烷基或含有支链的C4-C22亚烯基，优选为含有支链的C6-C16亚烷基； M2和M3各自独立地选自C4-C22亚烷基或C4-C22亚烯基，优选为C3-C8亚烷基。
更优选地，在本发明一些实施方式中，M2与M3相同，且为C4-C22亚烷基。
在本发明一些实施方式中，M1与M4相同，且为含有支链的C4-C22亚烷基。
在本发明的一些实施方式中，R1、R2彼此相同或不同，并且各自独立地选自C4-C22烷基或C4-C22烯基，优选为C5-C12烷基。
在优选的实施方式中，R1-L1-M1-L2-M2-片段为R1-C(＝O)O-M1-OC(＝O)-M2-，R2-L4-M4-L3-M3-片段R2-C(＝O)O-M4-OC(＝O)-M3-。进一步优选地，M1、M2、M3、M4彼此相同或不同，并且M1与M4各自独立地选自含有支链的C4-C22亚烷基或含有支链的C4-C22亚烯基；M2和M3各自独立地选自C4-C22亚烷基或C4-C22亚烯基；R1、R2彼此相同或不同，并且各自独立地选自C4-C22烷基或C4-C22烯基。
本领域人员可以依照常识对上述针对不同基团的优选实施方式进行组合，得到本发明化合物的更优实施例。
在本发明的优选实施方式中，通式(I)结构选自以下结构中的一种：
在本发明的优选实施方式中，通式(I)结构选自以下结构中的一种：


本领域人员可以依照常识对上述针对不同基团的优选实施方式进行组合，得到本发明化合物的更优实施例。
氨基脂质的制备方法
本发明提供上述氨基脂质的制备方法，包括以下步骤：
S1：环氧化合物和羧酸发生开环反应，制备中间体1 R1-L1-M1-OH；
S2：中间体1和羧酸化合物原料在缩合剂下发生缩合反应，制备中间体2 R1-L1-M1-L2-M2-离去基团；
S3：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物；
或包括以下步骤：
S1’：二醇经TBS保护后，发生氧化反应，氧化产物与格氏试剂发生加成反应，制备中间体1’HO-M1-OTBS；
S2’：中间体1’和羧酸化合物在缩合剂下发生缩合反应，制备中间体2’TBSO-M1-L2-M2-离去基团；
S3’：中间体2’TBSO-M1-L2-M2-离去基团脱去TBS保护，并与羧酸化合物在缩合剂下发生缩合反应，生成中间体2 R1-L1-M1-L2-M2-离去基团；
S4’：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物。
优选地，步骤S2’中的羧酸为H-L2-M2-离去基团。
优选地，步骤S3’中的羧酸为R1-L1-H。
优选地，所述离去基团为卤素。
优选地，制备的各中间体和目标产物均经过柱层析纯化。
在一些实施方式中，所述制备方法包括以下步骤：
开环反应：将羧酸化合物(1.0eq)、环氧化合物原料(0.6～3.0eq)、三氯化铁(0.5～10mol％)、吡啶(0.2～20mol％)混合后，置于室温下搅拌过夜，反应结束后，经柱层析纯化得到中间体1，收率56％～99.0％。
缩合反应：依次将中间体1(1.0eq)、含卤羧酸(0.6～3.0eq)、EDCI-HCl(1.0～6.0eq)、DMAP(0.05～0.5eq)、DIPEA(1.0～8.0eq)、DCM混合后，置于室温搅拌过夜，经柱层析纯化得到中间体2，收率46.0％～96.4％。
取代反应：依次将中间体2(1.0-5.0eq)、碳酸钾(1.0～5.0eq)、氨类化合物(1.0eq)、碘化钠(1.0-3.0eq)和乙腈混合后，置于20～100℃条件下搅拌过夜，经柱层析纯化得到氨基脂质，收率32.0％～92.1％。
在一些实施方式中，所述制备方法包括以下步骤：
加成反应：二醇经TBS保护后，与PCC发生氧化反应，氧化产物0.9～1.2eq、在无水四氢呋喃中于-20℃下搅拌2-20分钟，加入格氏试剂1.2～1.5eq，在-20℃至0℃搅拌1-6小时，经柱层析纯化得到中间体1；
缩合反应1：依次将中间体1’(1.0eq)、含卤羧酸(0.6～3.0eq)、EDCI-HCl(1.0～6.0eq)、DMAP(0.05～0.5eq)、DIPEA(1.0～8.0eq)、DCM混合后，置于室温搅拌过夜，经柱层析纯化得到中间体2’；
缩合反应2：将中间体2’(1.0eq)用氟化铵(5.0～20.0eq)脱去TBS保护，并且与羧酸(0.6～3.0eq)、EDCI-HCl(1.0～6.0eq)、DMAP(0.05～0.5eq)、DIPEA(1.0～8.0eq)、DCM混合后，置于室温搅拌过夜，经柱层析纯化得到中间体2；
取代反应：依次将中间体2(1.0-5.0eq)、碳酸钾(1.0～5.0eq)、氨类化合物(1.0eq)、碘化钠(1.0-3.0eq)和乙腈混合后，置于20～100℃条件下搅拌过夜，经柱层析纯化得到氨基脂质。
脂质纳米颗粒
本发明提供一种脂质纳米颗粒，包括上述任一氨基脂质。
可以理解的是，上述氨基脂质的所有选择和优选项本身也适用于本发明的包含氨基脂质的脂质纳米颗粒。
在本发明的一些实施方式中，所述脂质纳米颗粒还包括类固醇、中性脂质和/或聚合物结合的脂质。
类固醇
“类固醇”是一种有机化合物，其具有以特定分子构型排列的四个环。它包含以下碳骨架：
类固醇和中性类固醇包括天然存在的类固醇及其类似物(如两亲性脂质胆固醇半琥珀酸酯(CHEMS)，组成为琥珀酸酯化到胆固醇的β-羟基基团作为胆固醇衍生物)。中性类固醇可以是在生理条件下不具有可电离的原子或基团的类固醇，或者可以是两性离子类固 醇。在一个优选实施方式中，中性类固醇不含在生理条件下可电离的原子或基团。在一些优选的实施方式中，类固醇或类固醇类似物是胆固醇。术语“类固醇”和“中性类固醇”在本文中可交换使用。
中性脂质
本发明的“中性脂质”，也称为“辅助脂质”，优选为磷脂或中性磷脂。如本文中所用，“中性磷脂”是一种两亲性化合物，由通常具有两个疏水性脂肪酸“尾”和包含磷酸盐基团的亲水性“头”的分子组成。磷酸盐基团可以用简单的有机分子如胆碱、乙醇胺或丝氨酸修饰。磷脂在自然界中大量存在。本发明中“磷脂”或“中性磷脂”既包括天然磷脂，也包括合成磷脂。
聚合物结合的脂质
术语“聚合物结合的脂质”指同时包含脂质部分和聚合物部分的分子。优选地，聚合物结合的脂质是聚乙二醇化脂质或PEG-脂质。术语“聚乙二醇化脂质”或“PEG-脂质”是指同时包含脂质部分和聚乙二醇部分的分子。聚乙二醇化脂质是本领域已知的并且包括PEG-DMG等。
在具体的实施方式中，所述聚合物结合的脂质其化学式为P-Y-L，其中P是亲水性聚合物部分，Y是任选的连接物，L是脂质部分。
具体地，亲水性聚合物部分P可以是聚乙二醇PEG。在具体的实施方式中，PEG部分的平均分子质量在1kDa和3kDa之间，例如在1.5kDa至2.5kDa之间、1.7kDa至2.3kDa之间、1.8kDa至2.2kDa之间、1.9kDa至2.1kDa之间或2kDa。因此，PEG可以是通常被称为“PEG 2000”的PEG。
在另一个实施方式中，聚合物结合的脂质中的亲水性聚合物部分P也可以是与上述亲水性聚合物部分不同的实质上亲水性聚合物，即，聚合物结合的脂质中的亲水性聚合物部分P可基于聚(环氧丙烷)、聚(乙烯吡咯烷酮)、聚(乙烯醇)、聚-N-(2-羟丙基)甲基丙烯酰胺、羟乙基淀粉化(HESylation)过程(根据PMID 24681396)、PAS化(PASylation)法(即脯氨酸-丙氨酸-丝氨酸)、本领域已知的XTEN法(即基于PEG的肽)、聚肌氨酸(polysarcosin)或聚(乙酸乙烯酯)。
具体地，任选的连接物Y可以是任何有用的间隔基结构，例如选自那些通常在聚乙二醇化脂质中发现有用的间隔基，例如，但不限于琥珀酰亚胺、胺、醚、酯、酸酐、醛、酮、酰胺、氨基甲酸酯连接物或其组合。
具体地，脂质部分L可以是源自磷脂、鞘脂或神经酰胺。如本文中所用，术语“源自磷脂或神经酰胺”包括磷脂和神经酰胺的自由基。实例为包含磷脂酰乙醇胺或磷脂酰甘油 部分的聚合物结合的脂质。
在优选的实施方式中，聚合物结合的脂质是聚乙二醇化脂质。所述聚乙二醇化脂质包括但不限于以下聚乙二醇化脂质：聚乙二醇化二酰甘油脂质(PEG-DAG)；聚乙二醇化神经酰胺脂质(PEG-Cer)；聚乙二醇化磷脂酰乙醇胺脂质(PEG-PE)；聚乙二醇化琥珀酸二酰甘油脂质(PEG-S-DAG)；聚乙二醇化二烷氧基丙基氨基甲酸酯脂质；1，2-二肉豆蔻基-rac-甘油-3-甲氧基聚乙二醇(“PEG-DMG”或“DMG-PEG”)。
在更优选的实施方式中，所述聚合物结合的脂质为DMG-PEG2000。
优选地，如本领域中所用，“DMG-PEG 2000”被认为是比率为约97∶3的1，2-DMG PEG2000和1，3-DMG PEG2000的混合物。
在本发明的一些实施方式中，所述脂质纳米颗粒中，氨基脂质、类固醇、中性脂质和聚合物结合的脂质的摩尔比为30-70∶30-65∶0-30∶0.2-5。进一步优选为30-60∶35-60∶0-20∶0.3-3。
在本发明的优选实施方式中，所述脂质纳米颗粒中，所述氨基脂质为前述优选的氨基脂质，所述类固醇为胆固醇，所述中性脂质为磷脂，所述聚合物结合的脂质为聚乙二醇化脂质；氨基脂质、胆固醇、磷脂和聚乙二醇化脂质的摩尔比为40-50∶40-45∶10-15∶0.5-2。
在一个优选实施方式中，所述脂质纳米颗粒中，所述聚乙二醇化脂质为DMG-PEG2000。
本发明的脂质纳米颗粒不限于任何特定形态，并且应被解释为包括当氨基脂质和任选的一种或多种其他的脂质结合时产生的任何形态，例如在水相环境和/或在存在核酸化合物的情况下。例如，脂质体、脂质复合物、lipoplex等在脂质纳米颗粒的范围内。
