Compound or salt thereof, lipid composition, pharmaceutical composition, and delivery carrier

Abstract

The present invention addresses the problem of providing: a compound or a salt thereof that is to constitute a lipid composition that makes it possible to achieve high expression of a protein when an mRNA is used and dose-responsive protein production when administered at a high dose; and a lipid composition, a pharmaceutical composition, and a delivery carrier that use the compound or salt thereof. The present invention provides a compound represented by formula (1) or a salt thereof. Each group in the formula is defined in the description.

Description

Compounds or salts thereof, lipid compositions, pharmaceutical compositions and delivery carriers

[0001] The present invention relates to a compound or a salt thereof that facilitates the introduction of nucleic acids into cells, etc. The present invention further relates to a lipid composition, a pharmaceutical composition, and a delivery carrier that contain the compound or a salt thereof.

[0002] Nucleic acid drugs have a clear mechanism of action against diseases and few side effects, and are expected to be next-generation pharmaceuticals. For example, nucleic acid drugs using siRNA (small interfering RNA) can inhibit the expression of target genes in a sequence-specific manner within cells. As a result, diseases and symptoms caused by the abnormal expression of specific genes or groups of genes can be alleviated or treated. In order to express the functions of these nucleic acids, it is necessary to deliver the nucleic acid drugs into cells.

[0003] As a method for efficiently delivering nucleic acids into cells, there is a method using a viral vector such as a retrovirus or an adenovirus. The method using a viral vector has high gene transfer efficiency, but there is a limit to the size of the gene to be transferred, and there are concerns about immunogenicity and safety. On the other hand, gene transfer using a lipid composition has no limit to the gene to be transferred and can solve the above problems, so that its development is being actively carried out.

[0004] Patent Document 1 describes an amino lipid or a salt thereof represented by a specific formula (1), and also describes a lipid composition containing the amino lipid or a salt thereof. Patent Document 1 also describes the delivery of nucleic acids to mouse cells using the lipid composition.

[0005] Patent Document 2 describes a lipid composition containing an amino lipid represented by a specific formula (1) or a salt thereof, a nonionic lipid, a lipid having a nonionic hydrophilic polymer structure, and nucleic acid, with or without a zwitterionic lipid. Patent Document 2 also describes the use of the lipid composition to deliver nucleic acid to mouse cells.

[0006] Patent Document 3 describes a lipid composition comprising a first lipid, which is a lipid represented by a predetermined formula (1) or a salt thereof, sterols, and nucleic acid, wherein the ratio of the number of moles of the first lipid in the lipid composition to the number of moles of the sterols in the lipid composition is 0.300 or more and less than 1.299. Patent Document 3 also describes the delivery of nucleic acid to mouse cells using the lipid composition.

[0007] International Publication No. WO2019 / 235635 International Publication No. WO2020 / 246581 International Publication No. WO2021 / 095876

[0008] In the prior art, it was difficult to achieve high protein expression when using mRNA and dose-responsive protein production at high doses. The present invention aims to provide a compound or a salt thereof constituting a lipid composition that can achieve a high nucleic acid encapsulation rate and excellent nucleic acid delivery when using mRNA. Another aim of the present invention is to provide a lipid composition, a pharmaceutical composition, and a delivery carrier that use the above compound or a salt thereof and can achieve a high nucleic acid encapsulation rate and excellent nucleic acid delivery when using mRNA.

[0009] As a result of intensive research to solve the above problems, the present inventors have confirmed that a lipid composition prepared using a compound represented by the following formula (1) or a salt thereof can achieve a high nucleic acid encapsulation rate and excellent nucleic acid delivery when mRNA is used, and have completed the present invention. According to the present invention, the following inventions are provided.

[0010] <1> A compound represented by the following formula (1) or a salt thereof: In the formula, R １ represents a hydrocarbon group having 1 to 24 carbon atoms, R ２ represents a hydrogen atom or a hydrocarbon group having 1 to 24 carbon atoms; R ３ represents a hydrocarbon group having 6 to 24 carbon atoms and having a branched chain, R ４ and R ５ each independently represents an optionally substituted hydrocarbon group having 1 to 6 carbon atoms; R ４ and R ５The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently -O-R ６ , -OC(O)OR ６ , —OC(O)—R ６ , —CO(O)—R ６ , —C(O)—R ６ , -C(O)N(R ６ ) R ７ , or -N(R ６ ) R ７ indicates, R ６ and R ７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, L represents *-O-C(O)- or *-C(O)O-, * represents R １ a represents an integer of 1 to 12, b represents an integer of 1 to 4, c represents an integer of 1 to 4, and d represents an integer of 1 to 4. <2> R １ represents a hydrocarbon group having 2 to 12 carbon atoms; R ２ represents a hydrocarbon group having 2 to 12 carbon atoms; R ３ represents a hydrocarbon group having 8 to 22 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 6 carbon atoms, L represents *-O-C(O)-, * represents R １ a represents an integer of 2 to 10, b represents an integer of 2 to 4, c represents an integer of 2 to 4, and d represents an integer of 2 to 4. The compound according to <1> or a salt thereof. <3> R １ represents a hydrocarbon group having 3 to 10 carbon atoms; R ２ represents a hydrocarbon group having 3 to 10 carbon atoms; R ３ represents a hydrocarbon group having 10 to 20 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 3 carbon atoms, L represents *-O-C(O)-, * represents R １a represents an integer of 3 to 8, b represents 2 or 3, c represents 2 or 3, and d represents 2 or 3. The compound according to <1> or <2>, or a salt thereof. <4> R ３ is -CH(R ３１ ) (R ３２ ), or -CH ２ CH (R ３３ ) (R ３４ ), and R ３１ , R ３２ , R ３３ , and R ３４ and each independently represent a linear hydrocarbon group having 3 to 12 carbon atoms, or a salt thereof. <5> A compound or a salt thereof selected from the following compounds: heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyloctanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-7-(2-((2-heptylnonanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-nonylundecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-7-(2-((3-heptyldecanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-hexylooctanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-6-(2-((2-heptylnonanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-octyldecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-nonylundecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-hexyldecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-6-(2-((3-heptyldecanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; <6> A lipid composition comprising the compound or salt thereof according to any one of <1> to <5> and a lipid. <7> The lipid composition according to <6>, wherein the lipid is at least one type of lipid selected from the group consisting of neutral lipids and lipids having a nonionic hydrophilic polymer chain. <8> The lipid composition according to <6> or <7>, further comprising a sterol. <9> The lipid composition according to any one of <6> to <8>, further comprising at least one selected from the group consisting of nucleic acids, proteins, peptides, and small molecules. <10> A pharmaceutical composition comprising the lipid composition according to any one of <6> to <9>. <11> A delivery carrier comprising the lipid composition according to any one of <6> to <9>.

[0011] By using the compound of the present invention, it is possible to produce a lipid composition, a pharmaceutical composition and a delivery carrier that can achieve a high nucleic acid encapsulation rate and excellent nucleic acid delivery when mRNA is used. The lipid composition, the pharmaceutical composition and the delivery carrier of the present invention can achieve a high nucleic acid encapsulation rate and excellent nucleic acid delivery when mRNA is used.

[0012] FIG. 1 shows the results of measuring the dose-response of each nucleic acid-lipid composition.

[0013] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively. <Compound of the Present Invention> The present invention relates to a compound represented by the following formula (1) or a salt thereof. By using the compound represented by formula (1) or a salt thereof as a constituent component of a lipid composition, high protein expression when mRNA is used and dose-responsive protein production at high doses can be achieved. In the formula, R １ represents a hydrocarbon group having 1 to 24 carbon atoms, R ２ represents a hydrogen atom or a hydrocarbon group having 1 to 24 carbon atoms; R ３ represents a hydrocarbon group having 6 to 24 carbon atoms and having a branched chain, R ４ and R ５ each independently represents an optionally substituted hydrocarbon group having 1 to 6 carbon atoms; R ４ and R ５ The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently -O-R ６ , -OC(O)OR ６ , —OC(O)—R ６ , —CO(O)—R ６ , —C(O)—R ６ , -C(O)N(R ６ ) R ７ , or -N(R ６ ) R ７ indicates, R ６ and R ７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, L represents *-O-C(O)- or *-C(O)O-, * represents R１ a represents an integer of 1 to 12; b represents an integer of 1 to 4; c represents an integer of 1 to 4; and d represents an integer of 1 to 4.

[0014] R １ represents preferably a hydrocarbon group having 2 to 18 carbon atoms, more preferably a hydrocarbon group having 2 to 12 carbon atoms, more preferably a hydrocarbon group having 3 to 10 carbon atoms, and even more preferably a hydrocarbon group having 3 to 8 carbon atoms; R ２ represents preferably a hydrocarbon group having 2 to 12 carbon atoms, more preferably a hydrocarbon group having 3 to 10 carbon atoms, more preferably a hydrocarbon group having 3 to 6 carbon atoms, more preferably a hydrocarbon group having 4 to 6 carbon atoms, and particularly preferably a hydrocarbon group having 6 carbon atoms.

[0015] R ３ represents a hydrocarbon group having preferably 8 to 20 carbon atoms and having a branched chain, and more preferably represents a hydrocarbon group having 11 to 17 carbon atoms and having a branched chain.

