Method for producing lipid nanoparticles encapsulating nucleic acid, method for producing pharmaceutical composition comprising said production method, and method for introducing nucleic acid into cell or target cell

Abstract

Provided is a method for producing nucleic acid-encapsulated lipid nanoparticles capable of efficiently and simply encapsulating an arbitrary nucleic acid. A method for producing nucleic acid-encapsulated lipid nanoparticles, comprising the steps of: a) preparing a nucleic acid-free lipid nanoparticle suspension by mixing an alcoholic solution containing an ionic lipid, a sterol, and a PEG lipid with an acidic buffer solution having a buffering effect at a pH of 1-6.5; and b) a step of mixing the lipid nanoparticle suspension obtained in step a) without freeze-drying with an aqueous solution containing nucleic acid and optionally containing 0-25 v / v% of alcohol, and optionally incubating the mixture at 0-95 DEG C for 0-60 minutes to obtain nucleic acid-encapsulated lipid nanoparticles.

Description

Technical Field

[0001] The present invention relates to a method for producing a lipid nanoparticle encapsulating nucleic acid, a method for producing a pharmaceutical composition comprising the production method, and a method for introducing nucleic acid into a cell or a target cell. The production method for the lipid nanoparticle encapsulating nucleic acid comprises the step of adding nucleic acid after preparing lipid nanoparticles not containing nucleic acid. Background Art

[0002] In order to put nucleic acid therapy using oligonucleic acids such as siRNA and gene therapy using mRNA or pDNA into practical use, effective and safe nucleic acid delivery vectors are needed. Viral vectors are nucleic acid delivery vectors with high expression efficiency, but non-viral nucleic acid delivery vectors that can be used more safely are being developed.

[0003] Since cationic liposomes using cationic lipids with quaternary ammonium are positively charged, they can form lipid complexes (lipoplexes) with negatively charged nucleic acids through electrostatic interactions, thereby delivering nucleic acids into cells. In addition, lyophilized compositions of cationic liposomes without nucleic acids are prepared by utilizing electrostatic interactions between quaternary ammonium and nucleic acids, and lipid complexes can also be formed by rehydration with an aqueous solution of nucleic acids, thus showing that they can also be used as gene introduction reagents (for example, refer to Patent Documents 1 and 2).

[0004] However, it is difficult to control the particle size of lipoplexes prepared by this method, and cytotoxicity from positively charged cationic lipids becomes a problem.

[0005] Therefore, lipid nanoparticles (called Lipid Nanoparticles or LNPs) using ionic lipids having tertiary amines in the molecule that are positively charged under acidic conditions and uncharged near neutrality have been developed and are becoming the most commonly used non-viral nucleic acid delivery vectors (for example, see Non-Patent Document 1).

[0006] As lipid nanoparticles using ionic lipids having a tertiary amine in the molecule, there are also examples in which a degradable group is imparted to the ionic lipid (for example, see Non-Patent Document 3).

[0007] As described above, various non-viral vectors have been developed, but nucleic acids are generally unstable compounds, and thus their stability as formulations remains a problem. Prior art literature Patent Literature

[0008] Patent Document 1: Japanese Patent No. 4919397 Patent Document 2: Japanese Patent No. 4598908 Patent Document 3: Japanese Patent No. 6093710 Patent Document 4: International Publication No. 2017 / 218704 Patent Document 5: International Publication No. 2021 / 060440 Non-patent literature:

[0009] Non-patent literature 1: Gene Therapy 6: 271-281, 1999 Non-patent document 2: Biol. Pharm. Bull. 41, 1291-1294 (2018) Summary of the invention Problems to be solved by the invention

[0010] As one of the methods for improving the stability of the preparation, attempts have been made to freeze-dry lipid nanoparticles encapsulating nucleic acids and rehydrate them at the time of use to reconstitute the lipid nanoparticles (Patent Document 4 and Non-Patent Document 2).

[0011] Although these methods are useful as methods for improving the storage stability of lipid nanoparticles encapsulating specific nucleic acids, they have problems as methods for more easily encapsulating any nucleic acid in lipid nanoparticles.

[0012] As a method for simply encapsulating any nucleic acid in lipid nanoparticles, there can be mentioned a method of preparing a lyophilized composition not containing nucleic acid and then rehydrating it with an aqueous solution of nucleic acid, as described in Patent Documents 1 and 2.

[0013] However, lipid nanoparticles obtained using ionic lipids having tertiary amines in the molecule do not undergo electrostatic interaction with nucleic acids because the surface charge after preparation is weakly negative to neutral. It is therefore impossible to prepare lipid nanoparticles encapsulating nucleic acids by the method disclosed in Patent Documents 1 and 2 of preparing a freeze-dried composition that does not contain nucleic acids and then rehydrating it with an aqueous nucleic acid solution.

[0014] In this regard, Patent Document 5 shows that lipid nanoparticles that do not contain nucleic acids are prepared in an acidic buffer, further freeze-dried by adding a cryoprotectant, and then rehydrated with an aqueous solution containing nucleic acids, thereby enabling any nucleic acid to be efficiently and simply encapsulated in lipid nanoparticles. However, the above-mentioned technique requires a freeze-drying step, and thus there is room for improvement in terms of simplicity.

[0015] In view of the above problems, the present invention aims to provide a method for producing lipid nanoparticles encapsulating nucleic acids that can efficiently and simply encapsulate any nucleic acid, which is not achievable in the prior art, a method for producing a pharmaceutical composition comprising the production method, and a method for introducing nucleic acids into cells or target cells. Technical solutions to the problem

[0016] In view of the above problems, the inventors have made unremitting efforts and found that by preparing lipid nanoparticles without nucleic acid in an acidic buffer and adding an aqueous solution containing nucleic acid, any nucleic acid can be efficiently and simply encapsulated in the lipid nanoparticles. In addition, using the lipid nanoparticles prepared by this method to carry out gene introduction experiments in cells, it was unexpectedly found that the gene introduction efficiency was improved compared with the prior art, thereby completing the present invention.

[0017] That is, the present invention includes the following contents: [1] A method for producing lipid nanoparticles encapsulating nucleic acid, comprising the following steps: a) mixing an alcohol solution containing ionic lipids, sterols and PEG lipids with an acidic buffer having a buffering effect at pH 1 to 6.5 to prepare a lipid nanoparticle suspension that does not contain nucleic acids, and b) mixing the lipid nanoparticle suspension obtained in step a with an aqueous solution containing nucleic acid and optionally containing 0-25 v / v% of alcohol without freeze-drying, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain lipid nanoparticles encapsulating nucleic acid.

[0018] [2] The method for producing nucleic acid-encapsulating lipid nanoparticles according to [1], further comprising the following step c after step b: c) replacing the external aqueous phase of the obtained nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution.

[0019] [3] The method for producing nucleic acid-encapsulating lipid nanoparticles according to [1] or [2], wherein in step a, the method further comprises: freezing the nucleic acid-free lipid nanoparticles at -80 to 0°C and then thawing them at 0 to 95°C.

[0020] [4] A method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [3], wherein in step a, the method further comprises: after preparing the lipid nanoparticle suspension, replacing the external aqueous phase with another acidic buffer having a buffering effect at pH 1 to 6.5 by dialysis, ultrafiltration or dilution.

[0021] [5] The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [4], wherein in step a, the alcohol solution further contains a phospholipid.

[0022] [6] The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [5], wherein the ionic lipid is a compound represented by formula (1).

[0023] [Chemical formula 1]

[0024] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or a group represented by formula (3): R 9 -O-CO-(CH2)a- (3) (In formula (3), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. )).

[0025] [7] The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of [1] to [5], wherein the ionic lipid is a compound represented by formula (2).

[0026] [Chemical formula 2]

[0027] (Where, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen, R 1 represents an aliphatic hydrocarbon group having 8 or less carbon atoms, L 1 represents an ester bond, an amide bond, a carbamate bond, an N-alkyl carbamate bond, a carbonate bond or a urea bond, k represents 0 or 1, R x and R y each independently represents an alkylene group having 2 to 5 carbon atoms, L 2 represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond, R 2 represents an alkylene group having 8 or less carbon atoms or does not exist, Y represents a group, which (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) contains at least one selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue and a residue of a sterol derivative.).

[0028] [8] A method for introducing a nucleic acid into a cell, comprising the step of bringing a nucleic acid-encapsulating lipid nanoparticle produced by the method of any one of [1] to [7] into contact with a cell in vitro.

[0029] [9] A method for introducing a nucleic acid into a target cell, comprising the step of administering to a living organism a nucleic acid-encapsulating lipid nanoparticle produced by the method of any one of [1] to [7].

[0030]

[10] A method for producing a pharmaceutical composition, comprising the method according to any one of [1] to [7]. Effects of the Invention

[0031] In the manufacturing method of the lipid nanoparticles of the encapsulated nucleic acid of the present invention, since the nucleic acid aqueous solution is added without freeze-drying after the preparation of the lipid nanoparticles without nucleic acid, the lipid nanoparticles encapsulated with any nucleic acid can be efficiently and simply manufactured. In addition, compared with the prior art, the nucleic acid introduction efficiency of the lipid nanoparticles of the encapsulated nucleic acid prepared by the manufacturing method of the present invention is high, which is conducive to the gene introduction in cells or organisms, and is particularly useful as a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a graph evaluating the effect of buffer on encapsulation efficiency. Figure 2 This is a graph evaluating the effect of buffer on particle size. Figure 3 The figure evaluates the effect of sucrose concentration and incubation temperature on encapsulation efficiency. The incubation temperature is expressed in °C. Figure 4 This is a graph evaluating the effects of sucrose concentration and incubation temperature on particle size. The incubation temperature is expressed in °C. Figure 5 The graph evaluates the effect of sucrose concentration and incubation temperature on in vitro activity. The incubation temperature is expressed in °C. Figure 6 It is a graph evaluating the effect of incubation time on encapsulation efficiency. Figure 7 This is a graph evaluating the effect of incubation time on particle size. Figure 8 This is a graph evaluating the effect of incubation time on in vitro activity. Fig. 9 Yes means Figure 8 A plot of the area under the curve (AUC) of . Fig.10 This is a graph evaluating the effect of buffer pH on the encapsulation efficiency. Fig.11 This is a graph evaluating the effect of buffer pH on particle size. Fig.12 This is a graph evaluating the effect of the salt concentration of the buffer on the encapsulation efficiency. Fig.13 This is a graph evaluating the effect of the salt concentration of the buffer on the particle size. Fig.14 This is a graph comparing the encapsulation efficiency of the nucleic acid encapsulating nanoparticles of the present invention and a lyophilized product. Fig.15 This is a graph comparing the particle sizes of the nucleic acid-encapsulating nanoparticles of the present invention and a lyophilized product. Fig.16 This is a graph comparing the in vitro activities of the nucleic acid encapsulating nanoparticles of the present invention and the lyophilized product. Fig.17 Yes means Fig.16 A plot of the area under the curve (AUC) of . Fig.18 This is a graph comparing the in vivo activities of the nucleic acid encapsulating nanoparticles of the present invention and the lyophilized product. Fig.19 It is a graph for evaluating the effect of mixing ratio and mixing method on encapsulation efficiency. Fig. 20 This is a graph that evaluates the effects of the mixing ratio and mixing method on the particle size. Fig.21 It is a graph evaluating the effects of mixing ratio and mixing method on in vitro activity. Fig. 22is a graph evaluating the effect of buffer on encapsulation efficiency. Fig.23 This is a graph evaluating the effect of buffer on particle size. Fig.24 It is a graph evaluating the effect of incubation temperature on encapsulation efficiency. Fig.25 This is a graph evaluating the effect of the incubation temperature on the particle size. Fig.26 This is a graph evaluating the effect of incubation temperature on in vitro activity. Fig. 27 It is a graph evaluating the effect of incubation time on encapsulation efficiency. Fig.28 This is a graph evaluating the effect of incubation time on particle size. Fig.29 This is a graph evaluating the effect of incubation time on in vitro activity. Fig.30 This is a graph evaluating the effect of buffer pH on the encapsulation efficiency. Fig.31 This is a graph evaluating the effect of buffer pH on particle size. Fig.32 This is a graph evaluating the effect of buffer pH on in vitro activity. Fig.33 This is a graph showing the relationship between pH when mRNA is mixed and the zeta potential at each pH. Fig.34 This is a graph evaluating the effect of the salt concentration of the buffer on the encapsulation efficiency. Fig.35 This is a graph evaluating the effect of the salt concentration of the buffer on the particle size. Fig.36 This is a graph comparing the encapsulation efficiency of the nucleic acid encapsulating nanoparticles of the present invention and a lyophilized product. Fig.37 This is a graph comparing the particle sizes of the nucleic acid-encapsulating nanoparticles of the present invention and a lyophilized product. Fig.38 This is a graph comparing the in vivo activities of the nucleic acid encapsulating nanoparticles of the present invention and the lyophilized product. Fig.39 This is a graph comparing the encapsulation efficiency of the nucleic acid encapsulating nanoparticles of the present invention and the nucleic acid encapsulating nanoparticles (MF) prepared by microchannels. Fig.40 Graphs showing expression distribution in nucleic acid encapsulating nanoparticles (MF) prepared by microchannels. Fig.41 This is a diagram showing the expression distribution in the nucleic acid-encapsulating nanoparticles of the present invention. Fig.42 This is a graph comparing the in vitro activities of the nucleic acid encapsulating nanoparticles of the present invention and the lyophilized product. DETAILED DESCRIPTION

[0033] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0034] The present invention relates to a method for preparing nucleic acid-encapsulating lipid nanoparticles by mixing a lipid solution containing ionic lipids, sterols and PEG lipids without nucleic acid with an acidic buffer having a buffering effect at pH 1 to 6.5, and then adding a nucleic acid aqueous solution.

[0035] Lipid nanoparticles refer to particles with a membrane structure in which the hydrophilic groups of amphiphilic lipids are arranged toward the aqueous phase side of the interface. "Amphiphilic lipids" refer to lipids having both a hydrophilic group showing hydrophilicity and a hydrophobic group showing hydrophobicity. Examples of amphiphilic lipids include ionic lipids, phospholipids, and PEG lipids. The lipid nanoparticles used in the present invention contain ionic lipids, sterols and PEG lipids as membrane constituents, and may also contain phospholipids. The particle size of the lipid nanoparticles is not particularly limited, preferably 10nm to 500nm, more preferably 30nm to 300nm. The determination of the particle size can be carried out using a particle size distribution measuring device such as Zetasizer Nano (Malvern Company). The particle size of the lipid nanoparticles can be appropriately adjusted by the method for manufacturing lipid nanoparticles. In the present invention, the particle size refers to the average particle size (number average) measured by the dynamic light scattering method. In the present invention, "total lipids" refers to the total amount of lipids. Examples of lipids include ionic lipids, sterols, PEG lipids, and phospholipids. In the present invention, "containing no nucleic acid" or "no nucleic acid" means substantially containing no nucleic acid, and means that the nucleic acid content is below the detection limit. In the present invention, the "nucleic acid-encapsulating lipid nanoparticle" refers to a lipid nanoparticle in which a nucleic acid is encapsulated inside the lipid nanoparticle.

[0036] Ionic lipids The ionic lipid that can be used in the present invention is any one that is composed of a tertiary amino group and a hydrophobic group and can form lipid nanoparticles. Specific examples of ionic lipids include: 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLi-n-KC2-DMA), 4-(dimethylamino)butyric acid-heptatriacontahedral-6,9,28,31-tetraen-19-yl ester (referred to as DLi-MC3-DMA or MC3), 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid 17-carbon-9-yl ester (lipid 5), 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)octanoic acid 17-carbon-9-yl ester (lipid 8), compounds of the following formula (1) or (2), preferably compounds of the following formula (1) or (2), DODMA, MC3, lipid 5, lipid 8, and most preferably compounds of the following formula (1) or (2). Formula (1)

[0037] [Chemical formula 3]

[0038] (In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3bEach independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or a group represented by formula (3). R 9 -O-CO-(CH2)a-(3) (In formula (3), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer from 2 to 10. ))

[0039] R 1a and R 1b Each independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, preferably linear. The alkylene group preferably has 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms. Specific examples of the alkylene group having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. 1a and R 1b Preferably, each is independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and most preferably each is an ethylene group.

[0040] R 1a Can be used with R 1b The same or different, but preferably R 1a is with R 1b Same group.

[0041] X a and X b Each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, and is preferably a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups.

[0042] The alkyl group having 1 to 6 carbon atoms in the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group may be linear, branched, or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl, preferably methyl, ethyl, propyl, or isopropyl, and most preferably methyl.

[0043] The preferred specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is represented by X 1 express.

[0044] [Chemical formula 4]

[0045] X 1 R 5 The alkyl group having 1 to 6 carbon atoms may be straight-chain, branched or cyclic. The alkyl group preferably has 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and cyclohexyl. Methyl, ethyl, propyl or isopropyl is preferred, and methyl is most preferred.

[0046] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include aziridinyl, aziridinyl, pyrrolidinyl, piperidinyl, imidazolinyl, and piperazinyl, with pyrrolidinyl, piperidinyl, and piperazinyl being preferred, and piperidinyl being most preferred.

[0047] A preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group is represented by X 2 express.

[0048] [Chemical formula 5]

[0049] X 2 p is 1 or 2. When p is 1, X 2 When p is 2, X 2 Preferably, p is 2.

[0050] The preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is represented by X 3 express.

[0051] [Chemical formula 6]

[0052] X 3 w is 1 or 2. When w is 1, X 3 When X is imidazoline, w is 2, 3 It is piperazinylene.

[0053] X a Can be used with X b The same or different, but preferably X a With X b are the same group.

[0054] R 2a and R 2b Each independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group, and each independently represents an alkylene group having 8 or less carbon atoms.

[0055] The alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear. The number of carbon atoms contained in the alkylene group is preferably 6 or less, and most preferably 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include methylene, ethylene, trimethylene, isopropylidene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, etc., preferably methylene, ethylene, trimethylene, tetramethylene, and most preferably ethylene.

[0056] In the present specification, "oxydialkylene having 8 or less carbon atoms" is an alkylene group linked via an ether bond (alkylene-O-alkylene, in other words, "alkyleneoxyalkylene"), and refers to a group in which the sum of the carbon atoms of the two alkylene groups present is 8 or less. The two alkylene groups present may be the same or different, but are preferably the same. Specific examples of oxydialkylene having 8 or less carbon atoms include oxydimethylene, oxydiethylene, oxybis(trimethylene) groups (i.e., trimethyleneoxytrimethylene), oxybis(tetramethylene) groups (i.e., tetramethyleneoxytetramethylene), and the like. Oxydimethylene, oxydiethylene, and oxybis(trimethylene) groups are preferred, and oxydiethylene is most preferred.

[0057] R 2a Can be used with R 2b The same or different, but preferably R 2a is with R 2b Same group.

