WO2023236953A1 - 含氮链状化合物、制备方法、包含其的组合物和应用 - Google Patents

含氮链状化合物、制备方法、包含其的组合物和应用 Download PDF

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WO2023236953A1
WO2023236953A1 PCT/CN2023/098621 CN2023098621W WO2023236953A1 WO 2023236953 A1 WO2023236953 A1 WO 2023236953A1 CN 2023098621 W CN2023098621 W CN 2023098621W WO 2023236953 A1 WO2023236953 A1 WO 2023236953A1
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mol
alkyl group
lipid
formula
independently
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PCT/CN2023/098621
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French (fr)
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林金钟
卢静
俞航
姜婷
张凡
张博阳
郭俊香
陈林
王冰
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上海蓝鹊生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids

Definitions

  • the present invention relates to a nitrogen-containing chain compound, preparation methods, compositions containing the same and applications.
  • Nucleic acid drugs are an important direction in current basic and applied research. Nucleic acid drugs can be used for the prevention and/or treatment of viral and bacterial infectious diseases, tumors, metabolic diseases, etc. Their production costs are lower and the cycle is shorter, which is conducive to the rapid development of personalized drugs. However, nucleic acids are negatively charged macromolecules that are difficult to penetrate through cell membranes. At the same time, nucleic acids have poor stability. By developing various nucleic acid packaging and delivery systems, the instability of nucleic acid drugs can be overcome to a certain extent and their delivery efficiency can be improved.
  • Lipid nanoparticles have been shown to be useful as vehicles for delivering bioactive substances, such as small molecule drugs, proteins, and nucleic acids, into cells and/or intracellular compartments.
  • Optimizing the nucleic acid drug delivery system by designing and optimizing the types and amounts of each component in lipid nanoparticles is of great significance to improve the efficacy of nucleic acid drug prevention and treatment, especially for the delivery of RNA preventive and/or therapeutic agents.
  • Lipid compounds and related methods and compositions are of great significance to improve the efficacy of nucleic acid drug prevention and treatment, especially for the delivery of RNA preventive and/or therapeutic agents.
  • the present invention aims to provide a new ionizable lipid compound that can be used to deliver nucleic acid drugs, and to increase the types of ionizable lipid compounds and the selection of nucleic acid preventive and/or therapeutic agent delivery carriers.
  • the present invention provides a nitrogen-containing chain compound, a preparation method, a composition containing the same and applications.
  • the composition of the present invention can be used to efficiently deliver nucleic acid drugs.
  • the technical solution of the present invention is as follows:
  • the present invention provides a nitrogen-containing chain compound shown in formula I or a pharmaceutically acceptable salt thereof,
  • Z and W are independently C 3 -C 10 alkylene
  • Y and Q are independently
  • A is a C 2 -C 6 alkylene group
  • Each RA -1 and RA -2 is independently a C 2 -C 6 alkylene group
  • M is a C 1 -C 6 alkylene group
  • R 1 and R 2 are independently C 6 -C 20 alkyl
  • R 5 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 5-1 ;
  • Each R 5-1 is independently hydroxy or
  • Each R 5-1-1 is independently a C 6 -C 20 alkyl group
  • R 6 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 6-1 ;
  • Each R 6-1 is independently hydroxyl or
  • Each R 6-1-1 is independently a C 6 -C 20 alkyl group.
  • the present invention provides a nitrogen-containing chain compound shown in formula I or a pharmaceutically acceptable salt thereof,
  • Z and W are independently C 3 -C 10 alkylene
  • Y and Q are independently
  • A is a C 2 -C 6 alkylene group
  • Each RA -1 and RA -2 is independently a C 2 -C 6 alkylene group
  • M is a C 1 -C 6 alkylene group
  • R 1 and R 2 are independently C 6 -C 20 alkyl
  • R 5 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 5-1 ;
  • Each R 5-1 is independently hydroxy or
  • R 5-1-1 is independently a C 6 -C 20 alkyl group
  • R 6 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 6-1 ;
  • Each R 6-1 is independently hydroxyl or
  • R 6-1-1 is independently a C 6 -C 20 alkyl group.
  • the present invention provides a nitrogen-containing chain compound shown in formula I or a pharmaceutically acceptable salt thereof,
  • Z and W are independently C 4 -C 10 alkylene
  • Y and Q are independently
  • A is a C 2 -C 6 alkylene group
  • Each RA -1 and RA -2 is independently a C 2 -C 6 alkylene group
  • M is a C 1 -C 6 alkylene group
  • R 1 and R 2 are independently C 6 -C 20 alkyl
  • R 5 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 5-1 ;
  • Each R 5-1 is independently hydroxy or
  • R 5-1-1 is independently a C 6 -C 20 alkyl group
  • R 6 is a C 2 -C 10 alkyl group that is unsubstituted or substituted by 1, 2 or 3 R 6-1 ;
  • Each R 6-1 is independently hydroxyl or
  • R 6-1-1 is independently a C 6 -C 20 alkyl group.
  • the C 4 -C 10 alkylene group may be a C 5 -C 8 alkylene group, preferably a straight chain alkane, for example
  • the C 3 -C 10 alkylene group may be a C 3 -C 8 alkylene group, preferably a straight chain alkane, for example
  • the C 4 -C 10 alkylene group can be a C 4 -C 10 alkylene group, or it can also be a C 5 -C 8 alkylene group, preferably a straight chain alkane.
  • the C 4 -C 10 alkylene group can be a C 4 -C 10 alkylene group, or it can also be a C 5 -C 8 alkylene group, preferably a straight chain alkane.
  • the C 3 -C 10 alkylene group may be a C 3 -C 8 alkylene group, preferably a straight chain alkane, for example
  • the C 2 -C 6 alkylene group can be For example
  • the C 2 -C 6 alkylene group can be For example
  • the C 2 -C 6 alkylene group can be For example
  • the C 2 -C 6 alkylene group can be
  • the C 2 -C 6 alkylene group can be
  • the C 1 -C 6 alkylene group can be
  • the C 1 -C 6 alkylene group can be
  • the C 6 -C 20 alkyl group in R 1 , can be C 10 -C 18 , for example
  • the C 6 -C 20 alkyl group in R 1 , can be C 10 -C 19 , for example
  • the C 6 -C 20 alkyl group in R 2 , can be C 10 -C 18 , for example
  • the C 6 -C 20 alkyl group in R 2 , can be C 10 -C 19 , for example
  • the C 2 -C 10 alkyl group may be a C 2 -C 8 alkyl group, for example Also for example
  • the C 6 -C 20 alkyl group in R 5-1-1 , can be C 11 -C 18 , for example
  • the C 2 -C 10 alkyl group may be a C 2 -C 8 alkyl group, for example Also for example
  • the C 6 -C 20 alkyl group in R 6-1-1 , can be C 11 -C 18 , for example
  • the nitrogen-containing chain compound represented by formula I is a nitrogen-containing chain compound represented by formula Ia.
  • Y is Where a is connected to R 2 and b is connected to Z.
  • Q is Where a is connected to R 1 and b is connected to W.
  • Q and Y are the same.
  • Z and W are the same.
  • R 1 and R 2 are the same.
  • R 5 and R 6 are the same.
  • Z and W are independently C 5 -C 8 alkylene groups.
  • Z and W are independently C 3 -C 8 alkylene groups.
  • A is a C 2 -C 6 alkylene group or
  • RA -1 and RA-2 are independently C 2 -C 4 alkylene groups.
  • M is methylene
  • R 1 and R 2 are independently C 10 -C 18 , such as C 10 -C 12 , also such as
  • R 1 and R 2 are independently a C 10 -C 20 alkyl group, preferably More preferably
  • R 5 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 5-1 .
  • R 5-1-1 is a C 10 -C 18 alkyl group, such as a C 14 -C 18 alkyl group, also such as
  • R 6 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 6-1 .
  • R 6-1-1 is a C 10 -C 18 alkyl group, such as a C 14 -C 18 alkyl group, also such as
  • Y is Among them, a is connected to R 2 , and b is connected to Z;
  • Q is Among them, a is connected to R 1 , and b is connected to W;
  • Z and W are independently C 3 -C 8 alkylene
  • A is C 2 -C 6 alkylene
  • R A-1 and R A-2 are independently C 2 -C 4 alkylene
  • M is methylene
  • R 1 and R 2 are independently C 10 -C 20 alkyl
  • R 5 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 5-1 ;
  • R 5-1-1 is a C 10 -C 18 alkyl group
  • R 6 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 6-1 ;
  • R 6-1-1 is a C 10 -C 18 alkyl group.
  • Y is Among them, a is connected to R 2 , and b is connected to Z;
  • Q is Among them, a is connected to R 1 , and b is connected to W;
  • Z and W are independently C 5 -C 8 alkylene
  • A is C 2 -C 6 alkylene
  • R A-1 and R A-2 are independently C 2 -C 4 alkylene
  • M is methylene
  • R 1 and R 2 are independently C 10 -C 18 ;
  • R 5 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 5-1 ;
  • R 5-1-1 is a C 10 -C 18 alkyl group
  • R 6 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 6-1 ;
  • R 6-1-1 is a C 10 -C 18 alkyl group.
  • R 1 and R 2 are the same;
  • R 5 and R 6 are the same;
  • Z and W are independently C 5 -C 8 alkylene
  • A is C 2 -C 6 alkylene
  • R A-1 and R A-2 are independently C 2 -C 4 alkylene
  • M is methylene
  • R 1 and R 2 are independently
  • R 5 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 5-1 ;
  • R 5-1-1 is a C 14 -C 18 alkyl group
  • R 6 is a C 2 -C 8 alkyl group substituted by 1, 2 or 3 R 6-1 ;
  • R 6-1-1 is a C 14 -C 18 alkyl group.
  • the nitrogen-containing chain compound represented by Formula I is a bilaterally symmetrical compound.
  • W can be
  • W can be
  • R 1 can be
  • R 1 can be
  • R 2 can be
  • R 2 can be
  • R 5 can be
  • R 6 can be
  • A can be any organic compound selected from the group consisting of:
  • A can be any organic compound selected from the group consisting of:
  • the nitrogen-containing chain compound shown in Formula I is any of the following compounds:
  • the invention also provides a method for preparing a nitrogen-containing chain compound represented by formula I, which includes the following steps: in a solvent, in the presence of a base and an iodide salt, a compound represented by formula I-1 is mixed with a compound represented by formula I
  • the compound represented by -2 can undergo a coupling reaction as shown in the following formula;
  • X is halogen
  • A is C 2 -C 6 alkylene
  • Y, Q, Z, W, R 5 , R 6 , R 1 and R 2 are as mentioned above, and Y is the same as Q, R 1 and R 2 is the same, Z is the same as W.
  • the halogen may be fluorine, chlorine, bromine or iodine, such as bromine.
  • the molar ratio of the compound represented by Formula I-2 to the compound represented by Formula I-2 may be 1:(1-3), for example, 1:2.6.
  • the base can be a conventional base in this field.
  • the base may be a basic carbonate (the cation in the salt is an alkali metal ion and the anion is carbonate), such as K 2 CO 3 .
  • the molar ratio of the compound represented by Formula I-2 to the base may be 1:(1-5); for example, 1:3.5.
  • the solvent can be a conventional solvent in this field, and the solvent can be an ether solvent or/and a nitrile solvent.
  • the ether solvent may be methyl tert-butyl ether.
  • the nitrile solvent may be acetonitrile.
  • the volume ratio of the nitrile solvent to the ether solvent may be 1:1.
  • the mass volume ratio of the compound represented by Formula I-2 to the solvent may be 10-50 mg/mL; for example, 16 mg/mL.
  • the iodized salt can be a conventional iodized salt in this field.
  • the iodized salt may be a basic iodized salt, such as KI.
  • the molar ratio of the compound represented by Formula I-2 to the iodide salt may be 1:(1-2); for example, 1:1.2.
  • the reaction temperature of the coupling reaction can be a common reaction temperature in this field, preferably 50-100°C, such as 80°C.
  • the present invention also provides a lipid carrier, which includes substance Z, which is a compound represented by Formula I as described above or a pharmaceutically acceptable salt thereof.
  • the lipid carrier further includes a diluent.
  • the diluent may be phosphate buffer or Tris buffer, etc.
  • the lipid carrier further includes phospholipids.
  • the phospholipid can be a conventional phospholipid in the art, which is an amphoteric auxiliary molecule that contributes to the fusion of lipid particles and cell membranes.
  • the phospholipid may be a phospholipid molecule with a charged polar end and a non-polar end of the fatty chain, such as distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphocholine (DMPC), dioleoyl phosphocholine alkali (DOPC), palmitoyl phosphocholine (DPPC), 1,2-distearoyl phosphocholine (DSPC), undecanoyl phosphocholine (DUPC) or palmitoyl phosphocholine (POPC), etc.
  • DSPC distearoyl phosphatidylcholine
  • DMPC dimyristoyl phosphocholine
  • DOPC dioleoyl phosphocholine alkali
  • DPPC palmitoyl phosphocholine
  • DUPC unde
  • the lipid carrier also includes PEG lipid (polyethylene glycol modified lipid).
  • the PEG lipid may be a lipid molecule modified with a polyethylene glycol hydrophilic end.
  • the PEG lipid is preferably selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.
  • PEG-modified dimyristoylglycerol DMG-PEG2000
  • the lipid carrier further includes sterols.
  • the sterols can be conventional sterols in this field, and the sterols include animal, plant or fungal sterols.
  • the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassinosterol, tomatine, ursolic acid and ⁇ -tocopherol, such as cholesterol and the like.
  • the molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, such as 0.6-2:1.
  • the molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, such as 0.68:1, 0.69:1, 0.71:1, 0.74:1, 0.76:1, 0.77:1, 0.79:1, 0.83:1, 0.84:1, 0.85:1, 0.86:1, 0.88:1, 0.89:1, 0.9:1, 0.91:1, 0.94: 1. 0.99:1, 1.04:1, 1.07:1 or 1.28:1.
  • the molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, such as 0.6-2:1; another example is 0.66:1, 0.68:1, 0.69:1, 0.70:1, 0.71:1, 0.72:1, 0.74:1, 0.76:1, 0.77:1, 0.79:1, 0.82:1, 0.83:1, 0.84:1, 0.85: 1.
  • the molar ratio of the substance Z to the phospholipid is 1-15:1, preferably 2-8:1, such as 3-6:1.
  • the molar ratio of the substance Z to the phospholipid is 1-25:1, preferably 2-25:1, such as 22.5:1, 20:1, 17.5:1, 15:1, 11.25:1, 10:1, 8.75:1, 7.5:1, 6.67:1, 5:1, 4.75:1, 4.5:1, 4:1, 3.9:1, 3.6:1, 3.3: 1, 3:1, 2.86:1, 2.5:1 or 2.2:1.
  • the molar ratio of the substance Z to the PEG lipid is 20-130:1, preferably 20-80:1, for example, 20-40:1.
  • the molar ratio of the substance Z to the PEG lipid is 16-130:1, preferably 16-80:1, such as 16-40:1; another example is 16: 1, 18:1, 20:1, 22.5:1, 25:1, 27.5:1, 28.1:1, 31.25:1 or 33.3:1.
  • the molar ratio of the substance Z to the PEG lipid is 16-130:1, preferably 16-80:1, such as 16-40:1; another example is 16: 1. 18:1, 18.8:1, 20:1, 21.9:1, 22.5:1, 25:1, 26.9:1, 27.5:1, 28.1:1, 29.6:1, 30:1, 31.25:1, 33.3:1 or 37.5:1.
  • the molar content of substance Z is about 30 mol% to 60 mol%.
  • the molar content of substance Z is about 30 mol% to 60 mol%; preferably 40 mol% to 55 mol%; for example, 40 mol%, 43 mol%, 45 mol%, 47.4 mol%, 50 mol%, 50 mol% or 55 mol%. %.
  • the molar content means the percentage of a certain substance in the total mass of the lipid carrier, and the sum of the molar contents of each component in the lipid carrier does not exceed 100 mol%. In a certain preferred embodiment, the molar content of the phospholipid is about 0 mol% to 30 mol%.
  • the molar content of the phospholipid is about 0 mol% to 30 mol%; preferably 0 mol% to 18 mol%; for example, 0 mol%, 2 mol%, 4 mol%, 6 mol%, 8 mol%, 10 mol%, 11 mol%, 12mol%, 14mol%, 16mol% or 18mol%.
  • the molar content of the sterol is about 15 mol% to 55 mol%.
  • the molar content of the sterol is about 15 mol% to 60 mol%, preferably 40.4 mol% to 58.4 mol%, For example, 42.4 mol%, 44.4 mol%, 46.4 mol%, 48.4 mol%, 50.4 mol%, 52.4 mol% or 56.4 mol%.
  • the molar content of the sterol is about 15 mol% to 60 mol%, preferably 40.4 mol% to 58.4 mol%, such as 40.4 mol%, 41 mol%, 42.4 mol%, 43 mol%, 43.4 mol%, 44.4mol%, 46.4mol%, 47.4mol%, 48mol%, 48.4mol%, 49mol%, 49.4mol%, 49.5mol%, 50mol%, 50.4mol%, 50.5mol%, 51mol%, 51.4mol%, 51.5mol %, 52mol%, 52.25mol%, 52.4mol%, 52.5mol%, 52.75mol%, 53mol%, 53.4mol%, 54mol%, 54.25mol%, 54.4mol%, 54.5mol%, 54.75mol%, 55mol%, 56 mol%, 56.4 mol%, 56.5 mol%, 57 mol%, 57.5 mol%, 58 mol% or 58.4 mol%.
