cationic lipids

Abstract

This invention provides techniques for efficiently delivering active ingredients, particularly nucleic acids, into cells, and cationic lipids used in these techniques. The cationic lipids of this invention are compounds represented by formula (I) or their salts. n1 represents an integer from 2 to 6, n2 represents an integer from 0 to 2, n3 represents an integer from 0 to 2, L represents -C(O)O- or -NHC(O)O-, and Ra represents a linear C 5‑13 Alkyl, straight-chain C 13‑17 Alkenyl or straight-chain C 17 Dienyl, Rb represents straight-chain C 2‑9 Alkyl group, Rc represents hydrogen atom or straight-chain C. 2‑9 Alkyl group, Rd represents hydrogen atom or straight-chain C 2‑9 Alkyl, Re indicates straight-chain C 2‑9 Alkyl, Rf indicates straight-chain C 2‑9 alkyl.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980051986.6 (International Application No. PCT / JP2019 / 031411), with the Chinese national phase entry date of February 4, 2021 (international application date of August 8, 2019) and the invention title "Catonic Lipids". Technical Field

[0002] This invention relates to cationic lipids capable of delivering nucleic acids as active ingredients into various cells, tissues, or organs. Furthermore, this invention relates to lipid particles containing such cationic lipids, and compositions containing such lipid particles and nucleic acids. Background Technology

[0003] In recent years, research and development of nucleic acid drugs containing nucleic acids as active ingredients has been actively pursued. For example, numerous studies have been conducted on nucleic acid drugs containing siRNA, miRNA, miRNA mimics, or antisense nucleic acids, exhibiting degradative or inhibitory effects on target mRNAs. Additionally, research has been conducted on nucleic acid drugs containing mRNA encoding target proteins for intracellular expression of those proteins. Related to these research and developments, technologies for efficiently delivering nucleic acids into cells, tissues, or organs have been developed as drug delivery systems (DDS) technologies.

[0004] As for the aforementioned DDS technology, techniques are conventionally known that involve mixing nucleic acids and lipids to form a complex, and then using the complex to mediate the uptake of nucleic acids into cells. As for the lipids used in the formation of the aforementioned complex, cationic lipids, hydrophilic polymeric lipids, and accessory lipids are conventionally known. Among the aforementioned cationic lipids, compounds described in the prior art literature below are known, for example.

[0005] Patent Document 1 describes compounds or salts thereof represented by the following formulas.

[0006]

[0007] It is specified that: In the formula, R 1 Each can independently choose C8 to C, which can be replaced. 24 Alkyl groups and C8-C groups that can be substituted 24 Group composed of alkenyl groups; R 2 and R 3 Each is independently selected from the group consisting of hydrogen, substituted C1-C8 alkyl groups, substituted arylalkyl groups, etc.; Y 1 and Y 2 Each is independently selected from the group consisting of hydrogen, substituted C1-C6 alkyl groups, substituted arylalkyl groups, etc.; Y3 Each of the following groups is independently selected from hydrogen, substituted C1-C8 alkyl groups, substituted arylalkyl groups, etc., where it exists; m is any integer from 1 to 4, n is any integer from 0 to 3, p is 0 or 1, and the sum of m, n and p is 4; k is any integer from 1 to 5; q is 0 or 1; etc.

[0008] Patent document 2 describes compounds or salts thereof represented by the following formulas.

[0009]

[0010] In the formula, W represents -NR 1 R 2 or formula -N + R 3 R 4 R 5 (Z - ), R 1 and R 2 Each represents C independently. 1-4 Alkyl or hydrogen atom, R 3 R 4 and R 5 Each represents C independently. 1-4 Alkyl, Z - The symbol represents an anion, and X represents a C that can be substituted. 1-6 Alkylene, Y A Y B and Y C Each can independently represent a substituted methine, L A L B and L C Each independently represents a substituted methylene group or bond, R A1 R A2 R B1 R B2 R C1 and R C2 Each can be independently represented as a replaceable C. 4-10 alkyl.

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: Booklet No. WO2003 / 102150

[0014] Patent Document 2: Booklet No. WO2016 / 021683 Summary of the Invention

[0015] The problem that the invention aims to solve

[0016] It is hoped that cationic lipids capable of efficiently delivering nucleic acids into cells will contribute to the development of nucleic acid drugs with excellent efficacy, safety (low toxicity), and therapeutic benefits. Furthermore, it is hoped that cationic lipids capable of delivering nucleic acids into various cells will enable the development of nucleic acid drugs targeting various diseases occurring in various tissues. However, as of now, no cationic lipids have been found that fully meet the above requirements.

[0017] The object of this invention is to provide a technology capable of efficiently introducing nucleic acids into cells, and the use of cationic lipids, etc., in this technology. Furthermore, from another perspective, the object of this invention is to provide a technology capable of introducing nucleic acids into various types of cells, and the use of compounds, etc., in this technology.

[0018] Methods for solving problems

[0019] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that the above-mentioned problems could be solved by using the compound represented by the following formula or its salt, thereby completing the present invention.

[0020] That is, the present invention relates to at least the following inventions. [1]

[0022] The compound or its salt represented by formula (I).

[0023]

[0024] [In the formula,

[0025] n1 represents an integer from 2 to 6, n2 represents an integer from 0 to 2, and n3 represents an integer from 0 to 2.

[0026] L represents -C(O)O- or -NHC(O)O-.

[0027] Ra represents linear C. 5-13 Alkyl, straight-chain C 13-17 Alkenyl or straight-chain C 17 Dieneyl chain,

[0028] Rb represents linear C. 2-9 alkyl,

[0029] Rc represents hydrogen atoms or straight-chain C. 2-9 alkyl,

[0030] Rd represents hydrogen atoms or straight-chain C atoms. 2-9 alkyl,

[0031] Re represents linear C. 2-9 alkyl,

[0032] Rf represents linear C. 2-9 alkyl.] [2]

[0034] 4,5-Dibutylnonanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester or its salt. [3]

[0036] Didecanoic acid 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-dimethyl ester or its salt. [4]

[0038] 4,5-Dibutylnonanoic acid 3-((6-(dimethylamino)hexanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester or its salt. [5]

[0040] 4,5-Dipentyldecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyloxy)methyl)propyl ester or its salt. [6]

[0042] 4-Heptyl undecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyloxy)methyl)propyl ester or its salt. [7]

[0044] A lipid particle containing the compound described in item 1 or a salt thereof. [8]

[0046] A composition for nucleic acid delivery, comprising nucleic acid and lipid particles as described in item 7. [9]

[0048] The composition as described in item 8, wherein the nucleic acid is RNA.

[10]

[0050] The composition as described in item 9, wherein the RNA is mRNA or siRNA.

[0051] It should be noted that in this specification, "the compound represented by formula (I)" is sometimes referred to as "compound (I)". Additionally, "the compound represented by formula (I) or its salt" is sometimes referred to as "the compound of the present invention". "Lipid particles containing the compound represented by formula (I) or its salt (the compound of the present invention)" is sometimes referred to as "lipid particles of the present invention". "A nucleic acid delivery composition containing nucleic acids and the lipid particles of the present invention" is sometimes referred to as "the composition of the present invention".

[0052] Invention Effects

[0053] According to the present invention, nucleic acids can be introduced into cells, tissues, or organs with excellent efficiency. Furthermore, according to the present invention, nucleic acids can be introduced into various cells, tissues, or organs (e.g., cancer cells). According to the present invention, drugs or research reagents for introducing nucleic acids into various cells, tissues, or organs can be obtained. Moreover, according to the present invention, when nucleic acids are introduced into cells, tissues, or organs, the activity (e.g., pharmaceutical efficacy) of the nucleic acid is highly manifested. Detailed Implementation

[0054] The definitions of each substituent used in this specification are described in detail below. Unless otherwise specified, each substituent has the following definition.

[0055] In this specification, "linear C" is used as a reference. 5-13 Alkyl groups can be listed as pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and tridecyl.

[0056] In this specification, "linear C" is used as a reference. 13-17"Alkenyl" can be listed as follows: 1-tetracene-alkenyl, 2-tetracene-alkenyl, 3-tetracene-alkenyl, 4-tetracene-alkenyl, 5-tetracene-alkenyl, 6-tetracene-alkenyl, 7-tetracene-alkenyl, 8-tetracene-alkenyl, 9-tetracene-alkenyl, 10-tetracene-alkenyl, 11-tetracene-alkenyl, 12-tetracene-alkenyl; 1-tetradecene-alkenyl, 2-tetradecene-alkenyl, 3-tetradecene-alkenyl, 4-tetradecene-alkenyl, 5-tetradecene-alkenyl. 6-Tetradecenyl, 7-Tetradecenyl, 8-Tetradecenyl, 9-Tetradecenyl, 10-Tetradecenyl, 11-Tetradecenyl, 12-Tetradecenyl, 13-Tetradecenyl; 1-Pentadecenyl, 2-Pentadecenyl, 3-Pentadecenyl, 4-Pentadecenyl, 5-Pentadecenyl, 6-Pentadecenyl, 7-Pentadecenyl, 8-Pentadecenyl, 9-Pentadecenyl, 10-Pentadecenyl, 11 -Pentadecenyl, 12-Pentadecenyl, 13-Pentadecenyl, 14-Pentadecenyl; 1-Hexadecenyl, 2-Hexadecenyl, 3-Hexadecenyl, 4-Hexadecenyl, 5-Hexadecenyl, 6-Hexadecenyl, 7-Hexadecenyl, 8-Hexadecenyl, 9-Hexadecenyl, 10-Hexadecenyl, 11-Hexadecenyl, 12-Hexadecenyl, 13-Hexadecenyl, 14-Hexadecenyl 15-Hexadecenyl; 1-Heptadecanenyl, 2-Heptadecanenyl, 3-Heptadecanenyl, 4-Heptadecanenyl, 5-Heptadecanenyl, 6-Heptadecanenyl, 7-Heptadecanenyl, 8-Heptadecanenyl, 9-Heptadecanenyl, 10-Heptadecanenyl, 11-Heptadecanenyl, 12-Heptadecanenyl, 13-Heptadecanenyl, 14-Heptadecanenyl, 15-Heptadecanenyl, 16-Heptadecanenyl. These straight-chain C 13-17 Alkenes contain one carbon-carbon double bond, so they can form cis and trans structures, and can be any structure.

[0057] In this specification, "linear C" is used as a reference. 17"Heptadecanyl" can be listed as, for example, 1,3-heptadecadienyl, 1,4-heptadecadienyl, 1,5-heptadecadienyl, 1,6-heptadecadienyl, 1,7-heptadecadienyl, 1,8-heptadecadienyl, 1,9-heptadecadienyl, 1,10-heptadecadienyl, 1,11-heptadecadienyl, 1,12-heptadecadienyl, 1,13-heptadecadienyl, 1,14-heptadecadienyl, 1,15-heptadecadienyl, 1,16-heptadecadienyl, 2,4-heptadecadienyl, 2,5-heptadecadienyl, 2,6-heptadecadienyl, 2,7-heptadecadienyl, 2,8-heptadecadienyl, 2,9-heptadecadienyl, 2,10-heptadecadienyl... Alkenyl, 2,11-heptadecadienyl, 2,12-heptadecadienyl, 2,13-heptadecadienyl, 2,14-heptadecadienyl, 2,15-heptadecadienyl, 2,16-heptadecadienyl, 3,5-heptadecadienyl, 3,6-heptadecadienyl, 3,7-heptadecadienyl, 3,8-heptadecadienyl, 3,9-heptadecadienyl, 3,10-heptadecadienyl, 3,11-heptadecadienyl, 3,12-heptadecadienyl, 3,13-heptadecadienyl, 3,14-heptadecadienyl, 3,15-heptadecadienyl, 3,16-heptadecadienyl, 4,6-heptadecadienyl, 4,7-heptadecadienyl, 4,8-heptadecadienyl, 4, 9-Heptadecanediyl, 4,10-Heptadecanediyl, 4,11-Heptadecanediyl, 4,12-Heptadecanediyl, 4,13-Heptadecanediyl, 4,14-Heptadecanediyl, 4,15-Heptadecanediyl, 4,16-Heptadecanediyl, 5,7-Heptadecanediyl, 5,8-Heptadecanediyl, 5,9-Heptadecanediyl, 5,10-Heptadecanediyl, 5,11-Heptadecanediyl, 5,12-Heptadecanediyl, 5,13-Heptadecanediyl, 5,14-Heptadecanediyl, 5,15-Heptadecanediyl, 5,16-Heptadecanediyl, 6,8-Heptadecanediyl, 6,9-Heptadecanediyl, 6,10-Heptadecanediyl, 6,11 -Heptadecanyl, 6,12-Heptadecanyl, 6,13-Heptadecanyl, 6,14-Heptadecanyl, 6,15-Heptadecanyl, 6,16-Heptadecanyl, 7,9-Heptadecanyl, 7,10-Heptadecanyl, 7,11-Heptadecanyl, 7,12-Heptadecanyl, 7,13-Heptadecanyl, 7,14-Heptadecanyl, 7,15-Heptadecanyl, 7,16-Heptadecanyl, 8,10-Heptadecanyl, 8,11-Heptadecanyl, 8,12-Heptadecanyl, 8,13-Heptadecanyl, 8,14-Heptadecanyl, 8,15-Heptadecanyl, 8,16-Heptadecanyl, 9,11-Heptadecanediyl, 9,12-Heptadecanediyl, 9,13-Heptadecanediyl, 9,14-Heptadecanediyl, 9,15-Heptadecanediyl, 9,16-Heptadecanediyl, 10,12-Heptadecanediyl, 10,13-Heptadecanediyl, 10,14-Heptadecanediyl, 10,15-Heptadecanediyl, 10,16-Heptadecanediyl, 11,13-Heptadecanediyl, 11,14-Heptadecanediyl, 11,15-Heptadecanediyl, 11,16-Heptadecanediyl, 12,14-Heptadecanediyl, 12,15-Heptadecanediyl, 12,16-Heptadecanediyl, 13,15-Heptadecanediyl, 13,16-Heptadecanediyl, 14,16-Heptadecanediyl. These linear C, 17 The dienyl group contains two carbon-carbon double bonds, so each can independently form cis and trans structures, and each can be any structure.