[0058] Y a and Y b Each is independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each is independently an ester bond, an amide bond or a carbamate bond, more preferably each is independently an ester bond or an amide bond, and most preferably each is an ester bond. a and Y b There is no restriction on the bonding direction, but a and Y b In the case of an ester bond, it is preferred to present -Z a -CO-OR 2a -and-Zb -CO-OR 2b -structure.

[0059] Y a Can be used with Y b The same or different, but preferably Y a Yes and Y b Same group.

[0060] Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. The number of carbon atoms contained in the aromatic compound is preferably 6 to 12, and most preferably 6 to 7. In addition, the aromatic compound preferably has one aromatic ring.

[0061] As the types of aromatic rings contained in the aromatic compound having 3 to 16 carbon atoms, the aromatic hydrocarbon rings include a benzene ring, a naphthalene ring, and an anthracene ring. The aromatic heterocyclic rings include an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furothiophenol ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring. Preferred are a benzene ring, a naphthalene ring, and an anthracene ring, and the most preferred is a benzene ring.

[0062] The aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylparaaminobenzenesulfonyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a carbon The ureido group having 1 to 4 carbon atoms, the alkoxy group having 1 to 4 carbon atoms, the aryl group having 6 to 10 carbon atoms, the aryloxy group having 6 to 10 carbon atoms, and the like. Preferred examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylparaaminobenzenesulfonyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0063] As Z a and Z b The preferred specific structure of Z1 .

[0064] [Chemical formula 7]

[0065] In the formula, s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and u R 4 Each independently represents a substituent.

[0066] Z 1 The s is preferably an integer of 0 to 1, and more preferably 0. Z 1 The t is preferably an integer of 0 to 2, and more preferably 1. Z 1 The u is preferably an integer of 0-2, and more preferably an integer of 0-1.

[0067] Z 1 R 4 It is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylparaaminobenzenesulfonyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a 4 urea groups, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, aryloxy groups having 6 to 10 carbon atoms, and the like. Preferred examples include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylparaaminobenzenesulfonyl, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, urea, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, and phenoxy. In the case of multiple R 4 In the presence of 4 It can be the same or different.

[0068] Z a Can be used with Z b The same or different, but preferably Z a Yes and Z b Same group.

[0069] n a and n b Each independently is 0 or 1.

[0070] n a Can be used with n b The same or different, but preferably n a With n b same.

[0071] R 3a and R 3b Each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and a cyclopropane ring, or the formula (3): R 9 -O-CO-(CH2)a- (3) (In formula (3), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer of 2 to 10. The groups represented by ) are preferably each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and most preferably each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0072] The residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride represents a group having the following structure: the hydroxyl group of the fat-soluble vitamin having a hydroxyl group is substituted with *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2-. * represents the bonding position to the fat-soluble vitamin. The residue derived from the reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride represents a group having the following structure: the hydroxyl group of the sterol derivative having a hydroxyl group is substituted with *-O-CO-CH2-CH2- or *-O-CO-CH2-CH2-CH2-. * represents the bonding position to the sterol derivative.

[0073] Examples of the fat-soluble vitamin having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0074] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, and ergosterol, and cholesterol and cholestanol are preferred.

[0075] The aliphatic hydrocarbon group having 1 to 40 carbon atoms may be straight-chain or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, but carbon-carbon double bonds are preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 12 to 22, more preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, etc., but preferably includes an alkyl group or an alkenyl group. Specific examples of the aliphatic hydrocarbon group having 1 to 40 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, triacontyl, tetracontyl, dodecenyl, tridecenyl, tetradecenyl, 1-decenyl, 1-pentadecenyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, 1-eicosadienyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, and the like. The aliphatic hydrocarbon group having 1 to 40 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecyl, heptadecenyl, heptadecadienyl, and 1-hexylnonyl, and particularly preferably tridecyl, heptadecyl, heptadecenyl, heptadecadienyl, and heptadecadienyl.

[0076] In one embodiment of the present invention, R 3a and R 3b The aliphatic hydrocarbon group having 1 to 40 carbon atoms (preferably 12 to 22 carbon atoms) represented by is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is contained in -CO-O- in formula (1). As a specific example of the aliphatic hydrocarbon group, when linoleic acid is used as the fatty acid, it is heptadecadienyl, and when oleic acid is used as the fatty acid, it is heptadecenyl.

[0077] R 3a and R 3bThe alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring in the alkyl chain refers to an alkyl group having 3 to 40 carbon atoms and having at least one cyclopropane ring in the alkyl chain. The 3 to 40 carbon atoms of the alkyl group do not include the carbon atoms of the cyclopropane ring. The number of cyclopropane rings in the alkyl group is preferably 1. 3a and R 3b The alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring is preferably a group represented by formula (4).

[0078] [Chemical formula 8]

[0079] (In formula (4), b and c are each independently an integer, and the sum of b and c is 2 to 39.) Preferably, b is an integer of 1 to 20, and c is an integer of 1 to 19. More preferably, b is an integer of 2 to 18, more preferably an integer of 3 to 17, and still more preferably an integer of 4 to 12. More preferably, c is an integer of 3 to 15, more preferably an integer of 3 to 11, and still more preferably an integer of 3 to 9. Examples of the group represented by formula (4) include 7-(2-octylcyclopropyl)heptyl and the like.

[0080] In formula (3), R 9The aliphatic hydrocarbon group having 2 to 20 carbon atoms may be straight-chain or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, preferably 1 to 3, and more preferably 1 to 2. Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, but carbon-carbon double bonds are preferred. The number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 8 to 20, more preferably 9 to 19, further preferably 13 to 19, and most preferably 13 to 17. The aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, etc., but preferably includes an alkyl group or an alkenyl group, and more preferably an alkyl group. Specific examples of the aliphatic hydrocarbon group having 2 to 20 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecyl, nonadecyl, eicosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadienyl, octadecadienyl, nonadecadienyl, eicosadienyl, eicosatoenyl, eicosatetraenyl, eicosapentaenyl, isostearyl, 1-hexylheptyl, 1-ethylnonyl, 1-butylnonyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 3-octylundecyl, etc. The aliphatic hydrocarbon group having 2 to 20 carbon atoms is preferably tridecyl, pentadecyl, heptadecyl, nonadecenyl, heptadecenyl, heptadecadienyl, 1-hexylnonyl, and particularly preferably tridecyl, heptadecyl, heptadecenyl, heptadecadienyl, heptadecadienyl.

[0081] In formula (3), a is preferably an integer of 3 to 9, more preferably an integer of 3 to 7, further preferably an integer of 5 to 7, and most preferably 5 or 7.

[0082] R 3a Can be used with R 3b The same or different, but preferably R 3a is with R 3b Same group.

[0083] In one embodiment of the present invention, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y b Same, Za With Z b Same, R 3a With R 3b same.

[0084] Preferred examples of the ionic lipid represented by formula (1) (may be simply referred to as "ionic lipid (1)" in this specification) include the following ionic lipids. [Ionic lipids (1-1)] Ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 6 carbon atoms (e.g., methylene, ethylene); X a and X b Each is independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g. -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g. piperidinylene); R 2a and R 2b Each is independently an alkylene group having 8 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0085] [Ionic lipids (1-2)] Ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene, ethylene); X a and X bEach is independently a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and 1 tertiary amino group (e.g. -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g. piperidinylene); R 2a and R 2b Each is independently an alkylene group having 6 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); n a and n b Each is independently 0 or 1; R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (eg, tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (eg, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0086] [Ionic lipids (1-3)] Ionic lipid (1), wherein R 1a and R 1b Each is independently an alkylene group having 1 to 2 carbon atoms (i.e., methylene or ethylene); X a and X b Each independently is X 1 :

[0087] [Chemical formula 9]

[0088] (Where R 5 is an alkyl group having 1 to 3 carbon atoms (e.g., methyl group). ), or X 2 :

[0089] [Chemical formula 10]

[0090] (where p is 1 or 2); R 2a and R 2bEach is independently an alkylene group having 4 or less carbon atoms (e.g., methylene, ethylene, trimethylene); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each independently is Z 1 :

[0091] [Chemical formula 11]

[0092] (wherein, s is an integer of 0 to 1, t is an integer of 0 to 2, u is an integer of 0 to 2 (preferably 0), and u R 4 Each independently represents a substituent. ); n a and n b Each is independently 0 or 1; R 3a and R 3b Each is independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (eg, tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (eg, heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0093] Specific examples of ionic lipids (1) include the following: O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M, B-2, B-2-5, TS-P4C2, L-P4C2, and O-P4C2.

[0094] In addition, specific examples of the ionic lipid (1) include lipids 1 to 20 described in WO2021 / 195529A2.

[0095] [Table 1-1]

[0096] [Table 1-2]

[0097] [Table 1-3]

[0098] Ionic lipid (1) can be produced by a known method (for example, the method described in WO2019 / 188867A1 (US2021 / 0023008A1), US9708628B2, and WO2021 / 195529A2).

[0099] A compound represented by formula (2).

[0100] [Chemical formula 12]

[0101] (Where, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen, R 1 represents an aliphatic hydrocarbon group having 8 or less carbon atoms, L 1 represents an ester bond, an amide bond, a carbamate bond, an N-alkyl carbamate bond, a carbonate bond or a urea bond, k represents 0 or 1, R x and R y each independently represents an alkylene group having 2 to 5 carbon atoms, L 2 represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond, R 2 represents an alkylene group having 8 or less carbon atoms or does not exist, Y represents (i) one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) at least one group selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) at least one group selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue and a residue of a sterol derivative. ).

[0102] One preferred example of X can be represented by the following general formula (5). R α -N(R β )- (5) (Where, R α is an aliphatic hydrocarbon group, R β is an aliphatic hydrocarbon group, an aliphatic group containing one or more heteroatoms other than nitrogen, or an aliphatic group containing one or more tertiary amines, or R α and R β It can also be bonded to form a 3- to 8-membered nitrogen-containing alicyclic ring)

[0103] More preferably, X is a dialkylamino group (the carbon number of the two alkyl groups of the dialkylamino group is independently 1 to 8), a 3- to 6-membered cyclic amino group which may have a hetero atom, or -N(R a )-R b , R a is an alkyl group having 1 to 8 carbon atoms, R b -(CH2)qOR c , R c is hydrogen or an alkyl group having 1 to 8 carbon atoms, q is an integer from 2 to 4.

[0104] The carbon number of the two alkyl groups in the dialkylamino group is preferably 1 to 5, and more preferably 1 to 4. The alkyl group may be linear, branched or cyclic. Specific examples include methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, cyclopentyl, etc. Preferably, each is independently methyl, ethyl, propyl or isopropyl, and more preferably, each is independently methyl or ethyl.

[0105] The 3- to 6-membered cyclic amino group which may have a hetero atom refers to a group in which the substituents of the amino group are bonded to form a ring, the number of atoms forming the above-mentioned ring is 3 to 6, and the group optionally contains hetero atoms such as oxygen. The 3- to 6-membered cyclic amino group which may have a hetero atom is preferably a 5- or 6-membered cyclic amino group which may have a hetero atom, and more preferably a 6-membered cyclic amino group which may have a hetero atom. As the cyclic amino group, it is preferably an amino group whose ring is formed only by a nitrogen atom and a methylene group (-CH2-), and the ring optionally contains an oxygen atom. Specifically, it is 1-pyrrolidinyl, 1-piperidinyl, morpholinyl (4-morpholinyl), preferably 1-piperidinyl, morpholinyl.

[0106] As R 1 The aliphatic hydrocarbon group having 8 or less carbon atoms is preferably an alkylene group, an alkenylene group or an alkynylene group, and more preferably an alkylene group or an alkenylene group.

[0107] The alkylene group having a carbon number of 8 or less may be either linear or branched. The carbon number of the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having a carbon number of 8 or less is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylidene group (-CH(CH3)CH2-, -CH2CH(CH3)-), an isobutylidene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group or a hexamethylene group, more preferably a methylene group, an ethylene group, a trimethylene group, an isopropylidene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group or an isobutylidene group (-C(CH3)2CH2-, -CH2C(CH3)2-).

[0108] The alkenylene group having 8 or less carbon atoms may be either linear or branched. The number of carbon atoms in the alkenylene group is preferably 6 or less, more preferably 4 or less. The alkenylene group having 8 or less carbon atoms is preferably propenylene (-CH2CH=CH-, -CH=CHCH2-), butenylene, isopropenylene, isobutenylene, pentenylene or hexenylene, more preferably propenylene (-CH2CH=CH-, -CH=CHCH2-), butenylene, isopropenylene or isobutenylene.

[0109] L 1 It is an ester bond, an amide bond, a carbamate bond, an N-alkyl carbamate bond, a carbonate bond or a urea bond, and is preferably an ester bond, an amide bond, a carbamate bond, an N-methyl carbamate bond or a carbonate bond.

[0110] In this specification, N-alkyl carbamate bond means -NR 10 -CO-O- or -O-CO-NR 10 -, R 10 It represents an alkyl group having 1 to 8 carbon atoms. The alkyl group having 1 to 8 carbon atoms may be any of linear, branched or cyclic. The number of carbon atoms in the above alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specifically, it includes: methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, cyclopentyl, etc. Methyl, ethyl, propyl or isopropyl is preferred, and methyl is more preferred.

[0111] k represents 0 or 1. Here, k being 0 means that there is no R 1 -L 1 , that is, X and R x Direct bonding. When l, m, n, etc. are 0, the same meaning applies.

[0112] As for R x and R yThe alkylene group having 2 to 5 carbon atoms may be linear or branched, but is preferably linear. The alkylene group preferably has 2 to 4 carbon atoms, and more preferably 2 carbon atoms. Specifically, it is ethylene, trimethylene, tetramethylene, isopropylene (-CH(CH3)CH2-, -CH2CH(CH3)-) or isobutylene (-C(CH3)2CH2-, -CH2C(CH3)2-), preferably ethylene or trimethylene, more preferably ethylene.

[0113] L 2 It is an ester bond, an amide bond, a urethane bond, a carbonate bond, an ether bond or a urea bond, preferably an ester bond or an amide bond, and more preferably an ester bond.

[0114] R 2 represents an alkylene group having 8 or less carbon atoms, or does not exist. 2 Does not exist means L 2 Directly bonded to Y. R 2 The alkylene group having 8 or less carbon atoms may be either straight-chain or branched, but is preferably straight-chain. The number of carbon atoms of the alkylene group is preferably 6 or less, and more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably methylene, ethylene, trimethylene, tetramethylene, isopropylidene (-CH(CH3)CH2-, -CH2CH(CH3)-), isobutylene (-C(CH3)2CH2-, -CH2C(CH3)2-), pentamethylene or hexamethylene, and more preferably methylene, ethylene, trimethylene, isopropylidene (-CH(CH3)CH2-, -CH2CH(CH3)-), tetramethylene or isobutylene (-C(CH3)2CH2-, -CH2C(CH3)2-).

[0115] R a and R c The alkyl group having 1 to 8 carbon atoms may be straight-chain or branched, but is preferably straight-chain. The alkyl group preferably has 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Specific examples of the alkyl group having 1 to 8 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, and 2-methylbutyl. Methyl, ethyl, propyl, or isopropyl is preferred, and methyl is more preferred.

[0116] One preferred example of Y can be represented by the following general formula (6).

[0117] [Chemical formula 13]

[0118] (Where, R 3 represents an alkylene group having 8 or less carbon atoms, L 3 represents an ester bond or a carbonate bond, S 1 represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R e -L a -R 7 ' or -R e -L a -Z'-L b -R 7 ', S 2 Indicates -R e '-L a -R 7 " or -R e '-L a -Z"-L b -R 7 ”, R e and R e ' each independently represents an alkylene group having 8 or less carbon atoms, L a represents an ester bond or a carbonate bond, L b represents an ester bond or a carbonate bond, l means 0 or 1, Z, Z' and Z" each independently represent a divalent group having 3 to 16 carbon atoms, having at least one aromatic ring and derived from an aromatic compound which may have a heteroatom, L x represents an ester bond or a carbonate bond, R 4 represents an alkylene group having 8 or less carbon atoms or does not exist, R 5 represents hydrogen or an alkyl group having 1 to 8 carbon atoms, S 3 Indicates -R 6 '-L 4 '-R 7 ”', m represents 0 or 1, R 6 and R 6 'Each independently represents an alkylene group having 8 or less carbon atoms or does not exist, L 4 and L 4 ' each independently represents an ester bond or a carbonate bond, R 7 , R 7 '、R 7 ” and R7 "' each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms or -(CH2)pC(=O)-R f , R f represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, p represents 2 or 3, n represents 0 or 1.

[0119] R 4 represents an alkylene group having 8 or less carbon atoms or does not exist. 4 Absence refers to R 5 and S 3 The carbon atom bonded to L x Direct bonding.

[0120] R 6 and R 6 ' each independently represents an alkylene group having 8 or less carbon atoms or is absent. When m=0, R 6 Does not exist means L x With L 4 Direct bonding, when m=1, R 6 Absence refers to R 5 and S 3 The carbon atom bonded to L 4 Direct bonding. 6 'Non-existence means R 4 and R 5 The carbon atom bonded to L 4 'Direct bonding.

[0121] R 3 , R 4 , R 6 , R 6 '、R e and R e The alkylene group having 8 or less carbon atoms in the ' may be either straight-chain or branched, but is preferably straight-chain. The number of carbon atoms in the alkylene group is preferably 6 or less, and more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylidene group (-CH(CH3)CH2-, -CH2CH(CH3)-), an isobutylidene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group or a hexamethylene group, and more preferably a methylene group, an ethylene group, a trimethylene group, an isopropylidene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group or an isobutylidene group (-C(CH3)2CH2-, -CH2C(CH3)2-).

[0122] R 5 and S1 The alkyl group having 1 to 8 carbon atoms may be straight-chain or branched, but is preferably straight-chain. The alkyl group preferably has 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Specific examples of the alkyl group having 1 to 8 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, and 2-methylbutyl. Methyl, ethyl, propyl, or isopropyl is preferred, and methyl is more preferred.

[0123] L x , L 3 , L 4 , L 4 ',L a and L b It is an ester bond or a carbonate bond, preferably an ester bond.

[0124] Z, Z' and Z" each independently represent a divalent group having 3 to 16 carbon atoms, having at least one aromatic ring and derived from an aromatic compound which may have a heteroatom. Here, the divalent group refers to a divalent group having a structure obtained by removing two hydrogen atoms from the above aromatic compound. The number of carbon atoms in the above aromatic compound is preferably 6 to 12, more preferably 6 to 7. In addition, the number of aromatic rings in the above aromatic compound is preferably 1. Z, Z' and Z" may be the same or different, but preferably Z, Z' and Z" are the same.