本发明的脂质纳米颗粒可以与至少一种药学上可接受的载体或赋形剂结合，得到药物组合物。因此，组合物可以是干燥组合物，例如粉末或颗粒，或固体单元，例如冻干形式或片剂。作为选择，组合物可以是液体形式，并且每种赋形剂可以以溶解或分散(例如悬浮或乳化)形式独立地加入。在一个优选实施方式中组合物被配制成无菌固体组合物，例如粉末或冻干形式，用于与水性液体载体重组。对于包含下文进一步详细描述的包含生物活性组分的组合物，也优选这种制剂。
如本文中所用，“纳米颗粒”是具有任何结构或形态的亚微米颗粒。亚微米颗粒也可以称为胶体或胶质。亚微米颗粒也可以称为胶体或胶体。关于纳米颗粒所基于的材料，以及结构或形态，可以将纳米颗粒分类，例如，为纳米胶囊、囊泡、脂质体、脂质纳米颗粒、胶束、交联胶束、lipoplex、多聚物、混合或混合复合物，仅提及特定类型纳米颗粒的几种可能命名。“脂质纳米颗粒”(LNP)是由脂质形成的纳米颗粒，通常包含至少一种两 亲性、成膜脂质和任选的其他脂质，进一步任选地包含载荷材料如核酸化合物。如本文中所用，表述“脂质纳米颗粒”或“LNP”包括由脂质形成或共同形成的纳米颗粒的任何亚类型和形态，例如脂质体和lipoplex。
如上文定义，脂质纳米颗粒包括由脂质形成或共同形成的任何类型的纳米颗粒。特别是，脂质纳米颗粒可由包含至少一种两亲性、囊泡形成脂质的脂质组合共同形成。脂质体和lipoplexe是脂质纳米颗粒的实例。
优选地，在本发明的一些实施方式中，所述脂质纳米颗粒还包含生物活性组分。
生物活性组分是指具有生物活性的任何化合物或材料，由于该活性，化合物或材料可用于受试者(如动物，特别是人类受试者)的疾病或状况的预防、管理、改善、治疗或疗法。
在一些实施方式中，所述生物活性组分是选自由人工mRNA、包含至少一个编码序列的化学修饰或未修饰的信使RNA、自复制RNA、环状RNA、病毒RNA和复制子RNA组成的组的核酸化合物；或其任何组合。优选地，其中生物活性组分为mRNA或mRNA化合物。
在一些实施方式中，所述生物活性组分选自小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、微小RNA(miRNA)、Dicer-底物RNA(dsRNA)、小发夹RNA(shRNA)、信使RNA(mRNA)及其混合物。
在本发明的实施方式中，mRNA包括茎环、链终止核苷、polyA序列、多腺苷酸化信号和/或5′帽结构中的一种或多种。
在本发明的实施方式中，所述生物活性组分的包封率至少为50～90％。进一步优选地，所述生物活性组分的包封率至少为60～80％。
在本发明的实施方式中，所述脂质纳米颗粒中，脂质组分与生物活性组分的重量/重量比为约10∶1至约60∶1。进一步优选地，脂质组分与生物活性组分的重量/重量比约为20∶1。
在本发明的实施方式中，所述脂质纳米颗粒中N∶P比率为约2∶1至约30∶1。进一步优选地，N∶P比约为5.67∶1。
在本发明的实施方式中，所述脂质纳米颗粒的平均尺寸为约70nm至约100nm。
在本发明的实施方式中，所述脂质纳米颗粒的多分散指数为约0.10至约0.20。
在本发明的实施方式中，所述脂质纳米颗粒具有约-10mV至约+20mV的zeta电位。
在优选的实施方式中，所述生物活性组分与一种或多种脂质(例如氨基脂质和/或中性脂质)复合或结合，从而形成脂质体、脂质纳米颗粒(LNP)、lipoplex和/或纳米脂质体。在上下文中，术语“复合”或“结合”是指生物活性组分与一种或多种脂类基本上稳 定的组合成未共价结合的更大的复合物或组装体。
应用
本发明提供利用上述氨基脂质或脂质纳米颗粒或药物组合物进行治疗或预防传染病、癌症、肿瘤疾病、遗传疾病、过敏、毒性、自身免疫性疾病的方法。
相应地，本发明提供上述氨基脂质或脂质纳米颗粒或药物组合物在制备用于治疗或预防传染病、癌症、肿瘤疾病、遗传疾病、过敏、毒性、自身免疫性疾病药物中的应用。
其中，所述传染病包括病毒、细菌或原生动物传染病。所述病毒包括但不限于SARS冠状病毒2(SARS-CoV-2)、nCoV-2019冠状病毒、SARS冠状病毒(SARS-CoV)、布尼亚病毒、巨细胞病毒(CMV)、登革热病毒(DEN-1、DEN-2、DEN-3和DEN-4)、埃博拉病毒、黄病毒、乙型肝炎病毒(HBV)、单纯疱疹病毒(HSV)、人类免疫缺陷病毒(HIV)、人类偏肺病毒(hMPV)、人乳头瘤病毒(HPV)、人类副流感病毒(HPIV)、流感病毒、肠外致病性大肠杆菌、拉沙病毒(LASV)、MERS冠状病毒、结核分枝杆菌、尼帕病毒、诺如病毒、狂犬病病毒、呼吸道合胞病毒(RSV)、鼻病毒、轮状病毒、痘苗病毒、黄热病病毒、寨卡病毒。
所述癌症包括肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌和前列腺癌。
本发明还提供利用上述氨基脂质或脂质纳米颗粒或药物组合物进行基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的方法。
相应地，本发明还提供上述氨基脂质或脂质纳米颗粒在制备用于基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的药物中的应用。
本发明还提供一种向受试者递送药剂的方法，包括：向受试者施用配制在上述脂质纳米颗粒中的药剂。
药物施用时，根据药物的剂型确定施药方式。药物的剂型与赋形剂和/或载体有关。全身施用的一般途径包括：透皮、口服、肠外途径，包括皮下、静脉、肌肉内、动脉内、皮内和腹腔内注射和/或鼻内施用途径。局部施用的途径一般包括皮内、透皮、皮下或肌肉内注射，或病灶内、颅内、肺内、心内、肿瘤内和舌下注射。当药物为疫苗形式时，优选施用途径是肌肉内注射和皮内注射。
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
为了说明的目的，以下实施例中显示了制备本发明提供的化合物的一般方法。对于各 个反应步骤的更详细描述，参见以下实施例部分。本领域技术人员能够理解，其它合成途径可用于合成本发明化合物。尽管在方案中描述了并在下面讨论了具体的原料和试剂，但其它原料和试剂可以容易地替代以提供各种衍生物和/或反应条件。此外，结合以下实施例的公开，使用本领域技术人员熟知的常规化学方法，可以进一步修饰通过下述方法制备的许多化合物，并得到本发明限定范围内的其他化合物。
文中的缩写对应物质如下：
Py吡啶
EDCI 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐
DMAP 对二甲氨基吡啶
DIPEA 二异丙基乙胺
DCM 二氯甲烷
MeCN 乙腈
DSPC 二硬脂酰磷脂酰胆碱
DMG-PEG2000 1，2-二肉豆寇酰基-rac-甘油-3-甲氧基聚乙二醇2000。
氨基脂质化合物的制备
实施例1 氨基脂质E8LA12B6O3的合成
Step 1：E8LA12的合成：
在25mL反应管中依次加入FeCl3(16.2mg，2.5mol％)，Py(4mg，1.25mmol％)，月桂酸(200.32g/mol，800mg，4.0mmol)和1，2-环氧辛烷(128.22g/mol，1.02g，8mmol)，室温下搅拌反应过夜，反应结束后，经柱层析分离纯化得到1.2g中间体E8LA12(无色油状液体)，收率91％。
Step 2：E8LA12B6的合成：
在25mL反应管中依次加入E8LA12(328.54g/mol，1.2g，3.65mmol)，6-溴已酸(195.06g/mol，855mg，4.38mmol)，EDCI(191.70g/mol，2.8g，14.6mmol)，DMAP(45mg，0.365mmol)和DIPEA(2.83g，21.9mmol)和DCM(10mL)，室温下搅拌反应过夜。反应结束 后，经柱层析分离纯化得到1.65g中间体E8LA12B6(无色油状液体)，收率89％。
Step 3：E8LA12B6O3的合成：
在25mL的反应管中依次加入E8LA12B6(505.58g/mol，1.65g，3.26mmol)，碳酸钾(138.21g/mol，902mg，6.52mmol)，碘化钠(149.89g/mol，489mg，3.26mmol)，氨基丙醇(75.11g/mol，113mg，1.5mmol)和乙腈(10mL)，在75℃下搅拌反应过夜。反应结束后，经柱层析分离纯化得到0.94g E8LA12B6O3(无色油状液体)，收率68％。1H NMR(400MHz，CDCl3)：δ5.08-5.03(m，2H)，4.22(dd，J1＝11.6Hz，J2＝3.6Hz，2H)，4.02(dd，J1＝12.0Hz，J2＝6.4Hz，2H)，3.82(t，J＝5.6Hz，2H)，2.96(t，J＝7.6Hz，2H)，2.77(t，J＝6.0Hz，4H)，2.34-2.28(m，8H)，1.89-1.88(m，2H)，1.72-1.56(m，12H)，1.40-1.25(m，56H)，0.87(t，J＝7.0Hz，12H).13C NMR(100MHz，CDCl3)：δ173.1，70.7，65.5，59.0，58.5，57.2，34.2，33.9，31.9，31.8，30.7，30.3，29.6，29.3，29.0，28.0，26.7，25.0，25.3，24.7，22.7，14.1.ESI-MS C55H106NO9 +[M+H]+计算值924.7862，实测值924.7850.