[0058] In this specification, "linear C" is used as a reference. 2-9 Alkyl groups can be exemplified by, for example, ethyl, butyl, propyl, pentyl, hexyl, heptyl, octyl, and nonyl.

[0059] The preferred examples of n1, n2, n3, L, Ra, Rb, Rc, Rd, Re and Rf in equation (I) are described below.

[0060] n1 is preferably an integer from 3 to 5.

[0061] n2 is preferably an integer from 0 to 2.

[0062] n3 is preferably an integer from 0 to 2.

[0063] L is preferably -C(O)O-.

[0064] Ra is preferably a linear C. 5-9 Alkyl, straight-chain C 13-17 Alkenyl or straight-chain C 17 Dieneyl chain.

[0065] Rb is preferably a linear C-chain. 4-8 alkyl.

[0066] Rc is preferably a hydrogen atom or a straight-chain C. 4-7 alkyl.

[0067] Rd is preferably hydrogen atoms or straight-chain C. 3-6 alkyl.

[0068] Re is preferably linear C. 3-6 alkyl.

[0069] Rf is preferably linear C. 3-7 alkyl.

[0070] Preferred examples of compound (I) are described below.

[0071] Compound (IA): n1 is an integer from 3 to 5, n2 is 0, n3 is an integer from 0 to 2, L is -C(O)O-, Ra is a straight-chain C 5-9 Alkyl groups and Rb are straight-chain C 4-8 Alkyl group, Rc is a hydrogen atom, Rd is a hydrogen atom, and Re is a straight-chain carbon atom. 3-6 Alkyl groups and Rf are straight-chain C 3-7 Alkyl compounds.

[0072] Compound (IB): n1 is an integer from 3 to 5, n2 is 0 or 1, n3 is 0 or 1, L is -C(O)O-, Ra is a straight-chain C 5-9 Alkyl groups and Rb are straight-chain C atoms. 4-8 Alkyl groups and Rc are straight-chain C groups. 5-7 Alkyl group, Rd is a hydrogen atom, and Re is a straight-chain carbon atom. 3-6 Alkyl groups and Rf are straight-chain C 3-7 Alkyl compounds.

[0073] Compound (IC): n1 is an integer from 3 to 5, n2 is 0 or 1, n3 is an integer from 0 to 2, L is -C(O)O-, Ra is a straight-chain C 5-9 Alkyl groups and Rb are straight-chain C atoms. 4-8 Alkyl group, Rc is a hydrogen atom, and Rd is a straight-chain carbon atom. 3-6 Alkyl groups and Re are straight-chain C 3-6 Alkyl groups and Rf are straight-chain C 3-7 Alkyl compounds.

[0074] Compound (ID): n1 is an integer from 3 to 5, n2 is 0 or 1, n3 is an integer from 0 to 2, L is -C(O)O-, Ra is a straight-chain C 13-17 Alkenyl or straight-chain C 17 Dienyl groups and Rb are straight-chain C. 3-6 Alkyl groups and Rc are straight-chain C groups. 3-6 Alkyl group, Rd is a hydrogen atom, and Re is a straight-chain carbon atom. 2-6 Alkyl groups and Rf are straight-chain C 2-7 Alkyl compounds.

[0075] More preferred examples of compound (I) are described below.

[0076] Compound (a): n1 is 3 or 4, n2 is 0, n3 is 0 or 2, L is -C(O)O-, Ra is a straight-chain C7 alkyl group, Rb is a straight-chain C6 alkyl group, Rc is a hydrogen atom, Rd is a hydrogen atom, Re is a straight-chain C 5-6Alkyl groups and Rf are straight-chain C 6-7 Alkyl compounds.

[0077] Compound (b): n1 is an integer from 3 to 5, n2 is 0 or 1, n3 is 0 or 1, L is -C(O)O-, Ra is a straight-chain C 6-7 Alkyl groups and Rb are straight-chain C atoms. 5-6 Alkyl groups and Rc are straight-chain C groups. 5-6 Alkyl group, Rd is a hydrogen atom, and Re is a straight-chain carbon atom. 4-5 Alkyl groups and Rf are straight-chain C 5-6 Alkyl compounds.

[0078] Compound (c): n1 is an integer from 3 to 5, n2 is 0, n3 is 2, L is -C(O)O-, Ra is a straight-chain C 5-9 Alkyl groups and Rb are straight-chain C atoms. 4-8 Alkyl group, Rc is a hydrogen atom, and Rd is a straight-chain carbon atom. 3-5 Alkyl groups and Re are straight-chain C 3-5 Alkyl groups and Rf are straight-chain C 3-5 Alkyl compounds.

[0079] Compound (d): n1 is an integer from 3 to 5, n2 is 0 or 1, n3 is an integer from 0 to 2, L is -C(O)O-, Ra is a straight-chain C 13-17 Alkenyl or straight-chain C 17 Dienyl groups and Rb are straight-chain C. 3-5 Alkyl groups and Rc are straight-chain C groups. 3-5 Alkyl group, Rd is a hydrogen atom, and Re is a straight-chain carbon atom. 3-6 Alkyl groups and Rf are straight-chain C 3-7 Alkyl compounds.

[0080] Specific examples of particularly preferred compounds (I) are described below:

[0081] 4,5-Dibutylnonanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester;

[0082] 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-dimethyl ester;

[0083] 4,5-Dibutylnonanoic acid 3-((6-(dimethylamino)hexanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester;

[0084] 4,5-Dipentyldecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester;

[0085] 4-Heptylundecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester.

[0086] As a salt of compound (I), a pharmacologically acceptable salt is preferred, such as a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, or a salt with a basic or acidic amino acid.

[0087] Preferred examples of salts with inorganic bases include: alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; aluminum salts and ammonium salts. Sodium, potassium, calcium, and magnesium salts are preferred, and sodium and potassium salts are even more preferred.

[0088] Preferred examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, methylpyridine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine.

[0089] Preferred examples of salts with inorganic acids include salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid. Salts with hydrochloric acid and salts with phosphoric acid are preferred.

[0090] Preferred examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0091] Preferred examples of salts with basic amino acids include salts with arginine, lysine, and ornithine.

[0092] Preferred examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid.

[0093] In this invention, the compounds of this invention can be used as cationic lipids. Cationic lipids can form complexes with various molecules in a solvent or dispersion medium. These complexes may contain other components besides the compounds of this invention. Examples of these other components include other lipid components and nucleic acids.

[0094] As other lipid components mentioned above, structural lipids that can constitute lipid particles can be listed. Such structural lipids can be selected from, for example, sterols (e.g., cholesterol, cholesterol esters, cholesterol hemisuccinate, etc.), phospholipids (e.g., phosphatidylcholine (e.g., dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, lysophosphatidylcholine, dioleoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dilinoleoylphosphatidylcholine, MC-1010 (NOF), MC-2020 (NOF), MC-4040 (NOF), MC-6060 (NOF), MC-8080 (NOF) etc.), phosphatidylserine (e.g., dipalmitoylphosphatidylserine, distearylphosphatidylserine)). The invention comprises at least one of the following groups: dioleoylphosphatidylserine, palmitoylphosphatidylserine, etc.; phosphatidylethanolamine (e.g., dipalmitoylphosphatidylethanolamine, distearylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, palmitoylphosphatidylethanolamine, lysophosphatidylethanolamine, etc.); phosphatidylinositol, phosphatidic acid, etc.; and polyethylene glycol lipids (PEG lipids) (e.g., PEG-DAA, PEG-DAG, PEG-phospholipid conjugates, PEG-Cer, PEG-cholesterol, PEG-C-DOMG, 2KPEG-CMG, GM-020 (NOF), GS-020 (NOF), GS-050 (NOF), etc.). In this invention, all three types of sterols (especially cholesterol), phospholipids (especially phosphatidylcholine), and polyethylene glycol lipids are preferably used as structural lipids.

[0095] The ratio of the compound of the present invention to the structural lipid in the mixed lipid component forming the lipid particles of the present invention can be appropriately adjusted according to the purpose and use. For example, the ratio of structural lipid to 1 mole of the compound of the present invention is typically 0.008 to 4 moles, preferably 0.4 to 1.5 moles. Alternatively, if other specified methods are used, the ratio of the compound of the present invention to 1 to 4 moles, sterols to 0 to 3 moles, phospholipids to 0 to 2 moles, and polyethylene glycol lipids to 0 to 1 mole in the mixed lipid component is typically 1 to 4 moles, sterols to 0 to 3 moles, phospholipids to 0 to 2 moles, and polyethylene glycol lipids to 0 to 1 mole. A more preferred method when using the compound of the present invention mixed with other lipid components is a ratio of 1 to 1.5 moles of the compound of the present invention, 0 to 1.25 moles of sterols, 0 to 0.5 moles of phospholipids, and 0 to 0.125 moles of polyethylene glycol lipids.

[0096] The compounds of the present invention can be used to manufacture the lipid particles of the present invention. The lipid particles of the present invention refer to complexes that do not contain nucleic acids. The shape of the lipid particles of the present invention is not particularly limited, and includes, for example, complexes formed by the aggregation of the compounds of the present invention in a spherical manner, complexes formed by the aggregation of the compounds of the present invention without a specific shape, complexes formed by dissolving the compounds of the present invention in a solvent, and complexes formed by uniformly or non-uniformly dispersing the compounds of the present invention in a dispersion medium.

[0097] The lipid particles of the present invention (e.g., lipid particles composed of the compounds of the present invention and other structural lipids) can be used to manufacture compositions of the present invention, for example, containing the lipid particles and nucleic acids (especially nucleic acids as substances useful for pharmaceutical or research purposes). The compositions of the present invention can be used as pharmaceuticals or reagents. In the compositions of the present invention, preferably, as many proportions of nucleic acids as possible are encapsulated by the lipid particles (i.e., high encapsulation efficiency).

[0098] "Nucleic acid" can be any molecule, consisting of a nucleotide and a molecule with the same function as that nucleotide. Examples include RNA as a polymer of ribonucleotides, DNA as a polymer of deoxyribonucleotides, polymers of a mixture of ribonucleotides and deoxyribonucleotides, and nucleotide polymers containing nucleotide analogs. It can also be a nucleotide polymer containing nucleic acid derivatives. Furthermore, nucleic acids can be single-stranded or double-stranded. Double-stranded nucleic acids also include those in which one strand hybridizes to the other strand under stringent conditions.

[0099] Nucleotide analogs can be any molecule that modifies ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance compared to RNA or DNA, to increase affinity for complementary nucleic acids, to improve cell penetration, or to make them visible. Nucleotide analogs can be naturally occurring or non-natural molecules; examples include glycosidic-modified nucleotide analogs and phosphodiester-modified nucleotide analogs.

[0100] As a glycosidic modified nucleotide analog, any substance can be any substance formed by adding or replacing any chemical structural substance to or in part or all of the chemical structure of the sugar in a nucleotide. Specific examples include nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidinyl)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, nucleic acid analogs with a morpholino ring replacing the sugar portion (morpholinonucleotides), bridged nucleotides (BNA) having two cyclic structures by introducing a bridging structure into the sugar portion, and more specifically, locked nucleotides formed by bridging the oxygen atom at the 2' position and the carbon atom at the 4' position via a methylene group. Artificial nucleic acids include LNA (acid) and ethylene-bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)], and amide-bridged nucleic acid (AmNA) formed by bridging the carbon atoms at the 2' and 4' positions via an amide bond. Other examples include peptide nucleic acid (PNA) [Acc.Chem.Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J.Am.Chem.Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J.Am.Chem.Soc., 122, 6900 (2000)].

[0101] As a phosphodiester bond modified nucleotide analog, any substance can be any substance that is formed by adding or replacing any chemical substance to part or all of the chemical structure of the phosphodiester bond of the nucleotide. Specific examples include nucleotide analogs that are replaced by thiophosphate bonds, nucleotide analogs that are replaced by N3'-P5' phosphoramide bonds, etc. [Cell Engineering, 16, 1463-1473 (1997)] [RNAi and Antisense Methods, Kodansha (2005)].

[0102] As a nucleic acid derivative, any molecule can be formed by adding other chemical substances to the nucleic acid to improve its resistance to nucleases, stabilize it, increase its affinity for complementary strand nucleic acids, improve its cell penetration, or make it visible. Specific examples include 5'-polyamine derivatives, cholesterol derivatives, steroid derivatives, bile acid derivatives, vitamin derivatives, Cy5 derivatives, Cy3 derivatives, 6-FAM derivatives, and biotin derivatives.

[0103] The nucleic acid used in this invention is not particularly limited. For example, it can be a nucleic acid intended to improve a disease, symptom, injury or pathology, or to alleviate a disease, symptom, injury or pathology or to prevent its onset (sometimes referred to as "treatment of disease, etc." in this specification). It can also be a nucleic acid that, although not helpful for the treatment of disease, is useful for research purposes and is used to regulate the expression of a desired protein.

[0104] Disease-related genes or polynucleotides (sometimes referred to as “disease-related genes” in this specification) can be obtained from, for example, the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Maryland), the National Center for Biotechnology Information, and the National Library of Medicine (Bethesda, Maryland).

[0105] Specific examples of nucleic acids in this invention include, for example, siRNA, miRNA, miRNA mimics, antisense nucleic acids, ribozymes, mRNA, decoy nucleic acids, and aptamers. Preferably, siRNA, mRNA, or analogs or derivatives thereof obtained through artificial modification are preferred nucleic acids.

[0106] In this invention, "siRNA" refers to a double-stranded RNA or analogue of 10 to 30 bases, preferably 15 to 25 bases, containing a complementary sequence. The siRNA preferably has a prominent 1 to 3 bases at its 3' end, more preferably 2 bases. The complementary sequence portion can be completely complementary or may contain non-complementary bases, but is preferably completely complementary.

[0107] The siRNA used in this invention is not particularly limited, and siRNAs for knocking down the expression of disease-related genes, for example, can be used. Disease-related genes refer to any gene or polynucleotide that produces transcriptional or translational products at abnormal levels or in abnormal forms in cells derived from diseased tissues compared to non-disease control tissues or cells. Additionally, siRNAs used to regulate the expression of desired proteins useful for research purposes can also be used as siRNAs in this invention.