[0125] As the aromatic ring of the above-mentioned aromatic compound, it can be any one of an aromatic hydrocarbon ring or an aromatic heterocyclic ring. As the aromatic hydrocarbon ring, for example, benzene ring, naphthalene ring, anthracene ring, etc. can be mentioned. As the aromatic heterocyclic ring, for example, benzene ring, naphthalene ring, anthracene ring, etc. can be mentioned. As the aromatic heterocyclic ring, for example, benzene ring, pyrazole ring, oxazole ring, isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furanthiophenol ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring, quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine ring, phenoxazine ring, phenothiazine ring, acridine ring, etc. The aromatic ring of the above-mentioned aromatic compound is preferably benzene ring, naphthalene ring or anthracene ring, more preferably benzene ring.

[0126] The aromatic ring of the aromatic compound may have a substituent. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkyl-p-aminobenzenesulfonyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Preferred examples of the substituents include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylparaaminobenzenesulfonyl, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, ureido, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, phenoxy and the like.

[0127] Z, Z' and Z" are preferably each independently of the formula (7):

[0128] [Chemical formula 14]

[0129] (Where, t represents an integer from 0 to 3, u represents an integer from 0 to 3, v represents an integer from 0 to 4, v R 8 Each independently represents a substituent, *Indicates the same as L 3 or L a The bonding position of . t is preferably 0 or 1, more preferably 1. u is preferably an integer of 0 to 2, and more preferably 0. v is preferably an integer of 0 to 2, more preferably 0 or 1, and even more preferably 0.

[0130] As R 8Examples of R include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkyl-p-aminobenzenesulfonyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. 8 Preferred examples of include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, propionyloxy, butyryloxy, pentanoyloxy, hexanoyloxy, heptanoyloxy, octanoyloxy, nonanoyloxy, decanoyloxy, undecanoyloxy, dodecanoyloxy, tridecanoyloxy, tetradecanoyloxy, pentadecanoyloxy, hexadecanoyloxy, heptadecanoyloxy, octadecanoyloxy, octadecanoyloxy, octadecadienoyloxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylparaaminobenzenesulfonyl, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, tert-butyl, ureido, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, phenyl, phenoxy, and the like. In the case where there are a plurality of R 8 In the case of multiple R 8 They may be the same as or different from each other.

[0131] R 7 , R 7 '、R 7 ” and R 7 "' each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms or -(CH2)pC(=O)-R f , R f represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents 2 or 3. 7 , R 7 '、R 7 ” and R 7 ”'respectively the same or different.

[0132] In this specification, the residue of a fat-soluble vitamin having a hydroxyl group refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the fat-soluble vitamin. In addition, the residue of a sterol derivative having a hydroxyl group refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the sterol derivative.

[0133] The aliphatic hydrocarbon group having 10 to 37 carbon atoms may be linear or branched. The aliphatic hydrocarbon group preferably has 12 to 37 carbon atoms, more preferably 13 to 37 carbon atoms, and even more preferably 15 to 37 carbon atoms.

[0134] The aliphatic hydrocarbon group may be saturated or unsaturated. When the aliphatic hydrocarbon group is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds is preferably 1 to 6, more preferably 1 to 3, and further preferably 1 to 2. The unsaturated bond may be a carbon-carbon double bond or a carbon-carbon triple bond, but a carbon-carbon double bond is preferred. The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group.

[0135] Examples of the aliphatic hydrocarbon group having 10 to 37 carbon atoms include a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an eicosyl group, a heneicosyl group, a docosyl group, a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecyl group, an eicosyl group, a heneicosyl group, a docosyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecyl group, an eicosadienyl group, a heneicosadienyl group, group), docosadienyl, octadecatrienyl, eicosatrienyl, eicosatetraenyl, eicosapentaenyl, eicosahexenyl, isostearyl, 1-hexylheptyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, 1-dodecyltridecyl, 1-tetradecylpentadecyl, 1-hexadecylheptadecyl, 1-octadecylnonadecyl, and the like.

[0136] The aliphatic hydrocarbon group having 10 to 37 carbon atoms is preferably undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosyl, docosyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosenyl, heneicosenyl, didec ... Dodecenyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadienyl, octadecadienyl, nonadecadienyl, eicosadienyl, heneicosadienyl, heneicosadienyl, docosadienyl, octadecatrienyl, eicosatrienyl, eicosatetraenyl, eicosapentaenyl, eicosahexenyl, isostearyl, 1-hexylheptyl, 1-hexylnonyl, 1-octylnonyl, 1-octyldecadienyl Preferably, the present invention comprises the following: monoalkyl, 1-decylundecyl, 1-dodecyltridecyl, 1-tetradecylpentadecyl, 1-hexadecylheptadecyl, and 1-octadecylnonadecyl, and particularly preferably pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, heptadecenyl, dodecadienyl, tridecadienyl, tetradecadienyl, pentadecadienyl, hexadecadienyl, heptadecadienyl, octadecadienyl, nonadecadienyl, eicosadienyl, heneicosadienyl, heneicosadienyl, docosadienyl, octadecadienyl, eicostrienyl, eicosatetraenyl, eicosopentenyl, eicosahexenyl, isostearyl, 1-hexylheptyl, 1-hexylnonyl, 1-octylnonyl, 1-octylundecyl, 1-decylundecyl, 1-dodecyltridecyl, 1-tetradecylpentadecyl, 1-hexadecylheptadecyl, and 1-octadecylnonadecyl.

[0137] Examples of the fat-soluble vitamin having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0138] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, etc. The sterol derivative having a hydroxyl group is preferably cholesterol or cholestanol. R f Preferably, it is a residue of a fat-soluble vitamin having a hydroxyl group.

[0139] l, m, and n represent 0 or 1. A preferred combination of l, m, and n is l = 0, m = 0, n = 0; l = 0, m = 0, n = 1; l = 0, m = 1, n = 1; l=1、m=0、n=0, Particularly preferred l = 0, m = 0, n = 0; l = 0, m = 1, n = 1; l=1, m=0, n=0.

[0140] Preferred examples of the ionic lipid represented by formula (2) (may be simply referred to as "ionic lipid (2)" in the present specification) include the following ionic lipids. Ionic lipids (2), wherein X represents a dialkylamino group (the carbon number of the two alkyl groups of the dialkylamino group is independently 1 to 8), a 3- to 6-membered cyclic amino group which may have a hetero atom, or -N(R a )-R b , R a represents an alkyl group having 1 to 8 carbon atoms, R b Represents -(CH2)qOR c , R c represents hydrogen or an alkyl group having 1 to 8 carbon atoms, q is an integer from 2 to 4; R 1 is an alkylene group having 6 or less carbon atoms or an alkenylene group having 6 or less carbon atoms; L 1 is an ester bond, an amide bond, a carbamate bond or a carbonate bond, K is 0 or 1; R x and R y Each independently represents an alkylene group having 2 to 4 carbon atoms; L 2 is an ester bond or an amide bond; R 2 is an alkylene group having 6 or less carbon atoms or does not exist; Y is a group represented by formula (Y):

[0141] [Chemical formula 15]

[0142] (Where, R 3 represents an alkylene group having 6 or less carbon atoms, L 3 Represents ester bond, S 1represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R e -L a -R 7 ' or -R e -L a -Z'-L b -R 7 ', S 2 Indicates -R e '-L a -R 7 " or -R e '-L a -Z"-L b -R 7 ”, R e and R e ' each independently represents an alkylene group having 6 or less carbon atoms, L a Represents ester bond, L b Represents ester bond, l means 0 or 1, L x represents an ester bond or a carbonate bond, R 4 represents an alkylene group having 6 or less carbon atoms or does not exist, R 5 represents hydrogen or an alkyl group having 1 to 8 carbon atoms, S 3 Indicates -R 6 '-L 4 '-R 7 ”', m represents 0 or 1, R 6 and R 6 'Each independently represents an alkylene group having 6 or less carbon atoms or does not exist, L 4 and L 4 ' all represent ester bonds, R 7 , R 7 '、R 7 ” and R 7 "' each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms or -(CH2)pC(=O)-R f , R f represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, p is 2 or 3, and n represents 0 or 1. ); Z, Z' and Z" are each independently a group represented by formula (Z):

[0143] [Chemical formula 16]

[0144] (Where, t represents an integer of 0 or 1, u represents an integer from 0 to 2, v represents an integer from 0 to 2, v R 8 Each independently represents an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkyl-p-aminobenzenesulfonyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aryloxy group having 6 to 10 carbon atoms.).

[0145] Preferred specific examples of the ionic lipid (2) include the compounds described in Preparation Examples 1 to 46 described below, but the present invention should not be construed as being limited thereto. The compounds described in Preparation Examples 1 to 46 are referred to as Compounds 1 to 46, respectively.

[0146] The ionic lipid (2) is preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 19, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 34, 35, 36 and 42, and further preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 21, 22, 24, 31, 32, 34, 35 and 36, and most preferably compound 31 or compound 34.

[0147] [Table 2-1]

[0148] [Table 2-2]

[0149] [Table 2-3]

[0150] [Table 2-4]

[0151] [Table 2-5]

[0152] [Table 2-6]

[0153] Next, the method for producing the ionic lipid (2) is described. The ionic lipid (2) has a -SS- (disulfide) bond. Therefore, the method for producing the ionic lipid (2) includes: (A) Manufacturing X-(R 1 -L 1 ) k -R X -SH represents thiol and HS-R y -L 2 -R 2 -Y represented by the thiol, oxidizing and coupling it, and (B) A method of synthesizing the desired parts in sequence on a starting compound containing a -SS- bond to finally obtain an ionic lipid (2), etc. The method for producing the ionic lipid (2) is preferably the method (B).

[0154] Hereinafter, the method of (B) will be described, but the method for producing the ionic lipid (2) is not limited thereto.

[0155] The raw materials, reagents and compounds obtained in each step of the following production methods may form salts. Examples of such salts include the same salts as those in the above-mentioned compounds of the present invention.

[0156] When the compound obtained in each step is a free compound, it can be converted into a target salt by a known method. On the contrary, when the compound obtained in each step is a salt, it can be converted into a free form or other types of salts as the target by a known method.

[0157] The compound obtained in each step can be used in the next reaction after being kept in the reaction solution or obtained as a crude product. Alternatively, the compound obtained in each step can be appropriately purified by common purification methods such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, ion exchange chromatography, etc. from the reaction mixture.

[0158] When the compounds of the raw materials and reagents in each step are commercially available, the commercially available products may be used as they are.

[0159] In the reaction of each step, unless otherwise specified, the reaction time is usually 1 minute to 48 hours, preferably 10 minutes to 22 hours.

[0160] In the reaction of each step, unless otherwise specified, the reaction temperature is usually -78°C to 300°C, preferably -78°C to 150°C.

[0161] In the reaction of each step, unless otherwise specified, the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 8 equivalents, relative to the substrate. When the reagent is used as a catalyst, the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.4 equivalents, relative to the substrate. When the reagent also serves as a reaction solvent, the reagent is used in an amount of solvent.

[0162] In the reaction of each step, when not particularly specified, these reactions are carried out by dissolving or suspending in a solvent-free or appropriate solvent. As specific examples of solvents, the solvents described in the embodiments or methanol, ethanol, isopropanol, tert-butyl alcohol, tetrahydrofuran, toluene, cyclohexane, hexane, heptane, N,N-dimethylformamide, N-methylpyrrolidone, chloroform, dichloromethane, acetonitrile, dimethyl sulfoxide, ethyl acetate, acetone, water, etc. can be cited. The above solvents can be mixed and used in two or more in an appropriate ratio.

[0163] When a base is used in the reaction of each step, examples include the bases described in the Examples, or sodium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, sodium hydrogen carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), imidazole, piperidine, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, lithium hydride and the like.

[0164] When an acid or an acidic catalyst is used in the reaction of each step, examples include the acids and acidic catalysts described in the Examples, or hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, boron trifluoride diethyl ether complex, and the like.

[0165] In each step, the protection or deprotection reaction of the functional group is carried out according to known reagents and methods (for example, reagents and methods described in GREENE'S PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th edition, WILEY-INTERSCIENCEM) or the methods described in Examples.

[0166] Examples of the protecting group for a hydroxyl group such as an alcohol or a phenolic hydroxyl group include ether-type protecting groups such as tetrahydropyranyl ether, methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, tert-butyldimethylsilyl ether, and tert-butyldiphenylsilyl ether; carboxylate-type protecting groups such as acetate; sulfonate-type protecting groups such as methanesulfonate; and carbonate-type protecting groups such as tert-butyl carbonate.

[0167] The protective group can be removed by a known method such as a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, a trialkylsilyl halide, or a reduction method.

[0168] In each step, when a carbonation reaction or a carbamate reaction is performed, the reagents used include N,N'-disuccinimidyl carbonate (DSC), 4-nitrophenyl chloroformate (pNPCl), and a base (basic salts, organic bases, etc.). When a carbonation reaction is performed, an additive such as 4-dimethylaminopyridine (DMAP) may be further added.

[0169] In each step, when a mesylation reaction is performed, methanesulfonyl chloride and a base (basic salts, organic bases, etc.) are used as reagents.

[0170] In each step, when an esterification reaction, an amidation reaction or a ureidation reaction is performed, examples of the reagents used include: halogenated acyl bodies such as acid chloride, acid anhydrides, activated carboxylic acids such as active esters. Examples of carboxylic acid activators include: carbodiimide condensation agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), triazine condensation agents such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride N hydrate (DMT-MM), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), or combinations thereof. When a carbodiimide condensation agent is used, additives such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), and 4-dimethylaminopyridine (DMAP) may be further added to the reaction.

[0171] In each step, when a nucleophilic substitution reaction such as amination is performed, a nucleophilic reagent (amines, etc.) and a base (basic salts, organic bases, etc.) are used as reagents. An additive such as potassium iodide (KI) or tetrabutylammonium iodide (TBAI) may be further added to the reaction.

[0172] The ionic lipid (2) can be produced, for example, by the following production method: In addition, the salt of the ionic lipid (2) can be obtained by appropriately mixing it with an inorganic acid or an organic acid.

[0173] Method A: k = 1, l = 0, m = 0, n = 0, R x and R y When both are ethylene

[0174] [Chemical formula 17]

[0175] Method B: k = 1, l = 0, m = 1, n = 1, R x and R y When both are ethylene

[0176] [Chemical formula 18]

[0177] Method C: k = 1, l = 1, m = 0, n = 0, R x and R y All are ethylene, S 1 When hydrogen or alkyl

[0178] [Chemical formula 19]

[0179] Method D: k = 0, l = 0, m = 0, n = 0, R x and R y When both are ethylene

[0180] [Chemical formula 20]

[0181] In the above formula, F 1 ~F 10 Each independently represents a reactive functional group, P 1 ~P 4 Each independently represents a protecting group.

[0182] Specific production methods are described in Production Examples 1 to 46 described below. A person skilled in the art can appropriately select raw materials and carry out reactions according to the methods described in Production Examples 1 to 46 to produce the desired ionic lipid (2).

[0183] Phospholipids Phospholipids can be used as lipid membrane components of lipid nanoparticles. Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphatidylcholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycero-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), or lysates thereof. Specifically, there are: 1,2-didecanoyl-sn-glycero-3-phosphatidylcholine (DDPC), 1,2-Dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC), 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphatidylcholine (DLoPC), 1,2-Dierucyl-sn-glycero-3-phosphatidylcholine (DEPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphatidylcholine (MPPC), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (PSPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (SOPC) can be used, and those obtained by appropriately converting these PCs into PE, PS, PG, or PA can be used.

[0184] As the phospholipid used in the present invention, PC or PE is preferred, DOPC, DSPC, DEPC, POPC, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) are more preferred, and DOPC, DSPC and DEPC are particularly preferred.

[0185] Sterols Sterol can be used as a component for regulating the fluidity of the lipid membrane of lipid nanoparticles. Examples of sterol include cholesterol, lanosterol, phytosterol, yeast sterol, enol, streptosterol, stigmasterol, dihydrolanosterol and 7-dehydrocholesterol, preferably cholesterol, lanosterol, phytosterol, and more preferably cholesterol.

[0186] PEG lipids PEG lipids are used as stabilizers to coat the surface of lipid nanoparticles with hydrophilic polyethylene glycol (PEG) to inhibit aggregation between particles, or to inhibit interactions between biological components and particles when administered to a living body. The PEG region can have any molecular weight. In some embodiments, the PEG region has a molecular weight of 200 to 10,000 Da and can be a linear or branched chain.

[0187] Examples of PEG lipids include PEG-phospholipids, PEG-ceramides, PEG-diacylglycerols, and PEG-cholesterols. Preferred PEGs are diacylglycerol PEGs having a molecular weight of 1,000 to 10,000, and more preferably dimyristoylglycerol PEGs or distearoylglycerol PEGs having a molecular weight of 1,000 to 10,000.

[0188] Acidic buffer Buffers having a buffering effect in the acidic range can be used. Specifically, HCl / KCl buffer, p-toluenesulfonic acid / p-toluenesulfonic acid sodium salt buffer, tartaric acid / NaOH buffer, citric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / sodium formate buffer, citric acid / sodium citrate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1MNaCl buffer, phenylacetic acid / phenylacetic acid sodium salt buffer, acetic acid / sodium acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, sodium dimethylarsenate / HCl buffer, maleic acid HNa / NaOH buffer , maleic acid / Tris / NaOH buffer, phosphate buffer, KH2PO4 / NaOH buffer, imidazole / HCl buffer, s-trimethylpyridine (2,4,6-trimethylpyridine) / HCl buffer, triethanolamine HCl / NaOH buffer, 5,5-diethylbarbiturate sodium / HCl buffer, N-methylmorpholine / HCl buffer, sodium pyrophosphate / HCl buffer, MES buffer, malic acid buffer, ADA buffer, PIPES buffer, ACES buffer, HEPES, BES, Bis-Tris buffer, Bis-Tris propane buffer, anhydrous sodium carbonate buffer, glycylglycine buffer, MOPS, MOPSO, TES.

[0189] Preferably, the acidic buffer has a buffering effect at pH 1 to 6.5, more preferably at pH 3 to 6.5, and examples thereof include tartaric acid / NaOH buffer, citric acid / NaOH buffer, phthalic acid HK / HCl buffer, glycine / HCl buffer, trans-aconitic acid / NaOH buffer, formic acid / sodium formate buffer, citric acid / sodium citrate buffer, 3,3-dimethylglutaric acid / NaOH buffer, 3,3-dimethylglutaric acid / NaOH / 0.1M NaCl buffer, phenylacetic acid / phenylacetic acid sodium salt buffer, acetic acid / sodium acetate buffer, succinic acid / NaOH buffer, phthalic acid HK / NaOH buffer, sodium dimethylarsenate / HCl buffer, maleic acid HNa / NaOH buffer, maleic acid / Tris / NaOH buffer, phosphate buffer, KH2PO4 / NaOH buffer, MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, citric acid buffer, Bis-Tris buffer, glycylglycine buffer, etc., more preferably malic acid buffer or MES buffer.

