Classifications

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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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  • 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
  • C07C219/16—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
• • 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
  • 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
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • 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
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

• the present invention relates to a lipid composition containing lipids and nucleic acids.
• nucleic acid delivery techniques a method of administering nucleic acid-containing particles in which nucleic acid is encapsulated in particles (liposomes or lipid particles) is known.
• nucleic acid-containing particles are prepared using lipids having an amino group or the like and becoming cations at a low pH, and delivery of nucleic acids is realized by imparting an appropriate charge to the particles.
• Patent Document 1 discloses a compound having an ester group, an acetal group or the like as a linking group connecting an aliphatic group and an amino group.
• Patent Document 2 discloses a compound having a vinyloxy group, an amide group, an oxime group or the like as a linking group connecting an aliphatic group and an amino group.
• the above-mentioned lipid having an amino group or the like and becoming a cation at a low pH may be referred to as a cationic lipid.
• Patent Document 3 describes a cationic lipid for delivering a biologically active agent to cells and tissues.
• Patent Document 4 describes lipid nanoparticles containing a compound called DLin-MC3-DMA as a cationic lipid.
• Patent Document 5 describes (a) nucleic acids; (b) cationic lipids constituting about 50 mol% to about 85 mol% of total lipids present in particles; (c) approximately 13 mol% of total lipids present in particles. Non-cationic lipids constituting up to about 49.5 mol%; and (d) complex lipids constituting about 0.5 mol% to about 2 mol% of the total lipids present in the particles, which inhibit the aggregation of the particles. Nucleic acid-lipid particles containing are described. Patent Document 6 contains 40-65% specific structural cationic lipids, 5-10% neutral lipids, 25-40% sterols, and 0.5-10% PEG or PEG-modified lipids. Lipid preparations are described.
• lipid compositions containing lipids and nucleic acids lipid compositions capable of delivering a wide variety of nucleic acids are desired. Further, since lipids having an amino group are known to have toxicity, a technique capable of delivering nucleic acid more efficiently is required.
• the present invention has made it a problem to be solved to provide a lipid composition capable of realizing excellent nucleic acid delivery for a wide variety of nucleic acids.
• lipids in a lipid composition containing the first lipid represented by the formula (1) or a salt thereof, sterols, and nucleic acids have found that excellent nucleic acid delivery could be achieved by making the ratio of the number of moles of the first lipid in the composition to the number of moles of sterols in the lipid composition greater than or equal to 0.300 and less than 1.299.
• the present invention has been completed. According to the present invention, the following inventions are provided.
• X represents -NR 1- or -O-
• R 1 is a hydrogen atom, a hydrocarbon group, or R 21 -L 1 -R 22, 6 to 24 carbon atoms - a group represented by, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, L 1 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 2 and R 3 independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31- L 2- R 32- , where R 31 is a hydrocarbon having 1 to 24 carbon atoms.
• L 2 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms that may be substituted.
• R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and any one or more pairs of R 8 may form a linked 4 may contain O atoms to 7-membered ring together, Substituents on alkyl groups having 1 to 18 carbon atoms that may be substituted are hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , substituted or unsubstituted aryl groups, substituted or unsubstituted heteros.
• R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• Substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46, and-.
• lipid composition according to ⁇ 1> or ⁇ 2> further comprising a lipid having a nonionic hydrophilic polymer structure.
• lipid composition according to ⁇ 3> wherein the lipid having a nonionic hydrophilic polymer structure is a lipid having a polyethylene glycol structure.
• lipid composition according to ⁇ 4> wherein the lipid having a polyethylene glycol structure is a lipid having a diacylglycerol structure and a polyethylene glycol structure.
• ⁇ 6> The lipid composition according to any one of ⁇ 3> to ⁇ 5>, wherein the content of the lipid having a nonionic hydrophilic polymer structure with respect to the total lipid is 0.2 to 10 mol%.
• ⁇ 7> The lipid composition according to any one of ⁇ 1> to ⁇ 6>, wherein the content of the first lipid with respect to the total lipid is 20 to 55 mol%.
• ⁇ 8> The lipid composition according to any one of ⁇ 1> to ⁇ 7>, wherein the content of sterols with respect to total lipid is 20 to 70 mol%.
• the compound represented by the formula (1) is a compound represented by the formula (2).
• R 2 and R 3 independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31- L 2- R 32- .
• R 31 represents a hydrocarbon group having 1 to 24 carbon atoms.
• L 2 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or Show, R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted.
• R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted.
• Substituents on alkyl groups having 1 to 18 carbon atoms that may be substituted are hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , substituted or unsubstituted aryl groups, substituted or unsubstituted heteros. It is a group represented by an aryl group, —O (CO) OR 41 , —O (CO) —R 42 , —— (CO) OR 43 , or —OR 44 , R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• Substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46, and-. It is a group represented by O (CO) OR 41 , -O (CO) -R 42 ,-(CO) OR 43 , or -OR 44 , and is a group represented by R 41 , R 42 , R 43 , R. 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms. e indicates 2 or 3.
• R 2 and R R 3 are each independently 31 -L 2 -R 32 - Indicates a group represented by: Or one of R 2 and R 3 indicates a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon group having 3 to 24 carbon atoms.
• R 5 represents an unsubstituted alkyl group having 1 to 18 carbon atoms or an alkyl group having 1 to 18 carbon atoms substituted with —O (CO) -R 42 or ⁇ (CO) OR 43 ;
• R 7 and R 8 each independently represent an alkyl group having 1 to 4 carbon atoms;
• R 31 , L 2 , R 32 , R 42 and R 43 are synonymous with the definitions in ⁇ 8>.
• ⁇ 12> The lipid composition according to any one of ⁇ 1> to ⁇ 11>, further comprising a pharmaceutically acceptable carrier.
• lipid composition according to any one of ⁇ 1> to ⁇ 12> which is a composition for introducing nucleic acid into cells.
• lipid composition according to any one of ⁇ 1> to ⁇ 12> which is a composition for nucleic acid delivery in vivo.
• the lipid composition of the present invention can realize excellent nucleic acid delivery.
• the lipid composition of the present invention is a lipid composition containing the first lipid, sterols, and nucleic acid which is the lipid represented by the formula (1) or a salt thereof, and is the first lipid in the lipid composition.
• the lipid composition is such that the ratio of the number of moles of sterols to the number of moles of sterols in the lipid composition is 0.300 or more and less than 1.299.
• the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of sterols in the lipid composition is 0.300 or more and less than 1.299.
• the lipid compositions of the invention can achieve excellent nucleic acid delivery.
• the lower limit of the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of sterols in the lipid composition is preferably 0.350 or more, preferably 0.400 or more, 0.500 or more, or 0. It may be .600 or more.
• the upper limit of the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of sterols in the lipid composition is preferably 1.250 or less, and may be 1.200 or less.
• the lipid composition of the present invention contains a lipid represented by the formula (1) or a salt thereof.
• X represents -NR 1- or -O-
• R 1 is a hydrogen atom, a hydrocarbon group, or R 21 -L 1 -R 22, 6 to 24 carbon atoms - a group represented by, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, L 1 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 2 and R 3 independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31- L 2- R 32- , where R 31 is a hydrocarbon having 1 to 24 carbon atoms.
• L 2 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms that may be substituted.
• R 4 and R 5 , R 10 and R 5 , R 5 and R 12 , R 4 and R 6 , R 5 and R 6 , R 6 and R 7 , R 6 and R 10 , R 12 and R 7 , and R 7 and any one or more pairs of R 8 may form a linked 4 may contain O atoms to 7-membered ring together, Substituents on alkyl groups having 1 to 18 carbon atoms that may be substituted are hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , substituted or unsubstituted aryl groups, substituted or unsubstituted heteros.
• R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• Substituents of the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46, and -O.
• R 41 , R 42 , R 43 , R 44. , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• a, b, c, and d each independently indicate an integer of 0 to 3, where a + b is 1 or more and c + d is 1 or more.
• the hydrocarbon group having 6 to 24 carbon atoms in R 1 and the hydrocarbon group having 3 to 24 carbon atoms in R 2 and R 3 are preferably an alkyl group, an alkenyl group or an alkynyl group, and are preferably an alkyl group or an alkenyl group. More preferably it is a group.
• the alkyl group having 6 to 24 carbon atoms and the alkyl group having 3 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 6 to 20 carbon atoms, and the alkyl group having 3 to 24 carbon atoms is more preferably an alkyl group having 6 to 20 carbon atoms.
• the alkenyl group having 6 to 24 carbon atoms and the alkenyl group having 3 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 6 to 20 carbon atoms, and the alkenyl group having 3 to 24 carbon atoms is more preferably an alkenyl group having 6 to 20 carbon atoms.
• the alkynyl group having 6 to 24 carbon atoms is preferably an alkynyl group having 6 to 20 carbon atoms, and the alkynyl group having 3 to 24 carbon atoms is more preferably an alkynyl group having 6 to 20 carbon atoms.
• a hexynyl group a heptynyl group, an octynyl group, a nonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group.
• All of the above alkenyl groups preferably have one or two double bonds, and all alkynyl groups preferably have one or two triple bonds.
• the hydrocarbon group having 1 to 24 carbon atoms for R 21 and R 31 is preferably an alkyl group having 10 to 24 carbon atoms, an alkenyl group having 10 to 24 carbon atoms, or an alkynyl group having 10 to 24 carbon atoms. ..
• the alkyl group having 10 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkyl group having 10 to 24 carbon atoms is preferably an alkyl group having 12 to 24 carbon atoms.
• heptadecyl group heptadecyl group, octadecyl group, 2-butylhexyl group, 2-butyloctyl group, 1-pentylhexyl group, 2-pentylheptyl group, 3- Pentyloctyl group, 1-hexylheptyl group, 1-hexylnonyl group, 2-hexyloctyl group, 2-hexyldecyl group, 3-hexylnonyl group, 1-heptyloctyl group, 2-heptylnonyl group, 2-heptylundecyl Group, 3-heptyldecyl group, 1-octylnonyl group, 2-octyldecyl group, 2-octyldodecyl group, 3-octylundecyl group, 2-nonylundecyl group, 2-nonylundecyl group,
• the alkenyl group having 10 to 24 carbon atoms may be linear or branched, and may be chain or cyclic. Specifically, a decenyl group, an undecenyl group, a dodecenyl group, a dodecadienyl group, a tridecenyl group (preferably (Z) -trideca-8-enyl group), a tetradecenyl group (preferably a tetradeca-9-enyl group), a pentadecenyl group.
• Group (preferably (Z) -pentadeca-8-enyl group), hexadecenyl group (preferably (Z) -hexadeca-9-enyl group), hexadecadienyl group, heptadecenyl group (preferably (Z)) -Heptadeca-8-enyl group), heptadecadienyl group (preferably (8Z, 11Z) -heptadeca-8,11-dienyl group), octadecenyl group (preferably (Z) -octadeca-9-enyl group) ), Octadecadienyl group (preferably (9Z, 12Z) -octadeca-9,12-dienyl group) and the like.
• the alkynyl group having 10 to 24 carbon atoms may be linear or branched, and may be chain or cyclic. Specific examples thereof include a decynyl group, an undecynyl group, a dodecinyl group, a tetradecynyl group, a pentadecynyl group, a hexadecynyl group, a heptadecynyl group, and an octadecynyl group. All of the above alkenyl groups preferably have one or two double bonds, and all alkynyl groups preferably have one or two triple bonds.
• the divalent linking group and the hydrocarbon linking group having 1 to 18 carbon atoms may be an alkylene group having 1 to 18 carbon atoms or an alkaneylene group having 2 to 18 carbon atoms. preferable.
• the alkylene group having 1 to 18 carbon atoms may be linear or branched, and may be chain or cyclic.
• the number of carbon atoms is preferably 1 to 12, more preferably 1 to 10, and even more preferably 2 to 10.
• alkenylene group having 2 to 18 carbon atoms may be linear or branched, and may be chain or cyclic.
• the number of carbon atoms is preferably 1 to 12, more preferably 2 to 10.
• L 1 a preferable range of L 1 , -O (CO) O-, -O (CO)-, or-(CO) O- is preferable, and -O (CO)-or- (CO) O- is more preferable.
• L 2 a preferable range of L 2 , -O (CO) O-, -O (CO)-, or-(CO) O- is preferable, and -O (CO)-or- (CO) O- is more preferable.
• Alkyl groups having 1 to 18 carbon atoms that may be substituted for R 4 , R 6 , R 9 , R 10 , R 11 and R 12 are linear or branched. It may be in the form of a chain or a ring. The number of carbon atoms is preferably 1 to 12.
• the substituent is a hydroxyl group, a carboxyl group, -O (CO) OR 41 , -O (CO) -R 42 ,-(CO) OR 43 , or -O.
• the group represented by —R 44 is preferred, and the group represented by —O (CO) —R 42 or ⁇ (CO) OR 43 is more preferred.
• Alkyl groups having 1 to 18 carbon atoms that may be substituted for R 5 , R 7 , and R 8 may be linear or branched, and may be chain or cyclic. May be.
• the number of carbon atoms is preferably 1 to 12, more preferably 1 to 8.
• the substituent is a hydroxyl group, a carboxyl group, -O (CO) OR 41 , -O (CO) -R 42 ,-(CO) OR 43 , or -O.
• the group represented by —R 44 is preferred, and the group represented by —O (CO) —R 42 , ⁇ (CO) OR 43 , or —OR 44 is more preferred.
• Examples of the 4- to 7-membered ring that may contain an O atom include an azetidine ring, a pyrrolidine ring, a piperidine ring, a morpholine ring, and an azepane ring, and a 6-membered ring is preferable, and a piperidine ring and a morpholine ring are preferable.
• Substituents in alkyl groups with 1-18 carbon atoms that may be substituted are substituted or unsubstituted for R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12.
• the aryl group in the case of the aryl group of the number of carbon atoms is preferably 6 to 22, more preferably 6 to 18, and even more preferably 6 to 10. Specific examples thereof include a phenyl group, a naphthyl group, an anthrasenyl group and a phenanthrenyl group.
• Substituents on the aryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , -O (CO) OR 41 , and -O (CO).
• the groups represented by —R 42 , — (CO) OR 43 , or —OR 44 are preferred, with hydroxyl or carboxyl groups being more preferred.
• Specific examples of the substituted aryl group include a hydroxyphenyl group and a carboxyphenyl group.
• Substituents in alkyl groups with 1-18 carbon atoms that may be substituted are substituted or unsubstituted for R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12.
• the heteroaryl group in the case of the heteroaryl group the number of carbon atoms is preferably 1 to 12, and more preferably 1 to 6. Specific examples thereof include a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzoimidazolyl group, a thiazolyl group and an oxazolyl group.
• Substituents on the heteroaryl group include an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , -O (CO) OR 41 , and -O (CO). ) -R 42 ,-(CO) OR 43 , or —OR 44 is preferred, with hydroxyl or carboxyl groups being more preferred.
• Specific examples of the substituted or unsubstituted heteroaryl group include a hydroxypyridyl group, a carboxypyridyl group, a pyridonyl group and the like.
• the hydrocarbon groups having 1 to 18 carbon atoms for R 41 , R 42 , R 43 , R 44 , R 45 and R 46 include alkyl groups having 1 to 18 carbon atoms, alkenyl groups having 2 to 18 carbon atoms or carbons. It is preferably an alkynyl group having a number of 2 to 18, and more preferably an alkyl group having 1 to 18 carbon atoms or an alkenyl group having 2 to 18 carbon atoms.
• the alkyl group having 1 to 18 carbon atoms may be linear or branched, and may be chain or cyclic.
• the number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18.
• the alkenyl group having 2 to 18 carbon atoms may be linear or branched, and may be chain or cyclic.
• the number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18.
• the alkynyl group having 2 to 18 carbon atoms may be linear or branched, and may be chain or cyclic.
• the number of carbon atoms is preferably 3 to 18, and more preferably 5 to 18.
• R 1 is a hydrocarbon group or R 21 -L 1 -R 22, 6 to 24 carbon atoms - - X is -NR 1 preferably exhibits a group represented by.