[0016] R ３ In the "hydrocarbon group having 6 to 24 carbon atoms and having a branched chain" represented by the formula (1), it is preferable that the branched chain is present at the α-position or β-position of the carbonyl group (-C(=O)- to which R3 is bonded in formula (1)). ３ The "hydrocarbon group having 6 to 24 carbon atoms and having a branched chain" is preferably -CH(R ３１ ) (R ３２ ), or -CH ２ CH (R ３３ ) (R ３４ ) is a group represented by the formula:

[0017] R ３１ and R ３２ each independently represents a hydrocarbon group, R ３１ and R ３２ The total number of carbon atoms in the hydrocarbon group represented by R is 5 to 23. ３１ and R ３２ R each independently represents a linear hydrocarbon group, and more preferably represents a linear alkyl group. ３１and R ３２ The number of carbon atoms in the hydrocarbon groups represented by R may be the same or different. ３１ and R ３２ The difference in the number of carbon atoms between the hydrocarbon groups represented by R is preferably 5 or less, more preferably 3 or less, and even more preferably 1 or less. ３１ and R ３２ The number of carbon atoms of the hydrocarbon groups represented by R is preferably the same. ３１ and R ３２ are each independently preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 4 to 10 carbon atoms, more preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 4 to 8 carbon atoms, and even more preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 6 to 8 carbon atoms.

[0018] R ３３ and R ３４ each independently represents a hydrocarbon group, R ３３ and R ３４ The total number of carbon atoms in the hydrocarbon group represented by R is 4 to 22. ３３ and R ３４ R each independently represents a linear hydrocarbon group, and more preferably represents a linear alkyl group. ３３ and R ３４ The number of carbon atoms in the hydrocarbon groups represented by R may be the same or different. ３３ and R ３４ The difference in the number of carbon atoms between the hydrocarbon groups represented by R is preferably 5 or less, more preferably 3 or less, and even more preferably 1 or less. ３３ and R ３４ The number of carbon atoms of the hydrocarbon groups represented by R is preferably the same. ３３ and R ３４ are each independently preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 4 to 10 carbon atoms, more preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 4 to 8 carbon atoms, even more preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 6 to 8 carbon atoms, and particularly preferably a straight-chain hydrocarbon group (preferably an alkyl group) having 7 carbon atoms.

[0019] R３ is preferably —CH(R ３１ ) (R ３２ ), or -CH ２ CH (R ３３ ) (R ３４ ), and R ３１ , R ３２ , R ３３ , and R ３４ each independently represents a linear hydrocarbon group having 3 to 12 carbon atoms.

[0020] R ４ and R ５ each independently represents a hydrocarbon group having 1 to 3 carbon atoms which may be substituted. ４ and R ５ each independently represents a hydrocarbon group having preferably 1 to 6 carbon atoms, more preferably a hydrocarbon group having 1 to 3 carbon atoms; R ４ and R ５ The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently -O-R ６ , -OC(O)OR ６ , —OC(O)—R ６ , —CO(O)—R ６ , —C(O)—R ６ , -C(O)N(R ６ ) R ７ , or -N(R ６ ) R ７ indicates R ６ and R ７ are each independently a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms. ６ and R ７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 12 carbon atoms, and more preferably represents a hydrogen atom or a hydrocarbon group having 7 to 12 carbon atoms.

[0021] L preferably represents *-O-C(O)-, where * is R １a represents the bonding position with a. a preferably represents an integer of 2 to 10, more preferably an integer of 3 to 8. b preferably represents an integer of 2 to 4, more preferably 2 or 3. c preferably represents an integer of 2 to 4, more preferably 2 or 3. d preferably represents an integer of 2 to 4, more preferably 2 or 3.

[0022] In formula (1), preferably, R １ represents a hydrocarbon group having 2 to 12 carbon atoms; R ２ represents a hydrocarbon group having 2 to 12 carbon atoms; R ３ represents a hydrocarbon group having 8 to 22 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 6 carbon atoms, L represents *-O-C(O)-, * represents R １ a represents an integer of 2 to 10; b represents an integer of 2 to 4; c represents an integer of 2 to 4; and d represents an integer of 2 to 4.

[0023] In formula (1), R is more preferably １ represents a hydrocarbon group having 3 to 10 carbon atoms; R ２ represents a hydrocarbon group having 3 to 10 carbon atoms; R ３ represents a hydrocarbon group having 10 to 20 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 3 carbon atoms, L represents *-O-C(O)-, * represents R １ a represents an integer of 3 to 8; b represents 2 or 3; c represents 2 or 3; and d represents 2 or 3.

[0024] The hydrocarbon group is preferably an alkyl group, an alkenyl group, or an alkynyl group.

[0025] The alkyl group may be linear or branched, and may be linear or cyclic. Specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a cyclopentyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a trimethyldodecyl group (preferably a 3,7,11-trimethyldodecyl group), a tetradecyl group, a pentadecyl group, a hexadecyl group, a tetramethylhexadecyl group (preferably a 3,7,11,15-tetramethylhexadecyl group), a heptadecyl group, an octadecyl group, a 2-butylhexyl group, and a 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, 3-nonyldodecyl group, 2-decyldodecyl group, 2-decyltetradecyl group, 3-decyltridecyl group, 2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl group, and the like.

[0026] The alkenyl group may be linear or branched, linear or cyclic. Specific examples include allyl, prenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl (preferably, (Z)-2-nonenyl or (E)-2-nonenyl), decenyl, undecenyl, dodecenyl, dodecadienyl, tridecenyl (preferably, (Z)-tridec-8-enyl), tetradecenyl (preferably, tetradec-9-enyl), and pentadecenyl (preferably, (Z)-pentadecen-8-enyl). , a hexadecenyl group (preferably a (Z)-hexadec-9-enyl group), a hexadecadienyl group, a heptadecenyl group (preferably a (Z)-heptadeca-8-enyl group), a heptadecadienyl group (preferably a (8Z,11Z)-heptadeca-8,11-dienyl group), an octadecenyl group (preferably a (Z)-octadec-9-enyl group), an octadecadienyl group (preferably a (9Z,12Z)-octadeca-9,12-dienyl group), and the like.

[0027] The alkynyl group may be linear or branched, open-chain or cyclic, and specific examples thereof include a propargyl group, a butynyl group, a pentynyl group, 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.

[0028] Preferably, all of the above alkenyl groups have one or two double bonds, and preferably, all of the alkynyl groups have one or two triple bonds.

[0029] The compound of the present invention may form a salt. Examples of salts of basic groups include salts with mineral acids such as hydrochloric acid, hydrobromic acid, nitric acid, and sulfuric acid; salts with organic carboxylic acids such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, 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. Salts of 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 salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Of the above-mentioned salts, preferred salts include pharmacologically acceptable salts.

[0030] Specific preferred examples of the compound of the present invention include Compounds 1 to 12 described in the Examples below, but the present invention is not limited thereto. Among Compounds 1 to 12, Compounds 1, 2, 3, 4, 5, 6, 9, and 11 are preferred, and Compounds 2, 3, 4, 9, and 11 are more preferred.

[0031] <Production Method> The production method of the compound of the present invention will be described. The compound of the present invention can be produced by combining known methods, and can be produced, for example, according to the production method shown below.

[0032] [Production Method 1] A method for producing a compound of formula [1] from a compound of formula [2]. "During the ceremony, R １ , R ２ , R ３ , R ４ , R ５, L, a, b, c and d have the same meanings as above; R ｂ and R ｃ each independently represents either no carbon atom or a hydrocarbon group having 1 to 3 carbon atoms; R Ａ means a hydrocarbon group having 1 to 7 carbon atoms; R ｐ means a protecting group; X １ means a hydroxyl group or a leaving group." Examples of leaving groups include a chloro group, a fluoro group, a bromo group, a trichloromethoxy group, a 4-nitro-phenoxy group, a 2,4-dinitrophenoxy group, a 2,4,6-trichlorophenoxy group, a pentafluorophenoxy group, a 2,3,5,6-tetrafluorophenoxy group, an imidazolyl group, a triazolyl group, a 3,5-dioxo-4-methyl-1,2,4-oxadiazolidyl group, an N-hydroxysuccinimidyl group, a methanesulfonyl group, and a 4-toluenesulfonyl group.

[0033] (1-1) The compound of formula [3] can be produced by reacting the compound of formula [2] in the presence of water and an acid, with or without a solvent. The acid used in this reaction can be an inorganic or organic acid. Organic acids are preferred, and specific examples include formic acid, acetic acid, trifluoroacetic acid, 4-toluenesulfonic acid, and methanesulfonic acid, with formic acid being more preferred. The amount of acid used can be 1 to 100 times (v / w), preferably 1 to 10 times (v / w), relative to the compound of formula [2]. The amount of water used can be 0.1 to 100 times (v / w), preferably 0.1 to 10 times (v / w), relative to the compound of formula [2]. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. The amount of the solvent used is not particularly limited, but may be 0 to 50 times (v / w) the amount of the compound of formula [2]. This reaction may be carried out at −30 to 150° C., preferably 0 to 100° C., for 5 minutes to 48 hours.

[0034] (1-2) The compound of formula [5] can be produced by reacting the compound of formula [3] with the compound of formula [4] in the presence of a reducing agent. Examples of known compounds of formula [4] include N,N-diethylethylenediamine and N,N-diethyl-1,3-diaminopropane. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples include halogenated hydrocarbons, alcohols, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include ethers and esters, with ethyl acetate being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [3]. Examples of reducing agents used in this reaction include sodium borohydride, sodium cyanoborohydride, pyridine borane, 2-picoline borane, and sodium triacetoxyborohydride, with sodium triacetoxyborohydride being more preferred. The amount of reducing agent used may be 0.1 to 100 times, preferably 0.1 to 10 times, the molar amount of the compound of formula [3]. The amount of compound of formula [4] used may be 0.1 to 1 times, the molar amount of the compound of formula [3]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0035] (1-3) The compound of formula [7] can be produced by reacting the compound of formula [5] with the compound of formula [6] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula [6] can be used in an amount of 1 to 10 times the molar amount of the compound of formula [5].