[0190] Method for preparing lipid nanoparticles In the method of the present invention, a lipid nanoparticle suspension containing no nucleic acid is prepared by mixing an alcohol solution containing an ionic lipid, a sterol and a PEG lipid with an acidic buffer having a buffering effect at pH 1 to 6.5. In order to prepare lipid nanoparticles, any operation inducing organization can be used. As alcohol, for example, ethanol, tert-butyl alcohol, etc. can be mentioned.

[0191] Examples of the "organization-inducing procedure" for preparing lipid nanoparticles include: alcohol dilution method using a microchannel or vortex, simple hydration method, ultrasonic treatment, heating, vortexing, ether injection method, French pressing method, bile acid method, Ca 2+, etc. 2+The fusion method, freeze-thaw method, reverse phase evaporation method and other methods known per se are preferably alcohol dilution methods using microchannels or vortices, and more preferably alcohol dilution methods using microchannels. The particle preparation in the alcohol dilution method using microchannels can be carried out, for example, using NanoAssemblr (Precision NanoSystems). The prepared lipid nanoparticles can replace the buffer of the external aqueous phase by ultrafiltration, dialysis, dilution and other operations. In a preferred embodiment, after preparing the lipid nanoparticle suspension, the external aqueous phase can be replaced with other acidic buffers having a buffering effect at pH 1 to 6.5 (preferably pH 3 to 6.5) by ultrafiltration, dialysis, dilution and other operations. In addition, monosaccharides, sugar alcohols, disaccharides, oligosaccharides, polysaccharides and the like can be added to the lipid nanoparticle suspension. As a saccharide, a disaccharide is preferably used, and sucrose is more preferably used. The concentration of the saccharide is preferably 0 to 320 mg / mL as the final concentration, and more preferably 0 to 160 mg / mL.

[0192] Storage conditions of lipid nanoparticles Lipid nanoparticles not containing nucleic acids can be stored as liquids in the temperature range of 0°C to 50°C, or can be stored frozen in the temperature range of -80°C to 0°C. From the viewpoint of the stability of the constituent components, the storage temperature when stored as a liquid is preferably 0°C to 30°C, more preferably 0°C to 10°C, and most preferably 0°C to 5°C. From the viewpoint of stability of the constituent components, the storage temperature when frozen and stored is preferably -80°C to -10°C, more preferably -80°C to -20°C. When stored frozen, it is thawed at 0°C to 100°C before use. From the viewpoint of stability of the constituent components, the melting temperature is preferably 0°C to 95°C, more preferably 0°C to 50°C, further preferably 0°C to 30°C, and most preferably 0°C to 10°C. Freezing and thawing are preferably carried out at atmospheric pressure.

[0193] Nucleic acid addition steps The nucleic acid adding step is a step of preparing nucleic acid-encapsulating lipid nanoparticles by mixing the nucleic acid-free lipid nanoparticles prepared in the previous step with the aqueous solution containing nucleic acid without freeze-drying. It should be noted that "no freeze drying" means that the nucleic acid adding step is performed immediately after the preparation step of the nucleic acid-free lipid nanoparticles. The preparation step of the nucleic acid-free lipid nanoparticles includes: storing the nucleic acid-free lipid nanoparticles as a liquid, or freezing and storing the nucleic acid-free lipid nanoparticles and thawing them. Add an aqueous solution containing the following nucleic acid, the amount of nucleic acid in the aqueous solution of the nucleic acid is the ratio of the total lipid and nucleic acid contained in the lipid nanoparticles, for example, total lipid / nucleic acid=1-1000nmol / μg, preferably total lipid / nucleic acid=50-500nmol / μg, and the lipid nanoparticles encapsulating nucleic acid can be prepared by pipetting or vortex mixing. In addition, in order to improve the encapsulation rate of nucleic acid, alcohol can be added, and methanol, ethanol, n-butanol, tert-butanol can be used as alcohol, preferably ethanol. The concentration of alcohol in the aqueous solution containing nucleic acid is preferably 0-50v / v%, more preferably 0-30v / v%, and further preferably 0-25v / v%. In addition, further in the nucleic acid addition step, in order to improve the nucleic acid encapsulation efficiency, the aqueous solution containing nucleic acid can be added to the liquid or frozen body-free lipid nanoparticles, and alcohol is further added to cultivate. The conditions of cultivation are, for example, 0-120 minutes at 0-100°C, preferably 0-60 minutes at 0-95°C. As the aqueous solution containing nucleic acid, it is preferred to use an acidic buffer containing nucleic acid. As the acidic buffer, a buffer having a buffering effect in an acidic range can be used, preferably an acidic buffer having a buffering effect at pH 1 to 6.5, more preferably at pH 3 to 6.5. Specifically, as the acidic buffer used in the preparation of lipid nanoparticles, the same buffer as exemplified can be cited, for example: MES buffer, malic acid buffer, phthalic acid buffer, maleic acid buffer, succinic acid buffer, tartaric acid buffer, citric acid buffer, etc. can be cited.

[0194] Steps to replace the external aqueous phase with a neutral buffer By replacing the external aqueous phase of the obtained nucleic acid-encapsulating lipid nanoparticles with a neutral buffer in the nucleic acid adding step, nucleic acid-encapsulating lipid nanoparticles that can be used as a nucleic acid-transferring agent can be prepared. Examples of a method for replacing the external aqueous phase with a neutral buffer include a method using dialysis, ultrafiltration or dilution. Examples of the neutral buffer include phosphate buffered saline (PBS), Tris-HCl buffer, ADA, PIPES, PIPES sesquisodium, ACES, MOPS, BES, MOPSO, TES, HEPES, TAPSO, POPSO, and HEPSO. The pH of the neutral buffer is 6 to 8.

[0195] By contacting the lipid nanoparticles of the encapsulated nucleic acid prepared in the method of the present invention with cells, the nucleic acid can be imported into the cell in vivo and / or in vitro. Therefore, the present invention provides a method for importing the nucleic acid into the cell in vivo and / or in vitro.

[0196] By the method of the present invention, any nucleic acid can be introduced into cells. As the type of nucleic acid, there can be mentioned: DNA, RNA, chimeric nucleic acid of RNA, DNA / RNA hybrid, etc., but it is not limited thereto. In addition, the nucleic acid can use any one of 1 to 3 chains, but is preferably single-stranded or double-stranded. The nucleic acid can be other types of nucleotides as N-glucosides of purine or pyrimidine bases, or other oligomers with non-nucleotide backbones (for example, commercially available peptide nucleic acids (PNA), etc.), or other oligomers with special bonds (wherein the oligomer contains base pairing found in DNA or RNA, nucleotides with a configuration that allows base attachment), etc. Furthermore, the nucleic acid may be, for example, a nucleic acid with a known modification, a nucleic acid with a label known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are substituted with an analog, a nucleic acid modified with an intramolecular nucleotide, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., a nuclease, a nuclease inhibitor, a toxin, an antibody, a signal peptide, poly-L-lysine, etc.), a sugar (e.g., a monosaccharide, etc.), a nucleic acid having an intercalating compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelate (e.g., a metal, a radioactive metal, boron, an oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., an α-isomer nucleic acid, etc.), and the like.

[0197] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. For example, plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligonucleotide, etc. can be mentioned, preferably plasmid DNA, cDNA, antisense DNA, and more preferably plasmid DNA. Circular DNA such as plasmid DNA can be digested by appropriate restriction endonucleases, etc., and can also be used as linear DNA.

[0198] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. can be mentioned, preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.

[0199] The nucleic acid used in the present invention is preferably purified by a method commonly used by those skilled in the art.

[0200] For the purpose of, for example, the prevention and / or treatment of a disease, the lipid nanoparticles of the encapsulated nucleic acid produced by the method of the present invention can be administered into a living body (in vivo). Therefore, the nucleic acid used in the present invention is preferably a nucleic acid (preventive / therapeutic nucleic acid) that has preventive and / or therapeutic activity for a certain specified disease. As such nucleic acid, for example, nucleic acids that can be used for gene therapy, etc. can be cited.

[0201] The lipid nanoparticles encapsulating nucleic acids produced by the method of the present invention can be used as a drug delivery system for selectively delivering nucleic acids, etc., into specific cells, and can be used, for example, for DNA vaccines based on the introduction of antigen genes into dendritic cells, gene therapy drugs for tumors, nucleic acid drugs that inhibit the expression of target genes using RNA interference, etc. Therefore, the present invention provides a method for producing a pharmaceutical composition.

[0202] The particle size of the lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. The particle size can be measured, for example, using a particle size distribution measuring device such as Zetasizer Nano (Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted by the method for producing the lipid nanoparticles.

[0203] The surface potential (zeta potential) of the lipid nanoparticles encapsulating nucleic acids is not particularly limited, but is preferably -15 to +15 mV, more preferably -10 to +10 mV. In previous gene introductions, particles with positively charged surface potential were mainly used. This is useful as a method for promoting electrostatic interaction with heparin sulfate on the cell surface with a negative charge, thereby promoting cell uptake, but it is possible that the positive surface charge interacts with the delivered nucleic acid in the cell and inhibits the release of nucleic acid from the vector, or the interaction between mRNA and the delivered nucleic acid and inhibits protein synthesis. This problem can be solved by adjusting the surface charge to the above range. The measurement of the surface charge can be carried out using a zeta potential measuring device such as ZetasizerNano, for example. The surface charge of lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.

[0204] The following specifically describes the step of bringing lipid nanoparticles encapsulating nucleic acid into contact with cells in vitro.

[0205] The cells are suspended in an appropriate culture medium several days before contacting the cells with the lipid nanoparticles and cultured under appropriate conditions. When contacting the cells with the lipid nanoparticles, the cells may or may not be in the proliferation phase.

[0206] The culture fluid during the contact can be a serum-containing culture medium or a serum-free culture medium, but the serum concentration in the culture medium is preferably below 30% by weight, more preferably below 20% by weight. When the culture medium includes excessive serum or other proteins, it is possible to hinder the contact of the lipid nanoparticle with the cell.

[0207] The cell density during contact is not particularly limited, but can be appropriately set in consideration of the type of cells, etc., and is generally 1×10 4 ~1×10 7 cells / mL range.

[0208] In the cell prepared in this way, for example, a lipid nanoparticle suspension encapsulated with the above-mentioned nucleic acid is added. The amount of the suspension added is not particularly limited, and can be appropriately set in consideration of the number of cells, etc. The concentration of the lipid nanoparticles when contacting the cells is not particularly limited as long as the target nucleic acid can be introduced into the cell, but as a lipid concentration, it is usually 1 to 300 nmol / mL, preferably 10 to 200 nmol / mL, and as a nucleic acid concentration, it is usually 0.01 to 100 μg / mL, preferably 0.05 to 10 μg / mL.

[0209] After the above suspension is added to the cells, the cells are cultured. The temperature, humidity, CO2 concentration, etc. during the culture are appropriately set in consideration of the type of cells. When the cells are mammalian cells, usually, the temperature is about 37°C, the humidity is about 95%, and the CO2 concentration is about 5%. In addition, the culture time can also be appropriately set in consideration of the conditions such as the type of cells used, but is usually within the range of 0.1 to 96 hours, preferably within the range of 0.2 to 72 hours, and more preferably within the range of 0.5 to 48 hours. When the above culture time is too short, the nucleic acid cannot be fully introduced into the cells, and when the culture time is too long, the cells sometimes become weak.

[0210] The nucleic acid is introduced into the cells by the above culture, but it is preferred to replace the medium with a fresh medium or add a fresh medium to the medium and continue the culture. When the cells are mammalian-derived cells, the fresh medium preferably contains serum or nutritional factors.

[0211] In addition, as mentioned above, by using the lipid nanoparticles encapsulated with nucleic acid, not only in vitro (invitro), but also in vivo (in vivo) can nucleic acid be introduced into cells. That is, by giving the lipid nanoparticles encapsulated with nucleic acid to the object, the lipid nanoparticles can be made to reach and contact the target cell, thereby in vivo the nucleic acid encapsulated in the lipid nanoparticles is introduced into the cell. As the object that can give the lipid nanoparticles, there is no particular limitation, for example, can be enumerated: vertebrates such as mammals (for example, people, monkeys, mice, rats, hamsters, cattle, etc.), birds (for example, chickens, ostriches, etc.), amphibians (for example, frogs, etc.), fish (for example, zebrafish, medaka, etc.), invertebrates such as insects (for example, silkworms, moths, fruit flies, etc.), plants, etc. As the object of giving the lipid nanoparticles encapsulated with nucleic acid, preferably people or other mammals.

[0212] The type of target cells is not particularly limited. By using lipid nanoparticles encapsulating nucleic acids, nucleic acids can be introduced into cells in various tissues (e.g., liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0213] As a method of administering lipid nanoparticles into a subject (e.g., vertebrates, invertebrates, etc.) into which nucleic acids and / or compounds other than nucleic acids are introduced, there is no particular limitation as long as the lipid nanoparticles can reach and contact target cells, thereby introducing the compound introduced into the lipid nanoparticles into the cells. Considering the type of the introduced compound, the type and site of the target cell, etc., a known administration method (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spraying, etc.) can be appropriately selected. The administration amount of the lipid nanoparticles is not particularly limited as long as it is within the range that can achieve the introduction of the compound into the cell, and can be appropriately selected considering the type of the administration subject, the administration method, the type of the introduced compound, the type and site of the target cell, etc.

[0214] When lipid nanoparticles encapsulating nucleic acid are used as the nucleic acid-transferring agent, the nucleic acid-transferring agent can be formulated according to conventional methods.

[0215] When providing the nucleic acid introduction agent as a research reagent, the nucleic acid introduction agent of the present invention can be provided as a sterile solution or suspension formed by using the lipid nanoparticles encapsulating the nucleic acid directly, or with water or other physiologically acceptable liquids (e.g., water-soluble solvents (e.g., malic acid buffer, etc.), organic solvents (e.g., ethanol, methanol, DMSO, tert-butyl alcohol, etc.), or a mixture of a water-soluble solvent and an organic solvent, etc.). The nucleic acid introduction agent of the present invention can appropriately contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0216] In addition, when the nucleic acid introduction agent is provided as a drug, the nucleic acid introduction agent of the present invention can be manufactured as an oral agent (e.g., tablets, capsules, etc.) or a non-oral agent (e.g., injections, sprays, etc.) by using the lipid nanoparticles encapsulating the nucleic acid directly or using them together with known pharmaceutically acceptable additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.), and mixing them in the unit dosage form required for conventionally recognized formulation implementations. It is preferably manufactured as a non-oral agent (more preferably an injection).

[0217] The nucleic acid-transfer agent of the present invention can be used as a preparation for children in addition to adults. Example

[0218] Hereinafter, embodiments of the present invention will be described in more detail, but the present invention is not limited to these embodiments.

[0219] In the following examples, the ionic lipids represented by formula (1) or formula (2) are shown by the names described in the above table. Sometimes, the lipid nanoparticles encapsulating nucleic acids are referred to as "nanoparticles encapsulating nucleic acids". In addition, the meanings of the abbreviations used in the following examples are as follows. Chol: Cholesterol DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (number average molecular weight (Mn) of PEG: 2000) DOPC: 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine DSPC: 1,2-distearoyl-sn-glycero-3-phosphatidylcholine DEPC: 1,2-dierucyl-sn-glycero-3-phosphatidylcholine MES: 2-morpholineethanesulfonic acid PBS: Phosphate buffered saline DDW: Deionized distilled water DCM: dichloromethane THF: Tetrahydrofuran IPA: 2-propanol DHP: 3,4-dihydro-2H-pyran PPTS: Pyridinium p-toluenesulfonate THP: 2-Tetrahydropyran DMAP: 4-dimethylaminopyridine DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride N-hydrate DSC: Bis(N-succinimidyl) carbonate EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride TEA: triethylamine TBAI: Tetrabutylammonium iodide DIPEA: N,N-diisopropylethylamine NPM: 4-phenylmorpholine MsCl: Methanesulfonyl chloride Ms:Methylsulfonyl pNPCl: 4-nitrophenyl chloroformate

[0220] Example 1 Effect of buffer SS-OP and MC3 were used as ionic lipids. When SS-OP was used as an ionic lipid, the lipid composition was SS-OP: DOPC: Chol: DMG-PEG2000 = 52.5: 7.5: 40: 1.5 in terms of molar ratio, and when MC3 was used as an ionic lipid, the lipid composition was MC3: DSPC: Chol: DMG-PEG2000 = 50: 10: 38.5: 1.5 in terms of molar ratio.

[0221] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 5) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 5), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 5) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0222] The particle solution (total lipid 20mM, 15μL) was diluted and mixed with a buffer (nucleic acid concentration 1.5μg / 135μL, 135μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) of MES (pH5), phthalic acid (pH5), maleic acid (pH5), succinic acid (pH5), DL-malic acid (pH5), DL-tartaric acid (pH5) or citric acid (pH5) under stirring conditions using a vortex mixer, and incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0223] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated in all buffers of MES, phthalic acid, maleic acid, succinic acid, DL-malic acid, DL-tartaric acid, or citric acid, and no difference in encapsulation efficiency was observed (see Table 3, Figure 1 and Figure 2). The particle size is expressed by Z-Ave (Z-average) and Number Mean (number mean) (the same applies hereinafter).

[0224] [Table 3]

[0225] Example 2 Effect of sucrose concentration and incubation temperature SS-OP was used as the ionic lipid, and the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000=52.5:7.5:40:1.5 in terms of molar ratio.

[0226] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution or DNase / RNase-free distilled water was added to a final sucrose concentration of 0 mg / mL, 160 mg / mL, or 320 mg / mL to prepare a particle solution.

[0227] A solution obtained by storing a particle solution (total lipid 20 mM, 15 μL) at 4°C (hereinafter also referred to as a liquid preparation) or a solution obtained by freezing a particle solution at -80°C and thawing it at 4°C (hereinafter also referred to as a freeze-thaw particle) was diluted and mixed with a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) under stirring conditions using a vortex mixer, and for each mixture of sucrose concentration, the liquid sample was incubated at 95°C, 75°C, 55°C, 35°C, and room temperature for 5 minutes using an aluminum block type incubator. Among them, the mixture with a sucrose concentration of 160 mg / mL was further incubated on ice for 5 minutes to obtain a sample. The freeze-thaw particle was incubated at 95°C, 35°C, and room temperature for 5 minutes. 150 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acid.

[0228] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulating nanoparticles were analyzed. The particle size and PdI were measured by the dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated at any incubation temperature, and no difference in encapsulation efficiency was observed. In addition, mRNA was encapsulated at any sucrose concentration, and no difference in encapsulation efficiency was observed. The particle size of the obtained nucleic acid-encapsulating nanoparticles incubated at 95°C was larger, and no difference in particle size was observed at other temperatures (75°C, 55°C, 35°C or room temperature) (see Table 4, Figure 3 and Figure 4 ).