实施例2 氨基脂质E12LA6B6O3的合成
Sten 1：E12LA6的合成：
在25mL反应管中依次加入FeCl3(16.2mg，2.5mol％)，Py(4mg，1.25mmol％)，正己酸(116.1600g/mol，464mg，4.0mmol)和1，2-环氧十二烷(184.18g/mol，1.47g，8mmol)，室温下搅拌反应过夜，反应结束后，经柱层析分离纯化得到1.02g中间体E12LA6(无色油状液体)，收率85％。
Step 2：E12LA6B6的合成：
在25mL反应管中依次加入E12LA6(300.48g/mol，901mg，3.0mmol)，6-溴已酸(195.06g/mol，703mg，3.6mmol)，EDCI(191.70g/mol，2.3g，12.0mmol)，DMAP(37mg，0.3mmol)和DIPEA(2.33g，18mmol)和DCM(10mL)，室温下搅拌反应过夜。反应结束后，经柱层析分离纯化得到1.08g中间体E12LA6B6(无色油状液体)，收率76％。
Step 3：E12LA6B6O3的合成：
在25mL的反应管中依次加入E12LA6B6(477.52g/mol，956mg，2.0mmol)，碳酸钾(138.21g/mol，221mg，1.6mmol)，碘化钠(149.89g/mol，120mg，0.8mmol)，氨基丙醇(75.11g/mol，60mg，0.8mmol)和乙腈(5mL)，在75℃下搅拌反应过夜。反应结束后，经柱层析分离纯化得到458mg E12LA6B6O3(无色油状液体)，收率66％。1H NMR(400MHz，CDCl3)：δ5.08-5.06(m，2H)，4.22(dd，J1＝12.0Hz，J2＝3.6Hz，2H)，4.02(dd，J1＝11.6Hz，J2＝6.4Hz，2H)，3.79(t，J＝5.2Hz，2H)，2.73-2.70(m，2H)，2.52-2.48(m，4H)，2.33-2.28(m，8H)，1.74-1.50(m，14H)，1.42-1.25(m，48H)，0.91-0.86(m，12H).(如图1所示)ESI-MS C51H98NO9 +[M+H]+计算值868.7236，实测值868.7234.
实施例3氨基脂质E8LA8B6O3的合成
将实施例1中的月桂酸替换为辛酸，按照相同的方法可制得氨基脂质E8LA8B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ5.08-5.02(m，2H)，4.22(dd，J1＝12.0Hz，J2＝3.6Hz，2H)，4.01(dd，J1＝11.6Hz，J2＝6.0Hz，2H)，3.81(t，J＝5.2Hz，2H)，2.98(t，J＝6.0Hz，2H)，2.79(t，J＝7.6Hz，4H)，2.34-2.27(m，8H)，1.92-1.86(m，2H)，1.73-1.56(m，12H)，1.40-1.26(m，40H)，0.88-0.85(m，12H).ESI-MS C47H90NO9 +[M+H]+计算值812.6610，实测值812.6598.
实施例4氨基脂质E8LA10B6O3的合成
将实施例1中的月桂酸替换为癸酸，按照相同的方法可制得氨基脂质E8LA10B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.89(m，12H)，1.25-1.40(m，48H)，1.56-1.72(m， 12H)，1.88-1.90(m，2H)，2.28-2.33(m，8H)，2.77-2.79(m，4H)，2.96-2.98(m，2H)，3.79-3.80(m，2H)，4.01-4.05(m，2H)，4.21-4.24(m，2H)，5.03-5.08(m，2H)。ESI-MSC51H98NO9 +[M+H]+计算值868.7，实测值868.7。
实施例5 氨基脂质E10LA6B6O3的合成
将实施例2中的1，2-环氧十二烷替换为1，2-环氧癸烷，按照相同的方法可制得氨基脂质E10LA6B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ5.08-5.02(m，2H)，4.22(dd，J1＝12.0Hz，J2＝3.2Hz，2H)，4.01(dd，J1＝11.6Hz，J2＝6.4Hz，2H)，3.79(t，J＝5.2Hz，2H)，2.82(t，J＝5.6Hz，2H)，2.62(t，J＝8.0Hz，4H)，2.36-2.27(m，8H)，1.81-1.76(m，2H)，1.68-1.53(m，12H)，1.37-1.24(m，40H)，0.90-0.84(m，12H).ESI-MS C47H90NO9 +[M+H]+计算值812.6610，实测值812.6634.
实施例6 氨基脂质E10LA8B6O3的合成
将实施例1中的1，2-环氧辛烷替换为1，2-环氧癸烷，将月桂酸替换为正辛酸，按照相同的方法可制得氨基脂质E10LA8B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.84-0.89(m，12H)，1.26-1.41(m，48H)，1.56-1.73(m，12H)，1.89-1.91(m，2H)，2.26-2.32(m，8H)，2.78-2.80(m，4H)，2.96-2.98(m，2H)，3.80-3.81(m，2H)，4.01-4.04(m，2H)，4.20-4.24(m，2H)，5.03-5.07(m，2H)。ESI-MSC51H98NO9 +[M+H]+计算值868.7，实测值868.7。
实施例7 氨基脂质E10LA10B6O3的合成
将实施例1中的1，2-环氧辛烷替换为1，2-环氧癸烷，将月桂酸替换为癸酸，按照相同的方法可制得氨基脂质E10LA10B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.92(m，12H)，1.25-1.40(m，56H)，1.56-1.71(m，12H)，1.87-1.89(m，2H)，2.27-2.32(m，8H)，2.77-2.80(m，4H)，2.97-2.99(m，2H)，3.79-3.80，(m，2H)，4.00-4.04(m，2H)，4.21-4.25(m，2H)，5.02-5.07(m，2H)。ESI-MSC55H106NO9 +[M+H]+计算值924.8，实测值924.9。
实施例8 氨基脂质E12LA8B6O3的合成
将实施例2中正己酸替换为正辛酸，按照相同的方法可制得氨基脂质E12LA8B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ5.07-5.04(m，2H)，4.23(dd，J1＝12.0Hz，J2＝3.6Hz，2H)，4.01(dd，J1＝12.0Hz，J2＝6.4Hz，2H)，3.80(t，J＝5.2Hz，2H)，2.86(t，J＝5.6Hz，2H)，2.66(t，J＝7.6Hz，4H)，2.33-2.28(m，8H)，1.83-1.80(m，2H)，1.69-1.56(m，12H)，1.38-1.25(m，56H)，0.89-0.85(m，12H).ESI-MS C55H106NO9 +[M+H]+计算值924.7862，实测值924.7891.