[0036] (1-4) The compound of formula [8] can be produced by deprotecting the compound of formula [7]. This reaction may be carried out, for example, according to the method described in T. W. Greene et al., Protective Groups in Organic Synthesis, 4th Edition, pp. 16-366, 2007, John Wiley & Sons, Inc.

[0037] (1-5) The compound of formula [1] can be produced by reacting a compound of formula [8] with a compound of formula [9] in the presence or absence of an acid, in the presence or absence of a condensing agent or acid halide, and in the presence or absence of a base. Known examples of compounds of formula [9] include 2-butyloctanoic acid. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include halogenated hydrocarbons and ethers, with dichloromethane and tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [8]. The acid used in this reaction can be an inorganic or organic acid. Sulfonic acids are preferred, and specific examples include sulfuric acid, 4-toluenesulfonic acid, and methanesulfonic acid. Examples of the condensing agent used in this reaction include carbodiimides such as N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; carbonyls such as carbonyldiimidazole; acid azides such as diphenylphosphoryl azide; acid cyanides such as diethylphosphoryl cyanide; 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; and uroniums such as O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The acid halide used in this reaction includes, for example, carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride, sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride, chloroformates such as ethyl chloroformate and isobutyl chloroformate, etc. The base used in this reaction includes an inorganic base or an organic base.Organic bases are preferred, and specific examples include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, N,N-dimethylaminopyridine, and 9-azajulolidine. The amount of base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of formula [8]. The amount of compound of formula [9] used is not particularly limited, but may be 0.8 to 10 times (v / w) the amount of compound of formula [8]. This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0038] (1-6) The compound of formula [1] can be produced by reacting the compound of formula [3] with the compound of formula

[10] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2), and the compound of formula

[10] can be used in an amount of 0.1 to 10 times by mole relative to the compound of formula [3].

[0039] [Production Method 2] A method for producing a compound of formula [2] from a compound of formula

[11] . "During the ceremony, R １ , R ２ , R ｂ , R Ａ , L, a and X １ has the same meaning as above."

[0040] The compound of formula [2] can be produced by reacting a compound of formula

[11] with a compound of formula

[12] 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. Examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include nitriles, with acetonitrile being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula

[11] . The base used in this reaction can be an inorganic base or an organic base. Specific examples include potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaminopyridine. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of formula

[11] . The amount of the compound of formula

[12] used is not particularly limited, but may be 0.1 to 10 times the molar amount of the compound of formula

[11] . This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0041] [Production Method 3] A method for producing a compound of formula

[11] from a compound of formula

[13] . "During the ceremony, R １ , R ２ ,L,a,X １ has the same meaning as above, and X ２ means a leaving group."

[0042] (3-1) The compound of formula [15A] can be produced by reacting a compound of formula [13A] with a compound of formula [14A] in the presence or absence of an acid, in the presence or absence of a condensing agent or acid halide, and in the presence or absence of a base. Known examples of compounds of formula [13A] include 6-bromohexanoic acid. Known examples of compounds of formula [14A] include 1-heptanol. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include aromatic hydrocarbons and ethers, with toluene and tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula [13A]. The acid used in this reaction may be an inorganic acid or an organic acid. The acid is preferably a sulfonic acid, and specific examples include sulfuric acid, 4-toluenesulfonic acid, methanesulfonic acid, etc. 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, acid azides such as diphenylphosphoryl azide, acid cyanides such as diethylphosphoryl cyanide, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, and uroniums such as O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Examples of the acid halide used in this reaction include carboxylic acid halides such as acetyl chloride and trifluoroacetyl chloride; sulfonic acid halides such as methanesulfonyl chloride and tosyl chloride; and chloroformates such as ethyl chloroformate and isobutyl chloroformate.The base used in this reaction may be an inorganic base or an organic base. Organic bases are preferred, and specific examples include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaminopyridine. The amount of the compound of formula [13A] used is not particularly limited, but may be 0.5 to 10 times (v / w) the amount of the compound of formula [14A]. This reaction may be carried out at -30 to 150°C, preferably 0 to 150°C, for 5 minutes to 48 hours.

[0043] (3-2) The compound of formula [15B] can be produced by reacting the compound of formula [13B] with the compound of formula [14B] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base. For example, octanoic acid is known as the compound of formula [13B]. For example, 6-bromo-1-hexanol is known as the compound of formula [14B]. This reaction may be carried out in accordance with Production Method (3-1).

[0044] (3-3) The compound of formula

[11] can be produced by reacting a compound of formula [15A] or formula [15B] with a compound of formula

[16] in the presence or absence of a base and in the presence or absence of an additive. Examples of compounds of formula

[16] include 1-butylamine and 1-hexylamine. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include nitriles and ethers, with acetonitrile and tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, and may be 1 to 500 times (v / w) the amount of the compound of formula [15A] or formula [15B]. The base used in this reaction may be an inorganic base or an organic base. Specific examples of the base include potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaminopyridine, with potassium carbonate being more preferred. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of Formula [15A] or Formula [15B]. The amount of the compound of Formula

[16] used is not particularly limited, but may be 1 to 10 times the molar ratio of the compound of Formula [15A] or Formula [15B]. Specific examples of additives used in this reaction include lithium iodide, sodium iodide, potassium iodide, benzyltriethylammonium iodide, and benzyltriethylammonium bromide. The amount of the additive used may be 0.1 to 10 times the molar amount of the compound of formula [15A] or formula [15B]. This reaction may be carried out at −30 to 150° C., preferably 0 to 100° C., for 5 minutes to 48 hours.

[0045] [Production Method 4] A method for producing a compound of formula

[12] from a compound of formula

[17] . "During the ceremony, R ｂ , R Ａ , a, X １ and X ２ has the same meaning as above."

[0046] (4-1) A compound of formula

[12] can be produced by reacting a compound of formula

[17] with a compound of formula

[18] in the presence or absence of a base. Known examples of compounds of formula

[18] include 1,1'-carbonyldi(1,2,4-triazole), 1,1'-carbonyldiimidazole, 4-nitrophenyl chloroformate, triphosgene, and phosgene. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include ethers, with tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, but may be 1 to 500 times (v / w) the amount of the compound of formula

[17] . The base used in this reaction includes inorganic and organic bases. The base is preferably an organic base, and specific examples include triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaminopyridine. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar amount of the compound of formula

[17] . The amount of the compound of formula

[18] used is not particularly limited, but may be 1 to 10 times the molar amount of the compound of formula

[17] . This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0047] [Production Method 5] A method for producing a compound of formula

[10] from a compound of formula

[17] . "During the ceremony, R ３ , R ４ , R ５ , R ｂ , RＡ , c, d and X １ has the same meaning as above."

[0048] (5-1) The compound of formula

[19] can be produced by reacting the compound of formula

[17] with the compound of formula [9] in the presence or absence of an acid, in the presence or absence of a condensing agent or an acid halide, and in the presence or absence of a base. This reaction may be carried out in accordance with Production Method (1-5).

[0049] (5-2) The compound of formula

[20] can be produced by reacting the compound of formula

[19] in the presence of water and an acid, with or without a solvent. This reaction can be carried out in accordance with Production Method (1-1).

[0050] (5-3) The compound of formula

[10] can be produced by reacting the compound of formula

[20] with the compound of formula [4] in the presence of a reducing agent. This reaction can be carried out in accordance with the production method (1-2).

[0051] [Production Method 6] A method for producing a compound of formula [9A] from a compound of formula

[21] . "During the ceremony, R ３ and X １ has the same meaning as above; R ３１ and R ３２ means a combination of hydrocarbon groups with a total carbon number of 5 to 23; and X means a leaving group.

[0052] The compound of formula [9A] can be produced by reacting a compound of formula

[21] with a compound of formula

[22] in the presence of a base, with or without an additive. Known examples of compounds of formula

[21] include octanoic acid. Known examples of compounds of formula

[22] include 1-iodohexane. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, and aromatic hydrocarbons. These solvents may be used in combination. Preferred solvents include ethers, with tetrahydrofuran being more preferred. The amount of solvent used is not particularly limited, and may be 1 to 500 times (v / w) the amount of the compound of formula

[21] . Examples of bases used in this reaction include inorganic bases and organic bases. Specific examples of the base include sodium hydride, calcium hydride, lithium diisopropylamide, n-butyllithium, 1,8-diazabicyclo[5.4.0]-7-undecene, and 4-dimethylaminopyridine. These bases may be used in combination. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of formula

[21] . The amount of the compound of formula

[22] used is not particularly limited, but may be 1 to 10 times the molar ratio of the compound of formula

[21] . Specific examples of additives used in this reaction include lithium iodide, sodium iodide, potassium iodide, and benzyltriethylammonium iodide. The amount of the additive used may be 0 to 10 times the molar ratio of the compound of formula

[22] . This reaction may be carried out at −30 to 150° C., preferably 0 to 100° C., for 5 minutes to 48 hours.

[0053] [Production Method 7] A method for producing a compound of formula [9B] from a compound of formula

[23] . "During the ceremony, R ３ , X １ and X has the same meaning as above; R ３３ represents a hydrocarbon group having 2 to 11 carbon atoms; and R represents a hydrocarbon group having 1 to 10 carbon atoms.

