New cationic lipids with a cystine backbone

Abstract

The present invention provides a cationic lipid represented by formula (1) (wherein each symbol is as defined in the description), a lipid membrane structure using the cationic lipid, a nucleic acid transfection agent using the cationic lipid, and a method of nucleic acid transfection using the nucleic acid transfection agent.

Description

[Technical field]

[0001] The present invention relates to a cationic lipid having a cystine skeleton, a lipid membrane structure containing the same, and uses thereof. [Background technology]

[0002] To put nucleic acid therapy into practical use, an effective and safe nucleic acid delivery carrier is required. Viral vectors are nucleic acid delivery carriers with high expression efficiency, but development of non-viral nucleic acid delivery carriers that are safer to use is underway, and among them, carriers using cationic lipids are the non-viral nucleic acid delivery carriers currently most commonly used.

[0003] Cationic lipids are broadly composed of an amine moiety and a lipid moiety. The cationic amine moiety interacts electrostatically with nucleic acid, which is a polyanion, to form a liposome or lipid membrane structure, facilitating cellular uptake and delivering the nucleic acid into the cell.

[0004] Examples of commonly used cationic lipids include DOTAP and DODAP. These cationic lipids, when combined with phospholipids, form positively charged liposomes or lipid membrane structures, which can electrostatically interact with nucleic acids to deliver nucleic acids to target cells (Non-Patent Document 1).

[0005] On the other hand, in order for lipid membrane structures using cationic lipids to exert practical effects in vivo as nucleic acid delivery carriers, they need to satisfy requirements such as good pharmacokinetics, specifically high stability in blood, high accumulation in target tissues such as liver and tumors, etc. In response to this issue, the following examples have been reported in which pharmacokinetics was improved by adjusting the pKa of the surface of lipid membrane structures.

[0006] Non-Patent Documents 2 and 3 show 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. Furthermore, these documents state that adjusting the surface pKa of the lipid membrane structure to a value favorable for endosomal escape promotes escape of the lipid membrane structure from the endosome, enabling efficient delivery of nucleic acid into the cytoplasm.

[0007] Thus, cationic lipids with improved pharmacokinetics have been developed, but due to the nature of nucleic acid delivery carriers, which generally introduce foreign substances into cells, it is desirable for them to exert a large effect with a small amount of uptake. That is, when a lipid membrane structure is used as a delivery carrier for an expression vector into a cell, it is desired to increase the expression amount per unit lipid membrane structure taken into the cell and to increase the expression efficiency within the cell. In order to increase the expression efficiency within the cell, in addition to the pharmacokinetics, it is necessary to improve the intracellular pharmacokinetics such as uptake into the cell, escape from the endosome, and permeation through the nuclear membrane (Non-Patent Document 4).

[0008] There are examples where intracellular dynamics have been improved by imparting biodegradability to cationic lipids (Patent Documents 1 to 4). These documents describe cationic lipids having a structure linked by disulfide bonds that exhibit biodegradability, and show that intracellular dynamics can be improved by dissociating nucleic acids from lipid membrane structures by utilizing the fact that disulfide bonds are cleaved within cells. In fact, since the cationic lipid exhibits a high nucleic acid delivery efficiency compared to known cationic lipids DOTAP and DODAP, it has been revealed that the cationic lipid can improve intracellular dynamics, such as improving the efficiency of delivery of nucleic acids into the cytoplasm, and furthermore, the effect of reducing toxicity by imparting degradability is expected.

[0009] Thus, there are several reports of improving the pharmacokinetics and intracellular pharmacokinetics of nucleic acid delivery carriers by adjusting the surface pKa of lipid membrane structures, introducing degradable disulfide bonds, etc. However, there are a wide variety of diseases targeted by nucleic acid therapy, and in order to establish treatments suited to each disease, it is necessary to increase the number of lipid types to choose from and further improve the effects. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US9708628 [Patent Document 2] International Publication No. 2016 / 121942 [Patent Document 3] International Publication No. 2019 / 188867 [Patent Document 4] US2018 / 0298379 [Non-patent literature]

[0011] [Non-Patent Document 1] Biomaterials 29(24-25):3477-96,2008 [Non-Patent Document 2] Molecular Therapy 24(4):788-795,2016 [Non-Patent Document 3] Angewante Chemie International Edition 51:8529-8533,2012 [Non-Patent Document 4] Molecular Therapy 13(4):786―794,2006 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a cationic lipid that can be used as a nucleic acid delivery carrier with good intracellular dynamics, a lipid membrane structure using the cationic lipid, a nucleic acid transfer agent using the cationic lipid, and a method for nucleic acid transfer using the nucleic acid transfer agent. [Means for solving the problem]

[0013] The present inventors have conducted extensive research in light of the above problems and have found a cationic lipid that can adjust the surface pKa of a lipid membrane structure and improve its in vivo and intracellular dynamics. Specifically, the cationic lipid has a lipid moiety and an amine moiety introduced into a cystine backbone, and since it has a disulfide bond, the disulfide bond is cleaved in cells, and it also has the effect of dissociating nucleic acid from the lipid membrane structure. It has been found that a lipid membrane structure containing this novel cationic lipid can efficiently deliver nucleic acid into the cytoplasm.

[0014] That is, the present invention encompasses the following.

[0015] [1] Formula (1):

[0016] [ka]

[0017] (In the formula, L 1a and L 1b each independently represents an amide bond, a carbamate bond, or a urea bond; R 1a and R 1b each independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group having 8 or less carbon atoms, ka and kb each independently represent 0 or 1; X a and X b each independently represents a dialkylamino group (the carbon number of the two alkyl groups is independently 1 to 5) or a 3- to 6-membered cyclic amino group, L4a and L 4b each independently represents an ester bond or an amide bond, R 2a and R 2b each independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group having 8 or less carbon atoms, L 2a and L 2b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, ma and mb each independently represent 0 or 1; R 3a and R 3b each independently represents 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 3a and L 3b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, na and nb each independently represent 0 or 1; R 4a and R 4b are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R 5 -CO-(CH2) p - represents R 5 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. A cationic lipid represented by the formula: [2] The cationic lipid according to [1] above, wherein na and nb are both 1.

[0018] [3] R 3a and R 3b are each independently expressed by the formula (2):

[0019] [ka]

[0020] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a , L 2b 、 L 4a or L 4b represents the bonding position with s represents an integer of 0 to 3; t represents an integer of 0 to 3; u represents an integer from 0 to 4, and u R 6 each independently represents a substituent. The cationic lipid according to the above [1] or [2], wherein the cationic lipid is a group represented by the formula: [4] The cationic lipid according to [3] above, wherein s is 0.

[0021] [5] The cationic lipid according to any one of [1] to [4] above, wherein ka and kb are both 0. [6] The cationic lipid according to any one of [1] to [4] above, wherein ka and kb are both 1.

[0022] [7] R 5 is a residue of a fat-soluble vitamin having a hydroxyl group. [8] R 4a and R 4b Each of the above is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms. [9] R 4a and R 4b However, each is independently R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin having a hydroxyl group.

[0023]

[10] A lipid membrane structure comprising the cationic lipid according to any one of the above [1] to [9] as a constituent lipid of the membrane.

[11] A nucleic acid transfer agent comprising the cationic lipid according to any one of [1] to [9] above, or the lipid membrane structure according to

[10] above.

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

[11] above, which contains a nucleic acid, ex vivo.

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

[11] encapsulating a nucleic acid to a living body so as to deliver the nucleic acid to a target cell. Effect of the Invention

[0024] The present invention relates to a cationic lipid having a dialkylamino group or a cyclic amino group and a lipid moiety introduced into a biodegradable cystine backbone, a lipid membrane structure containing the same, and uses thereof. The cationic lipid of the present invention can form a lipid membrane structure. The cationic lipid and lipid membrane structure of the present invention can be used for a nucleic acid transfer agent. The cationic lipid of the present invention has an amine moiety positioned at the end of the molecular structure, allowing for modification of the structure near the amine moiety, thereby allowing for a wide range of pKa adjustment. Furthermore, the disulfide bond contained in the cystine backbone of the cationic lipid of the present invention is cleaved in a reductive environment within the cell, promoting the release of the encapsulated substance (nucleic acid). Therefore, a nucleic acid transfer agent using the cationic lipid of the present invention can achieve high nucleic acid delivery efficiency into the cytoplasm.

[0025] When nucleic acid is transferred using the cationic lipid of the present invention or a lipid membrane structure containing the same, degradation of the nucleic acid by serum components is suppressed, which is advantageous for nucleic acid transfer in the presence of serum or in vivo. [Brief description of the drawings]

[0026] [Figure 1]FIG. 1 shows the in vitro gene expression activity in Jurkat cells of various lipid nanoparticles (sometimes abbreviated as "LNP" in this specification) prepared from the cationic lipid of the present invention. [Diagram 2] FIG. 1 shows the in vivo gene expression activity of various LNPs prepared from the cationic lipids of the present invention. [Diagram 3] FIG. 1 shows the in vitro gene expression activity in HeLa cells of various LNPs prepared from the cationic lipids of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these. The present invention relates to a compound represented by formula (1):

[0028] [ka]

[0029] The cationic lipid may be used alone or in combination of two or more. In the following, the "cationic lipid represented by formula (1)" may be abbreviated as "cationic lipid (1)". Compounds represented by other formulas may also be abbreviated in the same manner.

[0030] L 1a and L 1b are each independently an amide bond, a carbamate bond or a urea bond, preferably each independently an amide bond or a carbamate bond, and more preferably both are amide bonds.

[0031] L 1a L 1b may be the same as or different from, but preferably, L 1a L 1b is equivalent to.