  • the molar content of the sterol in the lipid carrier is about 15 mol% to 60 mol%, preferably 40.4% mol% to 58.4 mol%, for example 43mol%, 43.4mol%, 44.4mol%, 46.4mol%, 47.4mol%, 48mol%, 48.4mol%, 49mol%, 49.4mol%, 49.5mol%, 50mol%, 50.4mol%, 50.5mol%, 51mol% , 51.4mol%, 51.5mol%, 52mol%, 52.4mol%, 52.25mol%, 52.5mol%, 52.75mol%, 53mol%, 53.4mol%, 54mol%, 54.25mol%, 54.4mol%, 54.5mol%, 54.75mol%, 55mol%, 56mol%, 56.4mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol% or 58.4mol%; another example is 52.5mol% to 54.5
  • the molar content of the PEG lipid is about 0 mol% to 10 mol%.
  • the molar content of the PEG lipid is about 0 mol% to 10 mol%, such as 1.5 mol% to 2.5 mol%; such as 1.6 mol% or 2 mol%.
  • the molar content of the PEG lipid is about 0 mol% to 10 mol%, and the molar content of the PEG lipid can be 0.5 mol% to 2.5 mol%, or 0.5 mol% to 1.5 mol%. Or 1.5 mol% to 2.5 mol%, such as 1.6 mol% or 2 mol%.
  • the molar content of the PEG lipid is about 0 mol% to 10 mol%, for example, 0.5 mol% to 2.5 mol%, specifically, for example, 0.25 mol%, 0.5 mol%, 0.75 mol%, 1 mol%. %, 1.5 mol%, 1.6 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol% or 5 mol%; further 0.5 mol% to 2 mol%; it can also be 0.5 mol% to 1.5 mol% or 1.5 mol% to 2.5 mol%; it can also be 1.6 mol% or 2 mol%.
  • the molar content of the PEG lipid is about 0 mol% to 10 mol%, specifically, for example, 0.25 mol%, 0.5mol%, 0.75mol%, 1mol%, 1.5mol%, 1.6mol%, 2mol%, 2.5mol%, 3mol%, 3.5mol%, 4mol% or 5mol%; for example, 0.25mol% to 3mol% , further 0.5 mol% to 2.5 mol%, further 0.5 mol% to 2 mol%.
  • the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.
  • the lipid carrier consists of the substance Z, the phospholipid, the PEG lipid and the sterol.
  • the lipid carrier consists of the substance Z, the diluent, the PEG lipid and the sterol.
  • the lipid carrier consists of the substance Z, the PEG lipid and the sterol.
  • the lipid carrier does not contain phospholipids.
  • the molar ratio of substance Z to sterols in the lipid carrier can be 0.6-2:1; preferably 0.68:1, 0.69:1 , 0.7:1, 0.77:1, 0.85:1, 0.86:1, 1.04:1 or 1.28:1.
  • the molar ratio of substance Z to sterols in the lipid carrier is 0.6-2:1, such as 0.68:1, 0.69:1, 0.70 :1, 0.71:1, 0.72:1, 0.74:1, 0.76:1, 0.77:1, 0.79:1, 0.82:1, 0.83:1, 0.84:1, 0.85:1, 0.86:1, 0.87:1 , 0.88:1, 0.89:1, 0.9:1, 0.91:1, 0.92:1, 0.93:1, 0.94:1, 0.97:1, 0.99:1, 1.04:1, 1.07:1, 1.1:1, 1.16 :1, 1.23:1, 1.28:1, 1.30:1, 1.41:1, 1.52:1 or 1.58:1.
  • the molar ratio of substance Z to PEG lipid in the lipid carrier can be 16-35:1; preferably 16:1, 18 :1, 20:1, 22.5:1, 25:1, 27.5:1 or 28.1:1.
  • the molar ratio of the substance Z to the PEG lipid in the lipid carrier can be 16-35:1; another example is 16:1, 18 :1, 20:1, 21.9:1, 22.5:1, 25:1, 26.9:1, 27.5:1, 28.1:1, 29.6:1 or 30:1.
  • the present invention also provides a lipid nanoparticle, which includes a therapeutic agent and/or a preventive agent and the aforementioned lipid carrier.
  • the therapeutic agent and/or preventive agent may be one or two or more nucleic acids.
  • the nucleic acid may be a conventional nucleic acid in the art.
  • the therapeutic and/or preventive agent may be single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small RNA Clamp RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of nucleic acid molecules known in the art, preferably mRNA, such as firefly luciferase (Fluc) mRNA or SARS-CoV-2 spike protein (Spike) mRNA.
  • mRNA such as firefly luciferase (Fluc) mRNA or SARS-CoV-2 spike protein (Spike) mRNA.
  • the nitrogen to phosphorus ratio in the lipid nanoparticles can be 2:1-30:1, and the nitrogen to phosphorus ratio of the composition refers to the ionizable nitrogen in one or more ionizable lipid compounds.
  • the ratio of the number of moles of atoms to the number of moles of phosphate groups in RNA Preferably, it is 2:1-20:1, such as 3:1-20:1, and another example is 3:1-16:1.
  • the mass ratio of the lipid carrier to the therapeutic agent and/or preventive agent can be 3-80:1, preferably 6-60:1.
  • the particle size (average particle size) of the lipid nanoparticles can be 10-200 nm, preferably 40-150 nm, such as 60-150 nm.
  • the particle size (average particle size) of the lipid nanoparticles can be 10-200 nm, preferably 40-150 nm, such as 60-150 nm; another example, 50-150 nm.
  • the lipid carrier encapsulates the therapeutic agent and/or preventive agent.
  • the present invention also provides a composition, which includes substance Z, which is a compound represented by Formula I as described above or a pharmaceutically acceptable salt thereof.
  • the composition further includes one or more of diluent, phospholipid, PEG lipid, sterol and therapeutic and/or preventive agent.
  • the diluent, phospholipid, PEG lipid, sterol and therapeutic agent and/or preventive agent are as described above.
  • the substance Z and one or more of the diluent, phospholipid, PEG lipid and sterol form a lipid carrier as described above.
  • the lipid carrier and the therapeutic agent and/or preventive agent form lipid nanoparticles as described above.
  • the encapsulation rate of the therapeutic agent and/or preventive agent in the composition is at least 50%, preferably at least 70%.
  • the polydispersity index of the composition is not higher than 0.5, for example, not higher than 0.3.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • pharmaceutically acceptable means relatively nontoxic, safe, and suitable for use by patients.
  • pharmaceutically acceptable salt refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base.
  • base addition salts can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, etc.
  • acid addition salts can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent.
  • Pharmaceutically acceptable acid addition salts include, but are not limited to: hydrochloride, sulfate, methanesulfonate, etc.
  • hydrochloride sulfate, methanesulfonate, etc.
  • the "-" at the end of a group means that the group is connected to the rest of the molecule through that site.
  • alkyl refers to a linear or branched, saturated, monovalent hydrocarbon group having the specified number of carbon atoms (eg, C 1 -C 6 ).
  • Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.
  • alkylene is a divalent group connected to the rest of the molecule by two single bonds and is otherwise defined in the same manner as the term "alkyl”.
  • alkoxy refers to the group RX -O-, R Alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, etc.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effect of the present invention is that the present invention provides a nitrogen-containing chain compound shown in Formula I, which has a novel structure and can be used to prepare lipid nanoparticles.
  • Lipid nanoparticles containing nitrogen-containing chain compounds as shown in Formula I have a low polydispersity index and can deliver mRNA efficiently.
  • the nitrogen-containing chain compound of the present invention is used to prepare LNP preparations, it can still have good properties and delivery ability when the phospholipid component is as low as 4 mol% or less.
  • the nitrogen-containing chain compound of the present invention is used to prepare LNP preparations, the LNP preparations prepared with low PEG lipids still have good properties and delivery capabilities, thereby reducing the impact of high PEG lipids. of Efficacy and Safety Risks.
  • Figure 1 is a diagram showing the nucleic acid gel electrophoresis results of each LNP preparation prepared in Example 1;
  • Figure 2 shows the chemiluminescence intensity measured after co-culture of 293FT cells and the LNP preparation prepared in Example 1 for 18-24 hours in Example 2;
  • FIGS 3 and 4A show the total bioluminescence in the body measured at different times after mice were intravenously administered the LNP preparations LQ104-1 to LQ104-8 prepared in Example 1 in Example 3;
  • Figure 4B is the total antibody titer measured in mice after intramuscular injection of LNP preparations LQ104-9, LQ104-10 or LQ107 in Example 3;
  • Figures 5A to 5D show the total biomass in vivo of mice measured at different times after each LNP preparation in Example 4 was administered intravenously (Figure 5A, Figure 5B) or intramuscularly (Figure 5C, Figure 5D). The amount of luminescence or the total amount of luminescence at the administration site;
  • Figures 6A to 6C show the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 5 to mice;
  • Figures 7A and 7B show the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 6 to mice;
  • Figure 8 shows the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 7 to mice;
  • Figures 9A and 9B show the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 8 to mice;
  • FIGS 10A to 10C show the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 10 to mice;
  • Figures 11A to 11C show the total bioluminescence in the body measured at different times after intravenous administration of each LNP preparation in Example 11 to mice;
  • Figures 12A to 12D show the total biomass in vivo of mice measured at different times after intravenous injection (Figure 12A, Figure 12B) or intramuscular injection (Figure 12C, Figure 12D) of each LNP preparation in Example 12. The amount of luminescence or the total amount of luminescence at the administration site.
  • Figures 13A and 13B show the total bioluminescence amount in the body measured at different times after intravenous administration of each LNP preparation in Example 13 to mice.
  • reaction solution was filtered, spun to dryness, and purified by column chromatography to obtain 1.1 g of colorless oil.
  • reaction solution was filtered through diatomaceous earth and spun to dryness. After purification by column chromatography, 700 mg of colorless oil was obtained, with a yield of 70%.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 15 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 800 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 13.8 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 760 mg of colorless oil was obtained.
  • reaction solution was filtered, spun to dryness, and purified by column chromatography to obtain 15.3 g of colorless oil.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 820 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.5 g of colorless oil was obtained.
  • reaction solution was filtered, spun to dryness, and purified by column chromatography to obtain 730 mg of colorless oil.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.4 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 770 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 13.3 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 660 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.2 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 840 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.8 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 750 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 15.1 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 680 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 12.8 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 590 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.5 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 770 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 15.2 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 840 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.6 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 750 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 14.1 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 690 mg of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 13.7 g of colorless oil was obtained.
  • reaction solution was filtered and spun to dryness. After purification by column chromatography, 790 mg of colorless oil was obtained.
  • the reaction solution was filtered and spin-dried. After purification by column chromatography, 850 mg of colorless oil was obtained, which was the compound LQ104-H3. The yield was 43%.
  • N,N'-bis(2-hydroxyethyl)ethylenediamine purchased from Adamas Reagent Co., Ltd., product number: 013455310, purity: RG, 98%;
  • K 2 CO 3 purchased from Shanghai Yien Chemical Technology Co., Ltd., item number: RH425011, purity: AR, 99%;
  • Acetonitrile purchased from Shanghai Titan Technology Co., Ltd., item number: 01111797, purity: AR, ⁇ 99.0%;
  • Methyl tert-butyl ether purchased from Shanghai Titan Technology Co., Ltd., product number: 01030342, purity: AR, ⁇ 99.0%;
  • Malonic acid purchased from Adamas Reagent Co., Ltd., item number: 01022573, purity: RG, 99%;
  • DCC dicyclohexylcarbodiimide, purchased from Adamas Reagent Co., Ltd., product number: 012041444, purity: RG, 99%;
  • DMAP 4-dimethylaminopyridine, purchased from Adamas Reagent Co., Ltd., product number: 01271081, purity: RG, 99%;
  • DCM dichloromethane, purchased from Shanghai Titan Technology Co., Ltd., item number: 01111853, purity: AR, ⁇ 99.5%;
  • Diatomite purchased from Shanghai Titan Technology Co., Ltd., item number: 01589000, purity: premium grade, ⁇ 89.0%, 200 mesh;
  • 6-Bromohexanoic acid purchased from Adamas Reagent Co., Ltd., item number: 01073739, purity: RG, 98%+;
  • N,N'-bis(2-hydroxyethyl)ethylenediamine purchased from Adamas Reagent Co., Ltd., product number: 013455310, purity: RG, 98%;
  • 9-Heptadecanol purchased from Dalian Ruiying Technology Co., Ltd., purity: 98%;
  • 5-Bromovaleric acid purchased from Bide Pharmaceuticals, product number: BD9634, purity: 98%;
  • 4-Bromobutyric acid purchased from Shanghai Titan Technology Co., Ltd., item number: 011016520, purity: RG, 99%+;
  • 2-Hexyl undecanoic acid purchased from Bide Pharmaceutical, product number: BD75392, purity: 98%;
  • 8-Bromooctanoic acid purchased from Jiangsu Aikang, purity: 98%;
  • 6-Bromo-1-hexanol purchased from Adamas Reagent Co., Ltd., product number: 01074359, purity: RG, 98%;
  • N,N'-bis(2-hydroxyethyl)-1,3-propanediamine (purchased from Beijing Visail Chemical, purity: 95%).
  • Example 1 is used to verify whether the lipid nanoparticle (Lipid Nanoparticle, LNP) preparation prepared from the ionizable lipid compound disclosed in the present application can effectively encapsulate mRNA and maintain the structural integrity of the mRNA.
  • the ionizable lipid compound finally prepared in Preparation Example 1 and Preparation Example 2, distearoylphosphatidylcholine (DSPC, purchased from Nippon Seika Co., Ltd., product number: S01005), cholesterol (purchased from Nippon Seika Co., Ltd.
  • Ethanol was removed by dialysis against 0.01 M phosphate buffered saline (PBS) for 12 to 24 hours. Finally, the LNP solution was filtered through a sterile filter with a pore size of 0.22 ⁇ m (manufacturer: Millex, product number: SLGPR33RB), and concentrated by ultrafiltration (manufacturer: Amicon-Ultra, molecular weight cutoff: 10KDa) to obtain the ionizable ionizable membrane as described in this application. LNP preparation obtained by encapsulating Fluc mRNA or Spike mRNA with lipid, DSPC, cholesterol and DMG-PEG2000.
  • PBS phosphate buffered saline
  • the molar ratios of ionizable lipid compounds to DSPC, cholesterol and DMG-PEG2000, as well as the nitrogen-phosphorus ratios of ionizable lipids to mRNA are shown in Table 1.
  • the dynamic light scattering method was used to determine the particle size and polydispersity index (PDI) of each LNP preparation by the Malvern Zetasizer Ultra instrument (Manufacturer: Malvern); the Quant-it Ribogreen RNA quantitative assay kit (Manufacturer: ThermoFisher Scientific, Cat.
  • FIG. 1 is a nucleic acid gel electrophoresis diagram of each LNP preparation in this example. Wherein, the gel is a 1% agarose gel (manufacturer: Biowest; product number: BY-R0100), and the test condition is 160V electrophoresis for 20 minutes.
  • the encapsulation rate is used to indicate whether the LNP can effectively encapsulate mRNA.
  • the encapsulation rate is higher than 70%, which means that the LNP can effectively encapsulate mRNA.
  • Loading mRNA; single and bright bands in the agarose gel electrophoresis diagram indicate that the mRNA structure is complete. Among them, the closer the PDI is to 0, the better, and the closer the encapsulation rate is to 100%, the better.
