Cationic lipids containing aromatic ester bonds

Abstract

To provide cationic lipids that can be used as nucleic acid delivery carriers with good nucleic acid delivery efficiency. [Solution] A cationic lipid represented by the following formula (1) (the meaning of the symbols in the formula is as described in the specification). JPEG2026116229000074.jpg3577

Description

[Technical Field]

[0001] The present invention relates to cationic lipids having aromatic ester bonds, lipid membrane structures containing the same, nucleic acid delivery agents and pharmaceutical compositions containing any of the same, a method for introducing nucleic acids into cells or target cells, and a method for producing cell-based pharmaceuticals. In this specification, "aromatic ester bond" means an ester bond bonded to an aromatic ring. [Background technology]

[0002] To put nucleic acid therapy into practical use, there is a need for effective and safe nucleic acid delivery carriers. Viral vectors are nucleic acid delivery carriers with good delivery efficiency, but the development of non-viral nucleic acid delivery carriers that can be used more safely is progressing, and among these, carriers using cationic lipids are currently the most commonly used non-viral nucleic acid delivery carriers.

[0003] Cationic lipids are broadly composed of an amine moiety and a lipid moiety. In these cationic lipids, the cationic amine moiety and the polyanion nucleic acid interact electrostatically to form liposomes or lipid membrane structures, thereby promoting uptake into cells and delivering nucleic acids into the cell.

[0004] Commonly known cationic lipids include 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) and 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP). These known cationic lipids, when combined with phospholipids, can form positively charged liposomes or lipid membrane structures, which can electrostatically interact with nucleic acids to deliver them to target cells (see, for example, Non-Patent Document 1).

[0005] On the other hand, for lipid membrane structures using cationic lipids to exert practical effects in vivo as nucleic acid delivery carriers, they need to exhibit unique pharmacokinetics. Specifically, they need to meet requirements such as high stability in the blood. To address this challenge, it is known that lipid membrane structures with their surface pKa adjusted to near neutral and incorporating PEG lipids exhibit a long blood lifetime after intravenous injection and accumulate at tumor sites. Furthermore, there are examples of improving pharmacokinetics by adjusting the surface pKa of lipid membrane structures. There are also examples of improving pharmacokinetics by controlling the particle size of lipid membrane structures.

[0006] For example, Non-Patent Documents 2 and 3 show that the pharmacokinetics and distribution in individual cells within the liver can be controlled by adjusting the surface pKa of lipid membrane structures. These documents demonstrate that by adjusting the surface pKa of lipid membrane structures, the escape of lipid membrane structures from endosomes is promoted, allowing for efficient delivery of nucleic acids into the cytoplasm.

[0007] Non-patent document 4 shows that the particle size of lipid membrane structures plays an important role in various biological phenomena, including circulating half-life, extravascular migration, macrophage uptake, and in vivo distribution. In particular, non-patent document 4 shows that controlling the particle size of lipid membrane structures to 20-200 nm results in a high transfection effect in in vitro studies. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Biomaterials 29 (24-25): 3477-3496, 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] Pharmaceutics 2024, 16(12), 1521 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] While cationic lipids with improved pharmacokinetics have been developed, given their nature as nucleic acid delivery carriers that introduce foreign substances into cells, it is desirable for them to exert a significant effect with a small amount of uptake. In other words, when lipid membrane structures are used as delivery carriers for nucleic acids (expression vectors) into cells, it is necessary to increase the nucleic acid delivery efficiency and thereby increase the efficiency of gene expression within the cell.

[0010] The present invention has been made in view of the above problems, and aims to provide a cationic lipid that can produce a lipid membrane structure with good nucleic acid delivery efficiency. [Means for solving the problem]

[0011] As a result of diligent research by the present inventors, it has been found that by using the cationic lipid of the present invention described below, it is possible to produce a lipid membrane structure with good nucleic acid delivery efficiency.

[0012] The present invention, which can achieve the above objective, is as follows: [1] Formula (1):

[0013] [ka]

[0014] (In the formula, R 1 This represents an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group, a tertiary amino group, or a hydroxyl group as a substituent. R 2a and R 2bEach independently represents an alkylene group having 1 to 16 carbon atoms and containing at least one cleavable bond. R 3a and R 3b Each independently represents an alkoxy group having 1 to 4 carbon atoms or a halogen atom. ma and mb each independently represent an integer from 0 to 4. R 4a and R 4b Each independently represents R 5 -CO-O-*(where * represents the bonding position in the above formula), and R 5 represents a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms which may contain at least one cleavable bond, or R 6 -CO-(CH2) p -*(where * represents the bonding position, and R 6 represents the residue of a fat-soluble vitamin having a hydroxyl group or the residue of a sterol derivative having a hydroxyl group, and p represents an integer from 1 to 8.) represents.) The cationic lipid represented by.

[0015] [2] The nitrogen-containing heterocyclic group is a pyrrolidinyl group, a morpholinyl group, a piperazinyl group, a piperidyl group, an azepanyl group, a pyrrolyl group, a pyrazolyl group, a pyridyl group, or an indolyl group. The tertiary amino group is a di(alkyl)amino group, and The carbon numbers of the two alkyl groups in the di(alkyl)amino group are each independently 1 to 4. The cationic lipid according to [1] above. [3] R 2a and R 2b Each independently represents the formula (2): **-(CH2) a -CO-O-(CH2) b -(S-S) e -(CH2) c -O-CO-(NH) f -(CH2) d -*** (2) (where ** represents the bonding position with the nitrogen atom in the formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), a~d each independently represent integers from 1 to 4, and e and f each independently represent either 0 or 1. A cationic lipid according to [1] or [2] above, representing a divalent group indicated by . [4] A cationic lipid according to any one of [1] to [3] above, wherein ma and mb are both 0. [5] R 5 The cationic lipid according to any one of [1] to [4] above, wherein the cationic lipid is a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, which may contain at least one degradable bond.

[0016] [6] A lipid membrane structure comprising a cationic lipid as a constituent lipid of the membrane, as described in any one of [1] to [5] above. [7] The lipid membrane structure according to [6] further comprising nucleic acids.

[0017] [8] A nucleic acid delivery agent containing a cationic lipid as described in any one of [1] to [5] above. [9] The nucleic acid delivery agent according to [8] further comprising nucleic acid.

[0018]

[10] A pharmaceutical composition comprising a cationic lipid as described in any one of [1] to [5] above.

[11] The pharmaceutical composition according to

[10] further comprising nucleic acid.

[0019]

[12] A method for introducing nucleic acids contained in the nucleic acid introduction agent into cells, comprising bringing the nucleic acid introduction agent described in [9] into contact with cells in vitro.

[13] A method for introducing nucleic acids contained in the nucleic acid introduction agent into target cells in the living body, comprising administering the nucleic acid introduction agent described in [9] above to a living body.

[14] A method for producing a cell pharmaceutical product containing cells expressing the gene in the nucleic acid, comprising bringing the nucleic acid agent described in [9] into contact with cells to introduce the nucleic acid contained in the nucleic acid agent into the cells. [Effects of the Invention]

[0020] By using the cationic lipids of the present invention, lipid membrane structures with good nucleic acid delivery efficiency can be manufactured. [Modes for carrying out the invention]

[0021] The cationic lipid of the present invention is a cationic lipid represented by the following formula (1):

[0022] [ka]

[0023] In this specification and the claims (hereinafter referred to as "this specification"), "cationic lipid represented by formula (1)" may be abbreviated as "cationic lipid (1)". Groups represented by other formulas may also be abbreviated in the same way as "cationic lipid represented by formula (1)".