[0229] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the nucleic acid encapsulated particles added to the culture medium was 0.02 μg / 4 μL. After 16 hours, the amount of luciferase protein introduced was evaluated using the luminescence. Add 100 μL of Steady-Glo (registered trademark), shake for 5 minutes, dissolve the cells, and quantify the luminescence using a GloMax 20 / 20 luminescence detector. As a result, the introduction of luciferase protein was confirmed at any incubation temperature, and no difference in in vitro activity was observed. In addition, higher activity was observed in the freeze-thaw particles compared to the liquid preparation (refer to Figure 5 ).

[0230] [Table 4]

[0231] Example 3 Effect of incubation time SS-OP was used as the ionic lipid, and the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000=52.5:7.5:40:1.5 in terms of molar ratio.

[0232] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0233] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 1.5μg / 135μL, buffer condition is MES (pH6), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and incubated at room temperature for 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes or 1 minute using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids. Alternatively, for comparison, 150μL of PBS was first added to the particle solution (total lipid 20mM, 15μL), and then mRNA encoding luciferase (same as above) was added to a final concentration of 1.5μg / 300μL.

[0234] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, all mRNAs were encapsulated at any incubation time, and no difference in encapsulation efficiency was observed. In addition, a solution in which PBS was first added to the particle solution (PBS→mRNA) resulted in a lower encapsulation efficiency (refer to Figure 6 and Figure 7 ).

[0235] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the obtained nucleic acid-encapsulated particles added to the culture medium was 0.02 μg / 4 μL. Luciferin was added to the culture medium to 0.1 mM, and the luminescence was used to evaluate the amount of luciferase protein introduced over time. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in an incubation-type luminescence detector (Kronos), and the cumulative luminescence was measured for 2 minutes every 1 hour. As a result, the nucleic acid introduction efficiency did not change with the incubation time. In addition, in the solution in which PBS was first added to the particle solution (PBS→mRNA), the nucleic acid introduction efficiency was low (refer to Figure 8 and Fig. 9 ).

[0236] Example 4 Effect of pH As the ionic lipid, SS-OP, SS-OC, MC3 or DODAP was used. Furthermore, when SS-OP or SS-OC was used, DOPC, Chol and DMG-PEG2000 were used as other lipids, and when MC3 or DODAP was used, DSPC, Chol and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP or SS-OC:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP or SS-OC, DOPC and Chol. In addition, the molar ratio of MC3 or DODAP:DSPC:Chol:DMG-PEG2000 used was 50:10:38.5:1.5.

[0237] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) at a flow ratio of 7:1 of acidic buffer solution: lipid ethanol solution, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15, ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7), and concentrated to about 500 μL by ultrafiltration again under centrifugal conditions (25°C, 1000 g, 5 minutes). After performing the operation more than 2 times, the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0238] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 1.5μg / 135μL, buffer condition MES (pH6), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and the mixtures of each pH and ionic lipid combination were incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0239] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulating nanoparticles were analyzed. The particle size and PdI were measured by the dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, SS-OP and MC3 were encapsulated up to pH 6, and the encapsulation efficiency decreased at pH 6.5 and pH 7. On the other hand, SS-OC and DODAP were encapsulated up to pH 5.5, and the encapsulation efficiency decreased at pH 6, pH 6.5, and pH 7 (refer to Fig.10 and Fig.11 ).

[0240] Example 5 Effect of Salt Concentration SS-OP and MC3 were used as ionic lipids. When SS-OP was used as an ionic lipid, the lipid composition was SS-OP: DOPC: Chol: DMG-PEG2000 = 52.5: 7.5: 40: 1.5 in terms of molar ratio, and when MC3 was used as an ionic lipid, the lipid composition was MC3: DSPC: Chol: DMG-PEG2000 = 50: 10: 38.5: 1.5 in terms of molar ratio.

[0241] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0242] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 1.5μg / 135μL, buffer conditions are MES (pH6) containing NaCl of 0mM, 1mM, 10mM, 30mM, 50mM, 100mM, 150mM or 300mM, 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and the mixtures of each salt concentration and ionic lipid combination were incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0243] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, the mRNA encapsulation efficiency decreased as the salt concentration of the buffer increased (refer to Fig.12 and Fig.13 ).

[0244] Example 6: Effect of mixing ratio and mixing method SS-OP was used as the ionic lipid, and the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000=52.5:7.5:40:1.5 in terms of molar ratio.

[0245] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0246] The particle solution or the solution obtained by diluting the particle solution with MES (pH 6) buffer (lipid concentration 300 nmol / 15 μL, 300 nmol / 75 μL or 300 nmol / 135 μL) and the MES (pH 6) buffer (nucleic acid concentration 3 μg / 135 μL, 3 μg / 75 μL or 3 μg / 15 μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) were diluted and mixed under stirring conditions using a vortex mixer or by tapping after pipetting, and incubated at 37° C. for 5 minutes using an aluminum block type incubator. 150 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0247] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by a dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by a Ribogreen (registered trademark) assay. As a result, under stirring conditions using a vortex mixer, a high encapsulation efficiency was obtained at any mixing ratio, but when mixing was performed by tapping after pipetting, the smaller the volume of the nucleic acid aqueous solution, the lower the encapsulation efficiency (refer to Fig.19 , Fig. 20 and Fig.21 ).

[0248] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the nucleic acid encapsulated particles added to the culture medium was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated using the luminescence. 100 μL of Steady-Glo (registered trademark) was added, shaken for 5 minutes, the cells were dissolved, and the luminescence was quantified using a GloMax 20 / 20 luminescence detector. As a result, no difference in in vitro activity was observed under stirring conditions using a vortex mixer, mixing was performed by tapping after pipetting, and the in vitro activity decreased when the volume of the nucleic acid aqueous solution was small (reference Fig.21 ).

[0249] Example 7 Effect of Buffer SS-OP and MC3 were used as ionic lipids. When SS-OP was used as an ionic lipid, the lipid composition was SS-OP: DOPC: Chol: DMG-PEG2000 = 52.5: 7.5: 40: 1.5 in terms of molar ratio, and when MC3 was used as an ionic lipid, the lipid composition was MC3: DSPC: Chol: DMG-PEG2000 = 50: 10: 38.5: 1.5 in terms of molar ratio.

[0250] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 5) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 5), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 5) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0251] The particle solution (total lipid 20mM, 15μL) was diluted and mixed with a buffer (nucleic acid concentration 3μg / 135μL, 135μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) of MES (pH5), phthalic acid (pH5), maleic acid (pH5), succinic acid (pH5), DL-malic acid (pH5), DL-tartaric acid (pH5) or citric acid (pH5) under stirring conditions using a vortex mixer, and incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0252] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nanoparticles encapsulating nucleic acid were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, mRNA was encapsulated in all buffers of MES, phthalic acid, maleic acid, succinic acid, DL-malic acid, DL-tartaric acid, or citric acid, and no difference in encapsulation efficiency was observed (refer to Fig. 22 and Fig.23 ).

[0253] Example 8 Effect of Cultivation Temperature SS-OP was used as the ionic lipid, and the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000=52.5:7.5:40:1.5 in terms of molar ratio.

[0254] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution or DNase / RNase-free distilled water was added to a final sucrose concentration of 0 mg / mL, 160 mg / mL, or 320 mg / mL to prepare a particle solution.

[0255] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer condition MES (pH5), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and incubated at 95°C, 75°C, 55°C or 37°C for 5 minutes using an aluminum block incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0256] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulating nanoparticles were analyzed. The particle size and PdI were measured by the dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, the mRNA was encapsulated at any incubation temperature, and no difference in encapsulation efficiency was observed. It was observed that the particle size of the obtained nucleic acid-encapsulating nanoparticles tended to be larger as the temperature was increased (refer to Fig.24 and Fig.25 ).

[0257] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the nucleic acid encapsulated particles added to the culture medium was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated using the luminescence. 100 μL of Steady-Glo (registered trademark) was added, shaken for 5 minutes, the cells were dissolved, and the luminescence was quantified using a GloMax20 / 20 luminescence detector. As a result, the introduction of luciferase protein was confirmed at any incubation temperature, and no difference in in vitro activity was observed (refer to Fig.26 ).

[0258] Example 9 Effect of incubation time SS-OP was used as the ionic lipid, and the lipid composition was SS-OP:DOPC:Chol:DMG-PEG2000=52.5:7.5:40:1.5 in terms of molar ratio.

[0259] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0260] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer condition is MES (pH6), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and incubated at room temperature for 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes or 1 minute using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids. Alternatively, for comparison, 150μL of PBS was first added to the particle solution (total lipid 20mM, 15μL), and then mRNA encoding luciferase (same as above) was added to a final concentration of 1.5μg / 300μL.

[0261] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by dynamic light scattering using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, all mRNAs were encapsulated at any incubation time, and no difference in encapsulation efficiency was observed. In addition, a solution in which PBS was first added to the particle solution (PBS→mRNA) resulted in a lower encapsulation efficiency (refer to Fig. 27 and Fig.28 ).

[0262] The obtained nucleic acid-encapsulated nanoparticles were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the obtained nucleic acid-encapsulated particles added to the culture medium was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated using the luminescence. Add 100 μL of Steady-Glo (registered trademark), shake for 5 minutes, dissolve the cells, and quantify the luminescence using a GloMax 20 / 20 luminescence detector. As a result, the introduction of luciferase protein was confirmed at any incubation time, and no difference in in vitro activity was observed. In addition, in the solution in which PBS was first added to the particle solution (PBS→mRNA), a decrease in in vitro activity was observed (refer to Fig.29 ).

[0263] Example 10 Effect of pH As the ionic lipid, SS-OP, SS-OC, MC3 or DODAP was used. Furthermore, when SS-OP or SS-OC was used, DOPC, Chol and DMG-PEG2000 were used as other lipids, and when MC3 or DODAP was used, DSPC, Chol and DMG-PEG2000 were used as other lipids. The molar ratio of SS-OP or SS-OC:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG2000 was used relative to the total of SS-OP or SS-OC, DOPC and Chol. In addition, the molar ratio of MC3 or DODAP:DSPC:Chol:DMG-PEG2000 used was 50:10:38.5:1.5.

[0264] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) at a flow ratio of 7:1 of acidic buffer solution: lipid ethanol solution, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15, ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes), and concentrated to about 1500 μL. The concentrate was diluted to 15 mL using MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7), and concentrated to about 500 μL by ultrafiltration again under centrifugal conditions (25°C, 1000 g, 5 minutes). After performing the operation more than 2 times, the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0265] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer conditions MES (pH5, pH5.5, pH6, pH6.5 or pH7), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and the mixtures of each pH and ionic lipid combination were incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0266] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by the dynamic light scattering method using Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, SS-OP, SS-OC, MC3, and DODAP were all encapsulated up to pH 6, and the encapsulation efficiency decreased at pH 6.5 and pH 7 (refer to Fig.30 and Fig.31 ).

[0267] The obtained nucleic acid encapsulating nanoparticles were made using SS-OP and acted on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were cultured in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at a rate of 5×10 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the nucleic acid encapsulated particles added to the culture medium was 0.02 μg / 4 μL. After 13 hours, the amount of luciferase protein introduced was evaluated using the luminescence. Add 100 μL of Steady-Glo (registered trademark), shake for 5 minutes, dissolve the cells, and quantify the luminescence using a GloMax 20 / 20 luminescence detector. As a result, the highest in vitro activity was shown at pH 6, the activity was slightly reduced at pH 6.5, and the others showed low activity at pH 5, pH 5.5, and pH 7 (refer to Fig.32 ).

[0268] The zeta potential of the particles prepared using SS-OP in the obtained nucleic acid-encapsulating nanoparticles was analyzed. The zeta potential was measured by electrophoretic light scattering using a Zetasizer (registered trademark) using MES buffer (pH 5, pH 5.5, pH 6, pH 6.5, or pH 7). As a result, the particles prepared at pH 5 and pH 5.5 showed higher values ​​in a low pH environment than the particles prepared at pH 6, pH 6.5, and pH 7 (refer to Fig.33 ).

[0269] Example 11 Effect of Salt Concentration SS-OP and MC3 were used as ionic lipids. When SS-OP was used as an ionic lipid, the lipid composition was SS-OP: DOPC: Chol: DMG-PEG2000 = 52.5: 7.5: 40: 1.5 in terms of molar ratio, and when MC3 was used as an ionic lipid, the lipid composition was MC3: DSPC: Chol: DMG-PEG2000 = 50: 10: 38.5: 1.5 in terms of molar ratio.

[0270] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0271] The particle solution (total lipid 20mM, 15μL) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer conditions are MES (pH6) containing NaCl of 0mM, 1mM, 10mM, 30mM, 50mM, 100mM, 150mM or 300mM, 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and the mixtures of each salt concentration and ionic lipid combination were incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0272] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulated nanoparticles were analyzed. The particle size and PdI were measured by a dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by a Ribogreen (registered trademark) assay. As a result, there was a tendency for the mRNA encapsulation efficiency to decrease as the salt concentration of the buffer increased (see Fig.34 and Fig.35 ).

[0273] Example 12 Effect of Ionic Lipid Type LN-81 (Compound 34 in the Production Example described below) was used as the ionic lipid. The lipid composition was LN-81:DEPC:Chol:DMG-PEG2000=52.5:17.5:30:1 and LN-81:DEPC:Chol:DMG-PEG2000=60:5:35:1 in terms of molar ratio.

[0274] 2400 μL of acidic malic acid buffer (20 mM, pH 5.0) and 800 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 3:1, a total flow rate of 4 mL / min, and a syringe holder temperature of 25°C. 3000 μL of MES buffer (pH 6.0) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6.0), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. The mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6.0) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0275] The liquid or frozen-thawed particles were diluted and mixed with a MES (pH 6) buffer (nucleic acid concentration 11.1 μg / 180 μL, 180 μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLucmRNA-(L-7602)) under stirring conditions using a vortex mixer, and incubated at 37°C for 5 minutes using an aluminum block incubator. 100 μL of the incubated solution was dispensed, and 1010 μL of PBS was added thereto to obtain nanoparticles encapsulating nucleic acids.

[0276] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nanoparticles of encapsulated nucleic acid were analyzed. The particle size and PdI were measured by the dynamic light scattering method using Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by Ribogreen (registered trademark) determination method. As a result, mRNA was encapsulated (see Table 5).

[0277] [Table 5]

[0278] Comparative Example 1 Preparation method of lyophilized material SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0279] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. Sucrose solution was added to a final concentration of 160 mg / mL. The obtained solution was added to a sample vial (Mighty Vial) at a volume of 200 μL each, and freeze-dried to obtain a lyophilized product.

[0280] The lyophilized product was dissolved in a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 2 μg / 200 μL, dissolved in DDW). The mixture was incubated at 95°C for 5 minutes using an aluminum block incubator. 200 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acid.

[0281] Comparative Example 2 Preparation Method of Lyophilized Material SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0282] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. Sucrose solution was added to a final concentration of 160 mg / mL. The obtained solution was added to a sample vial (Mighty Vial) at a volume of 200 μL each, and freeze-dried to obtain a lyophilized product.

[0283] The lyophilized product was dissolved in a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 4 μg / 200 μL, dissolved in DDW). The mixture was incubated at 95°C for 5 minutes using an aluminum block incubator. 200 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acid.

[0284] Comparative Example 3 Preparation method of lyophilized product SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0285] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) at a flow ratio of 7:1 of acidic buffer solution: lipid ethanol solution, a total flow rate of 16 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. Sucrose solution was added to a final concentration of 160 mg / mL. The obtained solution was added to a sample vial (Mighty Vial) at a volume of 200 μL each, and freeze-dried to obtain a lyophilized product.

[0286] The lyophilized product was dissolved in a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 4 μg / 200 μL, dissolved in DDW). The mixture was incubated at 75°C for 5 minutes using an aluminum block incubator. 200 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acid.

[0287] Test Example 1 In vitro activity SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0288] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0289] A solution (liquid preparation) obtained by storing a particle solution (total lipid 20 mM, 15 μL) at 4°C or a solution (freeze-thaw particles) obtained by freezing a particle solution at -80°C and thawing it at 4°C was diluted and mixed with a solution (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) under stirring conditions using a vortex mixer, and incubated at 95°C for 5 minutes using an aluminum block type incubator. 150 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0290] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid encapsulating nanoparticles or the nucleic acid encapsulating nanoparticles prepared using the freeze-drying technique in Comparative Example 1 were analyzed. The particle size and PdI were measured by a dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by a Ribogreen (registered trademark) assay. As a result, in the liquid preparation and the solution obtained by freeze-thawing, the same mRNA encapsulation efficiency as that of the nucleic acid encapsulating nanoparticles using the freeze-drying technique was obtained. In addition, compared with the nucleic acid encapsulating nanoparticles using the freeze-drying technique, the particle size of the obtained nucleic acid encapsulating nanoparticles was smaller (refer to Fig.14 and Fig.15 ).

[0291] The obtained nucleic acid encapsulating nanoparticles or the nucleic acid encapsulating nanoparticles prepared by freeze-drying technology in Comparative Example 1 were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were incubated at 5×10 4% in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution). 3 Cells / 100 μL were seeded in a 96-well transparent flat-bottom plate. The nucleic acid concentration of the nucleic acid encapsulated particles added to the culture medium was 0.02 μg / 4 μL. Luciferin was added to the culture medium to 0.1 mM, and the luminescence was used to evaluate the amount of luciferase protein introduced over time. HeLa cells cultured in a culture medium containing nucleic acid-encapsulated nanoparticles and luciferin were placed in a culture-type luminescence detector (Kronos), and the cumulative luminescence was measured for 2 minutes every 1 hour. As a result, a higher nucleic acid introduction efficiency was obtained in the liquid preparation and the solution obtained by freeze-thawing compared to the nucleic acid-encapsulated nanoparticles using freeze-drying technology (refer to Fig.16 and Fig.17 ).

[0292] Test Example 2 In vivo activity SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0293] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0294] A solution (liquid preparation) obtained by storing a particle solution (total lipid 20 mM, 15 μL) at 4°C or a solution (freeze-thaw particles) obtained by freezing a particle solution at -80°C and thawing it at 4°C was diluted and mixed with a solution (nucleic acid concentration 1.5 μg / 135 μL, buffer condition MES (pH 5), 135 μL) containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) under stirring conditions using a vortex mixer, and incubated at 95°C for 5 minutes using an aluminum block type incubator. 150 μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0295] The obtained nucleic acid-encapsulating nanoparticles or the nucleic acid-encapsulating nanoparticles prepared using freeze-drying technology in Comparative Example 1 were applied to mice (Balb / c, 6 weeks old, female), and the gene expression activity in the liver was evaluated using a GloMax 20 / 20 luminescence detector. Each nucleic acid-encapsulating nanoparticle was administered to the mouse tail vein at 0.05 mg / kg. Six hours after administration, the liver was removed and the expression of luciferase protein was evaluated. As a result, higher gene expression activity in the liver was observed in the liquid and the solution obtained by freeze-thawing compared to the nucleic acid-encapsulating nanoparticles using freeze-drying technology (refer to Fig.18 ).