[0108] In this invention, "mRNA" refers to RNA containing a base sequence that can be translated into a protein. The mRNA used in this invention is not particularly limited to any mRNA capable of expressing the desired protein within cells. Preferably, the mRNA is useful for pharmaceutical purposes (e.g., for disease treatment) and / or for research purposes. Examples of such mRNAs include those used to induce intracellular expression of marker proteins such as luciferase.

[0109] The diseases mentioned above are not particularly limited, and examples such as those described below can be listed. Except where specific disease examples are listed, examples of disease-related genes are indicated in parentheses. As nucleic acids in this invention, nucleic acids that regulate the expression levels of these disease-related genes (or the proteins they encode) can also be listed.

[0110] (1) Hematologic disorders [anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT), naked lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5), hemorrhagic diseases (TBXA2R, P...] Factor 2RX1, P2X1), Factor H and H-like factor 1 deficiency (HF1, CFH, HUS), Factor V and Factor VIII deficiency (MCFD2), Factor VII deficiency (F7), Factor X deficiency (F10), Factor XI deficiency (F11), Factor XII deficiency (F12, HAF), Factor XIIIA deficiency (F13A1, F13A), Factor XIIIB deficiency (F13B), Fanconi anemia (FANCA, FACA, FA1, FA, FAA, FAAP95) FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FACE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCCJ, PHF9, FANCL, FANCM, KIAA1596), hemophagocytic lymphohistiocytosis (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3), HLH3, FHL3), hemophilia A (F8, F8C, HEMA), hemophilia B (F9, HEMB), bleeding disorders (PI, ATT, F5), leukopenia (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4), sickle cell anemia (HBB), thalassemia (HBA2, HBB, HBD, LCRB, HBA1), etc.

[0111] (2) Inflammatory / immune diseases [AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1), autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A), complex immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4), HIV infection (CCL5, SCYA5, D17S135E, TCP228, IL10, C] SIF, CMKBR2, CCR2, DMKBR5, CCCKR5, CCR5), immunodeficiency (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (IL10, IL-1, IL-13, IL-17, IL-15) -23, CTLA4), severe complex immunodeficiency syndrome (JAK3, JAKL, DCLRE1C, ATREMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4), rheumatoid arthritis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, etc.), Sjögren's syndrome, Behr's disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, discoid ulcers, etc. [Symptoms include lupus erythematosus, Castlemen's disease, ankylosing spondylitis, polymyositis, dermatomyositis, polyarteritis nodosa, mixed connective tissue disease, scleroderma, deep lupus erythematosus, chronic thyroiditis, Graves' disease, autoimmune gastritis, type I and type II diabetes mellitus, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, severe myasthenia gravis, graft-versus-host disease, Addison's disease, abnormal immune response, arthritis, dermatitis, radiation dermatitis, primary biliary cirrhosis, etc.]

[0112] (3) Metabolic / Liver / Kidney Diseases [Amyloid neuropathy (TTR, PALB), amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB), non-alcoholic steatohepatitis and liver fibrosis (COL1A1), cirrhosis (KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988), cystic fibrosis (CFTR, ABCC7, CF, MRP7), glycogen storage disease (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM), hepatocellular adenoma (TCF)] 1. HFN1A, MODY3, liver failure (SCOD1, SCO1), liver lipase deficiency (LIPC), hepatic granulomatous disease (CTNNB1, PDFGRL, PGRL, PRLTS, AXIN1, AXIN, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5), renal medullary cystic disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2), phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS), polycystic liver and polycystic kidney (FCYT, PKHD1, APRKD, PDK1, PDK2, PDK4, PDKTS, PRKCSH, G19P1, PCLD, SEC63), etc.

[0113] (4) Neurological disorders [ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF), Alzheimer's disease (APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), Autism (BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2), Fragile X staining] Syndrome (FMR2, FXR1, FXR2, mGLUR5), Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, DBH, NDUFV2), Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9), integrative disorders (GSK3, 5-HTT, COMT, DRD, SLC6A3, DAOA, DTNBP1), secretion-related disorders (APH-1), etc.

[0114] (5) Eye diseases [Age-related macular degeneration (Abcr, Ccl2, cp, Timp3, cathepsin D, Vldlr, Ccr2), cataracts (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BSFP2, CP] 49, CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1), corneal opacity (APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M 1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD), congenital hereditary flat cornea (KERA, CNA2), glaucoma (MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A), Liber Congenital cataracts (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3), macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2, etc.);

[0115] (6) Neoplastic diseases [malignant tumors, angiogenesis glaucoma, infantile hemangioma, multiple myeloma, chronic sarcoma, metastatic melanoma, Kaposi's sarcoma, angiogenesis, cachexia, metastatic breast cancer, etc., cancer (e.g., colorectal cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumors, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), mesothelioma, pancreatic cancer (e.g., pancreatic duct cancer, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), breast cancer (e.g., invasive...] Invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer, etc.), ovarian cancer (e.g., epithelial ovarian cancer, gonadal germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor, etc.), prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), liver cancer (e.g., primary liver cancer, extrahepatic bile duct cancer, etc.), thyroid cancer (e.g., medullary thyroid carcinoma, etc.), kidney cancer (e.g., renal cell carcinoma, transitional epithelial carcinoma of the renal pelvis and ureter, etc.), uterine cancer, brain tumors (e.g., pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, etc.). Cellular tumors, degenerative astrocytomas, melanoma, sarcoma, bladder cancer, hematologic malignancies including multiple myeloma, pituitary adenoma, glioma, acoustic neuroma, retinal sarcoma, pharyngeal cancer, laryngeal cancer, tongue cancer, thymoma, esophageal cancer, duodenal cancer, colon cancer, rectal cancer, hepatocellular carcinoma, pancreatic endocrine tumors, bile duct cancer, gallbladder cancer, penile cancer, ureteral cancer, testicular tumors, vulvar cancer, cervical cancer, uterine endometrial cancer, uterine sarcoma, choriocarcinoma, vaginal cancer, skin cancer, mycosis fungoides, basal cell carcinoma, soft tissue sarcoma, malignant lymphoma, Hodgkin's lymphoma. Chigger's disease, myelodysplastic syndrome, adult T-cell leukemia, chronic myeloproliferative disorders, pancreatic endocrine tumors, fibrous histiocytoma, leiomyosarcoma, diaphragmatic sarcoma, carcinoma of unknown primary origin, etc., leukemia (e.g., acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, etc.), chronic leukemia (e.g., chronic lymphoblastic leukemia, chronic myeloid leukemia, etc.), myeloproliferative syndrome, etc.), uterine sarcoma (e.g., mixed mesodermal tumor of the uterus, uterine leiomyosarcoma, endometrial stromal tumor, etc.), myelofibrosis, etc.

[0116] The compositions of the present invention, as pharmaceuticals, can be manufactured using pharmaceutically acceptable carriers and by methods known in the field of formulation technology. Examples of dosage forms for the aforementioned pharmaceuticals include, for instance, non-oral formulations (e.g., liquids such as injections) incorporating conventional adjuvants such as buffers and / or stabilizers, and topical formulations such as ointments, creams, liquids, or plasters incorporating conventional pharmaceutical carriers.

[0117] The compositions of the present invention can be used to deliver active ingredients into various types of cells, tissues, or organs. Examples of cells to which the compositions of the present invention can be applied include, for example, spleen cells, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibroblasts, muscle cells (e.g., skeletal muscle cells, cardiomyocytes, myoblasts, myosatellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, obesity cells, leukocytes, neutrophils, basophils, eosinophils, monocytes, megakaryocytes), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or interstitial cells, oocytes, sperm cells or precursor cells that can differentiate into these cells, stem cells (e.g., including artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)), hematopoietic cells, oocytes, and fertilized eggs. Furthermore, as tissues or organs to which the compositions of the present invention can be applied, examples include all tissues or organs in which the aforementioned cells are present, such as the brain, various parts of the brain (e.g., olfactory bulb, platysma, basal ganglia, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidneys, liver, gonads, thyroid gland, gallbladder, bone marrow, adrenal glands, skin, muscles, lungs, digestive tract (e.g., large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testes, testes, ovaries, placenta, uterus, bones, joints, and skeletal muscles. These cells, tissues, or organs may be cancerous cells or cancerous tissues that have become cancerous.

[0118] In one embodiment of the present invention, the composition of the present invention is used to introduce nucleic acids as active ingredients into cells other than cardiomyocytes, tissues or organs other than the heart.

[0119] The compositions of the present invention have particularly excellent efficiency in introducing nucleic acids into cancer cells.

[0120] The compounds, lipid particles, and compositions of the present invention can be used safely and with low toxicity. When using the compositions of the present invention in vivo or as a drug, the composition can be administered to a subject (e.g., human or non-human mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey) (preferably human)) in a manner that delivers an effective amount of nucleic acid to target cells.

[0121] When the compositions of the present invention are used in vivo or as pharmaceuticals, they can be safely administered orally or non-orally (e.g., locally, rectally, or intravenously) by formulations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, and orally disintegrating tablets), powders, granules, capsules (including soft capsules and microcapsules), liquids, lozenges, syrups, emulsions, suspensions, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), topical preparations (e.g., nasal administration, transdermal preparations, and ointments), suppositories (e.g., rectal suppositories and vaginal suppositories), pills, nasal preparations, inhaled preparations, and drops. These formulations can be controlled-release formulations such as immediate-release or sustained-release formulations (e.g., sustained-release microcapsules).

[0122] The method for manufacturing the compound of the present invention will be described below.

[0123] The raw materials and reagents used in each step of the following manufacturing method, as well as the resulting compounds, can each form a salt. Examples of such salts include, for instance, the same salts as those in the compounds of the present invention described above.

[0124] If the compound obtained in each step is a free compound, it can be converted into the target salt using known methods. Conversely, if the compound obtained in each step is a salt, it can be converted into a free form or another type of salt as the target using known methods.

[0125] The compounds obtained in each step can be used directly in the reaction solution for the next reaction or used in the next reaction after obtaining the crude product. Alternatively, the compounds obtained in each step can be separated and / or purified from the reaction mixture using conventional methods such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractionation, and chromatography.

[0126] If the compounds used for the raw materials and reagents in each step are commercially available, the commercially available products can be used directly.

[0127] The reaction time in each step can vary depending on the reagents and solvents used. Unless otherwise specified, it is usually 1 minute to 48 hours, preferably 10 minutes to 8 hours.

[0128] The reaction temperature in each step can vary depending on the reagents and solvents used. Unless otherwise specified, it is usually -78℃ to 300℃, preferably -78℃ to 150℃.

[0129] In each step of the reaction, the pressure may vary depending on the reagents and solvents used. Unless otherwise specified, it is usually 1 to 20 atmospheres, preferably 1 to 3 atmospheres.

[0130] In each step of the reaction, a microwave synthesis apparatus, such as the Initiator manufactured by Biotage, is sometimes used. The reaction temperature varies depending on the reagents and solvents used, and unless otherwise specified, it is typically room temperature to 300°C, preferably room temperature to 250°C, and more preferably 50°C to 250°C. The reaction time varies depending on the reagents and solvents used, and unless otherwise specified, it is typically 1 minute to 48 hours, preferably 1 minute to 8 hours.

[0131] In each step of the reaction, unless otherwise specified, a reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, relative to the matrix. When the reagent is used as a catalyst, a reagent in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents, relative to the matrix is ​​used. When the reagent also serves as a reaction solvent, the amount of reagent used is the same as the solvent amount.

[0132] In each step of the reaction, unless otherwise specified, these reactions are carried out under solvent-free conditions or after being dissolved or suspended in a suitable solvent. Specific examples of solvents may be listed, such as those described in the examples or the following solvents.

[0133] Alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butanol, 2-methoxyethanol, etc.

[0134] Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, cyclopentylmethyl ether, etc.;

[0135] Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.;

[0136] Saturated hydrocarbons: cyclohexane, hexane, heptane, etc.;

[0137] Amides: N,N-dimethylformamide, N-methylpyrrolidone, etc.;

[0138] Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.;

[0139] Nitriles: acetonitrile, etc.;

[0140] Sulfoxides: such as dimethyl sulfoxide;

[0141] Aromatic organic bases: pyridine, etc.;

[0142] Acid anhydrides: acetic anhydride, etc.;

[0143] Organic acids: formic acid, acetic acid, trifluoroacetic acid, etc.;

[0144] Inorganic acids: hydrochloric acid, sulfuric acid, etc.;

[0145] Esters: ethyl acetate, isopropyl acetate, etc.;

[0146] Ketones: acetone, methyl ethyl ketone, etc.;

[0147] water.

[0148] The above solvents can be used by mixing two or more in appropriate proportions.

[0149] When a base is used in the reaction at each step, the base shown below or the base described in the examples can be used.

[0150] Inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.;

[0151] Alkaline salts: sodium carbonate, calcium carbonate, sodium bicarbonate, etc.

[0152] Organic bases: triethylamine, diethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, imidazole, piperidine, etc.

[0153] Metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, etc.;

[0154] Alkali metal hydrides: sodium hydride, etc.;

[0155] Metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilamide, etc.;

[0156] Organic lithium compounds: n-butyllithium, sec-butyllithium, etc.

[0157] When an acid or acidic catalyst is used in the reaction at each step, the acid or acidic catalyst shown below, or the acid or acidic catalyst described in the examples, may be used.

[0158] Inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.;

[0159] Organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.;

[0160] Lewis acids: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous ferric chloride, etc.

[0161] Unless otherwise specified, the reactions in each step are carried out according to known methods, such as those described in the 5th Edition of the Experimental Chemistry Lecture Series, Volumes 13 - 19 (edited by the Chemical Society of Japan); the New Experimental Chemistry Lecture Series, Volumes 14 - 15 (edited by the Chemical Society of Japan); Revised Precision Organic Chemistry, 2nd Edition (L.F. Tietze, Th. Eicher, Nankodo); Revised Organic Reactions, Their Structures and Key Points (written by Hideo Togo, Kodansha); Organic Syntheses Collective, Volumes I - VII (John Wiley & Sons Inc); Modern Organic Synthesis in the Laboratory - A Collection of Standard Experimental Procedures (written by Jie Jack Li, Oxford University Press); Comprehensive Heterocyclic Chemistry III, Volumes 1 - 14 (Elsevier Japan K.K.); Organic Synthesis Strategies Learned from Named Reactions (translated by Kiyoshi Tomioka, published by Kagaku Dojin); Comprehensive Organic Transformations (VCH Publishers), published in 1989, etc., or the methods described in the examples.