  • each LNP in this application is between 70-120nm, the PDI is less than 0.3, and the encapsulation rate is higher than 80%; specifically, the encapsulation rate of the LQ104 series is stable at 90% Above, the encapsulation rate of the LNP preparation prepared from LQ107 was 83.7%. It can be seen that LNP preparations prepared according to the molar ratio and nitrogen to phosphorus ratio shown in Table 1 can effectively encapsulate mRNA and maintain the integrity of the mRNA structure. It can also be seen from Table 1 and the experimental data not listed in this application that the performance of the LQ104 series is better than that of the LQ107 series.
  • LQ104-1 to LQ104-8 (containing Fluc mRNA) of Example 1 were injected through the tail vein into 6- to 8-week-old female Balb/C mice (Viton) at a dose of 5 ⁇ g/mouse.
  • the total luminescence intensity is measured by bioluminescence imaging and is the luminescence intensity data of the luminescent site 6 to 15 minutes (min) after intraperitoneal injection of D-luciferin potassium salt.
  • the total luminescence intensity of the expression area in vivo was counted through Living Image software (manufacturer: PerkinElmer), and further, the area under the curve (AUC, unit: p/s*hour) was calculated through GraphPad software.
  • the AUC in each embodiment of the present application is The area under the curve connecting the total luminous intensity measurement points from the 4th hour or 6th hour (using the experimental method of this application, the peak value at 3-6 hours after the drug is the same) to the 48th hour after the drug.
  • the test results are shown in Figures 3 and 4A and Table 2.
  • E+10 is the expression of scientific notation, which means 10 raised to the 10th power; for example, E+11 means 10 raised to the 11th power, the same below.
  • the reading of the total luminescence intensity measured by an in vivo imager in mice that have not been treated with medication is of the order of 10 5 .
  • LQ104-1 to LQ104-8 are strongly expressed in mice.
  • we determined the LNP formulation with better expression by observing the area under the curve (AUC) of the line connecting the measurement points. The larger the area under the curve, the better the expression effect.
  • LQ104-9, LQ104-10, and LQ107 were injected intramuscularly into 6- to 8-week-old female Balb/C mice at a dose of 2 ⁇ g/mouse, and repeated on the 21st day of the first injection. Inject the same LNP preparation. and after the second dose The whole blood of mice was collected at a specific time point (the data in this example is the 7th day after the second administration). The collected blood was centrifuged at 4°C and 2000 ⁇ g for 10 min to separate the serum from the whole blood; subsequently, the serum was inactivated in a water bath at 56°C for 30 min and stored at -80°C for analysis. .
  • the total antibody titer in serum was determined by enzyme-linked immunosorbent (ELISA) method. Specifically, SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein (Manufacturer: Yiqiao Shenzhou, Product No.: 40589-V08B1) is used to coat the antigen, and SARS-CoV-2 (2019-nCoV ) Spike Neutralizing Antibody Mouse Mab (Manufacturer: Yiqiao Shenzhou, Catalog No.: 40591-MM43) as a control, blocked with 2% bovine serum albumin (BSA), Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, Catalog No.
  • BSA bovine serum albumin
  • H+L Peroxidase AffiniPure Goat Anti-Mouse IgG
  • the ionizable lipids prepared in Preparation Example 3 to Preparation Example 17 were selected, and LNP preparations (containing Fluc mRNA) were prepared according to the molar ratio and nitrogen to phosphorus ratio shown in Table 3 according to the same method as Example 1. .
  • Malvern Zetasizer Ultra was used to measure the particle size, PDI and surface potential of each LNP preparation; Quant-it Ribogreen RNA quantitative assay kit (Manufacturer: ThermoFisher Scientific, Cat.
  • the particle sizes of the LNP preparations prepared from the ionizable lipids described in Preparation Example 3 to Preparation Example 17 are all between 50-100 nm; the PDI is less than 0.3, specifically between 0.044 and 0.098; The encapsulation rates are all higher than 96%.
  • the LNP preparations in this example can effectively entrap mRNA; the surface potentials are all weakly negative, and the pKa is between 6-7.3, which is equivalent to the range recognized in the art.
  • each group of LNP preparations was injected into 6 to 8-week-old female Balb/C mice at a dose of 5 ⁇ g/mouse through tail vein injection or intramuscular injection of the lower limbs.
  • the total luminous intensity at the liver site or lower limb administration site was calculated.
  • the test results are shown in Figure 5A to Figure 5D and Table 4A to Table 4D.
  • Figures 5A and 5B show the statistical results of luminescence in the liver of mice after intravenous injection
  • Figure 5C and Figure 5D show the statistical results of luminescence in the lower limbs of mice after intramuscular injection.
  • LQ104-E16b-2 obtained in Preparation Example 7 LQ104-E17b-4 obtained in Preparation Example 14, and LQ104-E18b-3 obtained in Preparation Example 17 were selected, and each was divided into 8 groups, as described in Table 5 below.
  • the LNP preparation (containing Fluc mRNA) was prepared according to the preparation components and nitrogen to phosphorus ratio. And their particle size, PDI and encapsulation efficiency were measured, and the results are shown in Table 5.
  • the particle size of the LNP preparation prepared from the compounds LQ104-E16b-2, LQ104-E17b-4 or LQ104-E18b-3 according to the above components and nitrogen to phosphorus ratio is between 60-90nm; the PDI is less than 0.3, and most of them are concentrated below 0.1; the encapsulation rates are all higher than 90%, and they are mainly concentrated between 97% and 99%.
  • LQ104-E16b-2 obtained through Preparation Example 7 was selected as the ionizable lipid.
  • 21 kinds of LNP preparations (fluc-encapsulated) were prepared in the same manner as in Example 1. mRNA), and determine their particle size, PDI and encapsulation efficiency.
  • This example is mainly used to verify the preferred component contents of DSPC and cholesterol in LNP preparations. Among them, based on the total molar number of the four components being 100%, keeping the molar percentages of LQ104-E16b-2 and DMG-PEG unchanged, and verifying the changes in the molar percentages of DSPC and cholesterol, each LNP prepared Performance of the formulation.
  • the particle size of each group of LNP preparations prepared from LQ104-E16b-2 is between 60-110nm; the PDI is less than 0.3, specifically between 0.053 and 0.166; the encapsulation rate is higher than 90% , and both are higher than 96.8%.
  • Example 3 inject all the LNP preparations prepared in this example into 6 to 8 week old female Balb/C mice through the tail vein at a dose of 5 ⁇ g/mouse, and use The total luminous intensity of the liver region was calculated using the same method as Example 3.
  • the test results are shown in Figures 7A and 7B and Tables 8A to 8B. It can be seen that the LNP preparations tested in this example have strong expression in mice. Among them, the total luminescence intensity of LQ104-E16b-2 (DS-f1) to LQ104-E16b-2 (DS-f10) is higher, indicating that the molar percentage of DSPC is between 0% and 18% of the corresponding LNP preparation in vivo. Better delivery capabilities.
  • Example 6 replaces the phospholipid DSPC in the LNP preparation component with DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin, dioleoylphosphatidylethanolamine), and implements it in accordance with Example 1
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin, dioleoylphosphatidylethanolamine
  • the particle size of the LNP preparation prepared in this example is between 70-100 nm, the PDI is less than 0.3, and the encapsulation rate is higher than 96%.
  • each LNP preparation prepared in this example was injected into 6 to 8-week-old female Balb/C mice through the tail vein at a dose of 5 ⁇ g/mouse, and the living livers of the mice were counted.
  • the total luminous intensity of the site, the test results are shown in Figure 8 and Table 10. It can be seen that each LNP preparation prepared in this example has strong expression in mice.
  • we tested a variety of phospholipid molecules and the prepared LNP formulations can achieve in vivo delivery and expression.
  • This example selects LQ104-E16b-2 obtained through Preparation Example 7 as the ionizable lipid.
  • the LNP preparation fluc mRNA encapsulated
  • the mRNA in this example was diluted in a 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 was used during dialysis.
  • the particle size, PDI and encapsulation efficiency of all LNP formulations in this example were determined. This example is mainly used to verify the preferred component content of ionizable lipids in LNP preparations.
  • DSPC DSPC with molar percentages of 0%, 2%, 4%, and 10% and PEG lipids with molar percentages of 1.6% to simultaneously screen the content of ionizable lipids in several specific components. range (cholesterol is used in this example to make up the balance after the other three components are determined).
  • the particle size of the LNP preparation prepared in this example is between 55-120 nm, the PDI is less than 0.3, and the encapsulation rate is higher than 90%.
  • LNP preparations LQ104-E16b-2 (DS-f1) to LQ104-E16b-2 (DS-f7) prepared in Example 6 were stored in an environment of 4°C for 14 days, and their particle sizes and PDI were measured. The results See Table 13.
  • LNP preparations LQ104-E16b-2 (ION-f3), LQ104-E16b-2 (ION-f6), LQ104-E16b-2 (ION-f7), LQ104-E16b-2 (ION) prepared in Example 8 were used.
  • an LNP preparation (fluc mRNA-encapsulated) was prepared according to the molar ratio and nitrogen to phosphorus ratio in Table 15.
  • the mRNA solution of this example was diluted in a 25mM, pH 5.0 sodium acetate solution, and the solution was 20 during dialysis. mM, pH 7.5 Tris-acetic acid solution.
  • This example is mainly used to verify the performance of the LNP preparation of the present application prepared by using three components (ionizable lipid, cholesterol and DMG-PEG) and four components (ionizable lipid, phospholipid, cholesterol and DMG-PEG). , and to investigate the preferred component content of DMG-PEG in the LNP preparation.
  • the particle size, PDI and encapsulation efficiency of each LNP were measured, and the results are shown in Table 15.
  • the LQ104 series is prepared with three components or four components, its particle size is between 60-115nm, PDI is less than 0.3, and the encapsulation rate is higher than 96%.
  • each group of LNP preparations in this example was injected into 6 to 8-week-old female Balb/C mice through the tail vein at a dose of 5 ⁇ g/mouse, and the living livers of the mice were counted.
  • the total luminous intensity of the part, the test results are shown in Figure 10A, Figure 10B and Figure 10C and Table 16A to Table 16C. It can be seen that all LNP preparations in this example have strong expression in mice. Moreover, experiments have also shown that the expression effect is better when the molar percentage of PEG lipid is between 1.5% and 2.5%; more specifically, the molar percentage of PEG lipid is between 1.5% and 2.0%. .
  • Example 8 prepare the LNP preparation (containing Fluc mRNA) according to the molar ratio and nitrogen to phosphorus ratio in Table 17.
  • This example is mainly used to verify the performance of LNP preparations prepared with different nitrogen to phosphorus ratios. Based on the optimal molar percentage ranges of ionizable lipids and DSPC verified in the previous examples, we set the nitrogen to phosphorus ratio to 4:1 to 8:1 respectively to prepare the LNP preparation. The particle size, PDI and encapsulation efficiency of each LNP preparation were measured, and the results are shown in Table 17.
  • the particle size of the LNP preparation in this example is between 60-120 nm, the PDI is less than 0.3, and the encapsulation rate is higher than 96%.
  • each group of LNP preparations in this example was injected into 6 to 8-week-old female Balb/C mice through the tail vein at a dose of 5 ⁇ g/mouse, and the living mice were counted.
  • the total luminous intensity of the liver, the test results are shown in Figure 11A, Figure 11B and Figure 11C and Table 18A to Table 18C. It can be seen that when the nitrogen to phosphorus ratio is in the range of 4:1 to 8:1, LNP preparations have strong expression in mice.
  • Example 8 prepare the LNP preparation (containing Fluc mRNA) according to the molar ratio and nitrogen to phosphorus ratio in Table 19.
  • This example selects the ionizable lipid prepared from Preparation Example 3 to Preparation Example 17.
  • This example is mainly used to verify the performance of the three-component (ionizable lipid, cholesterol and DMG-PEG) LNP preparation in this application. .
  • the particle size, PDI and encapsulation efficiency of each LNP were measured, and the results are shown in Table 19.
  • the particle sizes of the LNP preparations in this example are all between 60-130 nm, the PDIs are all less than 0.3, and the encapsulation rates are all higher than 90%.
  • the LNP preparations of each group of this Example are injected into 6 to 8-week-old female Balb/C mice through tail vein injection or intramuscular injection of the lower limbs at a dose of 5 ⁇ g/mouse. And count the liver parts (when administering via tail vein injection) The main area of expression) and the total luminous intensity of the lower limb administration site (the main area of expression when intramuscular injection of the lower limbs is used for administration).
  • the test results are shown in Figure 12A to Figure 12D and Table 20A to Table 20D.
  • Figures 12A and 12B show the statistical results of luminescence in the liver of mice after intravenous injection
  • Figures 12C and 12D show the statistical results of luminescence in the lower limbs of mice after intramuscular injection. It can be seen from Figure 12A to Figure 12D that all LNP preparations in this example have strong expression in mice.
  • LQ104-E16b-2 obtained through Preparation Example 7 was selected as the ionizable lipid.
  • 16 kinds of LNP preparations fluc mRNA-encapsulated were prepared according to the molar ratio and nitrogen-phosphorus ratio in Table 21. ), and determine their particle size, PDI and package Closure rate.
  • Example 3 inject all the LNP preparations prepared in this example into 6 to 8 week old female Balb/C mice through the tail vein at a dose of 5 ⁇ g/mouse, and use The total luminescence intensity of the liver region was calculated using the same method as Example 3.
  • the test results are shown in Tables 22 and 23 and Figures 13A and 13B. It can be seen that the LNP preparations tested in this example have strong expression in mice.
  • the total luminescence intensity of LQ104-E16b-2 (PEG-f1) to LQ104-E16b-2 (PEG-f6) in the 0% DSPC group is higher, indicating that the molar percentage of PEG lipids in this group is between 0.25
  • the LNP formulation corresponding to % to 2% has better in vivo delivery ability;
  • the total luminescence intensity of LQ104-E16b-2 (PEG-f9) to LQ104-E16b-2 (PEG-f15) in the 2% DSPC group is higher, indicating that this In the group, the molar percentage of PEG lipids ranged from 0.25% to 2.5%, corresponding to LNP formulations with better in vivo delivery capabilities.
  • the phospholipid content in LNP preparations is below 4 mol%.
  • the prepared LNP preparation prepared using the ionizable lipid compound provided by the present application, even if the phospholipid component is as low as 4 mol% or less, for example, 0 mol% to 2 mol%, as shown in this example, the prepared LNP The formulation still has good properties and delivery capabilities.
  • the prepared LNP preparation without increasing the PEG lipid content, for example, within the content range of 0.5 mol% to 2.5 mol%, the prepared LNP preparation still has good properties and delivery ability, thus reducing the risk of The risk of affecting efficacy and safety due to elevated PEG lipids.