[0024] First, let's explain the groups included in equation (1). In formula (1), R 1 R represents an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group, a tertiary amino group, or a hydroxyl group as a substituent. 1 Preferably, the substituent is a C1-C4 alkyl group having a nitrogen-containing heterocyclic group or a tertiary amino group. The C1-C4 alkyl group may be linear or branched. Note that the number of carbon atoms in the alkyl group does not include the carbon atoms of the substituent nitrogen-containing heterocyclic group or tertiary amino group.

[0025] In this specification, the nitrogen-containing heterocyclic group may be non-aromatic or aromatic. Furthermore, the nitrogen-containing heterocyclic group may have substituents such as alkyl groups having 1 to 4 carbon atoms.

[0026] Examples of non-aromatic nitrogen-containing heterocyclic groups include pyrrolidinyl groups (e.g., the group shown in formula (N1) or formula (N2) below), morpholinyl groups (e.g., the group shown in formula (N3) below), piperazinyl groups (e.g., the group shown in formula (N4) below), piperidyl groups (e.g., the group shown in formula (N5) below), and azepanyl groups (e.g., the group shown in formula (N6) below) (wherein * represents a bond position). In this specification, as stated above, "*" etc. indicate a bond position, not a carbon atom. Therefore, for example, "-*" indicates a single bond.

[0027] [ka]

[0028] Examples of aromatic nitrogen-containing heterocyclic groups include pyrrolyl groups (e.g., the group shown in formula (N7) below), pyrazolyl groups (e.g., the group shown in formula (N8) below), pyridyl groups (e.g., the group shown in formula (N9) below), and indolyl groups (e.g., the group shown in formula (N10) below) (where * indicates the bond position).

[0029] [ka]

[0030] In one embodiment of the present invention, the nitrogen-containing heterocyclic group is preferably a pyrrolidinyl group, a morpholinyl group, a piperazinyl group, a piperidyl group, an azepanyl group, a pyrrolyl group, a pyrazolyl group, a pyridyl group, or an indolyl group, more preferably a pyrrolidinyl group, a morpholinyl group, or an indolyl group, and even more preferably a pyrrolidinyl group or a morpholinyl group.

[0031] In another embodiment of the present invention, the nitrogen-containing heterocyclic group is preferably any of the groups (N1) to (N10), more preferably (N1), (N3), or (N10), and even more preferably (N1) or (N3).

[0032] In this specification, a tertiary amino group is, for example, a di(alkyl)amino group. The number of carbon atoms in the two alkyl groups of the di(alkyl)amino group is preferably 1 to 4, more preferably 1 to 3, independently of each other. Examples of di(alkyl)amino groups include those represented by the following formulas (N11) to (N16) (wherein * represents a bond position).

[0033] [ka]

[0034] In one embodiment of the present invention, the di(alkyl)amino group is preferably any of groups (N11) to (N16), and more preferably group (N12).

[0035] In a preferred embodiment of the present invention, Nitrogen-containing heterocyclic groups include pyrrolidinyl, morpholinyl, piperazinyl, piperidyl, azepanyl, pyrrolyl, pyrazolyl, pyridyl, or indolyl groups. The tertiary amino group is a di(alkyl)amino group, and The number of carbon atoms in the two alkyl groups within the di(alkyl)amino group is independently 1 to 4.

[0036] In another preferred embodiment of the present invention, The nitrogen-containing heterocyclic group is a pyrrolidinyl group, a morpholinyl group, or an indolyl group. The tertiary amino group is a di(alkyl)amino group, and The number of carbon atoms in the two alkyl groups within the di(alkyl)amino group is independently 1 to 4.

[0037] In another preferred embodiment of the present invention, R 1 This is an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group or a tertiary amino group as a substituent. The nitrogen-containing heterocyclic group is either a pyrrolidinyl group or a morpholinyl group. The tertiary amino group is a di(alkyl)amino group, and The number of carbon atoms in the two alkyl groups within the di(alkyl)amino group is independently 1 to 4.

[0038] In another preferred embodiment of the present invention, A nitrogen-containing heterocyclic group is one of the groups (N1) to (N10), and A tertiary amino group is one of the groups (N11) to (N16).

[0039] In another preferred embodiment of the present invention, Nitrogen-containing heterocyclic groups are (N1), (N3), or (N10), and A tertiary amino group is a group (N12).

[0040] In another preferred embodiment of the present invention, R 1 This is an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group or a tertiary amino group as a substituent. Nitrogen-containing heterocyclic groups are (N1) or (N3) groups, and A tertiary amino group is a group (N12).

[0041] In another preferred embodiment of the present invention, R 1 The group is one of the groups shown in formula (3) to formula (23) below (where * represents the bond position in the formulas below).

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] In another preferred embodiment of the present invention, R 1 These are base(3), base(5), base(11), base(16), or base(23).

[0049] In another preferred embodiment of the present invention, R 1 It is base(3), base(5), or base(16).

[0050] In formula (1), R 2a and R 2b Each of these independently represents an alkylene group having 1 to 16 carbon atoms and containing at least one decomposable bond. In this specification, the carbon number of an "alkylene group" does not include the carbon number of decomposable bonds (e.g., ester bonds) that the "alkylene group" may contain. 2a and R 2b They may be the same or different. 2a and R 2b These are preferably the same group.

[0051] In this specification, alkylene groups may be linear or branched. Examples of alkylene groups include methylene, ethylene, trimethylene (-(CH2)3-), propylene (-CH(CH3)CH2-, -CH2CH(CH3)-), tetramethylene (-(CH2)4-), butylene (-CH(C2H5)CH2-, -CH2CH(C2H5)-), pentamethylene (-(CH2)5-), hexamethylene (-(CH2)6-), heptamethylene (-(CH2)7-), octamethylene (-(CH2)8-), nonamethylene (-(CH2)9-), and decamethylene (-(CH2)) 10 -), undecamethylene group (-(CH2) 11 -) dodecamethylene group (-(CH2) 12 -), tridecamethylene group (-(CH2) 13 -) Tetradecamethylene group (-(CH2) 14 -) Pentadecamethylene group (-(CH2) 15 -), hexadecamethylene group (-(CH2) 16 -) is one example (the "-" in the above formula represents a single bond).

[0052] In this specification, "degradable bond" means a bond that can be hydrolyzed by intracellular enzymes. Examples of degradable bonds include ester bonds, carbamate bonds, and disulfide bonds.

[0053] In this specification, "ester bond" means -CO-O- or -O-CO- (the "-" in the above formula represents a single bond).

[0054] In this specification, "carbamate bond" means -O-CO-NH- or -NH-CO-O- (where "-" represents a single bond).

[0055] In this specification, "disulfide bond" means -SS- (where "-" in the above formula represents a single bond).

[0056] In one embodiment of the present invention, R 2a and R 2bPreferably, each of these is a 1-16 carbon alkylene group containing at least one degradable bond independently selected from the group consisting of ester bonds, carbamate bonds, and disulfide bonds.

[0057] In another embodiment of the present invention, R 2a and R 2b Preferably, each is independently expressed in formula (2): **-(CH2) a -CO-O-(CH2) b -(SS) e -(CH2) c -O-CO-(NH) f -(CH2) d -*** (2) (In the formula, ** represents the bond position with the nitrogen atom in formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), a~d each independently represent integers from 1 to 4, and e and f each independently represent either 0 or 1. It is a divalent group represented by [this symbol].

[0058] In formula (2), a is preferably 2 or 3, more preferably 2. In formula (2), b and c are each independently preferably integers between 1 and 3, and more preferably 2 or 3. The sum of b and c in equation (2) is preferably an integer between 2 and 6, and more preferably an integer between 4 and 6. In formula (2), d is preferably 1 or 2, more preferably 1.

[0059] In equation (2), "e is 0" means that in equation (2), "(SS) e This means that there is no "-". That is, a divalent base (2) where e is 0 is given by equation (2e): **-(CH2) a -CO-O-(CH2) b -(CH2) c -O-CO-(NH) f -(CH2)d -*** (2e) (The definitions of the symbols used in the formulas are as specified herein.) It is a divalent group represented by [this symbol].

