Cationic lipid having disulfide bond, lipid membrane structure including same, nucleic acid introduction agent and pharmaceutical composition containing any one of same, method for introducing nucleic acid into cell or target cell, and method for producing cellular pharmaceutical

Abstract

The present invention provides: a cationic lipid that can be used as a nucleic acid delivery carrier having excellent intercellular behavior; a lipid membrane structure including the same; a nucleic acid introduction agent and a pharmaceutical composition containing any one of the same; a method for introducing nucleic acid into a cell or a target cell; and a method for producing a cellular pharmaceutical. The present invention pertains to: a cationic lipid represented by formula (1) (definitions of the symbols in the formula are as described in the description); a lipid membrane structure using the cationic lipid; a nucleic acid introduction agent and a pharmaceutical composition using the cationic lipid or the lipid membrane structure; a method for introducing nucleic acid into a cell or a target cell by using the nucleic acid introduction agent; and a method for producing a cellular pharmaceutical by using the nucleic acid introduction agent.

Description

Cationic lipid having a disulfide bond, lipid membrane structure containing the same, nucleic acid transfer agent and pharmaceutical composition containing any of these, method for transferring nucleic acid into cells or target cells, and method for producing cell medicine

[0001] The present invention relates to a cationic lipid having a disulfide bond, a lipid membrane structure containing the same, a nucleic acid transfer agent and pharmaceutical composition containing either of them, a method for transferring a nucleic acid into a cell or a target cell, and a method for producing a cellular pharmaceutical.

[0002] To commercialize nucleic acid therapy, effective and safe nucleic acid delivery carriers are required. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but safer non-viral nucleic acid delivery carriers have been developed, and among them, cationic lipid-based carriers are currently the most commonly used non-viral nucleic acid delivery carriers.

[0003] Cationic lipids are broadly composed of an amine moiety and a lipid moiety. The cationic amine moiety interacts electrostatically with polyanionic nucleic acids to form liposomes or lipid membrane structures, facilitating cellular uptake and delivering nucleic acids into cells.

[0004] Known cationic lipids that are widely used include 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) and 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP). These known cationic lipids, when combined with phospholipids, form positively charged liposomes or lipid membrane structures that interact electrostatically with nucleic acids, enabling delivery of nucleic acids to target cells (see, for example, Non-Patent Document 1).

[0005] On the other hand, for a lipid membrane structure using a cationic lipid to exert practical effects in vivo as a nucleic acid delivery carrier, it must have good pharmacokinetics. Specifically, it must meet requirements such as high stability in the blood and high accumulation in target tissues such as the liver and tumors. To address this issue, it is known that lipid membrane structures in which the pKa of the surface of the lipid membrane structure is adjusted to near neutral and PEG-lipids are introduced exhibit a long life in the blood after intravenous injection and accumulate at tumor sites. Furthermore, there are examples in which pharmacokinetics has been improved by adjusting the surface pKa of the lipid membrane structure.

[0006] For example, Non-Patent Documents 2 and 3 disclose that the in vivo kinetics and distribution in each cell in the liver can be controlled by adjusting the surface pKa of the lipid membrane structure. These documents show that adjusting the surface pKa of the lipid membrane structure for endosomal escape promotes the escape of the lipid membrane structure from the endosome, enabling efficient delivery of nucleic acid into the cytoplasm.

[0007] U.S. Patent No. 9,708,628 International Publication No. 2016 / 121942 International Publication No. 2019 / 188867 International Publication No. 2021 / 193397

[0008] Biomaterials 29(24-25):3477-96,2008Molecular Therapy 24(4):788-795,2016Angewante Chemie International Edition 51:8529-8533, 2012Molecular Therapy 13(4):786-794,2006

[0009] Although cationic lipids with improved in vivo kinetics have been developed, due to the nature of nucleic acid delivery carriers, which generally introduce foreign substances into cells, it is desirable for them to exert a significant effect with a small amount of uptake. That is, when lipid membrane structures are used as delivery carriers for expression vectors into cells, it is desirable to increase the expression level per unit lipid membrane structure uptaken into the cells and improve the expression efficiency within the cells. In other words, in order to increase the expression efficiency within the cells, it is necessary to improve not only in vivo kinetics but also intracellular kinetics such as uptake into the cells, escape from the endosome, and nuclear membrane penetration (see, for example, Non-Patent Document 4).

[0010] Furthermore, methods for improving expression efficiency include imparting biodegradability to cationic lipids (see, for example, Patent Documents 1 to 4). These patent documents disclose cationic lipids having a structure linked by disulfide bonds that exhibit biodegradability, and demonstrate that intracellular dynamics are improved by dissociating nucleic acids from lipid membrane structures by utilizing the cleavage of disulfide bonds within cells. Such cationic lipids exhibit higher nucleic acid delivery efficiency than known cationic lipids such as DOTAP and DODAP. That is, it has been revealed that the cationic lipids described in Patent Documents 1 to 4 can improve intracellular dynamics, such as improving the efficiency of nucleic acid delivery into the cytoplasm, and furthermore, the effect of reducing toxicity by imparting degradability is expected.

[0011] However, nucleic acid therapy targets a wide range of diseases, and further improvement of intracellular dynamics is required to establish treatments tailored to each disease.

[0012] In view of the above problems, the present invention aims to provide a cationic lipid that can be used as a nucleic acid delivery carrier with good intracellular dynamics, a lipid membrane structure containing the same, a nucleic acid transfer agent and pharmaceutical composition containing any of these, a method for transferring a nucleic acid into a cell or target cell, and a method for producing a cellular pharmaceutical.

[0013] In view of the above problems, the present inventors conducted extensive research and found that a cationic lipid represented by the following formula (1) can efficiently deliver nucleic acids to target cells. Specifically, the cationic lipid represented by the following formula (1) is an asymmetric cationic lipid in which an aromatic ring-containing lipid moiety and an amine moiety are introduced via a disulfide bond, and the disulfide bond is cleaved within the cell, thereby dissociating nucleic acids from the lipid membrane structure. Furthermore, the present inventors found that a lipid membrane structure containing this novel cationic lipid can efficiently deliver nucleic acids into the cytoplasm.

[0014] That is, the present invention includes the following: [1] Formula (1):

[0015]

[0016] (wherein, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms; R １ represents an aliphatic hydrocarbon group having 8 or less carbon atoms; １ represents an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond; k represents 0 or 1; R ｘ and R ｙ each independently represents an alkylene group having 2 to 5 carbon atoms; ２ represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond; R ２ represents an alkylene group having 8 or less carbon atoms or is absent, and Y (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one bond selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) represents a group containing at least one bond selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue, and a residue of a sterol derivative.

[0017] [2] X is represented by formula (2): R α -N(R β )-(2) (wherein, Rα represents an aliphatic hydrocarbon group; R β represents an aliphatic hydrocarbon group, an aliphatic group containing one or more heteroatoms other than nitrogen, or an aliphatic group containing one or more tertiary amines, or R α and R β may be bonded to form a 3- to 8-membered nitrogen-containing alicyclic ring.

[0018] [3] X is a dialkylamino group (the carbon number of the two alkyl groups of the dialkylamino group is each independently 1 to 8), a 3- to 6-membered cyclic amino group which may have a heteroatom, or -N(R ａ )-R ｂ represents R ａ represents an alkyl group having 1 to 8 carbon atoms, and R ｂ is -(CH ２ ) q-O-R ｃ represents R ｃ represents hydrogen or an alkyl group having 1 to 8 carbon atoms, and q represents an integer of 2 to 4. The cationic lipid having a disulfide bond according to [1].

[0019] [4] R １ represents an alkylene group having 8 or less carbon atoms, or an alkenylene group having 8 or less carbon atoms.

[0020] [5] Y is represented by formula (3):

[0021]

[0022] (In the formula, R ３ represents an alkylene group having 8 or less carbon atoms; ３ represents an ester bond or a carbonate bond; １ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ', S ２is -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ '', and R ｅ and R ｅ L ′ each independently represents an alkylene group having 8 or less carbon atoms; ａ represents an ester bond or a carbonate bond; ｂ represents an ester bond or a carbonate bond; l represents 0 or 1; Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; L ｘ represents an ester bond or a carbonate bond; R ４ represents an alkylene group having 8 or less carbon atoms or is absent, R ５ represents hydrogen or an alkyl group having 1 to 8 carbon atoms; ３ is -R ６ '-L ４ '-R ７ "'" represents, m represents 0 or 1, R ６ and R ６ L ′ each independently represents an alkylene group having 8 or less carbon atoms or is absent; ４ and L ４ R ′ each independently represents an ester bond or a carbonate bond; ７ , R ７ ', R ７ '' and R ７ "'" each independently represent an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents 2 or 3, and n represents 0 or 1. The cationic lipid having a disulfide bond according to any one of [1] to [4], wherein

[0023] [6] Z, Z′ and Z″ are represented by the formula (4):

[0024]

[0025] (Wherein, t represents an integer of 0 to 3, u represents an integer of 0 to 3, v represents an integer of 0 to 4, and v R ８ each independently represents a substituent, * represents L ３ or L ａ The cationic lipid having a disulfide bond according to any one of [1] to [5], wherein the group is represented by the formula:

[0026] [7] R ｘ and R ｙ and [6] are both ethylene groups.

[0027] [8] R ７ , R ７ ', R ７ '' and R ７ "'" each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ ) ２ -C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group. [1] - [7] The cationic lipid having a disulfide bond according to any one of [1] to [7].

[0028] [9] A lipid membrane structure comprising the cationic lipid having a disulfide bond according to any one of [1] to [8] as a constituent lipid of the membrane.

[0029]

[10] A lipid membrane structure comprising the cationic lipid having a disulfide bond according to any one of [1] to [8] as a constituent lipid of the membrane, and further comprising a nucleic acid.

[0030]

[11] A nucleic acid transfer agent comprising the cationic lipid having a disulfide bond according to any one of [1] to [8], or the lipid membrane structure according to

[10] .

[0031]

[12] A pharmaceutical composition comprising the cationic lipid having a disulfide bond according to any one of [1] to [8], or the lipid membrane structure according to

[10] .

[0032]

[13] A method for introducing a nucleic acid into a cell, comprising contacting the cell with the nucleic acid introduction agent according to

[11] in vitro, in which the nucleic acid is encapsulated.

[0033]

[14] A method for introducing a nucleic acid into a target cell, comprising administering the nucleic acid introduction agent according to

[11] encapsulating the nucleic acid to a living body so that the nucleic acid is delivered to the target cell.

[0034]

[15] A method for producing a cell pharmaceutical containing cells that express a specific gene, comprising contacting cells with the nucleic acid transfer agent according to

[11] encapsulating a nucleic acid, and transferring the nucleic acid into the cells.

[0035] The cationic lipid of the present invention has a biodegradable disulfide bond, which is cleaved in a reductive environment within the cell, promoting the release of the encapsulated substance (nucleic acid). Therefore, the cationic lipid of the present invention, or a lipid membrane structure containing the same, can achieve high nucleic acid delivery efficiency into the cytoplasm. Therefore, it is advantageous for gene transfer in cells and living organisms, and is particularly useful as a pharmaceutical composition. Furthermore, the above-mentioned nucleic acid transfer agent has excellent gene transfer efficiency into cells, so it can efficiently produce cells expressing specific genes, and is useful in the production of cell medicines containing cells expressing specific genes.

[0036] The present invention relates to a compound represented by formula (1):

[0037]

[0038] The present invention provides a cationic lipid represented by the formula (1). The above cationic lipids may be used alone or in combination of two or more. Hereinafter, the "cationic lipid having a disulfide bond represented by formula (1)" may be abbreviated as "cationic lipid (1)." Compounds represented by other formulas may also be abbreviated in the same manner. X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms, and R１ represents an aliphatic hydrocarbon group having 8 or less carbon atoms; １ represents an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond; k represents 0 or 1; R ｘ and R ｙ each independently represents an alkylene group having 2 to 4 carbon atoms; ２ represents an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond; R ２ represents an alkylene group having 8 or less carbon atoms or is absent, and Y (i) contains one or more divalent groups derived from an aromatic compound which may have a heteroatom, and (ii) has a group containing at least one bond selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) represents a group containing at least one bond selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a fat-soluble vitamin residue, and a residue of a sterol derivative.

[0039] A preferred example of X can be represented by the following general formula (2): α -N(R β )-(2) (wherein, R α is an aliphatic hydrocarbon group, R β is an aliphatic hydrocarbon group, an aliphatic group containing one or more heteroatoms other than nitrogen, or an aliphatic group containing one or more tertiary amines, or R α and R β may be bonded to form a 3- to 8-membered nitrogen-containing alicyclic ring)

[0040] More preferred examples of X include a dialkylamino group (the two alkyl groups of the dialkylamino group each independently have 1 to 8 carbon atoms), a 3- to 6-membered cyclic amino group which may have a heteroatom, or —N(R a )-R ｂ and R ａ is an alkyl group having 1 to 8 carbon atoms, and R ｂ is -(CH ２ ) q-O-R ｃ and Rｃ is hydrogen or an alkyl group having 1 to 8 carbon atoms; and q is an integer of 2 to 4.

[0041] The number of carbon atoms in the two alkyl groups in the dialkylamino group is preferably each independently 1 to 5, more preferably each independently 1 to 4. The alkyl group may be linear, branched, or cyclic. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, and a cyclopentyl group. Preferably, they are each independently a methyl group, an ethyl group, a propyl group, or an isopropyl group, and more preferably they are each independently a methyl group or an ethyl group.