• one of R 2 and R 3 is a hydrogen atom; the other of R 2 and R 3 is a hydrocarbon group having 6 to 24 carbon atoms, or a group represented by R 31- L 2- R 32-. It is preferable to show.
• R 2 and R 3 each independently preferably represent a hydrocarbon group having 6 to 24 carbon atoms or a group represented by R 31 ⁇ L 2 ⁇ R 32- .
• R 4 , R 6 , R 9 , R 10 , R 11 and R 12 are preferably hydrogen atoms.
• R 5 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms which may be substituted with —O (CO) -R 42 or ⁇ (CO) OR 43, and an aryl group. It is preferably an alkyl group having 1 to 18 carbon atoms which may be substituted with, an alkyl group having 1 to 18 carbon atoms which may be substituted with a hydroxyl group, and when it is an alkyl group, R 4 , R 6 , R 10 And R 12 may be linked to each other to form a ring which may contain an O atom.
• an alkyl group having 1 to 18 carbon atoms an alkyl group having 1 to 18 carbon atoms which may be substituted with —O (CO) -R 42 or ⁇ (CO) OR 43, or an aryl group is substituted. It is preferably an alkyl group having 1 to 12 carbon atoms, an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, and an alkyl group having 1 to 18 carbon atoms, —O (CO) —R 42. Alternatively, it is more preferably an alkyl group having 1 to 18 carbon atoms which may be substituted with ⁇ (CO) OR 43.
• R 7 and R 8 may be independently substituted with a hydrogen atom, a hydrocarbon group having 1 to 18 carbon atoms, —O (CO) -R 42 or ⁇ (CO) OR 43 , respectively.
• An alkyl group of 18, an alkyl group having 1 to 8 carbon atoms which may be substituted with an aryl group, or an alkyl group having 1 to 8 carbon atoms which may be substituted with a hydroxyl group, or R 7 and R 8 are mutually exclusive. It is preferable that they are linked to form a 4- to 7-membered ring which may contain an O atom.
• R 5 and R 7 or R 8 are not connected to each other and do not form a ring.
• a + b is preferably 1 or 2, more preferably 1.
• c + d is preferably 1 or 2, more preferably 1.
• the compound represented by the formula (1) is preferably a compound represented by the following formula (1-1).
• R 24 is a hydrogen atom, a hydrocarbon group, or R 21 -L 1 -R 22, 6 to 24 carbon atoms - a group represented by, R 21 represents a hydrocarbon group having 1 to 24 carbon atoms, L 1 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 22 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 25 represents a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31- L 2- R 32- , and R 31 represents a hydrocarbon group having 1 to 24 carbon atoms, and L 2 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 4 , R 5 , R 6 , R 7 , R 8 , R 10 , and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms that may be substituted.
• Substituents on alkyl groups having 1 to 18 carbon atoms that may be substituted are hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , substituted or unsubstituted aryl groups, substituted or unsubstituted heteros. It is a group represented by an aryl group, —O (CO) OR 41 , —O (CO) —R 42 , —— (CO) OR 43 , or —OR 44 , R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• Substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46, and-. It is a group represented by O (CO) OR 41 , -O (CO) -R 42 ,-(CO) OR 43 , or -OR 44 , and is a group represented by R 41 , R 42 , R 43 , R. 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• R 4, R 5, R 6 , R 7, R 8, R 10, and R 12 in the formula (1-1) are the same as those of the formula (1).
• R 24 of the formula (1-1) is preferably an alkyl group or an alkenyl group having 6 to 24 carbon atoms.
• the alkyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 8 to 20 carbon atoms.
• the alkenyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 8 to 20 carbon atoms.
• R 25 of the formula (1-1) is preferably an alkyl group or an alkenyl group having 6 to 24 carbon atoms.
• the alkyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkyl group having 6 to 24 carbon atoms is preferably an alkyl group having 7 to 20 carbon atoms.
• the alkenyl group having 6 to 24 carbon atoms may be linear or branched, and may be chain or cyclic.
• the alkenyl group having 6 to 24 carbon atoms is preferably an alkenyl group having 8 to 20 carbon atoms.
• X indicates -O-;
• R 2 , R 3 , R 31 , L 2 , and R 32 are synonymous with the definitions in equation (1).
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms that may be substituted.
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms that may be substituted.
• the definition of the substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted, the substituted or unsubstituted aryl group, and the substituent on the substituted or unsubstituted heteroaryl group is defined in the formula (1). Is synonymous with a + b is 1 and c +
• the compound represented by the formula (1) is a compound represented by the following formula (2).
• R 2 and R 3 independently represent a hydrogen atom, a hydrocarbon group having 3 to 24 carbon atoms, or a group represented by R 31- L 2- R 32- .
• R 31 represents a hydrocarbon group having 1 to 24 carbon atoms.
• L 2 is -O (CO) O-, -O (CO)-,-(CO) O-, -O-, or Show
• R 32 is a divalent linking group and represents a hydrocarbon linking group having 1 to 18 carbon atoms.
• R 5 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted.
• R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms which may be substituted.
• Substituents on alkyl groups having 1 to 18 carbon atoms that may be substituted are hydroxyl groups, carboxyl groups, amino groups represented by -NR 45 R 46 , substituted or unsubstituted aryl groups, substituted or unsubstituted heteros. It is a group represented by an aryl group, —O (CO) OR 41 , —O (CO) —R 42 , —— (CO) OR 43 , or —OR 44 , R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• Substituents on the substituted or unsubstituted aryl group and the substituted or unsubstituted heteroaryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46, and-. It is a group represented by O (CO) OR 41 , -O (CO) -R 42 ,-(CO) OR 43 , or -OR 44 , and is a group represented by R 41 , R 42 , R 43 , R. 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms. e indicates 2 or 3.
• the definitions of R 2 , R 3 , R 5 , R 7 and R 8 are the same as those in equation (1).
• R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms and may be substituted with 1 to 18 carbon atoms for R 5.
• Substituents on the alkyl group of are hydroxyl groups, substituted or unsubstituted aryl groups, —O (CO) OR 41 , —O (CO) —R 42 , ⁇ (CO) OR 43 , or ⁇ . It is a group represented by OR 44 , and R 41 , R 42 , R 43 , R 44 , R 45 and R 46 each independently represent a hydrocarbon group having 1 to 18 carbon atoms and are substituted or unsubstituted.
• Substituents on the aryl group are an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a carboxyl group, an amino group represented by -NR 45 R 46 , -O (CO) OR 41 , -O (CO)-. It is a group represented by R 42 ,-(CO) OR 43 , or -OR 44 , and R 41 , R 42 , R 43 , R 44 , R 45, and R 46 have 1 carbon atoms, respectively. It shows up to 18 hydrocarbon groups.
• R 2 and R 3 each independently represent a hydrocarbon group having 3 to 24 carbon atoms or a group represented by R 31- L 2- R 32- , where L 2 is. , -O (CO)-or-(CO) O-, where R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms and may be substituted. Substituents on the alkyl groups of numbers 1-18 are unsubstituted aryl groups, —O (CO) -R 42 , or ⁇ (CO) OR 43 , where R 42 and R 43 are independent, respectively. It shows a hydrocarbon group having 1 to 18 carbon atoms.
• R 2 and R 3 each independently represent a hydrogen atom or a hydrocarbon group having 3 to 24 carbon atoms
• R 7 and R 8 each independently represent a hydrogen atom.
• the substituent on the alkyl group having 1 to 18 carbon atoms and optionally substituted is an unsubstituted aryl group, -O (CO) -R 42 , or-(CO).
• ) is a group represented by O-R 43, R 42, and R 43 each independently represents a hydrocarbon group having 1 to 18 carbon atoms.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , where L 2 is -O (CO)-or-(CO).
• L 2 is -O (CO)-or-(CO).
• R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and the substituent on the alkyl group having 1 to 18 carbon atoms which may be substituted is ,
• An unsubstituted aryl group, a group represented by -O (CO) -R 42 , or-(CO) O-R 43 , and R 42 and R 43 are independently hydrocarbonized with 1 to 18 carbon atoms, respectively. Indicates a hydrogen group.
• R 2 and R 3 each independently represent a group represented by R 31- L 2- R 32- , where L 2 is -O (CO)-or-(. CO) O—, R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 18 carbon atoms and may be substituted on an alkyl group having 1 to 18 carbon atoms. Is a substituent represented by an unsubstituted aryl group, -O (CO) -R 42 , or-(CO) O-R 43 , and R 42 and R 43 are independently having 1 to 18 carbon atoms, respectively. Indicates a hydrocarbon group.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 3 to 24 carbon atoms.
• L 2 represents -O (CO)-or-(CO) O-
• R 7 and R 8 each independently have a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
• Substituents on alkyl groups with 1-18 carbon atoms that may be shown and substituted are the substituents represented by an unsubstituted aryl group, —O (CO) —R 42 , or ⁇ (CO) OR 43.
• R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• one of R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 6 carbon atoms.
• Group is indicated, L 2 indicates -O (CO)-or-(CO) O-, and R 7 and R 8 each independently indicate a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
• Substituents on alkyl groups with 1-18 carbon atoms that may be substituted are the groups represented by —O (CO) —R 42 , or ⁇ (CO) OR 43 , R 42 , and R.
• Each of 43 independently represents a hydrocarbon group having 1 to 18 carbon atoms.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 6 carbon atoms.
• Group is indicated, L 2 indicates -O (CO)-or-(CO) O-, R 5 indicates a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 7 and R 8 indicate a group.
• Independently indicate a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 6 carbon atoms.
• Group is indicated, L 2 indicates -O (CO)-or-(CO) O-, R 5 indicates a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 7 and R 8 indicate a group.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 has 3 to 5 carbon atoms.
• L 2 indicates -O (CO)-or-(CO) O-
• R 5 indicates a hydrogen atom or an alkyl group having 1 to 18 carbon atoms
• R 7 and R independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 has 3 to 5 carbon atoms.
• Indicates a hydrocarbon group L 2 indicates -O (CO)-or-(CO) O-, R 5 indicates a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 7 and R. 8 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms independently, and e represents 2.
• one of R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 6 carbon atoms.
• L 2 indicates -O (CO)-or-(CO) O-
• R 5 indicates a hydrogen atom or a substituted alkyl group having 1 to 18 carbon atoms
• R 7 and Each of R 8 independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms
• the substituted substituent on the alkyl group having 1 to 18 carbon atoms is -O (CO) -R 42
• -(CO) O-R 43 is a group
• R 42 and R 43 each independently represent a hydrocarbon group having 1 to 18 carbon atoms.
• R 2 and R 3 represents a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon having 6 carbon atoms.
• Group is indicated, L 2 indicates -O (CO)-or-(CO) O-, R 5 indicates a hydrogen atom or an substituted alkyl group having 1 to 18 carbon atoms, and R 7 and R 8 independently represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and the substituted substituent on the alkyl group having 1 to 18 carbon atoms is -O (CO) -R 42 , or -(CO) O-R 43 is a group, R 42 and R 43 independently represent a hydrocarbon group having 1 to 18 carbon atoms, and e represents 2.
• the preferred embodiment is R 2 and wherein at least one of R 3 represents one or more hydrocarbon group having 3 to 24 carbon atoms including an unsaturated bond;
• R 2 and R R 3 are each independently 31 -L 2 -R 32 - Indicates a group represented by: Or one of R 2 and R 3 indicates a group represented by R 31- L 2- R 32- , and the other of R 2 and R 3 is a hydrocarbon group having 3 to 24 carbon atoms.
• R 5 represents an unsubstituted alkyl group having 1 to 18 carbon atoms or an alkyl group having 1 to 18 carbon atoms substituted with —O (CO) -R 42 or ⁇ (CO) OR 43 ;
• R 7 and R 8 each independently represent an alkyl group having 1 to 4 carbon atoms; (Here, R 31 , L 2 , R 32 , R 42 , and R 43 are synonymous with the definition in equation (2)). If.
• the compound represented by the formula (1) may form a salt.
• Salts in the basic group include, for example, salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitrate and sulfuric acid; formic acid, acetic acid, citrate, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, Salts with organic carboxylic acids such as tartrate, aspartic acid, trichloroacetic acid and trifluoroacetic acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid and naphthalenesulfonic acid.
• mineral acids such as hydrochloric acid, hydrobromic acid, nitrate and sulfuric acid
• formic acid acetic acid, citrate, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid
• Salts in acidic groups include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and trimethylamine, triethylamine, tributylamine, pyridine, N, N- Nitrogen-containing organic bases such as dimethylaniline, N-methylpiperidin, N-methylmorpholin, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1-ephenamine and N, N'-dibenzylethylenediamine. And salt etc.
• preferred salts include pharmacologically acceptable salts.
• Preferred specific examples of the compound represented by the formula (1) include the compounds described in Examples 1 to 135 described later, but the present invention is not construed as being limited thereto.
• the compounds described in Examples 1 to 135 are referred to as Compounds 1 to 135, respectively.
• compound 24, compound 30, compound 31, compound 50, compound 56, compound 69, compound 88, compound 89, compound 91, compound 93, compound 103, compound 112, compound 118, compound 119, compound 134 and compound. 135 is particularly preferred.
• the compound represented by the formula (1) can be produced by combining known methods, and can be produced, for example, according to the production method shown below.
• R a and R b are leaving groups; R c , R d and Re are amino protecting groups or imino protecting groups; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 have the same meanings as above.
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 have the same meanings as above.
• As leaving groups for example, chloro group, fluoro group, bromo group, trichloromethoxy group.
• Examples of the amino-protecting group or the imino-protecting group include a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 2-nitrobenzenesulfonyl group, a benzyl group and the like.
• Known compounds of formula [3] include, for example, 4-nitrophenyl chloroformate, 1,1'-carbonyldiimidazole, triphosgene and phosgene.
• the compound of formula [4] can be produced by reacting the compound of formula [2] with the compound of formula [3] in the presence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons. Examples thereof include hydrogens, and these solvents may be mixed and used.
• Preferred solvents include ethers, with tetrahydrofuran being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [2].
• Examples of the base used in this reaction include inorganic bases and organic bases.
• the base is preferably an organic base, and specific examples thereof include triethylamine, N, N-diisopropylethylamine, 4-methylmorpholine, pyridine, and N, N-dimethylaminopyridine.
• the amount of the base used may be 1 to 50 times mol, preferably 1 to 10 times mol, of the compound of the formula [2].
• the amount of the compound of the formula [3] to be used is not particularly limited, but may be 0.3 to 10 times (v / w) the amount of the compound of the formula [2]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• Known compounds of formula [5] include, for example, (9Z, 12Z) -di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amines and dihexadecylamines.
• the compound of formula [6] can be produced by reacting the compound of formula [4] with the compound of formula [5] in the presence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons. Examples thereof include hydrogens, and these solvents may be mixed and used.
• Preferred solvents include ethers, with tetrahydrofuran being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [4].
• Examples of the base used in this reaction include inorganic bases and organic bases.
• the base is preferably an organic base, and specific examples thereof include triethylamine, N, N-diisopropylethylamine, 4-methylmorpholine, pyridine, and N, N-dimethylaminopyridine.
• the amount of the base used may be 1 to 50 times mol, preferably 1 to 10 times mol, of the compound of the formula [4].
• the amount of the compound of the formula [5] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [4]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• Compounds of formula [2A] include, for example, tert-butyl (2-((tert-butoxycarbonyl) amino) ethyl) (2-hydroxyethyl) carbamate and tert-butyl (2-((2-hydroxyethyl) (methyl)). ) Amino) Ethyl) Carbamate and the like are known.
• the compound of formula [6A] is prepared by reacting the compound of formula [2A] with the compound of formula [3] in the presence of a base, and then combining the compound of formula [4A] with the compound of formula [5] in the presence of a base. It can be produced by reacting with. This reaction may be carried out according to the production methods (1-1) and (1-2).
• the compound of formula [6] can be produced by deprotecting the compound of formula [6A]. This reaction is, for example, T.I. W. TW Greene et al., Protective Groups in Organic Synthesis, 4th Edition, pp. 696-926, 2007, John Wiley and Sons. Wiley & Sons, INC.) May be followed.
• R a and R b are leaving groups; R c , R d and Re are amino protecting groups or imino protecting groups; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 have the same meanings as above.