实施例9 化合物E12LA12B6O3的合成
步骤1：化合物E12LA12B6O3-2的合成
在25mL反应管中依次加入三氯化铁(16.2mg，0.1mmol，0.025eq)、吡啶(4mg，0.05mmol，0.0125eq)、月桂酸(800mg，4.0mmol，1.0eq)和1，2-环氧十二烷(884mg，4.8mmol，1.2eq)，室温下搅拌反应过夜，反应结束后，反应液浓缩，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝50∶1至10∶1)，得到化合物E12LA12B6O3-2(1.2g，86.0％)。ESI-MS C24H49O3 +[M+H]+计算值385.4，实测值385.4。
步骤2：化合物E12LA12B6O3-3的合成
在25mL反应管中依次加入E12LA12B6O3-2(1.2g，3.65mmol，1.0eq)、6-溴已酸(973mg，4.38mmol，1.2eq)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(2.8g，14.6mmol，4.0eq)、4-二甲氨基吡啶(45mg，0.365mmol，0.1eq)和N，N-二异丙基乙胺(2.83g，21.9mmol，6.0eq)和20mL二氯甲烷，室温下搅拌反应过夜，反应结束后，反应液浓缩，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至20∶1)，得到化合物E12LA12B6O3-3(1.65g，92.0％)。ESI-MS C30H58BrO4 +[M+H]+计算值561.4，实测值561.4。
步骤3：E12LA12B6O3的合成
在25mL的反应管中依次加入E12LA12B6O3-3(1.65g，3.36mmol，2.2eq)、碳酸钾(464mg，3.36mmol，2.2eq)、碘化钠(150mg，1.0mmol，0.67eq)，3-氨基丙醇(113mg，1.5mmol)，和10mL乙腈，70～80℃下搅拌反应过夜，反应结束后，经柱层析分离得到0.94g E12LA12B6O3(0.94g，68％)。1H NMR(400MHz，CDCl3)：δ0.87-0.91(m，12H)，1.24-1.38(m，68H)，1.42-1.58(m，4H)，1.55-1.69(m，14H)，2.29-2.34(m，8H)，2.38-2.42(m，4H)，2.61-2.64(m，2H)，3.78-3.81，(m，2H)，4.01-4.06(m，2H)，4.20-4.24(m，2H)，5.04-5.10(m，2H)。ESI-MS C63H122NO9 +[M+H]+计算值1036.9，实测值1037.0。
实施例10 氨基脂质E8CA5B6O3的合成
将实施例9中的1，2-环氧十二烷替换为1，2-环氧辛烷，月桂酸替换为2-戊基-庚酸，按照相同的方法可制得氨基脂质E8CA5B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.90(m，18H)，1.25-1.37(m，48H)，1.40-1.70(m，18H)，2.25-2.36(m，6H)，2.44-2.48(m，4H)，2.66-2.68(m，2H)，3.77-3.79，(m，2H)，4.00-4.05 (m，2H)，4.21-4.25(m，2H)，5.03-5.08(m，2H)。ESI-MS C55H106NO9 +[M+H]+计算值924.8，实测值924.8。
实施例11 氨基脂质E12LA6B609的合成
将实施例2中的3-氨基丙醇替换为正戊胺，按照相同的方法可制得氨基脂质E12LA6B6O9，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.90(m，15H)，1.24-1.36(m，54H)，1.53-1.74(m，16H)，2.27-2.33(m，8H)，2.83(s，6H)，3.98-4.03(m，2H)，4.21-4.25(m，2H)，5.02-5.07(m，2H)。ESI-MS C53H102NO8 +[M+H]+计算值880.8，实测值880.8
实施例12 氨基脂质E12LA6B6O10的合成
将实施例2中的3-氨基丙醇替换为反式对氨基环己醇，按照相同的方法可制得氨基脂质E12LA6B6O10，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.84-0.90(m，12H)，1.24-1.31(m，54H)，1.53-1.65(m，14H)，1.99-2.01(m，2H)，2.26-2.31(m，8H)，2.42-2.46(m，4H)，3.99-4.03(m，2H)，4.19-4.22(m，2H)，5.03-5.08(m，2H)。ESI-MS C54H102NO9 +[M+H]+计算值908.8，实测值908.7。
实施例13 氨基脂质E12LA6B6O12的合成
将实施例2中的3-氨基丙醇替换为3-二甲胺基丙胺，按照相同的方法可制得氨基脂质E12LA6B6O12，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.94(m，12H)，1.26-1.35(m，42H)，1.41-1.48(m，6H)，1.55-1.72(m，18H)，2.29-2.36(m，8H)，2.70-2.76(m，2H)，3.37-3.41(m，4H)，3.99-4.04(m，2H)，4.24-4.34(m，6H)，5.00-5.06(m，2H)。ESI-MS C53H103N2O8 +[M+H]+计算值895.8，实测值895.8。
实施例14 氨基脂质E12LA6B6O13的合成
将实施例2中的3-氨基丙醇替换为1-(2-氨乙基)哌啶，按照相同的方法可制得氨基脂质E12LA6B6O13，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.82-0.92(m，12H)，1.24-1.36(m，50H)，1.44-1.69(m，18H)，2.27-2.31(m，8H)，2.47-2.50(m，4H)，2.57-2.61(m，4H)，2.72-2.74(m，2H)，3.99-4.04(m，2H)，4.19-4.23(m，2H)，5.03-5.08(m，2H)。ESI-MS C55H105N2O8 +[M+H]+计算值921.8，实测值921.8。
实施例15 氨基脂质E12LA6B6O15的合成
将实施例2中的3-氨基丙醇替换为N-(2-氨乙基)-4-羟基哌啶，按照相同的方法可制得氨基脂质E12LA6B6O15，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.90(m，12H)，1.26-1.37(m，50H)，1.48-1.67(m，16H)，2.28-2.33(m，8H)，2.54-2.61(m，6H)，2.84-2.89(m，4H)，3.76(s，1H).4.00-4.07(m，2H)，4.20-4.24(m，2H)，5.04-5.09(m，2H)。ESI-MS C55H105N2O9 +[M+H]+计算值937.8，实测值937.9。
实施例16 氨基脂质E12LA12B6O30的合成
将实施例2中的3-氨基丙醇替换为β-丙氨酸，按照相同的方法可制得氨基脂质E12LA12B6O30，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.92(m，12H)，1.24-1.37(m，68H)，1.44-1.60(m，4H)，1.54-1.70(m，14H)，2.29-2.44(m，12H)，2.61-2.64(m，2H)，3.78-3.81，(m，2H)，4.01-4.05(m，2H)，4.21-4.24(m，2H)，5.02-5.08(m，2H)。ESI-MS C64H122NO10 +[M+H]+计算值1064.9，实测值1065.0。
实施例17 氨基脂质E12LA6B6O31的合成
将实施例2中的3-氨基丙醇替换为4-氨基丁腈，按照相同的方法可制得氨基脂质E12LA12B6O31，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.25-1.39(m，68H)，1.42-1.60(m，4H)，1.58-1.72(m，12H)，1.78-1.82(m，2H)，2.31-2.42(m，12H)，2.60-2.64(m，2H)，3.79-3.82，(m，2H)，4.00-4.04(m，2H)，4.18-4.22(m，2H)，5.02-5.09(m，2H)。ESI-MS C64H121N2O8 +[M+H]+计算值1045.9，实测值1045.9。
实施例18 氨基脂质K3LA6B6O3的合成
步骤1：化合物K3LA6B6O3-2的合成
往250mL圆底烧瓶中加入咪唑(6.54g，96mmol，1.2eq)，抽换气使圆底烧瓶内充满氮气，依次加入二氯甲烷(60mL)和N，N-二甲基甲酰胺(30mL)，1，3-丙二醇(6.09g，80mmol，1.0eq)，将混合物在0℃搅拌10分钟，将叔丁基二甲基氯硅烷(12.06g，80mmol，1.0eq)溶解在二氯甲烷(60mL)中滴加到混合物中，将所得混合物室温搅拌4小时。将反应混合物水洗3次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至10∶1)，得到化合物K3LA6B6O3-2(10.96g，72％)，ESI-MS C9H23O2Si+[M+H]+计算值191.1，实测值191.1。
步骤2：化合物K3LA6B6O3-3的合成
氯铬酸吡啶鎓盐(6.09g，80mmol，1.0eq)和化合物K3LA6B6O3-2(10.96g，57.6mmol，1.0eq)的二氯甲烷(100mL)溶液在室温下搅拌5小时。将反应混合物过滤，滤液浓缩，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至20∶1)，得到化合物K3LA6B6O3-3(8.69g，80％)，ESI-MS C9H21O2Si+[M+H]+计算值189.1，实测值189.1。
步骤3：化合物K3LA6B6O3-4的合成
取500mL圆底烧瓶，抽换气使圆底烧瓶内充满氮气，加入化合物K3LA6B6O3-3(8.69g，46.14mmol，1.0eq)，无水四氢呋喃(150mL)，将混合物在-20℃下搅拌10分钟，将70mL癸基溴化镁溶液(1mol/L无水四氢呋喃溶液，1.5eq)滴加到混合物中，混合物在-20℃至0℃搅拌3小时，加入50mL饱和氯化铵溶液淬灭，用乙酸乙酯萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至 20∶1)，得到化合物K3LA6B6O3-4(9.93g，65％)，ESI-MS C19H43O2Si+[M+H]+计算值331.3，实测值331.4。
步骤4：化合物K3LA6B6O3-5的合成