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Abstract

本发明公开了一种含氮链状化合物、制备方法、包含其的组合物和应用。本发明提供了一种如式(I)所示的含氮链状化合物或其药学上可接受的盐。本发明的如式I所示的含氮链状化合物可用于制备脂质载体。本发明制备的脂质载体能够包载核酸药物，可用于将核酸预防剂和/或治疗剂递送至哺乳动物细胞及器官并发挥作用。

Description

含氮链状化合物、制备方法、包含其的组合物和应用
本申请要求申请日为2022年6月6日的中国专利申请2022106524310的优先权，申请日为2023年3月17日的中国专利申请2023102724825的优先权，申请日为2023年5月25日的中国专利申请2023106034922的优先权，本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种含氮链状化合物、制备方法、包含其的组合物和应用。
背景技术
核酸药物为现今基础和应用研究的一个重要方向。核酸药物可用于对病毒及细菌感染性疾病、肿瘤、代谢性疾病等的预防和/或治疗，其生产成本更低且周期更短，有利于快速开发个性化药物。然而，核酸为荷负电的大分子，难以透过细胞膜，同时核酸稳定性不佳，通过开发各种核酸包装和递送***可一定程度上克服核酸药物的不稳定性，提高其递送效率。
脂质纳米颗粒已被证明可用作递送生物活性物质(如小分子药物、蛋白质和核酸)进入细胞和/或细胞内区室的载体。通过设计及优化脂质纳米颗粒中的各组分的种类和用量，从而优化核酸药物递送***对于提高核酸药物预防及治疗的功效具重要意义，尤其是可用于递送RNA预防剂和/或治疗剂的脂质化合物以及相关的方法和组合物。
发明内容
本发明旨在提供一种新的可用于递送核酸药物的可电离脂质化合物，增加可电离脂质化合物的种类及核酸预防剂和/或治疗剂递送载体的选择。为解决以上技术问题，本发明提供了一种含氮链状化合物、制备方法、包含其的组合物和应用。本发明的组合物可用于高效递送核酸药物。本发明的技术方案如下：
本发明提供了一种如式I所示的含氮链状化合物或其药学上可接受的盐，
其中，Z和W独立地为C3-C10的亚烷基；
Y和Q独立地为
A为C2-C6的亚烷基、
各个RA-1和RA-2独立地为C2-C6的亚烷基；
M为C1-C6的亚烷基；
R1和R2独立地为C6-C20的烷基；
R5为未取代或被1个、2个或3个R5-1取代的C2-C10的烷基；
各个R5-1独立地为羟基或
各个R5-1-1独立地为C6-C20的烷基；
R6为未取代或被1个、2个或3个R6-1取代的C2-C10的烷基；
各个R6-1独立地为羟基或
各个R6-1-1独立地为C6-C20的烷基。
本发明提供了一种如式I所示的含氮链状化合物或其药学上可接受的盐，
其中，Z和W独立地为C3-C10的亚烷基；
Y和Q独立地为
A为C2-C6的亚烷基、
各个RA-1和RA-2独立地为C2-C6的亚烷基；
M为C1-C6的亚烷基；
R1和R2独立地为C6-C20的烷基；
R5为未取代或被1个、2个或3个R5-1取代的C2-C10的烷基；
各个R5-1独立地为羟基或
R5-1-1独立地为C6-C20的烷基；
R6为未取代或被1个、2个或3个R6-1取代的C2-C10的烷基；
各个R6-1独立地为羟基或
R6-1-1独立地为C6-C20的烷基。
本发明提供了一种如式I所示的含氮链状化合物或其药学上可接受的盐，
其中，Z和W独立地为C4-C10的亚烷基；
Y和Q独立地为
A为C2-C6的亚烷基、
各个RA-1和RA-2独立地为C2-C6的亚烷基；
M为C1-C6的亚烷基；
R1和R2独立地为C6-C20的烷基；
R5为未取代或被1个、2个或3个R5-1取代的C2-C10的烷基；
各个R5-1独立地为羟基或
R5-1-1独立地为C6-C20的烷基；
R6为未取代或被1个、2个或3个R6-1取代的C2-C10的烷基；
各个R6-1独立地为羟基或
R6-1-1独立地为C6-C20的烷基。
某一优选方案中，如式I所示的含氮链状化合物或其药学上可接受的盐中，某些基团的定义可如下所述，其他基团的定义可如其他任一方案所述(以下简称“某一优选方案中”)：Z中，所述C4-C10的亚烷基可为C5-C8的亚烷基，优选为直链烷烃，例如
某一优选方案中，Z中，所述C3-C10的亚烷基可为C3-C8的亚烷基，优选为直链烷烃，例如
某一优选方案中，W中，所述C4-C10的亚烷基可为C4-C10的亚烷基，还可为C5-C8的亚烷基，优选为直链烷烃，例如
某一优选方案中，W中，所述C3-C10的亚烷基可为C3-C8的亚烷基，优选为直链烷烃，例如
某一优选方案中，A中，所述C2-C6的亚烷基可为 例如
某一优选方案中，A中，所述C2-C6的亚烷基可为 例如
某一优选方案中，RA-1中，所述C2-C6的亚烷基可为 例如
某一优选方案中，RA-2中，所述C2-C6的亚烷基可为 例如
某一优选方案中，M中，所述C1-C6的亚烷基可为 例如
某一优选方案中，R1中，所述C6-C20的烷基可为C10-C18，例如
某一优选方案中，R1中，所述C6-C20的烷基可为C10-C19，例如
某一优选方案中，R2中，所述C6-C20的烷基可为C10-C18，例如
某一优选方案中，R2中，所述C6-C20的烷基可为C10-C19，例如
某一优选方案中，R5中，所述C2-C10的烷基可为C2-C8的烷基，例如 还例如
某一优选方案中，R5-1-1中，所述C6-C20的烷基可为C11-C18，例如
某一优选方案中，R6中，所述C2-C10的烷基可为C2-C8的烷基，例如 还例如
某一优选方案中，R6-1-1中，所述C6-C20的烷基可为C11-C18，例如
某一优选方案中，所述如式I所示的含氮链状化合物为如式I-a所示的含氮链状化合物
某一优选方案中，Y为其中a与R2相连，b与Z相连。
某一优选方案中，Q为其中a与R1相连，b与W相连。
某一优选方案中，Q和Y相同。
某一优选方案中，Z和W相同。
某一优选方案中，R1和R2相同。
某一优选方案中，R5和R6相同。
某一优选方案中，Z和W独立地为C5-C8的亚烷基。
某一优选方案中，Z和W独立地为C3-C8的亚烷基。
某一优选方案中，A为C2-C6的亚烷基或
某一优选方案中，RA-1和RA-2独立地为C2-C4的亚烷基。
某一优选方案中，M为亚甲基。
某一优选方案中，R1和R2独立地为C10-C18，例如C10-C12，还例如
某一优选方案中，R1和R2独立地为C10-C20的烷基，优选为 更优选为
某一优选方案中，R5为被1个、2个或3个R5-1取代的C2-C8的烷基。
某一优选方案中，R5-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
某一优选方案中，R6为被1个、2个或3个R6-1取代的C2-C8的烷基。
某一优选方案中，R6-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
某一优选方案中，Y为其中a与R2相连，b与Z相连；
Q为其中a与R1相连，b与W相连；
Z和W独立地为C3-C8的亚烷基；
A为C2-C6的亚烷基或
RA-1和RA-2独立地为C2-C4的亚烷基；
M为亚甲基；
R1和R2独立地为C10-C20的烷基；
R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
R5-1-1为C10-C18的烷基；
R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
R6-1-1为C10-C18的烷基。
某一优选方案中，Y为其中a与R2相连，b与Z相连；
Q为其中a与R1相连，b与W相连；
Z和W独立地为C5-C8的亚烷基；
A为C2-C6的亚烷基或
RA-1和RA-2独立地为C2-C4的亚烷基；
M为亚甲基；
R1和R2独立地为C10-C18；
R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
R5-1-1为C10-C18的烷基；
R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
R6-1-1为C10-C18的烷基。
某一优选方案中，Q和Y相同；
Z和W相同；
R1和R2相同；
R5和R6相同；
Z和W独立地为C5-C8的亚烷基；
A为C2-C6的亚烷基或
RA-1和RA-2独立地为C2-C4的亚烷基；
M为亚甲基；
R1和R2独立地为
R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
R5-1-1为C14-C18的烷基；
R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
R6-1-1为C14-C18的烷基。
某一优选方案中，所述如式I所示的含氮链状化合物为左右对称的化合物。
某一优选方案中，Z可为
某一优选方案中，Z可为
某一优选方案中，W可为
某一优选方案中，W可为
某一优选方案中，R1可为
某一优选方案中，R1可为
某一优选方案中，R2可为
某一优选方案中，R2可为
某一优选方案中，R5可为
某一优选方案中，R6可为
某一优选方案中，A可为
某一优选方案中，A可为
某一优选方案中，所述如式I所示的含氮链状化合物为如下任一化合物：