[0060] In equation (2), "f is 0" means that in equation (2), "(NH) f This means that there is no "-". That is, a divalent base (2) where f is 0 is given by equation (2f): **-(CH2) a -CO-O-(CH2) b -(SS) e -(CH2) c -O-CO-(CH2) d -*** (2f) (The definitions of the symbols used in the formulas are as specified herein.) It is a divalent group represented by . f is preferably 0.

[0061] The preferred combinations of a to f in equation (2) are as follows: a is 2 or 3, b is an integer between 1 and 3, c is an integer between 1 and 3, the sum of b and c is an integer between 2 and 6, d is 1 or 2, and e and f are independently 0 or 1.

[0062] A more preferred combination of a to f in equation (2) is as follows: a is 2, b is 2 or 3, c is 2 or 3, the sum of b and c is preferably an integer between 4 and 6, d is 1, e is 0 or 1, and f is 0.

[0063] In formula (1), R 3a and R 3b Each of these independently represents an alkoxy group or halogen atom having 1 to 4 carbon atoms, and ma and mb independently represent integers from 0 to 4. 3a and R 3b They may be the same or different. 3a and R 3b These are preferably the same group.

[0064] In this specification, the alkoxy group may be linear or branched. Examples of alkoxy groups include the methoxy group, ethoxy group, propoxy group, isopropoxy group, and tert-butoxy group.

[0065] Examples of halogen atoms used herein include fluorine, chlorine, bromine, and iodine atoms.

[0066] ma and mb are substituents on the benzene ring, respectively. 3a and R 3b This is the number of R. In equation (1), "ma is 0" means that R in equation (1) 3a This means that does not exist. In equation (1), "mb is 0" means that R in equation (1) 3b This means that it does not exist. ma and mb are preferably both 0.

[0067] In formula (1), R 4a and R 4b Each of them is independent of R 5 -CO-O-* (wherein * represents the bond position) and R 5 This refers to a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, which may contain at least one degradable bond (hereinafter referred to as "R"). 5 It is sometimes written as "aliphatic hydrocarbon group of," or R 6 -CO-(CH2) p -*(In the above formula, * represents the bond position, R 6 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 an integer from 1 to 8. 4a and R 4b They may be the same or different. 4a and R 4b These are preferably the same group.

[0068] In this specification, examples of monovalent aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, and alkynyl groups. Monovalent aliphatic hydrocarbon groups are preferably linear or branched. In this specification, the number of carbon atoms in a "monovalent aliphatic hydrocarbon group" does not include the number of carbon atoms in decomposable bonds (e.g., ester bonds) that the "monovalent aliphatic hydrocarbon group" may contain. The number of carbon atoms in an "alkyl group," an "alkenyl group," and an "alkynyl group" is the same as the number of carbon atoms in a "monovalent aliphatic hydrocarbon group."

[0069] In this specification, alkyl groups may be linear or branched. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl groups. Examples include henicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, hentriacontyl group, dotriacontyl group, tritriacontyl group, tetratriacontyl group, pentatriacontyl group, hexatriacontyl group, tetracontyl group, hentetracontyl group, dotetracontyl group, tritetracontyl group, and tetratetracontyl group.

[0070] In this specification, the alkenyl group may be linear or branched. Also, in this specification, the number of olefinic carbon-carbon double bonds in the alkenyl group may be only one or two or more. Examples of alkenyl groups include etenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icocenyl, henicocenyl, dococenyl, tricocenyl, tetracocenyl, pentacocenyl, hexacocenyl, heptacocenyl, octacocenyl, nonacocenyl, triacontenyl, hentriacontenyl, and dotriacontenyl groups.

[0071] In this specification, the alkynyl group may be linear or branched. Also, in this specification, the number of carbon-carbon triple bonds in the alkynyl group may be only one or two or more. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, noninyl, desinyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadesinyl, heptadecynyl, octadecynyl, nonadesinyl, icosinyl, henicosinyl, docosinyl, tricosinyl, tetracosinyl, pentacosinyl, hexacosinyl, heptacosinyl, octacosinyl, nonacosinyl, triacontinyl, hentriacontinyl, and dotriacontinyl.

[0072] R 5 The number of carbon atoms in the aliphatic hydrocarbon group is preferably 3 to 30. If the aliphatic hydrocarbon group is an alkyl group which may contain at least one degradable bond, the number of carbon atoms in the alkyl group is preferably 3 to 30. If the aliphatic hydrocarbon group is an alkenyl group which may contain at least one degradable bond, the number of carbon atoms in the alkenyl group is preferably 3 to 30, more preferably 10 to 30.

[0073] R 5 The degradable bond that the aliphatic hydrocarbon group of 5 may contain is preferably at least one degradable bond selected from the group consisting of a disulfide bond and an ester bond, and more preferably a disulfide bond or an ester bond.

[0074] In the above-mentioned "R 6 -CO-(CH2) p -*", R 6 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 an integer of 1 to 8. p is preferably 2 or 3.

[0075] In the present specification, the "residue of a fat-soluble vitamin having a hydroxyl group" means a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the fat-soluble vitamin. Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, tocotrienol, and the like.

[0076] In the present specification, the "residue of a sterol derivative having a hydroxyl group" represents a monovalent group having a structure obtained by removing a hydrogen atom from the hydroxyl group of the sterol derivative. Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, and the like.

[0077] R 5 is preferably a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, which may contain at least one degradable bond.

[0078] In a preferred embodiment of the present invention, R 5is a monovalent aliphatic hydrocarbon group that may contain at least one dissociable bond selected from the group consisting of a disulfide bond, an ester bond, and The monovalent aliphatic hydrocarbon group is an alkyl group having 3 to 30 carbon atoms or an alkenyl group having 3 to 30 carbon atoms.

[0079] In another preferred embodiment of the present invention, R 5 is an alkyl group having 3 to 30 carbon atoms that may contain a disulfide bond, or an alkenyl group having 3 to 30 carbon atoms that may contain an ester bond. Examples of the alkyl group having 3 to 30 carbon atoms containing a disulfide bond include R 5 possessed by Compound 29 of the examples. Examples of the alkenyl group having 3 to 30 carbon atoms containing an ester bond include R 5 possessed by Compound 28 of the examples.

[0080] In another preferred embodiment of the present invention, R 5 is an alkenyl group having 10 to 30 carbon atoms.

[0081] Preferred examples of the cationic lipid (1) include the following cationic lipids.

[0082] [Cationic lipid (1-1)] R 1 is an alkyl group having 1 to 4 carbon atoms having a nitrogen-containing heterocyclic group, a tertiary amino group, or a hydroxy group as a substituent, The nitrogen-containing heterocyclic group is a pyrrolidinyl group, a morpholinyl group, a piperazinyl group, a piperidyl group, an azepanyl group, a pyrrolyl group, a pyrazolyl group, a pyridyl group, or an indolyl group, The tertiary amino group is a di(alkyl)amino group, and The carbon numbers of the two alkyl groups in the di(alkyl)amino group are each independently 1 to 4, R 2a and R 2b are each independently of formula (2): **-(CH2) a -CO-O-(CH2)b -(SS) e -(CH2) c -O-CO-(NH) f -(CH2) d -*** (2) (In the formula, ** represents the bond position with the nitrogen atom in formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), a~d each independently represent integers from 1 to 4, and e and f each independently represent either 0 or 1. It is a divalent group represented by, ma and mb are both 0. R 4a and R 4b , each independently, R 5 -CO-O-* (wherein * represents a bond position), and R 5 However, it is a monovalent aliphatic hydrocarbon group which may contain at least one degradable bond selected from the group consisting of disulfide bonds and ester bonds, and The monovalent aliphatic hydrocarbon group is an alkyl group having 3 to 30 carbon atoms or an alkenyl group having 3 to 30 carbon atoms. Cationic lipids (1-1).