[0032] L 1a and L 1b are preferably each independently *-NH-CO-**, *-NH-CO-O-** or *-NH-CO-NH-**, more preferably each independently *-NH-CO-** or *-NH-CO-O-**, and more preferably both are *-NH-CO-** (in the above formula, ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bond position with the carbon atom on the opposite side.) Here, "R 1a or R 1b The "bonding position with L 1a So, "R 1a The bond position with L 1b So, "R 1b Also, "R 1a or R 1b The bond position with the carbon atom on the opposite side to the L 1a So, "R 1a means the bonding position with the carbon atom on the opposite side, and L 1b So, "R 1b means the position of bonding to the carbon atom on the opposite side. Other similar expressions have similar meanings.

[0033] R 1a and R 1b R each independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group having 8 or less carbon atoms, and preferably each independently represents an alkylene group having 8 or less carbon atoms. 1a is R 1b may be the same as or different from, but preferably, R 1a is R 1b is equivalent to.

[0034] R 1a and R 1b The alkylene group having 8 or less carbon atoms may be either linear or branched, but is preferably linear. The alkylene group preferably has 6 or less carbon atoms, more preferably 4 or less carbon atoms.

[0035] R 1a and R 1b Examples of the alkylene group having 8 or less carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group, an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.

[0036] R 1a and R 1b 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(CH3)CH2-, -CH2CH(CH3)-), an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group, and more preferably a methylene group, an ethylene group, a trimethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-).

[0037] An oxydialkylene group having 8 or less carbon atoms means a divalent group (i.e., alkylene-O-alkylene) having a structure in which two alkylene groups are bonded via an oxy group (-O-), and the total number of carbon atoms in the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are preferably the same.

[0038] R 1a and R 1bExamples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group (methyleneoxymethylene group), an oxydiethylene group (ethyleneoxyethylene group), an oxydi(trimethylene) group (trimethyleneoxytrimethylene group), an oxydibutylene group (butyleneoxybutylene group), etc. The oxydialkylene group having 8 or less carbon atoms is preferably an oxydimethylene group (methyleneoxymethylene group), an oxydiethylene group (ethyleneoxyethylene group), or an oxydi(trimethylene) group (trimethyleneoxytrimethylene group), more preferably an (ethyleneoxyethylene group).

[0039] R 1a and R 1b are preferably each independently an alkylene group having 8 or less carbon atoms, more preferably each independently a methylene group, ethylene group, trimethylene group, tetramethylene group, isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), pentamethylene group, or hexamethylene group, even more preferably each independently a methylene group, ethylene group, trimethylene group, isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), tetramethylene group, or isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), and particularly preferably both are methylene groups.

[0040] ka and kb each independently represent 0 or 1. Here, when ka is 0, L 1a -R 1a does not exist. The same applies when kb, etc. are 0. It is preferable that both ka and kb are 1.

[0041] The cationic lipid (1) in which ka and kb are both 0 is represented by the following formula (1a).

[0042] [ka]

[0043] The cationic lipid (1) in which ka and kb are both 1 is represented by the following formula (1b).

[0044] [ka]

[0045] X a and X b each independently represents a dialkylamino group (the carbon number of each of the two alkyl groups is independently 1 to 5) or a 3- to 6-membered cyclic amino group. X a isX b may be the same as or different from, but preferably, X a isX b is equivalent to.

[0046] The number of carbon atoms in the two alkyl groups in the dialkylamino group is preferably each independently 1 to 3, and more preferably both are 1. The total number of carbon atoms in the two alkyl groups in the dialkylamino group is preferably 2 to 6, and more preferably 2 to 4.

[0047] Examples of the alkyl group in the dialkylamino group 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, a cyclopentyl group, etc. The two alkyl groups in the dialkylamino group are preferably each independently a methyl group, an ethyl group, a propyl group, or an isopropyl group, and more preferably both are methyl groups.

[0048] The 3- to 6-membered cyclic amino group means a group in which the substituents of the amino group are bonded to form a ring, and the number of atoms forming the ring is 3 to 6. The 3- to 6-membered cyclic amino group is preferably a 5- or 6-membered cyclic amino group, more preferably a 6-membered cyclic amino group. As the cyclic amino group, an amino group in which the ring is formed only from a nitrogen atom and a methylene group (-CH2-) is preferable.

[0049] Examples of the 3- to 6-membered cyclic amino group include a 1-aziridinyl group, a 1-azetidinyl group, a 1-pyrrolidinyl group, a 1-piperidyl group, a 4-morpholinyl group, a 4-thiomorpholinyl group, etc. The 3- to 6-membered cyclic amino group is preferably a 1-pyrrolidinyl group or a 1-piperidyl group, and more preferably a 1-piperidyl group.

[0050] X a and X b are preferably each independently a dialkylamino group (the carbon number of the two alkyl groups is independently 1 to 3) or a 5- or 6-membered cyclic amino group, and more preferably each independently a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, a dipropylamino group, a di(isopropyl)amino group, or a 1-piperidyl group. a and X b More preferably, X are the same as each other and are a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, a dipropylamino group, a di(isopropyl)amino group, or a 1-piperidyl group. a and X b and are particularly preferably both dimethylamino groups.

[0051] L 4a and L 4b are each independently an ester bond or an amide bond, and preferably both are ester bonds.

[0052] L 4a L 4bmay be the same as or different from, but preferably, L 4a L 4b is equivalent to.

[0053] L 4a and L 4b are preferably each independently *-O-CO-** or *-NH-CO-**, and more preferably both are *-O-CO-** (in the above formula, * represents R 2a , R 2b , R 3a , R 3b , R 4a or R 4b ** indicates the bond position with R 2a , R 2b , R 3a , R 3b , R 4a or R 4b represents the bond position with the carbon atom on the opposite side.) Here, "R 2a , R 2b , R 3a , R 3b , R 4a or R 4b The "bonding position with" refers to a position where ma is 1 (i.e., L 2a -R 2a exists), then L 4a So, "R 2a The bond position with the 2a -R 2a does not exist) and na is 1 (i.e., L 3a -R 3a exists), then L 4a So, "R 3a The bond position with the 2a -R 2a does not exist) and na is 0 (i.e., L 3a -R 3a does not exist), then L 4a So, "R 4a Other similar expressions have similar meanings.

[0054] R 2a and R 2bR each independently represents an alkylene group having 8 or less carbon atoms or an oxydialkylene group having 8 or less carbon atoms. 2a is R 2b may be the same as or different from, but preferably, R 2a is R 2b is equivalent to.

[0055] R 2a and R 2b The alkylene group having 8 or less carbon atoms may be either linear or branched, but is preferably linear. The alkylene group preferably has 6 or less carbon atoms, more preferably 4 or less carbon atoms.

[0056] R 2a and R 2b Examples of the alkylene group having 8 or less carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group, an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, a hexamethylene group, a heptamethylene group, and an octamethylene group.

[0057] R 2a and R 2b The alkylene group having 8 or less carbon atoms is preferably a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group, more preferably an ethylene group, a trimethylene group, a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), and even more preferably an ethylene group.

[0058] R 2a and R 2b The two alkylene groups in the oxydialkylene group having 8 or less carbon atoms may be the same or different, but are preferably the same.

[0059] R 2aand R 2b Examples of the oxydialkylene group having 8 or less carbon atoms include an oxydimethylene group (methyleneoxymethylene group), an oxydiethylene group (ethyleneoxyethylene group), an oxydi(trimethylene) group (trimethyleneoxytrimethylene group), and an oxydibutylene group.

[0060] R 2a and R 2b The oxydialkylene group having 8 or less carbon atoms is preferably an oxydimethylene group (methyleneoxymethylene group), an oxydiethylene group (ethyleneoxyethylene group), or an oxydi(trimethylene) group (trimethyleneoxytrimethylene group), more preferably an oxydiethylene group (ethyleneoxyethylene group).

[0061] R 2a and R 2b are preferably each independently an alkylene group having 8 or less carbon atoms, more preferably each independently a methylene group, ethylene group, trimethylene group, tetramethylene group, isobutylene group (-C(CH)CH-, -CHC(CH)-), pentamethylene group, or hexamethylene group, even more preferably each independently an ethylene group, trimethylene group, tetramethylene group, or isobutylene group (-C(CH)CH-, -CHC(CH)-), and particularly preferably both are ethylene groups.

[0062] L 2a and L 2b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably both are ester bonds.

[0063] L 2a L 2b may be the same as or different from, but preferably, L 2a L 2b is equivalent to.

[0064] L 2a and L 2b are preferably each independently *-CO-O-**, *-O-CO-**, *-CO-NH-**, *-NH-CO-**, *-NH-CO-O-**, *-O-CO-NH-**, *-O-** or *-NH-CO-NH-**, more preferably each independently *-CO-O-**, *-CO-NH-** or *-NH-CO-O-**, and further preferably both are *-CO-O-** (in the above formula, * represents R 3a , R 3b , R 4a or R 4b ** indicates the bond position with R 2a or R 2b )

[0065] ma and mb each independently represent 0 or 1, and preferably both are 1.

[0066] The cationic lipid (1) in which ma and mb are both 0 is represented by the following formula (1c).

[0067] [ka]

[0068] The cationic lipid (1) in which ma and mb are both 1 is represented by the following formula (1d).

[0069] [ka]

[0070] R 3a and R 3bR each independently represents a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally having a heteroatom. Here, the divalent group refers to a divalent group having a structure obtained by removing two hydrogen atoms from the aromatic compound. The number of carbon atoms in the aromatic compound is preferably 6 to 12, more preferably 6 to 7. The number of aromatic rings in the aromatic compound is preferably 1. 3a is R 3b may be the same as or different from, but preferably, R 3a is R 3b is equivalent to.

[0071] The aromatic ring of the aromatic compound may be either an aromatic hydrocarbon ring or an aromatic hetero ring. Examples of the aromatic hydrocarbon ring include a benzene ring, a naphthalene ring, and an anthracene ring. Examples of the aromatic hetero ring 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.