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 have the same meanings as above.
• As leaving groups for example, chloro group, fluoro group, bromo group, trichloromethoxy group.
• Examples of the amino-protecting group or the imino-protecting group include a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 2-nitrobenzenesulfonyl group, a benzyl group and the like.
• Known compounds of formula [3] include, for example, 4-nitrophenyl chloroformate, 1,1'-carbonyldiimidazole, triphosgene and phosgene.
• the compound of formula [8] can be produced by reacting the compound of formula [7] with the compound of formula [3] in the presence of a base. This reaction may be carried out according to the production method (1-1).
• the compound of formula [9] can be produced by reacting the compound of formula [8] with the compound of formula [2] in the presence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons. Examples thereof include hydrogens, and these solvents may be mixed and used. Preferred solvents include ethers, with tetrahydrofuran being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [8].
• Examples of the base used in this reaction include inorganic bases and organic bases.
• the base is preferably an organic base, and specific examples thereof include triethylamine, N, N-diisopropylethylamine, 4-methylmorpholine, pyridine, and N, N-dimethylaminopyridine.
• the amount of the base used may be 1 to 50 times mol, preferably 1 to 10 times mol, of the compound of the formula [8].
• the amount of the compound of the formula [2] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [8]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• Compounds of formula [2A] include, for example, tert-butyl (2-((tert-butoxycarbonyl) amino) ethyl) (2-hydroxyethyl) carbamate and tert-butyl (2-((2-hydroxyethyl) (methyl)). ) Amino) Ethyl) Carbamate and the like are known.
• the compound of formula [9] is produced by reacting the compound of formula [8] with the compound of formula [2A] in the presence of a base, and then deprotecting the compound of formula [9A] in the presence of a base. can do. This reaction may be carried out according to the production methods (2-2) and (1-4).
• R a , R b and R g are leaving groups;
• R f is an alkyl group having 1 to 18 carbon atoms;
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 42 have the same meanings as above.
• As leaving groups for example, chloro group, fluoro group, bromo group, trichloromethoxy.
• Known compounds of formula [3] include, for example, 4-nitrophenyl chloroformate, 1,1'-carbonyldiimidazole, triphosgene and phosgene.
• the compound of formula [8] can be produced by reacting the compound of formula [7] with the compound of formula [3] in the presence of a base. This reaction may be carried out according to the production method (1-1).
• Compounds of formula [2B] include, for example, 2,2'-((2- (diethylamino) ethyl) azandyl) bis (ethane-1-ol) and 2,2'-((3- (diethylamino) propyl) azandyl). ) Bis (ethane-1-all) is known.
• the compound of formula [9B] can be produced by reacting the compound of formula [8] with the compound of formula [2B] in the presence of a base. This reaction may be carried out according to the production method (2-2).
• Known compounds of formula [10A] include, for example, dodecanoic acid, decanoic acid, nonanoic acid and octanoic acid.
• the compound of formula [9C] can be produced by reacting the compound of formula [9B] with the compound of formula [10A] in the presence of a condensing agent or an acid halide in the presence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons. Examples thereof include hydrogens, and these solvents may be mixed and used.
• Preferred solvents include ethers, with tetrahydrofuran being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [9B].
• Examples of the base used in this reaction include inorganic bases and organic bases.
• the base is preferably an organic base, and specific examples thereof include triethylamine, N, N-diisopropylethylamine, 4-methylmorpholine, pyridine, and N, N-dimethylaminopyridine.
• the amount of the base used may be 1 to 50 times mol, preferably 1 to 10 times mol, of the compound of the formula [9B].
• Condensing agents used in this reaction include, for example, carbodiimides such as N, N'-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; carbonyls such as carbonyldiimidazole; diphenylphosphoryl.
• carbodiimides such as N, N'-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
• carbonyls such as carbonyldiimidazole
• diphenylphosphoryl diphenylphosphoryl.
• Acid azides such as azides
• Acid cyanides such as diethylphosphoryl cyanide
• Acid halides used in this reaction include, for example, carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride; sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride; ethyl chloroformate and isobutyl chloroformate. Chloroformates and the like can be mentioned.
• the amount of the compound of the formula [10A] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [9B]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• the compound of the formula [10B] for example, dodecanoic acid chloride, decanoic acid chloride, nonanoic acid chloride, octanoic acid chloride and the like are known.
• the compound of formula [9C] can be produced by reacting the compound of formula [9B] with the compound of formula [10B] in the presence of a base.
• the compound of the formula [10B] can be produced by reacting the compound of the formula [10A] with thionyl chloride, oxalyl chloride and the like.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides and aromatic hydrocarbons. Examples thereof include hydrogens, and these solvents may be mixed and used. Preferred solvents include ethers, with tetrahydrofuran being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [9B].
• Examples of the base used in this reaction include inorganic bases and organic bases. The amount of the base used may be 1 to 50 times mol, preferably 1 to 10 times mol, of the compound of the formula [9B].
• the amount of the compound of the formula [10B] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [2B]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• R h and R i are leaving groups; R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 have the same meanings as above.
• the leaving group include a chloro group, a bromo group, an iodo group, a methanesulfonyl group, a 4-toluenesulfonyl group, a chloromethanesulfonyl group, a trifluoromethanesulfonyl group, and the like.
• Compounds of formula [12] include, for example, 2-chloro-N, N-dimethylethane-1-amine, 4- (2-chloroethyl) morpholine and 2-chloro-N, N-diethylethane-1-amine, 2, -Bromo-N, N-diethylethane-1-amine, 3-chloro-N, N-diethylethane-1-amine and the like are known.
• the compound of formula [2] can be produced by reacting the compound of formula [11] with the compound of formula [12] in the presence or absence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, alcohols, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, etc. Aromatic hydrocarbons and water are mentioned, and these solvents may be mixed and used.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [11].
• Examples of the base used in this reaction include inorganic bases and organic bases. The amount of the base used may be 1 to 10000 times mol, preferably 1 to 5000 times mol, of the compound of the formula [11].
• the amount of the compound of the formula [12] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [11]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• the compound of formula [2] can be produced by reacting the compound of formula [13] with the compound of formula [14] in the presence or absence of a base. This reaction may be carried out according to the production method (4-1).
• R j is a leaving group
• R k is an alkyl group having 1 to 18 carbon atoms
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 43 have the same meaning as above.
• As leaving groups for example, chloro group, bromo group, iodo group, methanesulfonyl group, 4-toluenesulfonyl group, chloromethanesulfonyl group, trifluoromethanesulfonyl. Group, etc.
• the compound of formula [2] can be produced by reacting the compound of formula [2C] with the compound of formula [15A] in the presence or absence of a base.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, alcohols, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, etc. Aromatic hydrocarbons and water are mentioned, and these solvents may be mixed and used.
• Preferred solvents include ethers or nitriles, with tetrahydrofuran or acetonitrile being more preferred.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [2C].
• Examples of the base used in this reaction include inorganic bases and organic bases.
• the amount of the base used may be 1 to 10000 times mol, preferably 1 to 5000 times mol, of the compound of the formula [2C].
• the amount of the compound of the formula [15A] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [13].
• This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• R g and R l are leaving groups;
• R m is an alkyl group having 1 to 18 carbon atoms;
• R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 42 have the same meanings as above.
• ”As the leaving group for example, a chloro group, a bromo group, an iodo group, a methanesulfonyl group, a 4-toluenesulfonyl group, a chloromethanesulfonyl group, Trifluoromethanesulfonyl group, trichloromethoxy group, 4-nitro-phenoxy group, 2,4-dinitrophenoxy group, 2,4,6-trichlorophenoxy group, pentafluorophenoxy group, 2,3,5,6-tetrafluorophenoxy Examples thereof include a group, an imidazolyl group, a triazolyl group, a 3,5-dioxo-4-methyl-1,2,4-oxadiazolidyl group, and an N-hydroxysuccinimidyl group.
• Known compounds of formula [10A] include, for example, dodecanoic acid, decanoic acid, nonanoic acid and octanoic acid.
• the compound of formula [2] can be produced by reacting the compound of formula [2B] with the compound of formula [10A] in the presence of a condensing agent or an acid halide in the presence of a base. This reaction may be carried out according to the production method (3-3).
• the compound of formula [10B] for example, dodecanoic acid chloride, decanoic acid chloride, nonanoic acid chloride, octanoic acid chloride and the like are known.
• the compound of formula [2] can be produced by reacting the compound of formula [2B] with the compound of formula [10B] in the presence of a base. This reaction may be carried out according to the production method (3-4).
• the compound of formula [2] can be produced by reacting the compound of formula [2C] with the compound of formula [16] in the presence or absence of a base. This reaction may be carried out according to the production method (4-1).
• R n , Ro and R p are alkyl groups having 1 to 17 carbon atoms; R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 42 and R 43 have the same meaning as above. "
• Known compounds of formula [17A] include, for example, formaldehyde, acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal and octanal.
• the compound of the formula [2] is such that the compound of the formula [2C] is reacted with the compound of the formula [17A] in the presence of a reducing agent, the presence or absence of a reduction catalyst, and the presence or absence of an acid.
• the solvent used in this reaction is not particularly limited as long as it does not affect the reaction, but for example, alcohols, halogen hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, etc.
• Aromatic hydrocarbons and water are mentioned, and these solvents may be mixed and used.
• the amount of the solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of the formula [2C].
• Examples of the acid used in this reaction include inorganic acids and organic acids. The amount of the acid used may be 0.01 to 10000 times mol, preferably 0.05 to 100 times mol, of the compound of the formula [2C].
• Examples of the reducing agent used in this reaction include sodium triacetoxyborohydride, sodium cyanoborohydride, 2-picoline borane, formic acid and hydrogen.
• Examples of the reduction catalyst used in this reaction include palladium-carbon, palladium hydroxide-carbon, platinum-carbon, rhodium-carbon and ruthenium-carbon.
• the amount of the compound of the formula [17A] to be used is not particularly limited, but may be 1 to 10 times (v / w) the amount of the compound of the formula [13]. This reaction may be carried out at ⁇ 30 to 150 ° C., preferably 0 to 100 ° C. for 5 minutes to 48 hours.
• Known compounds of formula [17B] include, for example, 2-oxoethyl octanoate and 2-oxoethyl nonanoate.
• the compound of the formula [2] is such that the compound of the formula [2C] is reacted with the compound of the formula [17B] in the presence of a reducing agent, the presence or absence of a reduction catalyst, and the presence or absence of an acid. Can be manufactured by This reaction may be carried out according to the production method (7-1).
• the compound of the formula [17C] for example, heptyl 3-oxopropanoate and octyl 3-oxopropanoate are known.
• the compound of the formula [2] is such that the compound of the formula [2C] is reacted with the compound of the formula [17C] in the presence of a reducing agent, the presence or absence of a reduction catalyst, and the presence or absence of an acid. Can be manufactured by This reaction may be carried out according to the production method (7-1).
• isomers for example, optical isomers, geometric isomers, tautomers, etc.
• these isomers can also be used.
• solvates, hydrates and crystals of various shapes are present, these solvates, hydrates and crystals of various shapes can also be used.
• a compound having an amino group, a hydroxyl group, a carboxyl group or the like is known in advance after the reaction by protecting these groups with ordinary protecting groups. These protecting groups can be removed by the above method.
• the compound obtained by the above-mentioned production method is subjected to a reaction known per se such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration or hydrolysis, or a combination of these reactions is appropriately combined. This can lead to other compounds.
• the content of the lipid represented by the formula (1) or a salt thereof with respect to the total lipid is preferably 20 mol% or more and 55 mol% or less, and 22 mol% or more and 55 mol% or less. Is more preferable.
• the nucleic acid is mRNA
• the content of the lipid represented by the formula (1) or a salt thereof with respect to the total lipid is more preferably 25 mol% or more and 52 mol% or less, and 32 mol% or more and 48 mol. It is more preferably% or less.
• the content of the lipid represented by the formula (1) or a salt thereof with respect to the total lipid is more preferably 32 mol% or more and 55 mol% or less, and 37 mol% or more and 55 mol. % Or less, and particularly preferably 47 mol% or more and 55 mol% or less.
• the lipid composition of the present invention contains sterols.
• the sterols are not particularly limited, but are cholesterol, phytosterol (citosterol), stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanol, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl. Examples include ether, cholesteryl-4'-hydroxybutyl ether and the like. Among these, cholesterol or a derivative thereof is preferable.
• the content of sterols with respect to total lipid is preferably 20 mol% to 70 mol%.
• the content of sterols with respect to the total lipid is more preferably 30 mol% to 66 mol%, further preferably 30 to 60 mol%.
• the nucleic acid is siRNA
• the content of sterols with respect to the total lipid is more preferably 45 mol% to 68 mol%, further preferably 45 mol% to 63 mol%, 45 mol%. It is particularly preferably ⁇ 52 mol%.
• the lipid composition of the present invention contains a lipid having a nonionic hydrophilic polymer structure.
• a lipid having a nonionic hydrophilic polymer structure in the oil phase, the effect of stabilizing the dispersion of lipid particles can be obtained.
• nonionic hydrophilic polymer examples are not particularly limited, but are nonionic vinyl-based polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, and the like. Examples thereof include nonionic modified natural polymers, block polymers or graft copolymers having two or more of these polymers as constituent units.
• nonionic hydrophilic polymers preferably nonionic polyethers, nonionic polyesters, nonionic polyamino acids or nonionic synthetic polypeptides, more preferably nonionic polyethers or nonionic polyethers.
• the lipid having a nonionic hydrophilic polymer structure is preferably a lipid having a polyethylene glycol structure.
• the lipid having a nonionic hydrophilic polymer is not particularly limited, and examples thereof include PEG-modified phosphoethanolamine, diacylglycerol PEG derivative, dialkylglycerol PEG derivative, cholesterol PEG derivative, and ceramide PEG derivative.
• diacylglycerol PEG is preferable. That is, the lipid having a polyethylene glycol structure is preferably a lipid having a diacylglycerol structure and a polyethylene glycol structure.
• the acyl group of the diacylglycerol moiety is more preferably an acyl group having 12 to 22 carbon atoms.
• the weight average molecular weight of the PEG chain is preferably 500 to 5000, more preferably 750 to 3000.
• the nonionic hydrophilic polymer chain may be branched and may have a substituent such as a hydroxymethyl group.
• the content of the lipid having a nonionic hydrophilic polymer structure with respect to the total lipid is preferably 0.2 mol% to 10 mol%, preferably 0.2 mol% to 5 mol. % Is more preferable.
• the content of the lipid having a nonionic hydrophilic polymer structure with respect to the total lipid is more preferably 0.2 mol% to 5 mol%, more preferably 0.2 to 3 mol. It is more preferably%, and most preferably 0.5 mol% to 2.5 mol%.
• the content of the lipid having a nonionic hydrophilic polymer structure with respect to the total lipid is more preferably 0.2 mol% to 2.3 mol%, and 1.2 mol. It is more preferably% to 2.3 mol%.
• the lipid composition of the present invention may or may not contain a zwitterionic lipid.
• Phospholipid is preferable as the zwitterionic lipid.
• the phospholipid is not particularly limited, and examples thereof include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylcholine and phosphatidylethanolamine are preferable.
• the zwitterionic lipid may be used alone or in combination of a plurality of different zwitterionic lipids.
• the phosphatidylcholine is not particularly limited, but is soy lecithin (SPC), hydrogenated soy lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (EPC), 1,2-dipalmitoyl-sn-glycero-3- Phosphatidyl (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidyl (DSPC), 1-palmitoyl-2-oleoyl- Examples thereof include sn-glycero-3-phosphatidolin (POPC) and 1,2-dioreoil-sn-glycero-3-phosphatidolin (DOPC).
• SPC soy lecithin
• HSPC hydrogenated soy lecithin
• EPC egg yolk lecithin
• EPC hydrogenated egg yolk lecithin
• 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DMPC
• 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC
• 1,2-distearoyl-sn-glycero -3-Phosphocholine DSPC
• 1,2-dioreoil-sn-glycero-3-phosphocholine DOPC
• the phosphatidylethanolamine is not particularly limited, but is limited to 1,2-dimyristyl-sn-glycero-3-phoethanolamine (DMPE) and 1,2-dipalmitoyl-sn-glycero-3-phoethanolamine (DPPE).