[0162] In each step, the protection or deprotection reactions of functional groups are carried out according to known methods, such as those described in "Protective Groups in Organic Synthesis", 4th Edition (written by Theodora W. Greene, Peter G.M. Wuts), published by Wiley - Interscience in 2007; "Protecting Groups", 3rd Edition (written by P.J. Kocienski), published by Thieme in 2004, etc., or the methods described in the examples.

[0163] Examples of protecting groups for hydroxyl groups such as alcohols and phenolic hydroxyl groups include, for example, ether - type protecting groups such as methoxymethyl ether, benzyl ether, p - methoxybenzyl ether, tert - butyldimethylsilyl ether, tert - butyldiphenylsilyl ether, tetrahydropyranyl ether; carboxylic acid ester - type protecting groups such as acetate; sulfonic acid ester - type protecting groups such as methanesulfonate; carbonate - type protecting groups such as tert - butyl carbonate, etc.

[0164] Examples of protecting groups for the carbonyl group of aldehydes include, for example, acetal - type protecting groups such as dimethyl acetal; cyclic acetal - type protecting groups such as cyclic 1,3 - dioxane, etc.

[0165] Examples of protecting groups for the carbonyl group of ketones include: ketal-type protecting groups such as dimethyl ketal; cyclic ketal-type protecting groups such as cyclic 1,3-dioxane; oxime-type protecting groups such as O-methyl oxime; and hydrazone-type protecting groups such as N,N-dimethylhydrazone.

[0166] Examples of carboxyl protecting groups include ester-type protecting groups such as methyl esters and amide-type protecting groups such as N,N-dimethylamide.

[0167] Examples of protecting groups for thiols include ether-type protecting groups such as benzyl thioether; and ester-type protecting groups such as thioacetate, thiocarbonate, and thiocarbamate.

[0168] Protecting groups for aromatic heterocycles such as amino, imidazole, pyrrole, and indole include, for example: urethane-type protecting groups such as benzyl carbamate; amide-type protecting groups such as acetamide; alkylamine-type protecting groups such as N-triphenylmethylamine; and sulfonamide-type protecting groups such as methanesulfonamide.

[0169] The removal of the protecting group can be carried out using known methods, such as using acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkyl halosyl silanes (e.g., trimethyliodosilane, trimethylbromosilane), reduction methods, etc.

[0170] In reduction reactions at each step, the reducing agents used can include: lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), sodium borohydride, tetramethyltriacetoxyammonium borohydride, and other metal hydrides; boranes such as borane tetrahydrofuran complexes; Raney nickel; Raney cobalt; hydrogen; formic acid, etc. For example, Raney nickel or Raney cobalt can be used in the presence of hydrogen or formic acid. In the reduction of carbon-carbon double or triple bonds, methods using catalysts such as palladium-carbon or Lindlar catalysts are employed.

[0171] When oxidation reactions are carried out in each step, the oxidants used can be listed as follows: peracids such as m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, and tert-butylperoxide; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; hypochlorites such as sodium hypochlorite; periodates such as sodium periodate; high-valent iodine reagents such as iodobenzoylbenzene; manganese-containing reagents such as manganese dioxide and potassium permanganate; lead-containing reagents such as lead tetraacetate; chromium-containing reagents such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), and Jones' reagent; halogen compounds such as N-bromosuccinimide (NBS); oxygen; ozone; sulfur trioxide-pyridine complexes; osmium tetroxide; selenium dioxide; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), etc.

[0172] In cases where free radical cyclization reactions are carried out in each step, the free radical initiators used can include: azo compounds such as azobisisobutyronitrile (AIBN); water-soluble free radical initiators such as 4-4'-azobis-4-cyanopentanoic acid (ACPA); triethylboron in the presence of air or oxygen; and benzoyl peroxide. Additionally, the free radical reaction reagents used can include tributyltinane, tris(trimethylsilyl)silane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, and samarium iodide.

[0173] In the case of carrying out the Wittig reaction in each step, alkylphosphines can be listed as Wittig reagents. Alkylphosphines can be produced by known methods, for example, by... It is prepared by reacting salt with a strong base.

[0174] When the Horner-Emmons reaction is carried out in each step, the reagents used can include phosphoryl acetate esters such as dimethylphosphonoacetate and diethylphosphonoacetate; bases such as alkali metal hydrides and organolithium compounds.

[0175] When performing a Friedel-Crafts reaction in each step, the reagents used can include Lewis acids, acyl chlorides, or alkylating agents (e.g., haloalkanes, alcohols, alkenes, etc.). Alternatively, organic acids or inorganic acids can be used instead of Lewis acids, and acid anhydrides such as acetic anhydride can be used instead of acyl chlorides.

[0176] In the case of aromatic nucleophilic substitution reactions in each step, nucleophiles (e.g., amines, imidazoles, etc.) and bases (e.g., basic salts, organic bases, etc.) can be used as reagents.

[0177] In cases where nucleophilic addition reactions, nucleophilic 1,4-addition reactions (Michael addition reactions), or nucleophilic substitution reactions utilizing carbanions are carried out in each step, the bases used to generate carbanions can include organolithium compounds, metal alkoxides, inorganic bases, and organic bases.

[0178] When the Grignard reaction is carried out in each step, Grignard reagents can be listed as aryl magnesium halides such as phenyl magnesium bromide, and alkyl magnesium halides such as methyl magnesium bromide and isopropyl magnesium bromide. Grignard reagents can be prepared by known methods, for example, by reacting haloalkanes or haloaromatics with metallic magnesium using ethers or tetrahydrofurans as solvents.

[0179] When the Knoevenagel condensation reaction is carried out in each step, active methylene compounds (e.g., malonic acid, diethyl malonate, malononitrile, etc.) sandwiched between two electron-withdrawing groups and bases (e.g., organic bases, metal alkoxides, inorganic bases) can be used as reagents.

[0180] When the Vilsmeier-Haack reaction is carried out in each step, sulfonyl chlorides and amide derivatives (e.g., N,N-dimethylformamide, etc.) can be used as reagents.

[0181] When performing azidation reactions of alcohols, haloalkanes, and sulfonates in various steps, examples of azidating agents used include diphenyl azidophosphate (DPPA), trimethylsilane azide, and sodium azide. For instance, in the case of azidation of alcohols, methods using diphenyl azidophosphate and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) and methods using trimethylsilane azide and Lewis acids are available.

[0182] In cases where the amination reaction involves reduction in each step, reducing agents used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, and formic acid. When the matrix is ​​an amine compound, carbonyl compounds used include aldehydes such as acetaldehyde and ketones such as cyclohexanone, in addition to paraformaldehyde. When the matrix is ​​a carbonyl compound, primary amines such as ammonia and methylamine, and secondary amines such as dimethylamine, can be used.

[0183] When performing photoelongation reactions in each step, azodicarboxylic acid esters (e.g., diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.) and triphenylphosphine can be used as reagents.

[0184] When esterification, amidation, or ureaization reactions are carried out in various steps, the reagents used can include acyl halides such as acyl chlorides and acyl bromides; and activated carboxylic acids such as acid anhydrides, active esters, and sulfate esters. Activators of carboxylic acids can include: carbodiimide condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD); triazine condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride-n-hydrate (DMT-MM); carbonate condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenyl azide phosphate (DPPA); and benzotriazol-1-yloxy-tris(dimethylamino) Salts (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Xiangshan reagent); thionyl chloride; lower alkyl esters of haloformic acids such as ethyl chloroformate; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); sulfuric acid; or combinations thereof. When using carbodiimide-based condensing agents, additives such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), and dimethylaminopyridine (DMAP) can be further added to the reaction.

[0185] In the case of coupling reactions in each step, the metal catalysts used can include: palladium compounds such as palladium(II) acetate, tetra(triphenylphosphine)palladium(O), dichlorobis(triphenylphosphine)palladium(II), dichlorobis(triethylphosphine)palladium(II), tris(dibenzylpyrone)dipalladium(O), 1,1'-bis(diphenylphosphino)ferrocene palladium(II) chloride, and palladium(II) acetate; nickel compounds such as tetra(triphenylphosphine)nickel(O); rhodium compounds such as tri(triphenylphosphine)rhodium(III) chloride; cobalt compounds; copper compounds such as copper oxide and copper iodide(I); and platinum compounds. A base can be further added to the reaction; such bases include inorganic bases and basic salts.

[0186] When the sulfur carbonylation reaction is carried out in each step, phosphorus pentasulfide can be used as a representative sulfur carbonylating agent. However, in addition to phosphorus pentasulfide, reagents with the 1,3,2,4-dithiaphosphazene-2,4-disulfide structure, such as 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiaphosphazene-2,4-disulfide (Lowesson reagent), can also be used.

[0187] In the case of a Wohl-Ziegler reaction in each step, halogenating agents used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, and thioyl chloride. Furthermore, the reaction can be accelerated by adding free radical initiators such as heat, light, benzoyl peroxide, and azobisisobutyronitrile.

[0188] In cases where the halogenation of the hydroxyl group is carried out in each step, halogenating agents used can include acyl halides of hydrohalic acids and inorganic acids. Specifically, in chlorination, hydrochloric acid, thionyl chloride, and phosphorus oxychloride can be used, and in bromination, 48% hydrobromic acid can be used. Alternatively, a method can be used to obtain haloalkane bodies from alcohols via the reaction of triphenylphosphine with carbon tetrachloride or carbon tetrabromide. Alternatively, a two-stage reaction can be used to synthesize haloalkane bodies, involving the conversion of the alcohol to a sulfonate ester followed by reaction with lithium bromide, lithium chloride, or sodium iodide.

[0189] When the Arbuzov reaction is carried out in each step, the reagents used can be listed as follows: haloalkanes such as ethyl bromoacetate; phosphites such as triethyl phosphite and tri(isopropyl) phosphite.

[0190] When sulfonyl esterification reactions are carried out in each step, the sulfonating agents used can include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride, etc.

[0191] In the case of hydrolysis reactions in each step, acids or bases can be used as reagents. Additionally, in the case of acid hydrolysis of tert-butyl esters, formic acid, triethylsilane, etc., are sometimes added to reduce and capture the byproduct tert-butyl cations.

[0192] In cases where dehydration reactions are carried out in each step, the dehydrating agents used can include sulfuric acid, phosphorus pentoxide, phosphorus oxychloride, N,N'-dicyclohexylcarbodiimide, alumina, polyphosphoric acid, etc.

[0193] Compound (I) can be manufactured, for example, by the methods described below. In this invention, particularly during esterification, a compound (I) with the desired structure can be synthesized by using suitable starting materials corresponding to the structure of the target compound (I). Furthermore, salts of compound (I) can be obtained by suitable mixing with inorganic bases, organic bases, organic acids, or basic or acidic amino acids.

[0194] Preparation method A (when L is -C(O)O-)

[0195]

[0196] Preparation method B (in the case where L is -NHC(O)O-)

[0197]

[0198] Method C

[0199]

[0200] In the above formula, P 1 P 2 P 3 P 4 P 5 and P 6 Each independently represents a protective base.

[0201] Representation of compound (A):

[0202] Representation of compound (B): R1 indicates

[0203] Representation of compound (C): R2 indicates

[0204] Hereinafter, a method for manufacturing a composition for nucleic acid introduction (transfection) containing lipid particles of the compound of the present invention and a nucleic acid as an active ingredient will be described.

[0205] The lipid particles of the present invention can be manufactured using known methods for preparing lipid particles from lipid components by mixing the compound of the present invention, which is a cationic lipid, with other lipid components as needed. For example, the above-mentioned (mixed) lipid components can be dissolved in an organic solvent, and the resulting organic solvent solution can be mixed with water or a buffer solution (e.g., emulsification) to produce a lipid particle dispersion. The above mixing can be performed using a microfluidic mixing system (e.g., the NanoAssemblr device (Precision NanoSystems)). The resulting lipid particles can be desalted or dialyzed and sterilized by filtration. In addition, pH and osmotic pressure can be adjusted as needed.

[0206] Compound (I) can be obtained in various structures by combining the definitions of n1, n2, n3, L, Ra, Rb, Rc, Rd, Re, and Rf in formula (I). In the manufacture of lipid particles, compound (I) can be used alone as a single compound with a specific structure, or it can be used as a mixture of multiple compounds with different structures.

[0207] As "other lipid components," examples include structural lipids as described above, such as sterols, phospholipids, and polyethylene glycol lipids. "Other lipid components" are used, for example, in amounts of 0.008 to 4 moles relative to 1 mole of the compound of the present invention. The compound of the present invention is preferably used in combination with other lipid components (particularly cholesterol, phosphatidylcholine, and polyethylene glycol lipids). A preferred method for using the compound of the present invention in combination with other lipid components is a mixture of 1 to 4 moles of the compound of the present invention, 0 to 3 moles of sterols, 0 to 2 moles of phospholipids, and 0 to 1 mole of polyethylene glycol lipids. A more preferred method for using the compound of the present invention in combination with other lipid components is a mixture of 1 to 1.5 moles of the compound of the present invention, 0 to 1.25 moles of sterols, 0 to 0.5 moles of phospholipids, and 0 to 0.125 moles of polyethylene glycol lipids.

[0208] The concentration of the compound of the present invention in the above-mentioned organic solvent solution, or the mixture of the compound of the present invention with other lipid components, is preferably 0.5 to 100 mg / mL.

[0209] Examples of organic solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or mixtures thereof. Organic solvents may contain 0–20% water or buffer solutions.

[0210] Examples of buffer solutions include acidic buffers (e.g., acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer) and neutral buffers (e.g., 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, phosphate-buffered saline (PBS)).

[0211] When using a microfluidic mixing system, it is preferable to mix 1 to 5 volumes of water or buffer solution relative to 1 volume of organic solvent solution. Furthermore, in this system, the flow rate of the mixture (a mixture of organic solvent solution and water or buffer solution) is, for example, 0.01 to 20 mL / min, preferably 0.1 to 10 mL / min, and the temperature is, for example, 5 to 60°C, preferably 15 to 45°C.

[0212] The compositions of the present invention can be prepared as lipid particle dispersions containing nucleic acids by pre-adding nucleic acids to water or buffer solution during the preparation of lipid particles or lipid particle dispersions. The nucleic acids are preferably added in such a manner that the concentration of nucleic acids in the water or buffer solution is, for example, 0.01–20 mg / mL, preferably 0.05–2.0 mg / mL.