K3LA6B6O3-4(9.93g，30mmol)，6-溴己酸(7.02g，36mmol，1.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(11.50g，60mmol，2.0eq)，N，N-二异丙基乙胺(15.51g，120mmol，4.0eq)，4-二甲氨基吡啶(0.37g，3mmol，0.1eq)的二氯甲烷(100mL)溶液，室温搅拌过夜。TLC显示反应完全，反应液用二氯甲烷萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至20∶1)，得到化合物K3LA6B6O3-5(12.79g，84％)，ESI-MS C25H52BrO3Si+[M+H]+计算值507.3，实测值507.2。
步骤5：化合物K3LA6B6O3-6的合成
K3LA6B6O3-5(12.79g，25.2mmol，1.0eq)，氟化铵(9.33g，252mmol，10.0eq)，甲醇(75mL)，65℃回流搅拌3小时。TLC显示反应完全，反应液用乙酸乙酯萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝50∶1至5∶1)，得到化合物K3LA6B6O3-6(8.72g，88％)，ESI-MS C19H38BrO3 +[M+H]+计算值393.2，实测值393.2。
步骤6：化合物K3LA6B6O3-7的合成
K3LA6B6O3-6(8.72g，22.2mmol)，己酸(3.09g，26.64mmol，1.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(8.51g，44.4mmol，2.0eq)，N，N-二异丙基乙胺(11.48g，88.8mmol，4.0eq)，4-二甲氨基吡啶(0.27g，2.2mmol，0.1eq)的二氯甲烷(50mL)溶液，室温搅拌过夜。
TLC显示反应完全，反应液用二氯甲烷萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至20∶1)，得到化合物K3LA6B6O3-7(7.86g，72％)，ESI-MS C25H48BrO4 +[M+H]+计算值491.3，实测值491.2。
步骤7：化合物K3LA6B6O3的合成
化合物K3LA6B6O3-7(7.86g，15.99mmol，3.0eq)，3-氨基丙醇(0.40g，5.33mmol，3.0eq)，碳酸钾(1.47g，10.66mmol，2.0eq)，碘化钠(0.80g，5.33mmol，1.0eq)的乙腈(10mL)溶液，75℃搅拌过夜，反应浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝100∶1至20∶1)，得到化合物K3LA6B6O3-7(3.58g，75％)，1H NMR(400MHz，CDCl3)：δ0.85-0.90(m，12H)，1.24-1.36(m，48H)，1.56-1.73(m，18H)，2.25-2.29(m，8H)，2.48-2.52(m，4H)，2.70-2.72(m，2H)，3.76-3.79(m，2H)，4.03-4.11(m，4H)，4.93-4.96(m，2H)。ESI-MS C53H102NO9 +[M+H]+计算值896.8，实测值896.9。
实施例19 氨基脂质K4LA6B6O3的合成
将实施例18中的1，3-丙二醇替换为1，4-丁二醇，按照相同的方法可制得氨基脂质K4LA6B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.91(m，12H)，1.26-1.41(m，48H)，1.56-1.72(m，18H)，2.27-2.33(m，8H)，2.81-2.84(m，4H)，3.00-3.04(m，2H)，3.81-3.84(m，2H)，4.04-4.07(m，4H)，4.87-4.90(m，2H)。ESI-MS C55H106NO9 +[M+H]+计算值924.8，实测值924.8。
实施例20 氨基脂质K5LA6B6O3的合成
将实施例18中的1，3-丙二醇替换为1，5-戊二醇，按照相同的方法可制得氨基脂质K5LA6B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.26-1.41(m，54H)，1.50-1.67(m，18H)，2.27-2.34(m，8H)，2.91-2.96(m，4H)，3.10-3.14(m，2H)，3.83-3.85(m，2H)，4.03-4.06(m，4H)，4.85-4.89(m，2H)。ESI-MS C57H110NO9 +[M+H]+计算值952.8，实测值952.8。
实施例21 氨基脂质E12LA6B6O3A2的合成
步骤1：化合物E12LA6B6O3的合成
按照实施例2的方法，合成E12LA6B6O3。
步骤2：化合物E12LA6B6O3A2的合成
E12LA6B6O3(0.87g，1mmol，1.0eq)，N，N′-二环己基碳二亚胺(0.41g，1mmol，2.0eq)，DMAP(0.12g，0.1mmol，0.1eq)，3-(4-苯基-哌嗪-1-基)-丙酸(0.26g，1.5mmol，1.5eq)的二氯甲烷溶液，室温搅拌4小时，TLC显示反应完全，反应液过滤，滤液浓缩后，使用快速柱层 析系统纯化(二氯甲烷∶甲醇＝100∶1至10∶1)，得到化合物E12LA6B6O3A2(0.80g，78％)，1H NMR(400MHz，CDCl3)：δ0.85-0.90(m，12H)，1.25-1.32(m，48H)，1.36-1.49(m，4H)，1.53-1.66(m，12H)，2.27-2.50(m，25H)，2.67-2.71(m，2H)，4.00-4.04(m，2H)，4.08-4.11(m，2H)，4.19-4.23(m，2H)，5.04-5.09(m，2H)。ESI-MS C59H112N3O10 +[M+H]+计算值1022.8，实测值1022.9。
实施例22 氨基脂质E12LA6B6O3A3的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为4-(4-甲基-1-哌嗪基)丁酸，按照相同的方法可制得氨基脂质E12LA6B6O3A3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.90(m，12H)，1.25-1.45(m，50H)，1.55-1.66(m，12H)，1.69-1.83(m，4H)，2.27-2.47(m，27H)，4.00-4.04(m，2H)，4.07-4.10(m，2H)，4.19-4.23(m，2H)，5.04-5.10(m，2H)。ESI-MS C60H114N3O10 +[M+H]+计算值1036.8，实测值1036.8。
实施例23 氨基脂质E12LA6B6O3A4的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为3-(二甲氨基)丙酸，按照相同的方法可制得氨基脂质E12LA6B6O3A4，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.84-0.89(m，12H)，1.24-1.35(m，50H)，1.41-1.48(m，6H)，1.52-1.65(m，12H)，2.23(s，6H)，2.26-2.30(m，8H)，2.35-2.39(m，4H)，2.44-2.48(m，4H)，2.58-2.62(m，2H)，3.99-4.03(m，2H)，4.08-4.11(m，2H)，4.18-4.22(m，2H)，5.03-5.09(m，2H)。ESI-MS C56H107N2O10 +[M+H]+计算值967.8，实测值967.8。
实施例24 氨基脂质E12LA6B6O3A5的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为5-(二甲氨基)戊酸，按照相同的方法可制得氨基脂质E12LA6B6O3A5，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.84-0.89(m，12H)，1.24-1.35(m，48H)，1.41-1.75(m，18H)，2.21(s，6H)，2.22-2.30(m，12H)，2.35-2.37(m，4H)，2.42-2.45(m，2H)，3.98-4.03(m，2H)，4.05-4.09(m，2H)，4.18-4.22(m，2H)，5.03-5.09(m，2H)。ESI-MS C58H111N2O10 +[M+H]+计算值995.8，实测值995.9。
实施例25 氨基脂质E12LA6B6O3A6的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为7-(二甲氨基)庚酸，按照相同的方法可制得氨基脂质E12LA6B6O3A3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.84-0.91(m，12H)，1.23-1.37(m，50H)，1.39-1.49(m，6H)，1.52-1.64(m，14H)，2.21(s，6H)，2.25-2.30(m，12H)，2.33-2.37(m，4H)，2.42-2.45(m，2H)，3.98-4.03(m，2H)，4.05-4.08(m，2H)，4.18-4.22(m，2H)，5.03-5.08(m，2H)。ESI-MS C60H115N2O10 +[M+H]+计算值1023.8，实测值1023.8。
实施例26 氨基脂质E8LA6B6O3A6的合成
将实施例21中的E12LA6B6O3替换为E8LA6B6O3，按照相同的方法可制得氨基脂质E8LA6B6O3A6，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.26-1.43(m，36H)，1.39-1.49(m，6H)，1.56-1.79(m，12H)，2.23-2.48(m，24H)，4.00-4.11(m，4H)，4.20-4.24(m，2H)，5.07-5.11(m，2H)。ESI-MS C52H99N2O10 +[M+H]+计算值911.7，实测值911.9。
实施例27 氨基脂质E12LA6B6O3A7的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为1-甲基哌啶-4-甲酸，按照相同的方法可制得氨基脂质E12LA6B6O3A7，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.87-0.92(m，12H)，1.26-1.41(m，48H)，1.57-1.83(m，20H)，2.24-2.40(m，16H)，2.45-2.48(m，2H)，2.80-2.84(m，2H)，4.01-4.06(m，2H)，4.10-4.13(m，2H)，4.21-4.25(m，2H)，5.06-5.11(m，2H)。ESI-MS C58H109N2O10 +[M+H]+计算值993.8，实测值993.7。
实施例28 氨基脂质E12LA6B6O3A8的合成