[0042] The 3- to 6-membered cyclic amino group which may have a heteroatom means a group in which substituents of an amino group are bonded to form a ring, the number of atoms forming the ring is 3 to 6, and the group may contain a heteroatom such as oxygen. The 3- to 6-membered cyclic amino group which may have a heteroatom is preferably a 5- or 6-membered cyclic amino group which may have a heteroatom, more preferably a 6-membered cyclic amino group which may have a heteroatom. The cyclic amino group is one in which the ring contains a nitrogen atom and a methylene group (-CH ２ An amino group formed only from 1-(-) is preferred, and may contain an oxygen atom. Specifically, it is a 1-pyrrolidinyl group, a 1-piperidyl group, or a morpholino group (4-morpholinyl group), and preferably a 1-piperidyl group or a morpholino group.

[0043] R １ The aliphatic hydrocarbon group having 8 or less carbon atoms in the formula (I) is preferably an alkylene group, an alkenylene group, or an alkynylene group, and more preferably an alkylene group or an alkenylene group.

[0044] The alkylene group having 8 or less carbon atoms may be either linear or branched. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0045] The alkenylene group having 8 or less carbon atoms may be either linear or branched. The number of carbon atoms in the alkenylene group is preferably 6 or less, more preferably 4 or less. The alkenylene group having 8 or less carbon atoms is preferably a propenylene group (-CH ２ CH=CH-, -CH=CHCH ２ -), butenylene group, isopropenylene group, isobutenylene group, pentenylene group, or hexenylene group, and more preferably a propenylene group (-CH ２ CH=CH-, -CH=CHCH ２ -), butenylene group, isopropenylene group, or isobutenylene group.

[0046] L １is an ester bond, an amide bond, a carbamate bond, an N-alkylcarbamate bond, a carbonate bond or a urea bond, and is preferably an ester bond, an amide bond, a carbamate bond, an N-methylcarbamate bond or a carbonate bond.

[0047] As used herein, an N-alkylcarbamate bond is defined as —NR ９ —CO—O— or —O—CO—NR ９ represents -, and R ９ represents an alkyl group having 1 to 8 carbon atoms. The alkyl group having 1 to 8 carbon atoms may be linear, branched, or cyclic. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, and a cyclopentyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0048] k represents 0 or 1. Here, when k is 0, R １ -L １ does not exist, that is, X and R ｘ The same applies when l, m, n, etc. are 0.

[0049] R ｘ and R ｙ The alkylene group having 2 to 5 carbon atoms may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 2 to 4, more preferably 2. Specifically, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), or an isobutylene group (—C(CH ３ ) ２ CH２ -, -CH ２ C(CH ３ ) ２ -), preferably an ethylene group or a trimethylene group, more preferably an ethylene group.

[0050] L ２ is an ester bond, an amide bond, a carbamate bond, a carbonate bond, an ether bond or a urea bond, preferably an ester bond or an amide bond, more preferably an ester bond.

[0051] R ２ represents an alkylene group having 8 or less carbon atoms, or is absent. ２ does not exist, L ２ and Y are directly bonded. ２ The alkylene group having 8 or less carbon atoms in the above formula may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0052] R ａ and R ｃ The alkyl group having 1 to 8 carbon atoms in the formula (I) may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, and a 2-methylbutyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0053] A preferred example of Y can be represented by the following general formula (3).

[0054]

[0055] (In the formula, R ３ represents an alkylene group having 8 or less carbon atoms; ３ represents an ester bond or a carbonate bond; １ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ', S ２ is -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ '', and R ｅ and R ｅ L ′ each independently represents an alkylene group having 8 or less carbon atoms; ａ represents an ester bond or a carbonate bond; ｂrepresents an ester bond or a carbonate bond; l represents 0 or 1; Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom; L ｘ represents an ester bond or a carbonate bond; R ４ represents an alkylene group having 8 or less carbon atoms or is absent, R ５ represents hydrogen or an alkyl group having 1 to 8 carbon atoms; ３ is -R ６ '-L ４ '-R ７ "'" represents, m represents 0 or 1, R ６ and R ６ L ′ each independently represents an alkylene group having 8 or less carbon atoms or is absent; ４ and L ４ R ′ each independently represents an ester bond or a carbonate bond; ７ , R ７ ', R ７ '' and R ７ "'" each independently represent an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group; p represents 2 or 3; and n represents 0 or 1.

[0056] R ４ represents an alkylene group having 8 or less carbon atoms, or is absent. ４ does not exist, R ５ and S ３ and the carbon atom to which L is bonded ｘ means that the bond is direct.

[0057] R ６ and R ６ R' each independently represents an alkylene group having 8 or less carbon atoms, or is absent. ６ does not exist when m = 0, Lｘ and L ４ means that m is directly bonded, and when m=1, R ５ and S ３ and the carbon atom to which L is bonded ４ means that the bond is directly bonded. ６ ' does not exist, R ４ and R ５ and the carbon atom to which L is bonded ４ ' means direct binding.

[0058] R ３ , R ４ , R ６ , R ６ ', R ｅ and R ｅ The alkylene group having 8 or less carbon atoms in ' may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is preferably 6 or less, more preferably 4 or less. The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), isobutylene group (-C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -), pentamethylene group, or hexamethylene group, and more preferably methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH ３ ) CH ２ -, -CH ２ CH (CH ３ )-), a tetramethylene group, or an isobutylene group (—C(CH ３ ) ２ CH ２ -, -CH ２ C(CH ３ ) ２ -).

[0059] R ５ and S １The alkyl group having 1 to 8 carbon atoms in the formula (I) may be either linear or branched, but is preferably linear. The number of carbon atoms in the alkyl group is preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group having 1 to 8 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1,2-dimethylpropyl group, and a 2-methylbutyl group. A methyl group, an ethyl group, a propyl group, or an isopropyl group is preferred, and a methyl group is more preferred.

[0060] L ｘ , L ３ , L ４ , L ４ ', L ａ and L ｂ is an ester bond or a carbonate bond, preferably an ester bond.

[0061] Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. Here, the divalent group refers to a divalent group having a structure obtained by removing two hydrogen atoms from the above aromatic compound. The above aromatic compound preferably has 6 to 12 carbon atoms, more preferably 6 to 7. The above aromatic compound preferably has 1 aromatic ring. Z, Z', and Z'' may be the same or different, but preferably Z, Z', and Z'' are the same.

[0062] The aromatic ring of the aromatic compound may be either an aromatic hydrocarbon ring or an aromatic heterocycle. Examples of aromatic hydrocarbon rings include a benzene ring, a naphthalene ring, and an anthracene ring. Examples of aromatic heterocycles include an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring. The aromatic ring of the aromatic compound is preferably a benzene ring, a naphthalene ring, or an anthracene ring, and more preferably a benzene ring.

[0063] The aromatic ring of the aromatic compound may have a substituent. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Preferred examples of the substituent include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0064] Z, Z′ and Z″ are preferably each independently a group represented by formula (4):

[0065]

[0066] (Wherein, t represents an integer of 0 to 3, u represents an integer of 0 to 3, v represents an integer of 0 to 4, and v R ８ each independently represents a substituent, * represents L ３ or L ａ represents the bonding position with. t is preferably 0 or 1, more preferably 1. u is preferably an integer of 0 to 2, more preferably 0. v is preferably an integer of 0 to 2, more preferably 0 or 1, and even more preferably 0.

[0067] R ８ Examples of R include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. ８Preferred examples of the aryloxy group include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, a propanoyloxy group, a butanoyloxy group, a pentanoyloxy group, a hexanoyloxy group, a heptanoyloxy group, an octanoyloxy group, a nonanoyloxy group, a decanoyloxy group, an undecanoyloxy group, a dodecanoyloxy group, a tridecanoyloxy group, a tetradecanoyloxy group, a pentadecanoyloxy group, a hexadecanoyloxy group, a heptadecanoyloxy group, and an octadecanoyloxy group. Examples of R include an aryloxy group, an octadecenoyloxy group, an octadecadienoyloxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group. ８ If there are multiple R ８ may be the same or different from each other.

[0068] R ７ , R ７ ', R ７ '' and R ７ "'" each independently represents an aliphatic hydrocarbon group having 10 to 37 carbon atoms or -(CH ２ )p-C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents 2 or 3. ７ , R ７ ', R ７ '' and R ７ The "''' may be the same or different.

[0069] In this specification, the term "residue of a fat-soluble vitamin having a hydroxyl group" refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of a fat-soluble vitamin, and the term "residue of a sterol derivative having a hydroxyl group" refers to a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of a sterol derivative.

[0070] The aliphatic hydrocarbon group having 10 to 37 carbon atoms may be either linear or branched. The aliphatic hydrocarbon group preferably has 12 to 37 carbon atoms, more preferably 13 to 37 carbon atoms, and even more preferably 15 to 37 carbon atoms.

[0071] The aliphatic hydrocarbon group may be saturated or unsaturated. When the aliphatic hydrocarbon group is an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds is preferably 1 to 6, more preferably 1 to 3, and even more preferably 1 to 2. The unsaturated bond may be either a carbon-carbon double bond or a carbon-carbon triple bond, but is preferably a carbon-carbon double bond. The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group.

[0072] Examples of the aliphatic hydrocarbon group having 10 to 37 carbon atoms include a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a henicosyl group, a docosyl group, a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a henicosyl group, a heneicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecadienyl group, a pentadecadien ... Examples of such alkyl groups include a xadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadienyl group, a henicosadienyl group, a heneicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, an icosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, a 1-dodecyltridecyl group, a 1-tetradecylpentadecyl group, a 1-hexadecylheptadecyl group, and a 1-octadecylnonadecyl group.

[0073] The aliphatic hydrocarbon group having 10 to 37 carbon atoms is preferably an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a heneicosenyl group, a docosyl group, a decenyl group, an undecenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an icosenyl group, a heneicosenyl group, a heneicosenyl group, a docosenyl group, a dodecadecyl group, or a dodecadecyl group. enyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, heneicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group , 1-decylundecyl group, 1-dodecyltridecyl group, 1-tetradecylpentadecyl group, 1-hexadecylheptadecyl group, and 1-octadecylnonadecyl group, and particularly preferred are pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group, docosyl group, heptadecenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, and nonadecadienyl group. nyl group, icosadienyl group, henicosadienyl group, heneicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group, icosapentaenyl group, docosahexaenyl group, isostearyl group, 1-hexylheptyl group, 1-hexylnonyl group, 1-octylnonyl group, 1-octylundecyl group, 1-decylundecyl group, 1-dodecyltridecyl group, 1-tetradecylpentadecyl group, 1-hexadecylheptadecyl group, and 1-octadecylnonadecyl group.

[0074] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0075] Examples of the sterol derivative having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, etc. The sterol derivative having a hydroxyl group is preferably cholesterol or cholestanol. ｆ is preferably a residue of a fat-soluble vitamin having a hydroxyl group. p is preferably 2.

[0076] l, m, and n represent 0 or 1. Preferred combinations of l, m, and n are l=0, m=0, n=0, l=0, m=0, n=1, l=0, m=1, n=1, and l=1, m=0, n=0, and particularly preferred are l=0, m=0, n=0, l=0, m=1, n=1, and l=1, m=0, n=0.

[0077] Suitable examples of the cationic lipid (1) of the present invention include the following cationic lipids: X is a dialkylamino group (the two alkyl groups of the dialkylamino group each independently have 1 to 8 carbon atoms), a 3- to 6-membered cyclic amino group which may have a heteroatom, or -N(R ａ )-R ｂ represents R ａ represents an alkyl group having 1 to 8 carbon atoms, and R ｂ is -(CH ２ ) q-O-R ｃ represents R ｃ represents hydrogen or an alkyl group having 1 to 8 carbon atoms, q is an integer of 2 to 4; R １ is an alkylene group having 6 or less carbon atoms or an alkenylene group having 6 or less carbon atoms; L １is an ester bond, an amide bond, a carbamate bond or a carbonate bond, k is 0 or 1; R ｘ and R ｙ are each independently an alkylene group having 2 to 4 carbon atoms; ２ is an ester bond or an amide bond; R ２ is an alkylene group having 6 or less carbon atoms or is absent; Y is a group represented by the formula (Y):

[0078]

[0079] (In the formula, R ３ represents an alkylene group having 6 or less carbon atoms; ３ represents an ester bond, S １ represents hydrogen, an alkyl group having 1 to 8 carbon atoms, -R ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ', S ２ is -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ '', and R ｅ and R ｅ L ′ each independently represents an alkylene group having 6 or less carbon atoms; ａ represents an ester bond; ｂ represents an ester bond; l represents 0 or 1; L ｘ represents an ester bond or a carbonate bond; R ４ represents an alkylene group having 6 or less carbon atoms or is absent, R ５ represents hydrogen or an alkyl group having 1 to 8 carbon atoms; ３ is -R ６ '-L ４ '-R ７ "'" represents, m represents 0 or 1, R ６ and R６ L ′ each independently represents an alkylene group having 6 or less carbon atoms, or is absent; ４ and L ４ Both R and R′ represent an ester bond. ７ , R ７ ', R ７ '' and R ７ "'" each independently represent an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ represents R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents 2 or 3, and n represents 0 or 1; Z, Z', and Z'' are each independently a group represented by the formula (Z):

[0080]

[0081] (Wherein, t represents an integer of 0 or 1, u represents an integer of 0 to 2, v represents an integer of 0 to 2, and v R ８ each independently represent an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 18 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aryloxy group having 6 to 10 carbon atoms.) is a group represented by the formula (1); cationic lipid (1).

[0082] The cationic lipid (1) of the present invention may exist as geometric isomers, stereoisomers such as optical isomers, tautomers, etc., and the cationic lipid (1) of the present invention encompasses all possible isomers, including these, and mixtures thereof. In a mixture of multiple isomers, there is no particular limitation on the proportion of each isomer.