[0054] (7-1) A compound of formula

[25] can be produced by reacting a compound of formula

[23] with a compound of formula

[24] in the presence of a base. Known examples of compounds of formula

[23] include di-tert-butyl malonate. Known examples of compounds of formula

[24] include 1-bromooctane. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction, and examples include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in mixtures. Preferred solvents include mixed solvents of sulfoxides and aromatic hydrocarbons, with a mixed solvent of dimethyl sulfoxide and toluene being more preferred. The amount of solvent used is not particularly limited, and may be 1 to 500 times (v / w) the amount of the compound of formula

[23] . Examples of bases used in this reaction include inorganic bases and organic bases. Specific examples of the base include potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and 4-dimethylaminopyridine. Potassium hydroxide, sodium hydroxide, and lithium hydroxide are more preferred. The amount of the base used may be 1 to 50 times, preferably 1 to 10 times, the molar ratio of the compound of formula

[23] . The amount of the compound of formula

[24] used is not particularly limited, but may be 2 to 10 times the molar ratio of the compound of formula

[23] . This reaction may be carried out at −30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0055] (7-2) The compound of formula

[26] can be produced by reacting the compound of formula

[25] in the presence or absence of an acid and 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, and examples include water, halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may also be used in mixtures. A preferred solvent is a mixed solvent of water and aromatic hydrocarbons. The amount of solvent used is not particularly limited, but may be 0.1 to 10 times (v / w) the amount of the compound of formula

[25] . The acid used in this reaction may be an inorganic or organic acid. Specific examples include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, formic acid, and trifluoroacetic acid, with trifluoroacetic acid being preferred. The amount of acid used is not particularly limited, but may be 1 to 50 times (v / w) the amount of the compound of formula

[25] . The base used in this reaction may be an inorganic or organic base. Specific examples of the base include potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and 4-dimethylaminopyridine. The amount of the base used may be 2 to 50 times the molar amount of the compound of formula

[25] . This reaction may be carried out at -30 to 150°C, preferably 0 to 100°C, for 5 minutes to 48 hours.

[0056] (7-3) The compound of formula [9B] can be produced by reacting the compound of formula

[26] in the presence or absence of an acid, a base, or a solvent. The solvent used in this reaction is not particularly limited as long as it does not affect the reaction. Examples of the solvent include halogenated hydrocarbons, ethers, esters, amides, nitriles, sulfoxides, and aromatic hydrocarbons. These solvents may be used in combination. The amount of the solvent used is not particularly limited, but may be 0 to 10 times (v / w) the amount of the compound of formula

[26] . The acid used in this reaction can be an inorganic or organic acid. Specific examples include hydrochloric acid, phosphoric acid, sulfuric acid, formic acid, and trifluoroacetic acid. The amount of the acid used is not particularly limited, but may be 1 to 50 times (v / w) the amount of the compound of formula

[26] . The base used in this reaction can be an inorganic or organic base. Specific examples of the base include potassium hydroxide, sodium hydroxide, lithium hydroxide, potassium carbonate, sodium carbonate, lithium carbonate, potassium phosphate, sodium phosphate, lithium phosphate, triethylamine, N,N-diisopropylethylamine, 4-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene, and 4-dimethylaminopyridine, with pyridine being more preferred. The amount of base used may be 2 to 50 times the molar amount of the compound of formula

[26] . This reaction may be carried out at -30 to 250°C, preferably 0 to 200°C, for 5 minutes to 48 hours.

[0057] In the compounds used in the above-mentioned production methods, when isomers (e.g., optical isomers, geometric isomers, tautomers, etc.) exist, these isomers can also be used. In addition, when solvates, hydrates, and various forms of crystals exist, these solvates, hydrates, and various forms of crystals can also be used.

[0058]

[0033] In the compounds used in the above-mentioned production methods, for example, compounds having an amino group, a hydroxyl group, or a carboxyl group can have these groups protected in advance with a conventional protecting group, and after the reaction, these protecting groups can be removed by a method known per se. The compounds obtained by the above-mentioned production methods can be derived into other compounds by subjecting them to a reaction known per se, such as condensation, addition, oxidation, reduction, rearrangement, substitution, halogenation, dehydration, or hydrolysis, or by an appropriate combination of these reactions.

[0059] <Lipid Composition> In the present invention, a lipid composition containing the compound of the present invention or a salt thereof and a lipid can be prepared. When preparing the lipid composition, in addition to the compound of the present invention, at least one lipid selected from the group consisting of neutral lipids and lipids having nonionic hydrophilic polymer chains can be used. The lipid composition may further contain a sterol. The lipid composition may further contain at least one selected from the group consisting of nucleic acids, proteins, peptides, and small molecules.

[0060] In the lipid composition of the present invention, the amount of the compound represented by formula (1) of the present invention or a salt thereof is preferably 20 mol% to 80 mol%, more preferably 35 mol% to 70 mol%, and even more preferably 40 mol% to 65 mol%, based on the total lipid amount.

[0061] <Sterol> The lipid composition of the present invention preferably contains a sterol. By including a sterol in the lipid composition of the present invention, membrane fluidity can be reduced, and the lipid composition can be stabilized. Examples of sterols include, but are not limited to, cholesterol, phytosterols (sitosterol, β-sitosterol, stigmasterol, fucosterol, spinasterol, brassicasterol, etc.), ergosterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether. Among these, cholesterol is preferred. In the lipid composition of the present invention, the amount of sterol incorporated is preferably 10 mol% to 60 mol%, more preferably 20 mol% to 55 mol%, and even more preferably 25 mol% to 50 mol%, relative to the total lipid amount.

[0062] <Neutral lipid> The lipid composition of the present invention preferably contains a neutral lipid. The neutral lipid is not particularly limited, but includes phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, etc., and phosphatidylcholine is preferred. In addition, the neutral lipid may be used alone or in combination with a plurality of different neutral lipids.

[0063] The phosphatidylcholine is not particularly limited, but includes soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated egg yolk lecithin (HEPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), etc. Among the above, dipalmitoylphosphatidylcholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is preferred, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is more preferred.

[0064] The phosphatidylethanolamine is not particularly limited, and examples thereof include dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylethanolamine (DLoPE), diphytanoylphosphatidylethanolamine (D(Phy)PE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), ditetradecylphosphatidylethanolamine, dihexadecylphosphatidylethanolamine, dioctadecylphosphatidylethanolamine, and diphytanylphosphatidylethanolamine.

[0065] Examples of sphingomyelin include, but are not limited to, egg yolk-derived sphingomyelin, milk-derived sphingomyelin, etc. Examples of ceramide include, but are not limited to, egg yolk-derived ceramide, milk-derived ceramide, etc.

[0066] In the lipid composition of the present invention, the blending amount of the neutral lipid is preferably 3 mol % or more and 55 mol % or less, and more preferably 3 mol % or more and 45 mol % or less, based on the total amount of the constituent lipid components.

[0067] <Lipids Having Nonionic Hydrophilic Polymer Chains> The lipid composition of the present invention may contain a lipid having a nonionic hydrophilic polymer chain in the oil phase. In the present invention, by including a lipid having a nonionic hydrophilic polymer chain in the oil phase, the dispersion stabilization effect of the lipid composition can be obtained. Examples of nonionic hydrophilic polymers include, but are not limited to, nonionic vinyl polymers, nonionic polyamino acids, nonionic polyesters, nonionic polyethers, nonionic natural polymers, nonionic modified natural polymers, and block polymers or graft copolymers containing two or more of these polymers as building blocks. Among these nonionic hydrophilic polymers, preferred are nonionic polyethers, nonionic polyesters, nonionic polyamino acids, or nonionic synthetic polypeptides, more preferred are nonionic polyethers or nonionic polyesters, even more preferred are nonionic polyethers or nonionic monoalkoxy polyethers, and particularly preferred is polyethylene glycol (polyethylene glycol will also be referred to as PEG hereinafter).

[0068] Lipids having a nonionic hydrophilic polymer chain include, but are not limited to, PEG-modified phosphoethanolamine, diacylglycerol PEG derivatives, monoacylglycerol PEG derivatives, dialkylglycerol PEG derivatives, cholesterol PEG derivatives, and ceramide PEG derivatives. Among these, monoacylglycerol PEG or diacylglycerol PEG is preferred. The weight-average molecular weight of the PEG chain of the nonionic hydrophilic polymer derivative is preferably 500 to 5,000, more preferably 750 to 3,000. The nonionic hydrophilic polymer chain may be branched or may have a substituent such as a hydroxymethyl group.

[0069] In the lipid composition of the present invention, the amount of lipid having a nonionic hydrophilic polymer chain is preferably 0.25 mol% to 12 mol% of the total lipid amount, more preferably 0.5 mol% to 6 mol%, and even more preferably 1 mol% to 3 mol%.

[0070] <Nucleic acids, proteins, peptides, and small molecules> The lipid composition of the present invention may contain at least one selected from the group consisting of nucleic acids, proteins, peptides, and small molecules. Examples of nucleic acids include plasmid DNA, nanoplasmid DNA, single-stranded DNA, double-stranded DNA, siRNA (small interfering RNA), miRNA (micro RNA), mRNA, antisense oligonucleotides (also known as ASO), ribozymes, aptamers, dsRNA, saRNA, sgRNA, shRNA, tRNA, and cyclic RNA, and any of these may be included. Modified nucleic acids may also be included. RNA is particularly preferred as the nucleic acid, and RNA with a base count of 5 to 20,000 bases is preferred. In the lipid composition of the present invention, the mass ratio of lipid to nucleic acid is preferably 2 to 1,000, more preferably 3 to 500, even more preferably 5 to 200, and particularly preferably 5 to 100.