[0072] 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 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an 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.

[0073] R 3a and R 3b are preferably each independently represented by formula (2):

[0074] [ka]

[0075] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a , L 2b 、 L 4a or L 4b represents the bonding position with s represents an integer of 0 to 3; t represents an integer of 0 to 3; u represents an integer from 0 to 4, and u R 6 each independently represents a substituent. It is a group represented by the following formula:

[0076] s is preferably 0 or 1, and more preferably 0. t is preferably an integer of 0 to 2, and more preferably 1. u is preferably an integer of 0 to 2, more preferably 0 or 1, and further preferably 0.

[0077] R 6 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 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an 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. 6 Preferred examples of R 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, a isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, a isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group. 6 If there are multiple R 6 may be the same or different from each other.

[0078] L3a and L 3b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or an ether bond, more preferably both are ester bonds.

[0079] L 3a L 3b may be the same as or different from, but preferably, L 3a L 3b It is the same group as

[0080] L 3a and L 3b are preferably each independently *-CO-O-**, *-O-CO-**, *-CO-NH-**, *-NH-CO-**, *-NH-CO-O-**, *-O-CO-NH-**, *-O-** or *-NH-CO-NH-**, more preferably each independently *-CO-O-**, *-CO-NH-** or *-NH-CO-O-**, and further preferably both are *-CO-O-** (in the above formula, * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b )

[0081] Each of na and nb independently represents 0 or 1, and preferably both are 1.

[0082] The cationic lipid (1) in which na and nb are both 0 is represented by the following formula (1e).

[0083] [ka]

[0084] The cationic lipid (1) in which na and nb are both 1 is represented by the following formula (1f).

[0085] [ka]

[0086] R 4a and R 4b are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - represents R 5 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. 4a is R 4b may be the same as or different from, but preferably, R 4a is R 4b It is the same group as

[0087] 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 the 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 the sterol derivative.

[0088] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be either linear or branched. The aliphatic hydrocarbon group preferably has 13 to 19 carbon atoms, and more preferably has 13 to 17 carbon atoms.

[0089] 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, and is preferably a carbon-carbon double bond. The aliphatic hydrocarbon group is preferably an alkyl group or an alkenyl group.

[0090] Examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include 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 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 docosenyl group, a dodecadienyl group, a tridecadienyl group, a tetradecenyl group, a Examples of such alkyl groups include dienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl 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, and 1-decylundecyl group. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is preferably a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, and particularly preferably a tridecyl group, a heptadecyl group, a heptadecenyl group, or a heptadecadienyl group.

[0091] In one embodiment of the present invention, R 4a and R 4b The aliphatic hydrocarbon group with 12 to 22 carbon atoms is derived from a fatty acid. In this case, the carbonyl (-CO-) derived from the fatty acid is L 3a , L 3b , L 2a , L 2b , L 4a or L 4b It is contained in R using oleic acid as the fatty acid. 4a and R 4b If R 4a and R 4b are both 8-heptadecenyl groups.

[0092] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, etc. The fat-soluble vitamin having a hydroxyl group is preferably tocopherol.

[0093] 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.

[0094] R 5 is preferably a residue of a fat-soluble vitamin having a hydroxyl group. p is preferably 2.

[0095] R 4a and R 4b each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin having a hydroxyl group, and p is preferably 2 or 3; R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin having a hydroxyl group, and p is more preferably 2 or 3; R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol), and p is more preferably 2; R 4a and R 4bare the same and are particularly preferably aliphatic hydrocarbon groups having 12 to 22 carbon atoms.

[0096] In one embodiment of the present invention, L 1a L 1b is the same as R 1a is R 1b is the same as X a isX b is the same as L 4a L 4b is the same as R 2a is R 2b is the same as L 2a L 2b is the same as R 3a is R 3b is the same as L 3a L 3b is the same as and R 4a is R 4b It is preferably the same as

[0097] Suitable examples of the cationic lipid (1) of the present invention include the following cationic lipids. <Cationic lipid (1-1)> L 1a and L 1b are both *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are each independently an alkylene group having 8 or less carbon atoms; ka and kb are each independently 0 or 1; X a and X b are each independently a dialkylamino group (the carbon number of the two alkyl groups is each independently 1 to 3) or a 5- or 6-membered cyclic amino group; L 4a and L 4bIn both cases, *-O-CO-** (wherein * represents R 2a or R 2b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms; L 2a and L 2b Both of them are *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are each independently expressed by the formula (2):

[0098] [ka]

[0099] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents an integer from 0 to 3. t represents an integer of 0 to 3; u represents an integer from 0 to 4, and u R 6each independently represents an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an 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: L 3a and L 3b Both of them are *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; and R 4a and R 4b each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin (e.g., tocopherol) having a hydroxyl group, and p is 2 or 3; Cationic lipids (1).

[0100] <Cationic lipids (1-2)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1bare each independently a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group; ka and kb are each independently 0 or 1; X a and X b are each independently a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, a dipropylamino group, a di(isopropyl)amino group, or a 1-piperidyl group; L 4a and L 4b In both cases, *-O-CO-** (wherein * represents R 2a or R 2b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; R 2a and R 2b are each independently a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group; L 2a and L 2b Both of them are *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are each independently expressed by the formula (2):

[0101] [ka]

[0102] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0 or 1; t represents an integer of 0 to 2; u represents an integer from 0 to 2, and u R 6 each independently represents 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, or a phenoxy group. is a group represented by the formula: L 3a and L 3b Both of them are *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin (e.g., tocopherol) having a hydroxyl group, and p is 2 or 3; Cationic lipids (1).

[0103] <Cationic lipid (1-2a)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are each independently a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group; ka and kb are each independently 0 or 1; X a and X b are each independently a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, or a 1-piperidyl group; L 4a and L 4b In both cases, *-O-CO-** (wherein * represents R 2a or R 2b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; R 2a and R 2b are each independently a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-), a pentamethylene group, or a hexamethylene group; L 2a and L 2b Both of them are *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are each independently expressed by the formula (2):

[0104] [ka]

[0105] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0 or 1; t represents an integer of 0 to 2; u represents an integer from 0 to 2, and u R 6 each independently represents 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, or a phenoxy group. is a group represented by the formula: L 3a and L 3b Both of them are *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin (e.g., tocopherol) having a hydroxyl group, and p is 2 or 3; Cationic lipids (1).

[0106] <Cationic lipids (1-3)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-); ka and kb are each independently 0 or 1; X a and X b are the same as each other and are a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, a dipropylamino group, a di(isopropyl)amino group, or a 1-piperidyl group; L 4a and L 4b In both cases, *-O-CO-** (wherein * represents R 2a or R 2b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; R 2a and R 2b are each independently an ethylene group, a trimethylene group, a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-); L 2a and L 2b are both *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are each independently expressed by the formula (2):

[0107] [ka]

[0108] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0, t represents 1, and u represents 0.) is a group represented by the formula: L 3a and L 3b Both of them are *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; and R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin (e.g., tocopherol) having a hydroxyl group, and p is 2; Cationic lipids (1).

[0109] <Cationic lipid (1-3a)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1aor R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are each independently a methylene group, an ethylene group, a trimethylene group, an isopropylene group (-CH(CH3)CH2-, -CH2CH(CH3)-), a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-); ka and kb are each independently 0 or 1; X a and X b are the same as each other and are a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, or a 1-piperidyl group; L 4a and L 4b In both cases, *-O-CO-** (wherein * represents R 2a or R 2b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; R 2a and R 2b are each independently an ethylene group, a trimethylene group, a tetramethylene group, or an isobutylene group (-C(CH3)2CH2-, -CH2C(CH3)2-); L 2a and L 2b are both *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are each independently expressed by the formula (2):

[0110] [ka]

[0111] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0, t represents 1, and u represents 0.) is a group represented by the formula: L 3a and L 3b Both of them are *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; and R 4a and R 4b are the same as each other and are an aliphatic hydrocarbon group having 12 to 22 carbon atoms or R 5 -CO-(CH2) p - and R 5 is a residue of a fat-soluble vitamin (e.g., tocopherol) having a hydroxyl group, and p is 2; Cationic lipids (1).

[0112] <Cationic lipid (1-3b)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are both methylene groups; ka and kb are each independently 0 or 1; X a and X bare the same as each other and are a dimethylamino group, an N-methyl-N-ethylamino group, an N-methyl-N-propylamino group, a diethylamino group, or a 1-piperidyl group; R 2a and R 2b are both ethylene groups; L 2a and L 2b Both of them are *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are identical to each other and satisfy the formula (2):

[0113] [ka]

[0114] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0, t represents 1, and u represents 0.) is a group represented by the formula: L 3a and L 3b are both *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; R 4a and R 4b are the same and are aliphatic hydrocarbon groups having 12 to 22 carbon atoms; Cationic lipids (1).

[0115] <Cationic lipids (1-4)> L 1a and L 1b Both are *-NH-CO-** (wherein ** represents R 1a or R 1b represents the bonding position with R 1a or R 1b represents the bonding position to the carbon atom on the opposite side; R 1a and R 1b are both methylene groups; ka and kb are each independently 0 or 1; X a and X b are both dimethylamino groups; R 2a and R 2b are both ethylene groups; L 2a and L 2b Both of them are *-CO-O-** (wherein * represents R 3a or R 3b ** indicates the bond position with R 2a or R 2b ) represents the bond position with; ma and mb are both 1; R 3a and R 3b are identical to each other and satisfy the formula (2):

[0116] [ka]

[0117] (In the formula, *L 3a or L 3b represents the bonding position with ** is L 2a or L 2b represents the bonding position with s represents 0, t represents 1, and u represents 0.) is a group represented by the formula: L 3a and L 3b are both *-CO-O-** (wherein * represents R 4a or R 4b ** indicates the bond position with R 3a or R 3b ) represents the bond position with; na and nb are both 1; R 4a and R 4b are the same and are aliphatic hydrocarbon groups having 12 to 22 carbon atoms; Cationic lipids (1).