[0083] The cationic lipid (1) of the present invention may form a salt, which is not particularly limited as long as it is a pharmacologically acceptable salt, and examples thereof include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, maleic acid, fumaric acid, benzoic acid, and methanesulfonic acid.

[0084] Specific preferred examples of the compound of the present invention include the compounds described in Examples 1 to 46 below, but the present invention is not construed as being limited thereto. The compounds described in Examples 1 to 46 are referred to as Compounds 1 to 46, respectively.

[0085] The cationic lipid (1) of the present invention is preferably at least one selected from the group consisting of these, more preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 19, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 34, 35, 36, and 42, even more preferably at least one selected from the group consisting of compounds 5, 7, 8, 9, 10, 11, 15, 21, 22, 24, 31, 32, 34, 35, and 36, and most preferably compound 31 or compound 34.

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Next, a method for producing the cationic lipid (1) of the present invention will be described. The cationic lipid (1) of the present invention has an -S-S- (disulfide) bond. Therefore, the method for producing the cationic lipid (1) of the present invention is as follows: (A) X-(R １ -L １ ) ｋ -R Ｘ Thiol represented by —SH and HS—R ｙ -L ２-R ２ (B) a method in which necessary portions are sequentially synthesized from a starting compound containing an -S-S- bond to finally obtain the cationic lipid (1) of the present invention. The method for producing the cationic lipid (1) of the present invention is preferably method (B).

[0092] Method (B) will be explained below, but the method for producing the cationic lipid (1) of the present invention is not limited to this.

[0093] The raw materials, reagents used in each step of the following production methods, and the resulting compounds may each form a salt. Examples of such salts include the same salts as those of the compounds of the present invention described above.

[0094] When the compound obtained in each step is a free compound, it can be converted into the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted into the free form or another desired type of salt by a known method.

[0095] The compound obtained in each step can be used in the next reaction either as a reaction solution or as a crude product, or can be appropriately purified from the reaction mixture by a common purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0096] When the raw materials and reagent compounds for each step are commercially available, the commercially available products can be used as they are.

[0097] In the reaction of each step, the reaction time is usually 1 minute to 48 hours, preferably 10 minutes to 22 hours, unless otherwise specified.

[0098] In the reactions of each step, the reaction temperature is usually −78° C. to 300° C., preferably −78° C. to 150° C., unless otherwise specified.

[0099] In the reactions of each step, unless otherwise specified, the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 8 equivalents, relative to the substrate. When a reagent is used as a catalyst, the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.4 equivalents, relative to the substrate. When a reagent also serves as a reaction solvent, the reagent is used in the amount of the solvent.

[0100] Unless otherwise specified, the reactions in each step are carried out without solvent or by dissolving or suspending in an appropriate solvent. Specific examples of the solvent include those described in the examples, as well as methanol, ethanol, isopropanol, tert-butyl alcohol, tetrahydrofuran, toluene, cyclohexane, hexane, heptane, N,N-dimethylformamide, N-methylpyrrolidone, chloroform, dichloromethane, acetonitrile, dimethyl sulfoxide, ethyl acetate, acetone, and water. Two or more of the above solvents may be mixed in an appropriate ratio.

[0101] When a base is used in the reaction of each step, examples of the base include the bases described in the examples, as well as sodium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, sodium hydrogencarbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), imidazole, piperidine, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium hydride.

[0102] When an acid or an acidic catalyst is used in the reaction of each step, examples of the acid or acidic catalyst include those described in the Examples, as well as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate, and boron trifluoride diethyl ether complex.

[0103] In each step, protection or deprotection of a functional group is carried out using known reagents and methods (for example, the reagents and methods described in GREENE'S PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed., WILEY-INTERSCIENCEM) or in accordance with the methods described in the Examples.

[0104] Examples of the protecting group for a hydroxyl group of an alcohol or the like or a phenolic hydroxyl group include ether-type protecting groups such as tetrahydropyranyl ether, methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, and t-butyldiphenylsilyl ether; carboxylic acid ester-type protecting groups such as acetate ester; sulfonic acid ester-type protecting groups such as methanesulfonate ester; and carbonate-type protecting groups such as t-butyl carbonate.

[0105] The protecting group can be removed by a known method, such as a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, or a trialkylsilyl halide, or a reduction method.

[0106] In each step, when a carbonation reaction or a carbamate reaction is carried out, examples of the reagents used include N,N'-disuccinimidyl carbonate (DSC), 4-nitrophenyl chloroformate (pNPCl), and a base (basic salt, organic base, etc.). When carbonation is carried out, an additive such as 4-dimethylaminopyridine (DMAP) may be further added.

[0107] In each step, when a mesylation reaction is carried out, methanesulfonyl chloride and a base (basic salts, organic bases, etc.) are used as reagents.

[0108] When an esterification reaction, amidation reaction, or urea reaction is carried out in each step, examples of the reagent used include activated carboxylic acids such as acyl halides such as acid chlorides, acid anhydrides, and activated esters. Examples of carboxylic acid activators include carbodiimide condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), triazine condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and combinations thereof. When a carbodiimide condensing agent is used, an additive such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), or 4-dimethylaminopyridine (DMAP) may be further added to the reaction.

[0109] In each step, when a nucleophilic substitution reaction such as amination is carried out, a nucleophile (e.g., an amine) and a base (e.g., a basic salt, an organic base) are used as reagents. An additive such as potassium iodide (KI) or tetrabutylammonium iodide (TBAI) may also be added to the reaction.

[0110] The cationic lipid (1) can be produced, for example, by the following method: Furthermore, a salt of the cationic lipid (1) can be obtained by appropriately mixing it with an inorganic acid or an organic acid.

[0111] Manufacturing method A: k = 1, l = 0, m = 0, n = 0, R ｘ and R ｙ When both are ethylene groups

[0112]

[0113] Manufacturing method B: k = 1, l = 0, m = 1, n = 1, R ｘ and R ｙ When both are ethylene groups

[0114]

[0115] Manufacturing method C: k = 1, l = 1, m = 0, n = 0, R ｘ and R ｙ are both ethylene groups, and S １ When is hydrogen or an alkyl group

[0116]

[0117] Manufacturing method D: k = 0, l = 0, m = 0, n = 0, R ｘ and R ｙ When both are ethylene groups

[0118]

[0119] In the above formula, F １ ~F １０ each independently represents a reactive functional group; P １ ~P ４ each independently represents a protecting group.

[0120] Specific production methods are described below in Examples 1 to 46. Those skilled in the art can produce the desired cationic lipid (1) by appropriately selecting raw materials and carrying out reactions in accordance with the methods described in Examples 1 to 46.

[0121] Next, the lipid membrane structure of the present invention will be described. The lipid membrane structure of the present invention contains the cationic lipid (1) of the present invention as a membrane constituent (constituent lipid). Here, the "lipid membrane structure" in the present invention means a structure having a membrane structure in which the hydrophilic groups of the amphipathic lipid are aligned toward the aqueous phase side of the interface. The "amphipathic lipid" means a lipid having both hydrophilic and hydrophobic groups. Examples of amphipathic lipids include cationic lipids and phospholipids.

[0122] The form of the lipid membrane structure of the present invention is not particularly limited, but examples of forms in which the cationic lipid (1) of the present invention is dispersed in an aqueous solvent include liposomes (e.g., unilamellar liposomes, multilamellar liposomes, etc.), O / W emulsions, W / O emulsions, spherical micelles, worm-like micelles, lipid nanoparticles (sometimes abbreviated as "LNPs" in this specification), and unspecified layered structures. The lipid membrane structure of the present invention is preferably an LNP.

[0123] The lipid membrane structure of the present invention may further contain other constituent components in addition to the cationic lipid (1) of the present invention. Examples of other constituent components include lipids (phospholipids (phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylcholine, etc.), glycolipids, peptide lipids, cholesterol, cationic lipids other than the cationic lipid (1) of the present invention, PEG lipids, etc.), surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, cholic acid sodium salt, octylglycoside, N-D-gluco-N-methylalkanamides, etc.), polyethylene glycol, proteins, etc. The content of other constituent components in the lipid membrane structure of the present invention is preferably 5 to 95 mol%, more preferably 10 to 90 mol%, and even more preferably 30 to 80 mol%, based on all constituent components in the lipid membrane structure of the present invention (i.e., the total of the cationic lipid (1) of the present invention and other constituent components).

[0124] The content of the cationic lipid (1) of the present invention in the lipid membrane structure of the present invention is not particularly limited, but typically, when the lipid membrane structure is used as a nucleic acid transfer agent as described below, the content of the cationic lipid (1) of the present invention is sufficient for transferring a nucleic acid. For example, the content of the cationic lipid (1) of the present invention in the lipid membrane structure of the present invention is preferably 5 to 100 mol %, more preferably 10 to 90 mol %, and even more preferably 20 to 70 mol %, based on the total lipids in the lipid membrane structure of the present invention.

[0125] The lipid membrane structure of the present invention can be prepared by dispersing the cationic lipid (1) of the present invention and other constituent components (lipids, etc.) in an appropriate solvent or dispersion medium, such as an aqueous solvent or an alcoholic solvent, and, if necessary, performing a procedure to induce organization.

[0126] Examples of the "operation to induce organization" include the ethanol dilution method using a microchannel or a vortex, simple hydration, ultrasonic treatment, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋ Examples of methods known per se include, but are not limited to, fusion, freeze-thaw, and reverse phase evaporation.

[0127] By encapsulating a nucleic acid in a lipid membrane structure containing the cationic lipid of the present invention and contacting the structure with a cell, the nucleic acid can be introduced into the cell in vivo and / or ex vivo. Thus, the present invention provides a nucleic acid transfer agent containing the above-mentioned cationic lipid (1) or lipid membrane structure.

[0128] The nucleic acid transfer agent of the present invention can transfer any nucleic acid into cells. The nucleic acid may be mono-, tri- or tri-stranded, but is preferably single- or double-stranded.

[0129] Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, DNA / RNA hybrids, etc. Nucleic acids other than DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids (hereinafter referred to as "other nucleic acids") may also be used. Examples of other nucleic acids include nucleotides having N-glycosides of purine or pyrimidine bases, oligomers having non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs)), and oligomers having special linkages (provided that the oligomers contain nucleotides with configurations that allow base pairing or base attachment as found in DNA or RNA).

[0130] Furthermore, the nucleic acid may be, for example, a nucleic acid having a known modification added thereto, a nucleic acid labeled as known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are replaced with an analogue, a nucleic acid having intramolecularly bridged nucleotides, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid containing an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0131] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples of DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc. Among these, plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA can also be digested with appropriate restriction enzymes and used as linear DNA.

[0132] The type of RNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples of RNA include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. Among these, siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon are preferred.

[0133] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0134] The nucleic acid transfer agent of the present invention encapsulating a nucleic acid can be administered in vivo, for example, for the purpose of preventing and / or treating a disease. Therefore, the nucleic acid used in the present invention is preferably one that has preventive and / or therapeutic activity against a specific disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0135] When forming the lipid membrane structure of the present invention, the target nucleic acid can be coexisted with the constituent components (lipids, etc.) of the lipid membrane structure of the present invention, thereby forming the lipid membrane structure of the present invention encapsulating the nucleic acid (i.e., the nucleic acid transfer agent of the present invention encapsulating the nucleic acid).

[0136] For example, when the lipid membrane structure of the present invention is formed as a liposome by the ethanol dilution method, an aqueous solution of nucleic acid and an ethanol solution of the components of the lipid membrane structure are vigorously mixed using a vortex mixer, a microchannel, or the like, and the resulting mixture is then diluted with an appropriate buffer solution to form the lipid membrane structure of the present invention encapsulating nucleic acid (i.e., the nucleic acid transfer agent of the present invention encapsulating nucleic acid).

[0137] Furthermore, when the lipid membrane structure of the present invention is formed as a liposome by the simple hydration method, the components of the lipid membrane structure are dissolved in an appropriate organic solvent, the solution is placed in a glass container, and the solvent is evaporated by drying under reduced pressure to obtain a lipid thin film. Next, an aqueous solution of nucleic acid is added to the obtained lipid thin film, followed by hydration and ultrasonic treatment with a sonicator, thereby forming the lipid membrane structure of the present invention encapsulating the nucleic acid (i.e., the nucleic acid transfer agent of the present invention encapsulating the nucleic acid).

[0138] One form of the lipid membrane structure of the present invention encapsulating a nucleic acid is an LNP in which the nucleic acid is encapsulated by forming an electrostatic complex between the nucleic acid and a cationic lipid. This LNP can be used as a drug delivery system for selectively delivering nucleic acids, etc., into specific cells, and is useful, for example, for DNA vaccines and gene therapy drugs for tumors that involve antigen gene transfer into dendritic cells, and nucleic acid drugs (pharmaceutical compositions) that suppress the expression of target genes using RNA interference.

[0139] The particle size of the lipid membrane structures of the present invention encapsulating nucleic acids is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm. This particle size can be measured using a particle size distribution analyzer such as Zetasizer Nano (Malvern). The particle size of the lipid membrane structures can be adjusted appropriately depending on the method for preparing the lipid membrane structures.

[0140] The zeta potential of the lipid membrane structure of the present invention encapsulating nucleic acid is not particularly limited, but is preferably −15 to +15 mV, more preferably −5 to +15 mV. Previous gene transfer techniques primarily used positively charged particles. This is useful as a method for promoting electrostatic interaction with heparin sulfate on the negatively charged cell surface and facilitating cellular uptake. However, a positive surface charge can (a) inhibit the release of nucleic acid from the carrier due to intracellular interaction with the delivered nucleic acid, or (b) inhibit protein synthesis due to interaction between mRNA and the delivered nucleic acid. This problem can be solved by adjusting the zeta potential within the above range. Zeta potential can be measured using a zeta potential measuring device such as the Zetasizer Nano. The zeta potential of the lipid membrane structure can be adjusted by the composition of the components of the lipid membrane structure, including the cationic lipid (1) of the present invention.