本发明还提供一种如式I所示的含氮链状化合物的制备方法，其包括如下步骤：溶剂中，在碱和碘盐存在下，如式I-1所示的化合物与如式I-2所示的化合物进行如下式所示的偶联反应，即可；
X为卤素，A为C2-C6的亚烷基，Y、Q、Z、W、R5、R6、R1和R2如前所述，且Y与Q相同，R1与R2相同，Z与W相同。
所述偶联反应中，所述卤素可为氟、氯、溴或碘，例如溴。
所述偶联反应中，所述如式I-2所示的化合物与所述如式I-2所示的化合物的摩尔比可为1：(1-3)，例如1：2.6。
所述偶联反应中，所述碱可为本领域常规碱。所述碱可为碱式碳酸盐(盐中阳离子为碱金属离子，阴离子为碳酸根)，例如K2CO3。
所述偶联反应中，所述如式I-2所示的化合物与所述碱的摩尔比可为1：(1-5)；例如1：3.5。
所述偶联反应中，所述溶剂可为本领域常规溶剂，所述溶剂可为醚类溶剂或/和腈类溶剂。所述醚类溶剂可为甲基叔丁基醚。所述腈类溶剂可为乙腈。所述腈类溶剂与醚类溶剂的体积比可为1：1。
所述偶联反应中，所述如式I-2所示的化合物与所述溶剂的质量体积比可为10-50mg/mL；例如16mg/mL。
所述偶联反应中，所述碘盐可为本领域常规碘盐。所述碘盐可为碱式碘盐，例如KI。
所述偶联反应中，所述如式I-2所示的化合物与所述碘盐的摩尔比可为1：(1-2)；例如1：1.2。
所述偶联反应中，所述偶联反应的反应温度可为本领域常反应温度，优选为50-100℃，例如80℃。
本发明还提供一种脂质载体，其包括物质Z，所述物质Z为如前所述的如式I所示化合物或其药学可接受的盐。
某一优选方案中，所述脂质载体还包括稀释剂。所述稀释剂可为磷酸盐缓冲液或Tris缓冲液等。
某一优选方案中，所述脂质载体还包括磷脂。
某一优选方案中，所述磷脂可为本领域常规磷脂，其为两性辅助性分子，有助于脂质颗粒和细胞膜的融合。所述磷脂可为具有带电极性端和脂肪链非极性端的磷脂类分子，例如二硬脂酰基磷脂酰胆碱(DSPC)、二肉豆蔻酰磷酸胆碱(DMPC)、二油酰磷酸胆碱(DOPC)、棕榈酰磷酸胆碱(DPPC)、1,2-二硬脂酰磷酸胆碱(DSPC)、二十一烷酰磷酸胆碱(DUPC)或棕榈酰磷酸胆碱(POPC)等。
某一优选方案中，所述脂质载体还包括PEG脂质(聚乙二醇修饰的脂质)。
某一优选方案中，所述PEG脂质可为具有聚乙二醇亲水端修饰的脂质分子。所述PEG脂质优选选自PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油和PEG修饰的二烷基甘油中的一种或多种，例如PEG修饰的二肉豆蔻酰甘油(DMG-PEG2000)等。
某一优选方案中，所述脂质载体还包括甾醇。
某一优选方案中，所述甾醇可为本领域常规甾醇，所述甾醇包括动物性、植物性或菌类甾醇。所述甾醇选自胆固醇、谷甾醇、麦角甾醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸和α-生育酚中的一种或多种，例如胆固醇等。
某一优选方案中，所述脂质载体中，所述物质Z与甾醇的摩尔比为0.5-5：1，优选为0.5-3：1，例如0.6-2：1。
某一优选方案中，所述脂质载体中，所述物质Z与甾醇的摩尔比为0.5-5：1，优选为0.5-3：1，例如0.68:1、0.69:1、0.71:1、0.74:1、0.76:1、0.77:1、0.79:1、0.83:1、0.84:1、0.85:1、0.86:1、0.88:1、0.89:1、0.9:1、0.91:1、0.94:1、0.99:1、1.04:1、1.07:1或1.28:1。
某一优选方案中，所述脂质载体中，所述物质Z与甾醇的摩尔比为0.5-5：1，优选为0.5-3：1，例如0.6-2：1；再例如0.66:1、0.68:1、0.69:1、0.70:1、0.71:1、0.72:1、0.74:1、0.76:1、0.77:1、0.79:1、0.82:1、0.83:1、0.84:1、0.85:1、0.86:1、0.87:1、0.88:1、0.89:1、0.9:1、0.91:1、0.92:1、0.93:1、0.94:1、0.97:1、0.99:1、1.04:1、1.07:1、1.1:1、1.16:1、1.23:1、1.28:1、1.30:1、1.32:1、1.41:1、1.52:1、1.58:1、1.64:1、1.65:1、1.74:1、1.79:1或1.96:1。
某一优选方案中，所述脂质载体中，所述物质Z与磷脂的摩尔比为1-15：1，优选为2-8：1，例如3-6：1。
某一优选方案中，所述脂质载体中，所述物质Z与磷脂的摩尔比为1-25：1，优选为2-25：1，例如22.5:1、20:1、17.5:1、15:1、11.25:1、10:1、8.75:1、7.5:1、6.67:1、5:1、4.75:1、4.5:1、4:1、3.9:1、3.6:1、3.3:1、3:1、2.86:1、2.5:1或2.2:1。
某一优选方案中，所述脂质载体中，所述物质Z与PEG脂质的摩尔比为20-130：1，优选为20-80:1，例如20-40：1。
某一优选方案中，所述脂质载体中，所述物质Z与PEG脂质的摩尔比为16-130：1，优选为16-80:1，例如16-40：1；再例如16:1、18:1、20:1、22.5:1、25:1、27.5:1、28.1:1、31.25:1或33.3:1。
某一优选方案中，所述脂质载体中，所述物质Z与PEG脂质的摩尔比为16-130：1，优选为16-80:1，例如16-40：1；再例如16:1、18:1、18.8:1、20:1、21.9:1、22.5:1、25:1、26.9:1、27.5:1、28.1:1、29.6:1、30:1、31.25:1、33.3:1或37.5:1。
某一优选方案中，所述物质Z的摩尔含量约为30mol％至60mol％。
某一优选方案中，所述物质Z的摩尔含量约为30mol％至60mol％；优选为40mol％至55mol％；例如40mol％、43mol％、45mol％、47.4mol％、50mol％、50mol％或55mol％。
本发明中，摩尔含量的含义为某物质占脂质载体的总物质量的百分比，脂质载体中各组分的摩尔含量之和不超过100mol％。某一优选方案中，所述磷脂的摩尔含量约为0mol％至30mol％。
某一优选方案中，所述磷脂的摩尔含量约为0mol％至30mol％；优选为0mol％至18mol％；例如0mol％、2mol％、4mol％、6mol％、8mol％、10mol％、11mol％、12mol％、14mol％、16mol％或18mol％。
某一优选方案中，所述甾醇的摩尔含量约为15mol％至55mol％。
某一优选方案中，所述甾醇的摩尔含量约为15mol％至60mol％，优选为40.4％mol％至58.4mol％， 例如42.4mol％、44.4mol％、46.4mol％、48.4mol％、50.4mol％、52.4mol％或56.4mol％。
某一优选方案中，所述甾醇的摩尔含量约为15mol％至60mol％，优选为40.4％mol％至58.4mol％，例如40.4mol％、41mol％、42.4mol％、43mol％、43.4mol％、44.4mol％、46.4mol％、47.4mol％、48mol％、48.4mol％、49mol％、49.4mol％、49.5mol％、50mol％、50.4mol％、50.5mol％、51mol％、51.4mol％、51.5mol％、52mol％、52.25mol％、52.4mol％、52.5mol％、52.75mol％、53mol％、53.4mol％、54mol％、54.25mol％、54.4mol％、54.5mol％、54.75mol％、55mol％、56mol％、56.4mol％、56.5mol％、57mol％、57.5mol％、58mol％或58.4mol％。
某一优选方案中，当所述脂质载体不包含磷脂时，所述脂质载体中，所述甾醇的摩尔含量约为15mol％至60mol％，优选为40.4％mol％至58.4mol％，例如43mol％、43.4mol％、44.4mol％、46.4mol％、47.4mol％、48mol％、48.4mol％、49mol％、49.4mol％、49.5mol％、50mol％、50.4mol％、50.5mol％、51mol％、51.4mol％、51.5mol％、52mol％、52.4mol％、52.25mol％、52.5mol％、52.75mol％、53mol％、53.4mol％、54mol％、54.25mol％、54.4mol％、54.5mol％、54.75mol％、55mol％、56mol％、56.4mol％、56.5mol％、57mol％、57.5mol％、58mol％或58.4mol％；又例如为52.5mol％至54.5mol％，还例如53mol％至54.5mol％。
某一优选方案中，所述PEG脂质的摩尔含量约为0mol％至10mol％。
某一优选方案中，所述PEG脂质的摩尔含量约为0mol％至10mol％，例如为1.5mol％至2.5mol％；例如1.6mol％或2mol％。
某一优选方案中，所述PEG脂质的摩尔含量约为0mol％至10mol％，所述PEG脂质的摩尔含量可为0.5mol％至2.5mol％，还可为0.5mol％至1.5mol％或者1.5mol％至2.5mol％，例如1.6mol％或2mol％。
某一优选方案中，所述PEG脂质的摩尔含量约为0mol％至10mol％，例如为0.5mol％至2.5mol％，具体地，还例如0.25mol％、0.5mol％、0.75mol％、1mol％、1.5mol％、1.6mol％、2mol％、2.5mol％、3mol％、3.5mol％、4mol％或5mol％；进一步为0.5mol％至2mol％；还可为0.5mol％至1.5mol％或者1.5mol％至2.5mol％；还可为1.6mol％或2mol％。
某一优选方案中，当所述脂质载体不包含磷脂时，或者所述磷脂的含量为4mol％以下时，所述PEG脂质的摩尔含量约为0mol％至10mol％，具体地，例如0.25mol％、0.5mol％、0.75mol％、1mol％、1.5mol％、1.6mol％、2mol％、2.5mol％、3mol％、3.5mol％、4mol％或5mol％；例如为0.25mol％至3mol％，进一步为0.5mol％至2.5mol％，进一步为0.5mol％至2mol％。
某一优选方案中，所述脂质载体由所述物质Z、所述稀释剂、所述磷脂、所述PEG脂质和所述甾醇组成。
某一优选方案中，所述脂质载体由所述物质Z、所述磷脂、所述PEG脂质和所述甾醇组成。
某一优选方案中，所述脂质载体由所述物质Z、所述稀释剂、所述PEG脂质和所述甾醇组成。
某一优选方案中，所述脂质载体由所述物质Z、所述PEG脂质和所述甾醇组成。
某一优选方案中，所述脂质载体不包含磷脂。
某一优选方案中，当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与甾醇的摩尔比可为0.6-2：1；优选为0.68:1、0.69:1、0.7:1、0.77:1、0.85:1、0.86:1、1.04:1或1.28:1。
某一优选方案中，当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与甾醇的摩尔比为0.6-2：1，例如0.68:1、0.69:1、0.70:1、0.71:1、0.72:1、0.74:1、0.76:1、0.77:1、0.79:1、0.82:1、0.83:1、0.84:1、0.85:1、0.86:1、0.87:1、0.88:1、0.89:1、0.9:1、0.91:1、0.92:1、0.93:1、0.94:1、0.97:1、0.99:1、1.04:1、1.07:1、1.1:1、1.16:1、1.23:1、1.28:1、1.30:1、1.41:1、1.52:1或1.58:1。
某一优选方案中，当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与PEG脂质的摩尔比可为16-35：1；优选为16:1、18:1、20:1、22.5:1、25:1、27.5:1或28.1:1。
某一优选方案中，当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与PEG脂质的摩尔比可为16-35：1；再例如16:1、18:1、20:1、21.9:1、22.5:1、25:1、26.9:1、27.5:1、28.1:1、29.6:1或30:1。
本发明还提供一种脂质纳米颗粒，其包括治疗剂和/或预防剂以及前述脂质载体。
某一优选方案中，所述治疗剂和/或预防剂可为一种或两种及以上核酸。所述核酸可为本领域常规核酸。所述治疗剂和/或预防剂可为单链脱氧核糖核酸(DNA)、双链DNA、小干扰RNA(siRNA)、不对称双链小干扰RNA(aiRNA)、微小RNA(miRNA)、小发夹RNA(shRNA)、环状RNA(circRNA)、转运RNA(tRNA)、信使RNA(mRNA)和本领域已知的其他形式的核酸分子，优选为mRNA，例如萤火虫荧光素酶(Fluc)mRNA或SARS-CoV-2刺突蛋白(Spike)mRNA。
某一优选方案中，所述脂质纳米颗粒中氮磷比可为2：1-30：1，所述组合物的氮磷比是指一种或多种可电离脂质化合物中可电离氮原子的摩尔数与RNA中磷酸酯基的摩尔数的比率。优选为2：1-20：1，例如3：1-20：1，还例如3：1-16：1。
某一优选方案中，所述脂质纳米颗粒中，所述脂质载体与治疗剂和/或预防剂的质量比可为3-80:1，优选为6-60:1。
某一优选方案中，所述脂质纳米颗粒的粒径(平均粒径)可为10-200nm，优选为40-150nm，例如60-150nm。
某一优选方案中，所述脂质纳米颗粒的粒径(平均粒径)可为10-200nm，优选为40-150nm，例如60-150nm；再例如50-150nm。
某一优选方案中，所述脂质纳米颗粒中，所述脂质载体包裹所述治疗剂和/或预防剂。
本发明还提供一种组合物，其包括物质Z，所述物质Z为如前所述的如式I所示化合物或其药学可接受的盐。
某一优选方案中，所述组合物还包括稀释剂、磷脂、PEG脂质、甾醇和治疗剂和/或预防剂中的一种或多种。
某一优选方案中，所述组合物中，所述稀释剂、磷脂、PEG脂质、甾醇和治疗剂和/或预防剂如前所述。
某一优选方案中，所述组合物中，所述物质Z与所述稀释剂、磷脂、PEG脂质和甾醇中的一种或多种形成如前所述脂质载体。
某一优选方案中，所述组合物中，所述脂质载体与所述治疗剂和/或预防剂形成如前所述的脂质纳米颗粒。某一优选方案中，所述组合物中，所述治疗剂和/或预防剂的包封率为至少50％，优选为至少为70％。
某一优选方案中，所述组合物中，所述组合物的多分散指数不高于0.5，例如不高于0.3。
如无特别说明，本发明所用术语具有如下含义：
术语“一个或多个”是指1个、2个或3个。
术语“卤素”是指氟、氯、溴或碘。
术语“药学上可接受”是指相对无毒、安全、适合于患者使用。
术语“药学上可接受的盐”是指化合物与药学上可接受的酸或碱反应得到的盐。当化合物中含有相对酸性的官能团时，可以通过在合适的惰性溶剂中用足量的药学上可接受的碱与化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：钠盐、钾盐、钙盐、铝盐、镁盐、铋盐、铵盐等。当化合物中含有相对碱性的官能团时，可以通过在合适的惰性溶剂中用足量的药学上可接受的酸与化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐包括但不限于：盐酸盐、硫酸盐、甲磺酸盐等。具体可参见Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl,Camille G.Wermuth,2011,2nd Revised Edition)。
结构片段中的是指该结构片段通过该位点与分子其余部分相连。例如，是指环己基。
基团末端的“-”是指该基团通过该位点与分子其余部分相连。例如，CH3-C(＝O)-是指乙酰基。
术语“烷基”是指具有指定碳原子数(例如，C1-C6)的、直链或支链的、饱和的一价烃基。烷基包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、正己基等。
术语“亚烷基”为二价基团，其通过两个单键与分子其余部分相连，其余定义同术语“烷基”。
术语“烷氧基”是指基团RX-O-，RX的定义同术语“烷基”。烷氧基包括但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基等。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明提供了一种如式I所示的含氮链状化合物，其结构新颖，其可用做制备脂质纳米颗粒。包含如式I所示的含氮链状化合物的脂质纳米颗粒具有较低的多分散指数，可以高效的递送mRNA。本发明的含氮链状化合物用于制备LNP制剂时，可在磷脂组分低至4mol％以下仍具有很好的性状及递送能力。进一步，本发明的含氮链状化合物用于制备LNP制剂时，在PEG脂质较低情况下制得的LNP制剂仍具有很好的性状及递送能力，从而减少了因PEG脂质高而影响的 疗效及安全性的风险。
附图说明
图1为实施例1制备的各LNP制剂的核酸凝胶电泳结果图；
图2为实施例2中293FT细胞与实施例1制得的LNP制剂共培养18-24小时后测得的化学发光强度；
图3和图4A为实施例3中小鼠静脉注射给药实施例1制得的LNP制剂LQ104-1至LQ104-8后，在不同时间测得的体内总生物发光量；
图4B为实施例3中小鼠肌内注射LNP制剂LQ104-9、LQ104-10或LQ107后测得的体内总抗体滴度；
图5A至图5D为小鼠静脉注射给药(图5A、图5B)或肌内注射给药(图5C、图5D)实施例4中各LNP制剂后，在不同时间测得的体内总生物发光量或给药部位总发光量；
图6A至图6C为小鼠静脉注射给药实施例5中各LNP制剂后，在不同时间测得的体内总生物发光量；
图7A和图7B为小鼠静脉注射给药实施例6中各LNP制剂后，在不同时间测得的体内总生物发光量；
图8为小鼠静脉注射给药实施例7中各LNP制剂后，在不同时间测得的体内总生物发光量；
图9A和图9B为小鼠静脉注射给药实施例8中各LNP制剂后，在不同时间测得的体内总生物发光量；
图10A至图10C为小鼠静脉注射给药实施例10中各LNP制剂后，在不同时间测得的体内总生物发光量；
图11A至图11C为小鼠静脉注射给药实施例11中各LNP制剂后，在不同时间测得的体内总生物发光量；
图12A至图12D为小鼠静脉注射给药(图12A、图12B)或肌内注射给药(图12C、图12D)实施例12中各LNP制剂后，在不同时间测得的体内总生物发光量或给药部位总发光量。
图13A和图13B为小鼠静脉注射给药实施例13中各LNP制剂后，在不同时间测得的体内总生物发光量。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
制备例1制备化合物LQ104
LQ104的制备
物料配比：
操作过程：
反应瓶中加入LQ001-1、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI、甲基叔丁基醚和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全。
后处理：
反应液过滤后旋干，柱层析纯化后得到1.1g无色油状物。
1HNMR(400MHz，CDCl3)δ：4.86(p，2H)，3.65-3.59(m，4H)，2.68-2.59(m，8H)，2.57-2.49(m，4H)，2.27(t，4H)，1.61(p，5H)，1.49(dt，12H)，1.28(d，61H)，0.87(t，12H)。
制备例2制备化合物LQ107
反应式：
物料配比：

操作过程：
反应瓶中加入LQ107-1、丙二酸、DCC、DMAP和DCM，室温下搅拌反应12h，TLC(DCM:MeOH＝20:1)显示反应完全。
后处理：
反应液用硅藻土过滤后旋干，柱层析纯化后得700mg无色油状物，收率70％。
1HNMR(400MHz，CDCl3)δ：4.86(p，2H)，4.07(dt，8H)，2.66(t，4H)，2.49-2.38(m，8H)，2.28(q，8H)，2.05(s，5H)，1.62(q，17H)，1.83-1.36(m，17H)，1.27(d，90H)，0.87(t，18H)。
制备例3制备化合物LQ104-E15b-1
E15b-1的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入6-溴己酸、DCC、DMAP和DCM，再加入8-十五醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到15g无色油状物。
LQ104-E15b-1的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E15b-1、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到800mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.86(p,J＝6.2Hz,2H),3.62(t,J＝4.9Hz,4H),2.67–2.59(m,8H),2.55(t,J＝8.0Hz,4H),2.28(t,J＝7.5Hz,4H),1.64(p,J＝7.5Hz,4H),1.50(tt,J＝8.5,4.5Hz,12H),1.34–1.20(m,46H),0.87(t,J＝7.0Hz,12H)。
制备例4制备化合物LQ104-E15b-2
E15b-2的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入5-溴戊酸、DCC、DMAP和DCM，再加入9-十七醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到13.8g无色油状物。
LQ104-E15b-2的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E15b-2、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到760mg无色油状物。
1H NMR(600MHz,CDCl3)δ:4.85(p,J＝6.2Hz,2H),3.70(t,J＝4.9Hz,4H),2.85–2.69(m,12H),2.32(t,J＝7.0Hz,4H),1.65–1.54(m,9H),1.49(q,J＝6.4Hz,8H),1.25(d,J＝9.9Hz,50H),0.87(t,J＝7.0Hz,12H)。
制备例5制备化合物LQ104-E15b-3
E15b-3的制备
反应式：
物料配比：

操作过程：
在1L的反应瓶中加入4-溴丁酸、DCC、DMAP和乙腈，再加入10-十九醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到15.3g无色油状物。
LQ104-E15b-3的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E15b-3、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到820mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.85(p,J＝6.2Hz,2H),3.69(t,J＝4.8Hz,4H),2.76(s,8H),2.66(t,J＝8.2Hz,4H),2.27(t,J＝7.5Hz,4H),1.61(p,J＝7.3Hz,4H),1.50(dq,J＝12.3,6.4,5.3Hz,12H),1.35–1.20(m,54H),0.87(t,J＝7.0Hz,12H)。
制备例6制备化合物LQ104-E16b-1
E16b-1的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入7-溴庚酸、DCC、DMAP和DCM，再加入8-十五醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.5g无色油状物。
LQ104-E16b-1的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E16b-1、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到730mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.85(p,J＝6.2Hz,2H),3.61(t,J＝4.9Hz,4H),2.67–2.60(m,8H),2.54(t,J＝7.9Hz,4H),2.27(t,J＝7.5Hz,4H),1.61(p,J＝7.5Hz,4H),1.50(qd,J＝7.6,3.4Hz,12H),1.37–1.20(m,50H),0.87(t,J＝7.0Hz,12H)。
制备例7制备化合物LQ104-E16b-2
E16b-2的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入6-溴己酸、DCC、DMAP和DCM，再加入9-十七醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.4g无色油状物。
LQ104-E16b-2的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E16b-2、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到770mg无色油状物。
1H NMR(600MHz,CDCl3)δ:4.85(p,J＝6.2Hz,2H),3.65(t,J＝4.8Hz,4H),2.73–2.65(m,8H),2.61(t,J＝8.1Hz,4H),2.28(t,J＝7.4Hz,4H),1.64(p,J＝7.5Hz,4H),1.51(dq,J＝19.0,7.3,6.8Hz,12H),1.35–1.20(m,54H),0.87(t,J＝6.9Hz,12H)。
制备例8制备化合物LQ104-E16b-3
E16b-3的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入6-溴己酸、DCC、DMAP和DCM，再加入7-十五醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到13.3g无色油状物。
LQ104-E16b-3的制备
反应式：
物料配比：

操作过程：
反应瓶中加入E16b-3、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到660mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.85(p,J＝6.3Hz,2H),3.65(t,J＝4.8Hz,4H),2.69(d,J＝5.2Hz,8H),2.62(d,J＝8.2Hz,4H),2.28(t,J＝7.4Hz,4H),1.64(p,J＝7.6Hz,4H),1.51(dq,J＝18.9,6.9,6.0Hz,12H),1.35–1.18(m,46H),0.87(t,J＝6.9Hz,12H)。
制备例9制备化合物LQ104-E16b-3R
E16b-3R的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入2-己基十一酸、DCC、DMAP和DCM，再加入5-溴-1-戊醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.2g无色油状物。
LQ104-E16b-3R的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E16b-3R、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到840mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.06(t,J＝6.6Hz,4H),3.70(t,J＝4.8Hz,4H),2.73(d,J＝51.1Hz,12H),2.30(tt,J＝8.8,5.3Hz,2H),1.66(p,J＝6.9Hz,4H),1.57(p,J＝7.9Hz,8H),1.46–1.39(m,4H),1.35(p,J＝7.5Hz,4H),1.32–1.19(m,42H),0.87(t,J＝6.9Hz,12H)。
制备例10制备化合物LQ104-E17b-1
E17b-1的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入8-溴辛酸、DCC、DMAP和DCM，再加入8-十五醇，加完后室温下搅拌 反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.8g无色油状物。
LQ104-E17b-1的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E17b-1、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到750mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.86(p,J＝6.2Hz,2H),3.64(t,J＝4.8Hz,4H),2.68(d,J＝6.8Hz,8H),2.58(t,J＝8.0Hz,4H),2.27(t,J＝7.5Hz,4H),1.61(p,J＝7.2Hz,4H),1.49(qd,J＝7.7,5.2,4.2Hz,12H),1.36–1.20(m,54H),0.87(t,J＝7.0Hz,12H)。
制备例11制备化合物LQ104-E17b-2
E17b-2的制备
反应式：
物料配比：