将实施例21中的3-(4-苯基-哌嗪-1-基)-丙酸替换为1-哌啶丙酸，按照相同的方法可制得氨基脂质E12LA6B6O3A8，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.85-0.92(m，12H)，1.24-1.36(m，44H)，1.40-1.45(m，6H)，1.54-1.69(m，12H)，2.27-2.30(m，8H)，2.34-2.52(m，12H)，2.63-2.67(m，2H)，4.00-4.04(m，2H)，4.07-4.10(m，8H)，4.19-4.23(m，2H)，5.03-5.09(m，2H)。ESI-MS C59H111N2O10 +[M+H]+计算值1007.8，实测值1007.9。
实施例29 氨基脂质E12LA12B6O30A9的合成
步骤1：化合物E12LA12B6O30A9-1的合成
按照实施例16的方法，合成E12LA12B6O30A9-1。
步骤2：化合物E12LA12B6O3A9的合成
E12LA12B6O30A9-1(1.06g，1mmol，1.0eq)，N，N′-二环己基碳二亚胺(0.41g，2mmol，2.0eq)，DMAP(0.12g，0.1mmol，0.1eq)，3-二甲氨基丙醇(0.16g，1.5mmol，1.5eq)的二氯甲烷溶液，室温搅拌4小时，TLC显示反应完全，反应液过滤，滤液浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝50∶1至10∶1)，得到化合物E12LA12B6O3A9-1(0.92g，80％)，1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.24-1.42(m，68H)，1.45-1.72(m，20H)，1.84-1.86(m，2H)，2.26(s，6H)，
2.30-2.42(m，12H)，2.60-2.63(m，2H)，3.78-3.81，(m，2H)，4.01-4.15(m，4H)，4.22-4.26(m，2H)，5.06-5.12(m，2H)。ESI-MS C69H133N2O10 +[M+H]+计算值1050.0，实测值1050.3。
实施例30 氨基脂质E12LA12B6O30A10的合成
将实施例29中的3-二甲氨基丙醇替换为3-二甲氨基丙胺，按照相同的方法可制得氨基脂质E12LA12B6O30A10，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.24-1.42(m，68H)，1.45-1.84(m，22H)，2.28(s，6H)，2.32-2.44(m，12H)，2.60-2.63(m，2H)，3.50-3.54，(m，2H)，3.78-3.82(m，2H)，4.00-4.04(m，2H)，4.22-4.26(m，2H)，5.06-5.12(m，2H)。ESI-MS C69H134N3O9 +[M+H]+计算值1149.0，实测值1149.0。
实施例31 氨基脂质E12LA12B6O3A11的合成
步骤1：化合物E12LA12B6O3A11-1的合成
按照实施例9的方法，合成E12LA6B6O3A11-1。
步骤2：化合物E12LA6B6O3A11-1-2的合成
取50mL圆底烧瓶，抽换气使圆底烧瓶内充满氮气，加入化合物E12LA6B6O3A11-1(5.18g，5mmol，1.0eq)，无水二氯甲烷(20mL)，将混合物在0℃下搅拌10分钟，滴加三氟甲磺酸酐(2.12g，7.5mmol，1.5eq)，混合物在0℃下搅拌10分钟，加入2，6二甲基吡啶(0.80g，7.5mmol，1.5eq)，反应室温搅拌过夜。反应液用水和饱和氯化钠溶液各洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝50∶1至20∶1)，得到化合物E12LA6B6O3A11-2(4.38g，75％)，ESI-MS C64H121F3NO11S+[M+H]+计算值1168.9，实测值1169.0。
步骤3：化合物E12LA6B6O3A11-3的合成
取100mL耐压瓶，加入E12LA6B6O3A11-2(4.38g，3.75mmol，1.0eq)，20mL四氢呋喃，5mL氨水(质量分数25％的水溶液)，混合物在100℃搅拌过夜，反应液用水和饱和氯化钠溶液各洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝20∶1至5∶1)，得到化合物E12LA6B6O3A11-3(1.71g，44％)，ESI-MS C63H123N2O8 +[M+H]+计算值1035.9，实测值1035.9。
步骤4：化合物E12LA6B6O3A11的合成
E12LA6B6O3A11-3(1.71g，1.65mmol，1.0eq)，N，N′-二环己基碳二亚胺(0.68g，3.3mmol，2.0eq)，DMAP(0.20g，0.16mmol，0.1eq)，4-二甲氨基丁酸(0.32g，2.48mmol，1.5eq)的二氯甲烷溶液，室温搅拌4小时，TLC显示反应完全，反应液过滤，滤液浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝50∶1至10∶1)，得到化合物E12LA6B6O3A11(1.48g，78％)，1H NMR(400MHz，CDCl3)：δ0.85-0.91(m，12H)，1.24-1.41(m，68H)，1.45-1.82(m，22H)，2.27(s，6H)，2.32-2.42(m，12H)，2.61-2.63(m，2H)，3.36-3.42，(m，2H)，3.77-3.79(m，2H)，4.01-4.04(m，2H)，4.23-4.26(m，2H)，5.04-5.10(m，2H)。ESI-MS C69H134N3O9 +[M+H]+计算值1049.0，实测值1049.1。
实施例32 氨基脂质E12N1LA6B6O3的合成
步骤1：化合物E12N1LA6B6O3-2的合成
1，2环氧十二烷(3.69g，20mmol，1.0eq)，20mL氨水(质量分数25％的水溶液)，乙醇10mL，水10mL，混合物在60℃搅拌16小时。反应液旋干，加入3mL乙醇和30mL正庚烷60℃打浆4小时，过滤，滤饼50℃烘干10小时，得到化合物E12N1LA6B6O3-2(3.46g，86％)，ESI-MS C12H28NO+[M+H]+计算值202.2，实测值202.2。
步骤2：化合物E12N1LA6B6O3-3的合成
化合物E12N1LA6B6O3-2(3.46g，17.2mmol，1.5eq)，己酸(1.34g，11.5mmol，1.0eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.30g，17.2mmol，1.5eq)，N，N-二异丙基乙胺(2.22g，17.2mmol，1.5eq)，4-二甲氨基吡啶(2.10g，17.2mmol，1.5eq)的二氯甲烷(40mL)溶液，室温搅拌过夜。反应液用二氯甲烷萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝10∶1至3∶1)，得到化合物E12N1LA6B6O3-3(1.10g，32％)，ESI-MS C18H38NO2 +[M+H]+计算值300.3，实测值300.2。
步骤3：化合物E12N1LA6B6O3-4的合成
化合物E12N1LA6B6O3-3(1.10g，3.68mmol，1.0eq)，己酸(0.51g，4.42mmol，1.2eq)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1.41g，7.36mmol，2.0eq)，N，N-二异丙基乙胺(1.90g，14.2mmol，4.0eq)，4-二甲氨基吡啶(0.04g，0.37mmol，0.1eq)的二氯甲烷(20mL)溶液，室温搅拌过夜。TLC显示反应完全，反应液用二氯甲烷萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至20∶1)，得到化合物E12N1LA6B6O3-4(1.42g，81％)，ESI-MS C24H47BrNO3 +[M+H]+计算值476.3，实测值476.3。
步骤4：化合物E12N1LA6B6O3的合成
化合物E12N1LA6B6O3-4(1.42g，2.99mmol，3.0eq)，3-氨基丙醇(0.075g，1.00mmol， 1.0eq)，碳酸钾(0.28g，2.00mmol，2.0eq)，碘化钠(0.15g，1.00mmol，1.0eq)的乙腈(10mL)溶液，75℃搅拌过夜，反应浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝100∶1至20∶1)，得到化合物E12N1LA6B6O3(0.68g，78％)。1H NMR(400MHz，CDCl3)：δ0.86-0.93(m，12H)，1.25-1.37(m，48H)，1.41-1.69(m，16H)，2.14-2.18(m，8H)，2.30-2.43(m，6H)，3.34-3.48(m，4H)，4.90-4.96(m，2H)。ESI-MS C51H100N3O7 +[M+H]+计算值866.8，实测值866.7。
实施例33氨基脂质E12N1LA8B6O3的合成
将实施例32中的己酸替换为辛酸，按照相同的方法可制得氨基脂质E12N1LA8B6O3，其结构式如下：
1H NMR(400MHz，CDCl3)：δ0.86-0.93(m，12H)，1.25-1.37(m，48H)，1.42-1.70(m，18H)，2.14-2.18(m，8H)，2.31-2.43(m，6H)，3.35-3.48(m，4H)，4.90-4.96(m，2H)。ESI-MS C55H108N3O7 +[M+H]+计算值922.8，实测值923.0。
实施例34 氨基脂质E12N2LA6B6O3的合成
步骤1：化合物E12N2LA6B6O3-1的合成
按照实施例32的步骤1和步骤2，合成E12N2LA6B6O3-1。
步骤2：化合物E12N2LA6B6O3-2的合成
取50mL圆底烧瓶，抽换气使圆底烧瓶内充满氮气，加入化合物E12N2LA6B6O3-1-1(1.50g，5mmol，1.0eq)，无水二氯甲烷(20mL)，将混合物在0℃下搅拌 10分钟，滴加三氟甲磺酸酐(2.12g，7.5mmol，1.5eq)，混合物在0℃下搅拌10分钟，加入2，6二甲基吡啶(0.80g，7.5mmol，1.5eq)，反应室温搅拌过夜。反应液用水和饱和氯化钠溶液各洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝50∶1至20∶1)，得到化合物E12N2LA6B6O3-2(1.51g，70％)，ESI-MS C19H37F3NO4S+[M+H]+计算值432.2，实测值432.2。
步骤3：化合物E12N2LA6B6O3-3的合成