[0213] Alternatively, the compositions of the present invention can also be manufactured as lipid particle dispersions containing active ingredients by mixing lipid particles or lipid particle dispersions with nucleic acids or aqueous solutions thereof using known methods. Lipid particle dispersions can be prepared by dispersing lipid particles in a suitable dispersion medium. Furthermore, aqueous solutions of the active ingredients can be prepared by dissolving the active ingredients in a suitable solvent.

[0214] The content of the compound of the present invention in the composition of the present invention, excluding the dispersion medium and solvent, is generally 10 to 70% by weight, preferably 40 to 70% by weight.

[0215] The nucleic acid content in the compositions of the present invention, excluding the dispersion medium and solvent, is typically 0.1 to 25% by weight, preferably 1 to 20% by weight.

[0216] The dispersion medium of lipid particle dispersions or dispersions containing the composition can be replaced with water or buffer solution by dialysis. Dialysis is performed using an ultrafiltration membrane with a molecular weight cutoff of 10–20 K at 4°C to room temperature. Dialysis can be repeated. Tangential flow filtration (TFF) can be used for dispersion medium replacement. Furthermore, after dispersion medium replacement, pH and osmotic pressure can be adjusted as needed. Examples of pH adjusters include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. Examples of osmotic pressure adjusters include inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol; and sugars such as glucose, fructose, lactose, and sucrose. The pH is typically adjusted to 6.5–8.0, preferably to 7.0–7.8. The osmotic pressure is preferably adjusted to 250–350 Osm / kg.

[0217] The compositions of the present invention may contain components other than lipid particles and nucleic acids, as needed. Examples of such components include, for instance, appropriate amounts of stabilizers and antioxidants.

[0218] As a stabilizer, there are no particular limitations; examples include sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose.

[0219] Examples of antioxidants include ascorbic acid, uric acid, cysteine, tocopherol isoforms (vitamin E, tocopherol α, β, γ, δ isomers, etc.), EDTA, and cysteine.

[0220] Hereinafter, analytical methods for lipid particles containing the compounds of the present invention, and for compositions containing the lipid particles and nucleic acids as active ingredients, are described.

[0221] The particle size of the lipid particles (in the composition) can be determined using known methods. For example, a particle size measuring device based on dynamic light scattering measurement technology, such as the Zetasizer Nano ZS (Malvern Instruments), can be used to calculate the Z-mean particle size through cumulative analysis of the autocorrelation function. The particle size (mean particle size) of the lipid particles (in the composition) is, for example, 10–200 nm, preferably 60–170 nm.

[0222] The concentration and encapsulation efficiency of nucleic acids (e.g., siRNA, mRNA) in the compositions of the present invention can be determined using known methods. For example, using Quant-iT... TMRibo Green (registered trademark) (Invitrogen) is used to fluorescently label nucleic acids, and their fluorescence intensity is measured to determine the concentration and encapsulation efficiency. The concentration of nucleic acids in the composition can be calculated using a standard curve prepared from an aqueous solution of nucleic acids with known concentrations. The encapsulation efficiency can be calculated based on the difference in fluorescence intensity caused by the presence or absence of Triton-X100 (a surfactant used to disintegrate lipid particles). It should be noted that the concentration of nucleic acids in the composition refers to the total concentration of nucleic acids encapsulated by lipid particles and unencapsulated nucleic acids, and the encapsulation efficiency refers to the proportion of nucleic acids encapsulated by lipid particles out of all nucleic acids in the composition.

[0223] Example

[0224] The present invention will be further described in detail through the following embodiments, manufacturing examples and test examples, but the present invention is not limited thereto and may be varied without departing from the scope of the present invention.

[0225] In the following examples, "room temperature" generally refers to about 10°C to about 35°C. Ratios shown in mixed solvents are volume ratios unless otherwise specified. % are weight percentages unless otherwise specified.

[0226] Unless otherwise specified, the elution in the column chromatography of the examples was performed under TLC (Thin Layer Chromatography) observation. For TLC observation, a Merck 60F plate was used as the TLC plate. 254 The developing solvent is the same solvent used as the elution solvent in column chromatography. Additionally, detection is performed using a UV detector, and TLC colorimetric reagents are used for observation as needed. In silica gel column chromatography, aminopropylsilane-bonded silica gel is used when labeled NH, and 3-(2,3-dihydroxypropoxy)propylsilane-bonded silica gel is used when labeled Diol. In preparative HPLC (high performance liquid chromatography), octadecyl-bonded silica gel is used when labeled C18. Unless otherwise specified, ratios shown for elution solvents are volume ratios.

[0227] 1 H NMR was measured using Fourier transform NMR. 1 ¹H NMR analysis was performed using software such as ACD / SpecManager (trade name). Peaks with very flat proton frequencies, such as those for hydroxyl and amino groups, were sometimes not recorded.

[0228] MS measurements were performed by LC / MS and MALDI / TOFMS. As an ionization method, ESI, APCI, or MALDI were used. CHCA was used as the matrix. Found values ​​are recorded. A molecular ion peak is usually observed, but sometimes as fragment ions. In the case of salts, molecular ion peaks, cation species, anion species, or fragment ion peaks of the free ion are usually observed.

[0229] In the following embodiments, the following abbreviations are used.

[0230] MS: Mass Spectrometry

[0231] M: molar concentration

[0232] N: Equivalent concentration

[0233] CDCl3: Deuterated chloroform

[0234] DMSO-d6: Deuterated dimethyl sulfoxide

[0235] 1 H NMR: Proton nuclear magnetic resonance

[0236] LC / MS: Liquid Chromatography-Mass Spectrometry

[0237] ESI: Electrospray ionization

[0238] APCI: Atmospheric Pressure Chemical Ionization

[0239] MALDI: Matrix-assisted laser desorption / ionization

[0240] TOFMS: Time-of-flight mass spectrometry

[0241] CHCA: α-Cyano-4-hydroxycinnamic acid

[0242] DMF: N,N-dimethylformamide

[0243] THF: Tetrahydrofuran

[0244] DMAP: 4-Dimethylaminopyridine

[0245] TBAF: Tetrabutylammonium fluoride

[0246] DIBAL-H: Diisobutylaluminum hydride

[0247] DBU: 1,8-diazabicyclo[5,4,0]undec-7-ene

[0248] [Example 1] 4-Heptylundecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester

[0249] A) Methyl heptaylnonanoate

[0250] A suspension of 3.78 g of 60% sodium hydride (containing mineral oil) in 100 mL of dehydrated DMF was stirred for 10 minutes under a nitrogen stream. Then, 5.0 g of dimethyl malonate was added dropwise at below 10 °C. After stirring for 10 minutes at this temperature, 18.3 mL of 1-iodoheptane was added dropwise, and the mixture was heated to room temperature. After 4 hours, the reaction mixture was neutralized with 6N hydrochloric acid, diluted with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was dissolved in 75 mL of DMSO, and 0.68 mL of water and 3.21 g of lithium chloride were added. The mixture was heated to 165 °C. After stirring at this temperature for 16 hours, water was added, and the mixture was diluted with ethyl acetate. After washing twice with saturated brine, the mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (8.14 g).

[0251] 1 H NMR(500MHz, CDCl3)δppm 0.84-0.90(6H,m),1.20-1.32(20H,m),1.36-1.47(2H,m),1.54-1.62(2H,m),2.33(1H,tt,J=9.0,5.4Hz),3.67(3H,s)

[0252] B) 2-Heptylnonane-1-ol

[0253] Under a nitrogen atmosphere and ice-cold conditions, a solution of methyl 2-heptylnonanoate (7.67 g) in dehydrated THF (92 mL) was added dropwise to a suspension of lithium aluminum hydride (2.15 g) in dehydrated THF (50 mL), and the mixture was stirred at below 10 °C for 1 hour. The mixture was then heated to room temperature and stirred for 3 hours. After cooling again to below 10 °C, sodium sulfate decahydrate was added little by little. The solution was diluted with ethyl acetate, filtered through diatomaceous earth to remove the insoluble matter, and the solvent was removed by distillation under reduced pressure to give the title compound (6.91 g).

[0254] 1H NMR(500MHz, CDCl3)δppm 0.86-0.91(6H,m),1.16-1.34(25H,m),1.41-1.49(1H,m),3.54(2H,t,J=5.2Hz)

[0255] C)2-Heptylnonanal

[0256] Under a nitrogen atmosphere, a solution of 4.9 mL of oxalyl chloride in 30 mL of dichloromethane was cooled to -70 °C, and while maintaining the temperature below -60 °C, a solution of 6.1 mL of dichloromethane in 30 mL of dimethyl sulfoxide was added dropwise. After stirring at -70 °C for 15 minutes, a solution of 6.9 g of 2-heptylnonane-1-ol in 25 mL of dichloromethane was added dropwise while maintaining the temperature below -60 °C. After stirring at -70 °C for 2 hours, triethylamine (23.8 mL) was added, and the temperature was raised to room temperature. A saturated aqueous solution of ammonium chloride was added for separation, and the solution was dried over anhydrous sodium sulfate. The solvent was then removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (6.06 g).

[0257] 1 H NMR (500MHz, CDCl3) δppm 0.85-0.92(6H,m),1.19-1.33(20H,m),1.37-1.47(2H,m),1.56-1.65(2H,m),2.18-2.25(1H,m),9.55(1H,d,J=3.2Hz)

[0258] D) Ethyl-4-heptayl undec-2-enoate

[0259] A suspension of 1.4 g sodium hydride (containing mineral oil) in 70 mL of dehydrated THF was stirred for 10 minutes under a nitrogen atmosphere. Then, 16.8 g of ethyl (diethoxyphosphoryl)acetate was added dropwise below 10 °C. After stirring at this temperature for 10 minutes, a solution of 6.0 g of 2-heptylnonanal in 60 mL of dehydrated THF was added dropwise, and the mixture was heated to room temperature. After stirring for a short time, the temperature was raised to 50 °C. After stirring for 6 hours, the reaction mixture was cooled to below 5 °C, water was added, and the mixture was diluted with ethyl acetate. After washing twice with saturated brine and drying with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (5.4 g).

[0260] 1H NMR(500MHz,CDCl3)δppm 0.87(6H,t,J=6.0Hz),1.16-1.34(25H,m),1.37-1.45(2H,m)2.07-2.15(1H,m ),4.19(2H,q,J=7.5Hz),5.75(1H,d,J=16.0Hz),6.75(1H,dd,J=16.0,10.0Hz)

[0261] E)4-Hepylundecanoic acid

[0262] 10% palladium on carbon (1.08 g) was added to a 100 mL ethanol solution of ethyl-4-heptyl undec-2-enoate (5.40 g) at room temperature, and the mixture was stirred for 20 hours under a hydrogen atmosphere. After the reaction, the palladium on carbon was removed by filtration, and the solvent was removed by distillation under reduced pressure. A 20 mL ethanol solution of 6.38 mL of 8N sodium hydroxide aqueous solution was added to the residue, and the mixture was stirred at 60 °C for 5 hours. The solvent was removed by distillation under reduced pressure, and the mixture was acidified with 6N hydrochloric acid. The residue was diluted with hexane, washed once with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to give the title compound (4.73 g).

[0263] 1 H NMR(500MHz, CDCl3)δppm 0.88(6H,t,J=7.5Hz),1.17-1.41(25H,m),1.16-1.34(2H,m),2.22-2.34(2H,m)

[0264] F)2-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol

[0265] A solution of tert-butylchlorodiphenylsilane (5.1 g) in DMF (10 mL) was added to a mixture of 2,2-bis(hydroxymethyl)propane-1,3-diol (5.0 g), 1H-imidazole (2.5 g), and DMF (200 mL) at room temperature. After stirring for 18 hours, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed three times with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (6.4 g).

[0266] 1H NMR (500MHz, CDCl3) δppm 1.07(9H,s),2.34(3H,t,J=5.5Hz),3.67(2H,s),3.74(6H,d,J=5.7Hz),7.39-7.48(6H,m),7.63-7.67(4H,m)

[0267] G)(5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol

[0268] At room temperature, 89 mg of p-toluenesulfonic acid monohydrate was added to a solution of 3.5 g of 2-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol and 1.5 g of 2,2-dimethoxypropane in 35 mL of acetone. After stirring for 2 hours, dilute ammonia was added to the reaction mixture for neutralization, and then the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.7 g).

[0269] 1 H NMR (500MHz, CDCl3) δppm 1.07(9H,s),1.27(3H,s),1.41(3H,s),2.12-2.18(1H,m),3.69-3.78(8H,m),7.38-7.47(6H,m),7.65-7.69(4H,m)

[0270] H)4-Heptylundecanoic acid (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0271] At 50 °C, 2.47 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to a DMF (30 mL) solution of (5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol (3.56 g), DMAP (1.37 g), and 4-heptylundecanoic acid (3.18 g). After stirring for 6 hours, ethyl acetate was added to the reaction mixture, which was washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. At room temperature, a 1 M, 10.3 mL solution of TBAF in THF was added to a 20 mL solution of the resulting residue (6.15 g). After stirring for 4 hours, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.34 g).

[0272] 1 H NMR(500MHz,CDCl3)δppm 0.88(6H,t,J=6.9Hz),1.22-1.32(25H,m),1.42(6H,s),1.57-1.62(2H,m) ,2.30-2.35(2H,m),3.48(2H,d,J=6.6Hz),3.71-3.73(4H,m),4.25(2H,s)

[0273] I) 4-Heptylundecanoic acid (5-(((5-dimethylamino)pentanoyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0274] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.94 g) was added to a DMF (30 mL) solution of 4-heptylundecanoic acid (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl) methyl ester (1.2 g), DMAP (0.94 g), and 5-(dimethylamino)valerate (0.74 g) at 40 °C. After stirring for 4 hours, ethyl acetate was added to the reaction mixture, which was washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (NH4+, ethyl acetate / hexane) to give the title compound (1.37 g).