• DMPE 1,2-dimyristyl-sn-glycero-3-phoethanolamine
• DPPE 1,2-dipalmitoyl-sn-glycero-3-phoethanolamine
• 1,2-Distearoyl-sn-glycero-3-phoethanolamine DSPE
• 1,2-diore oil-sn-glycero-3-phoethanolamine DOPE
• 1,2-dilinole oil-sn -Glycero-3-phophoethanolamine DLoPE
• 1,2-difitanoyl-sn-glycero-3-phophoethanolamine D (Phy) PE
• 1-palmitoyl-2-oleoyl-sn-glycero- 3-Phosphoethanolamine POPE
• 1,2-ditetradecyl-sn-glycero-3-phophoethanolamine 1,2-dihexadecyl-sn-glycero-3-phophoethanolamine
• 1,2-dioctadecyl examples thereof include -sn-glycero-3-phophoethanolamine and 1,2-diphytanyl-sn-glycero-3-phophoethanolamine.
• the sphingomyelin is not particularly limited, and examples thereof include egg yolk-derived sphingomyelin and milk-derived sphingomyelin.
• the content of the zwitterionic lipid with respect to the total lipid is preferably 0 mol% to 35 mol%.
• the content of the zwitterionic lipid with respect to the total lipid is preferably 0 mol% to 35 mol%, more preferably 0 mol% to 30 mol%, and 0. It is more preferably mol% to 25 mol%.
• the nucleic acid is siRNA
• the content of zwitterionic lipids relative to total lipids is preferably 0 mol%.
• the lower limit of the content of the zwitterionic lipid with respect to the total lipid is not particularly limited, but is generally 0.5 mol% or more. , It is preferably 1 mol% or more, and more preferably 2 mol% or more.
• the lipid composition of the present invention contains nucleic acids.
• the nucleic acid include plasmids, single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (microRNA), mRNA, antisense nucleic acid, ribozyme, aptamer, saRNA, sgRNA, and the like. Good. It may also contain modified nucleic acids.
• the content of nucleic acid with respect to total lipid is preferably 0.5 to 50% by mass, more preferably 1 to 25% by mass, still more preferably 1.5 to 20% by mass. It is particularly preferably 2 to 15% by mass.
• the total lipid content in the nucleic acid is preferably 2 to 200, more preferably 4 to 200, still more preferably 6 to 100, and particularly preferably 8 to 75.
• the method for producing the lipid composition of the present invention will be described.
• the method for producing the lipid composition is not limited, but all or some of the constituents of the lipid composition are dissolved in an organic solvent or the like to form an oil phase, and the water-soluble component is dissolved in water to form an aqueous phase. And the aqueous phase can be mixed and produced.
• a micromixer may be used for mixing, or an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like may be used for emulsification.
• a dry mixture containing lipids is prepared by vacuum-drying the solution containing lipids with an evaporator or the like or spray-drying with a spray dryer or the like, adding this mixture to an aqueous solvent, and further emulsifying with the above-mentioned emulsifier or the like. It can also be manufactured by doing so.
• the step (a) includes dissolving a component classified as a lipid in an organic solvent (alcohol such as ethanol, ester, etc.).
• the total lipid concentration after dissolution in an organic solvent is not particularly limited, but is generally 1 mmol / L to 100 mmol / L, preferably 5 mmol / L to 50 mmol / L, and more preferably 10 mmol / L to. It is 30 mmol / L.
• the aqueous phase can be obtained by dissolving nucleic acids (eg, siRNA, antisense nucleic acid, miRNA (microRNA), mRNA, etc.) in water or buffer. If necessary, components such as antioxidants can be added.
• the mixing ratio (mass ratio) of the aqueous phase and the oil phase is preferably 5: 1 to 1: 1 and more preferably 4: 1 to 2: 1.
• step (d) the method for removing the organic solvent from the dispersion liquid of lipid particles is not particularly limited, and a general method can be used.
• a general method can be used.
• the organic solvent can be removed by performing dialysis using a solution such as a buffer solution.
• the concentration of the dispersion liquid obtained in the step (d) can be adjusted.
• a solution such as phosphate buffered saline, physiological saline, or sucrose / Tris buffer can be used as a diluent to dilute to an appropriate concentration.
• the dispersion liquid obtained in step (d) can be concentrated by ultrafiltration using an ultrafiltration membrane or the like. It is preferable to use the concentrated dispersion as it is, and it is also preferable to adjust the concentration to a desired concentration using the diluted solution after concentration. Excipients and buffers may be added as solutions that can be used for dialysis in step (d) and dilution in step (e).
• excipients include sugars.
• saccharides include sucrose, trehalose, maltose, glucose, lactose, fructose, mannitol, sorbitol, inositol, xylitol and the like.
• buffer include ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine and the like.
• Aseptic filtration is preferable in order to obtain the dispersion liquid of the lipid particles of the present invention into a pharmaceutical composition.
• a filtration method a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, or the like can be used to remove unnecessary substances from the dispersion liquid of lipid particles.
• it is not particularly limited, but it is preferable to filter with a filter having a pore size capable of sterilization (preferably a filtration sterilization filter of 0.2 ⁇ m).
• aseptic filtration is preferably performed after step (c) or step (d).
• the dispersion liquid of the lipid particles of the present invention can be frozen or freeze-dried.
• the dispersion liquid of the lipid particles of the present invention can be frozen or freeze-dried by a general method, and the method is not particularly limited.
• the composition of the present invention is preferably composed of lipid particles.
• the lipid particle means a particle composed of a lipid, and includes a composition having a structure selected from lipid aggregates, micelles, and liposomes in which the lipid is aggregated, and the composition containing the lipid.
• the structure of the lipid particles is not limited to these as long as it is.
• Liposomes include liposomes having a lipid bilayer structure, having an aqueous phase inside, and having a single-layered double membrane, and multi-phase liposomes in which multiple layers are stacked. Either liposome may be included in the present invention.
• the morphology of lipid particles can be confirmed by electron microscope observation or structural analysis using X-rays.
• the lipid particles have a lipid bilayer structure (lamella structure) and an inner aqueous layer like liposomes, or the electron density inside the particles. It can be confirmed whether or not it has a high core and has a structure packed with constituents such as lipids.
• the presence or absence of a lipid bilayer structure (lamellar structure) on lipid particles can also be confirmed by X-ray small-angle scattering (SAXS) measurement.
• SAXS X-ray small-angle scattering
• the particle size of the lipid particles is not particularly limited, but is preferably 10 to 1000 nm, more preferably 30 to 500 nm, still more preferably 50 to 250 nm, and particularly preferably 50 to 200 nm.
• the particle size of the lipid particles can be measured by a general method (for example, dynamic light scattering method, laser diffraction method, etc.).
• a nucleic acid for example, a gene
• the lipid composition in the present invention contains a nucleic acid having a medicinal use
• the lipid composition can be administered to a living body as a nucleic acid medicine. That is, the lipid composition of the present invention is preferably a composition for introducing nucleic acid into cells.
• the lipid composition of the present invention is a carrier alone or pharmaceutically acceptable (also referred to as an administration medium, for example, physiological saline or phosphate buffer). Can be mixed with and administered to a living body.
• concentration of the lipid composition (lipid particles) in the mixture with the pharmaceutically acceptable carrier is not particularly limited, and can generally be 0.05% by mass to 90% by mass.
• other pharmaceutically acceptable additives such as a pH adjustment buffer and an osmotic pressure adjustment agent may be added.
• the administration route when administering the nucleic acid drug containing the lipid composition of the present invention is not particularly limited, and can be administered by any method.
• Oral administration, parenteral administration intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intravitreal administration, intramuscular administration, intravaginal administration, intravesical administration) , Intravitreal administration, lung administration, rectal administration, colon administration, buccal administration, nasal administration, intravaginal administration, inhalation, etc.
• Parenteral administration is preferable, and intravenous injection, subcutaneous injection, intradermal injection or intramuscular injection is preferable as the administration method.
• the nucleic acid drug containing the lipid composition of the present invention can also be administered by direct injection into the diseased site.
• the dosage form of the lipid composition of the present invention is not particularly limited, but when orally administered, the lipid composition of the present invention can be used in combination with a suitable excipient, such as tablets, lozenges, capsules, and pills. It can be used in the form of agents, suspensions, syrups and the like.
• a suitable excipient such as tablets, lozenges, capsules, and pills. It can be used in the form of agents, suspensions, syrups and the like.
• an antioxidant, a buffer, a bacteriostatic agent, and an isotonic sterile injection, a suspending agent, a solubilizing agent, and a thickening agent , Stabilizers or additives such as preservatives can be combined as appropriate.
• the lipid composition of the present invention is very useful as a nucleic acid delivery carrier because it can retain nucleic acids at a high inclusion rate.
• a nucleic acid or the like can be introduced into a cell by transfecting the cell with the lipid composition in vitro or in vivo.
• the nucleic acid delivery carrier utilizing the present invention is also useful as a nucleic acid delivery carrier in nucleic acid medicine. That is, the lipid composition of the present invention is useful as a composition for nucleic acid delivery in vitro or in vivo (preferably in vivo).
• purification by column chromatography was carried out using an automatic purification device ISOLERA (Biotage) or a medium-pressure liquid chromatograph YFLC W-prep 2XY (Yamazen Corporation).
• the carrier in silica gel column chromatography is Chro [Example matorex Q-Pack SI 50 (Fuji Silysia Chemical Ltd.), High Flash Columns W001, W002, W003, W004 or W005 (Yamazen Corporation). used.
• NH silica gel Chromatolex Q-Pack NH 60 (Fuji Silysia Chemical Ltd.) was used.
• the NMR spectrum was measured using Bruker AV300 (manufactured by Bruker) or Bruker AV400 (manufactured by Bruker) using tetramethylsilane as an internal reference, and the total ⁇ value was shown in ppm.
• the MS spectrum was measured using ACQUITY SQD LC / MS System (manufactured by Waters).
• the reaction mixture was cooled to room temperature, hexane (400 mL), tert-butyl methyl ether (100 mL) and water (200 mL) were added, the organic layer was separated, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane), and (9Z, 12Z) -di ((9Z, 12Z) -octadeca-9,12-diene-1-yl) amine 57.7 g.
• the obtained oil was purified by silica gel column chromatography (ethyl acetate-hexane, NH silica gel) and 2-((2- (dimethylamino) ethyl) ( Methyl) amino) ethyldi ((9Z, 12Z) -octadeca-9,12-diene-1-yl) carbamate (11.2 g) was obtained.
• Example 1 (3) 3-((2- (dimethylamino) ethyl) (methyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 3-((2- (Dimethylamino) ethyl) (methyl) amino) propyldi ((9Z, 12Z)-) in the same manner as in Example 1 (3) except that amino) propane-1-ol is used. Octadeca-9,12-dien-1-yl) carbamate was obtained.
• Example 3 A 12.0 mol / L sodium hydroxide aqueous solution (12.0 mol / L sodium hydroxide aqueous solution) in a water (5 mL) solution of piperidine-4-ol (2.0 g), 2-chloro-N, N-dimethylethane-1-amine hydrochloride (5.69 g). 5 mL) was added, and the mixture was stirred at room temperature for 9 hours. Dichloromethane and water were added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with dichloromethane. The organic layer and the extract were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• Example 1 (3) In Example 1 (3), 1- (2- (dimethylamino) ethyl) piperidine-4-ol was used instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol.
• 1-Il) Carbamate was obtained.
• Example 4 (1) In Example 3 (1), 1- (2- (dimethylamino) ethyl) was used in the same manner as in Example 3 (1) except that piperidine-3-ol was used instead of piperidine-4-ol. ) Piperidine-3-ol was obtained. MSm / z (M + H): 173.
• Example 1 (3) In Example 1 (3), 1- (2- (dimethylamino) ethyl) piperidine-3- was used instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. 1- (2- (dimethylamino) ethyl) piperidine-3-yldi ((9Z, 12Z) -octadeca-9,12-diene-" in the same manner as in Example 1 (3) except that oar was used. 1-Il) Carbamate was obtained.
• Example 5 (2) 2- (Methylamino) ethane-1-ol (3.0 g), potassium carbonate (22.1 g) in ethanol (60 mL) suspension with 4- (2-chloroethyl) morpholine hydrochloride (14.9 g) In addition, the mixture was stirred at 60 ° C. for 4 hours and under reflux for 3 hours. After cooling the reaction mixture to room temperature, the insoluble material was filtered off, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane, NH silica gel) to obtain 2- (methyl (2-morpholinoethyl) amino) ethane-1-ol (5.5 g). MSm / z (M + H): 189.
• Example 1 (3) 2- (2- (methyl (2-morpholinoethyl) amino) ethane-1 was used instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol.
• 2- (Methyl (2-morpholinoethyl) amino) ethyldi ((9Z, 12Z) -octadeca-9,12-diene-1-" in the same manner as in Example 1 (3) except that ool was used. Ill) I got carbamate.
• Example 6 (1) In Example 5 (1), the same method as in Example 5 (1) except that 2- (ethylamino) ethane-1-ol was used instead of 2- (methylamino) ethane-1-ol. , 2- (Ethyl (2-morpholinoethyl) amino) ethane-1-ol was obtained. MSm / z (M + H): 203.
• Example 1 (3) 2- (ethyl (2-morpholinoethyl) amino) ethane-1 was used instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol.
• 2- (Ethyl (2-morpholinoethyl) amino) ethyldi ((9Z, 12Z) -octadeca-9,12-diene-1-" in the same manner as in Example 1 (3) except that ool was used. Ill) I got carbamate.
• Example 7 (1) Example 5 (1) except that 2-chloro-N, N-diethylethane-1-amine hydrochloride is used instead of 4- (2-chloroethyl) morpholine hydrochloride. 2-((2- (diethylamino) ethyl) (methyl) amino) ethane-1-ol was obtained in the same manner as in the above. MSm / z (M + H): 175.
• Example 1 (3) 2-((2- (diethylamino) ethyl) (methyl) amino instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. ) 2-((2- (Diethylamino) ethyl) (methyl) amino) ethyldi ((9Z, 12Z) -octadeca- in the same manner as in Example 1 (3) except that ethane-1-ol is used. 9,12-dien-1-yl) carbamate was obtained.
• Example 8 (1) In Example 2 (1), instead of using 3-bromopropane-1-ol, 2-bromoethane-1-ol was used instead of N, N, N'-trimethylethane-1,2-diamine. 2-((3- (Dimethylamino) propyl) (methyl) amino) ethane in the same manner as in Example 2 (1) except that N, N, N'-trimethylpropane-1,3-diamine is used. I got -1-all. MSm / z (M + H): 161.
• Example 1 (3) 2-((3- (dimethylamino) propyl) (methyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2-((3- (Dimethylamino) propyl) (methyl) amino) ethyldi ((9Z, 12Z)-) in the same manner as in Example 1 (3) except that amino) ethane-1-ol was used. Octadeca-9,12-dien-1-yl) carbamate was obtained.
• Example 9 (1) In Example 1 (3), tert-butyl (2-((tert-butoxycarbonyl) amino) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2-((tert-Butoxycarbonyl) (2-((tert-butoxycarbonyl) amino) ethyl) amino in the same manner as in Example 1 (3) except that ethyl) (2-hydroxyethyl) carbamate is used. ) Ethyl) di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) carbamate was obtained.
• the obtained residue was purified by silica gel column chromatography (methanol-chloroform, NH silica gel) and 2-((2-aminoethyl) amino) ethyldi ((9Z, 12Z) -octadeca-9,12-diene- 1-Il) Carbamate (0.3 g) was obtained.
• Example 10 Examples 1 (3) except that dihexadecylamine is used instead of (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine). 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl dihexadecylcarbamate was obtained in the same manner as in 1 (3).
• Example 11 In Example 1 (3), instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadecane-9,12-dien-1-yl) amine, it was synthesized according to the method described in WO2016 / 081029A1.
• Example 12 In Example 1 (3), instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadecane-9,12-dien-1-yl) amine, it was synthesized according to the method described in WO2016 / 081029A1.