[0071] Examples of proteins, peptides, and small molecules include intracellular proteins, intracellular peptides, transmembrane proteins, transmembrane peptides, secreted proteins, secreted peptides, synthetic proteins, synthetic peptides, natural small molecular weight compounds, synthetic small molecular weight compounds, and compounds with antitumor activity. More specifically, examples of proteins include gene editing-related proteins such as CRISPR-Cas proteins, Zinc Fingers, and TALENs, hormones such as erythropoietin, biological factors such as VEGF, and cancer antigen proteins or antibodies. Examples of peptides include functional domains and recognition domains of the above proteins. They may contain mRNA or DNA encoding the above proteins. Peptides are preferably natural. Furthermore, the peptide structure may be linear or cyclic. When the peptide is cyclic, the linking portion of the ring may be an amide bond, a disulfide bond, or the like. Examples of small molecular weight compounds include anticancer agents, antibacterial agents, and antifungal agents. These proteins, peptides, and small molecules may or may not have physiological activity in vivo. Here, the term "low molecular weight" refers to an organic compound having a molecular weight of approximately 1,000 or less.

[0072] <Method of manufacturing lipid composition> A method of manufacturing the lipid composition of the present invention will be described. The method of manufacturing the lipid composition is not limited, but it can be produced by dissolving all or some of the oil-soluble components of the lipid composition in an organic solvent or the like to form an oil phase, dissolving the water-soluble components in water to form an aqueous phase, and mixing the oil phase and the aqueous phase. A micromixer may be used for mixing, or emulsification may be performed using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure injection emulsifier, or the like. Alternatively, a solution containing lipids may be dried under reduced pressure using an evaporator or the like, or spray-dried using a spray dryer or the like to prepare a dried mixture containing lipids, and this mixture may be added to an aqueous solvent and further emulsified using the above-mentioned emulsifier or the like.

[0073] An example of a method for producing a lipid composition containing nucleic acid includes the following steps: step (a): dissolving the components of the lipid composition containing the compound of the present invention in an organic solvent to obtain an oil phase, and dissolving the nucleic acid in an aqueous solvent to obtain an aqueous phase; step (b): mixing the oil phase and aqueous phase obtained in step (a) to obtain a lipid particle dispersion; step (c): diluting the lipid particle dispersion obtained in step (b); step (d): removing the organic solvent from the lipid particle dispersion; and step (e): adjusting the concentration of the lipid particle dispersion.

[0074] In step (a), the components of the lipid composition containing the compound of the present invention are dissolved in an organic solvent (e.g., an alcohol such as ethanol, or an ester). The total lipid concentration 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 30 mmol / L. The aqueous phase can be obtained by dissolving nucleic acids (e.g., siRNA, mRNA, antisense nucleic acids, etc.) in water or a buffer solution. The nucleic acid concentration is not particularly limited, but is preferably 1 to 1000 μg / mL, more preferably 10 to 500 μg / mL. If necessary, components such as buffer components and antioxidants for pH adjustment can be added. The pH of the aqueous phase is preferably 2.0 to 7.0, more preferably 3.0 to 6.0. To adjust the pH to the above range, buffer components such as acetic acid, citric acid, malic acid, phosphoric acid, MES, and HEPES are preferably used. If necessary, salts such as sodium chloride and potassium chloride may be added to adjust the salt strength, and sugars or sugar alcohols such as sucrose, trehalose, and mannitol may be added to adjust the osmotic pressure.

[0075] In step (b), the oil phase and the aqueous phase may be mixed by any method, including a batch method and an in-line method using a flow channel device. For the in-line method, a micro-flow channel device is preferably used, and examples of the micro-flow channel device that can be used include a Y-mixer, a T-mixer, a herringbone mixer, a ring micromixer, and an impingement jet mixer. The mixing ratio (volume ratio) of the aqueous phase to the oil phase is preferably 5:1 to 1:1, and more preferably 4:1 to 2:1.

[0076] In step (c), the lipid particle dispersion is mixed with a diluent solution to reduce the organic solvent content and stabilize the lipid particles. The diluent may be water, but may also include adjusting the pH or salt strength. The components contained in the diluent may be selected arbitrarily depending on the purpose. For example, a buffer solution (e.g., citrate buffer, citrate-buffered saline, acetate buffer, acetate-buffered saline, phosphate-buffered saline, Tris buffer, MES buffer, HEPES buffer, etc.) may be used to adjust the pH. In addition, sodium chloride, potassium chloride, sucrose, trehalose, fructose, mannitol, etc. may be included to adjust the salt strength or osmotic pressure, and the above buffer solutions may also be used with the addition of these additives.

[0077] The lipid particle dispersion and the diluted solution may be mixed by any method, including a batch method or an in-line method using a flow path device. The flow path device used during mixing may be a Y-shaped mixer, a T-shaped mixer, or the like. The time required for mixing the oil phase and the aqueous phase after mixing the diluted solution is not particularly limited, but the dilution is preferably carried out within 30 seconds, and more preferably within 10 seconds, after mixing the oil phase and the aqueous phase. The mixing ratio (liquid volume ratio) of the lipid particle dispersion and the diluted solution is preferably 1:0.5 to 1:10, and more preferably 1:1 to 1:5.

[0078] In some embodiments, in step (c), the lipid particle dispersion may be mixed with the dilution solution multiple times depending on the purpose.The dilution solutions used may be the same or different.In the lipid particle dispersion, the particle size of the lipid particles may change depending on the pH, so adjusting the pH of the dispersion is important.Therefore, for example, in order to adjust the pH of the lipid particle dispersion after mixing with the dilution solution, a buffer solution having an appropriate concentration and pH, or a buffer solution containing other components, may be used.

[0079] Furthermore, multiple dilution steps may be carried out continuously, and the interval between one dilution step and the next dilution step may be set arbitrarily, for example, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, or 24 hours. Furthermore, the pH of the lipid particle dispersion after step (c) is preferably pH 3.0 to 10.0, more preferably pH 3.5 to pH 9.0, and particularly preferably pH 4.0 to pH 8.5.

[0080] The lipid composition can be subjected to sizing as needed. The sizing method is not particularly limited, but the particle size can be reduced using an extruder or the like. In addition, the dispersion containing the lipid composition of the present invention can be subjected to freezing or freeze-drying by a general method.

[0081] In step (d), the method for removing the organic solvent from the lipid particle dispersion is not particularly limited, and a common method can be used. For example, a pH buffer solution such as phosphate buffered saline or Tris buffer can be used as the dialysis solution, and additives such as any salt or sugar can be added as needed to adjust the osmotic pressure or protect from freezing.

[0082] In step (e), the concentration of the lipid particle dispersion obtained in step (d) can be adjusted.When diluting, it can be diluted to an appropriate concentration using a solution such as phosphate buffered saline, physiological saline, Tris buffer, or sucrose-containing Tris buffer as a diluent.When concentrating, it can be concentrated by ultrafiltration using an ultrafiltration membrane, etc., with the dispersion obtained in step (d).It is preferable to use the concentrated dispersion as it is, or it is also preferable to use the above-mentioned diluent after concentrating to adjust to a desired concentration.

[0083] In some embodiments, the organic solvent removal step (step (d)) and the concentration adjustment step (step (e)) can be carried out continuously using tangential flow filtration (TFF). In this process, the organic solvent removal step and the concentration adjustment step may be carried out in any order. If necessary, the organic solvent removal step and the concentration adjustment step may each be carried out multiple times.

[0084] The solution that can be used for dialysis in step (d) or dilution in step (e) may contain an excipient, cryoprotectant, buffer, or antioxidant. Examples of excipients and cryoprotectants include, but are not limited to, sugars and sugar alcohols. Examples of sugars include sucrose, trehalose, maltose, glucose, lactose, and fructose, and examples of sugar alcohols include mannitol, sorbitol, inositol, and xylitol. Examples of buffers include, but are not limited to, ACES, BES, Bicine, CAPS, CHES, DIPSO, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, TAPS, TAPSO, TES, Tricine, Tris, phosphoric acid, acetic acid, and citric acid. Examples of antioxidants include EDTA, ascorbic acid, and tocopherol.

[0085] In order to prepare the lipid particle dispersion of the present invention into a pharmaceutical composition, it is preferable to perform sterile filtration. As a filtration method, insoluble matters can be removed from the lipid particle dispersion using a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, etc. In the present invention, although not particularly limited, it is preferable to perform filtration using a filter having a sterilizable pore size (preferably a 0.2 μm filtration sterilization filter). In addition, it is preferable to perform sterile filtration after step (d) or step (e). Furthermore, if necessary, the lipid particle dispersion of the present invention can be subjected to freezing or lyophilization. The lipid particle dispersion of the present invention can be subjected to freezing or lyophilization by a general method, and the method is not particularly limited.

[0086] <Regarding Lipid Composition> In the present invention, the lipid composition may be a lipid particle. Lipid particles refer to particles composed of lipids, and include compositions having any structure selected from lipid aggregates in which lipids are aggregated, micelles, liposomes, lipid nanoparticles (LNPs), and lipoplexes. However, the structure of the lipid particle is not limited to these, as long as the composition contains lipids. Liposomes include liposomes having a lipid bilayer structure and an aqueous phase inside, with a single bilayer membrane, and multilayer liposomes with multiple layers stacked on top of each other. Either type of liposome may be included in the present invention.