操作过程：
在1L的反应瓶中加入7-溴庚酸、DCC、DMAP和DCM，再加入9-十七醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到15.1g无色油状物。
LQ104-E17b-2的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E17b-2、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到680mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.85(p,J＝6.2Hz,2H),3.67(t,J＝4.9Hz,4H),2.73(s,8H),2.64(t,J＝8.0Hz,4H),2.27(t,J＝7.5Hz,4H),1.61(p,J＝7.4Hz,4H),1.51(dd,J＝13.6,7.0Hz,12H),1.38–1.19(m,58H),0.87(t,J＝7.0Hz,12H)。
制备例12制备化合物LQ104-E17b-3
E17b-3的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入7-溴庚酸、DCC、DMAP和DCM，再加入7-十五醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到12.8g无色油状物。
LQ104-E17b-3的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E17b-3、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到590mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.86(p,J＝6.3Hz,2H),3.63(t,J＝4.8Hz,4H),2.66(dd,J＝9.9,5.1Hz,8H),2.56(t,J＝8.0Hz,4H),2.27(t,J＝7.4Hz,4H),1.62(p,J＝7.5Hz,4H),1.49(dd,J＝10.3,4.9Hz,12H),1.38–1.20(m,50H),0.87(t,J＝7.0Hz,12H)。
制备例13制备化合物LQ104-E17b-3R
E17b-3R的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入2-己基十一酸、DCC、DMAP和DCM，再加入6-溴-1-己醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.5g无色油状物。
LQ104-E17b-3R的制备
反应式：
物料配比：

操作过程：
反应瓶中加入E17b-3R、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到770mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.05(t,J＝6.6Hz,4H),3.66(t,J＝4.8Hz,4H),2.66(d,J＝52.0Hz,12H),2.30(tt,J＝8.9,5.3Hz,2H),1.66–1.48(m,12H),1.46–1.20(m,54H),0.87(td,J＝7.0,1.5Hz,12H)。
制备例14制备化合物LQ104-E17b-4
E17b-4的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入6-溴己酸、DCC、DMAP和乙腈，再加入8-十七醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到15.2g无色油状物。
LQ104-E17b-4的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E17b-4、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到840mg无色油状物。
1H NMR(600MHz,CDCl3)δ：4.85(p,J＝6.3Hz,2H),3.67(t,J＝4.8Hz,4H),2.74(s,8H),2.66(t,J＝8.0Hz,4H),2.29(t,J＝7.4Hz,4H),1.64(p,J＝7.5Hz,4H),1.52(dq,J＝26.5,6.9,6.0Hz,12H),1.37–1.19(m,54H),0.87(t,J＝6.9Hz,12H)。
制备例15制备化合物LQ104-E18b-2
E18b-2的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入8-溴辛酸、DCC、DMAP和DCM，再加入7-十五醇，加完后室温下搅拌 反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.6g无色油状物。
LQ104-E18b-2的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E18b-2、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到750mg无色油状物。
1H NMR(600MHz,CDCl3)δ:4.86(p,J＝6.3Hz,2H),3.62(t,J＝4.9Hz,4H),2.68–2.60(m,8H),2.55(t,J＝8.0Hz,4H),2.27(t,J＝7.5Hz,4H),1.61(t,J＝7.4Hz,4H),1.49(p,J＝7.7,6.7Hz,12H),1.35–1.21(m,54H),0.87(t,J＝7.0Hz,12H)。
制备例16制备化合物LQ104-E18b-2R
E18b-2R的制备
反应式：
物料配比：

操作过程：
在1L的反应瓶中加入2-己基十一酸、DCC、DMAP和DCM，再加入7-溴-1-庚醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到14.1g无色油状物。
LQ104-E18b-2R的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E18b-2R、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到690mg无色油状物。
1H NMR(600MHz,CDCl3)δ:4.05(t,J＝6.7Hz,4H),3.77(t,J＝4.8Hz,4H),2.96–2.87(m,8H),2.81(t,J＝8.2Hz,4H),2.30(tt,J＝8.9,5.3Hz,2H),1.65–1.52(m,12H),1.46–1.38(m,4H),1.37–1.19(m,54H),0.87(td,J＝7.1,1.5Hz,12H)。
制备例17制备化合物LQ104-E18b-3
E18b-3的制备
反应式：
物料配比：
操作过程：
在1L的反应瓶中加入7-溴庚酸、DCC、DMAP和DCM，再加入8-十七醇，加完后室温下搅拌反应12h。TLC(PE:EA＝20:1)显示反应完全(产物rf值为0.6)。
后处理：
反应液过滤后旋干，柱层析纯化后得到13.7g无色油状物。
LQ104-E18b-3的制备
反应式：
物料配比：
操作过程：
反应瓶中加入E18b-3、N,N'-双(2-羟乙基)乙二胺、K2CO3、KI和乙腈，加热至80℃搅拌反应12h。 TLC(DCM:MeOH＝10:1)显示反应完全(产物rf值为0.5)。
后处理：
反应液过滤后旋干，柱层析纯化后得到790mg无色油状物。
1H NMR(600MHz,CDCl3)δ:4.86(p,J＝6.2Hz,2H),3.61(t,J＝4.8Hz,4H),2.67–2.60(m,8H),2.54(t,J＝8.0Hz,4H),2.27(t,J＝7.5Hz,4H),1.62(p,J＝7.5Hz,4H),1.49(tt,J＝7.6,4.6Hz,12H),1.37–1.20(m,58H),0.87(t,J＝6.9Hz,12H)。
制备例18制备化合物LQ104-H3
LQ104-H3的制备路线为：
物料配比：
操作过程：
在1L的反应瓶中加入LQ001-1、N,N'-二(2-羟乙基)-1,3-丙二胺、K2CO3、KI、和乙腈，加热至80℃，搅拌反应12小时。TLC(DCM：MeOH＝10：1)显示反应完全。
后处理：
反应液过滤后旋干，柱层析纯化后得到850mg无色油状物，即为所述化合物LQ104-H3，收率为43％。
质谱分析：
ESI-MS正离子质谱图中在m/z924处和925处有较强离子峰，与该化合物923.5的分子量吻合。
1HNMR(400MHz，CDCl3)δ：4.86(p，2H)，3.65-3.59(m，4H)，2.68-2.59(m，8H)，2.57-2.49(m，4H)，2.27(t，4H)，1.61(p，7H)，1.49(dt，12H)，1.28(d，61H)，0.87(t，12H)。
本申请前述制备例的试剂来源如下：
LQ001-1：自制；
N,N'-双(2-羟乙基)乙二胺：购自阿达玛斯试剂有限公司，货号：013455310，纯度：RG，98％；
K2CO3：购自上海易恩化学技术有限公司，货号：RH425011，纯度：AR，99％；
KI：购自上海易恩化学技术有限公司，货号：RH432132，纯度：AR，99％；
乙腈：购自上海泰坦科技股份有限公司，货号：01111797，纯度：AR，≥99.0％；
甲基叔丁基醚：购自上海泰坦科技股份有限公司，货号：01030342，纯度：AR，≥99.0％；
丙二酸：购自阿达玛斯试剂有限公司，货号：01022573，纯度：RG，99％；
DCC：二环己基碳二亚胺，购自阿达玛斯试剂有限公司，货号：012041444，纯度：RG，99％；
DMAP：4-二甲氨基吡啶，购自阿达玛斯试剂有限公司，货号：01271081，纯度：RG，99％；
DCM：二氯甲烷，购自上海泰坦科技股份有限公司，货号：01111853，纯度：AR，≥99.5％；
硅藻土：购自上海泰坦科技股份有限公司，货号：01589000，纯度：特优级，≥89.0％，200目；
8-十五醇：自制；
6-溴己酸：购自阿达玛斯试剂有限公司，货号：01073739，纯度：RG，98％+；
N,N'-双(2-羟乙基)乙二胺：购自阿达玛斯试剂有限公司，货号：013455310，纯度：RG，98％；
9-十七醇：购自大连锐盈科技有限公司，纯度：98％；
5-溴戊酸：购自毕得医药，货号：BD9634，纯度：98％；
10-十九醇：自制；
4-溴丁酸：购自上海泰坦科技股份有限公司，货号：011016520，纯度：RG，99％+；
7-溴庚酸：购自上海泰坦科技股份有限公司，货号：012345536，纯度：RG，98％；
7-十五醇：自制
5-溴-1-戊醇：购自毕得医药，纯度：98％；
2-己基十一酸：购自毕得医药，货号：BD75392，纯度：98％；
8-溴辛酸：购自江苏艾康，纯度：98％；
6-溴-1-己醇：购自阿达玛斯试剂有限公司，货号：01074359，纯度：RG，98％；
8-十七醇：自制
7-溴-1-庚醇：购自阿达玛斯试剂有限公司，货号：01001821，纯度：RG，98％；
N,N'-双(2-羟乙基)-1,3-丙二胺(购自北京维赛化学，纯度：95％)。
实施例1
实施例1用于验证由本申请公开的可电离脂质化合物制得的脂质纳米粒(Lipid Nanoparticle，LNP)制剂是否能有效包载mRNA并维持mRNA的结构完整。将制备例1和制备例2最终制得的可电离脂质化合物、二硬脂酰基磷脂酰胆碱(DSPC，购自日本精化株式会社，货号：S01005)、胆固醇(购自日本精化株式会社，货号：O01001)和二肉豆蔻酰甘油-聚乙二醇2000(DMG-PEG2000，购自国邦药业，货号：O02005)分别溶于乙醇(厂家：南京化学试剂股份有限公司，纯度99.6％)溶液，继而依 据一定的摩尔比混合，制得混合脂质的乙醇溶液，其中脂质总浓度为12.5mM(本申请出现的计量单位“M”是指mol/L)。将自制的萤火虫荧光素酶(Fluc)mRNA或自制的SARS-CoV-2刺突蛋白(Spike)mRNA(SARS-CoV-2刺突蛋白mRNA参见Tan,S.等人，bioRxiv 2022.05.10.491301.)在pH为4.0的50mM的柠檬酸盐缓冲液中稀释得到mRNA溶液。通过使用微流控装置，控制流速为12mL/min，控制混合脂质的乙醇溶液与前述步骤制得的mRNA溶液的体积比为1：3，按可电离脂质与mRNA的氮磷比为3-15：1制备脂质纳米颗粒。经0.01M的磷酸盐缓冲液(PBS)透析12至24小时除去乙醇。最后，LNP溶液通过孔径为0.22μm的无菌过滤器(厂家：Millex，货号：SLGPR33RB)过滤，并经超滤浓缩(厂家：Amicon-Ultra，截留分子量：10KDa)得到由本申请所述的可电离脂质与DSPC、胆固醇及DMG-PEG2000包封Fluc mRNA或Spike mRNA得到的LNP制剂。其中，可电离脂质化合物与DSPC、胆固醇和DMG-PEG2000的摩尔比，以及可电离脂质与mRNA的氮磷比如表1所示。使用动态光散射法，通过Malvern Zetasizer Ultra仪器(厂家：马尔文)测定各LNP制剂的粒径大小及多分散指数(Polymer dispersity index，PDI)；使用Quant-it Ribogreen RNA定量测定试剂盒(厂家：ThermoFisher Scientific，货号：R11490)测定LNP的包封率；通过核酸凝胶电泳考察mRNA完整性(电泳仪，厂家：上海天能)，测试结果见表1及图1。图1为本实施例各LNP制剂的核酸凝胶电泳图。其中，所述凝胶为1％的琼脂糖凝胶(厂家：Biowest；货号：BY-R0100)，测试条件为160V电泳20分钟。
表1

在本领域中，PDI小于0.3，则说明该LNP制剂中纳米颗粒大小相对均匀；包封率用来说明LNP是否能有效包载mRNA，包封率高于70％，则说明该LNP可有效包载mRNA；琼脂糖凝胶电泳图中条带单一且明亮可说明mRNA结构完整。其中，PDI越趋于0越好，包封率越趋于100％越好。由表1及图1可见，本申请的各LNP粒径介于70-120nm之间，PDI均小于0.3，包封率均高于80％；具体地，LQ104系列的包封率稳定在90％以上，而由LQ107制得的LNP制剂包封率为83.7％。可见，按照表1所示的摩尔比及氮磷比制得的LNP制剂均可有效包载mRNA并维持mRNA结构完整。且表1以及本申请未穷举罗列的实验数据也可看出，由LQ104系列的性能表现好于LQ107系列。此外，根据本申请已验证但未穷举罗列的实验数据来看，无论包载何种mRNA，即便进行上述体外实验得到的数据略有差异，但是对上述结论并无影响。
实施例2
在该实施例中，我们通过体外细胞实验来验证本申请LNP制剂的体外细胞递送及表达。以每孔1万个293FT细胞铺种在96孔板中，过夜培养至细胞贴壁。将实施例1的LNP制剂LQ104-1至LQ104-8按每孔含100纳克(ng)的mRNA计，分别加入96孔板的细胞培养液中。加入LNP制剂前，将细胞培养液替换为无抗生素、含10％胎牛血清的DMEM培养液(厂家：Gibco，货号：C11995500BT)，继续培养24小时，然后弃去细胞培养液，使用添加含有D-荧光素钾盐(厂家：PerkinElmer，货号：122799，终浓度1mM)和ATP(厂家：ApexBio，货号：C6931，终浓度2mM)的细胞裂解液按100μL/孔的剂量裂解细胞。采用酶标仪(厂家：thermo scientific)检测化学发光强度。测试结果见图2。在图2中，PBS为阴性对照，此组对应的化学发光强度读数可视为背景读值。化学发光强度读数越高，说明表达的越高。由图2可见，相比于PBS组，LQ104-1至LQ104-8组读数明显增加，表明各LNP均可有效递送Fluc mRNA进入细胞并表达。
实施例3
在该实施例中，按5μg/只的剂量将实施例1的LQ104-1至LQ104-8(包载Fluc mRNA)通过尾静脉注射到6至8周龄的雌性Balb/C小鼠(维通利华)体内(n＝3，即每组采用3只小鼠进行注射和测试，所呈现的数据结果为各组的测定均值)，并在给药后特定的时间节点(本实施例中为第6小时、第24小时、第48小时)腹腔注射D-荧光素钾盐，然后经IVIS Spectrum小动物活体成像仪(厂家：PerkinElmer)检测发光情况，统计小鼠活体表达部位(例如肝脏等部位)的总发光强度，发光强度越高则代表荧光素酶表达越高，即相应的LNP制剂在小鼠体内表达越好。该总发光强度是通过生物发光成像测量，在腹腔内注射D-荧光素钾盐的6至15分钟(min)后，发光部位的发光强度数据。通过Living Image软件(厂家：PerkinElmer)统计活体表达区域的总发光强度，进一步，通过GraphPad软件计算曲线下面积(AUC，单位：p/s*小时)，本申请各实施例中的AUC为药后第4小时或第6小时(采用本申请的实验方法，药后3-6小时的峰值相同)至药后第48小时总发光强度测量点连线的曲线下面积。测试结果如图3和4A及表2所示。
表2(图4A的AUC数据)
注：E+10为科学计数法的表示方式，表示10的10次方；例如E+11表示10的11次方，下同。
通常情况下，未进行给药处理的小鼠经活体成像仪检测总发光强度的读数数量级为105。由如图3和4A及表2可见，LQ104-1至LQ104-8在小鼠体内均有较强表达。进一步地在本申请的各实施例中，我们通过观察测量点连线的曲线下面积(AUC)来确定表达更好的LNP制剂，曲线下面积越大，则表达效果越好。
按2μg/只的剂量将LQ104-9、LQ104-10、LQ107(包载Spike mRNA)通过肌肉注射到6至8周龄的雌性Balb/C小鼠体内，在第一次注射的第21天重复注射相同的LNP制剂。并在第二次给药后 特定的时间节点(本次实施例数据为第二次给药后的第7天)采集小鼠全血。将采集的血液在4℃、2000×g的条件下离心10min，从全血中分离出血清；随后，将血清在56℃的水浴中灭活30min，并在-80℃下保存以用于分析。经酶联免疫吸附(ELISA)的方法测定血清中总抗体滴度。具体地，使用SARS-CoV-2(2019-nCoV)Spike S1+S2 ECD-His Recombinant Protein(厂家：义翘神州，货号：40589-V08B1)包被抗原，用SARS-CoV-2(2019-nCoV)Spike Neutralizing Antibody Mouse Mab(厂家：义翘神州，货号：40591-MM43)做对照，2％牛血清白蛋白(BSA)封闭，Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L)(Jackson ImmunoResearch，货号：115-035-003)二抗孵育，按照说明书，使用TMB(Invitrogen，货号：00-4201-56)显色进行酶联免疫吸附测定(ELISA)分析，测得Spike抗体滴度(该数据为第二次给药后第7天测定的小鼠血清中的总抗体滴度，n＝8)，测试结果见图4。
由图4可见，与注射PBS的动物相比，注射LQ104-9和LQ104-10的动物的Spike蛋白总抗体滴度显著增加(通过ANOVA进行统计学分析，**p＜0.01，***p＜0.001，与注射PBS的动物组相比)，表明给药后载体递送的mRNA有效表达，并诱导免疫反应。注射LQ107的动物组总抗体滴度未见增加。
实施例4
本实施例选取制备例3至制备例17所制得的可电离脂质，按照与实施例1相同的方法，根据表3所示的摩尔比及氮磷比制备LNP制剂(包载Fluc mRNA)。并使用Malvern Zetasizer Ultra测定各LNP制剂的粒径大小、PDI及表面电势；使用Quant-it Ribogreen RNA定量测定试剂盒(厂家：ThermoFisher Scientific，货号：R11490)测定LNP的包封率；使用6-(p-Toluidino)-2-naphthalene sulfonic acid sodium salt(TNS，购自南京熙泽医药科技有限公司，货号：XZ0743)染料结合试验测量LNP的pKa。
表3