[0118] The cationic lipid (1) of the present invention may have stereoisomers and geometric isomers, and unless otherwise specified, the cationic lipid (1) of the present invention includes these isomers. The cationic lipid (1) of the present invention may also be a mixture of multiple isomers. In the mixture of multiple isomers, the ratio of each isomer is not particularly limited.

[0119] Specific examples of the cationic lipid (1) of the present invention include the following O-Ph-cys-C2-DMA, O-Ph-cys-C2-EMA, O-Ph-cys-C2-MPA, O-Ph-cys-C2-DEA, O-Ph-cys-C2-DPA, O-Ph-cys-C2-DIA, O-Ph-cys-C2-Pip, E-Ph-cys-C2-DMA, and O-Ph-cys-C4-DMA, O-Ph-cys-DMA. 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 O-Ph-cys-C2-DMA, O-Ph-cys-C2-EMA, O-Ph-cys-C2-MPA, O-Ph-cys-C2-DEA, and O-Ph-cys-C2-Pip, and most preferably O-Ph-cys-C2-DMA.

[0120] [Table 1]

[0121] [Table 2]

[0122] 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 a -SS- (disulfide) bond. Therefore, the method for producing the cationic lipid (1) of the present invention is as follows: (A){R 4a -(L 3a -R 3a ) na -(L 2a -R 2a ) ma -L 4a -}CH(CH2SH){-(L 1a -R 1a ) ka -X a} and thiols represented by {R 4b -(L 3b -R 3b ) nb -(L 2b -R 2b ) mb -L 4b -}CH(CH2SH){-(L 1b -R 1b ) kb -X b and a method for preparing thiols of the formula {} followed by oxidation and coupling thereof, (B) A method of sequentially synthesizing necessary portions from a starting compound containing an -SS- 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 the above method (B).

[0123] The above method (B) will be explained below, but the method for producing the cationic lipid (1) of the present invention is not limited thereto.

[0124] Examples of starting compounds containing an -SS- bond include cystine, L-cystine and salts thereof, N,N,N',N'-tetramethyl-L-cystine and salts thereof, compounds in which the amino groups at both ends of cystine are protected with a 9-fluorenylmethyloxycarbonyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, an acetyl group, or the like, and salts thereof, compounds in which the carboxy groups at both ends of cystine are protected with a methyl group, an allyl group, or the like, and salts thereof.

[0125] For example, L 1a and L 1b Both are L 1 and R 1a and R 1b Both are R 1 where ka and kb are both 1, and X a and X b Both are X and L 4a and L 4b Both are L 4 and R 2a and R 2b Both are R 2 And L 2a and L 2b Both are L 2 where ma and mb are both 1, and R 3a and R 3b Both are R 3 And L 3a and L 3b Both are L 3 where na and nb are both 1, and R 4a and R 4b Both are R 4 When producing a cationic lipid (1) represented by the following formula (1'), a cationic lipid represented by the following formula (1') can be obtained by the synthesis route shown below.

[0126] [ka]

[0127] R 3 and a reactive functional group (FG 1 and F.G.2 In the compound (I) having one reactive functional group (FG 1 ) is reacted with a reagent to introduce a protecting group to form a protected functional group (PG 1 Compound (II) having a reactive functional group (FG) of compound (II) is synthesized. 2 ), R 2 and a reactive functional group (FG 3 and F.G. 4 ) in compound (III) having one reactive functional group (FG 3 ) to synthesize compound (IV). The reactive functional group (FG 4 ) to the protected amino group (PG 2 The compound (VI) is synthesized by reacting the terminal carboxyl group of cystine (V) with the protected amino group (PG 2 The protecting group is removed from compound (VII) to synthesize compound (VII) having an amino group at the terminal. 1 and X and a reactive functional group (FG 5 Compound (VIII) having a reactive functional group (FG 5 ) to synthesize compound (IX). 1 ) to remove the protecting group, and to provide a reactive functional group (FG 6 Finally, the compound (X) having the reactive functional group (FG 6 ), R 4 and reactive functional groups (FG 7 ) of compound (XI) having a reactive functional group (FG 7 ) to form the cystine skeleton, L 1 , R 1 , X, L 4 , R 2 , L 2 , R 3 , L 3 and R 4 It is possible to obtain a cationic lipid (1') having the following structure.

[0128] Protection and deprotection of reactive functional groups in the above-mentioned synthetic routes can be carried out using known reagents and methods (e.g., those described in GREENE'S PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th Edition, WILEY-INTERSCIENCEM).

[0129] The reactive functional group (FG 1 The solvent used in the protection reaction of (I) (i.e., the reaction of forming compound (II) from compound (I)) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, etc. Among these, dichloromethane, chloroform, and toluene are preferred.

[0130] The reactive functional group (FG 1 Examples of the protecting group used for protecting the aryl group include acetal protecting groups, silyl protecting groups, ester protecting groups, and ether protecting groups. Among these, acetal protecting groups are preferred. Examples of the reagent that forms the protecting group include 3,4-dihydro-2H-pyran.

[0131] A catalyst may be used in the reaction of forming compound (II) from compound (I). The catalyst used can be appropriately selected depending on the type of compound to be reacted.

[0132] The reaction temperature for forming compound (II) from compound (I) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0133] The reactive functional group (FG 1The compound (II) obtained by the protection reaction of (III) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0134] The reaction between compound (II) and compound (III) may be carried out without using a catalyst or in the presence of a catalyst. The catalyst used in the reaction between compound (II) and compound (III) can be appropriately selected depending on the type of compound to be reacted. As the catalyst, a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, or 4-dimethylaminopyridine (hereinafter sometimes abbreviated as "DMAP") may be used, or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid may be used.

[0135] For the reaction of compound (II) with compound (III), a condensing agent such as dicyclohexylcarbodiimide (hereinafter sometimes abbreviated as "DCC"), diisopropylcarbodiimide (hereinafter sometimes abbreviated as "DIC"), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter sometimes abbreviated as "EDC"), etc. may be used. Alternatively, compound (II) may be converted to an acid anhydride or the like using a condensing agent, and then the resulting acid anhydride or the like may be reacted with compound (III).

[0136] The solvent used in the reaction of compound (II) and compound (III) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, etc. Among these, tetrahydrofuran is preferred.

[0137] The reaction temperature of compound (II) and compound (III) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0138] Compound (IV) obtained by the reaction of compound (II) with compound (III) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0139] The reaction between compound (IV) and compound (V) may be carried out without using a catalyst or in the presence of a catalyst. The catalyst used in the reaction between compound (IV) and compound (V) can be appropriately selected depending on the type of compound to be reacted. As the catalyst, a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, DMAP, etc. may be used, or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid may be used.

[0140] A condensing agent such as DCC, DIC, EDC, etc. may be used for the reaction of compound (IV) with compound (V). Compound (V) may be converted to an acid anhydride, etc. using a condensing agent, and then the resulting acid anhydride, etc. may be reacted with compound (IV).

[0141] The solvent used in the reaction of compound (IV) and compound (V) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, etc. Among these, tetrahydrofuran is preferred.

[0142] The reaction temperature of compound (IV) and compound (V) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0143] Compound (VI) obtained by the reaction of compound (IV) with compound (V) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0144] The solvent used in the reaction for removing the protecting group of the amino group of compound (VI) (i.e., the reaction for forming compound (VII) from compound (VI)) may be any solvent that does not inhibit the reaction and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, methanol, ethanol, isopropanol, etc. Among these, tetrahydrofuran is preferred.

[0145] The reagent used to remove the protecting group of the amino group of compound (VI) can be appropriately selected depending on the protecting group.

[0146] The reaction temperature for forming compound (VII) from compound (VI) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0147] Compound (VII) obtained by the reaction of removing the protecting group of the amino group of compound (VI) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, ion exchange chromatography, etc.

[0148] The reaction between compound (VII) and compound (VIII) may be carried out without using a catalyst or in the presence of a catalyst. The catalyst used in the reaction between compound (VII) and compound (VIII) can be appropriately selected depending on the type of compound to be reacted. As the catalyst, a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, DMAP, etc. may be used, or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid may be used.

[0149] A condensing agent such as DCC, DIC, EDC, etc. may be used for the reaction of compound (VII) with compound (VIII). Alternatively, compound (VIII) may be converted to an acid anhydride or the like using a condensing agent, and then the resulting acid anhydride or the like may be reacted with compound (VII).

[0150] The solvent used in the reaction of compound (VII) and compound (VIII) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, methanol, ethanol, isopropanol, etc. Among these, dichloromethane and chloroform are preferred.

[0151] The reaction temperature of compound (VII) and compound (VIII) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0152] Compound (IX) obtained by the reaction of compound (VII) with compound (VIII) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0153] The protected functional group (PG) of compound (IX) 1The solvent used in the reaction of removing the protecting group from compound (IX) (i.e., the reaction of forming compound (X) from compound (IX)) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, methanol, ethanol, isopropanol, etc. Among these, water is preferred.

[0154] The protected functional group (PG) of compound (IX) 1 The reagent used to remove the protecting group from ) can be appropriately selected depending on the protecting group.

[0155] The reaction temperature for forming compound (X) from compound (IX) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0156] The protected functional group (PG) of compound (IX) 1 The compound (X) obtained by the reaction of removing the protecting group from (X) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0157] The reaction between compound (X) and compound (XI) may be carried out without using a catalyst or in the presence of a catalyst. The catalyst used in the reaction between compound (X) and compound (XI) can be appropriately selected depending on the type of compound to be reacted. As the catalyst, a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, DMAP, etc. may be used, or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid may be used.