[0083] [Cationic lipids (1-2)] R 1 However, the substituent is a nitrogen-containing heterocyclic group, a tertiary amino group, or an alkyl group having 1 to 4 carbon atoms. The nitrogen-containing heterocyclic group is a pyrrolidinyl group, a morpholinyl group, or an indolyl group. The tertiary amino group is a di(alkyl)amino group, and The number of carbon atoms in the two alkyl groups in the di(alkyl)amino group is 1 to 4, independently of each other. R 2a and R 2b However, each is independent of equation (2): **-(CH2) a -CO-O-(CH2) b-(SS) e -(CH2) c -O-CO-(NH) f -(CH2) d -*** (2) (In the formula, ** represents the bond position with the nitrogen atom in formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), 'a' represents 2 or 3, b represents an integer between 1 and 3. c represents an integer between 1 and 3. The sum of b and c is an integer between 2 and 6. d represents 1 or 2, and e and f each independently represent either 0 or 1. It is a divalent group represented by, ma and mb are both 0. R 4a and R 4b , each independently, R 5 -CO-O-* (wherein * represents a bond position) and R 5 However, it is an alkyl group having 3 to 30 carbon atoms that may contain a disulfide bond, or an alkenyl group having 3 to 30 carbon atoms that may contain an ester bond. Cationic lipids (1-2).

[0084] [Cationic lipids (1-3)] R 1 However, the substituent is an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group or a tertiary amino group. The nitrogen-containing heterocyclic group is either a pyrrolidinyl group or a morpholinyl group. The tertiary amino group is a di(alkyl)amino group, and The number of carbon atoms in the two alkyl groups in the di(alkyl)amino group is 1 to 4, independently of each other. R 2a and R 2b However, each independently, equation (2): **-(CH2) a -CO-O-(CH2)b -(SS) e -(CH2) c -O-CO-(NH) f -(CH2) d -*** (2) (In the formula, ** represents the bond position with the nitrogen atom in formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), a represents 2, b represents 2 or 3, c represents 2 or 3, The sum of b and c is preferably an integer between 4 and 6. d represents 1, e represents 0 or 1, and f represents 0. It is a divalent group represented by, ma and mb are both 0. R 4a and R 4b , each independently, R 5 -CO-O-* (wherein * represents a bond position), and R 5 However, it is an alkenyl group with 10 to 30 carbon atoms. Cationic lipids (1-3).

[0085] Specific examples of cationic lipids (1) include compounds 1 to 30 synthesized in the examples described later.

[0086] In one embodiment of the present invention, cationic lipid (1) is Preferably, it is at least one selected from the group consisting of compound 1 to compound 30. More preferably, at least one selected from the group consisting of compound 1, compound 3, compound 6, compound 7, and compound 27. More preferably, it is at least one selected from the group consisting of compound 3, compound 7, and compound 27.

[0087] The cationic lipid (1) of the present invention can be synthesized using commercially available starting materials in the same manner as in the examples described below. For example, R 2a and R 2b A cationic lipid (1) in which both are divalent groups represented by formula (2), e in formula (2) is 1, and f in formula (2) is 0 can be synthesized using commercially available starting materials in the same manner as in Example 1 or Example 2 described below. For example, R 2a and R 2b A cationic lipid (1) in which both e and f are divalent groups represented by formula (2), and in which both e and f in formula (2) are 1, can be synthesized using commercially available starting materials in the same manner as in Example 22 described below. For example, R 2a and R 2b A cationic lipid (1) in which both e and f are divalent groups represented by formula (2), and in which both e and f in formula (2) are 0, can be synthesized using commercially available starting materials in the same manner as in Example 24 described below. The conditions for the synthesis reaction (e.g., reaction temperature, reaction time, etc.) can be appropriately set by those skilled in the art.

[0088] The present invention also provides a lipid membrane structure containing a cationic lipid (1) as a constituent lipid of the membrane. The lipid membrane structure of the present invention may further contain nucleic acids.

[0089] In this specification, "lipid membrane structure" means a particle having a membrane structure in which the hydrophilic groups of amphiphilic lipids are arranged toward the aqueous phase side of the interface. "Amphiphilic lipid" means a lipid that has both hydrophilic groups that exhibit hydrophilicity and hydrophobic groups that exhibit hydrophobicity. Examples of amphiphilic lipids include cationic lipids and phospholipids.

[0090] The morphology of the lipid membrane structure of the present invention is not particularly limited, but examples of morphologies in which cationic lipids (1) are dispersed in an aqueous solvent include liposomes (e.g., single-layer liposomes, multilayer liposomes, etc.), O / W emulsions, W / O emulsions, spherical micelles, string-like micelles, lipid nanoparticles (LNPs, which may be abbreviated as "LNPs" herein), or unspecified layered structures. The lipid membrane structure of the present invention is preferably a liposome or an LNP, and more preferably an LNP.

[0091] The lipid membrane structure of the present invention may further contain other components in addition to the cationic lipid (1). Examples of such other components include lipids (phospholipids (formhatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, formhatidylglycerol, phosphatidylcholine, etc.), glycolipids, peptide lipids, cholesterol, cationic lipids other than cationic lipid (1), PEG lipids, etc.), surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, sodium cholate, octyl glycoside, ND-gluco-N-methylalkaneamides, etc.), polyethylene glycol, proteins, etc. The content of such other components in the lipid membrane structure of the present invention is, for example, 5 to 95 mol%, preferably 10 to 90 mol%, and more preferably 30 to 80 mol%, relative to the total components in the lipid membrane structure of the present invention.

[0092] The content of cationic lipid (1) 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 delivery agent as described later, it contains a sufficient amount of cationic lipid (1) to deliver nucleic acids. The content of cationic lipid (1) in the lipid membrane structure of the present invention is, for example, 5 to 95 mol%, preferably 10 to 90 mol%, and more preferably 20 to 70 mol%, relative to the total components of the lipid membrane structure of the present invention.

[0093] The lipid membrane structure of the present invention can be prepared by dispersing cationic lipids (1) and other constituent components (lipids, etc.) in a suitable solvent or dispersion medium, such as an aqueous solvent or an alcoholic solvent, and performing an operation to induce organization as necessary.

[0094] "Operations that induce organization" include, for example, ethanol dilution using microfluidics or vortexing, simple hydration, sonication, heating, vortexing, ether injection, French press, cholic acid method, Ca 2+ Examples of known methods include, but are not limited to, the fusion method, the freeze-thaw method, and the reverse-phase evaporation method.

[0095] By bringing a lipid membrane structure containing nucleic acids into contact with cells, the nucleic acids can be introduced into the cells in vivo and / or extra vivo. Accordingly, the present invention provides a nucleic acid delivery agent containing a cationic lipid (1). The nucleic acid delivery agent of the present invention is preferably the lipid structure of the present invention. The nucleic acid delivery agent of the present invention preferably contains nucleic acids.

[0096] The nucleic acid delivery agent of the present invention can introduce any nucleic acid into cells. Examples of nucleic acid types include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, any nucleic acid with 1 to 3 strands can be used, but 1-stranded or 2-stranded nucleic acids are preferred. The nucleic acid may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (e.g., commercially available peptide nucleic acids (PNA), etc.), or other oligomers having special bonds (provided that the oligomer contains nucleotides that have a configuration that allows for base pairing and base attachment, as found in DNA and RNA). Furthermore, the nucleic acid may be, for example, a nucleic acid with known modifications, a nucleic acid with a label known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are replaced with analogs, a nucleic acid with intramolecular nucleotide modifications, a nucleic acid having an uncharged bond (e.g., methyl sulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid having an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0097] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc. Plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA can also be digested with restriction enzymes as appropriate and used as linear DNA.