[0141] The pKa of the lipid membrane surface of the lipid membrane structure of the present invention (hereinafter sometimes abbreviated as "liposomal pKa") is not particularly limited, but is preferably 5.0 to 9.0, more preferably 5.3 to 8.5. The liposomal pKa is an index indicating the susceptibility of a lipid membrane structure taken up by endocytosis to protonation in the weakly acidic environment within the endosome. As described in Non-Patent Documents 2 and 3, in order to escape from the endosome and deliver nucleic acid into the cytoplasm, it is important to set the liposomal pKa to a value favorable for endosomal escape. By adjusting the liposomal pKa within the above range, nucleic acid can be efficiently delivered into the cytoplasm. The liposomal pKa can be appropriately adjusted by selecting the cationic lipid (1) of the present invention used as a component of the lipid membrane structure.

[0142] The nucleic acid can be introduced into cells by contacting the nucleic acid transfer agent of the present invention encapsulating the nucleic acid with the cell. The type of cell is not particularly limited, and prokaryotic and eukaryotic cells can be used. The cell is preferably a eukaryotic cell. The type of eukaryotic organism is also not particularly limited, and examples include vertebrates such as mammals including humans (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), and fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), plants, and microorganisms (e.g., yeast, etc.). The cell is more preferably an animal or plant cell, and even more preferably a mammalian cell. The cell may be a cultured cell line containing a cancer cell, a cell isolated from an individual or tissue, or a cell of a tissue or tissue fragment. The cell may also be an adherent cell or a non-adherent cell.

[0143] The step of contacting cells with the nucleic acid transfer agent of the present invention encapsulating nucleic acid in vitro will be specifically described below.

[0144] The cells are suspended in an appropriate medium and cultured under appropriate conditions several days before contact with the nucleic acid transfer agent of the present invention, which may or may not be in the proliferation phase.

[0145] The culture medium used when cells are contacted with the nucleic acid transfer agent of the present invention encapsulating nucleic acid may be a serum-containing medium or a serum-free medium. The serum concentration in the medium is preferably 30% by weight or less, more preferably 20% by weight or less. If the medium contains excessive proteins such as serum, contact between the nucleic acid transfer agent and cells may be inhibited.

[0146] The cell density at the time of contact between the nucleic acid transfer agent of the present invention encapsulating nucleic acid and the cells is not particularly limited and can be appropriately set in consideration of the type of cells, etc., but is usually 1 × 10 ４ ~1 x 10 ７ cells / mL range.

[0147] For example, a dispersion of the nucleic acid transfer agent of the present invention encapsulating the above-described nucleic acid is added to cells. The amount of the dispersion added is not particularly limited and can be appropriately determined taking into account the number of cells, etc. The concentration of the nucleic acid transfer agent in the dispersion when contacted with cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved. However, the lipid concentration in the dispersion is typically 1 to 100 nmol / mL, preferably 10 to 50 nmol / mL, and the nucleic acid concentration in the dispersion is typically 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL.

[0148] After adding the dispersion liquid to the cells, the cells are cultured. ２ The concentration and other factors are appropriately set taking into consideration the type of cells. When the cells are mammalian cells, the temperature is usually about 37°C, the relative humidity is about 95%, and CO ２ The concentration is approximately 5% by volume. The incubation time can also be set appropriately taking into account conditions such as the type of cells used, but is usually in the range of 0.1 to 76 hours, preferably 0.2 to 24 hours, and more preferably 0.5 to 12 hours. If the incubation time is too short, the nucleic acid may not be sufficiently introduced into the cells, and if the incubation time is too long, the cells may become weak.

[0149] The nucleic acid is introduced into the cells by the above-mentioned culture, and preferably the culture medium is replaced with a fresh medium or fresh medium is added to the medium and the culture is continued. When the cells are mammalian cells, the fresh medium preferably contains serum or nutrient factors.

[0150] By using the nucleic acid transfer agent of the present invention encapsulating nucleic acid, it is possible to transfer nucleic acid into cells not only in vitro but also in vivo. That is, by administering the nucleic acid transfer agent to a subject, the nucleic acid transfer agent reaches and contacts target cells, and the nucleic acid is transferred into cells in vivo. The subjects to which the nucleic acid transfer agent can be administered are not particularly limited, and examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), plants, etc. The subjects to which the nucleic acid transfer agent is administered are preferably humans or other mammals.

[0151] The type of target cell is not particularly limited, and by using the nucleic acid transfer agent of the present invention, it is possible to transfer nucleic acids into cells in various tissues (e.g., liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0152] The method of administering the nucleic acid transfer agent of the present invention encapsulating a nucleic acid to a subject (e.g., a vertebrate, an invertebrate, etc.) is not particularly limited as long as it allows the nucleic acid transfer agent of the present invention to reach and contact the target cell and transfer the nucleic acid into the cell. Taking into consideration the type of nucleic acid to be transferred, the type and site of the target cell, etc., a known administration method (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the nucleic acid transfer agent is not particularly limited as long as it is within a range that allows transfer of the nucleic acid into the cell, and can be appropriately selected taking into consideration the type of subject, administration method, type of nucleic acid to be transferred, type and site of the target cell, etc.

[0153] The cationic lipid (1) and lipid membrane structure of the present invention can also be used to introduce compounds other than nucleic acids into cells.

[0154] A nucleic acid transfer agent containing the cationic lipid (1) or lipid membrane structure of the present invention can be formulated according to conventional methods.

[0155] When the nucleic acid transfer agent of the present invention is provided as a research reagent, it may be provided as a sterile solution or dispersion containing the cationic lipid (1) of the present invention or the lipid membrane structure of the present invention and water or another physiologically acceptable solvent (e.g., a water-soluble solvent (e.g., malic acid buffer, etc.), an organic solvent (e.g., ethanol, methanol, DMSO, tert-butanol, etc.), or a mixed solvent of a water-soluble solvent and an organic solvent), or it may be provided as a solvent-free nucleic acid transfer agent. The nucleic acid transfer agent of the present invention may optionally contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0156] When the nucleic acid transfer agent of the present invention is provided as a pharmaceutical, it may be provided as an oral preparation (e.g., tablet, capsule, etc.) or parenteral preparation (e.g., injection, spray, etc.) obtained by mixing the cationic lipid (1) of the present invention or the lipid membrane structure of the present invention with known pharmaceutically acceptable additives (e.g., carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc.), or it may be provided as a nucleic acid transfer agent that does not contain known additives. The nucleic acid transfer agent of the present invention as a pharmaceutical is preferably a parenteral preparation, more preferably an injection. Furthermore, the nucleic acid transfer agent of the present invention may be either a preparation for adults or a formulation for children.

[0157] The nucleic acid transfer agent of the present invention can also be provided in the form of a kit. The kit can contain, in addition to the cationic lipid (1) or lipid membrane structure of the present invention, reagents used during nucleic acid transfer. In one embodiment, the nucleic acid transfer agent (or kit) of the present invention further contains a polycation (e.g., protamine). By using the nucleic acid transfer agent (or kit) of the present invention in this embodiment, an electrostatic complex of a nucleic acid and a polycation (e.g., protamine) can be encapsulated in the lipid membrane structure of the present invention, thereby enabling more efficient transfer of nucleic acid into cells.

[0158] The present invention provides a method for producing a cellular pharmaceutical containing cells expressing a specific gene, which comprises contacting cells with the nucleic acid transfer agent of the present invention encapsulating a nucleic acid and introducing the nucleic acid into the cells.

[0159] Cells that express a specific gene refer to cells in which a gene of interest is expressed by introducing a nucleic acid into the cells.

[0160] By contacting cells with the nucleic acid transfer agent of the present invention encapsulating nucleic acid ex vivo, the nucleic acid can be introduced into the cells. By administering cells expressing a specific gene of interest to a subject, diseases can be treated or prevented. The step of contacting cells with the nucleic acid transfer agent of the present invention encapsulating nucleic acid ex vivo can be carried out in the same manner as described above.

[0161] The cells used in the production of cell medicines are not particularly limited as long as they are immune cells, and examples thereof include T cells, B cells, NK cells, dendritic cells, macrophages, and monocytes. The T cells used in the production of cell medicines may be T cells induced to differentiate from lymphocyte precursor cells, including pluripotent cells. Examples of lymphocyte precursor cells, including pluripotent cells, include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Undifferentiated cells, such as pluripotent cells, can be differentiated into T cells by known methods.

[0162] Nucleic acids used in the production of cell medicines include, for example, nucleic acids encoding chimeric antigen receptors (CARs) and T cell receptors (TCRs). The CAR-encoding nucleic acids used in the production of cell medicines include an antigen-binding domain of an antibody capable of specifically recognizing the surface antigen to be recognized by the target immune cell, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain. The TCR-encoding nucleic acids used in the production of cell medicines are nucleic acids encoding the α chain and β chain of a TCR capable of specifically recognizing the surface antigen to be recognized by the target T cell. Examples of nucleic acids encoding CARs and TCRs include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids.

[0163] The cellular pharmaceutical contains cells that express a specific gene, and may further contain pharmaceutically acceptable additives (e.g., carriers, excipients, vehicles, preservatives, stabilizers, etc.). The cellular pharmaceutical is preferably a parenteral preparation, more preferably an injectable preparation.

[0164] The cell pharmaceutical can be used for the treatment or prevention of diseases such as cancer. There are no particular limitations on the cancers to which the pharmaceutical of the present invention can be applied, and examples include, but are not limited to, lung cancer, breast cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, leukemia, malignant lymphoma, and myeloma.

[0165] The subjects to which the cellular pharmaceutical product can be administered are not particularly limited, and examples thereof include mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), etc. The subjects to which the cellular pharmaceutical product can be administered are preferably humans or other mammals.

[0166] The method of administration of the cellular pharmaceutical is not particularly limited as long as it allows the cells to express the target gene, and can be appropriately selected, for example, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.) taking into consideration the type of cell, target disease, etc. The dosage of the cellular pharmaceutical is not particularly limited as long as it is within a range that allows the cells to express the target gene, and can be appropriately selected taking into consideration the type of recipient, administration method, type of cell, target disease, etc.

[0167] Examples of the present invention will be described in more detail below, but the present invention is not limited to these examples.

[0168] The meanings of the abbreviations used in the description of the examples are as follows: DCM: dichloromethane THF: tetrahydrofuran IPA: 2-propanol DHP: 3,4-dihydro-2H-pyran PPTS: pyridinium p-toluenesulfonate THP: 2-tetrahydropyranyl DMAP: 4-dimethylaminopyridine DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate DSC: di(N-succinimidyl) carbonate EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride TEA: triethylamine TBAI: tetrabutylammonium iodide DIPEA: N,N-diisopropylethylamine NPM: 4-phenylmorpholine MsCl: methanesulfonyl chloride Ms: methanesulfonyl pNPCl: 4-nitrophenyl chloroformate mRNA: messenger RNA Chol: cholesterol DMG-PEG2k: 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (weight average molecular weight of polyethylene glycol chain: 2000) DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine PBS: phosphate buffered saline MES: 2-morpholinoethanesulfonic acid TNS: sodium 6-(p-toluidino)-2-naphthalenesulfonate

[0169] The names and structures of the cationic lipids produced in the following Examples and Comparative Examples are shown in Tables 2 and 3. The Comparative Examples were produced according to the production method described in Patent Document 3.

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Example 1 Synthesis of Compound 1 Compound 1 was produced by the following synthetic route, but the present invention is not limited to this synthetic route.

[0185]

[0186] <Synthesis of Intermediate 1> 4-Hydroxyphenylacetic acid (75.0 g) and PPTS (12.4 g) were dissolved in 400 g of dichloromethane at room temperature, and the resulting solution was cooled to 10-20°C. A solution obtained by dissolving DHP (207 g) in 100 g of dichloromethane was added dropwise thereto, and the reaction was carried out at 25°C for 2 hours. The reaction solution was cooled to 10-20°C, and DMAP (30.1 g) was added thereto to quench the reaction. 590 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10-20°C. A solution of 98.8 g of 400 g / L aqueous sodium hydroxide solution and 307 g of ion-exchanged water was added dropwise to the cooled solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, and the dichloromethane and 2-propanol were distilled off. The resulting concentrate was washed twice with 750 g of chloroform, and then 6N hydrochloric acid was added to obtain a solution of pH 5.0. The resulting solution was extracted twice with 750 g of chloroform, and then 75.0 g of sodium sulfate was added to the organic layer to dehydrate it. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 98.2 g of Intermediate 1.

[0187] <Synthesis of Intermediate 2> Intermediate 1 (10.2 g), bis(2-hydroxyethyl) disulfide (9.99 g), and DMAP (1.06 g) were dissolved in 153 g of chloroform at room temperature. EDC (12.4 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 102 g of 20 wt% saline, and then 5.1 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 9.01 g of Intermediate 2.

[0188] <Synthesis of Intermediate 3-a> Intermediate 2 (350 mg), dimethylglycine hydrochloride (144 mg), triethylamine (143 mg), and DMAP (23.0 mg) were dissolved in 5.25 mL of chloroform at room temperature. EDC (270 mg) was added to the resulting solution and the reaction was allowed to proceed at room temperature for 2 hours. The reaction solution was washed with 3.5 g of 0.5 M phosphate buffer (pH 4.0), 3.5 g of 7 wt % sodium bicarbonate water, and 3.5 g of 20 wt % saline, in that order, and then dehydrated by adding 175 mg of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 378 mg of Intermediate 3-a.