[0158] A condensing agent such as DCC, DIC, EDC, etc. may be used for the reaction of compound (X) with compound (XI). Alternatively, compound (XI) may be converted to an acid anhydride or the like using a condensing agent, and then the resulting acid anhydride or the like may be reacted with compound (X).

[0159] The solvent used in the reaction of compound (X) and compound (XI) may be any solvent that does not inhibit the reaction, and may be used without any particular limitation. Examples of the solvent include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, toluene, etc. Among these, dichloromethane and chloroform are preferred.

[0160] The reaction temperature of compound (X) and compound (XI) is usually 0 to 150° C., preferably 0 to 80° C., and more preferably 10 to 50° C. The reaction time is usually 1 to 48 hours, and preferably 1 to 24 hours.

[0161] The cationic lipid (1') obtained by the reaction of compound (X) with compound (XI) can be appropriately purified by a general purification method such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, or ion exchange chromatography.

[0162] Specific production methods are described below in Examples 1 to 10. 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 10.

[0163] 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. 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 arranged toward the aqueous phase side of the interface. The "amphipathic lipid" means a lipid having both a hydrophilic group and a hydrophobic group. Examples of the amphipathic lipid include cationic lipids and phospholipids.

[0164] 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 type emulsions, W / O type emulsions, spherical micelles, wormlike micelles, lipid nanoparticles (LNPs), and unspecified layered structures. The lipid membrane structure of the present invention is preferably an LNP.

[0165] The lipid membrane structure of the present invention may further contain other components in addition to the cationic lipid (1) of the present invention. Examples of other 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, sodium cholic acid, octyl glycoside, ND-gluco-N-methylalkanamides, etc.), polyethylene glycol, proteins, etc. The content of other 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 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 components).

[0166] 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 usually, when the lipid membrane structure is used as a nucleic acid transfer agent described below, the content of the cationic lipid (1) of the present invention in the lipid membrane structure 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 all the lipids in the lipid membrane structure of the present invention.

[0167] The lipid membrane structure of the present invention can be prepared by dispersing the cationic lipid (1) of the present invention and other components (lipids, etc.) in an appropriate solvent or dispersion medium, such as an aqueous solvent or an alcoholic solvent, and, if necessary, performing an operation to induce organization.

[0168] Examples of the "operation to induce organization" include the ethanol dilution method using a microchannel or vortex, the simple hydration method, ultrasonic treatment, heating, vortexing, ether injection, the French press method, the cholate method, and Ca 2+ Examples of methods that can be used include, but are not limited to, methods known per se, such as fusion method, freeze-thaw method, and reverse phase evaporation method.

[0169] 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.

[0170] The nucleic acid transfer agent of the present invention can transfer any nucleic acid into cells. Any one of single-stranded to triple-stranded nucleic acids can be used, but single-stranded or double-stranded nucleic acids are preferred.

[0171] Examples of nucleic acids include, but are not limited to, DNA, RNA, chimeric nucleic acids of RNA, and DNA / RNA hybrids. Nucleic acids other than DNA, RNA, chimeric nucleic acids of RNA, and DNA / RNA hybrids (hereinafter referred to as "other nucleic acids") may 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), etc.), and oligomers having special bonds (provided that the oligomers contain nucleotides having a configuration that allows base pairing or base attachment as found in DNA or RNA).

[0172] Further, the nucleic acid may be, for example, a nucleic acid having a modification known in the art, a labeled nucleic acid known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more naturally occurring nucleotides are replaced with an analogue, a nucleic acid having intramolecularly crosslinked nucleotides, a nucleic acid having an uncharged bond (e.g., methylsulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged or sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side 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., an α-anomeric form of a nucleic acid, etc.), etc.

[0173] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected according to the purpose of use. 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 appropriately digested with restriction enzymes and used as linear DNA.

[0174] The type of RNA that can be used in the present invention is not particularly limited, and can be appropriately selected according to the purpose of use. 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.

[0175] The nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.

[0176] 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 certain disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0177] When forming the lipid membrane structure of the present invention, the lipid membrane structure of the present invention encapsulating the nucleic acid (i.e., the nucleic acid introduction agent of the present invention encapsulating the nucleic acid) can be formed by coexisting a target nucleic acid with the components (lipids, etc.) of the lipid membrane structure of the present invention.

[0178] For example, when forming the lipid membrane structure of the present invention 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 or a microchannel, 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).

[0179] 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 removed 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, and after hydration, ultrasonic treatment is performed with a sonicator to form the lipid membrane structure of the present invention in which the nucleic acid is encapsulated (i.e., the nucleic acid transfer agent of the present invention in which the nucleic acid is encapsulated).

[0180] One form of the lipid membrane structure of the present invention in which a nucleic acid is encapsulated is 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 for a drug delivery system for selectively delivering a nucleic acid or the like into a specific cell, and is useful, for example, for DNA vaccines and gene therapy drugs for tumors by introducing an antigen gene into dendritic cells, and nucleic acid drugs that suppress the expression of a target gene by utilizing RNA interference.

[0181] The particle size of the lipid membrane structure of the present invention encapsulating nucleic acid 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 measuring device such as Zetasizer Nano (Malvern). The particle size of the lipid membrane structure can be appropriately adjusted depending on the preparation method of the lipid membrane structure.

[0182] 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 -10 to +10 mV. In conventional gene transfer, particles with a positively charged surface have been mainly used. This is useful as a method for promoting electrostatic interaction with heparin sulfate on the negatively charged cell surface and promoting uptake into cells. However, the positive surface charge may cause (a) inhibition of release of nucleic acid from the carrier due to interaction with the delivery nucleic acid in the cell, or (b) inhibition of protein synthesis due to interaction between mRNA and the delivery nucleic acid. This problem can be solved by adjusting the zeta potential within the above range. The zeta potential can be measured using a zeta potential measuring device such as Zetasizer Nano. The zeta potential of the lipid membrane structure can be adjusted by the composition of the components of the lipid membrane structure containing the cationic lipid (1) of the present invention.

[0183] 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 7.2, and more preferably 6.0 to 6.8. The liposomal pKa is an index showing the susceptibility of the lipid membrane structure taken up by endocytosis to protonation in the weakly acidic environment in the endosome. As described in Non-Patent Documents 2 and 3, in order to escape from the endosome and deliver the 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, the 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.

[0184] The nucleic acid can be introduced into the cell by contacting the nucleic acid transfer agent of the present invention encapsulating the nucleic acid with the cell. The type of the 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 thereof 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 (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 be an adherent cell or a non-adherent cell.

[0185] The step of contacting cells with the nucleic acid transfer agent of the present invention encapsulating a nucleic acid in vitro will be specifically described below.

[0186] 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 encapsulating a nucleic acid. The cells may or may not be in a proliferation phase at the time of contact with the nucleic acid transfer agent.

[0187] The culture medium when the nucleic acid transfer agent of the present invention containing nucleic acid is contacted with cells 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 excess proteins such as serum, the contact between the nucleic acid transfer agent and cells may be inhibited.

[0188] The cell density at the time of contacting the nucleic acid transfer agent of the present invention containing nucleic acid with 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 ~1×10 7 in the range of cells / mL.

[0189] For example, a dispersion of the nucleic acid transfer agent of the present invention encapsulating the above-mentioned nucleic acid is added to the cells. The amount of the dispersion added is not particularly limited, and can be appropriately set in consideration of the number of cells, etc. The concentration of the nucleic acid transfer agent in the dispersion when contacting the cells is not particularly limited as long as the transfer of the target nucleic acid into the cells can be achieved, but the lipid concentration in the dispersion is usually 1 to 100 nmol / mL, preferably 10 to 50 nmol / mL, and the nucleic acid concentration in the dispersion is usually 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL.

[0190] After adding the above-mentioned dispersion liquid to the cells, the cells are cultured. The temperature, humidity, CO2 concentration, etc. during culture are appropriately set in consideration of the type of cells. When the cells are mammalian cells, the temperature is usually about 37°C, the relative humidity is about 95%, and the CO2 concentration is about 5% by volume. The culture time can also be appropriately set in consideration of conditions such as the type of cells used, but is usually in the range of 0.1 to 76 hours, preferably in the range of 0.2 to 24 hours, and more preferably in the range of 0.5 to 12 hours. If the above-mentioned culture time is too short, the nucleic acid is not sufficiently introduced into the cells, and if the culture time is too long, the cells may become weak.

[0191] 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 a fresh medium is added to the medium and the culture is continued. When the cells are derived from a mammal, the fresh medium preferably contains serum or a nutrient factor.

[0192] 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 the target cell, and the nucleic acid is transferred into the cell in vivo. The subjects to which the nucleic acid transfer agent can be administered are not particularly limited, and examples thereof 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.), and fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The subjects to which the nucleic acid transfer agent is administered are preferably humans or other mammals.

[0193] 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.).

[0194] The method of administration of 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 is a method that 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, and can be appropriately selected from known administration methods (e.g., oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) taking into consideration the type of nucleic acid to be transferred, the type and site of the target cell, etc. 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 to be administered, the administration method, the type of nucleic acid to be transferred, the type and site of the target cell, etc.

[0195] 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.

[0196] 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.

[0197] When the nucleic acid transfer agent of the present invention is provided as a research reagent, the nucleic acid transfer agent of the present invention 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 other 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 may be provided as a solvent-free nucleic acid transfer agent. The nucleic acid transfer agent of the present invention may contain physiologically acceptable additives known per se (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.) as appropriate.

[0198] When the nucleic acid transfer agent of the present invention is provided as a medicine, the nucleic acid transfer agent of the present invention may be provided as an oral preparation (e.g., tablet, capsule, etc.) or a 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 pharma- ceutical acceptable additives (e.g., carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc.), or 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 medicine is preferably a parenteral preparation, more preferably an injection. The nucleic acid transfer agent of the present invention may be either a preparation for adults or a preparation for children.