[0275] 1H NMR(500MHz,CDCl3)δppm 0.88(6H,t,J=6.9Hz),1.20-1.32(25H,m),1.42(6H,s),1.45-1.52(2H,m),1.54-1.67(4H,m) ,2.21(6H,s),2.23-2.31(4H,m),2.35(2H,t,J=7.5Hz),3.75(4H,s),4.11(2H,s),4.12(2H,s)

[0276] J)4-Heptylundecanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester

[0277] Acetic acid (6.85 mL) and water (3.43 mL) were added to 1.37 g of 4-heptylundecanoic acid (5-(((5-dimethylamino)pentanoyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester. After stirring at 70 °C for 2 hours, the solvent was removed by distillation under reduced pressure. Ethyl acetate and saturated sodium bicarbonate aqueous solution were added to the residue, and the mixture was stirred for 2 hours. After washing twice with water and drying with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure. A solution of DMF (4 mL) containing DMAP (478 mg) and octanoic acid (327 mg) was added to the obtained residue (400 mg), followed by the addition of 478 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride at 50 °C. After stirring for 4 hours, ethyl acetate was added to the reaction mixture. The mixture was washed twice with saturated sodium carbonate aqueous solution and once with saturated brine. After drying with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (NH4+, ethyl acetate / hexane) to give the title compound (300 mg).

[0278] 1 H NMR(500MHz,CDCl3)δppm 0.86-0.91(12H,m),1.15-1.34(45H,m),1.45-1.52(2H,m),1.53-1.66(4H,m),2.20(6H,s),2.23-2.36(10H,m),4.11(8H,s)

[0279] [Example 8] Bis(2-hexyloctanoic acid)2-(((6-(dimethylamino)hexanoyl)oxy)methyl-2-((octanoyl)methyl)propane-1,3-diyl ester

[0280] A) 2-Hexyloctanoic acid

[0281] A suspension of 60% sodium hydride (containing mineral oil) in dehydrated DMF (90 mL) was stirred for 10 minutes under a nitrogen atmosphere. Then, dimethyl malonate (5.0 g) was added dropwise at below 10°C. After stirring for 10 minutes at this temperature, 1-iodohexane (16.8 mL) was added dropwise, and the mixture was heated to room temperature. After 8 hours, acetic acid (1 mL) was added to the reaction mixture, followed by dilution with ethyl acetate, washing twice with water and once with saturated brine, drying with anhydrous sodium sulfate, and removing the solvent by distillation under reduced pressure. The residue was dissolved in ethanol (EtOH) (80 mL), and 25 mL of 8N sodium hydroxide aqueous solution was added. The mixture was stirred at 60°C for 6 hours. After neutralization with 6N hydrochloric acid, the mixture was diluted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, and removing the solvent by distillation under reduced pressure. The residue was heated at 160°C for 1.5 hours, cooled to room temperature, and purified by silica gel column chromatography (ethyl acetate / hexane) to obtain the title compound (7.45 g).

[0282] 1 H NMR(500MHz, CDCl3)δppm 0.83-0.92(6H,m),1.22-1.35(16H,m),1.38-1.52(2H,m),1.56-1.67(2H,m),2.34(1H,ddd,J=8.7,5.4,3.3Hz)

[0283] B)(2-(4-methoxyphenyl)-1,3-dioxane-5,5-diyl)diethanol

[0284] A 2.0 L aqueous solution of 506 g of bis(hydroxymethyl)propane-1,3-diol was stirred at 50 °C. Concentrated hydrochloric acid (18 mL) was added, followed by dropwise addition of 474 mL of p-methoxybenzaldehyde over 3 hours at approximately 30 °C. The reaction mixture was then brought to 25 °C and stirred for 5 hours. 120 mL of 2N sodium hydroxide aqueous solution was added, and the mixture was stirred for 1 hour. The crystals were filtered, washed with water, and recrystallized from ethyl acetate / hexane to give the title compound (769 g).

[0285] 1 H NMR(500MHz,DMSO-d6)δppm 3.24(2H,d,J=5.0Hz), 3.67(2H,d,J=5.4Hz), 3.74(3H,s), 3.77(2H,d,J=11.3Hz), 3.88(2H,t,J=11.3Hz ),4.53(1H,t,J=5.4Hz),4.62(1H,t,J=5.0Hz),5.34(1H,s),6.90(2H,d,J=8.9Hz),7.33(2H,d,J=8.9Hz)

[0286] C)9-(4-methoxyphenyl)-3,3-dimethyl-2,4,8,10-tetraoxaspiro[5.5]undecane

[0287] Pyridine was added to an 8 mL solution of (2-(4-methoxyphenyl)-1,3-dioxane-5,5-diyl)diethanol (2.00 g) and 2,2-dimethoxypropane (2.46 g) in DMF at room temperature. p-Toluenesulfonate (20 mg). After stirring for 4 hours, the reaction mixture was diluted with ethyl acetate, washed twice with saturated sodium bicarbonate aqueous solution and twice with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was recrystallized from ethyl acetate / hexane to give the title compound (1.62 g).

[0288] 1 H NMR(500MHz,DMSO-d6)δppm 1.34(6H,s),3.33(2H,s),3.63(2H,d,J=11.7Hz),3.74(3H,s),3.99(2H,s),4. 12(2H,d,J=11.7Hz),5.37(1H,s),6.90(2H,d,J=8.8Hz),7.34(2H,d,J=8.8Hz)

[0289] D)(5-(((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol

[0290] 1.5 M DIBAL-H solution (60 mL) was added dropwise to a suspension of 22.0 g of 9-(4-methoxyphenyl)-3,3-dimethyl-2,4,8,10-tetraoxaspiro[5.5]undecane in toluene (200 mL) at 5–20 °C, and the mixture was stirred at 15 °C for 3 hours. After adding 22 mL of methanol, 100 mL of 2 N sodium hydroxide aqueous solution and 200 mL of 4 N sodium hydroxide aqueous solution were added dropwise. After stirring for 1.5 hours, the toluene layer was separated and washed with 5% brine. The solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (14.7 g).

[0291] 1H NMR(500MHz,DMSO-d6)δppm 1.29(3H,s),1.29(3H,s),3.35(2H,s),3.39(2H,d,J=5.1Hz),3.61(4H,s),3.74( 3H,s),4.38(2H,s),4.59(1H,t,J=5.1Hz),6.90(2H,dlike,J=7.5Hz),7.24(2H,d like,J=7.5Hz)

[0292] E) Octanoic acid (5-((((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0293] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.94 g) was added to a DMF (20 mL) solution of (5-(((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol (2.00 g), DMAP (412 mg), and octanoic acid (1.27 g). After stirring for 4 hours, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.78 g).

[0294] 1 H NMR(500MHz,CDCl3)δppm 0.84-0.91(3H,m),1.22-1.33(8H,m),1.40(6H,s),1.53-1.61(2H,m),2.26(2H,t,J=7.6Hz),3.39(2H,s),3.68- 3.74(2H,m),3.76-3.80(2H,m),3.80(3H,s),4.15(2H,s),4.42(2H,s),6.87(2H,d,J=7.8Hz),7.20-7.24(2H,m)

[0295] F) Octanoic acid 3-hydroxy-2-(hydroxymethyl)-2-(((4-methoxybenzyl)oxy)methyl)propyl ester

[0296] To a THF solution (6 mL) of 754 mg of methyl octanoate (5-((((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester (754 mg), 6 mL of 1 N hydrochloric acid was added, and the mixture was stirred at room temperature for 6 hours. After adding 4 mL of saturated aqueous sodium bicarbonate or 2 N aqueous sodium hydroxide solution, the mixture was extracted with ethyl acetate and washed with water and saturated brine. This series of operations was repeated four times until deprotection was complete. After the reaction was complete, the solvent was removed by distillation under reduced pressure to give the title compound (608 mg).

[0297] 1 H NMR(500MHz,DMSO-d6)δppm 0.85(3H,t,J=7.3Hz),1.15-1.30(9H,m),1.40-1.50(2H,m),2.22(2H,t,J=7.5Hz),3.31(2H,s),3.39(4H,d,J=5 .4Hz),3.76(3H,s),3.95(2H,s),4.35(2H,s),4.43(2H,t,J=5.4Hz),6.89(2H,d,J=6.6Hz),7.21(2H,d,J=6.6Hz)

[0298] G) Bis(2-hexyloctanoic acid)2-(((4-methoxybenzyl)oxy)methyl)-2-((octanoyl)methyl)propane-1,3-dimethyl ester

[0299] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.41 g) was added to a DMF (20 mL) solution of 3-hydroxy-2-(((4-methoxybenzyl)oxy)methyl)propyl octanoate (2.0 g), DMAP (0.64 g), and 2-hexyloctanoic acid (2.63 g). After stirring at room temperature for 15 hours, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (3.48 g).

[0300] 1H NMR(500MHz,CDCl3)δppm 0.83-0.91(15H,m),1.19-1.33(42H,m),1.37-1.47(4H,m),1.51-1.61(4H,m),2.24(2H,t,J=7.6Hz),2.32(2H,brt ,J=5.4Hz),3.41(2H,s),3.80(3H,s),4.08-4.16(6H,m),4.39(2H,s),6.86(2H,d,J=7.6Hz),7.19(2H,d,J=8.8Hz)

[0301] H) bis(2-hexyloctanoic acid) 2-(hydroxymethyl)-2-((octanoyloxy)methyl)propane-1,3-dimethyl ester

[0302] 10% palladium on carbon (280 mg) was added to a 30 mL ethanol solution of bis(2-hexyloctanoic acid)-2-(((4-methoxybenzyl)oxy)methyl)-2-((octanoyloxy)methyl)propane-1,3-diyl ester (3.48 g) at room temperature, and the mixture was stirred for 7 hours under a hydrogen atmosphere. After the reaction, the palladium on carbon was removed by filtration, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (1.68 g).

[0303] 1 H NMR(500MHz,CDCl3)δppm 0.84-0.91(15H,m),1.19-1.33(42H,m),1.41-1.51(4H,m),1.54-1.63(4H,m), 2.30-2.38(4H,m),2.61-2.64(1H,m),3.48(2H,d,J=7.3Hz),4.08-4.14(6H,m)

[0304] I) Bis(2-hexyloctanoic acid)2-(((6-(dimethylamino)hexanoyl)oxy)methyl-2-((octanoyl)methyl)propane-1,3-diyl ester

[0305] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (303 mg) was added to a DMF (6 mL) solution of bis(2-hexyloctanoic acid)-2-(hydroxymethyl)-2-((octanoyloxy)methyl)propane-1,3-diyl ester (600 mg), DMAP (54 mg), and 6-(dimethylamino)hexanoic acid (280 mg) at room temperature. After stirring at 40 °C for 15 hours, ethyl acetate was added to the reaction mixture, followed by washing twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (NH4, ethyl acetate / hexane) to give the title compound (513 mg).

[0306] 1 H NMR(500MHz,CDCl3)δppm 0.83-0.92(15H,m),1.19-1.34(42H,m),1.39-1.50(6H,m),1.52-1.73(8H,m),2.21(6H,s),2.21-2.35(8H,m),4.10(8H,s)

[0307] [Example 10] 4,5-Dibutylnonanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester

[0308] A) Ethyl 3-butylhept-2-enoate

[0309] A suspension of 3.94 g of 60% sodium hydride (containing mineral oil) in 100 mL of dehydrated THF was stirred for 10 minutes under a nitrogen stream. Then, 23.7 g of ethyl (diethoxyphosphoryl)acetate was added dropwise below 10 °C. After stirring at this temperature for 10 minutes, 10.0 g of nonane-5-one was added, and the mixture was heated to room temperature. After stirring for a short time, the temperature was raised to 50 °C. After stirring for 10 hours, the reaction mixture was cooled to below 5 °C, water was added, and the mixture was diluted with ethyl acetate. After washing twice with saturated brine and drying with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (4.47 g).

[0310] 1 H NMR(500MHz,CDCl3)δppm 0.92(6H,td,J=7.3,3.2Hz),1.25-1.47(11H,m),2.14(2H,td,J=7.6,1.1Hz),2.57-2.62(2H,m),4.14(2H,q,J=7.1Hz),5.62(1H,s)

[0311] B) Ethyl 3-butylheptaate

[0312] 10% palladium on carbon (1.50 g) was added to a 25 mL ethanol solution of ethyl 3-butylhept-2-enoate (5.80 g) at room temperature, and the mixture was stirred for 5 hours under a hydrogen atmosphere. After the reaction, the palladium on carbon was removed by filtration, and the solvent was removed by distillation under reduced pressure to give the title compound (5.49 g).

[0313] 1 H NMR (500MHz, CDCl3) δppm 0.84-0.93(6H,m),1.21-1.33(15H,m),1.80-1.88(1H,m),2.22(2H,d,J=6.9Hz),4.12(2H,q,J=7.1Hz)

[0314] C) Ethyl 2,3-dibutylheptanoate

[0315] Under a nitrogen atmosphere, a solution of diisopropylamine (11.8 mL) in dehydrated THF (59 mL) was cooled to -10 °C, and a 1.6 M n-butyllithium (n-BuLi)hexane solution (35.2 mL) was slowly added dropwise. After the addition was complete, the reaction mixture was brought to 0 °C and stirred for 10 minutes. The mixture was then cooled again to -10 °C, and a solution of ethyl 3-butylheptanate (5.49 g) in dehydrated THF (16 mL) was added dropwise, while stirring at approximately -5 °C for 30 minutes. Then, 1-iodobutane (9.43 g) was added dropwise, stirred briefly, and then brought to room temperature. After stirring for 3 hours, the mixture was neutralized with 6N hydrochloric acid, diluted with ethyl acetate, washed twice with 10% sodium thiosulfate aqueous solution, washed once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (5.57 g).

[0316] 1 H NMR(500MHz, CDCl3)δppm 0.84-0.93(9H,m),1.17-1.41(20H,m),1.51-1.65(2H,m),2.34(1H,ddd,J=10.6,6.5,3.8Hz),4.07-4.19(2H,m)

[0317] D) 2,3-Dibutylheptane-1-ol

[0318] Under a nitrogen atmosphere and ice-cold conditions, a solution of 10 mL of dehydrated THF containing 5.50 g of ethyl 2,3-dibutylheptanoate was added dropwise to a suspension of 1.54 g of lithium aluminum hydride in 66 mL of dehydrated THF. After the addition was complete, the mixture was stirred for 10 minutes and allowed to return to room temperature. After stirring for 2 hours, the mixture was cooled to below 5°C, and sodium sulfate decahydrate was added little by little. After no foaming was observed, the mixture was diluted with ethyl acetate, and the insoluble matter was filtered through diatomaceous earth. The solvent was removed by distillation under reduced pressure to obtain the title compound (4.67 g).