[0087] The morphology of lipid particles can be confirmed by electron microscope observation or X-ray structural analysis.For example, by using a cryo-transmission electron microscope (cryo-TEM) method, it can be confirmed whether the lipid particles have a lipid bilayer structure (lamellar structure) and an inner water layer, like liposomes, or whether the particles have a core with high electron density inside and a structure packed with lipids and other components.Small-angle X-ray scattering (SAXS) measurement can also be used to confirm whether the lipid particles have a lipid bilayer structure (lamellar structure).

[0088] The particle size of the lipid particles of the present invention is not particularly limited, but is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and even more preferably 50 to 250 nm. The particle size of the lipid particles can be measured by a general method (e.g., dynamic light scattering method, laser diffraction method, etc.).

[0089] <Use of lipid composition> As an example of the use of the lipid composition of the present invention, a lipid composition containing a nucleic acid, a protein, a peptide, or a small molecule can be introduced into a cell by introducing the lipid composition containing the nucleic acid, a protein, a peptide, or a small molecule into the cell. Furthermore, when the lipid composition of the present invention contains a nucleic acid, a protein, a peptide, or a small molecule having a pharmaceutical use, the lipid composition can be administered to a living body as a pharmaceutical composition. That is, according to the present invention, a pharmaceutical composition containing the lipid composition of the present invention is provided.

[0090] When the lipid composition of the present invention is used as a pharmaceutical composition, the lipid composition of the present invention can be administered to a living body alone or mixed with a pharmaceutically acceptable administration medium (e.g., physiological saline or phosphate buffer). The concentration of the lipid composition in the mixture with the pharmaceutically acceptable administration medium is not particularly limited, and can generally be 0.05% to 90% by mass. In addition, other pharmaceutically acceptable additives, such as pH adjusting buffers, osmotic pressure adjusting agents, etc. may be added to the pharmaceutical composition containing the lipid composition of the present invention.

[0091] The administration route of the pharmaceutical composition containing the lipid composition of the present invention is not particularly limited, and can be administered by any method. Administration methods include oral administration and parenteral administration (intra-articular administration, intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intravitreal administration, intraperitoneal administration, intramuscular administration, intravaginal administration, intravesical administration, intrathecal administration, pulmonary administration, rectal administration, colonic administration, buccal administration, nasal administration, intracisternal administration, inhalation, etc.). Parenteral administration is preferred, and the preferred administration methods are intravenous injection, subcutaneous injection, intradermal injection, or intramuscular injection. The pharmaceutical composition containing the lipid composition of the present invention can also be administered by direct injection at the disease site.

[0092] The dosage form of the lipid composition of the present invention is not particularly limited, but when administered orally, the lipid composition of the present invention can be combined with an appropriate excipient and used in the form of tablets, troches, capsules, pills, suspensions, syrups, etc. Furthermore, formulations suitable for parenteral administration can contain additives such as antioxidants, buffers, bacteriostatic agents, and isotonic sterile injections, suspending agents, solubilizers, thickeners, stabilizers, or preservatives, as appropriate.

[0093] <Delivery Carrier> The lipid composition of the present invention is capable of retaining nucleic acids at a high encapsulation rate, making it extremely useful as a delivery carrier for nucleic acids. That is, according to the present invention, a delivery carrier comprising the lipid composition of the present invention is provided. With a delivery carrier utilizing the present invention, for example, the obtained lipid composition can be mixed with nucleic acids or the like and transfected in vitro or in vivo, thereby introducing nucleic acids or the like into cells. In addition, the delivery carrier utilizing the present invention is also useful as a nucleic acid delivery carrier for nucleic acid medicines. 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).

[0094] The present invention will now be described with reference to examples, but the present invention is not limited to these examples.

[0095] Unless otherwise specified, purification by column chromatography was performed using an automatic purification system ISOLERA (Biotage), a medium-pressure fractionation and purification system Purif-espoir-2 (Shoko Science Co., Ltd.), or a medium-pressure liquid chromatograph YFLC W-prep 2XY (Yamazen Corporation).

[0096] Unless otherwise specified, the carrier used in silica gel column chromatography was Chromatorex Q-Pack SI 50 (Fuji Silysia Chemical Ltd.), Hi-Flash Column W001, W002, W003, W004, or W005 (Yamazen Corporation). The NH silica gel used was Chromatorex Q-Pack NH 60 (Fuji Silysia Chemical Ltd.).

[0097] The NMR spectrum was measured using tetramethylsilane as an internal standard with a Bruker AVNEO400 (manufactured by Bruker), and all δ values ​​are shown in ppm.

[0098] MS spectra were measured using an ACQUITY SQD LC / MS System (manufactured by Waters).

[0099] [Synthesis Example 1] (1) To a mixture of 50 mL of dimethyl sulfoxide and 30 mL of toluene, 6.6 g of powdered potassium hydroxide was added, and under ice-cooling, a solution of 10 g of di-tert-butyl malonate in 10 mL of toluene and a solution of 19.9 g of 1-bromoheptane in 10 mL of toluene were added, followed by stirring overnight at 30°C or below. The reaction mixture was stirred in an ice bath and neutralized with hydrochloric acid, after which the organic layer was separated. The resulting organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain 22.9 g of di-tert-butyl 2,2-diheptylmalonate as a pale yellow oil. 1 H-NMR(CDCl3)δ:1.79-1.74 (4H, m), 1.44 (18H, s), 1.32-1.10 (20H, m), 0.87 (6H, t, J=6.8Hz).

[0100] (2) To a mixture of 22.9 g of di-tert-butyl 2,2-diheptylmalonate, 23 mL of toluene, and 2.3 mL of water, 45 mL of trifluoroacetic acid was added, followed by stirring at room temperature for 2 hours, and the solvent was distilled off under reduced pressure. 23 mL of hexane was added to the residue, and after stirring under ice cooling, the resulting solid was collected by filtration. The resulting solid was dried to obtain 11.7 g of 2,2-diheptylmalonic acid as a white solid. 1 H-NMR(CDCl3)δ:1.97-1.92 (4H, m), 1.32-1.17 (20H, m), 0.87 (6H, t, J=7.3Hz).

[0101] (3) 11.7 g of 2,2-diheptylmalonic acid was stirred at 165° C. for 3 hours to obtain 11.7 g of pale yellow oily 2-heptylnonanoic acid (A2). 1 H-NMR(CDCl3)δ:2.39-2.32 (1H, m), 1.96-1.93 (1H, m), 1.68-1.41 (4H, m), 1.36-1.16 (20H, m), 0.89-0.85 (6H, m).

[0102] (4) In the same manner as in Synthesis Example 1 (1) to (3), the compounds in Table 1, 2-hexyloctanoic acid (A1), 2-octyldecanoic acid (A3), and 2-nonylundecanoic acid (A4), were obtained.

[0103]

[0104] [Synthesis Example 2]

[0105] (1) To a solution of 5.0 g of 1-heptanol in 50 mL of toluene, 8.8 g of 6-bromohexanoic acid and 370 mg of 4-toluenesulfonic acid monohydrate were added, and the mixture was stirred under reflux for 6 hours. The solvent in the reaction mixture was distilled off under reduced pressure, and the residue was purified by NH silica gel column chromatography (hexane-ethyl acetate) to obtain 12.5 g of heptyl 6-bromohexanoate (B2) as a pale yellow oil. 1H-NMR(CDCl3)δ: 4.06 (2H, t, J=6.7Hz), 4.06 (2H, t, J=6.7Hz), 2.32 (2H, t, J=7.4Hz), 1.92-1.83 (2H, m), 1.72-1.56 (4H, m), 1.55-1.41 (4H, m), 1.40-1.20 (6H, m), 0.93-0.83 (3H, m).

[0106] (2) To a mixture of 3.0 g of heptyl 6-bromohexanoate (B2), 4.1 mL of n-hexylamine, and 30 mL of acetonitrile, 4.2 g of potassium carbonate was added and stirred at 60°C for 4 hours. After the reaction mixture was cooled to room temperature, the solvent was distilled off under reduced pressure. Ethyl acetate and water were added to the residue, and the organic layer was separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol-ethyl acetate) and then by NH silica gel column chromatography (ethyl acetate-hexane) to yield 2.5 g of heptyl 6-(hexylamino)hexanoate (B3) as a colorless oil. 1 H-NMR(CDCl3)δ: 4.05 (2H, t, J=6.7Hz), 2.63-2.54 (4H, m), 2.30 (2H, t, J=7.5Hz), 1.70-1.56 (4H, m), 1.55-1.41 (4H, m), 1.40-1.20 (17H, m), 0.93-0.84 (6H, m).

[0107] (3) A mixture of 2.5 g of heptyl 6-(hexylamino)hexanoate (B3), 2.2 g of 2,2-diethoxyethyl 1H-1,2,4-triazole-1-carboxylate described in WO 2024 / 014430, 25 mL of acetonitrile, and 2.2 mL of triethylamine was stirred at 50°C for 4 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. Ethyl acetate (20 mL) and water (20 mL) were added to the residue, and the organic layer was separated. The resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate-hexane) to yield 3.9 g of heptyl 6-(((2,2-diethoxyethoxy)carbonyl)(hexyl)amino)hexanoate (B4) as a colorless oil. 1 H-NMR(CDCl3)δ: 4.72-4.67 (1H, m), 4.17-4.02 (4H, m), 3.77-3.51 (4H, m), 3.26-3.12 (4H, m), 2.30 (2H, t, J=7.5Hz), 1.70-1.45 (10H, m), 1.37-1.17 (20H, m), 0.93-0.84 (6H, m)..