[0189] <Synthesis of Intermediate 4-a> Intermediate 3-a (363 mg) was dissolved in 3.27 g of THF, and then 13.1 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at room temperature for 2 hours. 5.45 g of chloroform was added to the reaction solution for washing. After washing, a 1 N aqueous sodium hydroxide solution was added to the aqueous layer to adjust the pH to 6.0, and the pH-adjusted aqueous layer was extracted with 5.45 g of chloroform. 180 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 187 mg of Intermediate 4-a.

[0190] <Synthesis of Compound 1> Intermediate 4-a (187 mg), 2-hexyldecanoic acid (128 mg), and DMAP (12.0 mg) were dissolved in 2.81 g of chloroform at room temperature. EDC (144 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.87 g of 20 wt% saline, and then 93.0 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 159 mg of Compound 1. <Synthesis of Compound 1> １ H-NMR (600MHz, CDCl ３)> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.8 8-2.97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0191] [Example 2] Synthesis of Compound 2 <Synthesis of Compound 2> Compound 2 was synthesized using intermediate 4-a and 2-decyldodecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0192] [Example 3] Synthesis of Compound 3 <Synthesis of Compound 3> Compound 3 was synthesized using intermediate 4-a and 2-tetradecylhexadecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.75 (m, 2H), 2.36 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2 .97 (m, 4H), 3.19 (s, 2H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0193] Example 4 Synthesis of Compound 4 <Synthesis of Intermediate 3-b> Intermediate 3-b represented by the following formula was synthesized in the same manner as in Example 1, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0194]

[0195] <Synthesis of Intermediate 4-b> Intermediate 4-b represented by the following formula was synthesized in the same manner as in Example 1, except that intermediate 3-b was used.

[0196]

[0197] <Synthesis of Compound 4> Compound 4 was synthesized in the same manner as in Example 1, except that intermediate 4-b was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.86-0.90 (m, 6H), 1.20-1.45 (m, 22H), 1.69-1.82 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.5 2-2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0198] [Example 5] Synthesis of Compound 5 <Synthesis of Compound 5> Compound 5 was synthesized using intermediate 4-b and 2-decyldodecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 34H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0199] [Example 6] Synthesis of Compound 6 <Synthesis of Compound 6> Compound 6 was synthesized using intermediate 4-b and 2-dodecyltetradecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 42H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0200] [Example 7] Synthesis of Compound 7 <Synthesis of Compound 7> Compound 7 was synthesized using intermediate 4-b and 2-tetradecylhexadecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 50H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0201] [Example 8] Synthesis of Compound 8 <Synthesis of Compound 8> Compound 8 was synthesized using intermediate 4-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0202] [Example 9] Synthesis of Compound 9 <Synthesis of Compound 9> Compound 9 was synthesized using intermediate 4-b and 2-octadecyleicosanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0203]

[0204] Example 10 Synthesis of Compound 10 <Synthesis of Intermediate 5> Intermediate 2 (5.10 g), 4-bromobutyric acid (2.52 g), and DMAP (335 mg) were dissolved in 76.5 g of chloroform at room temperature. EDC (3.94 g) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 51.0 g of 20 wt% saline, and then 2.55 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 6.07 g of Intermediate 5.

[0205] <Synthesis of Intermediate 6-a> Diethylamine (258 mg) was dissolved in 1.04 g of THF at room temperature. A solution obtained by dissolving Intermediate 5 (230 mg) in 780 mg of THF was added dropwise thereto, and the mixture was allowed to react at 50°C for 15 hours. 3.45 g of chloroform was added to the reaction solution, and the mixture was washed successively with 2.30 g of 0.5 M acetate buffer (pH 4.0), 2.30 g of 7 wt% sodium bicarbonate water, and 2.30 g of 20 wt% saline. 130 mg of sodium sulfate was added and the mixture was dehydrated. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 130 mg of Intermediate 6-a.

[0206] <Synthesis of Intermediate 7-a> Intermediate 7-a was synthesized using intermediate 6-a in the same manner as in the synthesis of intermediate 4-a in Example 1.

[0207] <Synthesis of Compound 10> Compound 10 was synthesized in the same manner as in Example 1 using intermediate 7-a and 2-hexadecyl octadecanoic acid. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0208] Example 11 Synthesis of Compound 11 <Synthesis of Intermediate 6-b> Intermediate 6-b represented by the following formula was synthesized in the same manner as in Example 10, except that piperidine was used.

[0209]

[0210] <Synthesis of Intermediate 7-b> Intermediate 7-b represented by the following formula was synthesized in the same manner as in Example 10, except that intermediate 6-b was used.

[0211]

[0212] <Synthesis of Compound 11> Compound 11 was synthesized using intermediate 7-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 10. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 60H), 1.45-1.60 (m, 4H), 1.69-1.84 (m, 4H), 2.20-2.50 (m, 8H), 2.52-2 .59 (m, 1H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0213] Example 12 Synthesis of Compound 12 <Synthesis of Intermediate 6-c> Intermediate 6-c represented by the following formula was synthesized in the same manner as in Example 10, except that dipropylamine was used.

[0214]

[0215] <Synthesis of Intermediate 7-c> Intermediate 7-c represented by the following formula was synthesized in the same manner as in Example 10, except that intermediate 6-c was used.

[0216]

[0217] <Synthesis of Compound 12> Compound 12 was synthesized using intermediate 7-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 10. １ H-NMR (600MHz, CDCl ３ )> δ: 0.86-0.92 (m, 12H), 1.20-1.45 (m, 62H), 1.69-1.84 (m, 4H), 2.28-2.60 (m, 9H), 2.8 8-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0218]

[0219] [Example 13] Synthesis of Compound 13 <Synthesis of Compound 13> Compound 13 was synthesized using the same synthetic route as in Example 8, except that intermediate 2 and 3-(dimethylamino)-2-methylpropionic acid hydrochloride were used as starting materials. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.13 (d, 3H), 1.20-1.45 (m, 58H), 1.68-1.78 (m, 2H), 2.18-2.22 (m, 7H), 2.52-2.7 0 (m, 3H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0220]

[0221] Example 14 Synthesis of Compound 14 <Synthesis of Intermediate 8> 3,4-Dihydroxyphenylacetic acid (3.00 g) and PPTS (448 mg) were dissolved in 40.0 g of dichloromethane at room temperature, and the resulting solution was cooled to 20°C. DHP (7.50 g) was added dropwise thereto, and the reaction was carried out at room temperature for 2 hours. The reaction solution was cooled to 20°C, and DMAP (1.09 g) was added thereto to quench the reaction. 47.0 g of 2-propanol was added to the quenched solution, and the mixture was cooled to 10-20°C. 30.0 g of 1N aqueous sodium hydroxide solution was added dropwise to the cooled solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, and dichloromethane and 2-propanol were distilled off. The resulting concentrate was washed with 30.0 g of chloroform, and 6N hydrochloric acid was added to obtain a solution with a pH of 5.0. The resulting solution was extracted with 30.0 g of chloroform, and then the organic layer was dehydrated by adding 1.50 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 4.98 g of Intermediate 8.

[0222] <Synthesis of Intermediate 9> Intermediate 8 (800 mg), bis(2-hydroxyethyl)disulfide (550 mg), and DMAP (58.0 mg) were dissolved in 12.0 g of chloroform at room temperature. EDC (684 mg) was added to the resulting solution, and the mixture was allowed to react at 25°C for 2 hours. The reaction solution was washed with 8.00 g of 20 wt% saline, and then 400 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, and the resulting crude product was purified using a column to obtain 530 mg of Intermediate 9.

[0223] <Synthesis of Intermediate 10-a> Intermediate 9 (525 mg), dimethylglycine hydrochloride (171 mg), triethylamine (169 mg), and DMAP (27.0 mg) were dissolved in 7.90 g of chloroform at room temperature. EDC (319 mg) was added to the resulting solution and the reaction was allowed to proceed at room temperature for 2 hours. The reaction solution was washed with 5.25 g of 0.5 M phosphate buffer (pH 4.0), 5.25 g of 7 wt % sodium bicarbonate water, and 5.25 g of 20 wt % saline, in that order, and then 250 mg of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 558 mg of Intermediate 10-a.

[0224] <Synthesis of Intermediate 11-a> Intermediate 10-a (550 mg) was dissolved in 4.95 g of THF, and then 19.8 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at room temperature for 2 hours. 8.25 g of chloroform was added to the reaction solution for washing. After washing, a 1 N aqueous sodium hydroxide solution was added to the aqueous layer to adjust the pH to 6.5, and the pH-adjusted aqueous layer was extracted with 8.25 g of chloroform. 300 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 503 mg of Intermediate 11-a.

[0225] <Synthesis of Compound 14> Intermediate 11-a (190 mg), oleic acid (289 mg), and DMAP (24.0 mg) were dissolved in 2.85 g of chloroform at room temperature. EDC (234 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.90 g of 20 wt% saline, and then dehydrated by adding 100 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 291 mg of Compound 14. <Synthesis of Compound 14> １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.36 (s, 6H), 2.50-2.5 3 (m, 4H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 5.32-5.38 (m, 4H), 7.10-7.17 (m, 3H)

[0226] Example 15 Synthesis of Compound 15 <Synthesis of Intermediate 10-b> Intermediate 10-b represented by the following formula was synthesized in the same manner as in Example 14, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0227]

[0228] <Synthesis of Intermediate 11-b> Intermediate 11-b represented by the following formula was synthesized in the same manner as in Example 14, except that intermediate 10-b was used.

[0229]

[0230] <Synthesis of Compound 15> Compound 15 was synthesized using intermediate 11-b and oleic acid according to the same synthetic route as in Example 14. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.22-1.42 (m, 40H), 1.69-1.83 (m, 6H), 1.99-2.05 (m, 8H), 2.26 (s, 6H), 2.33-2.54 (m, 4H), 2.50-2.53 (m, 4H), 2.88-2.97 (m, 4H), 3.63 (s, 2H), 4.34-4.42 (m, 4H), 5.32-5.38 (m, 4H), 7.10-7.17 (m, 3H)

[0231]

[0232] Example 16 Synthesis of Compound 16 Synthesis of Intermediate 12 Intermediate 2 (5.00 g) and triethylamine (2.72 g) were dissolved in 125 g of chloroform at room temperature. Methanesulfonyl chloride (2.31 g) was added to the resulting solution and reacted at 25°C for 3 hours. The reaction solution was washed with 75.0 g of 7 wt% aqueous sodium bicarbonate solution, and 5.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 4.42 g of Intermediate 12.

[0233] <Synthesis of Intermediate 13-a> Intermediate 12 (600 mg) was dissolved in 5.40 g of dimethylamine (2.0 mol / L in THF), and TBAI (49.0 mg) was added, followed by a reaction at 25°C for 20 hours. 9.00 g of chloroform was added to the reaction solution, which was then washed with 6.00 g of 0.5 M acetate buffer (pH 4.0), and 300 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 470 mg of Intermediate 13-a.

[0234] <Synthesis of Intermediate 14-a> Intermediate 14-a was synthesized in the same manner as for intermediate 4-a in Example 1, except that intermediate 13-a was used.

[0235] <Synthesis of Compound 16> Compound 16 was synthesized in the same manner as in Example 1 using intermediate 14-a and 2-hexadecyl octadecanoic acid. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.60 (m, 3H), 2 .80 (t, 2H), 2.90 (t, 2H), 3.63 (s, 2H), 4.35 (t, 2H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0236] Example 17 Synthesis of Compound 17 Synthesis of Intermediate 13-b 4.20 g of THF was added to intermediate 12 (600 mg) and diethylamine (779 mg), and the mixture was dissolved at room temperature. TBAI (49.0 mg) was added to the resulting solution, and the mixture was reacted at 25°C for 3 hours. 9.00 g of chloroform was added to the reaction solution, and the mixture was washed with 6.00 g of 0.5 M acetate buffer (pH 4.0), and 300 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 473 mg of intermediate 13-b represented by the following formula:

[0237]

[0238] <Synthesis of Intermediate 14-b> Intermediate 14-b represented by the following formula was synthesized in the same manner as in Example 16, except that intermediate 13-b was used.

[0239]

[0240] <Synthesis of Compound 17> Compound 17 was synthesized using intermediate 14-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 16. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.53-2.57 (m, 5H), 2.7 5-2.80 (m, 4H), 2.91 (t, 2H), 3.63 (s, 2H), 4.35 (t, 2H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0241]

[0242] Example 18 Synthesis of Compound 18 Synthesis of Intermediate 15-a Cystamine dihydrochloride (227 mg), DIPEA (391 mg), and dimethylglycine hydrochloride (141 mg) were dissolved in 4.54 g of methanol at room temperature. DMT-MM (837 mg) was added to the resulting solution, and the reaction was allowed to proceed at 25°C for 2 hours. Intermediate 1 (239 mg) was then added to the reaction solution, and the reaction was allowed to proceed at 25°C for 2 hours. The reaction solution was concentrated, and then 3.40 g of chloroform was added. The mixture was washed sequentially with 2.27 g of 0.5 M phosphate buffer (pH 6.0), 2.27 g of 7 wt% aqueous sodium bicarbonate, and 2.27 g of 20 wt% saline. 100 mg of sodium sulfate was added to the resulting solution for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 748 mg of Intermediate 15-a as a crude product.

[0243] <Synthesis of Intermediate 16-a> Intermediate 16-a was synthesized in the same manner as for intermediate 4-a in Example 1, except that a crude product of intermediate 15-a was used.

[0244] <Synthesis of Compound 18> Compound 18 was synthesized using intermediate 16-a and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.30 (s, 6H), 2.52-2.59 (m, 1H), 2.76-2.82 (m , 4H), 2.95 (s, 2H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0245] Example 19 Synthesis of Compound 19 <Synthesis of Intermediate 15-b> Intermediate 15-b represented by the following formula was synthesized in the same manner as in Example 18, except that 3-(dimethylamino)propionic acid hydrochloride was used.