[0098] The types of RNA that can be used in the present invention are not particularly limited and can be appropriately selected depending on the purpose of use. Examples include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. Preferably, siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

[0099] In the present invention, the nucleic acids used are preferably purified by methods commonly used by those skilled in the art.

[0100] The nucleic acid-containing nucleic acid-transfer agents of the present invention can be administered in vivo, for example, for the purpose of preventing and / or treating diseases. Therefore, the nucleic acids used in the present invention are preferably those that have preventive and / or therapeutic activity against a certain disease (preventive and therapeutic nucleic acids). Examples of such nucleic acids include those used in so-called gene therapy.

[0101] To introduce nucleic acids into cells using the nucleic acid delivery agent of the present invention, the target nucleic acid is made to coexist with the lipid membrane structure of the present invention when forming the lipid membrane structure of the present invention, thereby forming a lipid membrane structure of the present invention containing the nucleic acid (i.e., the nucleic acid delivery agent of the present invention containing the nucleic acid). For example, when forming liposomes as a lipid membrane structure by the ethanol dilution method, an aqueous solution of nucleic acid and an ethanol solution of the components of the lipid membrane structure of the present invention (lipids, etc.) are vigorously mixed using a vortex or microchannel, and then the mixture is diluted with an appropriate buffer. When forming liposomes as a lipid membrane structure by the simple hydration method, the components of the lipid membrane structure of the present invention (lipids, etc.) are dissolved in an appropriate organic solvent, the solution is placed in a glass container, and the solvent is removed by vacuum drying to obtain a lipid thin film. An aqueous solution of nucleic acid is then added and hydrated, and then sonicated with a sonicator.

[0102] One form of the lipid membrane structure of the present invention containing nucleic acids is an LNP in which nucleic acids are encapsulated by forming an electrostatic complex between nucleic acids and cationic lipids. This LNP can be used as a drug delivery system for selectively delivering nucleic acids into specific cells, and is useful, for example, for DNA vaccines and tumor gene therapies that introduce antigen genes into dendritic cells, and for nucleic acid drugs that suppress the expression of target genes using RNA interference.

[0103] The particle size of the lipid membrane structure containing nucleic acids according to the present invention is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 200 nm. The particle size can be measured using a particle size distribution analyzer such as the Zetasizer Nano (Malvern). The particle size of the lipid membrane structure can be appropriately adjusted by the method of preparing the lipid membrane structure.

[0104] The surface potential (zeta potential) of the lipid membrane structure of the present invention containing nucleic acids is not particularly limited, but is preferably -15mV to +15mV, more preferably -10mV to +10mV. In conventional gene transfer, particles with a positively charged surface potential have been mainly used. This is useful as a method to promote electrostatic interaction with heparin sulfate on the negatively charged cell surface and promote uptake into cells, but a positive surface potential may suppress the release of nucleic acids from carriers through interaction with delivery nucleic acids within the cell, and may also suppress protein synthesis through interaction between mRNA and delivery nucleic acids. This problem can be solved by adjusting the surface potential within the above range. Surface potential can be measured using a zeta potential measuring device such as a Zetasizer Nano. The surface potential of the lipid membrane structure can be adjusted by the composition of the components of the lipid membrane structure containing cationic lipids (1).

[0105] The lipid membrane surface pKa (hereinafter sometimes abbreviated as "Liposomal pKa") of the lipid membrane structure of the present invention is not particularly limited, but is preferably 4.5 to 9.5. Liposomal pKa is considered an indicator of how easily a lipid membrane structure taken up by endocytosis is protonated in the weakly acidic environment within an endosome. As described in Angewante Chemie International Edition 51: 8529-8533, 2012 or Molecular Therapy 24(4): 786-795, 2016, in order to escape from an endosome and deliver nucleic acids into the cytoplasm, it is important to set the Liposomal pKa to a value favorable for endosomal escape, and by adjusting it within the above range, nucleic acids can be efficiently delivered into the cytoplasm. Liposomal pKa can be adjusted by the composition of the components of the lipid membrane structure, which includes cationic lipids (1).

[0106] By bringing a nucleic acid delivery agent of the present invention (preferably a lipid membrane structure of the present invention containing nucleic acids) into contact with cells, the nucleic acids contained in the nucleic acid delivery agent can be introduced into the cells. Accordingly, the present invention also provides a method for introducing nucleic acids contained in the nucleic acid delivery agent into cells, which includes bringing the nucleic acid delivery agent of the present invention containing nucleic acids into contact with cells.

[0107] The type of cell is not particularly limited, and prokaryotes and eukaryotes can be used, but eukaryotes are preferred. The type of eukaryote is also not particularly limited, and examples include vertebrates such as mammals including humans (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (silkworms, moths, fruit flies, etc.), plants, microorganisms (e.g., yeast, etc.). More preferably, the cells targeted in the present invention are animal or plant cells, and even more preferably mammalian cells. The cells may be cultured cell lines including cancer cells, cells isolated from individuals or tissues, or cells from tissues or tissue fragments. Furthermore, the cells may be adherent cells or non-adherent cells.

[0108] The process of bringing the lipid membrane structure of the present invention, which contains nucleic acids, into contact with cells in vitro will be described in detail below.

[0109] The cells are suspended in a suitable culture medium several days before contact with the lipid membrane structure and cultured under appropriate conditions. At the time of contact with the lipid membrane structure, the cells may or may not be in the proliferation phase.

[0110] The culture medium used at the time of contact may be either a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% by weight or less, and more preferably 20% by weight or less. If the medium contains an excess of serum or other proteins, contact between the lipid membrane structure and the cells may be inhibited.

[0111] The cell density at the time of contact is not particularly limited and can be set appropriately considering the type of cell, etc., but is usually 1 × 10⁻⁶ 4 ~1 × 10 7 This is within the range of cells / mL.

[0112] A suspension of the lipid membrane structure of the present invention (i.e., the nucleic acid delivery agent of the present invention containing nucleic acid), for example, is added to the cells. The amount of the suspension added is not particularly limited and can be set appropriately considering the number of cells, etc. The concentration of the lipid membrane structure when it is brought into contact with the cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved, but the lipid concentration is usually 1 to 100 nmol / mL, preferably 10 to 50 nmol / mL, and the nucleic acid concentration is usually 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL.

[0113] After adding the above-mentioned suspension to the cells, the cells are cultured. The temperature, humidity, CO2 concentration, etc., during culture should be set appropriately considering the type of cells. If the cells are of mammalian origin, the temperature is usually about 37°C, the humidity is about 95%, and the CO2 concentration is about 5% by volume. The culture time can also be set appropriately considering the conditions such as the type of cells used, but it 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 culture time is too short, nucleic acids may not be sufficiently introduced into the cells, and if the culture time is too long, the cells may weaken.

[0114] Through the culture described above, nucleic acids are introduced into the cells. Preferably, the culture medium is replaced with fresh medium, or fresh medium is added to the medium and the culture is continued. If the cells are of mammalian origin, the fresh medium preferably contains serum or trophic factors.

[0115] Furthermore, as described above, by using the nucleic acid delivery agent of the present invention which contains nucleic acids, it is possible to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering the nucleic acid delivery agent of the present invention which contains nucleic acids to a living organism, the nucleic acids contained in the nucleic acid delivery agent can be introduced into target cells in the living organism. Accordingly, the present invention also provides a method for introducing nucleic acids contained in the nucleic acid delivery agent into target cells in the living organism, which includes administering the nucleic acid delivery agent of the present invention which contains nucleic acids to a living organism.

[0116] The organisms to which the nucleic acid-containing nucleic acid-introducing agent of the present invention can be administered are not particularly limited, and include, for example, vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The organism to which the nucleic acid-containing nucleic acid-introducing agent of the present invention can be administered is preferably a human or other mammal.