由表3可见，由制备例3至制备例17所述的可电离脂质各自制备的LNP制剂的粒径均介于50-100nm之间；PDI均小于0.3，具体在0.044至0.098之间；包封率均高于96％。说明该实施例中的LNP制剂均可有效包载mRNA；表面电势均为弱负电，pKa介于6-7.3之间，与本领域所公认的范围相当。
进一步地，参考实施例3的小鼠体内试验方法，对该实施例中各组LNP制剂按5μg/只的剂量通过尾静脉注射或下肢肌内注射到6至8周龄雌性Balb/C小鼠体内，统计肝脏部位或下肢给药部位的总发光强度，测试结果见图5A至图5D及表4A至表4D。其中图5A和图5B显示静脉注射给药后小鼠肝脏部位发光统计结果，图5C及图5D显示肌内注射给药后小鼠下肢给药部位发光统计结果。由图5A至图5D及表4A至表4D可见，该实施例测试的LNP制剂在小鼠体内均有较强表达，说明制备例3至制备例17所述的可电离脂质对应的LNP制剂均可有效递送mRNA至体内并表达。我们也测试了其他给药途径，如腹腔注射给药及皮下注射给药等，结果显示多种给药途径下该实施例中的LNP制剂的均能表达。
表4A(图5A的AUC数据)
表4B(图5B的AUC数据)
表4C(图5C的AUC数据)
表4D(图5D的AUC数据)
实施例5
本实施例选择根据制备例7得到的LQ104-E16b-2、制备例14得到的LQ104-E17b-4以及制备例17得到的LQ104-E18b-3，各分成8组，按照下表5所述的制剂组分和氮磷比制备LNP制剂(包载Fluc mRNA)。并使测定它们的粒径、PDI和包封率，结果如表5所示。
表5


由表5可见，由化合物LQ104-E16b-2、LQ104-E17b-4或LQ104-E18b-3按照上述组分及氮磷比制备的LNP制剂的粒径介于60-90nm之间；PDI均小于0.3，且大部分集中在0.1以下；包封率均高于90％，且主要集中在97％-99％之间。
同样地，参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例制备的所有LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，并统计小鼠活体肝脏部位总发光强度，测试结果见图6A至图6C及表6A至表6B。可见，该实施例中的LNP制剂在小鼠体内均有较强表达。
表6A(图6A AUC数据)
表6B(图6B AUC数据)
实施例6
该实施例选取通过制备例7得到的LQ104-E16b-2作为可电离脂质，按照表7中的摩尔比及氮磷比，采用与实施例1相同的方式制备21种LNP制剂(包载Fluc mRNA)，并测定它们的粒径、PDI及包封率。该实施例主要用于验证DSPC和胆固醇在LNP制剂中的优选组分含量。其中，以四个组分的总摩尔数为100％计，保持LQ104-E16b-2和DMG-PEG的摩尔百分含量不变，验证DSPC和胆固醇的摩尔百分含量变化所制得的各LNP制剂的表现。
表7

由表7可见，由LQ104-E16b-2制得的各组LNP制剂粒径介于60-110nm之间；PDI均小于0.3，具体介于0.053至0.166之间；包封率均高于90％，且均高于96.8％。
同样地，参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例制备的所有LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，并采用与实施例3相同的方法统计肝脏区域的总发光强度，测试结果见图7A及图7B及表8A至表8B。可见，该实施例测试的LNP制剂在小鼠体内均有较强表达。其中，LQ104-E16b-2(DS-f1)至LQ104-E16b-2(DS-f10)的总发光强较高，表明DSPC的摩尔百分含量介于0％-18％对应的LNP制剂的体内递送能力更好。
表8A(图7A的AUC数据)
表8B(图7B的AUC数据)

实施例7
与实施例6不同的是，本实施例将LNP制剂组分中的磷脂DSPC更换为DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin，二油酰磷脂酰乙醇胺)，并按照与实施例1同样的方法制备LNP制剂(包载Fluc mRNA)，以测试采用DOPE作为磷脂，组分从0％至22％变化所制得的LNP制剂的粒径、PDI和包封率，如表9所示。
表9

由表9可见，该实施例制备的LNP制剂粒径介于70-100nm之间，PDI均小于0.3，包封率均高于96％。
参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例制备的各LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，统计小鼠活体肝脏部位总发光强度，测试结果见图8及表10。可见，该实施例制备的各LNP制剂在小鼠体内均有较强表达。此外，我们测试了多种磷脂分子，所制LNP制剂均可实现体内递送及表达。
表10(图8的AUC数据)

实施例8
该实施例选取通过制备例7得到的LQ104-E16b-2作为可电离脂质，按照表11中的摩尔比及氮磷比，参考实施例1的方式制备LNP制剂(包载Fluc mRNA)。与实施例1不同的是，该实施例的mRNA稀释于25mM、pH为5.0的醋酸钠溶液，透析时采用20mM、pH为7.5的Tris-醋酸溶液。测定该实施例中所有LNP制剂的粒径、PDI及包封率。该实施例主要用于验证可电离脂质在LNP制剂中的优选组分含量。我们采用摩尔百分含量分别为0％、2％、4％、10％的DSPC固定搭配摩尔百分含量为1.6％的PEG脂质，同步筛选了几个特定组分的可电离脂质的含量范围(胆固醇在该实施例中用于在另外三个组分确定后补足余量)。
表11


由表11可见，该实施例制备的LNP制剂粒径介于55-120nm之间，PDI均小于0.3，包封率均高于90％。
参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例制备的所有LNP试剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，并统计小鼠活体肝脏部位总发光强度，测试结果见图9A及图9B及表12A至表12B。可见，该实施例制备的所有LNP制剂在小鼠体内均有较强表达。综合我们对系列可电离脂质组分含量的筛选测试，结果表明，可电离脂质介于40％-55％范围内对应LNP制剂的体内递送效果更好。
表12A(图9A的AUC数据)
表12B(图9B的AUC数据)
实施例9
将实施例6制备的LNP制剂LQ104-E16b-2(DS-f1)至LQ104-E16b-2(DS-f7)在4℃的环境下储存14天，并测定它们的粒径和PDI)，结果见表13。
表13
由表13可见，LQ104-E16b-2按照不同的DSPC含量制备得到的LNP制剂在4℃的条件下放置14天后，粒径变量小于5nm，表明样品稳定性较佳。同样地，我们选取本申请的其他LNP制剂，也能获得相当的效果。
另外，将实施例8制备的LNP制剂LQ104-E16b-2(ION-f3)、LQ104-E16b-2(ION-f6)、LQ104-E16b-2(ION-f7)、LQ104-E16b-2(ION-f8)、LQ104-E16b-2(ION-f13)、LQ104-E16b-2(ION-f14)、LQ104-E16b-2(ION-f20)按0.3mL分装后，在-80℃下储存。需要说明的是，本申请的各实验在样品冻存时需要补加终浓度8％的蔗糖作为保护剂，冻存时间为6h以上，以确保样品能够完全冻住；样品 复融时，将样品置于4℃下的时间不短于90min，通过观察，采用该条件能够确保LNP制剂完全解冻。将所有样品冻融5次，测定它们的粒径和PDI等。结果见表14。
表14
由表14可见，除LQ104-E16b-2(ION-f13)组外，其他各组LNP制剂在-80℃下冻融5次后，粒径变化均较小，变量小于10nm，初步表明样品稳定性较佳。
实施例10
参考实施例8的方式，按照表15中的摩尔比及氮磷比，制备LNP制剂(包载Fluc mRNA)。与实施例8同样地，该实施例的mRNA溶液稀释于25mM、pH 5.0的醋酸钠溶液，透析时溶液为20 mM、pH 7.5Tris-醋酸溶液。该实施例主要用来验证本申请的LNP制剂采用三组分(可电离脂质、胆固醇和DMG-PEG)制备和四组分(可电离脂质、磷脂、胆固醇和DMG-PEG)制备的性能，并考察DMG-PEG在LNP制剂中的优选组分含量，在该实施例中我们选取DMG-PEG的摩尔百分含量在1.5％-5％的范围内进行实验。测定各LNP的粒径、PDI和包封率，结果见表15。
表15


由表15可见，LQ104系列无论采用三组分还是四组分制备，其粒径介于60-115nm之间，PDI均小于0.3，包封率均高于96％。
参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例的各组LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，统计小鼠活体肝脏部位总发光强度，测试结果见图10A、图10B及图10C及表16A至表16C。可见，该实施例的所有LNP制剂在小鼠体内均有较强表达。并且，通过已进行的实验也表明，当PEG脂质的摩尔百分含量在1.5％-2.5％之间时表达效果更好；更加地，PEG脂质的摩尔百分含量为1.5％-2.0％。此外，我们测试了多种PEG脂质，所制LNP制剂均可实现体内递送及表达。
表16A(图10A的AUC数据)
表16B(图10B的AUC数据)
表16C(图10C的AUC数据)
实施例11
参考实施例8的方式，按照表17中的摩尔比及氮磷比，制备LNP制剂(包载Fluc mRNA)。该实施例主要用来验证采用不同的氮磷比制备的LNP制剂的性能。我们基于前述实施例中验证得到的较佳的可电离脂质及DSPC的摩尔百分含量的范围，将氮磷比分别设置为4：1至8：1制备LNP制剂。测定各LNP制剂的粒径、PDI和包封率，结果见表17。
表17

由表17可见，该实施例的LNP制剂粒径介于60-120nm之间，PDI均小于0.3，包封率均高于96％。
参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例的各组LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，并统计小鼠活体肝脏部位总发光强度，测试结果见图11A、图11B及图11C及表18A至表18C。可见，氮磷比在4：1至8：1范围内时，LNP制剂在小鼠体内均有较强表达。
表18A(图11A的AUC数据)
表18B(图11B的AUC数据)
表18C(图11C的AUC数据)
实施例12
参考实施例8的方式，按照表19中的摩尔比及氮磷比，制备LNP制剂(包载Fluc mRNA)。该实施例选取制备例3至制备例17所制得的可电离脂质，该实施例主要用来验证本申请中的三组分(可电离脂质、胆固醇和DMG-PEG)LNP制剂的性能。测定各LNP的粒径、PDI和包封率，结果见表19。
表19

由表19可见，该实施例的LNP制剂的粒径均介于60-130nm之间，PDI均小于0.3，包封率均高于90％。
参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例的各组LNP制剂通过尾静脉注射或下肢肌内注射到6至8周龄的雌性Balb/C小鼠体内，并统计肝脏部位(采用尾静脉注射给药时 表达的主要区域)及下肢给药部位(采用下肢肌内注射给药时主要关注的表达区域)总发光强度，测试结果见图12A至图12D及表20A至表20D。其中图12A和图12B显示静脉注射给药后小鼠肝脏部位的发光统计结果，图12C及图12D显示肌内注射给药后小鼠下肢给药部位的发光统计结果。由图12A至图12D可见，该实施例的所有LNP制剂在小鼠体内均有较强表达。
表20A(图12A的AUC数据)
表20B(图12B的AUC数据)
表20C(图12C的AUC数据)
表20D(图12D的AUC数据)
实施例13
该实施例选取通过制备例7得到的LQ104-E16b-2作为可电离脂质，参考实施例8的方式，按照表21中的摩尔比及氮磷比，制备16种LNP制剂(包载Fluc mRNA)，并测定它们的粒径、PDI及包 封率。
表21

由表21可见，当DMG-PEG2000介于0.5mol％至3mol％时，由LQ104-E16b-2制得的各组LNP制剂粒径介于70至150nm之间；PDI均小于0.3；包封率均高于90％，且均高于95.6％。
同样地，参考实施例3的小鼠体内试验方法，按5μg/只的剂量将该实施例制备的所有LNP制剂通过尾静脉注射到6至8周龄的雌性Balb/C小鼠体内，并采用与实施例3相同的方法统计肝脏区域的总发光强度，测试结果见表22和23及图13A和13B。可见，该实施例测试的LNP制剂在小鼠体内均有较强表达。其中，0％DSPC组中LQ104-E16b-2(PEG-f1)至LQ104-E16b-2(PEG-f6)的总发光强度较高，表明此组中PEG脂质的摩尔百分含量介于0.25％～2％对应的LNP制剂的体内递送能力更好；2％DSPC组中LQ104-E16b-2(PEG-f9)至LQ104-E16b-2(PEG-f15)的总发光强度较高，表明此组中PEG脂质的摩尔百分含量介于0.25％至2.5％对应的LNP制剂的体内递送能力更好。
表22(图13A的AUC数据)

表23(图13B的AUC数据)
在现有技术中，LNP制剂中的磷脂的含量在4mol％以下很难取得好的递送效果。但是，在采用本申请提供的可电离脂质化合物制备的LNP制剂中，即便磷脂组分低至4mol％以下，例如为0mol％至2mol％时，如本实施例展示的，所制得的LNP制剂仍具有很好的性状及递送能力。与此同时，在不升高PEG脂质含量的情况下，例如在0.5mol％至2.5mol％的含量范围内，所制得的LNP制剂仍具有很好的性状及递送能力，从而减少了因PEG脂质升高而影响的疗效及安全性的风险。

Claims (18)