[0246]

[0247] <Synthesis of Intermediate 16-b> Intermediate 16-b represented by the following formula was synthesized in the same manner as in Example 18, except that intermediate 15-b was used.

[0248]

[0249] <Synthesis of Compound 19> Compound 19 was synthesized using intermediate 16-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 18. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.33-2.38 (m, 2H), 2.52-2.59 (m, 3H) ), 2.76-2.82 (m, 4H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0250] Example 20 Synthesis of Compound 20 <Synthesis of Intermediate 15-c> Intermediate 15-c represented by the following formula was synthesized in the same manner as in Example 18, except that 4-(dimethylamino)butanoic acid hydrochloride was used.

[0251]

[0252] <Synthesis of Intermediate 16-c> Intermediate 16-c represented by the following formula was synthesized in the same manner as in Example 18, except that intermediate 15-c was used.

[0253]

[0254] <Synthesis of Compound 20> Compound 20 was synthesized using intermediate 16-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 18. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.80 (m, 4H), 2.25-2.38 (m, 8H), 2.52-2.59 (m, 1H), 2.7 6-2.82 (m, 4H), 3.50-3.60 (m, 6H), 6.34 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H), 7.50 (s, 1H)

[0255]

[0256] Example 21 Synthesis of Compound 21 <Synthesis of Intermediate 17-a> 7.10 g of dichloromethane was added to intermediate 2 (393 mg) and triethylamine (235 mg) and dissolved at room temperature. DSC (541 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, N,N-dimethylethylenediamine (112 mg) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. The reaction solution was washed with 5.90 g of 0.5 M acetate buffer (pH 4.0), 5.90 g of 7 wt% sodium bicarbonate water, and 5.90 g of 20 wt% saline, in that order, and then 200 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 472 mg of intermediate 17-a.

[0257] <Synthesis of Intermediate 18-a> Intermediate 18-a was synthesized in the same manner as for intermediate 4-a in Example 1, except that intermediate 17-a was used.

[0258] <Synthesis of Compound 21> Compound 21 was synthesized in the same manner as in Example 1 using intermediate 18-a and 2-hexadecyl octadecanoic acid. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.22 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.26 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0259] Example 22 Synthesis of Compound 22 <Synthesis of Intermediate 17-b> Intermediate 17-b represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-diethylethylenediamine was used.

[0260]

[0261] <Synthesis of Intermediate 18-b> Intermediate 18-b represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-b was used.

[0262]

[0263] <Synthesis of Compound 22> Compound 22 was synthesized using intermediate 18-b and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 4H) ), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0264] Example 23 Synthesis of Compound 23 <Synthesis of Intermediate 17-c> Intermediate 17-c represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-dimethyl-1,3-propanediamine was used.

[0265]

[0266] <Synthesis of Intermediate 18-c> Intermediate 18-c represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-c was used.

[0267]

[0268] <Synthesis of Compound 23> Compound 23 was synthesized using intermediate 18-c and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2 97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.70 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0269] Example 24 Synthesis of Compound 24 <Synthesis of Intermediate 17-d> Intermediate 17-d represented by the following formula was synthesized in the same manner as in Example 21, except that N,N,N'-trimethyl-1,3-propanediamine was used.

[0270]

[0271] <Synthesis of Intermediate 18-d> Intermediate 18-d represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-d was used.

[0272]

[0273] <Synthesis of Compound 24> Compound 24 was synthesized using intermediate 18-d and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 4H), 2.24 (s, 6H), 2.38-2.42 (m, 2H), 2.52-2.59 (m, 1H), 2.88-2.97 (m, 7H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0274] Example 25 Synthesis of Compound 25 <Synthesis of Intermediate 17-e> Intermediate 17-e represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-dimethyl-1,4-butanediamine was used.

[0275]

[0276] <Synthesis of Intermediate 18-e> Intermediate 18-e represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-e was used.

[0277]

[0278] <Synthesis of Compound 25> Compound 25 was synthesized using intermediate 18-e and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.50-1.62 (m, 4H), 1.69-1.84 (m, 2H), 2.20-2.42 (m, 8H), 2.52-2.59 (m, 1H), 2.8 8-2.97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.70 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0279] Example 26 Synthesis of Compound 26 <Synthesis of Intermediate 17-f> Intermediate 17-f represented by the following formula was synthesized in the same manner as in Example 21, except for using 4-(2-aminoethyl)morpholine.

[0280]

[0281] <Synthesis of Intermediate 18-f> Intermediate 18-f represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-f was used.

[0282]

[0283] <Synthesis of Compound 26> Compound 26 was synthesized using intermediate 18-f and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.40-2.46 (m, 6H), 2.52-2.59 (m, 1H), 2.88-2.97 (m , 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.26 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0284] Example 27 Synthesis of Compound 27 <Synthesis of Intermediate 17-g> Intermediate 17-g represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-dibutylethylenediamine was used.

[0285]

[0286] <Synthesis of Intermediate 18-g> Intermediate 18-g represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-g was used.

[0287]

[0288] <Synthesis of Compound 27> Compound 27 was synthesized using intermediate 18-g and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.87-0.93 (m, 12H), 1.20-1.45 (m, 66H), 1.69-1.84 (m, 2H), 2.39-2.57 (m, 7H), 2.88-2.97 (m, 4H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0289] Example 28 Synthesis of Compound 28 <Synthesis of Intermediate 17-h> Intermediate 17-h represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-diisopropylethylenediamine was used.

[0290]

[0291] <Synthesis of Intermediate 18-h> Intermediate 18-h represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-h was used.

[0292]

[0293] <Synthesis of Compound 28> Compound 28 was synthesized using intermediate 18-h and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 0.95-1.03 (d, 12H), 1.20-1.45 (m, 58H), 1.60-1.84 (m, 6H), 2.52-2.59 (m, 1H), 2.88-3.00 (m , 6H), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0294] Example 29 Synthesis of Compound 29 <Synthesis of Intermediate 17-i> Intermediate 17-i represented by the following formula was synthesized in the same manner as in Example 21, except that N,N-diethyl-N'-methylethylenediamine was used.

[0295]

[0296] <Synthesis of Intermediate 18-i> Intermediate 18-i represented by the following formula was synthesized in the same manner as in Example 21, except that intermediate 17-i was used.

[0297] <Synthesis of Compound 29> Compound 29 was synthesized using intermediate 18-i and 2-hexadecyl octadecanoic acid according to the same synthetic route as in Example 21. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.00 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.47-2.57 (m, 7H), 2.88-2.97 (m, 7H) ), 3.22-3.26 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0298]

[0299] Example 30 Synthesis of Compound 30 <Synthesis of Intermediate 19> 5.10 g of dichloromethane was added to intermediate 2 (510 mg) and NPM (670 mg) and dissolved at room temperature. pNPCl (690 mg) was added to the resulting solution and allowed to react at 25°C for 2 hours. DMAP (33.0 mg) and 2-(dimethylamino)ethanol (976 mg) were added to this reaction solution and allowed to react at 25°C for 6 hours. The reaction solution was washed with 5.10 g of 0.5 M acetate buffer (pH 4.0), 5.10 g of 7 wt% sodium bicarbonate water, and 5.10 g of 20 wt% saline, in that order, and then dehydrated by adding 250 mg of sodium sulfate. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 287 mg of intermediate 19.

[0300] <Synthesis of Intermediate 20> Intermediate 20 was synthesized in the same manner as for intermediate 4-a in Example 1, except that intermediate 19 was used.

[0301] <Synthesis of Compound 30> Compound 30 was synthesized using intermediate 20 and 2-hexadecyl octadecanoic acid in the same manner as in the synthesis of compound 1 in Example 1. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.28 (s, 6H), 2.58-2.61 (m, 3H), 2.91-2 94 (m, 4H), 3.63 (s, 2H), 4.23 (t, 2H), 4.32-4.38 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0302]

[0303] Example 31 Synthesis of Compound 31 <Synthesis of Intermediate 21> Intermediate 18-b (1.00 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (445 mg), and DMAP (57.0 mg) were dissolved in 15.0 g of chloroform at room temperature. EDC (668 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 10.0 g of 20 wt% saline, and then dehydrated by adding 500 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.25 g of Intermediate 21.

[0304] <Synthesis of Intermediate 22> Intermediate 21 (1.25 g) was dissolved in 11.3 g of THF, and then 45.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 22 hours. 18.8 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 18.8 g of chloroform. 600 mg of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 908 mg of Intermediate 22.

[0305] <Synthesis of Compound 31> Intermediate 22 (200 mg), myristic acid (175 mg), and DMAP (18.0 mg) were dissolved in 3.00 g of chloroform at room temperature. EDC (175 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator and purified using a column to obtain 106 mg of Compound 31. <Synthesis of Compound 31> １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0306] [Example 32] Synthesis of Compound 32 <Synthesis of Compound 32> Compound 32 was synthesized using intermediate 22 and palmitic acid according to the same synthetic route as in Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 48H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0307] [Example 33] Synthesis of Compound 33 <Synthesis of Compound 33> Compound 33 was synthesized using intermediate 22 and stearic acid according to the same synthetic route as in Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 56H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.28 (s, 1H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0308] [Example 34] Synthesis of Compound 34 <Synthesis of Compound 34> Compound 34 was synthesized using intermediate 22 and oleic acid according to the same synthetic route as in Example 31. １ H-NMR (600MHz, CDCl ３)> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0309] [Example 35] Synthesis of Compound 35 <Synthesis of Compound 35> Compound 35 was synthesized using intermediate 22 and linoleic acid according to the same synthetic route as in Example 31. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 28H), 1.42 (s, 3H), 1.55-1 .65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.75- 2.78 (m, 4H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30 -4.40 (m, 8H), 5.32-5.38 (m, 9H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0310]

[0311] Example 36 Synthesis of Compound 36 Synthesis of Intermediate 23 36.0 g of dichloromethane was added to Intermediate 2 (2.0 g) and triethylamine (1.20 g) and dissolved at room temperature. DSC (2.75 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, ethanolamine (394 mg) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. After the reaction, the solution was washed with 30.0 g of 0.5 M acetate buffer (pH 4.0), and then 1.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 1.41 g of Intermediate 23.

[0312] <Synthesis of Intermediate 24> Intermediate 23 (1.35 g) and triethylamine (594 mg) were dissolved in 20.0 g of chloroform at room temperature. Methanesulfonyl chloride (505 mg) was added to the resulting solution and reacted at room temperature for 3 hours. The reaction solution was washed with 15.0 g of 7 wt % aqueous sodium bicarbonate solution, and 500 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 1.34 g of Intermediate 24.

[0313] <Synthesis of Intermediate 25> Intermediate 2 (1.31 g) was dissolved in a mixed solvent of 11.8 g of THF and 13.1 g of IPA, and then 47.2 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 19.7 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 19.7 g of chloroform was added to the aqueous layer for extraction. 3.0 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 939 mg of Intermediate 25.

[0314] <Synthesis of Intermediate 26> Intermediate 25 (930 mg), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (393 mg), and DMAP (50.0 mg) were dissolved in 15 g of chloroform at room temperature. EDC (590 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 9.30 g of 20 wt% saline, and then 500 mg of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator and purified using a column to obtain 1.06 g of Intermediate 26.

[0315] <Synthesis of Intermediate 27> Intermediate 26 (1.00 g) was dissolved in a mixed solvent of 9.00 g of THF and 9.00 g of IPA, and then 40.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 15.0 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 15.0 g of chloroform was added to the aqueous layer for extraction. 500 mg of sodium sulfate was added to the organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 785 mg of Intermediate 27.

[0316] <Synthesis of Intermediate 28> Intermediate 27 (770 mg), oleic acid (802 mg), and DMAP (66.0 mg) were dissolved in 11.6 g of chloroform at room temperature. EDC (648 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 8.00 g of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.15 g of Intermediate 28.

[0317] <Synthesis of Compound 36> Intermediate 28 (250 mg) was dissolved in 2.50 g of THF, and 2-(methylamino)ethanol (137 mg) and TBAI (17.0 mg) were added, followed by a reaction at 40°C for 8 hours. 3.75 g of chloroform was added to the reaction solution, and the mixture was washed with 2.50 g of 0.5 M acetate buffer (pH 4.0), 2.50 g of 7 wt% sodium bicarbonate water, and 2.50 g of 20 wt% saline, in that order. The filtrate was concentrated using an evaporator and purified using a column to obtain 79 mg of Compound 36. <Synthesis of Compound 36> １ H-NMR (600MHz, CDCl ３)> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.58-3.63 (m, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0318] [Example 37] Synthesis of Compound 37 <Synthesis of Compound 37> Compound 37 was synthesized using intermediate 28 and 3-(methylamino)-1-propanol according to the same synthetic route as in Example 36. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.72 (m, 6H), 1.99-2.05 (m, 8H), 2.30-2.40 (m, 7H), 2.52-2.58 (m, 4H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.58-3.63 (m, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0319]

[0320] Example 38 Synthesis of Compound 38 Synthesis of Intermediate 29 Intermediate 2 (2.00 g), (E)-4-bromocrotonic acid (974 mg), and DMAP (131 mg) were dissolved in 30.0 g of chloroform at room temperature. EDC (1.54 g) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 2.26 g of Intermediate 29.

[0321] <Synthesis of Intermediate 30> Intermediate 29 (2.25 g) was dissolved in a mixed solvent of 20.23 g of THF and 22.5 g of IPA, and then 81.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 2 hours. 34.0 g of chloroform was added to the reaction solution for washing. After washing, a 400 g / L aqueous sodium hydroxide solution was added to the aqueous layer for neutralization, and then 34.0 g of chloroform was added to the aqueous layer for extraction. 1.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 1.51 g of Intermediate 30.