[0117] The type of target cell is not particularly limited, and by using the nucleic acid delivery agent of the present invention, which contains nucleic acids, it is possible to introduce nucleic acids into cells in various tissues (for example, liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0118] The method of administering the nucleic acid-containing nucleic acid-transfer agent of the present invention to a living organism is not particularly limited, and known administration methods (for example, oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the nucleic acid-containing nucleic acid-transfer agent of the present invention is not particularly limited, and can be appropriately selected considering the type of living organism to be administered to, the method of administration, the type and site of target cells, etc.

[0119] When cationic lipids (1) or lipid membrane structures are used as nucleic acid delivery agents, they can be formulated according to conventional methods.

[0120] When the nucleic acid delivery agent of the present invention, which is a lipid membrane structure of the present invention, is provided as a research reagent, it may be provided as is, or as a sterile solution or suspension of the lipid membrane structure of the present invention with, for example, water or other physiologically acceptable liquid (e.g., water-soluble solvent (e.g., malic acid buffer), organic solvent (e.g., ethanol, methanol, DMSO, tert-butanol), or a mixture of a water-soluble solvent and an organic solvent). The nucleic acid delivery agent of the present invention may optionally contain physiologically acceptable additives known to the present (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0121] Furthermore, when the nucleic acid delivery agent of the present invention, which is a lipid membrane structure of the present invention, is provided as a pharmaceutical product, the nucleic acid delivery agent of the present invention can be produced as an oral preparation (e.g., tablets, capsules, etc.) or a parenteral preparation (e.g., injection, spray, etc.), preferably as a parenteral preparation (more preferably as an injection), by using the lipid membrane structure of the present invention as is, or by using the lipid membrane structure of the present invention together with pharmaceutically acceptable known additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.) and mixing them in a unit dose form required for generally accepted formulation.

[0122] The nucleic acid delivery agent of the present invention can also be provided in the form of a kit. The kit may include a cationic lipid (1) or the lipid membrane structure of the present invention, as well as reagents used for nucleic acid delivery. In one embodiment, the nucleic acid delivery agent (or kit) of the present invention further comprises a polycation (e.g., protamine). By using the nucleic acid delivery agent (or kit) of the present invention in this embodiment, an electrostatic complex between the nucleic acid and the polycation (e.g., protamine) can be easily encapsulated in the lipid membrane structure of the present invention, and the nucleic acid can be delivered into cells.

[0123] The present invention also provides a pharmaceutical composition comprising a cationic lipid (1). The pharmaceutical composition of the present invention may further contain nucleic acids. Furthermore, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable known additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0124] The pharmaceutical composition of the present invention may be a powdered composition obtained by removing the solvent by freeze-drying or the like, or it may be a liquid composition. The powdered composition may be produced by removing the solvent from the liquid composition by filtration, centrifugation or the like, or the liquid composition may be produced by freeze-drying.

[0125] The pharmaceutical composition of the present invention can be manufactured as an oral preparation (e.g., tablets, capsules, etc.) or a parenteral preparation (e.g., injection, spray, etc.), preferably as a parenteral preparation (more preferably as an injection). The pharmaceutical composition of the present invention can be manufactured not only as a formulation for adults but also as a formulation for children.

[0126] The present invention also provides a method for producing a cell-based pharmaceutical product containing cells expressing a gene in the nucleic acid, which includes contacting a nucleic acid delivery agent containing the nucleic acid of the present invention with cells to introduce the nucleic acid contained in the nucleic acid delivery agent into the cells.

[0127] The term "cells expressing the gene in the nucleic acid" refers to cells that have expressed the target gene by introducing nucleic acid into the cell. By bringing the nucleic acid-containing nucleic acid delivery agent of the present invention into contact with cells outside the body, the nucleic acid can be introduced into the cells.

[0128] Examples of cells used in the manufacture of cell-based pharmaceuticals include T cells, B cells, NK cells, dendritic cells, macrophages, and monocytes. T cells used in the manufacture of cell-based pharmaceuticals may be T cells differentiated from lymphocyte precursor cells, including pluripotent cells. Examples of lymphocyte precursor cells, including pluripotent cells, include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Undifferentiated cells, such as pluripotent cells, can be differentiated into T cells by known methods.

[0129] Nucleic acids used in the manufacture of cell-based pharmaceuticals include, for example, nucleic acids that encode chimeric antigen receptors (CARs) and T cell receptors (TCRs). Nucleic acids encoding CARs used in the manufacture of cell-based therapies include an antigen-binding domain, an extracellular hinge domain, a transmembrane domain, and an intracellular T-cell signaling domain of the antibody, which can specifically recognize surface antigens that target immune cells should recognize. The nucleic acids encoding TCRs used in the manufacture of cell-based therapies are nucleic acids that encode the α and β chains of the TCR, which can specifically recognize the surface antigens that target T cells should recognize. The nucleic acids that encode CARs and TCRs are not particularly limited and include, for example, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids.

[0130] Cell-based pharmaceuticals contain cells expressing a specific gene and may also contain pharmaceutically acceptable excipients (e.g., carriers, excipients, vehicles, preservatives, stabilizers, etc.). Cell-based pharmaceuticals are preferably parenteral preparations, and more preferably injectable preparations.

[0131] Cellular therapies can be used to treat or prevent diseases such as cancer. There are no particular restrictions on the types of cancer that can be treated with cellular therapies, and examples include lung cancer, breast cancer, stomach cancer, colorectal cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, leukemia, malignant lymphoma, and myeloma.

[0132] The target population for which cell-based therapies can be administered is not particularly limited, and examples include mammals (e.g., humans, monkeys, mice, rats, hamsters, cattle, etc.). Preferably, the target population for cell-based therapies is humans or other mammals.

[0133] The method of administering cell-based pharmaceuticals is not particularly limited as long as it allows the cells to express the target gene. Considering the type of cells, the target disease, etc., appropriate methods such as parenteral administration (e.g., intravenous, intramuscular, topical, transdermal, subcutaneous, intraperitoneal, spray, etc.) can be selected as appropriate. The dosage of cell-based pharmaceuticals is not particularly limited as long as it allows the cells to express the target gene. It can be selected as appropriate considering the type of recipient, the method of administration, the type of cells, the target disease, etc. [Examples]

[0134] The present invention will be described in more detail below with reference to examples and test examples, but the present invention is not limited thereto.

[0135] The meanings of the abbreviations used in the description of the examples are as follows: Chol: Cholesterol DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide DMG-PEG2k:1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG chain number-average molecular weight: 2000) DMSO: Dimethyl sulfoxide DOPC:1,2-Dioleoyl-sn-glycero-3-phosphocholine DSC: N,N'-disuccinimimidyl carbonate EDC hydrochloride: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride LNP: Lipid nanoparticles MeOH: methanol MES: 2-Morpholinoethanesulfonic acid mRNA: Messenger RNA PBS: Phosphate-buffered saline TBS: tert-butyldimethylsilyl tBuOH:tert-butanol TMS-Cl: Trimethylsilyl chloride TNS: Sodium 6-(p-toluidino)-2-naphthalenesulfonate

[0136] The structures of compounds 1 to 30 synthesized in Examples 1 to 30, described later, are shown in Tables 1-1 to 1-5 below, and the structures of the comparative compounds are shown in Table 2 below. For the comparative compounds, reagents purchased from Fujifilm Wako Pure Chemical Industries, Ltd. were used.

[0137] [Table 1-1]

[0138] [Table 1-2]

[0139] [Table 1-3]

[0140] [Table 1-4]

[0141] [Table 1-5]

[0142] [Table 2]

[0143] [Example 1] Synthesis of Compound 1 Compound 1 was synthesized using the following synthetic route.