  1. 一种如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，
    其中，Z和W独立地为C3-C10的亚烷基；
    Y和Q独立地为
    A为C2-C6的亚烷基、
    各个RA-1和RA-2独立地为C2-C6的亚烷基；
    M为C1-C6的亚烷基；
    R1和R2独立地为C6-C20的烷基；
    R5为未取代或被1个、2个或3个R5-1取代的C2-C10的烷基；
    各个R5-1独立地为羟基或
    各个R5-1-1独立地为C6-C20的烷基；
    R6为未取代或被1个、2个或3个R6-1取代的C2-C10的烷基；
    各个R6-1独立地为羟基或
    各个R6-1-1独立地为C6-C20的烷基。
  2. 一种如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，
    其中，Z和W独立地为C4-C10的亚烷基；
    Y和Q独立地为
    A为C2-C6的亚烷基、
    各个RA-1和RA-2独立地为C2-C6的亚烷基；
    M为C1-C6的亚烷基；
    R1和R2独立地为C6-C20的烷基；
    R5为未取代或被1个、2个或3个R5-1取代的C2-C10的烷基；
    各个R5-1独立地为羟基或
    R5-1-1独立地为C6-C20的烷基；
    R6为未取代或被1个、2个或3个R6-1取代的C2-C10的烷基；
    各个R6-1独立地为羟基或
    R6-1-1独立地为C6-C20的烷基。
  3. 如权利要求1所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z中，所述C3-C10的亚烷基为C3-C8的亚烷基，优选为直链烷烃，例如
    (2)W中，所述C3-C10的亚烷基为C3-C8的亚烷基，优选为直链烷烃，例如
    (3)A中，所述C2-C6的亚烷基为 例如
    (4)R1中，所述C6-C20的烷基为C10-C19，例如
    (5)R2中，所述C6-C20的烷基为C10-C19，例如
  4. 如权利要求2所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z中，所述C4-C10的亚烷基为C5-C8的亚烷基，优选为直链烷烃，例如
    (2)W中，所述C4-C10的亚烷基为C5-C8的亚烷基，优选为直链烷烃，例如
    (3)A中，所述C2-C6的亚烷基为 例如
    (4)RA-1中，所述C2-C6的亚烷基为 例如
    (5)RA-2中，所述C2-C6的亚烷基为 例如
    (6)M中，所述C1-C6的亚烷基为 例如
    (7)R1中，所述C6-C20的烷基为C10-C18，例如
    (8)R2中，所述C6-C20的烷基为C10-C18，例如
    (9)R5中，所述C2-C10的烷基为C2-C8的烷基，例如 还例如
    (10)R5-1-1中，所述C6-C20的烷基为C11-C18，例如
    (11)R6中，所述C2-C10的烷基为C2-C8的烷基，例如 还例如
    (12)R6-1-1中，所述C6-C20的烷基为C11-C18，例如
    和(13)所述如式I所示的含氮链状化合物为如式I-a所示的含氮链状化合物
  5. 如权利要求1或2所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z和W相同；
    (2)R1和R2相同；
    (3)R5和R6相同；
    和(4)Q和Y相同。
  6. 如权利要求2所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z和W独立地为C5-C8的亚烷基；
    (2)A为C2-C6的亚烷基或
    (3)RA-1和RA-2独立地为C2-C4的亚烷基；
    (4)M为亚甲基；
    (5)R1和R2独立地为C10-C18，例如C10-C12，还例如
    (6)R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
    (7)R5-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
    (8)R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
    (9)R6-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
    (10)所述如式I所示的含氮链状化合物为左右对称的化合物；
    (11)Y为其中a与R2相连，b与Z相连；
    和(12)Q为其中a与R1相连，b与W相连；
    较佳地，所述如式I所示的含氮链状化合物为方案1或方案2：
    方案1、
    Y为其中a与R2相连，b与Z相连；
    Q为其中a与R1相连，b与W相连；
    Z和W独立地为C5-C8的亚烷基；
    A为C2-C6的亚烷基或
    RA-1和RA-2独立地为C2-C4的亚烷基；
    M为亚甲基；
    R1和R2独立地为C10-C18；
    R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
    R5-1-1为C10-C18的烷基；
    R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
    R6-1-1为C10-C18的烷基；
    方案2、
    Q和Y相同；
    Z和W相同；
    R1和R2相同；
    R5和R6相同；
    Z和W独立地为C5-C8的亚烷基；
    A为C2-C6的亚烷基或
    RA-1和RA-2独立地为C2-C4的亚烷基；
    M为亚甲基；
    R1和R2独立地为
    R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
    R5-1-1为C14-C18的烷基；
    R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
    R6-1-1为C14-C18的烷基。
  7. 如权利要求1所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z和W独立地为C3-C8的亚烷基；
    (2)A为C2-C6的亚烷基或
    (3)RA-1和RA-2独立地为C2-C4的亚烷基；
    (4)M为亚甲基；
    (5)R1和R2独立地为C10-C20的烷基，优选为 更优选为
    (6)R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
    (7)R5-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
    (8)R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
    (9)R6-1-1为C10-C18的烷基，例如C14-C18的烷基，还例如
    (10)所述如式I所示的含氮链状化合物为左右对称的化合物；
    (11)Y为其中a与R2相连，b与Z相连；
    和(12)Q为其中a与R1相连，b与W相连；
    较佳地，所述如式I所示的含氮链状化合物为方案3：
    Y为其中a与R2相连，b与Z相连；
    Q为其中a与R1相连，b与W相连；
    Z和W独立地为C3-C8的亚烷基；
    A为C2-C6的亚烷基或
    RA-1和RA-2独立地为C2-C4的亚烷基；
    M为亚甲基；
    R1和R2独立地为C10-C20的烷基；
    R5为被1个、2个或3个R5-1取代的C2-C8的烷基；
    R5-1-1为C10-C18的烷基；
    R6为被1个、2个或3个R6-1取代的C2-C8的烷基；
    R6-1-1为C10-C18的烷基。
  8. 如权利要求1或2中所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在 于，所述如式I所示的含氮链状化合物满足如下条件中的一种或多种：
    (1)Z为
    (2)W为
    (3)R1为
    (4)R2为
    (5)R5为
    (6)R6为
    和(7)A为
  9. 如权利要求1所述的如式I所示的含氮链状化合物或其药学上可接受的盐，其特征在于，所述 如式I所示的含氮链状化合物为如下任一化合物：


  10. 一种如式I所示的含氮链状化合物的制备方法，其特征在于，其包括如下步骤：溶剂中，在碱和碘盐存在下，如式I-1所示的化合物与如式I-2所示的化合物进行如下式所示的偶联反应，即可；
    X为卤素，A为C2-C6的亚烷基，Y、Q、Z、W、R5、R6、R1和R2如权利要求1-9任一项所述；且Y与Q相同，R1与R2相同，Z与W相同；
    较佳地，所述如式I所示的含氮链状化合物的制备方法满足如下条件中的一种或多种：
    (1)所述偶联反应中，所述卤素为氟、氯、溴或碘，例如溴；
    (2)所述偶联反应中，所述如式I-2所示的化合物与所述如式I-2所示的化合物的摩尔比为1： (1-3)，例如1：2.6；
    (3)所述偶联反应中，所述碱为碱式碳酸盐，例如K2CO3；
    (4)所述偶联反应中，所述如式I-2所示的化合物与所述碱的摩尔比为1：(1-5)；例如1：3.5；
    (5)所述偶联反应中，所述溶剂为醚类溶剂或/和腈类溶剂；所述醚类溶剂可为甲基叔丁基醚；所述腈类溶剂可为乙腈；所述腈类溶剂与醚类溶剂的体积比可为1：1；
    (6)所述偶联反应中，所述如式I-2所示的化合物与所述溶剂的质量体积比为10-50mg/mL；例如16mg/mL；
    (7)所述偶联反应中，所述碘盐为碱式碘盐，例如KI；
    (8)所述偶联反应中，所述如式I-2所示的化合物与所述碘盐的摩尔比为1：(1-2)；例如1：1.2；
    和(9)所述偶联反应中，所述偶联反应的反应温度为50-100℃，例如80℃。
  11. 一种脂质载体，其特征在于，其包括物质Z，所述物质Z选自于如权利要求1-9任一项所述的如式I所示化合物或其药学可接受的盐中的一种或多种。
  12. 如权利要求11所述的脂质载体，其特征在于，所述脂质载体满足如下条件中的一种或多种：
    (1)所述脂质载体还包括稀释剂；
    (2)所述脂质载体还包括磷脂；
    (3)所述脂质载体还包括PEG脂质；
    和(4)所述脂质载体还包括甾醇。
  13. 如权利要求12所述的脂质载体，其特征在于，所述脂质载体满足如下条件中的一种或多种：
    (1)所述稀释剂为磷酸盐缓冲液或Tris缓冲液；
    (2)所述磷脂为具有带电极性端和脂肪链非极性端的磷脂类分子，例如二硬脂酰基磷脂酰胆碱、二肉豆蔻酰磷酸胆碱、二油酰磷酸胆碱、棕榈酰磷酸胆碱、1,2-二硬脂酰磷酸胆碱、二十一烷酰磷酸胆碱或棕榈酰磷酸胆碱；
    (3)所述PEG脂质为具有聚乙二醇亲水端修饰的脂质分子；较佳地，所述PEG脂质优选选自PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油和PEG修饰的二烷基甘油中的一种或多种，例如所述PEG脂质为具PEG修饰的二肉豆蔻酰甘油；
    (4)所述甾醇包括动物性、植物性或菌类甾醇；较佳地，所述甾醇选自胆固醇、谷甾醇、麦角甾醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸和α-生育酚中的一种或多种，例如胆固醇；
    (5)所述脂质载体中，所述物质Z与甾醇的摩尔比为0.5-5：1，优选为0.5-3：1，例如0.6-2：1；再例如0.66:1、0.68:1、0.69:1、0.70:1、0.71:1、0.72:1、0.74:1、0.76:1、0.77:1、0.79:1、0.82:1、0.83:1、0.84:1、0.85:1、0.86:1、0.87:1、0.88:1、0.89:1、0.9:1、0.91:1、0.92:1、0.93:1、0.94:1、0.97:1、0.99:1、1.04:1、1.07:1、1.1:1、1.16:1、1.23:1、1.28:1、1.30:1、1.32:1、1.41:1、1.52:1、1.58:1、1.64:1、1.65:1、1.74:1、1.79:1或1.96:1；
    (6)所述脂质载体中，所述物质Z与磷脂的摩尔比为1-25：1，优选为2-25：1，例如22.5:1、 20:1、17.5:1、15:1、11.25:1、10:1、8.75:1、7.5:1、6.67:1、5:1、4.75:1、4.5:1、4:1、3.9:1、3.6:1、3.3:1、3:1、2.86:1、2.5:1或2.2:1；
    (7)所述脂质载体中，所述物质Z与PEG脂质的摩尔比为16-130：1，优选为16-80:1，例如16-40：1；再例如16:1、18:1、18.8:1、20:1、21.9:1、22.5:1、25:1、26.9:1、27.5:1、28.1:1、29.6:1、30:1、31.25:1、33.3:1或37.5:1；
    (8)所述物质Z的摩尔含量为30mol％至60mol％；优选为40mol％至55mol％；例如40mol％、43mol％、45mol％、47.4mol％、50mol％或55mol％；
    (9)所述磷脂的摩尔含量为0mol％至30mol％；优选为0mol％至18mol％；例如0mol％、2mol％、4mol％、6mol％、8mol％、10mol％、11mol％、12mol％、14mol％、16mol％或18mol％；
    (10)所述甾醇的摩尔含量为15mol％至60mol％；优选为40.4％mol％至58.4mol％，例如40.4mol％、41mol％、42.4mol％、43mol％、43.4mol％、44.4mol％、46.4mol％、47.4mol％、48mol％、48.4mol％、49mol％、49.4mol％、49.5mol％、50mol％、50.4mol％、50.5mol％、51mol％、51.4mol％、51.5mol％、52mol％、52.25mol％、52.4mol％、52.5mol％、52.75mol％、53mol％、53.4mol％、54mol％、54.25mol％、54.4mol％、54.5mol％、54.75mol％、55mol％、56mol％、56.4mol％、56.5mol％、57mol％、57.5mol％、58mol％或58.4mol％；
    和(11)所述PEG脂质的摩尔含量为0mol％至10mol％；例如0.5mol％至2.5mol％，还例如0.25mol％、0.5mol％、0.75mol％、1mol％、1.5mol％、1.6mol％、2mol％、2.5mol％、3mol％、3.5mol％、4mol％或5mol％；进一步为0.5mol％至2mol％；可为0.5mol％至1.5mol％，或者1.5mol％至2.5mol％；还可为1.6mol％或2mol％；
    较佳地，所述脂质载体为方案1、方案2、方案3或方案4：
    方案1、所述脂质载体由所述物质Z、所述稀释剂、所述磷脂、所述PEG脂质和所述甾醇组成；
    方案2、所述脂质载体由所述物质Z、所述稀释剂、所述PEG脂质和所述甾醇组成；
    方案3、所述脂质载体由所述物质Z、所述磷脂、所述PEG脂质和所述甾醇组成；
    方案4、所述脂质载体由所述物质Z、所述PEG脂质和所述甾醇组成。
  14. 如权利要求11所述的脂质载体，其特征在于，所述脂质载体不包含磷脂；
    较佳地，所述脂质载体满足如下条件中的一种或多种：
    (1)当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与甾醇的摩尔比为0.6-2：1；优选为0.68:1、0.69:1、0.70:1、0.71:1、0.72:1、0.74:1、0.76:1、0.77:1、0.79:1、0.82:1、0.83:1、0.84:1、0.85:1、0.86:1、0.87:1、0.88:1、0.89:1、0.9:1、0.91:1、0.92:1、0.93:1、0.94:1、0.97:1、0.99:1、1.04:1、1.07:1、1.1:1、1.16:1、1.23:1、1.28:1、1.30:1、1.41:1、1.52:1或1.58:1；
    (2)当所述脂质载体不包含磷脂时，所述脂质载体中，所述物质Z与PEG脂质的摩尔比为16-35：1；优选为16:1、18:1、20:1、21.9:1、22.5:1、25:1、26.9:1、27.5:1、28.1:1、29.6:1或30:1；
    (3)当所述脂质载体不包含磷脂时，所述脂质载体中，所述甾醇的摩尔含量约为15mol％至60mol％，优选为40.4％mol％至58.4mol％，例如43mol％、43.4mol％、44.4mol％、45mol％、46.4mol％、 47.4mol％、48mol％、48.4mol％、49mol％、49.4mol％、49.5mol％、50mol％、50.4mol％、50.5mol％、51mol％、51.4mol％、51.5mol％、52mol％、52.4mol％、52.25mol％、52.5mol％、52.75mol％、53mol％、53.4mol％、54mol％、54.2mol％、53.4mol％、54mol％、54.25mol％、54.4mol％、54.5mol％、54.75mol％、55mol％、56mol％、56.4mol％、56.5mol％、57mol％、57.5mol％、58mol％或58.4mol％；又例如为52.5mol％至54.5mol％，还例如53mol％至54.5mol％；
    和(4)当所述脂质载体不包含磷脂时，或者所述磷脂的含量为4mol％以下时，所述脂质载体中，所述PEG脂质的摩尔含量约为0mol％至10mol％，例如0.25mol％、0.5mol％、0.75mol％、1mol％、1.5mol％、1.6mol％、2mol％、2.5mol％、3mol％、3.5mol％、4mol％或5mol％；优选为0.25mol％至3mol％，又例如为0.5mol％至2.5mol％，或0.5mol％至2mol％。
  15. 一种脂质纳米颗粒，其特征在于，其包括治疗剂和/或预防剂以及如权利要求11-14任一项所述的脂质载体。
  16. 如权利要求15所述的脂质纳米颗粒，其特征在于，所述脂质纳米颗粒满足如下条件中的一种或多种：
    (1)所述治疗剂和/或预防剂为一种或两种及以上核酸；较佳地，所述治疗剂和/或预防剂为单链脱氧核糖核酸、双链DNA、小干扰RNA、不对称双链小干扰RNA、微小RNA、小发夹RNA、环状RNA、转运RNA或信使RNA，优选为mRNA；例如萤火虫荧光素酶mRNA或SARS-CoV-2刺突蛋白mRNA；
    (2)所述脂质纳米颗粒中氮磷比为2：1-30：1，优选为2：1-20：1，例如3：1-20：1，还例如3：1-16：1；
    (3)所述脂质纳米颗粒的粒径为10-200nm，优选为40-150nm，例如60-150nm；再例如50-150nm；
    (4)所述脂质纳米颗粒中，所述脂质载体与治疗剂和/或预防剂的质量比为3-80:1，优选为6-60:1；
    和(5)所述脂质纳米颗粒中，所述脂质载体包裹所述治疗剂和/或预防剂。
  17. 一种组合物，其特征在于，其包括物质Z，所述物质Z为如权利要求1-9任一项所述的如式I所示化合物或其药学可接受的盐。
  18. 如权利要求17所述的组合物，其特征在于，所述组合物还包括稀释剂、磷脂、PEG脂质、甾醇和治疗剂和/或预防剂中的一种或多种；
    较佳地，所述组合物中，所述稀释剂、磷脂、PEG脂质和甾醇如权利要求13或14所述；和或，所述治疗剂和/或预防剂如权利要求16所述；
    更佳地，所述组合物中，所述物质Z与所述稀释剂、磷脂、PEG脂质和甾醇中的一种或多种形成如权利要求11-14任一项所述的脂质载体；和或，所述组合物中，所述治疗剂和/或预防剂的包封率为至少50％，优选为至少为70％；
    还佳地，所述组合物中，所述脂质载体与所述治疗剂和/或预防剂形成如权利要求15或16所述的脂质纳米颗粒；和或，所述组合物中，所述组合物的多分散指数为不高于0.5，例如不高于0.3。
PCT/CN2023/098621 2022-06-06 2023-06-06 含氮链状化合物、制备方法、包含其的组合物和应用 WO2023236953A1 (zh)

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WO2017112865A1 (en) * 2015-12-22 2017-06-29 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
CN110520409A (zh) * 2017-03-15 2019-11-29 摩登纳特斯有限公司 用于细胞内递送治疗剂的化合物和组合物
WO2023091787A1 (en) * 2021-11-22 2023-05-25 Senda Biosciences, Inc. Novel ionizable lipids and lipid nanoparticles and methods of using the same

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WO2017112865A1 (en) * 2015-12-22 2017-06-29 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
CN110520409A (zh) * 2017-03-15 2019-11-29 摩登纳特斯有限公司 用于细胞内递送治疗剂的化合物和组合物
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