[0199] 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, a reagent used for 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 allowing the nucleic acid to be transferred into cells more efficiently. EXAMPLES

[0200] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0201] The meanings of the abbreviations used in the description of the examples are as follows: DCM: dichloromethane THF: tetrahydrofuran DHP: 3,4-dihydro-2H-pyran PPTS: Pyridinium p-toluenesulfonate DBU: Diazabicycloundecene DMAP: 4-dimethylaminopyridine EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Fmoc: 9-fluorenylmethyloxycarbonyl group 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 TEA: Triethylamine

[0202] Tables 3 and 4 show the names and structures of the cationic lipids prepared in the following examples.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Example 1] Synthesis of O-Ph-cys-C2-DMA O-Ph-cys-C2-DMA was prepared by the following synthetic route, but the present invention is not limited to such synthetic route.

[0206] [ka]

[0207] [ka]

[0208] <Synthesis of intermediate 1> 75.0g (493mmol) of 4-hydroxyphenylacetic acid (Kanto Chemical Co., Ltd.) and 12.4g (49.3mmol) of pyridinium paratoluenesulfonate (Kanto Chemical Co., Ltd.) were dissolved in 400g of dichloromethane at room temperature, and the resulting solution was cooled to 10-20°C. A solution obtained by dissolving 207g (2.46mol) of DHP (Fujifilm Wako Pure Chemical Industries Co., Ltd.) in 100g 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 30.1g (246mmol) of DMAP (Koei Chemical Co., Ltd.) was added to perform quenching. 590g of 2-propanol was added to the solution after quenching, and the solution was cooled to 10-20°C. A mixture of 98.8 g of 400 g / L sodium hydroxide aqueous 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 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 mixture with a pH of 5.0. The resulting mixture was extracted twice with 750 g of chloroform, and the organic layer was dehydrated by adding 75.0 g of sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 98.2 g of intermediate 1.

[0209] <Synthesis of intermediate 2> 25.0g (106mmol) of intermediate 1 and 2.60g (21.3mmol) of DMAP were added to 225g of THF and dissolved at room temperature, and 222g (3.58mol) of dehydrated ethylene glycol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the obtained mixture. 40.6g (212mmol) of EDC (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the obtained mixture and reacted at 25°C for 2 hours. The reaction solution was concentrated by an evaporator to distill off THF. After adding 250g of chloroform to the obtained concentrate and extracting, the organic layer was washed twice with 250g of 0.5M phosphate buffer (pH 3.0) and once with 250g of 20% by weight saline, and then dehydrated by adding 50g of sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated by an evaporator to obtain 28.4g of intermediate 2.

[0210] <Synthesis of intermediate 3> 16.4g (58.5mmol) of intermediate 2, 20g (29.2mmol) of N,N'-bis(9-fluorenylmethyloxycarbonyl)-L-cystine (manufactured by Watanabe Chemical Industry Co., Ltd.) and 1.43g (11.7mmol) of DMAP were added to 100g of THF and dissolved at room temperature. 16.8g (87.6mmol) of EDC was added to the obtained solution and reacted at 25°C for 2 hours. After adding 250g of chloroform to the reaction solution for extraction, the organic layer was washed twice with 250g of 0.5M phosphate buffer (pH 3.0) and once with 250g of 20% by weight saline, and then dehydrated by adding 40.0g of sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 31.5g of intermediate 3.

[0211] <Synthesis of intermediate 4> To 15.0 g (12.4 mmol) of Intermediate 3, 333 g of THF was added and dissolved at room temperature. To the resulting solution, 1.42 g (9.33 mmol) of DBU (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the reaction was carried out at 25 °C for 1 hour. The reaction solution was diluted with 750 g of 150 mM phosphate buffer (pH 3.0) and washed twice with 487 g of hexane. After washing, the diluted solution was extracted twice with 1120 g of chloroform, and then the organic layer was washed with 750 g of 20 wt% brine. After adding 30.0 g of sodium sulfate to the washed organic layer for dehydration, the sodium sulfate was removed by filtration, and then the filtrate was concentrated using an evaporator to obtain 6.30 g of Intermediate 4.

[0212] <Synthesis of Intermediate 5> To 600 mg (0.784 mmol) of Intermediate 4 and 194 mg (1.88 mmol) of N,N-dimethylglycine (manufactured by Tokyo Chemical Industry Co., Ltd.), 89.0 g of chloroform was added and dissolved at room temperature. 601 mg (3.14 mmol) of EDC was added, and the reaction was carried out at 25 °C for 2 hours. The reaction solution was washed twice with 60.0 g of 0.5 M phosphate buffer (pH 4.0), once with 60.0 g of 7 wt% sodium bicarbonate solution, and once with 60.0 g of 20 wt% brine, and then 7.00 g of sodium sulfate was added for dehydration. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 695 mg of Intermediate 5.

[0213] <Synthesis of Intermediate 6> To 680 mg (0.727 mmol) of Intermediate 5, 68.0 g of 0.5 M phosphate buffer (pH 2.0) was added, and the reaction was carried out at 25 °C for 2 hours. 100 g of chloroform was added to the reaction solution for washing. To the washed aqueous layer, an aqueous solution of sodium hydroxide at 400 g / L was added to adjust its pH to 6.0, and then the pH-adjusted aqueous layer was extracted twice with 100 g of chloroform. The organic layer was dehydrated by adding 1.36 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 278 mg of Intermediate 6.

[0214] <Synthesis of O-Ph-cys-C2-DMA> To 250 mg (0.326 mmol) of Intermediate 6, 184 mg (0.652 mmol) of oleic acid (manufactured by NOF Corporation), and 15.9 mg (0.130 mmol) of DMAP, 17.6 g of chloroform was added and dissolved at room temperature. 187 mg (0.976 mmol) of EDC was added to the resulting solution, and the reaction was carried out at 25 °C for 2 hours. The reaction solution was washed twice with 12.0 g of 20 wt% saline, and then 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 369 mg of a crude product. The obtained crude product was purified by column chromatography to obtain 222 mg of O-Ph-cys-C2-DMA.

[0215] <Of O-Ph-cys-C2-DMA 1 1H-NMR (600 MHz, CDCl3) δ: 0.86 - 0.90 (t, 6H), 1.22 - 1.42 (m, 40H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.32 (s, 12H), 2.52 - 2.56 (t, 4H), 2.90 - 3.03 (m, 4H), 3.09 - 3.43 (m, 4H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.88 - 4.97 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 7.81 - 7.88 (d, 2H)

[0216] [Example 2] Synthesis of O-Ph-cys-C2-EMA <Synthesis of N-ethyl-N-methylglycine> 8.50 g (144 mmol) of N-ethylmethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to 45 g of THF and dissolved at room temperature, and then cooled to 10 °C or lower. To this, a solution obtained by dissolving 2.00 g (14.4 mmol) of bromoacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) in 9.00 g of THF was added dropwise, and the reaction was carried out at 25 °C for 2 hours. After removing the white precipitate by filtration, the filtrate was concentrated by an evaporator to remove THF. After adding 10.0 g of ion-exchanged water to the obtained concentrate to prepare a solution, its pH was adjusted to 12.0 using a 400 g / L aqueous sodium hydroxide solution. The solution with adjusted pH was washed three times with 15.0 g of chloroform, and then the washed aqueous layer was neutralized to pH 7.0 with 6N hydrochloric acid, and the neutralized aqueous layer was concentrated by an evaporator. After adding 50.0 g of acetonitrile to the obtained residue to prepare a solution, 4.00 g of sodium sulfate was added for dehydration. After removing sodium sulfate by filtration, the filtrate was concentrated by an evaporator to obtain 1.35 g of N-ethyl-N-methylglycine represented by the following formula.

[0217] [Chemical formula]

[0218] <Synthesis of O-Ph-cys-C2-EMA> Using Intermediate 4 and N-ethyl-N-methylglycine, O-Ph-cys-C2-EMA was synthesized by the same synthetic route as in Example 1.

[0219] <O-Ph-cys-C2-EMA's 1 H-NMR (600 MHz, CDCl3) δ: 0.86 - 0.90 (t, 6H), 1.05 - 1.09 (t, 6H), 1.22 - 1.42 (m, 40H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.31 (s, 6H), 2.49 - 2.56 (m, 8H), 2.96 - 3.07 (m, 4H), 3.10 - 3.43 (m, 4H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.88 - 4.97 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 7.94 - 8.01 (d, 2H)

[0220] [Example 3] Synthesis of O-Ph-cys-C2-MPA <Synthesis of N-methyl-N-propylglycine> N-methylpropylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used, and N-methyl-N-propylglycine represented by the following formula was synthesized in the same manner as in Example 2 except for this.

[0221] [Chemical formula]

[0222] <Synthesis of O-Ph-cys-C2-MPA> Using Intermediate 4 and N-methyl-N-propylglycine, O-Ph-cys-C2-MPA was synthesized by the same synthetic route as in Example 1.

[0223] <O-Ph-cys-C2-MPA's 1 H-NMR (600 MHz, CDCl3)> δ: 0.86 - 0.93 (m, 12H), 1.22 - 1.42 (m, 40H), 1.47 - 1.52 (m, 4H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.30 (s, 6H), 2.38 - 2.42 (m, 4H), 2.52 - 2.56 (t, 4H), 2.96 - 3.07 (m, 4H), 3.11 - 3.43 (m, 4H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.88 - 4.97 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 7.94 - 8.01 (d, 2H)

[0224] [Example 4] Synthesis of O-Ph-cys-C2-DEA Using Intermediate 4 and sodium N,N-diethylglycinate (manufactured by Tokyo Chemical Industry Co., Ltd.), O-Ph-cys-C2-DEA was synthesized in the same synthetic route as in Example 1.