• Example 13 In Example 1 (3), instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadecane-9,12-dien-1-yl) amine, it was synthesized according to the method described in WO2016 / 081029A1.
• Example 14 (1) Same as Example 1 (1) except that 6-bromohexane-1-ol is used instead of (6Z, 9Z) -18-bromooctadecar 6,9-diene. N, N-bis (6-hydroxyhexyl) -2-nitrobenzenesulfonamide was obtained by the above method. (Z) -Non-2-ene was added to the obtained mixture of N, N-bis (6-hydroxyhexyl) -2-nitrobenzenesulfonamide (2.13 g), triethylamine (0.58 mL) and tetrahydrofuran (5 mL).
• Example 1 (3) In Example 1 (3), azaneylbis (hexane-6,1-diyl) diyl instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine). 2-((2- (Dimethylamino) ethyl) (methyl) in the same manner as in Example 1 (3) except that ((Z) -non-2-ene-1-yl) bis (carbonate) is used. ) Amino) ethylbis (6-((((Z) -non-2-ene-1-yl) oxy) carbonyl) oxy) hexyl) carbamate was obtained.
• Example 15 (1) In a suspension of N, N-dimethylformamide (20 mL) of nonane-1-amine (1.95 g) and potassium carbonate (1.87 g), (Z) -1-bromooctadeca-9-ene (4.53 g). ) was added, and the mixture was stirred at 80 ° C. for 9 hours. The reaction mixture was cooled to room temperature and water (40 mL) and hexane (40 mL) were added.
• Example 1 (3) instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine), (Z) -N-nonyl octadeca-9 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl (Z) -nonyl (octadeca-9) in the same manner as in Example 1 (3) except that -ene-1-amine is used. -En-1-yl) carbamate was obtained.
• Example 16 Examples 15 (1) except that (6Z, 9Z) -18-bromooctadeca-6,9-diene is used instead of (Z) -1-bromooctadeca-9-ene. (9Z, 12Z) -N-nonyl octadeca-9,12-diene-1-amine was obtained in the same manner as in 15 (1). MSm / z (M + H): 392.
• Example 1 (3) instead of using (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine), (9Z, 12Z) -N-nonyl octadeca 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl nonyl ((9Z,) 12Z) -octadeca-9,12-diene-1-yl) carbamate was obtained.
• Example 17 Example 1 (3), except that dioctylamine is used instead of (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine). 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl dioctyl carbamate was obtained in the same manner as in 3).
• Example 18 In Example 1 (3), except that dinonylamine is used instead of (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine). 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl dinonyl carbamate was obtained in the same manner as in).
• Example 19 In Example 1 (3), except that didecylamine is used instead of (9Z, 12Z9-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine). 2-((2- (Dimethylamino) ethyl) (methyl) amino) ethyl didecylcarbamate was obtained in the same manner as in).
• Example 20 (1) Synthesized according to the method described in WO2010 / 054401A1 (6Z, 9Z, 28Z, 31Z) -Heptatriaconta-6,9,28,31-tetraene-19-ol (5.0 g), triethylamine (4.0 mL) and tetrahydrofuran ( 4-Nitrophenyl chloroformate (3.8 g) was added to the mixture (25 mL), and the mixture was stirred at room temperature for 6 hours. Water and ethyl acetate were added to the reaction mixture, the organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the reaction mixture was cooled to room temperature, water and ethyl acetate were added, the organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) and 2-((2- (dimethylamino) ethyl) (methyl) amino) ethyl ((6Z, 9Z, 28Z, 31Z) -hepta). Triaconta-6,9,28,31-tetraene-19-yl) carbonate (0.36 g) was obtained.
• Example 21 In Example 20 (2), 2- (methyl (2) synthesized in Example 5 (1) instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2- (Methyl (2-morpholinoethyl) amino) ethyl ((6Z, 9Z, 28Z, 31Z)) in the same manner as in Example 20 (2) except that -morpholinoethyl) amino) ethane-1-ol was used. ) -Heptatria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 22 In Example 20 (2), 2- (ethyl (2) synthesized in Example 6 (1) instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2- (Ethyl (2-morpholinoethyl) amino) ethyl ((6Z, 9Z, 28Z, 31Z)) in the same manner as in Example 20 (2) except that -morpholinoethyl) amino) ethane-1-ol was used. ) -Heptatria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 23 In Example 20 (2), 2-((2- (2-)) synthesized in Example 7 (1) instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. 2-((2- (Diethylamino) ethyl) (methyl) amino) ethyl (in the same manner as in Example 20 (2) except that (diethylamino) ethyl) (methyl) amino) ethane-1-ol is used. (6Z, 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) Carbonate was obtained.
• Example 24 (1) In Example 5 (1), 2-chloro-N, N-dimethylethane-1-amine hydrochloride was used instead of 4- (2-chloroethyl) morpholine hydrochloride, and 2- (methylamino) ethane-1 was used. 2-((2- (Dimethylamino) ethyl) (ethyl) in the same manner as in Example 5 (1) except that 2- (ethylamino) ethane-1-ol was used instead of -ol. Amino) ethane-1-ol was obtained. MSm / z (M + H): 161.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (ethyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 25 (1) In Example 5 (1), 2-chloro-N, N-dimethylethane-1-amine hydrochloride was used instead of 4- (2-chloroethyl) morpholine hydrochloride, and 2- (methylamino) ethane-1 was used. 2-((2- (Dimethylamino) ethyl) (isopropyl) in the same manner as in Example 5 (1) except that 2- (isopropylamino) ethane-1-ol was used instead of -ol. Amino) ethane-1-ol was obtained. MSm / z (M + H): 175.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (isopropyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 26 (1) In Example 5 (1), tert-butyl (2-bromoethyl) carbamate is used instead of 4- (2-chloroethyl) morpholine hydrochloride in the same manner as in Example 5 (1). -Butyl (2-((2-hydroxyethyl) (methyl) amino) ethyl) carbamate was obtained. MSm / z (M + H): 219.
• Example 20 (2) In Example 20 (2), tert-butyl (2-((2-hydroxyethyl) (methyl)) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• Example 9 (2) 2-((tert-butoxycarbonyl) (2-((tert-butoxycarbonyl) amino) ethyl) amino) ethyl) di ((9Z, 12Z) -octadeca-9,12-diene Instead of using -1-yl) carbamate, tert-butyl (2-((((((6Z, 9Z, 28Z, 31Z))-heptatopia contour-6, 2-((2-Aminoethyl)) in the same manner as in Example 9 (2) except that 9,28,31-tetraene-19-yl) oxy) ethyl) (methyl) amino) ethyl) carbamate was used.
• Example 27 (2) 2-Bromoethane-1-ol (14.2 g) is added to an ethanol (50 mL) suspension of N, N'-dimethylethane-1,2-diamine (5.0 g) and potassium carbonate (17.2 g). , 60 ° C. for 5 hours. The reaction mixture was cooled to room temperature, the insoluble material was filtered off, the solvent was distilled off under reduced pressure, and 2,2'-(ethane-1,2-diylbis (methylazaneyl)) bis (ethane-1-ol) (10). .2 g) was obtained. MSm / z (M + H): 177.
• Example 20 (2) In Example 20 (2), instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol, 2,2'-(ethane-1,2-diylbis (methyl azaneyl)) )) (6Z, 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene in the same manner as in Example 20 (2) except that bis (ethane-1-ol) is used. -19-Il (2-((2-((2-hydroxyethyl) (methyl) amino) ethyl) (methyl) amino) ethyl) carbonate was obtained.
• Example 28 (1) In Example 5 (1), instead of using 2- (methylamino) ethane-1-ol, 2,2'-azanesilbis (ethane-1-ol) and 4- (2-chloroethyl) morpholine hydrochloride were used. 2,2'-((2- (dimethylamino)) in the same manner as in Example 5 (1) except that 2-chloro-N, N-dimethylethane-1-amine hydrochloride was used instead. Ethyl) azaneyl) bis (ethane-1-ol) was obtained. MSm / z (M + H): 177.
• Example 20 (2) In Example 20 (2), 2,2'-((2- (dimethylamino) ethyl)) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2-((2- (Dimethylamino) ethyl) (2-hydroxyethyl) amino) ethyl) (in the same manner as in Example 20 (2) except that azaneyl) bis (ethane-1-ol) is used. (6Z, 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) Carbonate was obtained.
• Example 29 (1) Synthesized according to the method described in WO2015 / 005253A1 (in a mixture of (19Z, 22Z) -octacosa-19,22-diene-11-ol (1.0 g), triethylamine (1.0 mL) and tetrahydrofuran (5.0 mL)). , 4-Nitrophenyl chloroformate (1.0 g) was added, and the mixture was stirred at room temperature for 2 hours. Water and ethyl acetate were added to the reaction mixture, the organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was reduced under reduced pressure. Distilled off.
• the obtained residue was purified by silica gel column chromatography (ethyl-hexane acetate) and 4-nitrophenyl ((19Z, 22Z) -octacosa-19,22-diene-11-yl) carbonate (2). 0.0 g) was obtained.
• Example 20 (2) In Example 20 (2), 4-nitro instead of using (6Z, 9Z, 28Z, 31Z) -heptatriaconta-6,9,28,31-tetraene-19-yl (4-nitrophenyl) carbonate. 2-((2- (Dimethylamino) ethyl) in the same manner as in Example 20 (2) except that phenyl ((19Z, 22Z) -octacosa-19,22-diene-11-yl) carbonate is used. (Methyl) Amino) Ethyl ((19Z, 22Z) -octacosa-19,22-diene-11-yl) carbonate was obtained.
• Example 30 (1) Potassium carbonate in a mixture of 2- (ethylamino) ethane-1-ol (4.0 g), 2-bromo-N, N-diethylethane-1-amine hydrobromide (17.6 g) and ethanol (80 mL) (18.6 g) was added, and the mixture was stirred under heating and reflux for 7 hours. The reaction mixture was cooled to room temperature, unnecessary substances were filtered off, and the solvent was distilled off under reduced pressure.
• Example 20 (2) 2-((2- (diethylamino) ethyl) (ethyl) amino instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. ) 2-((2- (Diethylamino) ethyl) (ethyl) amino) ethyl ((6Z, 9Z,), a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol is used. 28Z, 31Z) -heptateria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 31 (1) Potassium carbonate (8.0 g) in a mixture of 2- (propylamino) ethane-1-ol (2.0 g), 2-chloro-N, N-dimethylethane-1-amine hydrochloride (4.2 g) and ethanol (40 mL). ) was added, and the mixture was stirred under heating and reflux for 9 hours. The reaction mixture was cooled to room temperature, unnecessary substances were filtered off, and the solvent was distilled off under reduced pressure.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (propyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 32 (1) In Example 31 (1), the same method as in Example 31 (1) except that 2- (cyclohexylamino) ethane-1-ol is used instead of 2- (propylamino) ethane-1-ol. Then, 2- (cyclohexyl (2- (dimethylamino) ethyl) amino) ethane-1-ol as a yellow oil was obtained. MSm / z (M + H): 215.
• Example 20 (2) In Example 20 (2), 2- (cyclohexyl (2- (dimethylamino) ethyl) amino) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2- (Cyclohexyl (2- (dimethylamino) ethyl) amino) ethyl ((6Z, 9Z, 28Z,), a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol was used. 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 33 In Example 29 (2), 2-((2- (dimethylamino) ethyl) (ethyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2-((2- (Dimethylamino) ethyl) (ethyl) amino) ethyl ((19Z,), a colorless oil, in the same manner as in Example 29 (2) except that amino) ethane-1-ol was used. 22Z) -octacosa-19,22-diene-11-yl) carbonate was obtained.
• Example 35 the colorless oil 2-(((2)) was carried out in the same manner as in Example 34 except that (9Z, 12Z) -octadeca-9,12-dienoic acid was used instead of oleic acid. -((2- (Dimethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) propane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12) -Gienoart) was obtained.
• Example 36 (1) A mixture of benzaldehyde (30.0 g), 6-bromohexane-1-ol (56.1 g), triethylsilane (67.5 mL) and toluene (300 mL) in an ice-cooled trifluorinated boron diethyl ether complex (46.2 mL) was added, and the mixture was stirred at the same temperature for 40 minutes. Water was added to the reaction mixture, the organic layer was separated, washed with saturated aqueous sodium hydrogen carbonate solution, and the solvent was evaporated under reduced pressure.
• the reaction mixture was poured into a 10% aqueous sulfuric acid solution (330 mL) under ice-cooling, hexane (300 mL) was added, the organic layer was separated, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure.
• Tetrahydrofuran (200 mL), ethanol (100 mL) and a 10 mol / L potassium hydroxide aqueous solution were added to the obtained residue, and the mixture was stirred at 40 ° C. for 1 hour.
• Hexane (200 mL) and water (100 mL) were added to the reaction mixture, the organic layer was separated, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure.
• Example 37 (1) Sodium borohydride in a mixture of 2-((2- (dimethylamino) ethyl) amino) ethane-1-ol (250 mg), hexanal (0.35 mL), acetic acid (0.16 mL) and tetrahydrofuran (2.5 mL). (1.8 g) was added, and the mixture was stirred at room temperature for 2 hours.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (hexyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 9Z, 28Z, 31Z) -heptateria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 38 (1) In Example 31 (1), the same method as in Example 31 (1) except that 2- (butylamino) ethane-1-ol is used instead of 2- (propylamino) ethane-1-ol. Then, a yellow oily 2- (butyl (2- (dimethylamino) ethyl) amino) ethane-1-ol was obtained. MSm / z (M + H): 189.
• Example 20 (2) In Example 20 (2), 2- (butyl (2- (dimethylamino) ethyl) amino) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. 2- (Butyl (2- (dimethylamino) ethyl) amino) ethyl ((6Z, 9Z, 28Z,), a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol was used. 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 39 (1) In Example 30 (1), the same method as in Example 30 (1) except that 2- (butylamino) ethane-1-ol was used instead of 2- (ethylamino) ethane-1-ol. Then, 2- (butyl (2- (diethylamino) ethyl) amino) ethane-1-ol as a pale yellow oil was obtained. MSm / z (M + H): 217.
• Example 20 (2) 2- (butyl (2- (diethylamino) ethyl) amino) ethane instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol.
• -Heptatria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 40 (1) In Example 31 (1), the same method as in Example 31 (1) except that 2- (pentylamino) ethane-1-ol is used instead of 2- (propylamino) ethane-1-ol. Then, 2-((2- (dimethylamino) ethyl) (pentyl) amino) ethane-1-ol as a brown oil was obtained. MSm / z (M + H): 203.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (pentyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 9Z, 28Z, 31Z) -heptateria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 41 (1) In a mixture of tridecane-1,7,13-triol (5.0 g), oleic acid (13.4 g), triethylamine (18.2 mL), 4-dimethylaminopyridine (0.26 g) and N, N-dimethylformamide (25 mL). 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (10.3 g) was added, and the mixture was stirred at room temperature for 15 hours. Water and ethyl acetate were added to the reaction mixture, the organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane), and the pale yellow oil 7-(((4-nitrophenoxy) carbonyl) oxy) tridecane-1,13-diyldiolate ( 4.1 g) was obtained.
• the reaction mixture was cooled to room temperature, water and ethyl acetate were added, the organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate) and silica gel column chromatography (ethyl acetate-hexane, NH silica gel), and the colorless oil 7-(((2-((2- (2- ().
• Example 42 (2) Potassium carbonate in a mixture of 2- (isopropylamino) ethane-1-ol (2.0 g), 2-bromo-N, N-diethylethane-1-amine hydrobromide (7.6 g) and ethanol (20 mL) (8.0 g) was added, and the mixture was stirred under heating and reflux for 7 hours. The reaction mixture was cooled to room temperature, unnecessary substances were filtered off, and the solvent was distilled off under reduced pressure.
• Example 20 (2) 2-((2- (diethylamino) ethyl) (isopropyl) amino instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. ) 2-((2- (Diethylamino) ethyl) (isopropyl) amino) ethyl ((6Z, 9Z,), a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol is used. 28Z, 31Z) -heptateria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 43 In Example 41 (2), 2-((2- (dimethylamino) ethyl) (hexyl) amino instead of using 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol. ) Ethane-1-ol 7-(((2-((2- (dimethylamino) ethyl) (hexyl) amino) ethoxy) in the same manner as in Example 41 (2) except that ethane-1-ol is used. ) Carbonyl) oxy) tridecane-1,13-diyldiolate) was obtained.