[0144] [ka]

[0145] <Synthesis of Intermediate 1> 500 mg (3.10 mmol) of 3,3'-iminodipropionic acid was dissolved in 3.50 g of methanol, and then 674 mg (6.20 mmol) of TMS-Cl was added and the mixture was reacted at room temperature for 1 hour and 30 minutes. The resulting solution was concentrated using an evaporator, and the concentrate was vacuum-dried to obtain 666 mg of intermediate 1.

[0146] <Synthesis of Intermediate 2> 500 mg (2.22 mmol) of intermediate 1 was dissolved in 5.00 g of DMF, then 560 mg (5.55 mmol) of triethylamine, 36.8 mg (0.022 mmol) of potassium iodide, and 561 mg (2.22 mmol) of (3-bromopropoxy)(tert-butyl)dimethylsilane were added and the mixture was reacted at 70°C for 11 hours. 7.50 g of chloroform was added to the reaction solution, and the mixture was washed with 5% by weight sodium dihydrogen phosphate aqueous solution, 7% by weight sodium bicarbonate aqueous solution, and 20% by weight sodium chloride aqueous solution. The mixture was then dehydrated with sodium sulfate, and the resulting filtrate was concentrated using an evaporator. After concentration, silica gel column purification using ethanol / chloroform was performed to obtain 548 mg of intermediate 2.

[0147] <Synthesis of Intermediate 3> Intermediate 2 was dissolved in 1.38 g of tBuOH by adding 200 mg (0.528 mmol) of intermediate 2, and then 845 mg of 100 g / mL sodium hydroxide aqueous solution was added and the mixture was reacted at room temperature for 2 hours. The resulting solution was neutralized with 1.0 M sodium dihydrogen phosphate aqueous solution, and 8.60 g of acetonitrile was added. The mixture was concentrated using an evaporator, and the concentrate was vacuum-dried to obtain intermediate 3.

[0148] Intermediate 4 was synthesized using the following synthetic route.

[0149] [ka]

[0150] <Synthesis of 4-Oleoyloxyphenylacetic acid> 43.1 g (78.9 mmol) of oleic anhydride and 6.00 g (39.4 mmol) of 4-hydroxyphenylacetic acid were dissolved in 647 g of chloroform. 1.93 g (15.8 mmol) of DMAP was added, and the mixture was reacted at room temperature for 9 hours. The reaction mixture was washed with 216 g of 10 wt% aqueous acetic acid solution and 216 g of deionized water, and then treated with magnesium sulfate. 12.9 g was added and stirred for 30 minutes. The reaction mixture was dehydrated with magnesium sulfate, and the magnesium sulfate was removed by filtration. The resulting filtrate was concentrated using an evaporator. The concentrate was dissolved in 284 g of hexane, and insoluble matter was removed by filtration. The resulting filtrate was extracted with 168 g of acetonitrile. The acetonitrile layer after extraction was concentrated using an evaporator. The obtained concentrate was purified by silica gel column using ethanol / chloroform to obtain 3.80 g of 4-oleyloxyphenylacetic acid.

[0151] <Synthesis of Intermediate 4> 4-Oleoyloxyphenylacetic acid 3.80 g (9.12 mmol) in chloroform After dissolving 22.2g, 1.48g (9.60 mmol) of bis(2-hydroxyethyl) disulfide, 235mg (1.92 mmol) of DMAP, and 4.60g (24.0 mmol) of EDC hydrochloride were added and the mixture was reacted at room temperature for 2 hours. The resulting solution was washed with 5% by weight sodium dihydrogen phosphate aqueous solution, 7% by weight sodium bicarbonate aqueous solution, and 20% by weight sodium chloride aqueous solution, then dehydrated with sodium sulfate, and the resulting filtrate was concentrated using an evaporator. After concentration, the solution was purified by silica gel column using ethanol / chloroform to obtain 2.12g of intermediate 4.

[0152] <Synthesis of Intermediate 5> Intermediate 4 was dissolved in 22.2 g of chloroform, then 1.48 g (9.60 mmol) of bis(2-hydroxyethyl) disulfide, 235 mg (1.92 mmol) of DMAP, and 4.60 g (24.0 mmol) of EDC hydrochloride were added and the mixture was reacted at room temperature for 2 hours. The resulting solution was washed with 5 wt% aqueous sodium dihydrogen phosphate, 7 wt% aqueous sodium bicarbonate, and 20 wt% aqueous sodium chloride, and then dehydrated with sodium sulfate. The filtrate was concentrated using an evaporator. After concentration, the solution was purified by silica gel column using ethanol / chloroform to obtain 392 mg of intermediate 5.

[0153] <Synthesis of Compound 1> 285 mg (0.203 mmol) of intermediate 5 was mixed with 4.56 g of isopropyl alcohol, followed by 158 mg (0.831 mmol) of p-toluenesulfonic acid monohydrate, and the mixture was reacted at room temperature for 1 hour. After the reaction, 4.56 g of chloroform was added to the solution, and it was washed with a 7% by weight sodium bicarbonate aqueous solution and a 20% by weight sodium chloride aqueous solution. The solution was dehydrated with sodium sulfate, and the filtrate was concentrated using an evaporator. After concentration, the compound 1 was purified by silica gel column using ethanol / chloroform to obtain 170 mg of compound 1.

[0154] < Compound 1 1 H-NMR (600MHz, DMSO-d6) δ: 0.79-0.87(t, 6H), 1.17-1.38(m, 42H), 1.44-1.55(m, 2H), 1.57-1.66 (t, 4H), 1.92-2.04(t, 8H), 2.34-2.44(m, 6H), 2.51-2.59(m, 5H), 2.63 -2.68(m, 2H), 2.91-3.02(m, 8H), 3.34-3.40(m, 2H), 3.69(s, 4H), 4.18 -4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0155] [Example 2] Synthesis of Compound 2 Compound 2 was synthesized using the following synthetic route.

[0156]

Chem.

[0157] <Synthesis of Intermediate 6> After dissolving 1.15 g (7.55 mmol) of bis(2-hydroxyethyl) disulfide in 11.5 g of chloroform, 893 mg (7.08 mmol) of acrylic anhydride and 2.26 g (22.3 mmol) of triethylamine were added, and the mixture was reacted at room temperature for 1 hour. The reaction solution was washed with 5 wt% aqueous sodium dihydrogen phosphate solution, 7 wt% aqueous sodium hydrogen carbonate solution, and 20 wt% aqueous sodium chloride solution, dehydrated with sodium sulfate, and the filtrate after dehydration was concentrated using an evaporator. After concentration, silica gel column purification using ethanol / chloroform was carried out to obtain 670 mg of Intermediate 6.

[0158] <Synthesis of Intermediate 7> 153 mg (0.735 mmol) of Intermediate 6 was added to 30.0 mg (0.294 mmol) of N,N-dimethyl-1,3-propanediamine, and the mixture was reacted at room temperature for 10 hours to obtain 193 mg of a crude product of Intermediate 7.

[0159] <Synthesis of Compound 2> After dissolving 193 mg (0.372 mmol) of the crude product of Intermediate 7 in 1.93 g of chloroform, 387 mg (0.929 mmol) of 4-oleoyloxyphenylacetic acid, 9.1 mg (0.074 mmol) of DMAP, and 214 mg (1.12 mmol) of EDC hydrochloride were added, and the mixture was reacted at room temperature for 2 hours. The reaction solution was subjected to silica gel column purification using ethanol / chloroform to obtain 230 mg of Compound 2.