[0319] 1 H NMR(500MHz, CDCl3)δppm 0.86-0.93(9H,m),1.12-1.34(19H,m),1.37-1.46(1H,m),1.47-1.61(1H,m),3.49-3.62(2H,m)

[0320] E)2,3-Dibutylheptanal

[0321] A solution of 4.60 g of 2,3-dibutylheptane-1-ol and 6.02 mL of DBU in dichloromethane (46 mL) was cooled to -10 °C under a nitrogen atmosphere. While maintaining the temperature below -5 °C, a solution of 6.52 g of N-tert-butylphenylthionyl chloride in dichloromethane (20 mL) was added dropwise. After stirring at -10 °C for 3 hours, the solution was acidified with 1 N hydrochloric acid. After separation, the solvent was removed by distillation under reduced pressure. The residue was diluted with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (4.11 g).

[0322] 1 H NMR(500MHz, CDCl3)δppm 0.83-0.96(9H,m),1.15-1.39(16H,m),1.52-1.60(1H,m),1.62-1.74(2H,m),2.22-2.27(1H,m),9.64(1H,d,J=2.8Hz)

[0323] F) Ethyl 4,5-dibutylnon-2-enoate

[0324] A suspension of 1.0 g sodium hydride (containing mineral oil) in dehydrated THF (41 mL) was stirred for 10 minutes under a nitrogen stream and ice-cold. Then, 6.10 g of ethyl (diethoxyphosphoryl)acetate was added dropwise below 10 °C. After stirring at this temperature for 10 minutes, 8 mL of 4.10 g of dehydrated THF in 2,3-dibutylheptanal was added dropwise, and the mixture was heated to room temperature. After stirring for a short time, the temperature was raised to 50 °C. After stirring for 5 hours, the reaction mixture was cooled to below 5 °C, water was added, and the mixture was diluted with ethyl acetate. After washing twice with saturated brine and drying with anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (4.14 g).

[0325] 1 H NMR(500MHz,CDCl3)δppm 0.83-0.97(9H,m),1.14-1.38(22H,m),2.16-2.30(1H,m),4.10-4.22(2H,m),5.72-5.78(1H,m),6.80(1H,dd,J=15.6,9.6Hz)

[0326] G)4,5-Dibutylnon-2-enone acid

[0327] A solution of 4,5-dibutylnon-2-enoate ethyl ester (4.10 g) and 6.1 mL of 8N sodium hydroxide aqueous solution in 30 mL of ethanol was stirred at 60 °C for 2 hours. The solvent was removed by distillation under reduced pressure, and the solution was acidified with 1N hydrochloric acid. The residue was diluted with ethyl acetate, washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.80 g).

[0328] 1 H NMR (500MHz, CDCl3) δppm 0.81-0.93(9H,m),1.06-1.47(19H,m),2.16-2.33(1H,m),5.75-5.80(1H,m),6.92(1H,dd,J=15.8,9.8Hz)

[0329] H)4,5-Dibutylnon-2-enoic acid 3-hydroxy-2-(hydroxymethyl)-2-(((4-methoxybenzyl)oxy)methyl)propyl ester

[0330] At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (582 mg) was added to a DMF (4 mL) solution of (5-(((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol (600 mg), DMAP (240 mg), and 4,5-dibutylnon-2-enone acid (706 mg). After stirring for one night, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was dissolved in THF (12 mL), and then 1N hydrochloric acid (6 mL) was added, and the mixture was stirred for 3 days. Ethyl acetate was added to the reaction mixture, which was washed twice with 5% sodium bicarbonate aqueous solution and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Purification was performed using silica gel column chromatography (ethyl acetate / hexane) to obtain the title compound (870 mg).

[0331] 1 H NMR(500MHz,CDCl3)δppm 0.84-0.93(9H,m),1.13-1.36(18H,m),1.40-1.47(1H,m),2.21(1H,dt,J=9.3,4.8Hz),2.69(2H,td,J=6.6,2.5Hz),3.48(2H,s) ,3.55-3.67(4H,m),3.80-3.82(3H,m),4.23-4.32(2H,m),4.45(2H,s),5.74-5.79(1H,m),6.82-6.91(3H,m),7.21-7.25(2H,m)

[0332] I) 4,5-Dibutylnon-2-enoic acid 3-((4-methoxybenzyl)oxy)-2,2-bis((octanoyl)methyl)propyl ester

[0333] At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (757 mg) was added to a DMF (6 mL) solution of 4,5-dibutylnon-2-enoic acid 3-hydroxy-2-(hydroxymethyl)-2-(((4-methoxybenzyl)oxy)methyl)propyl ester (870 mg), DMAP (210 mg), and octanoic acid (545 mg). After stirring at 60 °C for 4 hours, ethyl acetate was added to the reaction mixture, followed by washing twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Purification was performed by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (1.26 g).

[0334] 1H NMR(500MHz,CDCl3)δppm 0.83-0.93(15H,m),1.13-1.45(35H,m),1.52-1.61(4H,m),2.17-2.31(5H,m),3.43(2H,s),3.80(3H ,s),4.10-4.22(6H,m),4.40(2H,s),5.73(1H,d,J=15.4Hz),6.78-6.90(3H,m),7.19(2H,d,J=7.9Hz)

[0335] J)4,5-Dibutylnonanoic acid 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis((octanoyl)methyl)propyl 4,5-dibutylnonanoic acid

[0336] At room temperature, 10% palladium on carbon (110 mg) was added to a mixed solution of 4,5-dibutylnon-2-enoic acid 3-((4-methoxybenzyl)oxy)-2,2-bis((octanoyloxy)methyl)propyl ester (1.26 g) in ethanol (10 mL) and ethyl acetate (10 mL), and the mixture was stirred overnight under a hydrogen atmosphere. After the reaction, the palladium on carbon was filtered off, and the solvent was removed by distillation under reduced pressure. At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (194 mg) was added to a DMF (4 mL) solution of the residue (500 mg), DMAP (95 mg), and 5-(dimethylamino)valerate (170 mg). After stirring at 50 °C for 7 hours, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (NH, ethyl acetate / hexane) to obtain the title compound (250 mg).

[0337] 1 H NMR(500MHz,CDCl3)δppm 0.84-0.91(15H,m),1.09-1.31(37H,m),1.42-1.52(2H,m),1.54-1.66(6H ,m),1.76-1.97(1H,m),2.21(6H,s),2.24-2.35(10H,m),4.07-4.13(8H,m)

[0338] [Example 14] 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-dimethyl ester of didecanoic acid

[0339] A) Ethyl 4,5-dibutylnonanoate

[0340] 10% palladium on carbon (0.54 g) was added to an ethanol (20 mL) solution of ethyl 4,5-dibutylnon-2-enoate (2.50 g) at room temperature, and the mixture was stirred for 5 hours under a hydrogen atmosphere. After the reaction, the palladium on carbon was removed by filtration, and the solvent was removed by distillation under reduced pressure to give the title compound (2.49 g).

[0341] 1 H NMR(500MHz, CDCl3)δppm 0.82-1.00(9H,m),1.10-1.33(23H,m),1.46-1.63(2H,m),2.19-2.36(2H,m),4.06-4.19(2H,m)

[0342] B) 4,5-Dibutylnonanoic acid

[0343] A solution of ethyl 4,5-dibutylnonanoate (2.49 g) and 3.55 mL of 8N sodium hydroxide aqueous solution in 12.5 mL of ethanol was stirred at 60 °C for 7 hours. The solvent was removed by distillation under reduced pressure, and the solution was acidified with 1N hydrochloric acid. The residue was diluted with ethyl acetate, washed twice with water and once with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to give the title compound (2.36 g).

[0344] 1 H NMR(500MHz, CDCl3)δppm 0.83-0.94(9H,m),0.95-1.33(20H,m),1.49(1H,ddt,J=13.7,9.3,6.8,6.8Hz),1.56-1.74(1H,m),2.26-2.42(2H,m)

[0345] C) 4,5-Dibutylnonanoic acid (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0346] At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (555 mg) was added to an 8 mL solution of (5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol (800 mg), DMAP (306 mg), and 4,5-dibutylnonanoic acid (678 mg). After stirring at 50 °C for 8 hours, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). At room temperature, a 2.32 mL solution of TBAF in THF was added to a 4 mL solution of the compound. After stirring at room temperature for one night, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed once with saturated sodium bicarbonate aqueous solution and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (680 mg).

[0347] 1 H NMR(500MHz,CDCl3)δppm 0.89(9H,t,J=6.8Hz),1.09-1.31(20H,m),1.42(6H,s),1.45-1.54(1H,m),1.57-1.62(1 H,m),2.29-2.37(3H,m),3.48(2H,d,J=6.6Hz),3.70-3.75(4H,m),4.25(2H,d,J=1.9Hz)

[0348] D) 4,5-Dibutylnonanoic acid (5-(((5-(dimethylamino)pentanoyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0349] At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (669 mg) was added to a DMF (7 mL) solution of 4,5-dibutylnonanoic acid (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl) methyl ester (680 mg), DMAP (388 mg), and 5-(dimethylamino)valerate (576 mg). After stirring at 50 °C for 7 hours, ethyl acetate was added to the reaction mixture, which was washed twice with water and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (740 mg).

[0350] 1H NMR(500MHz,CDCl3)δppm 0.89(9H,t,J=6.9Hz),1.09-1.31(21H,m),1.42(6H,s),1.48(3H,dt,J=15.1,7.6Hz),1.60-1.6 6(2H,m),2.21(6H,s),2.22-2.32(4H,m),2.35(2H,t,J=7.6Hz),3.75(4H,s),4.09-4.13(4H,m)

[0351] E) Didecanoic acid 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl ester

[0352] Acetic acid (8.4 mL) and water (4.2 mL) were added to 1.68 g of 4,5-dibutylnonanoic acid (5-(((5-(dimethylamino)pentanoyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester, and the mixture was stirred at 75 °C for 2 hours. The solvent was removed by distillation under reduced pressure, and ethyl acetate and saturated sodium hydroxide aqueous solution were added, followed by stirring for 2 hours. The organic layer was washed with saturated sodium hydroxide aqueous solution and saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. At room temperature, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (859 mg) was added to a DMF (7 mL) solution of residue (700 mg), DMAP (497 mg), and decanoic acid (701 mg). After stirring at 50°C for 7 hours, ethyl acetate was added to the reaction mixture. The mixture was washed twice with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane, ethyl acetate / methanol, and NH4+, ethyl acetate / hexane) to give the title compound (405 mg).

[0353] 1 H NMR(500MHz,CDCl3)δppm 0.84-0.93(15H,m),1.10-1.32(44H,m),1.40-1.54(3H,m),1.54-1.66(7H,m),2.21(6H,s),2.23-2.35(10H,m),4.11(8H,s)

[0354] [Example 18] 3-(((4-(dimethylamino)butyl)carbamoyl)oxy)-2,2-bis((octanoyl)methyl)propyl 4,5-dibutylnonanoic acid

[0355] To a tetrahydrofuran solution (7.5 mL) of methyl 4,5-dibutylnonanoic acid (5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxane-5-yl) ester (0.50 g), 1,1-carbonyldiimidazole (0.28 g) was added. After stirring for 1 hour, the reaction mixture was concentrated under reduced pressure. The residue was diluted with hexane to remove insoluble matter, and the filtrate was concentrated under reduced pressure. To the residue, tetrahydrofuran (10 mL), (4-aminobutyl)dimethylamine (0.20 g), and triethylamine (0.24 mL) were added. After stirring for 20 hours, the mixture was diluted with ethyl acetate and washed successively with water, aqueous ammonium chloride solution, and aqueous sodium bicarbonate solution. The solvent was removed by distillation under reduced pressure. Acetic acid (3.3 mL) and water (1.7 mL) were added to the residue, and the mixture was stirred at 65 °C for 5 hours. After cooling to room temperature, the solvent was removed by distillation under reduced pressure. The residue was diluted with ethyl acetate and washed successively with sodium bicarbonate aqueous solution and water. The solvent was removed by distillation under reduced pressure. N,N-dimethylformamide (4 mL), DMAP (61 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (288 mg), and octanoic acid (0.19 mL) were added to the residue. After stirring at 60 °C for 3 hours, ethyl acetate was added to the reaction mixture, and the mixture was washed successively with water and brine. The solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (NH4+, ethyl acetate / hexane) to give the title compound (215 mg).

[0356] 1 H NMR(500MHz,CDCl3)δppm 0.88(15H,t,J=7.09Hz)1.10-1.33(32H,m)1.40-1.62(14H,m)2.21(6H,s)2.25-2.32(8H,m)3.16(2H,m)4.10(8H,s)5.84(1H,m)

[0357] [Example 20] (9Z)-hexadec-9-enoic acid 3-((3-butylheptanoyl)oxy)-2-(((3-butylheptanoyl)oxy)methyl)-2-(((4-(dimethylamino)butanoyl)oxy)methyl)propyl ester

[0358] A)(9Z)-hexadec-9-enoic acid (5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester

[0359] Under a nitrogen atmosphere, at 50 °C, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.83 g) was added to a DMF (15 mL) solution of (5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methanol (1.50 g), DMAP (0.49 g), and palmitoleic acid (1.01 g). After stirring for 21 hours, ethyl acetate was added to the reaction mixture, which was washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.11 g).