• Example 44 (1) In Example 30 (1), the same method as in Example 30 (1) except that 2- (propylamino) ethane-1-ol was used instead of 2- (ethylamino) ethane-1-ol. Then, 2-((2- (diethylamino) ethyl) (propyl) amino) ethane-1-ol as a pale yellow oil was obtained. MSm / z (M + H): 203.
• Example 20 (2) 2-((2- (diethylamino) ethyl) (propyl) amino was used instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. ) 2-((2- (Diethylamino) ethyl) (propyl) amino) ethyl ((6Z, 9Z,), a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol is used. 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 45 Similar to Examples 41 (1) and (2) except that (9Z, 12Z) -octadeca-9,12-dienoic acid is used instead of oleic acid in Examples 41 (1) and (2). 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diyl (9Z, 9'Z, 12Z) , 12'Z) -bis (octadecane-9,12-dienoart) was obtained.
• Example 46 In Examples 41 (1) and (2), in the same manner as in Examples 41 (1) and (2), except that (Z) -hexadeca-9-enoic acid is used instead of oleic acid. Colorless oil 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diyl (9Z, 9'Z) -bis (hexadeca-) 9-Enoart) was obtained.
• Example 47 In Examples 41 (1) and (2), in the same manner as in Examples 41 (1) and (2), except that (Z) -tetradec-9-enoic acid is used instead of oleic acid. Colorless oil 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diyl (9Z, 9'Z) -bis (tetradeca-) 9-Enoate) was obtained.
• Example 48 In Examples 41 (1) and (2), 2-((2- (diethylamino) ethyl)) (instead of using 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol.
• the colorless oil 7-(((2-((2- (diethylamino) ethyl)) is the same as in Examples 41 (1) and (2) except that isopropyl) amino) ethane-1-ol is used.
• (Isopropyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diyldiolate was obtained.
• Example 49 (1) In Example 30 (1), N- (2-bromoethyl) -N-propylpropan-1-amine bromide was used instead of 2-bromo-N, N-diethylethane-1-amine hydrobromide. 2-((2- (Dipropylamino) ethyl) (ethyl) amino) ethane-1-ol as a colorless oil was obtained in the same manner as in Example 30 (1) except that a hydroxide was used. .. MSm / z (M + H): 217.
• Example 20 (2) In Example 20 (2), 2-((2- (dipropylamino) ethyl) (ethyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• Amino) Ethane-1-ol is the same method as in Example 20 (2) except that 2-((2- (dipropylamino) ethyl) (ethyl) amino) ethyl (( 6Z, 9Z, 28Z, 31Z) -heptatoria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 50 A mixture of 10-ethoxy-10-oxodecanoic acid (22.0 g), thionyl chloride (22.0 mL) and N, N-dimethylformamide (0.1 mL) was stirred under heating under reflux for 1 hour and 30 minutes. The solvent was evaporated under reduced pressure to give the pale yellow oil ethyl 10-chloro-10-oxodecanoate as a crude product.
• Tetraisopropyl orthotitanium (1.7 g) was added to a mixture of ethyl 10-oxodocosanoic acid (22.0 g) and 2-butyloctane-1-ol (31.9 g), and the mixture was stirred at 110 ° C. for 17 hours. Water and ethyl acetate were added to the reaction mixture, the organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-butyloctyl 10-oxodocosanoic acid (11.7 g) as a pale yellow solid.
• Example 41 (2) 2-butyloctyl10-(((4-nitrophenoxy)) instead of using 7-(((4-nitrophenoxy) carbonyl) oxy) tridecane-1,13-diyldiolate. ) Carbonyl) Oxy) Docosanoate is used in the same manner as in Example 41, except that the colorless oil 2-butyloctyl 12-dodecyl-3,6-diethyl-10-oxo-9,11-dioxa-3, 6-Diazahenicosan-21-Oate was obtained.
• Example 51 (1) In Example 30 (1), the same method as in Example 30 (1) except that 2- (benzylamino) ethane-1-ol was used instead of 2- (ethylamino) ethane-1-ol. Then, 2- (benzyl (2- (diethylamino) ethyl) amino) ethane-1-ol as a pale yellow oil was obtained. MSm / z (M + H): 251.
• Example 20 (2) 2- (benzyl (2- (diethylamino) ethyl) amino) ethane instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol.
• -Heptatria contour-6,9,28,31-tetraene-19-yl) carbonate was obtained.
• Example 52 (2) 2-((2-Dimethylamino) ethyl) (octyl) as a colorless oil in the same manner as in Example 37 (1) except that octanal is used instead of hexanal in Example 37 (1). Amino) ethane-1-ol was obtained. MSm / z (M + H): 245.
• Example 20 (2) 2-((2-dimethylamino) ethyl) (octyl) amino instead of using 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol. ) 2-((2- (Dimethylamino) ethyl) (octyl) amino) ethyl ((6Z, 9Z)) of the colorless oil in the same manner as in Example 20 (2) except that ethane-1-ol is used. , 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 53 (1) In Example 37 (1), the colorless oil 2-((2- (dimethylamino) ethyl) (dodecyl) was used in the same manner as in Example 37 (1) except that dodecanal was used instead of hexanal. ) Amino) Ethane-1-ol was obtained. MSm / z (M + H): 301.
• Example 20 (2) 2-((2- (dimethylamino) ethyl) (dodecyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used.
• 2-((2- (Dimethylamino) ethyl) (dodecyl) amino) ethyl) ((6Z) in the same manner as in Example 20 (2) except that amino) ethane-1-ol is used.
• 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 54 In Example 41 (2), 2-((2- (dipropylamino) ethyl) (ethyl) instead of 2-((2- (dipropylamino) ethyl) (ethyl) amino) ethane-1-ol was used.
• the colorless oil 7-(((2-((2- (dipropylamino) ethyl) (ethyl) amino) is the same method as in Example 41 (2) except that amino) ethane-1-ol is used.
• Ethoxy) carbonyl) oxy) tridecane-1,13-diyldiolate was obtained.
• Example 55 In Example 41 (2), 2-(benzyl (2- (diethylamino) ethyl) amino) ethane- instead of using 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol. 7-(((2- (benzyl (2- (diethylamino) ethyl) amino) ethoxy) carbonyl) oxy) tridecane of colorless oil in the same manner as in Example 41 (2) except that 1-ol is used. A -1,13-diylgeolate was obtained.
• Example 56 (1) In Example 41 (1), the colorless oil 7-(((4-nitrophenoxy)) was prepared in the same manner as in Example 41 (1) except that 2-hexyldecanoic acid was used instead of oleic acid. Carbonyl) oxy) tridecane-1,13-diylbis (2-hexyldecanoate) was obtained.
• Example 41 (2) 7-(((4-nitrophenoxy) carbonyl) oxy) instead of using 7-(((4-nitrophenoxy) carbonyl) oxy) tridecane-1,13-diyldiolate.
• Tridecane-1,13-diylbis (2-hexyldecanoate) was used in the same manner as in Example 41 (2), except that the colorless oil 7-(((2-((2- (diethylaminoate))).
• Ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-hexyldecanoate) was obtained.
• Example 57 8- (2-octylcyclopropyl) octanoic acid synthesized in Examples 41 (1) and (2) according to the method described in European Journal of Medicinal Chemistry, 2016,109, p134-145 instead of using oleic acid. 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) of colorless oil in the same manner as in Examples 41 (1) and (2) except that Oxy) tridecane-1,13-diylbis (8- (2-octylcyclopropyl) octanoic acid)) was obtained.
• Example 58 the colorless oil 7 was carried out in the same manner as in Examples 41 (1) and (2) except that 2-heptylundecanoic acid was used instead of oleic acid. -(((2-((2- (Diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-heptylundecanoic acid) was obtained.
• the colorless oil 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13 -Diylbis (2- (4,4-dimethylpentane-2-yl) -5,7,7-trimethyloctanoate) was obtained.
• Example 60 the colorless oil 7 was carried out in the same manner as in Examples 41 (1) and (2) except that 2-pentylheptanic acid was used instead of oleic acid. -(((2-((2- (Diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-pentylheptanoate) was obtained.
• Example 61 In Example 50 (3), 2-((2- (diethylamino) ethyl) (isopropyl) amino) instead of 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol was used. In the same manner as in Example 50 (3) except that ethane-1-ol was used, the colorless oil 2-butyloctyl 12-dodecyl-3-ethyl-6-isopropyl-10-oxo-9,11- Dioxa-3,6-diazahenicosan-21-ioate was obtained.
• Example 62 In Example 56 (2), 2-((2- (diethylamino) ethyl) (isopropyl) amino) instead of 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol was used. 7-(((2-((2- (diethylamino) ethyl) (isopropyl) amino) ethoxy) carbonyl) of colorless oil in the same manner as in Example 56 (2) except that ethane-1-ol is used. ) Oxy) Tridecane-1,13-diylbis (2-hexyldecanoate) was obtained.
• Example 63 A mixture of 2- (methylamino) ethane-1-ol (3 g), potassium carbonate (6.6 g), 1-bromopropane (5.6 mL) and acetonitrile (30 mL) was stirred at 60 ° C. for 9 hours and 30 minutes. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain 2- (methyl (propyl) amino) ethane-1-ol (4.3 g) as a colorless oil. MSm / z (M + H): 118.
• Example 20 (2) In Example 20 (2), 2- (isopropyl (2- (methyl (propyl) amino) ethyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. ) Amino) Ethane-1-ol in the same manner as in Example 20 (2) except that the colorless oil (6Z, 9Z, 28Z, 31Z) -heptatoria contour-6,9,28,31 -Tetraene-19-yl (2- (isopropyl (2- (methyl (propyl) amino) ethyl) amino) ethyl) carbonate was obtained.
• Example 64 (1) Methyl iodide (1.9 mL) was added dropwise to a solution of 2- (isopropylamino) ethane-1-ol (3 g) in dichloromethane (30 mL) under ice-cooling. After stirring at the same temperature for 1 hour and 15 minutes, the mixture was stirred at room temperature for 6 hours and 50 minutes. Potassium carbonate and water were added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate.
• Methanesulfonic anhydride (2.6 g) in a mixture of 2- (isopropyl (methyl) amino) ethane-1-ol (1.5 g), N, N-diisopropylethylamine (2.5 mL) and acetonitrile (15 mL) under ice-cooling. ) was added, and the mixture was stirred at room temperature for 4 hours and 50 minutes. To the reaction mixture was added 2- (propylamino) ethane-1-ol (4.3 mL), and the mixture was stirred at 70 ° C. for 23 hours and 30 minutes. The reaction mixture was cooled to room temperature, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with ethyl acetate.
• Example 20 (2) In Example 20 (2), 2-((2- (isopropyl (methyl) amino) ethyl) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. In the same manner as in Example 20 (2) except that (propyl) amino) ethane-1-ol was used, the colorless oil (6Z, 9Z, 28Z, 31Z) -heptatoria contour-6,9,28 , 31-Tetraene-19-yl (2-((2- (isopropyl (methyl) amino) ethyl) (propyl) amino) ethyl) carbonate was obtained.
• Example 65 (1) Ethyl 2- (diethoxyphosphoryl) ethyl acetate (9.4 mL) was added dropwise to a suspension of 60% wt sodium hydride (1.7 g) in tetrahydrofuran (60 mL) under ice-cooling, and the mixture was stirred at the same temperature for 30 minutes. Heptadecane-9-one (1.5 g) was added to the reaction mixture, and the mixture was stirred under heating under reflux for 16 hours. The reaction mixture was cooled to room temperature, the reaction mixture was poured into ice water, and then ethyl acetate was added.
• Example 41 (1) and (2) the colorless oil 7 was carried out in the same manner as in Examples 41 (1) and (2) except that 3-octylundecanoic acid was used instead of oleic acid. -(((2-((2- (Diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (3-octylundecanoic acid) was obtained.
• Example 66 In Examples 50 (1), (2) and (3), 12-ethoxy-12-oxododecanoic acid was used instead of 10-ethoxy-10-oxodecanoic acid in 1.0 mol / L dodecylmagnesium bromide-diethyl ether. 2-Butyloctyl as a colorless oil in the same manner as in Examples 50 (1), (2) and (3) except that a 1.0 mol / L decylmagnesium bromide-diethyl ether solution is used instead of the solution. 12-Decil-3,6-diethyl-10-oxo-9,11-dioxa-3,6-diazatricosan-23-oate was obtained.
• Example 67 Colorless oil in Examples 65 (1) and (2) in the same manner as in Examples 65 (1) and (2), except that tridecane-7-one was used instead of heptadecane-9-one. A 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (3-hexylnonanoic acid) was obtained.
• Example 68 Examples 50 (1), (2) and (3) except that a 1.0 mol / L decylmagnesium bromide-diethyl ether solution is used instead of the 1.0 mol / L dodecyl magnesium bromide-diethyl ether solution.
• the colorless oil 2-butyloctyl-12-decyl-3,6-diethyl-10-oxo-9,11-dioxa-3,6- Diazahenicosan-21-Oate was obtained.
• Example 69 In Example 68, 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1 was used instead of 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol.
• Diazahenicosan-21-Oate was obtained.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane), and the colorless oil 7-hydroxytridecane-1,13-diylbis (3-heptyl decanoate) (1.03 g) and the colorless oil were purified. 7,13-Dihydroxytridecyl 3-heptyl decanoate (1,03 g) was obtained.
• Example 71 (1) In Example 36 (1), a white solid was prepared in the same manner as in Example 36 (1) except that 5-bromopentane-1-ol was used instead of 6-bromohexane-1-ol. Undecane-1,6,11-triol was obtained. 1 1 H-NMR (CDCl 3 ) ⁇ : 3.70-3.55 (5H, m), 1.64-1.24 (16H, m).
• Examples 56 (1) and (2) In Examples 56 (1) and (2), Examples 56 (1) and (2) except that undecane-1,6,11-triol was used instead of tridecane-1,7,13-triol. 6-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) undecane-1,11-diylbis (2-hexyldeca) Noate) was obtained.
• Example 72 A mixture of diethyl 3-oxopentanate (4.0 g) and a 20% sodium ethoxide-ethanol solution (6.7 g) is stirred at 80 ° C. for 20 minutes, then ethyl 8-bromooctanoate (5.0 g) is added. , Stirred for 4 hours. A 20% sodium ethoxide-ethanol solution (6.7 g) was added to the reaction mixture, and the mixture was stirred for 5 minutes, ethyl 8-bromooctanoate (5.0 g) was added, and the mixture was stirred for 3 hours.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane), and the colorless oily bis (2-butyloctyl) 10-(((4-nitrophenoxy) carbonyl) oxy) nonadecane diate ( 436 mg) was obtained.
• Nonadecandioate is used in the same manner as in Example 41 (2), except that the colorless oily bis (2-butyloctyl) 10-(((2-((2) 2)). -(Diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) nonadecandioate was obtained.
• Example 73 Nonane as a white solid in Example 36 (1) in the same manner as in Example 36 (1) except that 4-bromobutane-1-ol was used instead of 6-bromohexane-1-ol. -1,5,9-triol was obtained.
• Examples 56 (1) and (2) In Examples 56 (1) and (2), Examples 56 (1) and (2) except that nonane-1,5,9-triol was used instead of tridecane-1,7,13-triol. ), The colorless oil 5-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) nonane-1,9-diylbis (2-hexyldeca) Noate) was obtained.
• Example 74 (1) Decanoic acid (3.0 g) was added dropwise to a suspension of 60% wt sodium hydride in tetrahydrofuran (30 mL) under ice-cooling, and the mixture was stirred at the same temperature for 30 minutes. A 1.5 mol / L lithium diisopropylamide-tetrahydrofuran-heptane-ethylbenzene solution (13.9 mL) was added to the reaction mixture at the same temperature, and after stirring at room temperature for 30 minutes, 1-iodooctane (3.8 mL) was added dropwise at 45 ° C. Stirred for 6 hours.