[0296] Test Example 3 In vivo activity SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0297] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0298] The particle solution (total lipid 20mM, 15μL) (liquid) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer condition MES (pH6), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0299] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid encapsulating nanoparticles or the nucleic acid encapsulating nanoparticles prepared using the freeze-drying technique in Comparative Example 2 and Comparative Example 3 were analyzed. The particle size and PdI were measured by a dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, the same mRNA encapsulation efficiency as that of the nucleic acid encapsulating nanoparticles using the freeze-drying technique was obtained in the liquid. In addition, compared with the nucleic acid encapsulating nanoparticles using the freeze-drying technique, the particle size of the obtained nucleic acid encapsulating nanoparticles was smaller (refer to Fig.36 and Fig.37 ).exist Fig.36 , Fig.37 and Fig.38 In the figure, Comparative Example 2 in which the total flow rate during the preparation of lipid nanoparticles was 1 mL / min was indicated as "freeze drying (FR=1)", and Comparative Example 3 in which the total flow rate was 16 mL / min was indicated as "freeze drying (FR=16)".

[0300] The obtained nanoparticles encapsulating nucleic acid or the nanoparticles encapsulating nucleic acid produced by freeze-drying technology in Comparative Example 2 and Comparative Example 3 were applied to mice (Balb / c, 6 weeks old, female), and the gene expression activity in the liver was evaluated using the IVIS imaging system. Each nanoparticle encapsulating nucleic acid was administered to the mouse tail vein at 0.1 mg / kg. Six hours after administration, the expression level of luciferase protein was evaluated. As a result, higher gene expression activity in the liver was observed in the liquid preparation compared to the nanoparticles encapsulating nucleic acid using freeze-drying technology (refer to Fig.38 ).

[0301] Test Example 4 In vivo activity SS-OP was used as the ionic lipid, and DOPC, Chol, and DMG-PEG 2000 were used as other lipids. The molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40, and 1.5 mol% of DMG-PEG 2000 was used relative to the total of SS-OP, DOPC, and Chol.

[0302] 2100 μL of acidic malic acid buffer (20 mM, pH 3.0) and 300 μL of lipid ethanol solution were measured in syringes respectively. LNPs were prepared using NanoAssmblr (registered trademark) ultra-high-speed nanomedicine preparation device (manufactured by Precision NanoSystems) under the conditions of a flow ratio of acidic buffer solution: lipid ethanol solution of 7:1, a total flow rate of 1 mL / min, and a syringe holder temperature of 25°C. 9000 μL of MES buffer (pH 6) was added to the collection, and the resulting mixture was transferred to Amicon Ultra 15 and ultrafiltered under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 1500 μL. The resulting concentrate was diluted to 15 mL using MES buffer (pH 6), and ultrafiltered again under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 500 μL. This operation was repeated twice or more, and the entire mixture obtained by concentration was transferred to Amicon Ultra 4, and 3000 μL of MES buffer (pH 6) was further added, and then ultrafiltration was performed under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 100 μL. Sucrose solution was added to a final concentration of 160 mg / mL to prepare a particle solution.

[0303] The particle solution (total lipid 20mM, 15μL) (liquid) and a solution containing mRNA encoding luciferase (CleanCap (registered trademark) FLuc mRNA-(L-7602)) (nucleic acid concentration 3μg / 135μL, buffer condition MES (pH6), 135μL) were diluted and mixed under stirring conditions using a vortex mixer, and incubated at 37°C for 5 minutes using an aluminum block type incubator. 150μL of PBS was added to the incubated solution to obtain nanoparticles encapsulating nucleic acids.

[0304] The particle size, polydispersity index (PdI), and mRNA encapsulation efficiency of the obtained nucleic acid-encapsulating nanoparticles (liquid) or the nucleic acid-encapsulating nanoparticles (MF) prepared by microchannels (LNPs made with the same lipid composition using MES buffer (20 mM, pH 6.5) containing nucleic acids and an ethanol solution of lipids) were analyzed. The particle size and PdI were measured by a dynamic light scattering method using a Zetasizer (registered trademark). The mRNA encapsulation efficiency was measured by the Ribogreen (registered trademark) assay. As a result, the same mRNA encapsulation efficiency as that of the nucleic acid-encapsulating nanoparticles prepared by microchannels was obtained in the liquid (refer to Fig.39 ).

[0305] The obtained nucleic acid-encapsulating nanoparticles (liquid) or the nucleic acid-encapsulating nanoparticles (MF) prepared by microchannels were applied to mice (Balb / c, 6 weeks old, female), and the gene expression activity in each organ (liver, heart, spleen, kidney and lung) was evaluated using an IVIS imaging system. Each nucleic acid-encapsulating nanoparticle was administered to the mouse tail vein at 0.1 mg / kg. After 6 hours from administration, the expression of luciferase protein was evaluated after dissection, and photographs were taken. As a result, the same expression distribution was observed in the liquid compared with the nucleic acid-encapsulating nanoparticles prepared by microchannels (refer to Fig.40 and Fig.41 ).

[0306] Test Example 5 In vitro activity of the particles of Example 12 The nucleic acid encapsulating nanoparticles obtained in Example 12 or the nucleic acid encapsulating nanoparticles prepared by freeze-drying technology in Comparative Example 1 were allowed to act on HeLa cells to evaluate the efficiency of nucleic acid introduction. HeLa cells were incubated at 3×10 4% in D-MEM medium (high glucose) (containing L-glutamine and phenol red) (supplemented with 10% FBS and 1% penicillin-streptomycin solution). 4 Cells / 600 μL were seeded in a 48-well transparent flat-bottom plate. The next day, potassium luciferin was added to the culture medium, and the nucleic acid concentration of the obtained nucleic acid-encapsulated particles added to the culture medium was 0.12 μg / 60 μL, and the amount of luciferase protein introduced for 24 hours was evaluated using Kronos HT. As a result, compared with the nucleic acid-encapsulated nanoparticles using freeze-drying technology, the liquid and freeze-thaw particles achieved higher nucleic acid introduction efficiency. In addition, when the liquid was compared with the freeze-thaw particles, the freeze-thaw particles had a higher nucleic acid introduction efficiency. On the other hand, as a composition, the liquid prepared with LN-81:DEPC:Chol:DMG-PEG2000=52.5:17.5:30:1 showed the highest nucleic acid introduction efficiency (refer to Fig.42 ).

[0307] Production of ionic lipids (2) Table 6 shows the names and structures of the ionic lipids produced in the following production examples.

[0308] [Table 6-1]

[0309] [Table 6-2]

[0310] [Table 6-3]

[0311] [Table 6-4]

[0312] [Table 6-5]

[0313] [Table 6-6]

[0314] [Table 6-7]

[0315] [Table 6-8]

[0316] [Table 6-9]

[0317] [Table 6-10]

[0318] [Table 6-11]

[0319] [Table 6-12]

[0320] [Table 6-13]

[0321] [Production Example 1] Synthesis of Compound 1 Compound 1 was produced by the following synthetic route, but the present invention is not limited to this synthetic route.

[0322] [Chemical formula 21]

[0323] <Synthesis of Intermediate 1> 4-Hydroxyphenylacetic acid (75.0 g) and PPTS (12.4 g) were dissolved in 400 g of dichloromethane at room temperature, and the obtained solution was cooled to 10-20°C. A solution obtained by dissolving DHP (207 g) in 100 g of dichloromethane was added dropwise thereto, and the mixture was reacted at 25°C for 2 hours. The reaction solution was cooled to 10-20°C, and DMAP (30.1 g) was added to quench the mixture. 590 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10-20°C. A solution of 98.8 g of 400 g / L sodium hydroxide aqueous solution and 307 g of ion exchange water was added dropwise to the cooled solution, and the mixture was reacted at 25°C for 2 hours. The reaction solution was concentrated with an evaporator, and dichloromethane and 2-propanol were distilled off. The obtained concentrate was washed twice with 750 g of chloroform, and 6N hydrochloric acid was added to obtain a solution with a pH of 5.0. The obtained solution was extracted twice with 750 g of chloroform, and then 75.0 g of sodium sulfate was added to dehydrate the organic layer. After the sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 98.2 g of Intermediate 1.

[0324] <Synthesis of Intermediate 2> Intermediate 1 (10.2 g), bis(2-hydroxyethyl) disulfide (9.99 g) and DMAP (1.06 g) were dissolved in 153 g of chloroform at room temperature. EDC (12.4 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 102 g of 20% by weight brine, and then 5.1 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator, and the obtained crude product was purified by a chromatographic column to obtain 9.01 g of Intermediate 2.

[0325] <Synthesis of Intermediate 3-a> At room temperature, intermediate 2 (350 mg), dimethylglycine hydrochloride (144 mg), triethylamine (143 mg) and DMAP (23.0 mg) were dissolved in chloroform 5.25 mL. EDC (270 mg) was added to the obtained solution and reacted at room temperature for 2 hours. The reaction solution was washed with 0.5 M phosphate buffer (pH 4.0) 3.5 g, 7 wt% sodium bicarbonate solution 3.5 g, and 20 wt% saline solution 3.5 g, and then sodium sulfate 175 mg was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator to obtain 378 mg of intermediate 3-a.

[0326] <Synthesis of Intermediate 4-a> After dissolving the intermediate 3-a (363 mg) in 3.27 g of THF, 13.1 g of 0.5 M phosphate buffer (pH 2.0) was added and reacted at room temperature for 2 hours. 5.45 g of chloroform was added to the reaction solution for washing. 1N sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 6.0. The pH-adjusted water layer was extracted with 5.45 g of chloroform. 180 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 187 mg of intermediate 4-a.

[0327] <Synthesis of Compound 1> Intermediate 4-a (187 mg), 2-hexyldecanoic acid (128 mg) and DMAP (12.0 mg) were dissolved in 2.81 g of chloroform at room temperature. EDC (144 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 1.87 g of 20% by weight saline, and then 93.0 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator, and the crude product obtained was purified by a chromatographic column to obtain 159 mg of compound 1. <Compound 1 1 H-NMR (600MHz, CDCl3)> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.8 8-2.97(m, 4H), 3.19(s, 2H), 3.63(s, 2H), 4.34-4.42(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0328] [Production Example 2] Synthesis of Compound 2 <Synthesis of Compound 2> Compound 2 was synthesized by the same synthesis route as in Production Example 1 using intermediate 4-a and 2-decyldodecanoic acid. <Compound 2 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97(m, 4H), 3.19(s, 2H), 3.63(s, 2H), 4.34-4.42(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0329] [Production Example 3] Synthesis of Compound 3 <Synthesis of Compound 3> Compound 3 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-a and 2-tetradecylhexadecanoic acid. <Compound 3 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97(m, 4H), 3.19(s, 2H), 3.63(s, 2H), 4.34-4.42(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0330] [Production Example 4] Synthesis of Compound 4 <Synthesis of Intermediate 3-b> Intermediate 3-b represented by the following formula was synthesized in the same manner as in Preparation 1 except that 4-(dimethylamino)butyric acid hydrochloride was used.

[0331] [Chemical formula 22]

[0332] <Synthesis of Intermediate 4-b> Intermediate 4-b represented by the following formula was synthesized in the same manner as in Preparation 1 except that intermediate 3-b was used.

[0333] [Chemical formula 23]

[0334] <Synthesis of Compound 4> Compound 4 was synthesized in the same manner as in Preparation 1 except that intermediate 4-b was used. <Compound 4 1 H-NMR (600MHz, CDCl3)> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.82 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.5 2-2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0335] [Production Example 5] Synthesis of Compound 5 <Synthesis of Compound 5> Compound 5 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-b and 2-decyldodecanoic acid. <Compound 5 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0336] [Production Example 6] Synthesis of Compound 6 <Synthesis of Compound 6> Compound 6 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-b and 2-dodecyltetradecanoic acid. <Compound 6 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 42H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0337] [Production Example 7] Synthesis of Compound 7 <Synthesis of Compound 7> Compound 7 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-b and 2-tetradecylhexadecanoic acid. <Compound 7 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0338] [Production Example 8] Synthesis of Compound 8 <Synthesis of Compound 8> Compound 8 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-b and 2-hexadecyloctadecanoic acid. <Compound 8 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0339] [Production Example 9] Synthesis of Compound 9 <Synthesis of Compound 9> Compound 9 was synthesized by the same synthesis route as in Preparation Example 1 using intermediate 4-b and 2-octadecyleicosanoic acid. <Compound 9 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0340] [Chemical formula 24]

[0341] [Production Example 10] Synthesis of Compound 10 <Synthesis of Intermediate 5> Intermediate 2 (5.10 g), 4-bromobutyric acid (2.52 g) and DMAP (335 mg) were dissolved in 76.5 g of chloroform at room temperature. EDC (3.94 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 51.0 g of 20% by weight saline solution, and then 2.55 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 6.07 g of intermediate 5.

[0342] <Synthesis of Intermediate 6-a> Diethylamine (258 mg) was dissolved in THF 1.04 g at room temperature. A solution obtained by dissolving intermediate 5 (230 mg) in THF 780 mg was added dropwise thereto, and the mixture was reacted at 50° C. for 15 hours. 3.45 g of chloroform was added to the reaction solution, and then washed with 0.5 M acetic acid buffer (pH 4.0) 2.30 g, 7 wt % sodium bicarbonate solution 2.30 g, and 20 wt % saline 2.30 g in sequence. 130 mg of sodium sulfate was added for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 130 mg of intermediate 6-a.

[0343] <Synthesis of Intermediate 7-a> Intermediate 7-a was synthesized by the same method as Intermediate 4-a in Production Example 1 using Intermediate 6-a.

[0344] <Synthesis of Compound 10> Compound 10 was synthesized in the same manner as Compound 1 in Preparation Example 1 using Intermediate 7-a and 2-hexadecanoic acid. <Compound 10 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.88-2.97(m, 4H), 3.63(s, 2H), 4.28-4.38(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0345] [Production Example 11] Synthesis of Compound 11 <Synthesis of Intermediate 6-b> Intermediate 6-b represented by the following formula was synthesized in the same manner as in Production Example 10 except that piperidine was used.

[0346] [Chemical formula 25]

[0347] <Synthesis of Intermediate 7-b> Intermediate 7-b represented by the following formula was synthesized in the same manner as in Production Example 10 except that Intermediate 6-b was used.

[0348] [Chemical formula 26]

[0349] <Synthesis of Compound 11> Compound 11 was synthesized by the same synthesis route as in Preparation Example 10 using Intermediate 7-b and 2-hexadecanoic acid. <Compound 11 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 60H), 1.45-1.60 (m, 4H), 1.69-1.84 (m, 4H), 2.20-2.50 (m, 8H), 2.52-2 .59(m,1H),2.88-2.97(m,4H),3.63(s,2H),4.28-4.38(m,4H),7.02-7.05(m,2H),7.27-7.30(m,2H)

[0350] [Production Example 12] Synthesis of Compound 12 <Synthesis of Intermediate 6-c> Intermediate 6-c represented by the following formula was synthesized in the same manner as in Production Example 10 except that dipropylamine was used.

[0351] [Chemical formula 27]

[0352] <Synthesis of Intermediate 7-c> Intermediate 7-c represented by the following formula was synthesized in the same manner as in Production Example 10 except that Intermediate 6-c was used.

[0353] [Chemical formula 28]

[0354] <Synthesis of Compound 12> Compound 12 was synthesized by the same synthesis route as in Preparation Example 10 using Intermediate 7-c and 2-hexadecyloctadecanoic acid. <Compound 12 1 H-NMR (600MHz, CDCl3)> δ: 0.86-0.92 (m, 12H), 1.20-1.45 (m, 62H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.8 8-2.97(m, 4H), 3.63(s, 2H), 4.28-4.38(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0355] [Chemical formula 29]

[0356] [Production Example 13] Synthesis of Compound 13 <Synthesis of Compound 13> Compound 13 was synthesized according to the same synthetic route as in Preparation Example 8, except that Intermediate 2 and 3-(dimethylamino)-2-methylpropionic acid hydrochloride were used as starting materials. <Compound 13 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.13 (d, 3H), 1.20-1.45 (m, 58H), 1.68-1.78 (m, 2H), 2.18-2.22 (m, 7H), 2.52-2.7 0 (m, 3H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0357] [Chemical formula 30]

[0358] [Production Example 14] Synthesis of Compound 14 <Synthesis of Intermediate 8> 3,4-Dihydroxyphenylacetic acid (3.00 g) and PPTS (448 mg) were dissolved in 40.0 g of dichloromethane at room temperature, and the obtained solution was cooled to 20°C. DHP (7.50 g) was added dropwise thereto, and the mixture was reacted at room temperature for 2 hours. The reaction solution was cooled to 20°C, and DMAP (1.09 g) was added to quench the mixture. 47.0 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10 to 20°C. 30.0 g of 1N aqueous sodium hydroxide solution was added dropwise to the cooled solution, and the mixture was reacted at 25°C for 2 hours. The reaction solution was concentrated with an evaporator, and dichloromethane and 2-propanol were distilled off. The obtained concentrate was washed with 30.0 g of chloroform, and then 6N hydrochloric acid was added to obtain a solution with a pH of 5.0. The obtained solution was extracted with 30.0 g of chloroform, and 1.50 g of sodium sulfate was added to the organic layer for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 4.98 g of Intermediate 8.

[0359] <Synthesis of Intermediate 9> Intermediate 8 (800 mg), bis(2-hydroxyethyl) disulfide (550 mg) and DMAP (58.0 mg) were dissolved in 12.0 g of chloroform at room temperature. EDC (684 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 8.00 g of 20 wt % saline, and then 400 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator, and the obtained crude product was purified by a chromatographic column to obtain 530 mg of intermediate 9.

[0360] <Synthesis of Intermediate 10-a> Intermediate 9 (525 mg), dimethylglycine hydrochloride (171 mg), triethylamine (169 mg) and DMAP (27.0 mg) were dissolved in chloroform 7.90 g at room temperature. EDC (319 mg) was added to the obtained solution and reacted at room temperature for 2 hours. The reaction solution was washed with 0.5 M phosphate buffer (pH 4.0) 5.25 g, 7 wt% sodium bicarbonate solution 5.25 g, and 20 wt% saline solution 5.25 g in sequence, and then sodium sulfate 250 mg was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator to obtain 558 mg of intermediate 10-a.

[0361] <Synthesis of Intermediate 11-a> After dissolving the intermediate 10-a (550 mg) in 4.95 g of THF, 19.8 g of 0.5 M phosphate buffer (pH 2.0) was added and reacted at room temperature for 2 hours. 8.25 g of chloroform was added to the reaction solution for washing. 1N sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 6.5, and the pH-adjusted water layer was extracted with 8.25 g of chloroform. 300 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 503 mg of intermediate 11-a.