[0108] (4) A mixture of 2.0 g of heptyl 6-(((2,2-diethoxyethoxy)carbonyl)(hexyl)amino)hexanoate (B4), 8 mL of formic acid, and 2 mL of water was stirred at 50°C for 2 hours, after which toluene was added and the mixture was evaporated under reduced pressure. This procedure of adding toluene again and evaporating under reduced pressure was repeated twice, yielding 1.67 g of crude colorless oily heptyl 6-(hexyl((2-oxoethoxy)carbonyl)amino)hexanoate (B5). 1 H-NMR(CDCl3)δ: 9.63 (1H, s), 4.61 (2H, s), 4.06 (2H, t, J=6.7Hz), 3.31-3.11 (4H, m), 2.31 (2H, t, J=7.3Hz), 1.72-1.42 (8H, m), 1.40-1.20 (14H, m), 0.94-0.83 (6H, m).

[0109] (5) To a solution of 1.6 g of heptyl 6-(hexyl((2-oxoethoxy)carbonyl)amino)hexanoate (B5) in 16 mL of ethyl acetate, 1.3 mL of N,N-diethyl-1,3-diaminopropane, 0.1 mL of acetic acid, and 2.5 g of sodium triacetoxyborohydride were added at room temperature, and the mixture was stirred at room temperature for 4 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the organic layer was separated and washed with saturated brine. Anhydrous sodium sulfate was added for drying, and the solvent was distilled off under reduced pressure. The resulting residue was purified by NH silica gel column chromatography (ethyl acetate-hexane) to give 1.26 g of heptyl 3-ethyl-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B6) as a colorless oil. 1 H-NMR(CDCl3)δ: 4.17 (2H, t, J=5.6Hz), 4.05 (2H, t, J=6.8Hz), 3.27-3.09 (4H, m), 2.85 (2H, t, J=5.6Hz), 2.67 (2H,t, J=7.0Hz), 2.55-2.43 (6H, m), 2.30 (2H, t, J=7.5Hz), 1.73-1.43 (10H, m), 1.38-1.20 (16H, m), 1.01 (6H, t, J=7.1Hz), 0.93-0.83 (6H, m). MS m / z(M+H): 515.

[0110] (6) To a solution of 1.26 g of heptyl 3-ethyl-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B6) in 13 mL of ethyl acetate, 0.58 g of 2-(benzyloxy)acetaldehyde and 1.04 g of sodium triacetoxyborohydride were added at room temperature and stirred for 4 hours. Ethyl acetate and saturated aqueous sodium bicarbonate were added to the reaction mixture, and the organic layer was separated and washed with saturated brine. Anhydrous sodium sulfate was added to the organic layer for drying, and the solvent was distilled off under reduced pressure. The resulting residue was purified by NH silica gel column chromatography (ethyl acetate-hexane) to yield 1.44 g of heptyl 7-(2-(benzyloxy)ethyl)-3-ethyl-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B7) as a colorless oil. 1 H-NMR(CDCl3)δ: 7.36-7.22 (5H, m), 4.51 (2H, s), 4.11 (2H, t, J=6.0Hz), 4.05 (2H, t, J=6.8Hz), 3.54 (2H, t, J=6.2Hz), 3.25-3.08 (4H, m), 2.81-2.72 (4H, m), 2.59-2.45 (6H, m), 2.43-2.36 (2H, m), 2.33-2.24 (2H, m), 1.70-1.41 (10H, m), 1.39-1.18 (16H, m), 1.00 (6H, t, J=7.2Hz), 0.92-0.84 (6H, m).MS m / z(M+H): 649.

[0111] (7) 1.44 g of heptyl 7-(2-(benzyloxy)ethyl)-3-ethyl-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B7), 44 mL of methanol, 0.43 g of 10% palladium / carbon (approximately 55% wet with water), and 1.4 g of ammonium formate were mixed and stirred under heating and reflux for 2 hours. 0.43 g of 10% palladium / carbon and 1.4 g of ammonium formate were added again, and the mixture was stirred under heating and reflux for 2 hours. The mixture was cooled to room temperature, and insoluble matter was removed by filtration through Celite, and the solvent was then distilled off under reduced pressure. The obtained residue was purified by NH silica gel column chromatography (methanol-ethyl acetate-hexane) to obtain 1.07 g of a colorless oily substance, heptyl 3-ethyl-12-hexyl-7-(2-hydroxyethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B8). 1 H-NMR(CDCl3)δ: 4.13 (2H, t, J=6.0Hz), 4.05 (2H, t, J=6.8Hz), 3.62-3.49 (2H, m), 3.27-3.08 (4H, m), 2.73 (2H, t, J=6.0Hz), 2.66-2.61 (2H, m), 2.59 (2H, t, J=6.8Hz), 2.55-2.42 (6H, m), 2.30 (2H, m), 1.72-1.41 (10H, m), 1.39-1.19 (16H, m), 1.01 (6H, t, J=7.2Hz), 0.92-0.84 (6H, m). MS m / z(M+H):559.

[0112] (8) To a solution of 150 mg of heptyl 3-ethyl-12-hexyl-7-(2-hydroxyethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (B8) in 1.5 mL of dichloromethane, 93 mg of 2-hexyloctanoic acid (A1), 84 μL of N,N'-diisopropylcarbodiimide, 141 mg of 9-azajulolidine, and 0.23 mL of triethylamine were added and stirred at room temperature overnight. After the solvent was removed from the reaction mixture under reduced pressure, ethyl acetate and water were added, and the organic layer was separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (methanol-ethyl acetate) and then by NH silica gel column chromatography (ethyl acetate-hexane) to obtain 140 mg of a colorless oily substance, heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyloctanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 1). 1 H-NMR(CDCl3)δ: 4.15-4.01 (6H, m), 3.26-3.09 (4H, m), 2.76 (4H, t, J=6.4Hz), 2.59-2.46 (6H, m), 2.45-2.37 (2H, m), 2.35-2.25 (3H, m), 1.70-1.38 (16H, m), 1.37-1.18 (30H, m), 1.01 (6H, t, J=7.1Hz), 0.92-0.82 (12H, m). MS m / z(M+H):769.

[0113] [Synthesis Example 3] Using intermediate (B8) as a raw material, intermediates (A2) to (A4), 2-hexyldecanoic acid, and 3-heptyldecanoic acid described in WO2019 / 235635, the compounds shown in Table 2, heptyl 3-ethyl-7-(2-((2-heptylnonanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 2), heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 3), heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 4), heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 5), heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 6), heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound Heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 4), heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 5), and heptyl 3-ethyl-7-(2-((3-heptyldecanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate (Compound 6) were obtained.

[0114]

[0115] [Synthesis Example 4]

[0116] (1) Heptyl 3-ethyl-11-hexyl-6-(2-hydroxyethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (B9) was obtained in the same manner as in Synthesis Example 2 (5) and (6), except that N,N-diethylethylenediamine was used instead of N,N-diethyl-1,3-diaminopropane. 1H-NMR(CDCl3)δ: 5.21 (1H, brs), 4.15 (2H, t, J=6.1Hz), 4.05 (2H, t, J=6.8Hz), 3.56-3.49 (2H, m), 3.26-3.10 (4H, m), 2.84 (2H, t, J=6.0Hz), 2.73-2.63 (4H, m), 2.59-2.44 (6H, m), 2.30 (2H, t, J=7.5Hz), 1.72-1.41 (10H, m), 1.39-1.19 (14H, m), 1.01 (6H, t, J=7.2Hz), 0.92-0.84 (6H, m). MS m / z(M+H):545.

[0117] (2) Using intermediate (B9) as a raw material, intermediates (A1) to (A4), 2-hexyldecanoic acid, and 3-heptyldecanoic acid described in WO2019 / 235635, the compounds in Table 3, heptyl 3-ethyl-11-hexyl-6-(2-((2-hexylooctanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-trimethyl-11-hexyl-6-(2-((2-hexyl ... Triazaheptadecan-17-oate (compound 7), heptyl 3-ethyl-6-(2-((2-heptylnonanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (compound 8), heptyl 3-ethyl-11-hexyl-6-(2-((2-octyldecanoyl)oxy)ethyl)-1 0-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (compound 9), heptyl 3-ethyl-11-hexyl-6-(2-((2-nonylundecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (compound 10), heptyl 3-ethyl-11-hexyl-6-(2-(( As a result, heptyl 3-ethyl-6-(2-((3-heptyldecanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (Compound 11), and heptyl 3-ethyl-6-(2-((3-heptyldecanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate (Compound 12) were obtained.

[0118]

[0119] Comparative Example 1 It was synthesized according to the method described in WO2020 / 246581 ([Example 136] 2-hexyloctyl 3-ethyl-11-heptyl-6-(2-(octanoyloxy)ethyl)-10-oxo-9-oxa-3,6,11-triazahenicosan-21-oate).

[0120] Comparative Example 2 It was synthesized according to the method described in WO2020 / 246581 ([Example 134] 2-hexyloctyl 3-ethyl-6-(2-(octanoyloxy)ethyl)-11-octyl-10-oxo-9-oxa-3,6,11-triazahenicosan-21-oate).