取100mL耐压瓶，加入E12N2LA6B6O3-2(1.51g，3.50mmol，1.0eq)，20mL四氢呋喃，5mL氨水(质量分数25％的水溶液)，混合物在100℃搅拌过夜，反应液用水和饱和氯化钠溶液各洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝20∶1至5∶1)，得到化合物E12N2LA6B6O3-3(0.47g，45％)，ESI-MS C18H39N2O+[M+H]+计算值299.3，实测值299.3。
步骤4：化合物E12N2LA6B6O3-4的合成
化合物E12N2LA6B6O3-3(0.74g，1.58mmol，3.0eq)，N，N′-二环己基碳二亚胺(0.65g，3.15mmol，2.0eq)，DMAP(0.20g，0.16mmol，0.1eq)，N，N-二异丙基乙胺(0.82g，6.32mmol，4.0eq)，6-溴己酸(0.46g，2.37mmol，1.5eq)的二氯甲烷溶液，室温搅拌4小时，TLC显示反应完全，反应液过滤，滤液浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝50∶1至1∶1)，得到化合物E12N2LA6B6O3-4(1.48g，88％)，ESI-MS C24H48BrN2O2 +[M+H]+计算值475.3，实测值475.3。
步骤5：化合物E12N2LA6B6O3的合成
化合物E12N2LA6B6O3-4(1.48g，1.39mmol，3.0eq)，3-氨基丙醇(0.035g，0.46mmol，1.0eq)，碳酸钾(0.13g，0.92mmol，2.0eq)，碘化钠(0.069g，0.46mmol，1.0eq)的乙腈(10mL)溶液，75℃搅拌过夜，反应浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝100∶1至20∶1)，得到化合物E12N2LA6B6O3(0.97g，81％)。1HNMR(400MHz，CDCl3)：δ0.86-0.92(m，12H)，1.25-1.37(m，42H)，1.41-1.69(m，18H)，2.06-2.18(m，12H)，2.30-2.42(m，4H)，3.32-3.48(m，4H)，3.70-3.74(m，2H)，4.90-4.96(m，2H)。ESI-MS C51H102N5O5 +[M+H]+计算值864.8，实测值864.7。
实施例35 氨基脂质E12S1LA6B6O3的合成
步骤1：化合物E12S1LA6B6O3-2的合成
在25mL反应管中依次加入三氯化铁(16.2mg，0.1mmol，0.025eq)、吡啶(4mg，0.05mmol，0.0125eq)、己酸(464mg，4.0mmol，1.0eq)和1，2-环氧十二烷(884mg，4.8mmol，1.2eq)，室温下搅拌反应过夜，反应结束后，反应液浓缩，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝50∶1至10∶1)，得到化合物E12S1LA6B6O3-2(1.2g，90.0％)。ESI-MS C18H37O3 +[M+H]+计算值301.3，实测值301.3。
步骤2：化合物E12S1LA6B6O3-3的合成
在25mL反应管中加入E12S1LA6B6O3-2(1.2g，3.6mmol，1.0eq)，三乙胺(0.36g，3.6mmol，1.0eq)，甲磺酰氯(0.49g，4.32mmol，1.2eq)，二氯甲烷10mL，混合物室温搅拌2小时，反应液用水和饱和氯化钠溶液各洗涤一次，无水硫酸钠干燥，过滤，滤液浓缩，加入10mLN，N-二甲基甲酰胺和硫氢化钠(0.24g，4.32mmol，1.2eq)，混合物45℃搅拌过夜，反应液用乙酸乙酯萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至10∶1)，得到化合物E12S1LA6B6O3-3(0.69g，61％)，ESI-MS C18H37O28+[M+H]+计算值317.3，实测值317.2。
步骤3：化合物E12S1LA6B6O3-4的合成
在25mL反应管中加入E12S1LA6B6O3-3(0.69g，2.2mmol，1.0eq)，二溴海因(0.13g，0.44mmol，0.2eq)，3-溴丙烷-1-硫醇(0.41g，2.64mmol，1.2eq)，二氯甲烷10mL，混合物室温搅拌1小时，反应液用乙酸乙酯萃取三次，饱和食盐水洗涤一次，使用无水硫酸钠干燥，浓缩后，使用快速柱层析系统纯化(正庚烷∶乙酸乙酯＝100∶1至10∶1)，得到化合物E12S1LA6B6O3-4(0.70g，68％)，ESI-MS C21H42BrO2S2 +[M+H]+计算值469.2，实测值469.2。
步骤4：化合物E12S1LA6B6O3的合成
化合物E12S1LA6B6O3-4(0.70g，1.50mmol，3.0eq)，3-氨基丙醇(0.038g，0.50mmol， 1.0eq)，碳酸钾(0.14g，1.00mmol，2.0eq)，碘化钠(0.075g，0.50mmol，1.0eq)的乙腈(10mL)溶液，75℃搅拌过夜，反应浓缩后，使用快速柱层析系统纯化(二氯甲烷∶甲醇＝100∶1至20∶1)，得到化合物E12S1LA6B6O3(1.09g，80％)。1H NMR(400MHz，CDCl3)：δ0.86-0.91(m，12H)，1.25-1.35(m，40H)，1.41-1.54(m，12H)，1.59-1.67(m，10H)，2.09-2.18(m，10H)，2.34-2.40(m，4H)，2.50-2.54(m，2H)，3.64-3.72(m，2H)，4.00-4.06(m，4H)。ESI-MS C49H98NO5S4 +[M+H]+计算值908.6，实测值908.7。
脂质纳米颗粒的制备
实施例36 脂质纳米颗粒的制备
将本发明的氨基脂质化合物(或DLin-MC3或SM-102(均购自艾韦拓(上海)医药科技有限公司))、磷脂脂质(DSPC)、胆固醇、聚乙二醇化脂质(DMG-PEG2000)按47.5∶10∶41∶1.5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)的柠檬酸缓冲液(50mM，pH＝4.0)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40∶1。
对所得脂质纳米颗粒进行表征，结果见表1。
其中，所制备的脂质纳米颗粒的粒径和PDI(多分散指数)通过Nano-ZSZEN3600(Malvern)测定。取脂质纳米颗粒(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1使用代表性氨基脂质化合物制备的LNP的表征数据

由以上结果可知，本发明提供的脂质纳米颗粒包封率都高于DLin-MC3，其中绝大多数的脂质纳米颗粒包封率都高于SM-102。
实施例37 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
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％CO2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％CO2浓度的培养箱中12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-GloTMLuciferase，1min后用多功能酶标仪(Biorek SynergyH1)进行检测。由代表性氨基脂质化合物组成的脂质纳米颗粒(LNP)在BMDC上转染Luc mRNA的表达强度见表2，以DLin-MC3和SM0102作为对照。
表2代表性氨基脂质化合物在BMDC上的转染的表达强度

可以看出，本发明提供的氨基脂质表达强度显著优于DLin-MC3，且其中一些氨基脂质表达强度优于SM-102。
实施例38 脂质纳米颗粒的萤光素酶mRNA体内递送性能评价
脂质纳米颗粒的制备：同实施例36。
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5只C57BL/6小鼠，按0.5mg/kg mRNA的用量，使用肌肉注射的给药方式注射脂质纳米颗粒溶液。12小时后，往每只小鼠的腹腔内注射200μL 10mg/mL的D-萤光素钾盐，5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组成的LNP肌注给药方式递送的Luc mRNA的表达强度见表3，以DLin-MC3和SM-102作为对照。
表3代表性氨基脂质组成的LNP肌注给药递送的Luc mRNA的表达强度
实施例39 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：将本发明的氨基脂质化合物与DSPC、胆固醇、DMG-PEG2000按摩尔比47.5∶10∶41∶1.5的比例混合溶解在无水乙醇中。使用微流控制备系统使所得的乙醇溶液和溶解有OVA-mRNA的柠檬酸缓冲液(50mM，pH＝4.0)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40∶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(1.5×105)皮下注射到小鼠大腿外侧。当肿瘤大小长到50mm3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×宽度。当肿瘤体积达到1500mm3时对小鼠实施安乐死。E12LA6B6O3组和E10LA6B6O3组的肿瘤生长速度显著慢于MC3组(如图2所示)，且分别有100％(E12LA6B6O3组)和80％(E10LA6B6O3组)的小鼠达到了完全缓解的效果，显著优于MC3组(如图3所示)。
实施例40不同磷脂脂质比例组成LNP的荧光素酶mRNA体内递送性能评价
脂质纳米颗粒的制备：保持可离子化脂质(E12LA6B6O3)含量为42.5％、聚乙二醇化脂质DMG-PEG2000含量为1.5％，调整磷脂脂质比例为15、12.5、10、7.5、5.0、2.5、0，将调整的磷脂脂质摩尔比增加或减少至胆固醇比例上，验证其对脂质纳米颗粒质控评价及荧光素酶mRNA体内递送性能影响。各组编号B1-B7，具体比例见表4，同时以ALC-0315和SM-102作为阳性对照。
表4：不同磷脂脂质下的组分比例
将E12LA6B6O3、磷脂脂质(DSPC)、胆固醇、聚乙二醇化脂质DMG-PEG2000按表4的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)的柠檬酸缓冲液(50mM，pH＝4.0)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0.22微米的微孔滤膜进行过滤。氨基脂质化合物与萤光素酶mRNA(Luc mRNA)的碱基摩尔比比约为6.5∶1。在马尔文粒度仪及酶标仪上检测脂质纳米颗粒的质控，同时加入SM-102和ALC-0315(均购自艾韦拓(上海)医药科技有限公司)作为对照组。