[0362] <Synthesis of Compound 14> Intermediate 11-a (190 mg), oleic acid (289 mg) and DMAP (24.0 mg) were dissolved in 2.85 g of chloroform at room temperature. EDC (234 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 1.90 g of 20 wt % saline solution, and then 100 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 291 mg of compound 14. <Compound 14 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.36 (s, 6H), 2.50-2.5 3(m, 4H), 2.88-2.97(m, 4H), 3.63(s, 2H), 4.34-4.42(m, 4H), 5.32-5.38(m, 4H), 7.10-7.17(m, 3H)

[0363] [Production Example 15] Synthesis of Compound 15 <Synthesis of Intermediate 10-b> Intermediate 10-b represented by the following formula was synthesized in the same manner as in Production Example 14 except that 4-(dimethylamino)butyric acid hydrochloride was used.

[0364] [Chemical formula 31]

[0365] <Synthesis of Intermediate 11-b> Intermediate 11-b represented by the following formula was synthesized in the same manner as in Production Example 14 except that Intermediate 10-b was used.

[0366] [Chemical formula 32]

[0367] <Synthesis of Compound 15> Compound 15 was synthesized by the same synthesis route as in Preparation Example 14 using Intermediate 11-b and oleic acid. <Compound 15 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.26 (s, 6H), 2.33-2.54 (m, 4H), 2.50-2.53(m, 4H), 2.88-2.97(m, 4H), 3.63(s, 2H), 4.34-4.42(m, 4H), 5.32-5.38(m, 4H), 7.10-7.17(m, 3H)

[0368] [Chemical formula 33]

[0369] [Production Example 16] Synthesis of Compound 16 <Synthesis of Intermediate 12> Intermediate 2 (5.00 g) and triethylamine (2.72 g) were dissolved in 125 g of chloroform at room temperature. Methanesulfonyl chloride (2.31 g) was added to the obtained solution and reacted at 25°C for 3 hours. The reaction solution was washed with 75.0 g of 7 wt% sodium bicarbonate solution and dehydrated by adding 5.00 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 4.42 g of intermediate 12.

[0370] <Synthesis of Intermediate 13-a> Intermediate 12 (600 mg) was dissolved in 5.40 g of dimethylamine (2.0 mol / L in THF), TBAI (49.0 mg) was added, and the mixture was reacted at 25° C. for 20 hours. 9.00 g of chloroform was added to the reaction solution, and 6.00 g of 0.5 M acetic acid buffer (pH 4.0) was used for washing, and 300 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated using an evaporator to obtain 470 mg of intermediate 13-a.

[0371] <Synthesis of Intermediate 14-a> Intermediate 14-a was synthesized in the same manner as Intermediate 4-a in Production Example 1 except that Intermediate 13-a was used.

[0372] <Synthesis of Compound 16> Compound 16 was synthesized in the same manner as Compound 1 in Preparation Example 1 using Intermediate 14-a and 2-hexadecanoic acid. <Compound 16 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.60 (m, 3H), 2 .80(t, 2H), 2.90(t, 2H), 3.63(s, 2H), 4.35(t, 2H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0373] [Production Example 17] Synthesis of Compound 17 <Synthesis of Intermediate 13-b> THF 4.20 g was added to intermediate 12 (600 mg) and diethylamine (779 mg) at room temperature and dissolved. TBAI (49.0 mg) was added to the obtained solution and reacted at 25° C. for 3 hours. Chloroform 9.00 g was added to the reaction solution, washed with 0.5 M acetic acid buffer (pH 4.0) 6.00 g, and sodium sulfate 300 mg was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator to obtain 473 mg of intermediate 13-b represented by the following formula.

[0374] [Chemical formula 34]

[0375] <Synthesis of Intermediate 14-b> Intermediate 14-b represented by the following formula was synthesized in the same manner as in Production Example 16 except that Intermediate 13-b was used.

[0376] [Chemical formula 35]

[0377] <Synthesis of Compound 17> Compound 17 was synthesized by the same synthesis route as in Preparation Example 16 using Intermediate 14-b and 2-hexadecanoic acid. <Compound 17 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.53-2.57 (m, 5H), 2.7 5-2.80 (m, 4H), 2.91 (t, 2H), 3.63 (s, 2H), 4.35 (t, 2H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0378] [Chemical formula 36]

[0379] [Production Example 18] Synthesis of Compound 18 <Synthesis of Intermediate 15-a> Cystine dihydrochloride (227 mg), DIPEA (391 mg) and dimethylglycine hydrochloride (141 mg) were dissolved in 4.54 g of methanol at room temperature. DMT-MM (837 mg) was added to the obtained solution, and the mixture was reacted at 25°C for 2 hours. Then, intermediate 1 (239 mg) was added to the reaction solution, and the mixture was reacted at 25°C for 2 hours. After the reaction solution was concentrated, 3.40 g of chloroform was added, and the mixture was washed with 2.27 g of 0.5 M phosphate buffer (pH 6.0), 2.27 g of 7 wt% sodium bicarbonate solution, and 2.27 g of 20 wt% saline solution in sequence. 100 mg of sodium sulfate was added to the obtained solution for dehydration, and after sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 748 mg of intermediate 15-a as a crude product.

[0380] <Synthesis of Intermediate 16-a> Intermediate 16-a was synthesized in the same manner as Intermediate 4-a in Production Example 1 except that the crude product of Intermediate 15-a was used.

[0381] <Synthesis of Compound 18> Compound 18 was synthesized by the same synthesis route as in Preparation Example 1 using Intermediate 16-a and 2-hexadecanoic acid. <Compound 18 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.30 (s, 6H), 2.52-2.59 (m, 1H), 2.76-2.82 (m , 4H), 2.95 (s, 2H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0382] [Production Example 19] Synthesis of Compound 19 <Synthesis of Intermediate 15-b> Intermediate 15-b represented by the following formula was synthesized in the same manner as in Production Example 18 except that 3-(dimethylamino)propionic acid hydrochloride was used.

[0383] [Chemical formula 37]

[0384] <Synthesis of Intermediate 16-b> Intermediate 16-b represented by the following formula was synthesized in the same manner as in Production Example 18 except that Intermediate 15-b was used.

[0385] [Chemical formula 38]

[0386] <Synthesis of Compound 19> Compound 19 was synthesized by the same synthesis route as in Preparation Example 18 using Intermediate 16-b and 2-hexadecyloctadecanoic acid. <Compound 19 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.33-2.38 (m, 2H), 2.52-2.59 (m, 3H ), 2.76-2.82(m, 4H), 3.50-3.60(m, 6H), 6.34(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H), 7.50(s, 1H)

[0387] [Production Example 20] Synthesis of Compound 20 <Synthesis of Intermediate 15-c> Intermediate 15-c represented by the following formula was synthesized in the same manner as in Production Example 18 except that 4-(dimethylamino)butyric acid hydrochloride was used.

[0388] [Chemical formula 39]

[0389] <Synthesis of Intermediate 16-c> Intermediate 16-c represented by the following formula was synthesized in the same manner as in Production Example 18 except that Intermediate 15-c was used.

[0390] [Chemical formula 40]

[0391] <Synthesis of Compound 20> Compound 20 was synthesized by the same synthesis route as in Preparation Example 18 using Intermediate 16-c and 2-hexadecanoic acid. <Compound 20 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.80 (m, 4H), 2.25-2.38 (m, 8H), 2.52-2.59 (m, 1H), 2.7 6-2.82(m, 4H), 3.50-3.60(m, 6H), 6.34(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H), 7.50(s, 1H)

[0392] [Chemical formula 41]

[0393] [Production Example 21] Synthesis of Compound 21 <Synthesis of Intermediate 17-a> 7.10 g of dichloromethane was added to intermediate 2 (393 mg) and triethylamine (235 mg) at room temperature to dissolve them. DSC (541 mg) was added to the obtained solution and reacted at 25°C for 2 hours. After removing the DSC remaining in the reaction solution by filtration, N,N-dimethylethylenediamine (112 mg) was added to the filtrate and reacted at 25°C for 1 hour. The reacted solution was washed with 5.90 g of 0.5 M acetate buffer (pH 4.0), 5.90 g of 7 wt% sodium bicarbonate solution, and 5.90 g of 20 wt% saline in sequence, and then 200 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 472 mg of intermediate 17-a.

[0394] <Synthesis of Intermediate 18-a> Intermediate 18-a was synthesized in the same manner as Intermediate 4-a in Production Example 1 except that Intermediate 17-a was used.

[0395] <Synthesis of Compound 21> Compound 21 was synthesized in the same manner as Compound 1 in Preparation Example 1 using Intermediate 18-a and 2-hexadecyloctadecanoic acid. <Compound 21 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.22 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 .97(m, 4H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.26(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0396] [Production Example 22] Synthesis of Compound 22 <Synthesis of Intermediate 17-b> Intermediate 17-b represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-diethylethylenediamine was used.

[0397] [Chemical formula 42]

[0398] <Synthesis of Intermediate 18-b> Intermediate 18-b represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-b was used.

[0399] [Chemical formula 43]

[0400] <Synthesis of Compound 22> Compound 22 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-b and 2-hexadecanoic acid. <Compound 22 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 4H) ), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.28(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0401] [Production Example 23] Synthesis of Compound 23 <Synthesis of Intermediate 17-c> Intermediate 17-c represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-dimethyl-1,3-propylenediamine was used.

[0402] [Chemical formula 44]

[0403] <Synthesis of Intermediate 18-c> Intermediate 18-c represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-c was used.

[0404] [Chemical formula 45]

[0405] <Synthesis of Compound 23> Compound 23 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-c and 2-hexadecyloctadecanoic acid. <Compound 23 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 .97(m, 4H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.70(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0406] [Production Example 24] Synthesis of Compound 24 <Synthesis of Intermediate 17-d> Intermediate 17-d represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N,N′-trimethyl-1,3-propylenediamine was used.

[0407] [Chemical formula 46]

[0408] <Synthesis of Intermediate 18-d> Intermediate 18-d represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-d was used.

[0409] [Chemical formula 47]

[0410] <Synthesis of Compound 24> Compound 24 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-d and 2-hexadecanoic acid. <Compound 24 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2.97(m, 7H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0411] [Production Example 25] Synthesis of Compound 25 <Synthesis of Intermediate 17-e> Intermediate 17-e represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-dimethyl-1,4-butanediamine was used.

[0412] [Chemical formula 48]

[0413] <Synthesis of Intermediate 18-e> Intermediate 18-e represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-e was used.

[0414] [Chemical formula 49]

[0415] <Synthesis of Compound 25> Compound 25 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-e and 2-hexadecanoic acid. <Compound 25 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.50-1.62 (m, 4H), 1.69-1.84 (m, 2H), 2.20-2.42 (m, 8H), 2.52-2.59 (m, 1H), 2.8 8-2.97(m, 4H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.70(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0416] [Production Example 26] Synthesis of Compound 26 <Synthesis of Intermediate 17-f> Intermediate 17-f represented by the following formula was synthesized in the same manner as in Production Example 21 except that 4-(2-aminoethyl)morpholine was used.

[0417] [Chemical formula 50]

[0418] <Synthesis of Intermediate 18-f> Intermediate 18-f represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-f was used.

[0419] [Chemical formula 51]

[0420] <Synthesis of Compound 26> Compound 26 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-f and 2-hexadecyloctadecanoic acid. <Compound 26 1 H-NMR (600MHz, CDCl3)> δ: 0.88(t, 6H), 1.20-1.45(m, 58H), 1.69-1.84(m, 2H), 2.40-2.46(m, 6H), 2.52-2.59(m, 1H), 2.88-2.97(m , 4H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.26(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0421] [Production Example 27] Synthesis of Compound 27 <Synthesis of Intermediate 17-g> Intermediate 17-g represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-dibutylethylenediamine was used.

[0422] [Chemical formula 52]

[0423] <Synthesis of Intermediate 18-g> Intermediate 18-g represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-g was used.

[0424] [Chemical formula 53]

[0425] <Synthesis of Compound 27> Compound 27 was synthesized according to the same synthesis route as in Preparation Example 21 using Intermediate 18-g and 2-hexadecyloctadecanoic acid. <Compound 27 1H-NMR (600MHz, CDCl3)> δ: 0.87-0.93 (m, 12H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 2H), 2.39-2.57 (m, 7H), 2.88-2.97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0426] [Production Example 28] Synthesis of Compound 28 <Synthesis of Intermediate 17-h> Intermediate 17-h represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-diisopropylethylenediamine was used.

[0427] [Chemical formula 54]

[0428] <Synthesis of Intermediate 18-h> Intermediate 18-h represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-h was used.

[0429] [Chemical formula 55]

[0430] <Synthesis of Compound 28> Compound 28 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-h and 2-hexadecanoic acid. <Compound 28 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 0.95-1.03 (d, 12H), 1.20-1.45 (m, 58H), 1.60-1.84 (m, 6H), 2.52-2.59 (m, 1H), 2.88-3.00 (m , 6H), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.28(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0431] [Production Example 29] Synthesis of Compound 29 <Synthesis of Intermediate 17-i> Intermediate 17-i represented by the following formula was synthesized in the same manner as in Production Example 21 except that N,N-diethyl-N′-methylethylenediamine was used.

[0432] [Chemical formula 56]

[0433] <Synthesis of Intermediate 18-i> Intermediate 18-i represented by the following formula was synthesized in the same manner as in Production Example 21 except that Intermediate 17-i was used.

[0434] [Chemical formula 57] <Synthesis of Compound 29> Compound 29 was synthesized by the same synthesis route as in Preparation Example 21 using Intermediate 18-i and 2-hexadecanoic acid. <Compound 29 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 7H) ), 3.22-3.26(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.28(s, 1H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0435] [Chemical formula 58]

[0436] [Production Example 30] Synthesis of Compound 30 <Synthesis of Intermediate 19> 5.10 g of dichloromethane was added to intermediate 2 (510 mg) and NPM (670 mg) at room temperature to dissolve them. pNPCl (690 mg) was added to the obtained solution, and the mixture was reacted at 25°C for 2 hours. DMAP (33.0 mg) and 2-(dimethylamino)ethanol (976 mg) were added to the reaction solution, and the mixture was reacted at 25°C for 6 hours. The solution after the reaction was washed with 5.10 g of 0.5 M acetate buffer (pH 4.0), 5.10 g of 7 wt% sodium bicarbonate solution, and 5.10 g of 20 wt% saline solution in sequence, and then 250 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator to obtain 287 mg of intermediate 19.

[0437] <Synthesis of Intermediate 20> Intermediate 20 was synthesized in the same manner as Intermediate 4-a in Production Example 1 except that Intermediate 19 was used.

[0438] <Synthesis of Compound 30> Compound 30 was synthesized in the same manner as Compound 1 in Preparation Example 1 using Intermediate 20 and 2-hexadecanoic acid. <Compound 30 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.58-2.61 (m, 3H), 2.91-2 .94(m, 4H), 3.63(s, 2H), 4.23(t, 2H), 4.32-4.38(m, 4H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0439] [Chemical formula 59]

[0440] [Production Example 31] Synthesis of Compound 31 <Synthesis of Intermediate 21> Intermediate 18-b (1.00 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (445 mg) and DMAP (57.0 mg) were dissolved in 15.0 g of chloroform at room temperature. EDC (668 mg) was added to the obtained solution and reacted at 25°C for 2 hours. The reaction solution was washed with 10.0 g of 20 wt% saline solution, and then 500 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 1.25 g of intermediate 21.

[0441] <Synthesis of Intermediate 22> After dissolving the intermediate 21 (1.25 g) in THF 11.3 g, 0.5 M phosphate buffer (pH 2.0) 45.0 g was added and reacted at 40° C. for 22 hours. 18.8 g of chloroform was added to the reaction solution for washing. 400 g / L of sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 6.0, and the water layer was extracted with 18.8 g of chloroform. 600 mg of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 908 mg of intermediate 22.

[0442] <Synthesis of Compound 31> Intermediate 22 (200 mg), myristic acid (175 mg) and DMAP (18.0 mg) were dissolved in 3.00 g of chloroform at room temperature. EDC (175 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was concentrated with an evaporator and purified with a chromatographic column to obtain 106 mg of compound 31. <Compound 31 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0443] [Production Example 32] Synthesis of Compound 32 <Synthesis of Compound 32> Compound 32 was synthesized by the same synthesis route as in Preparation Example 31 using Intermediate 22 and palmitic acid. <Compound 32 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 48H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0444] [Production Example 33] Synthesis of Compound 33 <Synthesis of Compound 33> Compound 33 was synthesized by the same synthesis route as in Preparation Example 31 using Intermediate 22 and stearic acid. <Compound 33 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 56H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0445] [Production Example 34] Synthesis of Compound 34 <Synthesis of Compound 34> Compound 34 was synthesized by the same synthesis route as in Preparation Example 31 using Intermediate 22 and oleic acid. <Compound 34 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6 H), 2.92-2.96(m, 4H), 3.22-3.26(m, 2H), 3.66(s, 2H), 4.30-4.40(m, 8H), 5.32-5.38(m, 5H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0446] [Production Example 35] Synthesis of Compound 35 <Synthesis of Compound 35> Compound 35 was synthesized by the same synthesis route as in Preparation Example 31 using Intermediate 22 and linoleic acid. <Compound 35 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 28H), 1.42 (s, 3H), 1.55-1 .65(m, 4H), 1.99-2.05(m, 8H), 2.36(t, 4H), 2.52-2.58(m, 6H), 2.75- 2.78 (m, 4H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30 -4.40(m,8H),5.32-5.38(m,9H),7.02-7.05(m,2H),7.27-7.30(m,2H)

[0447] [Chemical formula 60]

[0448] [Production Example 36] Synthesis of Compound 36 <Synthesis of Intermediate 23> 36.0 g of dichloromethane was added to intermediate 2 (2.0 g) and triethylamine (1.20 g) at room temperature to dissolve them. DSC (2.75 g) was added to the obtained solution and reacted at 25° C. for 2 hours. After removing the DSC remaining in the reaction solution by filtration, ethanolamine (394 mg) was added to the filtrate and reacted at 25° C. for 1 hour. The reacted solution was washed with 30.0 g of 0.5 M acetic acid buffer (pH 4.0), and then 1.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 1.41 g of intermediate 23.

[0449] <Synthesis of Intermediate 24> Intermediate 23 (1.35 g) and triethylamine (594 mg) were dissolved in 20.0 g of chloroform at room temperature. Methanesulfonyl chloride (505 mg) was added to the obtained solution and reacted at room temperature for 3 hours. The reaction solution was washed with 15.0 g of 7 wt% sodium bicarbonate solution and dehydrated by adding 500 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated by an evaporator to obtain 1.34 g of intermediate 24.

[0450] <Synthesis of Intermediate 25> After dissolving intermediate 2 (1.31 g) in a mixed solvent of THF 11.8 g and IPA 13.1 g, 0.5 M phosphate buffer (pH 2.0) 47.2 g was added and reacted at 40° C. for 2 hours. 19.7 g of chloroform was added to the reaction solution for washing. 400 g / L sodium hydroxide aqueous solution was added to the washed water layer for neutralization, and then 19.7 g of chloroform was added to the water layer for extraction. 3.0 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 939 mg of intermediate 25.