[0225] <Of O-Ph-cys-C2-DEA 1 1H-NMR (600 MHz, CDCl3)> δ: 0.86 - 0.90 (t, 6H), 1.03 - 1.06 (t, 12H), 1.22 - 1.42 (m, 40H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.52 - 2.61 (m, 12H), 3.00 - 3.43 (m, 8H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.88 - 4.97 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 8.13 - 8.20 (d, 2H)

[0226] [Example 5] Synthesis of O-Ph-cys-C2-DPA <Synthesis of N,N-dipropylglycine> Except for using dipropylamine (manufactured by Tokyo Chemical Industry Co., Ltd.), N,N-dipropylglycine represented by the following formula was synthesized in the same manner as in Example 2.

[0227]

Chemical formula

[0228] <Synthesis of O-Ph-cys-C2-DPA> Using Intermediate 4 and N,N-dipropylglycine, O-Ph-cys-C2-DPA was synthesized by the same synthetic route as in Example 1.

[0229] <Of O-Ph-cys-C2-DPA 1 1H-NMR (600 MHz, CDCl3) δ 0.86 - 0.92 (m, 18H), 1.22 - 1.51 (m, 48H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.43 - 2.46 (t, 8H), 2.52 - 2.55 (t, 4H), 3.02 - 3.43 (m, 8H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.86 - 4.95 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 8.09 - 8.16 (d, 2H)

[0230] [Example 6] Synthesis of O-Ph-cys-C2-DIA <Synthesis of N,N-diisopropylglycine> N,N-Diisopropylglycine represented by the following formula was synthesized in the same manner as in Example 2, except that diisopropylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used.

[0231] [Chemical formula]

[0232] <Synthesis of O-Ph-cys-C2-DIA> Using Intermediate 4 and N,N-diisopropylglycine, O-Ph-cys-C2-DIA was synthesized by the same synthetic route as in Example 1.

[0233] <Of O-Ph-cys-C2-DIA 1 1H-NMR (600 MHz, CDCl3) δ: 0.86 - 0.90 (t, 6H), 1.02 (s, 24H), 1.22 - 1.42 (m, 40H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.52 - 2.60 (m, 8H), 3.02 - 3.43 (m, 8H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.86 - 4.95 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 8.09 - 8.16 (d, 2H)

[0234] [Example 7] Synthesis of O-Ph-cys-C2-Pip <Synthesis of 2-(1-Piperidyl)acetic Acid> 2-(1-Piperidyl)acetic acid represented by the following formula was synthesized in the same manner as in Example 2, except that piperidine (manufactured by Fujifilm Wako Pure Chemical Corporation) was used.

[0235] [Chemical formula]

[0236] <Synthesis of O-Ph-cys-C2-Pip> Using Intermediate 4 and 2-(1-piperidyl)acetic acid, O-Ph-cys-C2-Pip was synthesized by the same synthetic route as in Example 1.

[0237] <Of O-Ph-cys-C2-Pip 1 1H-NMR (600 MHz, CDCl3) δ: 0.86 - 0.90 (t, 6H), 1.22 - 1.48 (m, 44H), 1.60 - 1.63 (m, 8H), 1.72 - 1.77 (m, 4H), 1.99 - 2.05 (m, 8H), 2.43 - 2.56 (m, 12H), 2.94 - 3.02 (m, 4H), 3.13 - 3.43 (m, 4H), 3.63 (s, 4H), 4.30 - 4.37 (m, 8H), 4.88 - 4.97 (m, 2H), 5.32 - 5.38 (m, 4H), 7.02 - 7.05 (m, 4H), 7.27 - 7.30 (m, 4H), 8.02 - 8.09 (d, 2H)

[0238] [Example 8] Synthesis of E-Ph-cys-C2-DMA E-Ph-cys-C2-DMA was synthesized using intermediate 6 and D-α-tocopherol succinate (Tokyo Chemical Industry Co., Ltd.) via the same synthetic route as in Example 1.

[0239] <E-Ph-cys-C2-DMAの 1 H-NMR (600MHz, CDCl3) δ:0.83-0.88(m, 24H), 1.00-1.81(m, 52H), 1.98(s, 6H), 2.01(s, 6H), 2.08(s, 6H), 2.32(s, 12H), 2.55-2.60(t, 4H), 2.80-2.83(m, 4H), 2.90 -3.03(m, 8H), 3.09-3.43(m, 4H), 3.63(s, 4H), 4.30-4.37(m, 8H), 4.88 -4.97(m, 2H), 7.02-7.05(m, 4H), 7.27-7.30(m, 4H), 7.81-7.88(d, 2H)

[0240] [Example 9] Synthesis of O-Ph-cys-C4-DMA O-Ph-cys-C4-DMA was synthesized using intermediate 4 and 4-(dimethylamino)butyric acid hydrochloride (manufactured by SIGMA-ALDRICH) via the same synthetic route as in Example 1.

[0241] <O-Ph-cys-C4-DMAの 1 H-NMR (600MHz, CDCl3) δ:0.86-0.90(t, 6H), 1.22-1.42(m, 40H), 1.72-1.83(m, 8H), 1.99-2.05(m, 8H), 2.20-2.40(m, 20H), 2.52-2.56(t, 4H), 3.02-3.39(m , 4H), 3.63(s, 4H), 4.30-4.37(m, 8H), 4.80-4.89(m, 2H), 5.32-5.38(m, 4H), 7.02-7.05(m, 4H), 7.27-7.30(m, 4H), 7.68-7.78(d, 2H)

[0242] [Example 10] Synthesis of O-Ph-cys-DMA O-Ph-cys-DMA was produced by the following synthetic route, but the present invention is not limited to such a synthetic route.

[0243]

Chemical formula

[0244] <Synthesis of Intermediate 7> 15.0 g of chloroform was added to 757 mg (2.70 mmol) of Intermediate 2, 500 mg (1.35 mmol) of N,N,N’,N’-tetramethyl-L-cystine dihydrochloride (manufactured by Toronto Research Chemicals), 820 mg (8.10 mmol) of triethylamine (manufactured by Kanto Chemical Co., Inc.), and 198 mg (1.62 mmol) of DMAP, and the mixture was dissolved at room temperature. 1.04 g (5.40 mmol) of EDC was added to the resulting solution, and the reaction was carried out at 25 °C for 2 hours. The reaction solution was washed twice with 10.0 g of 0.5 M phosphate buffer (pH 3.0) and once with 10.0 g of 7 wt% saline, 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 to obtain 960 mg of Intermediate 7.

[0245] <Synthesis of Intermediate 8> 90.0 g of 0.5 M phosphate buffer (pH 2.0) was added to 900 mg (1.10 mmol) of Intermediate 7, and the reaction was carried out at 25 °C for 2 hours. 130 g of chloroform was added to the reaction solution for washing. After washing, an aqueous solution of sodium hydroxide at 400 g / L was added to the aqueous layer to adjust its pH to 6.0, and then the mixture was extracted twice with 130 g of chloroform. The organic layer was dehydrated by adding 1.80 g of sodium sulfate. After removing the sodium sulfate by filtration, the filtrate was concentrated using an evaporator to obtain 359 mg of Intermediate 8.

[0246] <Synthesis of O-Ph-cys-DMA> 340 mg (0.521 mmol) of intermediate 8, 294 mg (1.04 mmol) of oleic acid, and 25.4 mg (0.208 mmol) of DMAP were added to 23.9 g of chloroform and dissolved at room temperature. 400 mg (2.08 mmol) of EDC was added to the obtained solution and reacted at 25 ° C for 2 hours. The reaction solution was washed twice with 16.0 g of 20 wt % saline, and then dehydrated by adding 680 mg of sodium sulfate. After removing sodium sulfate by filtration, the filtrate was concentrated with an evaporator to obtain 501 mg of crude product. The obtained crude product was purified by column to obtain 303 mg of O-Ph-cys-DMA.

[0247] <O-Ph-cys-DMAの 1 H-NMR spectrum (600MHz, CDCl3)>δ:0.86-0.90(t, 6H), 1.22-1.42(m, 40H), 1.72-1.77(m, 4H), 1.99-2.05(m, 8H), 2.33(s, 12H), 2.5 2-2.56(t, 4H), 2.93-3.04(m, 4H), 3.55-3.61(m, 6H), 4.30-4.37(m, 8H), 5.32-5.38(m, 4H), 7.02-7.05(m, 4H), 7.27-7.30(m, 4H)

[0248] [Test Example 1] Measurement of liposomal pKa Lipid nanoparticles (LNPs) containing no nucleic acid were used to evaluate liposomal pKa.

[0249] 1. Preparation of LNPs by Microfluidic Method (1) Preparation of lipid ethanol solution 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 in an Eppendorf tube at the desired ratio (cationic lipid:DOPC:Chol=52.5:7.5:40 (molar ratio)) so that the total amount of lipid was 720 nmol. To the resulting mixture, DMG-PEG2k (2 mM ethanol solution) was further added in an amount of about 1.5 mol per 100 mol of the total amount of cationic lipid, DOPC, and Chol, and then ethanol was added to prepare an ethanol solution of lipid (total amount: 360 μL).

[0250] (2) Preparation of LNPs using microfluidics 1080 μL of acidic malic acid buffer (20 mM, pH 3.0) containing a final concentration of 30 mM NaCl and 360 μL of ethanol solution of lipid were weighed into a syringe. Using an ultrafast nanomedicine preparation device NanoAssmblr (manufactured by Precision NanoSystems), LNP was prepared under the conditions of an acidic buffer solution addition rate of 3 mL / min, an ethanol solution of lipid: 1 mL / min, and a syringe holder temperature of 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 ultrafiltered under centrifugation conditions (25°C, 1000 g, 6 min) to concentrate to about 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 in total. The obtained concentrate was diluted with PBS to a lipid concentration of 0.5 mM to obtain a dispersion containing LNPs.