• Example 41 (1) and (2) the colorless oil 7 was carried out in the same manner as in Examples 41 (1) and (2) except that 2-octyldecanoic acid was used instead of oleic acid. -(((2-((2- (Diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-octyldecanoate) was obtained.
• Example 75 a colorless oily substance was prepared in the same manner as in Example 50 except that a 1.0 mol / L nonylmagnesium bromide-diethyl ether solution was used instead of the 1.0 mol / L dodecyl magnesium bromide-diethyl ether solution. 2-Butyloctyl 3,6-diethyl-12-nonyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 76 In Examples 74 (1) and (2), Examples 74 (1) and (2) except that nonanoic acid was used instead of decanoic acid and 1-iodoheptane was used instead of 1-iodooctane. ), The colorless oil 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-heptanenonanoic acid) ) was obtained.
• Example 77 In Examples 74 (1) and (2), Examples 74 (1) and (2) except that octanoic acid was used instead of decanoic acid and 1-iodohexane was used instead of 1-iodooctane. ), The colorless oil 7-(((2-((2- (diethylamino) ethyl) (ethyl) amino) ethoxy) carbonyl) oxy) tridecane-1,13-diylbis (2-hexyl octa) Noate) was obtained.
• Example 78 (1) 7,13-Dihydroxytridecyl3-heptyldecanoate (500 mg), decanoic acid (195 mg), triethylamine (0.43 mL), 4-dimethylaminopyridine (38 mg) synthesized in Examples 70 (1) and (2).
• Example 79 (1) Ethyl iodide (3.4 mL) was added dropwise to an acetonitrile solution (30 mL) of 2- (methylamino) ethane-1-ol (3.0 g) under ice-cooling, and the mixture was stirred at the same temperature for 1 hour and 45 minutes, and then 60. The mixture was stirred at ° C for 3 hours and 10 minutes. Potassium carbonate and water were added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain 2- (ethyl (methyl) amino) ethane-1-ol (3.4 g) as a colorless oil. MSm / z (M + H): 104.
• Example 20 (2) In Example 20 (2), 2- (tert-butyl (2- (ethyl (methyl) amino)) instead of 2-((2- (dimethylamino) ethyl) (methyl) amino) ethane-1-ol was used. ) Ethyl) amino) 2- (tert-butyl (2- (ethyl (methyl) amino) ethyl) amino, a colorless oil, in the same manner as in Example 20 (2) except that ethane-1-ol is used. ) Ethyl ((6Z, 9Z, 28Z, 31Z) -Heptatria Conta-6,9,28,31-Tetraene-19-yl) carbonate was obtained.
• Example 80 A 1 mol / L hexylmagnesium bromide-tetrahydrofuran solution (200 mL) was added dropwise to a solution of glutaric anhydride (27.3 g) in tetrahydrofuran (273 mL) under ice-cooling, and the mixture was stirred at the same temperature for 1 hour. After adding 2 mol / L hydrochloric acid aqueous solution (240 mL) to the reaction mixture under ice-cooling, ethyl acetate (270 mL) was added, the organic layer was separated, washed with water and saturated sodium chloride aqueous solution, and dried over anhydrous magnesium sulfate.
• the reaction mixture was cooled to room temperature, water and ethyl acetate were added, the organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate) and silica gel column chromatography (ethyl acetate-hexane, NH silica gel), and the colorless oil 2-butyloctyl 3,6-diethyl-12- Hexyl-10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate (356 mg) was obtained.
• Example 81 In Example 80 (2), 2-((2- (diethylamino) ethyl) (isopropyl) amino) instead of 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol was used. In the same manner as in Example 80 (2) except that ethane-1-ol was used, the colorless oil 2-butyloctyl3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11- Dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 82 (1) In Example 80 (1), the colorless oily substance 2 was prepared in the same manner as in Example 80 (1) except that 2-hexyldecane-1-ol was used instead of 2-butyloctane-1-ol. -Hexyldecyl 5-(((4-nitrophenoxy) carbonyl) oxy) undecanoic acid was obtained.
• Example 80 (2) 2-hexyldecyl 5-(((4-nitrophenoxy) carbonyl) oxy) instead of 2-butyloctyl 5-(((4-nitrophenoxy) carbonyl) oxy) undecanoic acid was used.
• Example 83 In Example 82 (2), 2-((2- (diethylamino) ethyl) (isopropyl) amino) instead of 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol was used.
• 2-Hexyldecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-, which is a colorless oil is carried out in the same manner as in Example 82 (2) except that ethane-1-ol is used. Dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 84 (1) A mixture of 10-methoxy-10-oxodecanoic acid (47.6 g), thionyl chloride (47.6 mL) and N, N-dimethylformamide (0.1 mL) was stirred under heating under reflux for 1 hour. The solvent was distilled off under reduced pressure to obtain a brown oily methyl 10-chloro-10-oxodecanoate (59.7 g).
• Tetraisopropyl orthotitanium (1.5 g) was added to a mixture of methyl 10-oxohexadecanoate (15.0 g) and 2-butyloctane-1-ol (14.7 g), and the mixture was stirred at 110 ° C. for 1 hour. Water (1 mL) is added to the reaction mixture, the mixture is stirred at room temperature for 15 minutes, purified by silica gel column chromatography (ethyl acetate-hexane), and the colorless oil 2-butyloctyl 10-oxohexadecanoate (21.6 g) is used. ) was obtained.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain 2-butyloctyl 10- (((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid (2.07 g) as a colorless oil. It was.
• the reaction mixture was cooled to room temperature, water and ethyl acetate were added, the organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate) and silica gel column chromatography (ethyl acetate-hexane, NH silica gel), and the colorless oil 2-butyloctyl 3-ethyl-12-hexyl- 6-Isopropyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate (296 mg) was obtained.
• Example 85 (1) In a mixture of 2,2'-azanediylbis (ethane-1-ol) (2.0 g), 2-bromo-N, N-diethylethane-1-amine hydrobromide (7.4 g) and ethanol (40 mL), Potassium carbonate (7.9 g) was added, and the mixture was stirred under heating and reflux for 8 hours. The reaction mixture was cooled to room temperature, unnecessary substances were filtered off, and the solvent was distilled off under reduced pressure.
• Example 84 (2) In Example 84 (2), 2,2'-((2- (diethylamino) ethyl) azandyl) instead of using 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1-ol) 2-Butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl)-, a colorless oil, in the same manner as in Example 84 (2) except that bis (ethane-1-ol) was used. 10-Oxo-9,11-dioxa-3,6-diazahenicosan-21-ioate was obtained.
• Example 86 2-Butyloctyl3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate synthesized in Example 85 (2) 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (142 mg) in a mixture of 250 mg), dodecanoic acid (112 mg), triethylamine (0.31 mL), 4-dimethylaminopyridine (136 mg) and dichloromethane (5 mL). ) was added, and the mixture was stirred at room temperature for 6 hours.
• Example 87 the colorless oil 2-butyloctyl 6- (2- (decanoyloxy) ethyl) -3 was used in the same manner as in Example 86 except that decanoic acid was used instead of dodecanoic acid. -Ethyl-12-hexyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 88 In Example 86, the colorless oil 2-butyloctyl 3-ethyl-12-hexyl-6- (2-(2-( Octanoyloxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 89 (1) In Example 84 (1), the colorless oily substance 2 was prepared in the same manner as in Example 84 (1) except that 2-hexyldecane-1-ol was used instead of 2-butyloctane-1-ol. -Hexyldecyl 10-((((4-nitrophenoxy) carbonyl) oxy) hexadecanoate was obtained.
• Example 84 (2) 2-hexyldecyl 10-((((4-nitrophenoxy) carbonyl)) instead of using 2-butyloctyl10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid.
• Oxy In the same manner as in Example 84 (2) except that hexadecanoate is used, the colorless oil 2-hexyldecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11 -Dioxa-3,6-diazahenicosan-21-ioate was obtained.
• Example 90 (1) Except for the use of 3-chloro-N, N-diethylpropan-1-amine instead of 2-bromo-N, N-diethylethane-1-amine hydrobromide in Example 85 (1). Obtained 2,2'-((3- (diethylamino) propyl) azandyl) bis (ethane-1-ol) as a colorless oil in the same manner as in Example 85 (1). MSm / z (M + H): 219.
• Example 85 (2) 2,2'-((3- (diethylamino) propyl) propyl, instead of using 2,2'-((2- (diethylamino) ethyl) azandyl) bis (ethane-1-ol) ) Azandiyl) Bis (ethane-1-ol) is used in the same manner as in Example 85 (2) except that the colorless oil 2-butyloctyl3-ethyl-13-hexyl-7- (2-hydroxy) is used. Ethyl) -11-oxo-10,12-dioxa-3,7-diazadocosan-22-oate was obtained.
• Example 91 the colorless oil 2-butyloctyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 86 except that oleic acid was used instead of dodecanoic acid. Ole oil oxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 92 With Examples 84 (1) and (2), except that 8-methoxy-8-oxooctanoic acid is used instead of 10-methoxy-10-oxodecanoic acid. In the same manner, a colorless oil 2-butyloctyl3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazanonadecane-19-ioate was obtained.
• Example 93 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate 2 -Butyloctyl 3-ethyl-13-hexyl-7- (2-hydroxyethyl) -11-oxo-10,12-dioxa-3,7-diazadocosan-22-oate, oleic acid instead of dodecanoic acid 2-Butyloctyl 3-ethyl-13-hexyl-7- (2- (oleic oxy) ethyl) -11-oxo-10,12, which is a colorless oil, in the same manner as in Example 86 except that -Dioxa-3,7-Diazadokosan-22-Oate was obtained.
• Example 94 In Example 92, 2-hexyldecyl 3-ethyl, a colorless oil, was used in the same manner as in Example 92 except that 2-hexyldecane-1-ol was used instead of 2-butyloctane-1-ol. 12-Hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazanonadecane-19-oate was obtained.
• Example 95 With Examples 84 (1) and (2), except that 6-methoxy-6-oxocaproic acid is used instead of 10-methoxy-10-oxodecanoic acid. In the same manner, a colorless oil 2-butyloctyl3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazaheptadecane-17-ioate was obtained. ..
• Example 96 In Example 95, the colorless oil 2-hexyldecyl 3-ethyl was used in the same manner as in Example 95 except that 2-hexyldecane-1-ol was used instead of 2-butyloctane-1-ol. 12-Hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazaheptadecane-17-oate was obtained.
• Example 97 In Example 85, 2-hexyldecyl 10-((((4-nitrophenoxy) carbonyl) oxy) hexa instead of using 2-butyloctyl10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid 2-Hexyldecyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-, a colorless oil, in the same manner as in Example 85 except that decanoate is used. Dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 98 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 99 In Example 98, the colorless oil 2-hexyldecyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 98 except that octanoic acid was used instead of decanoic acid. Octanoyloxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 100 the colorless oil 2-hexyldecyl 3-ethyl-6- (2- (hexanoyloxy)) was produced in the same manner as in Example 98 except that caproic acid was used instead of decanoic acid. Ethyl) -12-hexyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 101 In Example 80 (2), 2,2'-((2- (diethylamino) ethyl) azandyl) instead of using 2-((2- (diethylamino) ethyl) (ethyl) amino) ethane-1-ol) 2-Butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl)-, a colorless oil, in the same manner as in Example 80 (2) except that bis (ethane-1-ol) was used. 10-Oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 102 In Example 85 (2), 2-hexyldecyl 5-(((4-nitrophenoxy) carbonyl) oxy) instead of using 2-butyloctyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid ) 2-Hexyldecyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9, which is a colorless oil, in the same manner as in Example 85 (2) except that undecanoic acid is used. , 11-Dioxa-3,6-Diazahexadecane-16-Oate was obtained.
• Example 103 the colorless oil 2-butyloctyl 3-ethyl-12-hexyl-6- (2-(2-( Nonanoyloxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 104 the colorless oil 2-butyloctyl 3-ethyl-6- (2- (heptanoyloxy)) was produced in the same manner as in Example 86 except that heptanoic acid was used instead of dodecanoic acid. Ethyl) -12-hexyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 105 the colorless oil 2-butyloctyl 3-ethyl-6- (2- (hexanoyloxy)) was produced in the same manner as in Example 86 except that caproic acid was used instead of dodecanoic acid. Ethyl) -12-hexyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 106 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate. Example 86 except that 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate is used.
• Example 107 the colorless oil 2-butyloctyl 6- (2- (decanoyloxy) ethyl) -3 was used in the same manner as in Example 106 except that decanoic acid was used instead of dodecanoic acid. -Ethyl-12-hexyl-10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 108 In Example 106, the colorless oil 2-butyloctyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 106 except that octanoic acid was used instead of dodecanoic acid. Octanoyloxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 109 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate. Example 86 except that 2-hexyldecyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate is used.
• Example 110 the colorless oil 2-hexyldecyl 6- (2- (decanoyloxy) ethyl) -3 was used in the same manner as in Example 109 except that decanoic acid was used instead of dodecanoic acid. -Ethyl-12-hexyl-10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 111 In Example 109, the colorless oil 2-hexyldecyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 109 except that octanoic acid was used instead of dodecanoic acid. Octanoyloxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 112 In Examples 80 (1) and (2), 2-octyldodecane-1-ol was used instead of 2-butyloctane-1-ol, and 2-((2- (diethylamino) ethyl) (ethyl) amino. ) Similar to Examples 80 (1) and (2) except that 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1-ol is used instead of ethane-1-ol.
• Example 113 In Examples 80 (1) and (2), 2-decyltetradecane-1-ol was used instead of 2-butyloctane-1-ol, and 2-((2- (diethylamino) ethyl) (ethyl) amino. ) Similar to Examples 80 (1) and (2) except that 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1-ol is used instead of ethane-1-ol.
• Example 114 In Examples 84 (1) and (2), 4-ethoxy-4-oxobutanoic acid was used instead of 10-methoxy-10-oxodecanoic acid, and 2-butyloctane-1-ol was used instead of 2-butyloctane-1-ol. 2-Hexyldecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-, a colorless oil, in the same manner as in Examples 84 (1) and (2) except that hexyldecane-1-ol was used. 9,11-Dioxa-3,6-diazapentadecane-15-oate was obtained.
• Example 115 In Examples 80 (1) and (2), instead of using 2-butyloctane-1-ol, (Z) -octadeca-9-ene-1-ol was replaced with 2-((2- (diethylamino) ethyl).
• Ethane-1-ol was used instead of 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1-ol, except that Examples 80 (1) and ( In the same manner as in 2), the colorless oil (Z) -octadeca-9-ene-1-yl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6 -Diazahexadecane-16-Oate was obtained.
• Example 116 In Examples 84 (1) and (2), 7-ethoxy-7-oxoenanthic acid was used instead of 10-methoxy-10-oxodecanoic acid, and 2-butyloctane-1-ol was used instead of 2 2-Hexyldecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo in the same manner as in Examples 84 (1) and (2) except that -hexyldecane-1-ol is used. -9,11-Dioxa-3,6-diazooctadecane-18-oate was obtained.
• Example 117 In Examples 84 (1) and (2), 9-methoxy-9-oxononanoic acid was used instead of 10-methoxy-10-oxodecanoic acid, and 2-butyloctane-1-ol was used instead of 2-butyloctane-1-ol. 2-Hexyldecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-, a colorless oil, in the same manner as in Examples 84 (1) and (2) except that hexyldecane-1-ol was used. 9,11-Dioxa-3,6-diazaicosan-20-Oate was obtained.
• Example 118 In Example 98, the colorless oil 2-hexyldecyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 98 except that oleic acid was used instead of decanoic acid. Ole oil oxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 119 In Example 106, the colorless oil 2-butyloctyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 106 except that oleic acid was used instead of dodecanoic acid. Ole oil oxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 120 the colorless oil 2-hexyldecyl 3-ethyl-12-hexyl-6- (2-(2-() was carried out in the same manner as in Example 109 except that oleic acid was used instead of dodecanoic acid. Ole oil oxy) ethyl) -10-oxo-9,11-dioxa-3,6-diazahexadecane-16-oate was obtained.
• Example 121 Similar to Examples 84 (1) and (2) except that 2-octyldodecane-1-ol is used instead of 2-butyloctane-1-ol in Examples 84 (1) and (2). To obtain 2-octyldodecyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate as a colorless oil by the above method.