[0451] <Synthesis of Intermediate 26> Intermediate 25 (930 mg), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (393 mg) and DMAP (50.0 mg) were dissolved in 15 g of chloroform at room temperature. EDC (590 mg) was added to the obtained solution and reacted at 25°C for 2 hours. The reaction solution was washed with 9.30 g of 20 wt% saline solution, and then 500 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 1.06 g of intermediate 26.

[0452] <Synthesis of Intermediate 27> After dissolving the intermediate 26 (1.00 g) in a mixed solvent of THF 9.00 g and IPA 9.00 g, 40.0 g of 0.5 M phosphate buffer (pH 2.0) was added and reacted at 40° C. for 2 hours. 15.0 g of chloroform was added to the reaction solution for washing. 400 g / L sodium hydroxide aqueous solution was added to the washed water layer for neutralization, and then 15.0 g of chloroform was added to the water layer for extraction. 500 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 785 mg of intermediate 27.

[0453] <Synthesis of Intermediate 28> Intermediate 27 (770 mg), oleic acid (802 mg) and DMAP (66.0 mg) were dissolved in 11.6 g of chloroform at room temperature. EDC (648 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 8.00 g of 20 wt % saline solution, and then 2.0 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 1.15 g of intermediate 28.

[0454] <Synthesis of Compound 36> Intermediate 28 (250 mg) was dissolved in THF 2.50 g, 2-(methylamino)ethanol (137 mg) and TBAI (17.0 mg) were added and reacted at 40° C. for 8 hours. 3.75 g of chloroform was added to the reaction solution, and the mixture was washed with 0.5 M acetic acid buffer (pH 4.0) 2.50 g, 7 wt% sodium bicarbonate solution 2.50 g, and 20 wt% saline 2.50 g in sequence. The filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 79 mg of compound 36. <Compound 36 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96(m, 4H), 3.22-3.26(m, 2H), 3.58-3.63(m, 4H), 4.30-4.40(m, 8H), 5.32-5.38(m, 5H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0455] [Production Example 37] Synthesis of Compound 37 <Synthesis of Compound 37> Compound 37 was synthesized according to the same synthetic route as in Preparation Example 36 using Intermediate 28 and 3-(methylamino)-1-propanol. <Compound 37 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.72 (m, 6H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96(m, 4H), 3.22-3.26(m, 2H), 3.58-3.63(m, 4H), 4.30-4.40(m, 8H), 5.32-5.38(m, 5H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0456] [Chemical formula 61]

[0457] [Production Example 38] Synthesis of Compound 38 <Synthesis of Intermediate 29> Intermediate 2 (2.00 g), (E)-4-bromocrotonic acid (974 mg) and DMAP (131 mg) were dissolved in 30.0 g of chloroform at room temperature. EDC (1.54 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt % saline solution, and then 2.0 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 2.26 g of intermediate 29.

[0458] <Synthesis of Intermediate 30> After dissolving the intermediate 29 (2.25 g) in a mixed solvent of THF 20.23 g and IPA 22.5 g, 0.5 M phosphate buffer (pH 2.0) 81.0 g was added and reacted at 40° C. for 2 hours. 34.0 g of chloroform was added to the reaction solution for washing. 400 g / L sodium hydroxide aqueous solution was added to the washed water layer for neutralization, and then 34.0 g of chloroform was added to the water layer for extraction. 1.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 1.51 g of intermediate 30.

[0459] <Synthesis of Intermediate 31> Intermediate 30 (1.40 g), 2-hexadecyloctadecanoic acid (1.80 g) and DMAP (79.0 mg) were dissolved in 21.0 g of chloroform at room temperature. EDC (925 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was concentrated with an evaporator to obtain 2.98 g of a crude product of Intermediate 31.

[0460] <Synthesis of Compound 38> 500 mg of the crude product of intermediate 31 and 2.30 g of dimethylamine (2.0 mol / L in THF) were dissolved in 5.00 g of THF, potassium iodide (0.12 mmol) was added and reacted at 25° C. for 10 hours. After removing the insoluble matter by filtration, 7.50 g of chloroform was added to the filtrate, and washed with 5.00 g of 0.5 M acetic acid buffer (pH 4.0), 5.00 g of 7 wt % sodium bicarbonate solution, and 5.00 g of 20 wt % saline solution in sequence. After adding 250 mg of sodium sulfate for dehydration, sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 270 mg of compound 38. <Compound 38 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2.97 (m, 4H ), 3.07(t, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.97-6.00(m, 1H), 6.95-7.05(m, 3H), 7.27-7.30(m, 2H)

[0461] [Production Example 39] Synthesis of Compound 39 <Synthesis of Compound 39> Compound 39 was synthesized in the same manner as in Preparation Example 38 except that diethylamine was used. <Compound 39 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.50-2.59 (m, 5H), 2.88-2.97 (m, 4H ), 3.23(t, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.97-6.00(m, 1H), 6.95-7.05(m, 3H), 7.27-7.30(m, 2H)

[0462] [Production Example 40] Synthesis of Compound 40 <Synthesis of Compound 40> Compound 40 was synthesized in the same manner as in Preparation Example 38 except that 2-(ethylamino)ethanol was used. <Compound 40 1 H-NMR (600MHz, CDCl3)> δ: 0.88(t, 6H), 1.03(t, 3H), 1.20-1.45(m, 58H), 1.69-1.84(m, 2H), 2.50-2.59(m, 5H), 2.88-2.97(m, 4H), 3.23(t , 2H), 3.55-3.58(m, 2H), 3.63(s, 2H), 4.28-4.38(m, 4H), 5.97-6.00(m, 1H), 6.95-7.05(m, 3H), 7.27-7.30(m, 2H)

[0463] [Chemical formula 62]

[0464] [Production Example 41] Synthesis of Compound 41 <Synthesis of Compound 41> Compound 41 was synthesized by the same synthetic route as in Production Example 1 except that Intermediate 1, 4,4′-dithiobisbutane-1-ol and 4-(dimethylamino)butyric acid hydrochloride were used as starting materials. <Compound 41 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.20-1.50 (m, 64H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.59-2.70 (m, 4H), 3.63 (s, 2H), 4.22-4.33 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0465] [Chemical formula 63]

[0466] [Production Example 42] Synthesis of Compound 42 <Synthesis of Intermediate 32> At room temperature, bis(2-hydroxyethyl) disulfide (33.2 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (25.0 g) and DMAP (3.50 g) were dissolved in 250 g of chloroform. EDC (41.0 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt % saline solution, and then 25.0 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 21.8 g of intermediate 32.

[0467] <Synthesis of Intermediate 33> 180 g of dichloromethane was added to the intermediate 32 (10.0 g) and triethylamine (7.17 g) at room temperature to dissolve them. DSC (16.5 g) was added to the obtained solution, and the mixture was reacted at 25° C. for 2 hours. After removing the DSC remaining in the reaction solution by filtration, N,N-diethylethylenediamine (5.00 g) was added to the filtrate, and the mixture was reacted at 25° C. for 1 hour. The reaction solution was washed with 150 g of 0.5 M acetate buffer (pH 4.0), 150 g of 7 wt% sodium bicarbonate solution, and 150 g of 20 wt% saline in sequence, and then 5.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 13.1 g of the intermediate 33.

[0468] <Synthesis of Intermediate 34> After dissolving the intermediate 33 (13.0 g) in THF 49.0 g, 0.5 M phosphate buffer (pH 2.0) 195 g was added and reacted at 40° C. for 3 hours. 120 g of chloroform was added to the reaction solution for washing. 400 g / L sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 7.0, and the water layer was extracted with 195 g of chloroform. 6.50 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 5.01 g of intermediate 34.

[0469] <Synthesis of Intermediate 35> Intermediate 34 (5.00 g), Intermediate 1 (6.01 g) and DMAP (592 mg) were dissolved in 75.0 g of chloroform at room temperature. EDC (5.81 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 50.0 g of 20 wt % saline solution, and then 2.50 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 6.25 g of a crude product of Intermediate 35.

[0470] <Synthesis of Intermediate 36> After dissolving 6.25 g of the crude product of intermediate 35 in 56.3 g of THF, 225 g of 0.5 M phosphate buffer (pH 2.0) was added and reacted at 25° C. for 2 hours. 94.0 g of chloroform was added to the reaction solution for washing. 400 g / L of sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 6.0, and the water layer was extracted with 94.0 g of chloroform. 3.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 3.7 g of intermediate 36.

[0471] <Synthesis of Compound 42> Intermediate 36 (500 mg), oleic acid (436 mg) and DMAP (36.0 mg) were dissolved in 7.50 g of chloroform at room temperature. EDC (352 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 5.00 g of 20 wt % saline solution, and then 250 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 453 mg of compound 42. <Compound 42 1 H-NMR (600MHz, CDCl3)> δ: 0.88 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6 H), 2.92-2.96(m, 4H), 3.22-3.26(m, 2H), 3.66(s, 4H), 4.30-4.40(m, 8H), 5.32-5.38(m, 5H), 7.02-7.05(m, 4H), 7.27-7.30(m, 4H)

[0472] [Production Example 43] Synthesis of Compound 43 <Synthesis of Compound 43> Compound 43 was synthesized by the same synthesis route as in Preparation Example 42 using Intermediate 36 and myristic acid. <Compound 43 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32 (s, 1H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0473] [Chemical formula 64]

[0474] [Production Example 44] Synthesis of Compound 44 <Synthesis of Intermediate 37> Intermediate 34 (1.60 g), intermediate 1 (4.19 g) and DMAP (165 mg) were dissolved in 24.0 g of chloroform at room temperature. EDC (1.95 g) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 16.0 g of 20 wt % saline solution, and then 500 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator and purified by a chromatographic column to obtain 1.85 g of intermediate 37.

[0475] <Synthesis of Intermediate 38> Intermediate 37 (450 mg), oleic acid (241 mg) and DMAP (19.0 mg) were dissolved in 6.75 g of chloroform at room temperature. EDC (223 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 4.5 g of 20 wt % saline and then dehydrated by adding 2.0 g of sodium sulfate. After sodium sulfate was removed by filtration, the filtrate was concentrated by an evaporator to obtain 670 mg of a crude product of intermediate 38.

[0476] <Synthesis of Intermediate 39> The crude product (670 mg) of intermediate 38 was dissolved in a mixed solvent of 4.00 g of THF and 4.00 g of IPA, and 16.2 g of 0.5 M phosphate buffer (pH 2.0) was added to react at 40° C. for 3 hours. After adding 13.5 g of chloroform to the reaction solution for extraction, 9.00 g of 0.5 M phosphate buffer (pH 6.5) was added to the organic layer for washing. 300 mg of sodium sulfate was added to the washed organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 240 mg of intermediate 39.

[0477] <Synthesis of Compound 44> Intermediate 39 (150 mg), lauric acid (41.0 mg) and DMAP (5.0 mg) were dissolved in 2.25 g of chloroform at room temperature. EDC (53.0 mg) was added to the obtained solution and reacted at 25° C. for 2 hours. The reaction solution was washed with 1.50 g of 20 wt % saline solution, and then 50.0 mg of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 116 mg of compound 44. <Compound 44 1 H-NMR (600MHz, CDCl3)> δ: 0.80-0.92(m, 6H), 1.04(t, 6H), 1.25-1.38(m, 36H), 1.42(s, 3H), 1.55-1.65(m, 4H), 1.99-2.05(m, 4H), 2.36(t, 2H), 2.52-2.58(m , 6H), 2.92-2.96(m, 4H), 3.22-3.26(m, 2H), 3.66(s, 4H), 4.30-4.40(m, 8H), 5.32-5.36(m, 3H), 7.02-7.05(m, 2H), 7.27-7.30(m, 2H)

[0478] [Chemical formula 65]

[0479] [Production Example 45] Synthesis of Compound 45 <Synthesis of Intermediate 40> 30.0 g of dichloromethane was added to the intermediate 18-b (3.00 g) and NPM (3.41 g) at room temperature to dissolve them. pNPCl (3.51 g) was added to the obtained solution, and the mixture was reacted at 25°C for 10 hours. DMAP (170 mg) and 1,2-isopropylglycerol (7.37 g) were added to the reaction solution, and the mixture was reacted at 25°C for 8 hours. The reaction solution was washed with 30.0 g of 7 wt% sodium bicarbonate solution and 30.0 g of 20 wt% saline solution in sequence, and then 9.00 g of sodium sulfate was added for dehydration. After sodium sulfate was removed by filtration, the filtrate was concentrated with an evaporator and purified with a chromatographic column to obtain 2.16 g of intermediate 40.

[0480] <Synthesis of Intermediate 41> After dissolving the intermediate 40 (2.16 g) in THF 11.3 g, 0.5 M phosphate buffer (pH 2.0) 45.0 g was added and reacted at 40° C. for 2 hours. 19.0 g of chloroform was added to the reaction solution for washing. 400 g / L of sodium hydroxide aqueous solution was added to the washed water layer, and the pH was adjusted to 6.0, and the water layer was extracted with 19.0 g of chloroform. 1.20 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated with an evaporator to obtain 850 mg of intermediate 41.

[0481] <Synthesis of Compound 45> Compound 45 was synthesized according to the same synthetic route as in Preparation Example 42 except that Intermediate 41 and oleic acid were used. <Compound 45 1 H-NMR (600MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6 H), 2.92-2.96(m, 4H), 3.22-3.26(m, 2H), 3.66(s, 2H), 4.30-4.50(m, 8H), 5.28-5.38(m, 6H), 6.97-7.00(m, 2H), 7.27-7.30(m, 2H)

[0482] [Production Example 46] Synthesis of Compound 46 <Synthesis of Compound 46> Compound 46 was synthesized according to the same synthetic route as in Preparation Example 42 except that Intermediate 41 and myristic acid were used. <1H-NMR (600 MHz, CDCl3) of Compound 46 1 1H-NMR (600 MHz, CDCl3)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25 - 1.35 (m, 32H), 1.42 (s, 3H), 1.55 - 1.65 (m, 4H), 2.36 (t, 4H), 2.52 - 2.58 (m, 6H), 2.92 - 2.96 (m, 4H), 3.22 - 3.26 (m, 2H), 3.66 (s, 2H), 4.30 - 4.50 (m, 8H), 5.28 - 5.31 (m, 2H), 6.97 - 7.00 (m, 2H), 7.27 - 7.30 (m, 2H)

[0483] [Comparative Example] Synthesis of O-Ph-P4C2 Synthesis was carried out according to the synthesis route described in Production Example 3 of Patent Document 3. <1H-NMR (600 MHz, CDCl3) of O-Ph-P4C2 1 1H-NMR (600 MHz, CDCl3)> δ 0.88 (t, 6H), 1.22 - 1.42 (m, 46H), 1.54 - 1.76 (m, 12H), 1.94 - 2.03 (m, 12H), 2.52 - 2.56 (m, 4H), 2.62 - 2.66 (m, 4H), 2.80 - 2.89 (m, 8H), 3.59 (s, 4H), 4.13 (t, 4H), 5.34 - 5.37 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H) Industrial Applicability

[0484] According to the present invention, nucleic acids can be efficiently introduced into cells, and thus it can be used in nucleic acid drugs, gene therapy, and biochemical experiments.

[0485] This application is based on Japanese Patent Application No. 2022 - 051913 filed in Japan, the entire content of which is incorporated herein by reference.

Claims

1. A method for producing lipid nanoparticles encapsulating nucleic acid, characterized in that: The following steps are involved: a) mixing an alcohol solution containing ionic lipids, sterols and PEG lipids with an acidic buffer having a buffering effect at pH 1 to 6.5 to prepare a lipid nanoparticle suspension that does not contain nucleic acids, and b) mixing the lipid nanoparticle suspension obtained in step a with an aqueous solution containing nucleic acid and optionally containing 0-25 v / v% of alcohol without freeze-drying, and optionally incubating the mixture at 0-95° C. for 0-60 minutes to obtain lipid nanoparticles encapsulating nucleic acid.

2. The method for producing lipid nanoparticles encapsulating nucleic acid according to claim 1, comprising the following steps c after step b: c) replacing the external aqueous phase of the obtained nucleic acid-encapsulated lipid nanoparticles with a neutral buffer by dialysis, ultrafiltration or dilution.

3. The method for producing a nucleic acid-encapsulating lipid nanoparticle according to claim 1 or 2, wherein: In step a, the method further comprises the step of freezing the lipid nanoparticles without nucleic acid at -80 to 0°C and then melting them at 0 to 95°C.

4. The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of claims 1 to 3, wherein: In step a, the method further comprises: after preparing the lipid nanoparticle suspension, replacing the external aqueous phase with other acidic buffer having a buffering effect at pH 1 to 6.5 by dialysis, ultrafiltration or dilution.

5. The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of claims 1 to 4, wherein: In step a, the alcohol solution further comprises a phospholipid.

6. The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of claims 1 to 5, wherein: Ionic lipids are compounds represented by formula (1): In formula (1), R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, X a and X b each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, Y a and Y b each independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond, Z a and Z b Each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and having or not having a heteroatom, n a and n b Each independently is 0 or 1, R 3a and R 3b Each independently represents a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, a residue derived from a reaction product of a sterol derivative having a hydroxyl group and succinic anhydride or glutaric anhydride, an aliphatic hydrocarbon group having 1 to 40 carbon atoms, an alkyl group having 3 to 40 carbon atoms and having a cyclopropane ring, or a group represented by formula (3): R 9 -O-CO-(CH2)a-(3) In formula (3), R 9 represents an aliphatic hydrocarbon group having 2 to 20 carbon atoms, a represents an integer of 2-10.

7. The method for producing nucleic acid-encapsulating lipid nanoparticles according to any one of claims 1 to 5, wherein: Ionic lipids are compounds represented by formula (2): In the formula, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen, R 1 represents an aliphatic hydrocarbon group having 8 or less carbon atoms, L 1 represents an ester bond, an amide bond, a carbamate bond, an N-alkyl carbamate bond, a carbonate bond or a urea bond, k represents 0 or 1, R x and R y each independently represents an alkylene group having 2 to 5 carbon atoms, L 2 represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond, R 2 represents an alkylene group having 8 or less carbon atoms or does not exist, Y represents a group, which (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) contains at least one selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue and a residue of a sterol derivative.

8. A method for introducing a nucleic acid into a cell, comprising: A step of bringing the nucleic acid-encapsulating lipid nanoparticles produced by the method according to any one of claims 1 to 7 into contact with cells in vitro.

9. A method for introducing a nucleic acid into a target cell, comprising: A step of administering to a living body the nucleic acid-encapsulating lipid nanoparticles produced by the method according to any one of claims 1 to 7.

10. A method for producing a pharmaceutical composition, comprising the method according to any one of claims 1 to 7.

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