动物准备：选取体重约20g的6周龄的雌性Balb/C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3只Balb/C小鼠，按0.1mg/kg mRNA的用量，使用尾静脉注射的给药方式注射脂质纳米颗粒溶液。6小时后，往每只小鼠的腹腔内注射150μL 15mg/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 Spectrum)下，观察每只小鼠总的荧光强度并拍照记录，不同DSPC含量组成的LNP尾静脉注射给药方式递送的Luc mRNA的表达强度见表5。
表5：不同比例磷脂脂质含量组成的LNP质控数据及表达强度
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (32)

  1. 一种氨基脂质，具有通式(I)所示的结构，或其异构体、药学上可接受的盐、前药或溶剂合物：
    其中，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、-NR′R″或至少含有一个杂原子的环状烷基结构；所述环状烷基结构中可被取代的碳原子或杂原子未被取代或被一个或多个羟基、C1-C4烷基、C2-C4烯基、C3-C8环烷基或C3-C8环烯基取代；
    M1、M2、M3、M4彼此相同或不同，并且各自独立地选自C1-C24亚烷基、C3-C24亚环烷基、C2-C24亚烯基或C3-C24亚环烯基；
    R1、R2彼此相同或不同，并且各自独立地选自H、C1-C24烷基、C3-C24环烷基、C2-C24烯基或C3-C24环烯基；
    L1、L2、L3、L4彼此相同或不同，并且独立地选自-C(＝O)O-、-OC(＝O)-、-C(＝O)S-、-SC(＝O)-、-C(＝O)NR-、-NRC(＝O)-、-S(＝O)-、-OS(＝O)2-、-S(＝O)2O-、-O-、-S-或-S-S-；
    R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C10烷基、C3-C10环烷基、C3-C10烯基、C3-C10环烯基、末端连接叔胺基的C1-C10烷基、末端连接叔胺基的C3-C10环烷基、末端连接叔胺基的C3-C10烯基、或至少含有一个杂原子的环状烷基，所述环状烷基未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷基、C3-C8环烯基取代；
    M5各自独立地选自单键、C1-C16亚烷基、C2-C16亚烯基、C3-C8亚环烷基或C3-C8亚环烯基。
  2. 根据权利要求1所述的氨基脂质，其特征在于，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、-NR′R″或至少含有一个杂原子的环状烷基结构；所述环状烷基结构可被取代的碳原子或杂原子未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷基或C3-C8环烯基取代；R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C10烷基、C3-C10环烷基、C3-C10烯基或C3-C10环烯基、或至少含有一个杂原子的环状烷基，所述环状烷基未被取代或被一个或多个C1-C4烷基、C2-C4烯基、C3-C8环烷 基、C3-C8环烯基取代。
  3. 根据权利要求1或2所述的氨基脂质，其特征在于，通式(I)中，G选自H、OR、CN、-C(＝O)OR′、-OC(＝O)R′、-C(＝O)NR′R″、-NR′C(＝O)R″、-NR′R″或至少含有一个杂原子的环状烷基结构；其中杂原子为O或N；所述环状烷基未被取代或被一个或多个C1-C4烷基、C3-C8环烷基或羟基取代；R、R′、R″彼此相同或不同，并且各自独立地选自H、C1-C8烷基、C3-C8环烷基、C3-C8烯基或C3-C8环烯基，末端连接叔胺基的C1-C10烷基、末端连接叔胺基的C3-C10环烷基或至少含有一个杂原子的环状烷基，所述环状烷基未被取代或被一个或多个C1-C4烷基、C3-C8环烷基取代。
  4. 根据权利要求3所述的氨基脂质，其特征在于，通式(I)中，G选自H、OH或NR′R″、其中R′、R″彼此相同或不同，并且各自独立地选自H或C1-C4烷基；
    或者，G选自取代或未被取代的氧杂五元环烷基、氮杂五元环烷基、氮杂六元环烷基、双氮杂六元环烷基或氮杂氧杂六元环烷基。
  5. 根据权利要求4所述的氨基脂质，其特征在于，通式(I)中，当G选自取代的双氮杂六元环烷基时，取代基位置为不与M5相连的氮原子。
  6. 根据权利要求3所述的氨基脂质，其特征在于，通式(I)中，M5选自单键、C2-C16亚烷基、C2-C16亚烯基、C4-C8亚环烷基或C3-C8亚环烯基。
  7. 根据权利要求6所述的氨基脂质，其特征在于，通式(I)中，M5选自单键、C2-C16亚烷基或C4-C6亚环烷基。
  8. 根据权利要求7所述的氨基脂质，其特征在于，通式(I)中，M5和G相连形成的选自A1～A38中的一种：

  9. 根据权利要求7所述的氨基脂质，其特征在于，通式(I)中，M5和G相连形成的选自A39～A52中的一种：

  10. 根据权利要求8所述的氨基脂质，其特征在于，所述选自A1-A18、A22-A24、A28-A38中的一种。
  11. 根据权利要求10所述的氨基脂质，其特征在于，所述选自A15、A16、A17、A23、A29、A30、A33、A37、A38、A42中的一种。
  12. 根据权利要求1-11任一项所述的氨基脂质，其特征在于，通式(I)中，L1、L2、L3、L4彼此相同或不同，并且独立地选自-C(＝O)O-、-OC(＝O)-、-C(＝O)NR-或-NRC(＝O)-或-S-S-；
    优选的，L1与L4相同，且为-C(＝O)O-或-OC(＝O)-。
  13. 根据权利要求12所述的氨基脂质，其特征在于，当L1、L2、L3、L4独立地选自-C(＝O)NR-或-NRC(＝O)-时，其中R独立地选自H或C1-C10烷基。
  14. 根据权利要求1-13任一项所述的氨基脂质，其特征在于，通式(I)中，M1、M2、M3、M4彼此相同或不同，并且M1和M4各自独立地选自含有支链的C4-C22亚烷基、含有支链的C4-C22亚环烷基、含有支链的C4-C22亚烯基或含有支链的C4-C22亚环烯基，M2和M3各自独立地选自C4-C22亚烷基、C4-C22亚环烷基、C4-C22亚烯基或C4-C22亚环烯基。
  15. 根据权利要求14所述的氨基脂质，其特征在于，通式(I)中，M2与M3相同，且为C4-C22亚烷基；
    和/或，M1与M4相同，且为含有支链的C4-C22亚烷基。
  16. 根据权利要求2-15任一项所述的氨基脂质，其特征在于，通式(I)中，R1、R2彼此相同或不同，并且各自独立地选自C4-C22烷基或C4-C22烯基。
  17. 根据权利要求16所述的氨基脂质，其特征在于，通式(I)中，R1-L1-M1-L2-M2-片段为R1-C(＝O)O-M1-OC(＝O)-M2-，R2-L4-M4-L3-M3-片段为R2-C(＝O)O-M4-OC(＝O)-M3-。
  18. 根据权利要求17所述的氨基脂质，其特征在于，通式(I)结构选自以下结构中的一种：



  19. 权利要求1-18任一项所述的氨基脂质的制备方法，其特征在于，包括以下步骤：
    S1：环氧化合物和羧酸发生开环反应，制备中间体1 R1-L1-M1-OH；
    S2：中间体1和羧酸化合物原料在缩合剂下发生缩合反应，制备中间体2 R1-L1-M1-L2-M2-离去基团；
    S3：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物；
    或S1’：二醇经TBS保护后，发生氧化反应，氧化产物与格氏试剂发生加成反应，制备中间体1’HO-M1-OTBS；
    S2’：中间体1’和羧酸化合物原料在缩合剂下发生缩合反应，制备中间体2’TBSO-M1-L2-M2-离去基团；
    S3’：中间体2’TBSO-M1-L2-M2-离去基团脱去TBS保护，并且与羧酸反应，生成中间体2 R1-L1-M1-L2-M2-离去基团；
    S4’：中间体2和氨基化合物原料发生一次或多次取代反应，制备目标产物。
  20. 一种脂质纳米颗粒，其特征在于，包括权利要求1-18任一项所述的氨基脂质。
  21. 根据权利要求20所述的脂质纳米颗粒，其特征在于，所述脂质纳米颗粒还包括类固醇、中性脂质和/或聚合物结合的脂质；
    所述聚合物结合的脂质其化学式为P-Y-L，其中P是亲水性聚合物部分，Y是任选的连接物，L是脂质部分。
  22. 根据权利要求21所述的脂质纳米颗粒，其特征在于，所述类固醇为胆固醇；
    和/或，所述中性脂质为磷脂；
    和/或，所述聚合物结合的脂质为聚乙二醇化脂质。
  23. 根据权利要求21所述的脂质纳米颗粒，其特征在于，所述脂质纳米颗粒中，氨基脂质、类固醇、中性脂质和聚合物结合的脂质的摩尔比为30-70∶30-65∶0-30∶0.2-5。
  24. 一种药物组合物，其特征在于，包括权利要求20-23中任一项所述的脂质纳米颗粒和药学上可接受的载体。
  25. 一种利用权利要求1-18中任一项所述的氨基脂质或权利要求20-23中任一项所述的脂质纳米颗粒或权利要求24所述的药物组合物治疗或预防传染病、癌症、遗传疾病、 过敏、毒性、自身免疫性疾病的方法。
  26. 根据权利要求25所述的方法，其特征在于，所述癌症包括肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌和前列腺癌。
  27. 一种利用权利要求1-18中任一项所述的氨基脂质或权利要求20-23中任一项所述的脂质纳米颗粒或权利要求24所述的药物组合物进行基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的方法。
  28. 权利要求1-18中任一项所述的氨基脂质或权利要求20-23中任一项所述的脂质纳米颗粒或权利要求24所述的药物组合物在制备用于治疗或预防传染病、癌症、遗传疾病、过敏、毒性、自身免疫性疾病药物中的应用。
  29. 根据权利要求28所述的应用，其特征在于，所述癌症包括肺癌、胃癌、肝癌、食管癌、结肠癌、胰腺癌、脑癌、淋巴癌、血癌和前列腺癌。
  30. 权利要求1-18中任一项所述的氨基脂质或权利要求20-23中任一项所述的脂质纳米颗粒或权利要求24所述的药物组合物在制备用于基因治疗、基因疫苗接种、反义治疗、核酸转移或通过干扰RNA的治疗的药物中的应用。
  31. 一种向受试者递送药剂的方法，其特征在于，包括：向受试者施用配制在权利要求20-23中任一项所述的脂质纳米颗粒中的药剂。
  32. 如权利要求1-18中任一项所述的氨基脂质，用于治疗或预防传染病、癌症、遗传疾病、过敏、毒性、自身免疫性疾病。
PCT/CN2023/137365 2023-03-31 2023-12-08 氨基脂质、脂质纳米颗粒及其应用 WO2024198497A1 (zh)

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