[0322] <Synthesis of Intermediate 31> Intermediate 30 (1.40 g), 2-hexadecyl octadecanoic acid (1.80 g), and DMAP (79.0 mg) were dissolved in 21.0 g of chloroform at room temperature. EDC (925 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was concentrated using an evaporator, yielding 2.98 g of crude product Intermediate 31.

[0323] <Synthesis of Compound 38> 500 mg of the crude product of Intermediate 31 and 2.30 g of dimethylamine (2.0 mol / L in THF) were dissolved in 5.00 g of THF, and potassium iodide (0.12 mmol) was added and reacted at 25°C for 10 hours. After removing insoluble matter by filtration, 7.50 g of chloroform was added to the filtrate, which was then washed with 5.00 g of 0.5 M acetate buffer (pH 4.0), 5.00 g of 7 wt% sodium bicarbonate water, and 5.00 g of 20 wt% saline, in that order. 250 mg of sodium sulfate was added and dehydrated, after which the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator and purified using a column to obtain 270 mg of Compound 38. <Synthesis of Compound 38> １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.25 (s, 6H), 2.52-2.59 (m, 1H), 2.88-2.97 (m, 4H) ), 3.07 (t, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0324] [Example 39] Synthesis of Compound 39 <Synthesis of Compound 39> Compound 39 was synthesized in the same manner as in Example 38, except that diethylamine was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 6H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.50-2.59 (m, 5H), 2.88-2.97 (m, 4H) ), 3.23 (t, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0325] [Example 40] Synthesis of Compound 40 <Synthesis of Compound 40> Compound 40 was synthesized in the same manner as in Example 38, except that 2-(ethylamino)ethanol was used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.03 (t, 3H), 1.20-1.45 (m, 58H), 1.69-1.84 (m, 2H), 2.50-2.59 (m, 5H), 2.88-2.97 (m, 4H), 3.23 (t , 2H), 3.55-3.58 (m, 2H), 3.63 (s, 2H), 4.28-4.38 (m, 4H), 5.97-6.00 (m, 1H), 6.95-7.05 (m, 3H), 7.27-7.30 (m, 2H)

[0326]

[0327] Example 41 Synthesis of Compound 41 <Synthesis of Compound 41> Compound 41 was synthesized using the same synthetic route as in Example 1, except that intermediate 1, 4,4'-dithiobisbutan-1-ol, and 4-(dimethylamino)butanoic acid hydrochloride were used as starting materials. １ H-NMR (600MHz, CDCl ３)> δ: 0.88 (t, 6H), 1.20-1.50 (m, 64H), 1.69-1.84 (m, 4H), 2.21 (s, 6H), 2.28 (t, 2H), 2.35 (t, 2H), 2.52- 2.59 (m, 1H), 2.59-2.70 (m, 4H), 3.63 (s, 2H), 4.22-4.33 (m, 4H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0328]

[0329] Example 42 Synthesis of Compound 42 <Synthesis of Intermediate 32> Bis(2-hydroxyethyl)disulfide (33.2 g), 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (25.0 g), and DMAP (3.50 g) were dissolved in 250 g of chloroform at room temperature. EDC (41.0 g) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 9.0 mL of 20 wt% saline, and then 25.0 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 21.8 g of Intermediate 32.

[0330] <Synthesis of Intermediate 33> 180 g of dichloromethane was added to Intermediate 32 (10.0 g) and triethylamine (7.17 g) and dissolved at room temperature. DSC (16.5 g) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. After the DSC remaining in the reaction solution was removed by filtration, N,N-diethylethylenediamine (5.00 g) was added to the filtrate, and the reaction was carried out at 25°C for 1 hour. After the reaction, the solution was washed with 150 g of 0.5 M acetate buffer (pH 4.0), 150 g of 7 wt% sodium bicarbonate water, and 150 g of 20 wt% saline, in that order, and then 5.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator, yielding 13.1 g of Intermediate 33.

[0331] <Synthesis of Intermediate 34> Intermediate 33 (13.0 g) was dissolved in 49.0 g of THF, and then 195 g of 0.5 M phosphate buffer (pH 2.0) was added and the mixture was reacted at 40°C for 3 hours. 120 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 7.0. The aqueous layer was then extracted with 195 g of chloroform. 6.50 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 5.01 g of Intermediate 34.

[0332] <Synthesis of Intermediate 35> Intermediate 34 (5.00 g), Intermediate 1 (6.01 g), and DMAP (592 mg) were dissolved in 75.0 g of chloroform at room temperature. EDC (5.81 g) was added to the resulting solution and the mixture was allowed to react at 25°C for 2 hours. The reaction solution was washed with 50.0 g of 20 wt% saline, and then 2.50 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 6.25 g of crude Intermediate 35.

[0333] <Synthesis of Intermediate 36> 6.25 g of the crude product of Intermediate 35 was dissolved in 56.3 g of THF, and then 225 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 25°C for 2 hours. 94.0 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 94.0 g of chloroform. 3.00 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 3.7 g of Intermediate 36.

[0334] <Synthesis of Compound 42> Intermediate 36 (500 mg), oleic acid (436 mg), and DMAP (36.0 mg) were dissolved in 7.50 g of chloroform at room temperature. EDC (352 mg) was added to the resulting solution, and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 5.00 g of 20 wt% saline, and then dehydrated by adding 250 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 453 mg of compound 42. <Synthesis of Compound 42> １ H-NMR (600MHz, CDCl ３ )> δ: 0.88 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32-5.38 (m, 5H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0335] [Example 43] Synthesis of Compound 43 <Synthesis of Compound 43> Compound 43 was synthesized using intermediate 36 and myristic acid according to the same synthetic route as in Example 42. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.9 2-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32 (s, 1H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0336]

[0337] Example 44 Synthesis of Compound 44 Synthesis of Intermediate 37 Intermediate 34 (1.60 g), Intermediate 1 (4.19 g), and DMAP (165 mg) were dissolved in 24.0 g of chloroform at room temperature. EDC (1.95 g) was added to the resulting solution and reacted at 25°C for 2 hours. The reaction solution was washed with 16.0 g of 20 wt% saline, and then dehydrated by adding 500 mg of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 1.85 g of Intermediate 37.

[0338] <Synthesis of Intermediate 38> Intermediate 37 (450 mg), oleic acid (241 mg), and DMAP (19.0 mg) were dissolved in 6.75 g of chloroform at room temperature. EDC (223 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 4.5 g of 20 wt% saline, and then 2.0 g of sodium sulfate was added for dehydration. The sodium sulfate was removed by filtration, and the filtrate was concentrated using an evaporator to obtain 670 mg of crude product, Intermediate 38.

[0339] Synthesis of Intermediate 39: The crude product of Intermediate 38 (670 mg) was dissolved in a mixed solvent of 4.00 g of THF and 4.00 g of IPA, and then 16.2 g of 0.5 M phosphate buffer (pH 2.0) was added and the reaction was carried out at 40°C for 3 hours. 13.5 g of chloroform was added to the reaction solution for extraction, and then 9.00 g of 0.5 M phosphate buffer (pH 6.5) was added to the organic layer for washing. 300 mg of sodium sulfate was added to the washed organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator and purified using a column to obtain 240 mg of Intermediate 39.

[0340] <Synthesis of Compound 44> Intermediate 39 (150 mg), lauric acid (41.0 mg), and DMAP (5.0 mg) were dissolved in 2.25 g of chloroform at room temperature. EDC (53.0 mg) was added to the resulting solution and the reaction was carried out at 25°C for 2 hours. The reaction solution was washed with 1.50 g of 20 wt% saline, and then 50.0 mg of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 116 mg of Compound 44. <Synthesis of Compound 44>１ H-NMR (600MHz, CDCl ３ )> δ: 0.80-0.92 (m, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 36H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 4H), 2.36 (t, 2H), 2.52-2.58 (m , 6H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 4H), 4.30-4.40 (m, 8H), 5.32-5.36 (m, 3H), 7.02-7.05 (m, 2H), 7.27-7.30 (m, 2H)

[0341]

[0342] Example 45 Synthesis of Compound 45 <Synthesis of Intermediate 40> 30.0 g of dichloromethane was added to intermediate 18-b (3.00 g) and NPM (3.41 g) and dissolved at room temperature. pNPCl (3.51 g) was added to the resulting solution and reacted at 25°C for 10 hours. DMAP (170 mg) and 1,2-isopropylidene glycol (7.37 g) were added to this reaction solution and reacted at 25°C for 8 hours. The reaction solution was washed with 30.0 g of 7 wt% sodium bicarbonate water and 30.0 g of 20 wt% saline, in that order, and then dehydrated by adding 9.00 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator and purified using a column to obtain 2.16 g of intermediate 40.

[0343] <Synthesis of Intermediate 41> Intermediate 40 (2.16 g) was dissolved in 11.3 g of THF, and then 45.0 g of 0.5 M phosphate buffer (pH 2.0) was added and the mixture was reacted at 40°C for 2 hours. 19.0 g of chloroform was added to the reaction solution for washing. 400 g / L aqueous sodium hydroxide solution was added to the washed aqueous layer, and the pH was adjusted to 6.0. The aqueous layer was then extracted with 19.0 g of chloroform. 1.20 g of sodium sulfate was added to the extracted organic layer for dehydration, and the sodium sulfate was removed by filtration. The filtrate was concentrated using an evaporator to obtain 850 mg of Intermediate 41.

[0344] <Synthesis of Compound 45> Compound 45 was synthesized using the same synthetic route as in Example 42, except that intermediate 41 and oleic acid were used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.38 (m, 40H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 1.99-2.05 (m, 8H), 2.36 (t, 4H), 2.52-2.58 (m, 6) H), 2.92-2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.50 (m, 8H), 5.28-5.38 (m, 6H), 6.97-7.00 (m, 2H), 7.27-7.30 (m, 2H)

[0345] [Example 46] Synthesis of Compound 46 <Synthesis of Compound 46> Compound 46 was synthesized using the same synthetic route as in Example 42, except that intermediate 41 and myristic acid were used. １ H-NMR (600MHz, CDCl ３ )> δ: 0.89 (t, 6H), 1.04 (t, 6H), 1.25-1.35 (m, 32H), 1.42 (s, 3H), 1.55-1.65 (m, 4H), 2.36 (t, 4H), 2.52-2.58 (m, 6H), 2.92- 2.96 (m, 4H), 3.22-3.26 (m, 2H), 3.66 (s, 2H), 4.30-4.50 (m, 8H), 5.28-5.31 (m, 2H), 6.97-7.00 (m, 2H), 7.27-7.30 (m, 2H)

[0346] [Comparative Example] Synthesis of O-Ph-P4C2 O-Ph-P4C2 was synthesized according to the synthetic route described in Example 3 of Patent Document 3. １ H-NMR (600MHz, CDCl ３ )> δ0.88 (t, 6H), 1.22-1.42 (m, 46H), 1.54-1.76 (m, 12H), 1.94-2.03 (m, 12H), 2.52-2.56 (m, 4H), 2.62-2.66 (m , 4H), 2.80-2.89 (m, 8H), 3.59 (s, 4H), 4.13 (t, 4H), 5.34-5.37 (m, 4H), 7.02-7.05 (m, 4H), 7.27-7.30 (m, 4H)

[0347] Test Example 1 Measurement of Liposomal pKa Lipid nanoparticles (LNPs) containing no nucleic acid were used to evaluate liposomal pKa.

[0348] 1. Preparation of LNPs by Microchannel Method (1) Preparation of Lipid Ethanol Solution In an Eppendorf tube, a 10 mM ethanol solution of cationic lipid, a 5 mM ethanol solution of DOPC, and a 10 mM ethanol solution of chol were mixed at the desired ratio (cationic lipid: DOPC: chol = 52.5: 7.5: 40 (molar ratio)) so that the total lipid amount was 720 nmol. To the resulting mixture, DMG-PEG2k (2 mM ethanol solution) was added in an amount of about 1.5 moles per 100 moles of the total amount of cationic lipid, DOPC, and chol, and then ethanol was added to prepare a lipid ethanol solution (total volume: 360 μL).

[0349] (2) Preparation of LNP using a microchannel 1080 μL of acidic malic acid buffer (20 mM, pH 3.0) containing a final concentration of 30 mM NaCl and 360 μL of lipid ethanol solution were weighed into syringes. Using an ultrafast nanomedicine production device NanoAssmblr (manufactured by Precision NanoSystems), LNP was prepared under the following conditions: acidic buffer solution addition rate: 3 mL / min, lipid ethanol solution addition rate: 1 mL / min, and syringe holder temperature: 25 ° C., and collected in a 15 mL tube. After adding 3000 μL of MES buffer (pH 6.5) to the 15 mL tube, the resulting mixture was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugal conditions (25 ° C., 1000 g, 6 min) to concentrate to approximately 100 μL. The resulting concentrate was diluted to 4 mL with PBS and concentrated again under centrifugation conditions (25°C, 1000 g, 6 min) twice. The resulting concentrate was diluted with PBS to a lipid concentration of 0.5 mM to obtain a dispersion containing LNP.

[0350] 2. Measurement of Liposomal pKa 20 mM citrate buffer, sodium phosphate buffer, and Tris-HCl buffer containing a final concentration of 150 mM NaCl were prepared at various pH values ​​ranging from pH 3.0 to 10.0. TNS (Sigma) solution was diluted with ultrapure water to 0.6 mM. 2 μL of TNS solution, 12 μL of the LNP-containing dispersion prepared in [Test Example 1] 1., and 186 μL of buffer solutions adjusted to various pH values ​​were added to a black 96-well plate. The plate was shielded from light and shaken at 400 rpm for 10 minutes. Fluorescence intensity (excitation: 321 nm / emission: 447 nm) was measured using a plate reader (TECAN). The maximum fluorescence intensity for each LNP was set to 100% and the minimum to 0%, and the relative fluorescence intensity was calculated as a percentage. The pH at which the relative fluorescence intensity was 50% was defined as the liposomal pKa. The liposomal pKa of each LNP is shown in Table 4. For comparative examples, the cationic lipids listed in Table 3 above were used.