• Example 122 Acrylic acid chloride (0.45 mL) was added to a mixture of heptane-1-ol (0.86 mL), triethylamine (1.55 mL) and tetrahydrofuran (5.00 mL) under ice-cooling, and the mixture was stirred at room temperature for 2 hours. Water and ethyl acetate were added to the reaction mixture, the organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane) to obtain a colorless oily heptyl acrylate (0.57 g).
• Triethylamine (1.24 mL) was added to the resulting mixture of heptyl acrylate (0.57 g), 2-((2- (diethylamino) ethyl) amino) ethane-1-ol dihydrochloride (0.52 g) and tetrahydrofuran (10 mL).
• the mixture was stirred under heating and reflux for 8 hours.
• the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure.
• Example 84 (2) In Example 84 (2), heptyl 3-((2- (diethylamino) ethyl) (2-hydroxy) was used instead of 2-((2- (diethylamino) ethyl) (isopropyl) amino) ethane-1-ol. 2-Butyloctyl 3-ethyl-6- (3- (heptyloxy) -3-oxopropyl)-, a colorless oil, in the same manner as in Example 84 (2) except that ethyl) amino) propanoate was used. 12-Hexyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 123 Ethyl 2- (diethoxyphosphoryl) ethyl acetate (18.8 mL) was added dropwise to a suspension of 60% wt sodium hydride (3.3 g) in tetrahydrofuran (80 mL) under ice-cooling, and the mixture was stirred at the same temperature for 30 minutes. Undecane-6-one (2.0 g) was added to the reaction mixture, and the mixture was stirred under heating under reflux for 5 hours. The reaction mixture was cooled to room temperature, the reaction mixture was poured into ice water, and then ethyl acetate was added.
• Example 84 (1) a colorless oily substance was prepared in the same manner as in Example 84 (1) except that 3-pentyloctane-1-ol was used instead of 2-butyloctane-1-ol. 3-Pentyloctyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid was obtained.
• Example 85 (2) 3-pentyloctyl10-(((4-nitrophenoxy) carbonyl) oxy) instead of using 2-butyloctyl10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid.
• the colorless oil 3-pentyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9 , 11-Dioxa-3,6-Diazahenicosan-21-Oate was obtained.
• Example 125 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 126 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 127 (1) In Example 84 (1), instead of using a 1.0 mol / L hexylmagnesium bromide-diethyl ether solution, a 1.0 mol / L pentylmagnesium bromide-tetrahydrofuran solution was used, and instead of using 2-butyloctane-1-ol, 2 2-Hexyldecyl 10-(((4-nitrophenoxy) carbonyl) oxy) pentadecanoic acid as a colorless oil was obtained in the same manner as in Example 84 (1) except that -hexyldecane-1-ol was used. ..
• Example 84 (2) In Example 84 (2), 2-hexyldecyl 10-(((4-nitrophenoxy) carbonyl) oxy) instead of using 2-butyloctyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid. ) 2-Hexyldecyl 3-ethyl-6-isopropyl-10-oxo-12-pentyl-9,11-dioxa-, a colorless oil, in the same manner as in Example 84 (2) except that pentadecanoic acid is used. 3,6-Diazahenicosan-21-Oate was obtained.
• Example 84 (1) a colorless oily substance was prepared in the same manner as in Example 84 (1) except that 2-pentylheptane-1-ol was used instead of 2-butyloctane-1-ol. 2-Pentylheptyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid was obtained.
• Example 85 (2) 2-pentylheptyl 10-(((4-nitrophenoxy) carbonyl) oxy) instead of using 2-butyloctyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid.
• 11-Dioxa-3,6-Diazahenicosan-21-Oate was obtained.
• Example 129 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 130 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 131 In Example 85 (2), 2-hexyldecyl 10-(((4-nitrophenoxy) carbonyl) oxy) instead of using 2-butyloctyl 10-(((4-nitrophenoxy) carbonyl) oxy) hexadecanoic acid. ) 2-Hexyldecyl 3-ethyl-6- (2-hydroxyethyl) -10-oxo-12-pentyl-9, a colorless oil, in the same manner as in Example 85 (2) except that pentadecanoic acid is used. , 11-Dioxa-3,6-Diazahenicosan-21-Oate was obtained.
• Example 132 In Example 86, instead of using 2-butyloctyl 3-ethyl-12-hexyl-6- (2-hydroxyethyl) -10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate.
• Example 133 (1) Examples 124 (1) and (2), except that pelargonic acid is used instead of undecanoic acid and 1-iodoheptane is used instead of 1-iodononane in Examples 124 (1) and (2).
• Examples 84 (1) and (2) are the same as in Examples 84 (1) and (2) except that 2-heptylnonane-1-ol is used instead of 2-butyloctane-1-ol.
• 2-heptylnonane-1-ol is used instead of 2-butyloctane-1-ol.
• a colorless oil 2-heptylnonyl 3-ethyl-12-hexyl-6-isopropyl-10-oxo-9,11-dioxa-3,6-diazahenicosan-21-oate was obtained.
• Example 134 (2) 2-Hexyl-1-octanol (4.4 g), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.9 g), 4 in a solution of 10-bromodecanoic acid (4.0 g) in dichloromethane (80 mL). -Dimethylaminopyridine (0.9 g) and triethylamine (8.6 mL) were added at room temperature and stirred overnight at the same temperature. Ethyl acetate and water were added to the reaction mixture. The organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• N-octylamine (1.1 mL) and potassium carbonate (1.9 g) were added to a solution of 2-hexyloctyl 10-bromodecanoate (1.0 g) in N, N-dimethylformamide (5 mL) at room temperature, and the mixture was added at room temperature for 4 hours at 60 ° C. Stirred. After cooling the reaction mixture to room temperature, ethyl acetate and water were added. The organic layer was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure.
• Example 135 A 1 mol / L hexylmagnesium bromide-tetrahydrofuran solution (200 mL) was added dropwise to a solution of glutaric anhydride (27.3 g) in tetrahydrofuran (273 mL) under ice-cooling, and the mixture was stirred at the same temperature for 1 hour. After adding 2 mol / L hydrochloric acid aqueous solution (240 mL) to the reaction mixture under ice cooling, ethyl acetate (270 mL) was added, the organic layer was separated, and washed with water (80 mL) and saturated sodium chloride aqueous solution (80 m).
• Test Example 1 Preparation of nucleic acid lipid particles and measurement of reporter protein knockdown rate in mice ⁇ Preparation of nucleic acid lipid particles> The first compound 24, compound 30, compound 31, compound 50, compound 56, compound 69, compound 88, compound 89, compound 103, compound 112, compound 118, compound 119, and compound 134 produced in the above examples. Used as a lipid.
• the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of sterols in the lipid composition is shown in Table 1.
• the weight ratio of nucleic acids to total lipids at the time of mixing is also shown in Table 1.
• ⁇ Evaluation of siRNA inclusion rate> (Quantitative total nucleic acid concentration) To 60 ⁇ L of lipid particles holding nucleic acid, 30 ⁇ L of 3 mol / L sodium acetate aqueous solution and 9 ⁇ L of glycogen were added, and then 1.5 mL of ethanol was added to dissolve the lipid and precipitate only the nucleic acid. Then, centrifugation was performed to remove the supernatant. After air-drying for 15 minutes or more, water was added to redissolve the nucleic acid, and the concentration was measured using Nanodrop NF1000 (Thermo Fisher Scientific) to quantify the total nucleic acid concentration.
• Nanodrop NF1000 Thermo Fisher Scientific
• Quantum-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) was used and quantified according to the protocol.
• the 20 ⁇ TE buffer included in the above kit was diluted with water to obtain a 1 ⁇ TE buffer.
• TE represents Tris / EDTA (ethylenediaminetetraacetic acid).
• the lipid particle dispersion liquid holding the nucleic acid was diluted 10000 times with 1 ⁇ TE buffer.
• nucleic acid inclusion rate (total nucleic acid concentration-nucleic acid concentration in the outer aqueous phase) ⁇ total nucleic acid concentration x 100 The calculation results are shown in Table 2.
• FVII Factor VII
• the nucleic acid-lipid composition of the present invention showed a strong FVII inhibitory activity and an excellent nucleic acid delivery effect.
• Test Example 2 Measurement of pharmacokinetic (PK) data (liver accumulation) ⁇ Preparation of nucleic acid lipid particles> Compound 56, compound 88, and compound 89 produced in the above examples were used as the first lipid. The dispersions of the nucleic acid lipid particles shown in Table 3 were obtained in the same manner as in ⁇ Preparation of nucleic acid lipid particles> of Test Example 1.
• PK pharmacokinetic
• the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of sterols in the lipid composition is shown in Table 3.
• the weight ratio of nucleic acid to total lipid at the time of mixing is also shown in Table 3.
• ⁇ Measurement of liver accumulation> The dispersion of nucleic acid lipid particles prepared in ⁇ Preparation of Nucleic Acid Lipid Particles> was administered to C57BL6 / J mice by tail vein so as to be 0.1 mg / kg. Twenty-four hours after administration, the liver was removed, rapidly frozen in liquid nitrogen, and then frozen and pulverized with a multi-bead shocker. After thawing on ice, 0.25% Triton-PBS was added to obtain liver homogenate. The obtained liver homogenate was reverse transcribed using Taqman MicroRNA Reverse Transcription kit (Applied Biosystems, 4366597) and Factor VII Reverse Transcription Primer (Applied Biosystems). The reverse transcribed sample was quantified by Taqman MGB gene expression kit (Applied Biosystems, 4324036), Taqman Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, 4364 The measurement results are shown in Table 4.
• nucleic acid-lipid composition of the present invention strongly accumulates in the liver as compared with the nucleic acid-lipid composition of the comparative example.
• Test Example 3 Preparation of mRNA-encapsulating lipid particles and measurement of reporter protein expression rate in mice ⁇ Preparation of EPO mRNA-encapsulating lipid particles> The compounds listed in Table 5, 1,2-distearoyl-sn-glycero-3-phosphatidyl (product name: COATSOME (R) MC-8080; NOF corporation), L- ⁇ -diore oil phosphatidylethanolamine (product name: COATSOME).
• R MC-8181; NOF corporation
• cholesterol product name: Cholesterol HP; NOF CORPORATION
• 1,2-dimiristoyl-rac-glycero-3- (methylpolyoxyethylene 2000) hereinafter, DMG) -PEG2000
• PBS phosphate buffered saline
• Ethanol was removed by dialysis of this dispersion with a 10% sucrose aqueous solution using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to obtain EPO mRNA-encapsulating lipid particles.
• R MC-8181; NOF corporation
• cholesterol product name: Cholesterol HP; NOF CORPORATION
• 1,2-dimiristoyl-rac-glycero-3- (methylpolyoxyethylene 2000) hereinafter, DMG) -PEG2000
• PBS phosphate buffered saline
• Ethanol was removed by dialysis of this dispersion with a 10% sucrose aqueous solution using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to obtain FLuc mRNA-encapsulating lipid particles.
• ⁇ Evaluation of mRNA inclusion rate> (Quantification of total mRNA concentration) To 30 to 60 ⁇ L of lipid particles holding mRNA, 15 to 30 ⁇ L of 3 mol / L sodium acetate aqueous solution and 4.5 to 9 ⁇ L of glycogen were added, and then 0.75 to 1.5 mL of ethanol was added to dissolve the lipid. , Only mRNA was precipitated. Then, centrifugation was performed to remove the supernatant. After air-drying for 15 minutes or more, water was added to redissolve the mixture, and the concentration was measured using Nanodrop NF1000 (Thermo Fisher Scientific) to quantify the total mRNA concentration.
• Nanodrop NF1000 Thermo Fisher Scientific
• Quantum-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) was used and quantified according to the protocol.
• the 20 ⁇ TE buffer included in the above kit was diluted with water to obtain a 1 ⁇ TE buffer.
• TE represents Tris / EDTA (ethylenediaminetetraacetic acid).
• the lipid particle dispersion liquid holding the mRNA was diluted 10000 times with 1 ⁇ TE buffer.
• RNA concentration in the external aqueous phase was quantified by measuring fluorescence (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) using a plate reader Infinit EF200 (TECAN).
• mRNA inclusion rate (total mRNA concentration-mRNA concentration in the external aqueous phase) ⁇ total mRNA concentration ⁇ 100
• nucleic acid-lipid composition of the present invention has a stronger inhibitory activity on the expression rate of the reporter protein as compared with the nucleic acid-lipid composition of the comparative example.
• Test Example 4 Preparation of mRNA-encapsulating lipid particles and measurement of reporter protein expression rate in mice ⁇ Preparation of nucleic acid lipid particles> Compounds 107, 129 and 135 produced in the above examples were used as the first lipid.
• EPO mRNA (product name: CleanCap EPO mRNA (5 moU); TriLink) is mixed with a 50 mmol / L citrate buffer at pH 4 and the weight ratio of total lipid to mRNA becomes approximately 16: 1 to 64: 1.
• mRNA was dissolved in CA buffer to obtain an aqueous phase.
• PBS phosphate buffered saline
• Ethanol was removed by dialysis of this dispersion with a 20 mM Tris buffer containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific), and EPO mRNA was included. Lipid particles were obtained.
• DOPC 1,2-diore oil phosphatidylethanolamine
• FLuc mRNA (product name: CleanCap FLuc mRNA; TriLink) is mixed with 50 mmol / L citrate buffer at pH 4 so that the weight ratio of total lipid to mRNA is about 16: 1 to 64: 1 after mixing with the oil phase.
• the mRNA was dissolved in CA buffer to obtain an aqueous phase.
• PBS phosphate buffered saline
• Ethanol was removed by dialysis of this dispersion with a 20 mM Tris buffer containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific), and FLuc mRNA was included. Lipid particles were obtained.
• ⁇ Evaluation of mRNA inclusion rate> (Quantification of total mRNA concentration) The total mRNA concentration was quantified by adding 900 ⁇ L of methanol to 100 ⁇ L of mRNA and lipid particles containing mRNA to dissolve the lipid, and measuring the absorbance at 260 nm using a Thermo Fisher Scientific.
• Quantum-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific) was used and quantified according to the protocol.
• the 20 ⁇ TE buffer included in the above kit was diluted with water to obtain a 1 ⁇ TE buffer.
• TE represents Tris / EDTA (ethylenediaminetetraacetic acid).
• the lipid particle dispersion liquid holding the mRNA was diluted 50-fold with 1 ⁇ TE buffer.
• RNA concentration in the external aqueous phase was quantified by measuring fluorescence (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) using a plate reader Infinity F200 (TECAN).
• mRNA inclusion rate (total mRNA concentration-mRNA concentration in the external aqueous phase) ⁇ total mRNA concentration ⁇ 100
• EPO enzyme activity The dispersion of mRNA lipid particles prepared in the above ⁇ Preparation of EPO mRNA-encapsulating lipid particles> was intravenously administered to ICR mice so that the mRNA dose was 0.1 mg / kg. Blood was collected from the posterior vena cava 6 hours after administration to obtain plasma. Using the obtained plasma, human EPO enzyme activity was quantified using ab119522 Erythropoietin (EPO) Human Elisa Kit (Abcam). The results are shown in Table 11.
• nucleic acid-lipid composition of the present invention had a higher reporter protein expression rate than the nucleic acid-lipid composition of the comparative example.
• ⁇ Luciferase luminescence measurement> The dispersion of mRNA lipid particles prepared in the above ⁇ Preparation of FLuc mRNA-encapsulating lipid particles> was administered once to the ICR mice from the dorsal side into the rectus femoris muscle so as to have an mRNA dose of 1 ⁇ g. After 5 hours and 50 administration, 150 mg / kg of D-luciferin potassium (Fujifilm Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered, and 6 hours after administration under isoflurane gas anesthesia, luminescence was emitted using the IVIS Imaging System (PerkinElmer) in the prone position. It was measured. The ROI was set so that all the lower limbs on the side of administration were included, and the amount of luminescence (Photone / Sec) was quantified by Living Image Software (PerkinElmer). The results are shown in Table 12.
• the nucleic acid-lipid composition of the present invention showed better luminescence.

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