[0360] 1 H NMR (300MHz, CDCl3) δppm 0.85-0.94(3H,m),1.01-1.10(9H,m),1.24-1.35(16H,m),1.40(6H,d,J=10.3Hz),1.52-1.61(2H,m),1.97-2.07(4H,m),2.25(2H,t, J=7.6Hz),3.66(2H,s),3.77(4H,q,J=11.8Hz),4.18(2H,s),5.35(2H,ddd,J=5.8,3.4,2.7Hz),7.36-7.47(6H,m),7.65-7.69(4H,m)

[0361] B)(9Z)-Hexadec-9-enoic acid 3-((tert-butyl(diphenyl)silyl)oxy)-2,2-bis(((3-butylheptanoyl)oxy)methyl)propyl ester

[0362] Acetic acid (10.6 mL) and water (5.3 mL) were added to (9Z)-hexadec-9-enoic acid (5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane-5-yl)methyl ester (2.11 g) under a nitrogen atmosphere, and the mixture was stirred at 75 °C for 8 hours. After cooling to room temperature, the solvent was removed by distillation under reduced pressure. The residue was diluted with ethyl acetate, washed successively with saturated sodium bicarbonate aqueous solution and water, dried with anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain a residue (1.95 g). The residue (0.95 g) was weighed under a nitrogen atmosphere, dissolved in DMF (9.5 mL), and then DMAP (0.42 g) and 3-butylheptanoic acid (0.64 g) were added, and the mixture was stirred for a short time. Then, 0.69 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added at 50 °C. After stirring for 6 hours, ethyl acetate was added to the reaction mixture, which was washed once with water and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (0.71 g).

[0363] 1 H NMR (300MHz, CDCl3) δppm 0.84-0.93(15H,m),1.05(9H,s),1.18-1.37(40H,m),1.51-1.60(2H,m),1.78(2H,brd,J=5.2Hz),1.97-2.07(4H,m),2. 17-2.26(6H,m),3.63(2H,s),4.10-4.17(6H,m),5.35(2H,ddd,J=5.6,3.5,2.2Hz),7.35-7.48(6H,m),7.60-7.65(4H,m)

[0364] C)(9Z)-Hexadec-9-enoic acid 3-((3-Butylheptanoyl)oxy)-2-(((3-Butylheptanoyl)oxy)methyl)-2-(((4-(Dimethylamino)butanoyl)oxy)methyl)propyl ester

[0365] At room temperature, a THF solution (1M, 0.9mL) of TBAF was added to a THF solution (2.1mL) of (9Z)-hexadecano-9-enoic acid 3-((tert-butyl(diphenyl)silyl)oxy)-2,2-bis((((3-butylheptanyl)oxy)methyl)propyl)ester (0.71g). After stirring overnight at room temperature, the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed once with saturated sodium bicarbonate solution and once with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was dissolved in a DMF solution (5.3mL), and then DMAP (0.14g) and 4-(dimethylamino)butyrate salt (0.19g) were added, followed by stirring for a short time. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.24g) was added at 50°C. After stirring for 8 hours, ethyl acetate was added to the reaction mixture, which was washed once with water and once with saturated brine. The mixture was then dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (NH4, ethyl acetate / hexane) to give the title compound (0.31 g).

[0366] 1H NMR(300MHz,CDCl3)d ppm 0.85-0.94(15H,m),1.20-1.37(40H,m),1.55-1.66(2H,m),1.66-1.86(4H,m),1.97-2.08(4 H,m),2.21(6H,s),2.22-2.39(10H,m),4.08-4.17(8H,m),5.35(2H,ddd,J=5.6,3.5,2.1Hz)

[0367] Examples 2-7, 9, 11-13, 15-17, 19, and 21 in the table below were manufactured according to the methods shown in the above embodiments, or any method based thereon. For these examples, the name, structural formula, and... 1 The 1H NMR chemical shifts and the mass numbers implemented during manufacturing (in MS) are shown in Table 1 along with Examples 1, 8, 10, 14, 18 and 20.

[0368] [Table 1-1]

[0369]

[0370] [Table 1-2]

[0371]

[0372] [Table 1-3]

[0373]

[0374] [Manufacturing Example 1] Manufacturing Example of Lipid Nanoparticles Encapsulating siRNA

[0375] A lipid mixture (cationic lipids:DPPC:cholesterol:GS-020 = 60:10.6:28:1.4, molar ratio) was dissolved in 90% EtOH, 10% 25mM acetate buffer, pH 4.0, to obtain a lipid solution of 7.4 mg / ml. Luciferase (luc) siRNA (refer to Table 2) was dissolved in 25mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 0.15 mg / ml. The resulting lipid and nucleic acid solutions were mixed at room temperature using an Asia microfluidic system (Syrris) at a flow rate of 1 ml / min: 5 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed in water for 1 hour at room temperature and in PBS for 18 hours at room temperature using Slyde-A-Lyzer (20K molecular weight cutoff, Thermo Scientific). The solution was then filtered through a 0.2 μm syringe filter (Iwaki) and stored at 4°C. The results of the analysis are shown in Table 3. The particle size of the lipid particles in the composition was calculated as the Z-mean particle size using a Zetasizer Nano ZS (Malvern Instruments) particle size analyzer based on dynamic light scattering measurement technology, through cumulative autocorrelation function analysis.

[0376] [Table 2]

[0377] Luc siRNA sequence

[0378] Chain of Justice 5′-[mC][mU][mU]A[mC]G[mC][mU]GAG[mU]A[mC][mU][mU][mC]GA[ts]t-3′ antisense chain 5′-UCGAAGUACUCAGCGUAAG[ts]t-3′

[0379] N: RNA

[0380] n: DNA

[0381] [mN]:2′-OMe RNA

[0382] [ns]: Thiophosphate bond

[0383] [Table 3]

[0384] cationic lipids Average particle size (nm) siRNA concentration (μg / ml) Encapsulation efficiency (%) Example 2 71 415 96 Example 3 81 445 96 Example 4 73 121 91 Example 5 83 120 96 Example 6 77 389 93 Example 7 73 376 97

[0385] [Manufacturing Example 2] Manufacturing Example of Lipid Nanoparticles Encapsulating mRNA

[0386] A lipid mixture (cationic lipids:DPPC:cholesterol:GM-020 = 60:10.6:28:1.4, molar ratio) was dissolved in 90% EtOH and 10% water to obtain a lipid solution of 8.5 mg / ml. Luciferase mRNA (TriLink) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer at pH 4.0 to obtain a nucleic acid solution of 0.22 mg / ml. The lipid and nucleic acid solutions were mixed at room temperature using a Nanoassemblr device (Precision Nanosystems) at a flow rate of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed in water for 1 hour at room temperature and in PBS for 48 hours at 4°C using Slyde-A-Lyzer (20 kJ molecular weight cutoff, Thermo Scientific). The solution was filtered through a 0.2 μm syringe filter (Iwaki) and stored at 4°C. The results of the analysis are shown in Table 4.

[0387] [Table 4]

[0388] cationic lipids Average particle size (nm) mRNA concentration (μg / ml) Encapsulation efficiency (%) Example 1 112 145 96 Example 8 103 97 91 Example 9 96 77 94 Example 10 123 83 96 Example 11 122 78 93 Example 12 134 160 93 Example 13 119 155 99 Example 14 88 134 94 Example 15 164 127 99 Example 16 109 134 94 Example 17 152 116 87 Example 18 77 103 99

[0389] [Experimental Example 1] Experimental Example of siRNA Transfection into Cultured Cells

[0390] The Hep3B human hepatocellular carcinoma cell line, stably expressing luciferase, was cultured in 96-well plates at a density of 6000 cells / well. After 24 hours, 10 μl of lipid particles containing luciferase siRNA were added to the culture medium. Forty-eight hours after siRNA addition, the amount of luciferase expression reduction (knockdown) was determined using a Picagene LT2.0 kit (Toyobo). The siRNA concentration required for 50% knockdown, calculated from the assay results, is shown in Table 5.

[0391] [Table 5]

[0392] cationic lipids The required siRNA concentration (nM) for a 50% knockdown Example 2 14 Example 3 55 Example 4 190 Example 5 28 Example 6 41 Example 7 1.2

[0393] [Experimental Example 2] mRNA transfection experiment in cultured cells

[0394] The human colorectal cancer cell line HCT116 was cultured in 96-well plates at a cell density of 6000 cells / well. After 24 hours, 10 μl of lipid particles containing 10 ng of luciferase mRNA was added to the culture medium. 24 hours after the addition of mRNA, the Picagene LT2.0 kit (Toyobo) was added to the HCT116 culture plate, and the luciferase luminescence (count per sec (cps)) was measured using an EnVision (Perkin-Elmer) luminescent microplate reader. The results are shown in Tables 6–10.

[0395] [Table 6]

[0396] cationic lipids Luminescence (cps) average value of 3 wells PBS control 1040 Example 8 381693

[0397] [Table 7]

[0398] cationic lipids Luminescence (cps) average value of 3 wells PBS control 413 Example 9 63493

[0399] [Table 8]

[0400] cationic lipids Luminescence (cps) average value of 3 wells PBS control 1293 Example 10 500747 Example 11 572560

[0401] [Table 9]

[0402] cationic lipids Luminescence (cps) average value of 3 wells PBS control 80 Example 10 176813 Example 12 62587 Example 13 269467

[0403] [Table 10]

[0404] cationic lipids Luminescence (cps) average value of 3 wells PBS control 293 Example 1 110733 Example 10 112893 Example 14 178133 Example 15 3293 Example 16 141560 Example 17 7987 Example 18 12813

[0405] [Manufacturing Example 3] Manufacture of lipid nanoparticles encapsulating siRNA

[0406] A lipid mixture (cationic lipids:DPPC:cholesterol:GS-020 = 60:10.6:28:1.4, molar ratio) was dissolved in 90% EtOH, 10% 25mM acetate buffer, pH 4.0, to obtain a lipid solution of 7.4 mg / ml. Equal weights of siRNA targeting collagen 1a1 (Col1a1) and siRNA targeting factor VII (FVII) were dissolved in 25mM acetate buffer, pH 4.0, to obtain a nucleic acid solution of 0.15 mg / ml. It should be noted that the sequences of Col1a1 siRNA and FVII siRNA are cited from Hepatology, Vol. 672, No. 4, 2015 and Silence, Vol. 1, No. 16, 2010, respectively. The siRNA sequences are listed in Table 11. The obtained lipid solution and nucleic acid solution were mixed using a Nanoassemblr (Precision Nanosystems Inc.) at a flow rate of 1 ml / min: 5 ml / min to obtain a dispersion containing the composition. The resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at room temperature for 18 hours using Slyde-A-Lyzer (20 K molecular weight cutoff, Thermoscientific). The mixture was then filtered through a 0.2 μm syringe filter (Iwaki) and stored at 4 °C. The analytical results of the lipid nanoparticles encapsulating siRNA are shown in Table 12. The formation of lipid nanoparticles encapsulating siRNA using the compounds of this invention was excellent.

[0407] [Table 11]

[0408] Col1a1 siRNA sequence

[0409] Chain of Justice 5′-G[mU][mC][mU]AGA[mC]A[mU]G[mU][mU][mC]AG[mC][mU][mU][ts]t-3′ antisense chain 5′-AAGCUGAA[mC]AUGUC[mU]AGAC[ts]t-3′

[0410] FVII siRNA sequence

[0411] Chain of Justice 5′-GGA[fU][fC]A[fU][fC][fU][fC]AAG[fU][fC][fU][fU]A[fC][ts]t-3′ antisense chain 5′-G[fU]AAGA[fC][fU][fU]GAGA[fU]GA[fU][fC][fC][ts]t-3′

[0412] N: RNA

[0413] n: DNA

[0414] [mN]:2′-OMe RNA

[0415] [ns]: Phosphophosphate bond

[0416] [fN]: 2′-F RNA

[0417] [Table 12]

[0418] Analysis results of lipid nanoparticles encapsulating siRNA

[0419]

[0420] [Experimental Example 3] Knockdown of Hepatic Collagen 1a1 Gene in CCl4 Liver Fibrosis Model Mice

[0421] In 8-week-old male Balb / c mice, lipid nanoparticles encapsulated with 0.1 mg / kg Col1a1 siRNA were administered intravenously to the orbital venous plexus. Three hours later, a single forced oral administration of CCl4 mixed in corn oil was administered at a dose of 0.1 mL / kg (10 mL / kg) (n=6 for each group). Four days after administration of the siRNA-encapsulated lipid nanoparticles, livers were collected from euthanized mice under anesthesia for gene expression analysis by quantitative PCR. Col1a1 and FVII gene expression levels were normalized using GAPDH expression levels, and the reduction in Col1a1 gene expression relative to the siRNA-free group was defined as the knockdown rate. The results are shown in Table 13. In mice intravenously administered lipid nanoparticles encapsulated with siRNA formed using the compounds of this invention, a significant knockdown of the Col1a1 gene, a marker gene for activated astrocytes, was observed compared to the knockdown rate of FVII, a marker gene for hepatocytes.

[0422] [Table 13]

[0423] Results of knockdown experiments of the liver Col1a1 and FVII genes in CCI4 liver fibrosis model mice

[0424]

[0425] Industrial availability

[0426] The compounds, lipid particles, or compositions of the present invention can efficiently deliver nucleic acids into various cells, tissues, or organs. Therefore, the compounds, lipid particles, or compositions of the present invention can be utilized as part of the DDS (Direct Detection and Delivery) technology in nucleic acid drug development. Furthermore, the compounds, lipid particles, or compositions of the present invention can also be used as research nucleic acid delivery reagents.

[0427] Sequence List Free Text

[0428] Serial numbers 1 and 2: siRNA (sense and antisense strands, see Table 2) used in manufacturing example 2 to inhibit the expression of the luciferase gene.

[0429] Serial numbers 3 and 4: siRNA (sense and antisense strands, see Table 11) used in manufacturing example 3 to suppress the expression of the collagen 1A1 gene.

[0430] Serial numbers 5 and 6: siRNA (sense and antisense strands, see Table 11) used in manufacturing example 3 to suppress the expression of factor VII gene.

Claims

1. The compound represented by formula (I) or its salt, In formula (I), n1 represents an integer from 2 to 6, n2 represents an integer from 0 to 2, n3 represents an integer from 0 to 2, and L represents -C(O)O- or -NHC(O)O-. Ra represents linear C. 13-17 Alkenyl or straight-chain C 17 Dieneyl chain, Rb represents linear C. 2-9 alkyl, Rc represents hydrogen atoms or straight-chain C. 2-9 alkyl, Rd represents hydrogen atoms or straight-chain C. 2-9 alkyl, Re represents linear C. 2-9 alkyl, Rf represents linear C. 2-9 alkyl.

2. A lipid particle comprising the compound of claim 1 or a salt thereof.

3. A composition for nucleic acid delivery, comprising nucleic acid and the lipid particles of claim 2.

4. The composition of claim 3, wherein, Nucleic acid is RNA.

5. The composition of claim 4, wherein, The RNA can be either mRNA or siRNA.

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