[0121] Test Example 1: Preparation of mRNA-encapsulated lipid nanoparticles and measurement of reporter protein expression rate in mice <Preparation of EPO mRNA-encapsulated lipid nanoparticles> The compounds listed in Table 4, 1,2-distearoyl-sn-glycero-3-phosphocholine (product name: COATSOME® MC-8080; NOF corporation) (hereinafter referred to as DSPC), cholesterol (product name: Cholesterol HP; Nippon Fine Chemical Co., Ltd.), and 1,2-dimyristoyl-rac-glycero-3-(methylpolyoxyethylene 2000) (hereinafter referred to as DMG-PEG2000) (product name: SUNBRIGHT® GM-020; NOF corporation) were dissolved in ethanol in a molar ratio of 50:10:38.5:1.5, respectively, so that the total lipid concentration was 12.5 mmol / L, to obtain an oil phase.

[0122] EPO mRNA (product name: CleanCap EPO mRNA (5 moU); TriLink) was diluted with 50 mmol / L citrate buffer at pH 4.0 so that the weight ratio of total lipid concentration to mRNA concentration was approximately 20:1 to obtain an aqueous phase. Subsequently, the aqueous and oil phases were mixed using a NanoAssemblr (Precision NanoSystems) so that the volume ratio of aqueous phase:oil phase = 3:1, and the mixture was diluted 2-fold with phosphate-buffered saline (PBS) or water to obtain a dispersion of mRNA-lipid nanoparticles. This dispersion was dialyzed against 20 mM Tris buffer containing 8% sucrose using a dialysis cassette (Slide-A-Lyzer G2, MWCO: 10 kD, Thermo Fisher Scientific) to remove ethanol, thereby obtaining EPO mRNA-encapsulated lipid nanoparticles.

[0123] <Measurement of particle size> The particle size of the mRNA-encapsulated lipid nanoparticles was measured using a particle size analyzer NanoSAQLA (Otsuka Electronics). The lipid nanoparticles to be measured were diluted in phosphate-buffered saline (PBS) in advance. The results are shown in Table 4.

[0124] <Evaluation of mRNA Encapsulation Rate> (Quantification of Total mRNA Concentration) EPO mRNA was diluted with MilliQ water to prepare a 2-fold dilution series from 100 μg / mL to 3.1 μg / mL, and a calibration curve solution was prepared. Measurement solutions were prepared by mixing 50 μL of the calibration curve solution or mRNA lipid nanoparticles with 450 μL of methanol. The absorbance of each measurement solution at 260 nm and 330 nm was measured using a UV plate reader (Multiskan Go, Thermo Fisher Scientific), and the absorbance at 330 nm was subtracted from the absorbance at 260 nm to obtain the absorbance of each measurement solution. The total aqueous phase nucleic acid concentration was calculated from the calibration curve using the absorbance of each sample measurement solution.

[0125] (Quantification of mRNA concentration in the external aqueous phase) Using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific), the nucleic acid concentration in the external aqueous phase was quantified by the standard addition method. First, the 20x TE buffer included in the kit was diluted with water to prepare 1x TE buffer. TE stands for Tris / EDTA (ethylenediaminetetraacetic acid). EPO mRNA was diluted with TE buffer to final concentrations of 0 to 400 ng / mL, and a nucleic acid dilution series was prepared. 10 μL of mRNA lipid nanoparticles diluted 5-fold with TE buffer and 90 μL of the nucleic acid dilution series were mixed in a 96-well plate, and then 100 μL of RiboGreen reagent diluted 200-fold with TE buffer was added to each well. Fluorescence (excitation wavelength: 485 nm, emission wavelength: 535 nm) was measured using a fluorescent plate reader (Infitite 200 Pro M nano +, TECAN). The nucleic acid concentration in the external aqueous phase of each measurement solution was calculated from the results obtained using the standard addition method.

[0126] (Calculation of Encapsulation Rate) Using the quantitative results of the total mRNA concentration and the mRNA concentration in the external aqueous phase obtained in the above steps, the mRNA encapsulation rate of the mRNA lipid nanoparticles was calculated according to the following formula. The results are shown in Table 4. mRNA encapsulation rate (%) = (total mRNA concentration - mRNA concentration in the external aqueous phase) ÷ total mRNA concentration × 100

[0127]

[0128] <Measurement of EPO enzyme activity> The dispersion of mRNA lipid nanoparticles prepared in the above <Preparation of EPO mRNA-encapsulated lipid nanoparticles> was intravenously administered to ICR mice at an mRNA dose of 0.1 mg / kg. Six hours after administration, blood was collected from the posterior vena cava to obtain plasma. Human EPO enzyme activity was quantified using the obtained plasma using an Erythropoietin (EPO) Human Elisa Kit (Abcam). Quantitative values ​​were expressed as relative EPO protein amounts, with Comparative Example 2 set to 1. The results are shown in Table 5.

[0129]

[0130] It was shown that the nucleic acid-lipid composition of the present invention had a higher EPO protein expression rate than the nucleic acid-lipid composition of the comparative example.

[0131] <EPO Enzyme Activity Measurement (Dose Response)> The mRNA lipid nanoparticle dispersion prepared in the above <Preparation of EPO mRNA-Encapsulated Lipid Nanoparticles> was intravenously administered to ICR mice at mRNA doses of 0.1, 0.5, and 1.0 mg / kg. Six hours after administration, blood was collected from the posterior vena cava to obtain plasma. Human EPO enzyme activity was quantified using the obtained plasma using an Erythropoietin (EPO) Human Elisa Kit (Abcam). The quantified values ​​were expressed as relative EPO protein amounts, with the EPO protein amount at 0.1 mg / kg administration in Comparative Example 2 set at 1. The results are shown in Table 6.

[0132]

[0133] Furthermore, for each nucleic acid-lipid composition, the relative expression levels at 0.5 and 1.0 mg / kg relative to 0.1 mg / kg were calculated to determine the degree of increase in expression efficiency with increasing dose (hereinafter referred to as dose response). The dose response is shown in Figure 1.

[0134] FIG. 1 shows that the nucleic acid-lipid composition of the present invention exhibits a high dose-responsiveness in terms of EPO protein expression compared to the nucleic acid-lipid composition of the comparative example.

Claims

1. A compound represented by the following formula (1) or a salt thereof: In the formula, R １ represents a hydrocarbon group having 1 to 24 carbon atoms, R ２ represents a hydrogen atom or a hydrocarbon group having 1 to 24 carbon atoms; R ３ represents a hydrocarbon group having 6 to 24 carbon atoms and having a branched chain, R ４ and R ５ each independently represents an optionally substituted hydrocarbon group having 1 to 6 carbon atoms; R ４ and R ５ The substituents on the optionally substituted hydrocarbon group having 1 to 6 carbon atoms represented by the formula (I) are each independently -O-R ６ , -OC(O)OR ６ , —OC(O)—R ６ , —CO(O)—R ６ , —C(O)—R ６ , -C(O)N(R ６ ) R ７ , or -N(R ６ ) R ７ indicates, R ６ and R ７ each independently represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, L represents *-O-C(O)- or *-C(O)O-, * represents R １ a represents an integer of 1 to 12; b represents an integer of 1 to 4; c represents an integer of 1 to 4; and d represents an integer of 1 to 4.

2. R １ represents a hydrocarbon group having 2 to 12 carbon atoms; R ２ represents a hydrocarbon group having 2 to 12 carbon atoms; R ３ represents a hydrocarbon group having 8 to 22 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 6 carbon atoms, L represents *-O-C(O)-, * represents R １ a represents an integer of 2 to 10, b represents an integer of 2 to 4, c represents an integer of 2 to 4, and d represents an integer of 2 to 4. The compound or salt thereof according to claim 1, 3. R １ represents a hydrocarbon group having 3 to 10 carbon atoms; R ２ represents a hydrocarbon group having 3 to 10 carbon atoms; R ３ represents a hydrocarbon group having 10 to 20 carbon atoms and having a branched chain, R ４ and R ５ each independently represents a hydrocarbon group having 1 to 3 carbon atoms, L represents *-O-C(O)-, * represents R １ a represents a bonding position with a, a represents an integer of 3 to 8, b represents 2 or 3, c represents 2 or 3, and d represents 2 or 3. The compound or a salt thereof according to claim 1 .

4. R ３ is -CH(R ３１ ) (R ３２ ), or -CH ２ CH (R ３３ ) (R ３４ ), and R ３１ , R ３２ , R ３３ , and R ３４ and each independently represent a linear hydrocarbon group having 3 to 12 carbon atoms, or a salt thereof.

5. A compound selected from the following compounds or a salt thereof: heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyloctanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-7-(2-((2-heptylnonanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-octyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-nonylundecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-12-hexyl-7-(2-((2-hexyldecanoyl)oxy)ethyl)-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-7-(2-((3-heptyldecanoyl)oxy)ethyl)-12-hexyl-11-oxo-10-oxa-3,7,12-triazaoctadecane-18-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-hexylooctanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-6-(2-((2-heptylnonanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-octyldecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-nonylundecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-11-hexyl-6-(2-((2-hexyldecanoyl)oxy)ethyl)-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; Heptyl 3-ethyl-6-(2-((3-heptyldecanoyl)oxy)ethyl)-11-hexyl-10-oxo-9-oxa-3,6,11-triazaheptadecan-17-oate; 6. A lipid composition comprising the compound or salt thereof according to any one of claims 1 to 5 and a lipid.

7. The lipid composition according to claim 6, wherein the lipid is at least one type of lipid selected from the group consisting of neutral lipids and lipids having nonionic hydrophilic polymer chains.

8. The lipid composition of claim 6, further comprising a sterol.

9. The lipid composition of claim 6, further comprising at least one selected from the group consisting of nucleic acids, proteins, peptides and small molecules.

10. A pharmaceutical composition comprising the lipid composition according to claim 6.

11. A delivery carrier comprising the lipid composition of claim 6.

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