[0251] 2. Liposomal pKa Measurement 20 mM citrate buffer, sodium phosphate buffer and Tris-HCl buffer containing 150 mM NaCl at a final concentration were prepared, which were adjusted to various pH values ​​in the range of 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 dispersion containing LNP 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 value of the fluorescence intensity in each LNP was set to 100%, and the minimum value was set 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. Table 5 shows the liposomal pKa of each LNP.

[0252] [Table 5]

[0253] 3.Results The liposomal pKa of all LNPs was within the range of pKa (5.0-7.2) favorable for endosomal escape. In addition, the liposomal pKa of LNPs could be adjusted by changing the structure of the amino group (dialkylamino group or cyclic amino group) of the cationic lipid.

[0254] [Test Example 2] Preparation of mRNA-encapsulated particles and evaluation of their physical properties 1. Preparation of LNPs by Microfluidic Method (1) Preparation of lipid ethanol solution 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 in an Eppendorf tube at the desired ratio (cationic lipid:DOPC:Chol=55:5:40 (molar ratio)) so that the total lipid amount was 720 nmol. DMG-PEG2k (2 mM ethanol solution) was further added to the resulting mixture in an amount of about 1 mol per 100 mol of the total amount of cationic lipid, DOPC, and Chol, and then ethanol was added to prepare an ethanol solution of lipid (total amount: 360 μL).

[0255] (2) Preparation of acidic buffer solution of nucleic acid A 0.6 mg / mL solution of mRNA (CleanCap (registered trademark) FLuc mRNA (TriLink)) was weighed out to give 7.2 μg and placed in a 5 mL tube. An acidic malic acid buffer (20 mM, pH 3.0) containing a final concentration of 30 mM NaCl was added to this to prepare an acidic buffer solution of nucleic acid (total volume: 1080 μL).

[0256] (3) Preparation of LNPs using microfluidics The acidic buffer solution of nucleic acid and the ethanol solution of lipid were weighed into a syringe. Using an ultrafast nanomedicine preparation device NanoAssmblr (manufactured by Precision NanoSystems), LNP was prepared under the conditions of an addition rate of the acidic buffer solution of nucleic acid: 3 mL / min, an addition rate of the ethanol solution of lipid: 1 mL / min, and a 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 ultrafiltered under centrifugal conditions (25°C, 1000 g, 6 min) to concentrate to about 100 μL. The resulting concentrate was diluted to 4 mL with PBS and concentrated again under centrifugal conditions (25°C, 1000 g, 6 min) twice in total. The resulting concentrate was diluted with PBS to a lipid concentration of 2 mM to obtain a dispersion containing LNP.

[0257] 2. Measurement of particle size, PdI, and zeta potential of mRNA-encapsulated LNPs The particle size, PdI (polydispersity), and zeta potential of the mRNA-encapsulated LNP prepared by the method described above in 1 were measured by dynamic light scattering (Zetasizer Nano; Malvern). The results are shown in Table 6.

[0258] [Table 6]

[0259] 3.Results All mRNA-encapsulated LNPs had a particle diameter 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).

[0260] [Test Example 3] Evaluation of gene expression in Jurkat 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 (CleanCap (registered trademark) FLuc mRNA (TriLink)) were prepared by the method described in [Test Example 2] 1.

[0261] 2. Time-course Assessment of Gene Expression in Jurkat Cells In Vitro 24 hours before transfection, human leukemia T cell Jurkat cells were 5 The cells were seeded on a 3.5 cm dish at 1.8 cells / dish. After 24 hours, 200 μL of medium (RPMI1640) containing D-luciferin was added to the dish at a final concentration of 0.1 mM. 40 to 120 μL of each mRNA-encapsulated LNP dispersion (0.4 μg of mRNA) was added to the dish, and the dish was placed in an incubator-type luminometer KronosDio. Luciferase luminescence intensity was measured for 2 minutes every 3 hours. The cumulative luciferase luminescence intensity over 24 hours was calculated from the obtained time change in expression. The results are shown in Figure 1. Note that "E+0a'" (a': integer) in Figure 1 is an integer that is calculated from "10 a’". For example, "5.0E+05" represents "5.0 × 10 5 " represents.

[0262] 3.Results As shown in Figure 1, the LNP containing the cationic lipid of Example 1 exhibited excellent gene expression activity. Therefore, it was revealed that the LNP containing the cationic lipid of Example 1 is useful as an LNP that can promote the expression of mRNA.

[0263] [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 (CleanCap (registered trademark) FLuc mRNA (TriLink)) were prepared by the method described in [Test Example 2] 1.

[0264] 2. Assessing Gene Expression In Vivo Each of the prepared mRNA-encapsulated LNP dispersions was administered to the tail vein of Balb / c mice (male, 5 weeks old). The dose of mRNA was 0.05 mg per kg of mouse body weight, and the dose of the mRNA-encapsulated LNP dispersion was 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 to the abdominal cavity of the mouse. The dose of D-luciferin potassium was 3 mg per mouse, and the dose of the PBS solution of D-luciferin potassium was 200 μL per mouse. 15 minutes after administration of the PBS solution of D-luciferin potassium, the mouse was euthanized, and the liver, spleen, kidney, heart, and lung were removed. The luminescence in each organ was quantified using an In vivo imaging system (IVIS) with an exposure of 10 sec. The luminescence in each organ was quantified by image analysis using the Live Imaging software attached to 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 Figure 2. The bar graphs in Figure 2 show the results for the liver, spleen, kidney, heart, and lung from the left. In addition, "E+0a'" (a': integer) in Figure 2 is "10 a’ For example, "1.0E+02" represents "1.0×10 2 " represents.

[0265] 3.Results As shown in Figure 2, the LNP containing the cationic lipid of Example 1 exhibited excellent gene expression activity in the liver and spleen. Therefore, it was revealed that the LNP containing the cationic lipid of Example 1 is useful as an LNP that can promote the expression of mRNA.

[0266] [Test Example 5] 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 (CleanCap (registered trademark) FLuc mRNA (TriLink)) were prepared by the method described in [Test Example 2] 1.

[0267] 2. Time course evaluation of gene expression in HeLa cells 24 hours before transfection, 5.0 × 10 HeLa cells, a human cervical cancer cell line, were 4 The cells were seeded in a 3.5 cm dish at 1.5 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 8 μg / mL. The diluted mRNA-encapsulated LNP solution (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 Figure 3. Note that "E+0a'" (a': integer) in Figure 3 is a multiple of "10 a’ For example, "1.0E+06" represents "1.0×10 6 " represents.

[0268] 3.Results As shown in Figure 3, the cationic lipid-containing LNP of Example 1 exhibited excellent gene expression activity. Therefore, it was revealed that the cationic lipid-containing LNP of Example 1 is useful as an LNP that can promote mRNA expression. [Industrial Applicability]

[0269] The cationic lipid of the present invention is useful for nucleic acid medicines, gene therapy, biochemical experiments, and the like.

[0270] This application is based on Patent Application No. 2020-057383 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. Formula (1): 【Chemistry 1】 (In the formula, L １ａ and L １ｂ both represent an amide bond, R １ａ and R １ｂ each independently represents an alkylene group having 4 or less carbon atoms, k and k each independently represent 0 or 1; X ａ and X ｂ each independently represents a dialkylamino group (the two alkyl groups each independently have 1 to 3 carbon atoms) or a 5- or 6-membered cyclic amino group, L ４ａ and L ４ｂ Both represent an ester bond, R ２ａ and R ２ｂ each independently represents an alkylene group having 4 or less carbon atoms, L ２ａ and L ２ｂ Both represent an ester bond, ma and mb both represent 1; R ３ａ and R ３ｂ each independently represents the formula (2): 【Chemistry 2】 (In the formula, * represents the bonding position to L 3a or L 3b ; ** represents the bonding position to L 2a or L 2b ; s represents an integer of 0 to 3; t represents an integer from 0 to 3, and u represents 0. represents a group represented by L ３ａ and L ３ｂ Both represent an ester bond, na and nb both represent 1; R ４ａ and R ４ｂ each independently represents an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R ５ -CO-(CH ２ ) ｐ - represents R ５ 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. A cationic lipid represented by the formula:

2. The cationic lipid of claim 1, wherein s is 0.

3. The cationic lipid according to claim 1 or 2, wherein k and k are both 0.

4. The cationic lipid according to claim 1 or 2, wherein ka and kb are both 1.

5. R ５ The cationic lipid according to any one of claims 1 to 4, wherein is a residue of a fat-soluble vitamin having a hydroxyl group.

6. R ４ａ and R ４ｂ The cationic lipid according to any one of claims 1 to 4, wherein each of the is independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

7. R ４ａ and R ４ｂ are each independently R ５ -CO-(CH ２ ) ｐ - and R ５ The cationic lipid according to any one of claims 1 to 4, wherein is a residue of a fat-soluble vitamin having a hydroxyl group.

8. A lipid membrane structure comprising the cationic lipid according to any one of claims 1 to 7 as a constituent lipid of the membrane.

9. A nucleic acid transfer agent comprising the cationic lipid according to any one of claims 1 to 7 or the lipid membrane structure according to claim 8.

10. A method for introducing a nucleic acid into a cell, comprising contacting the cell with the nucleic acid introduction agent according to claim 9 encapsulating the nucleic acid in vitro.

11. A method for introducing a nucleic acid into a cell, comprising administering the nucleic acid introduction agent described in claim 9 encapsulating a nucleic acid to a living body (excluding humans) so as to deliver the nucleic acid to the target cell.

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