[0351]

[0352] 3. Results The liposomal pKa of all LNPs was within the pKa range (5.0 to 9.5) favorable for endosomal escape.

[0353] [Test Example 2] Preparation of mRNA Encapsulated Particles and Evaluation of Physical Properties 1. Preparation of LNPs by Microchannel Method (1) Preparation of Lipid Ethanol Solution In an Eppendorf tube, a 10 mM ethanol solution of cationic lipid, a 5 mM ethanol solution of DOPC, and a 10 mM ethanol solution of chol were mixed at the desired ratio (cationic lipid: DOPC: chol = 55:5:40 (molar ratio)) so that the total lipid amount was 720 nmol. To the resulting mixture, DMG-PEG2k (2 mM ethanol solution) was added in an amount of about 1 mole per 100 moles of the total amount of cationic lipid, DOPC, and chol, and then ethanol was added to prepare a lipid ethanol solution (total volume: 360 μL).

[0354] (2) Preparation of Acidic Buffer Solution of Nucleic Acid 7.2 μg of 0.6 mg / mL mRNA solution was weighed into a 5 mL tube, and an acidic malic acid buffer (20 mM, pH 3.0) containing a final concentration of 30 mM NaCl was added to prepare an acidic buffer solution of nucleic acid (total volume: 1080 μL).

[0355] (3) Preparation of LNP using a microchannel An acidic buffer solution of nucleic acid and an ethanol solution of lipid were weighed into syringes. Using an ultrafast nanomedicine production device NanoAssmblr (manufactured by Precision NanoSystems), LNP was prepared under the following conditions: an acidic buffer solution of nucleic acid addition rate: 3 mL / min, an ethanol solution of lipid addition rate: 1 mL / min, and a syringe holder temperature: 25 ° C. The LNP was collected in a 15 mL tube. After adding 3000 μL of MES buffer (pH 6.5) to the 15 mL tube, the resulting mixture was transferred to an Amicon Ultra 4 and subjected to ultrafiltration under centrifugation conditions (25 ° C., 1000 g, 6 min) to concentrate to approximately 100 μL. The resulting concentrate was diluted to 4 mL with PBS and concentrated again under centrifugation conditions (25°C, 1000 g, 6 min). This procedure was repeated twice. The resulting concentrate was diluted with PBS to a lipid concentration of 2 mM to obtain a dispersion containing LNP.

[0356] 2. Measurement of particle size, PdI, and zeta potential of mRNA-encapsulated LNP The particle size, PdI (polydispersity index), and zeta potential of the mRNA-encapsulated LNP prepared by the method described in 1 above were measured using dynamic light scattering (Zetasizer Nano; Malvern). The results are shown in Table 5. For comparative examples, the cationic lipids listed in Table 3 above were used.

[0357]

[0358] 3. Results All mRNA-encapsulated LNPs had a particle size of 30 to 300 nm, which is a preferred morphology, and the charge (zeta potential) at physiological pH was also in the preferred range (-15 to +15 mV).

[0359] Test Example 3 Evaluation of Gene Expression in HeLa Cells In Vitro 1. Preparation of mRNA-Encapsulated LNPs LNPs (cationic lipid:DOPC:Chol:DMG-PEG2k=55:5:40:1 (molar ratio)) encapsulating mRNA expressing luciferase were prepared by the method described in Test Example 2, 1.

[0360] 2. Time course evaluation of gene expression in HeLa cells 24 hours before transfection, 5.0 × 10 HeLa cells, which are human cervical cancer cells, were transfected. ４ The cells were seeded onto a 3.5 cm dish at a concentration of 1000 cells / 2 mL / dish. After 24 hours, the medium was replaced with a culture medium (D-MEM) containing 0.1 mM D-luciferin. The prepared mRNA-encapsulated LNP was diluted with PBS to an mRNA concentration of approximately 8 μg / mL. The diluted mRNA-encapsulated LNP solution (approximately 50 μL, mRNA: 0.4 μg) was added to a 3.5 cm dish and placed in an incubator-type luminometer KronosDio. The luciferase luminescence intensity was measured for 2 minutes every hour. The cumulative luciferase luminescence intensity over 24 hours was calculated from the obtained changes in expression over time. The results are shown in Table 6. Note that "E+0a" (a: integer) in Table 6 is a decimal fraction of "10 ａ For example, "1.0E+06" represents "1.0 x 10 ６ In the comparative examples, the cationic lipids listed in Table 3 above were used.

[0361]

[0362] 3. Results As shown in Table 6, the cationic LNPs of Examples 5, 7, 8, 9, 10, 11, 15, 19, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 34, 35, 36, and 42 exhibited superior gene expression activity compared to the comparative examples. Therefore, it was revealed that the cationic LNPs of Examples 5, 7, 8, 9, 10, 11, 15, 19, 21, 22, 23, 24, 25, 28, 29, 30, 31, 32, 34, 35, 36, and 42 are useful as LNPs that can promote mRNA expression.

[0363] Test Example 4 Evaluation of Gene Expression in Vivo 1. Preparation of mRNA-Encapsulated LNPs LNPs (cationic lipid:DOPC:Chol:DMG-PEG2k=55:5:40:1 (molar ratio)) encapsulating mRNA expressing luciferase were prepared by the method described in Test Example 2, 1.

[0364] 2. Evaluation of Gene Expression in Vivo Balb / c Mice (Male, 5 Weeks Old) were administered each of the prepared mRNA-encapsulated LNP dispersions via the tail vein. The mRNA dose was 0.05 mg per kg of mouse body weight, and the mRNA-encapsulated LNP dispersion was administered at a dose of 10 μL per g of mouse body weight. 4.5 hours after administration of the mRNA-encapsulated LNP dispersion, a PBS solution of D-luciferin potassium was administered intraperitoneally to the mice. The dose of D-luciferin potassium was 3 mg per mouse, and the PBS solution of D-luciferin potassium was administered at a dose of 200 μL per mouse. 15 minutes after administration of the PBS solution of D-luciferin potassium, the mice were euthanized, and the liver, spleen, kidneys, heart, and lungs were removed. The luminescence in each organ was quantified using an In Vivo Imaging System (IVIS) with an exposure time of 10 seconds. The amount of luminescence in each organ was quantified by image analysis using the Live Imaging software included with the IVIS. The amount of luminescence (photons / sec) was calculated from the acquired images and used as an index of gene expression activity. The results are shown in Tables 7 and 8. Note that "E+0a" (a: integer) in Tables 7 and 8 is "10 ａ ". For example, "1.0E+02" represents "1.0 x 10 ２ In the comparative examples, the cationic lipids listed in Table 3 above were used.

[0365]

[0366]

[0367] 3. Results As shown in Table 8, the LNPs containing the cationic lipids of Examples 31 and 34 exhibited superior gene expression activity, particularly in the spleen, compared to the comparative example. Therefore, it was revealed that the LNPs containing the cationic lipids of Examples 31 and 34 are useful as LNPs that can promote mRNA expression.

[0368] The cationic lipid of the present invention is useful for nucleic acid medicines, gene therapy, biochemical experiments, and the like.

[0369] This application is based on patent application No. 2022-051912 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. Formula (1): 【Chemistry 1】 (In the formula, X represents a nitrogen-containing aliphatic group containing one or more tertiary nitrogen atoms. R １ This represents an aliphatic hydrocarbon group with 8 or fewer carbon atoms. L １ This represents an ester bond, amide bond, carbamate bond, N-alkylcarbamate bond, carbonate bond, or urea bond. k represents 0 or 1, R ｘ and R ｙ Each of these independently represents an alkylene group with 2 to 5 carbon atoms. L ２ This represents an ester bond, amide bond, carbamate bond, carbonate bond, ether bond, or urea bond. R ２ This represents an alkylene group with 8 or fewer carbon atoms, or it does not exist. Y represents a group that (i) contains one or more divalent groups derived from an aromatic compound which may have heteroatoms, (ii) has at least one selected from the group consisting of an ester bond and a carbonate bond on the aromatic ring of the divalent group, and (iii) contains at least one selected from the group consisting of an aliphatic hydrocarbon group having 10 to 37 carbon atoms, a lipid-soluble vitamin residue, and a sterol derivative residue. A cationic lipid having a disulfide bond, represented by [the formula shown].

2. X is given by equation (2): R α -N(R β )- (2) (In the formula, R α represents an aliphatic hydrocarbon group, R β This represents an aliphatic hydrocarbon group, an aliphatic group containing one or more heteroatoms other than nitrogen, or an aliphatic group containing one or more tertiary amines, or R α and R β These may combine to form a 3- to 8-membered nitrogen-containing alicyclic ring. A cationic lipid having a disulfide bond as described in claim 1, wherein the group is represented by .

3. X is a dialkylamino group (the two alkyl groups of the dialkylamino group each have 1 to 8 carbon atoms independently), a cyclic amino group which may have a 3 to 6-membered heteroatom, or -N(R ａ )-R ｂ This represents, R ａ This represents an alkyl group with 1 to 8 carbon atoms. R ｂ is, -(CH ２ ) q-O-R ｃ This represents, R ｃ This represents hydrogen or an alkyl group having 1 to 8 carbon atoms. q represents an integer between 2 and 4. A cationic lipid having a disulfide bond as described in claim 1.

4. R １ However, a cationic lipid having a disulfide bond according to any one of claims 1 to 3, which represents an alkylene group having 8 or fewer carbon atoms, or an alkenylene group having 8 or fewer carbon atoms.

5. Y is given by equation (3): 【Chemistry 2】 (In the formula, R ３ This represents an alkylene group with 8 or fewer carbon atoms. L ３ This represents an ester bond or a carbonate bond. S １ This consists of hydrogen, an alkyl group having 1 to 8 carbon atoms, and -R. ｅ -L ａ -R ７ ', or -R ｅ -L ａ -Z'-L ｂ -R ７ ' represents, S ２ is, -R ｅ '-L ａ -R ７ '' or -R ｅ '-L ａ -Z''-L ｂ -R ７ Represents '', R ｅ and R ｅ Each of these independently represents an alkylene group with 8 or fewer carbon atoms. L ａ This represents an ester bond or a carbonate bond. L ｂ This represents an ester bond or a carbonate bond. l represents 0 or 1, Z, Z', and Z'' each independently represent a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, possessing at least one aromatic ring, and possibly containing a heteroatom. L ｘ This represents an ester bond or a carbonate bond. R ４ This represents an alkylene group with 8 or fewer carbon atoms, or it does not exist. R ５ This represents hydrogen or an alkyl group having 1 to 8 carbon atoms. S ３ is, -R ６ '-L ４ '-R ７ Represents ''', m represents 0 or 1, R ６ and R ６ Each of these independently represents an alkylene group with 8 or fewer carbon atoms, or it does not exist. L ４ and L ４ Each of these independently represents either an ester bond or a carbonate bond. R ７ , R ７ ', R ７ '' and R ７ Each of the '''s is independently an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ )p-C(=O)-R ｆ This represents, R ｆ represents a residue of a fat-soluble vitamin having a hydroxyl group or a residue of a sterol derivative having a hydroxyl group, and p represents 2 or 3. n represents either 0 or 1. A cationic lipid having a disulfide bond according to any one of claims 1 to 3, wherein the group is represented by .

6. Z, Z', and Z'' are given by equation (4): 【Transformation 3】 (In the formula, t represents an integer between 0 and 3. u represents an integer from 0 to 3. v represents an integer from 0 to 4, and v R ８ Each of these independently represents a substituent, * is L ３ or L ａ (This indicates the connection position with [the other element].) A cationic lipid having a disulfide bond according to any one of claims 1 to 3, wherein the group is represented by .

7. R ｘ and R ｙ A cationic lipid having a disulfide bond as described in any one of claims 1 to 3, wherein both are ethylene groups.

8. R ７ , R ７ ', R ７ '' and R ７ Each of these is independently an aliphatic hydrocarbon group having 10 to 37 carbon atoms, or -(CH ２ ) ２ -C(=O)-R ｆ This represents, R ｆ However, a cationic lipid having a disulfide bond according to any one of claims 1 to 3, which represents a residue of a fat-soluble vitamin having a hydroxyl group.

9. A lipid membrane structure comprising a cationic lipid having a disulfide bond as described in any one of claims 1 to 3 as a constituent lipid of the membrane.

10. A lipid membrane structure comprising a cationic lipid having a disulfide bond as described in claim 1 as a constituent lipid of the membrane, and further comprising nucleic acids.

11. A nucleic acid delivery agent comprising a cationic lipid having a disulfide bond as described in any one of claims 1 to 3, or a lipid membrane structure as described in claim 10.

12. A pharmaceutical composition comprising a cationic lipid having a disulfide bond as described in any one of claims 1 to 3, or a lipid membrane structure as described in claim 10.

13. A method for introducing nucleic acid into a cell, comprising bringing into contact with a cell a nucleic acid delivery agent according to claim 11, which contains the nucleic acid, in vitro.

14. A method for introducing nucleic acid into target cells, comprising administering a nucleic acid delivery agent according to claim 11, which contains nucleic acid, to a living organism (excluding humans) so as to deliver the nucleic acid to the target cells.

15. A method for producing a cell-based pharmaceutical product containing cells expressing a specific gene, comprising bringing a nucleic acid delivery agent according to claim 11, which contains a nucleic acid, into contact with cells to introduce the nucleic acid into the cells.

Images