[0160] <Compound 2's 1 1H-NMR (600 MHz, DMSO-d6)> δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.40-1.48(m, 2H), 1.59-1.66 (t, 4H), 1.92-2.04(t, 8H), 2.05-2.15(m, 6H), 2.31-2.42(m, 6H), 2.51 -2.59(m, 4H), 2.61-2.68(m, 4H), 2.90-3.02(m, 8H), 3.69(s, 4H), 4.18 -4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0161] [Example 3] Synthesis of Compound 3 <Synthesis of Intermediate 8> Using 142 mg (0.675 mmol) of intermediate 6 and 40.0 mg (0.307 mmol) of N,N-diethyl-1,3-diaminopropane, 182 mg of intermediate 8 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0162] [ka]

[0163] <Synthesis of Compound 3> 150 mg of compound 3 was synthesized using 381 mg (0.915 mmol) of 4-oleoyloxyphenylacetic acid and 182 mg (0.333 mmol) of intermediate 8, in the same manner as the synthesis of compound 2 described in Example 2.

[0164] [ka]

[0165] < Compound 3 1 H-NMR (600MHz, DMSO-d6) δ: 0.81 - 0.88 (t, 6H), 0.89 - 0.95 (m, 6H), 1.17 - 1.38 (m, 42H), 1.40 - 1.48 (m, 2H), 1.59 - 1.66 (m, 4H), 1.92 - 2.04 (t, 8H), 2.28 - 2.30 (m, 2H), 2.31 - 2.42 (m, 8H), 2.51 - 2.59 (m, 4H), 2.61 - 2.68 (m, 4H), 2.90 - 3.02 (m, 8H), 3.69 (s, 4H), 4.18 - 4.32 (m, 8H), 5.29 - 5.37 (m, 4H), 7.00 - 7.06 (m, 4H), 7.27 - 7.34 (m, 4H)

[0166] [Example 4] Synthesis of Compound 4 <Synthesis of Intermediate 9> Using 156 mg (0.748 mmol) of Intermediate 6 and 30.0 mg (0.340 mmol) of N,N - dimethylethylenediamine, 186 mg of Intermediate 9 was synthesized in the same manner as the synthesis of Intermediate 7 described in Example 2.

[0167]

Chemical formula

[0168] <Synthesis of Compound 4> Using 381 mg (0.915 mmol) of 4 - oleoyloxyphenylacetic acid and 186 mg (0.369 mmol) of Intermediate 9, 140 mg of Compound 4 was synthesized in the same manner as the synthesis of Compound 2 described in Example 2.

[0169]

Chemical formula

[0170] <Compound 4's 1 1H - NMR (600 MHz, DMSO - d6)> δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.59-1.66(t, 4H), 1.92-2.04 (t, 8H), 2.05-2.15(m, 4H), 2.18-2.24(m, 2H), 2.38-2.42(m, 6H), 2.51 -2.59(m, 4H), 2.61-2.68(m, 4H), 2.90-3.02(m, 8H), 3.69(s, 4H), 4.18 -4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0171] [Example 5] Synthesis of Compound 5 <Synthesis of Intermediate 10> Using 161 mg (0.773 mmol) of intermediate 6 and 45.0 mg (0.351 mmol) of 1-(3-aminopropyl)pyrrolidine, 206 mg of intermediate 10 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0172] [ka]

[0173] <Synthesis of Compound 5> 157 mg of compound 5 was synthesized using 354 mg (0.850 mmol) of 4-oleoyloxyphenylacetic acid and 206 mg (0.378 mmol) of intermediate 10, in the same manner as the synthesis of compound 2 described in Example 2.

[0174] [ka]

[0175] < Compound 5 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.42-1.52(m, 2H), 1.59-1.66(m, 8H), 1.92-2.04(m, 8H), 2.26-2.44(m, 12H), 2.51-2.59(m, 4H), 2.61-2.68(m, 4H), 2.90-3.02(m, 8H), 3.69(s, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0176] [Example 6] Synthesis of Compound 6 <Synthesis of Intermediate 11> Using 150 mg (0.720 mmol) of intermediate 6 and 52.5 mg (0.328 mmol) of tryptamine, 203 mg of intermediate 11 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0177] [ka]

[0178] <Synthesis of Compound 6> 231 mg of compound 6 was synthesized using 330 mg (0.792 mmol) of 4-oleoyloxyphenylacetic acid and 203 mg (0.351 mmol) of intermediate 11, in the same manner as the synthesis of compound 2 described in Example 2.

[0179] [ka]

[0180] < Compound 6 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.57-1.66(m, 4H), 1.92-2.04(m, 8 H), 2.40-2.48(m, 4H), 2.51-2.59(m, 2H), 2.61-2.83(m, 8H), 2.91-3.01(m, 8H), 3.69(s, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 6.90-6.98(m, 1H), 7.00-7.06(m, 5H), 7.08-7.13(m, 1H), 7.27-7.34(m, 5H), 7.48-7.52(m, 1H)

[0181] [Example 7] Synthesis of Compound 7 <Synthesis of Intermediate 12> Using 155 mg (0.744 mmol) of intermediate 6 and 44.0 mg (0.338 mmol) of 4-(2-aminoethyl)morpholine, 199 mg of intermediate 12 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0182] [ka]

[0183] <Synthesis of Compound 7> 140 mg of compound 7 was synthesized using 414 mg (0.994 mmol) of 4-oleoyloxyphenylacetic acid and 199 mg (0.364 mmol) of intermediate 12, in the same manner as the synthesis of compound 2 described in Example 2.

[0184] [ka]

[0185] < Compound 7 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.59-1.66(m, 4H), 1.92-2.04(m, 8H), 2.26-2.44(m, 8H), 2.54-2.59(m, 6H), 2.63-2.73(m, 4H), 2.90-3.02(m, 8H), 3.48-3.56(m, 4H), 3.69(s, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0186] [Example 8] Synthesis of Compound 8 <Synthesis of Intermediate 13> Using 156 mg (0.749 mmol) of intermediate 6 and 42.5 mg (0.340 mmol) of 1-(3-aminopropyl)imidazole, 199 mg of intermediate 13 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0187] [ka]

[0188] <Synthesis of Compound 8> 4-Oleoyloxyphenylacetic acid 502 mg (1.20 mol) and intermediate 13 Using 199 mg (0.367 mmol), 39 mg of compound 8 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0189] [ka]

[0190] < Compound 8 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 42H), 1.59-1.66(m, 4H), 1.83-1.95(m , 2H), 1.92-2.04(m, 8H), 2.33-2.46(m, 6H), 2.51-2.59(m, 4H), 2.63-2. 73(m, 4H), 2.90-3.02(m, 8H), 3.69(s, 4H), 4.18-4.32(m, 8H), 5.29-5.3 7(m, 4H), 6.95(s, 1H), 7.00-7.06(m, 5H), 7.27-7.34(m, 4H), 7.51(s, 1H)

[0191] [Example 9] Synthesis of Compound 9 <Synthesis of Intermediate 14> Using 156 mg (0.748 mmol) of intermediate 6 and 39.5 mg (0.340 mmol) of N,N-diethylethylenediamine, 196 mg of intermediate 14 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0192] [ka]

[0193] <Synthesis of Compound 9> 150 mg of compound 9 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 196 mg (0.347 mmol) of intermediate 14, in the same manner as the synthesis of compound 2 described in Example 2.

[0194] [ka]

[0195] < Compound 9 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 0.89-0.95(m, 6H), 1.17-1.38(m, 40H), 1.59-1.66(m, 4H), 2.14-2.18(t, 8H), 2.51-2.59(m, 16H), 2.90 -3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0196] [Example 10] Synthesis of Compound 10 <Synthesis of Intermediate 15> Using 156 mg (0.748 mmol) of intermediate 6 and 49.0 mg (0.340 mmol) of 3-(di-n-propylamino)propylamine, 205 mg of intermediate 15 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0197] [ka]

[0198] <Synthesis of Compound 10> 147 mg of compound 10 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 205 mg (0.366 mmol) of intermediate 15, in the same manner as the synthesis of compound 2 described in Example 2.

[0199] [ka]

[0200] < Compound 10 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 12H), 1.17-1.38(m, 40H), 1.43-1.45(m, 4H), 1.59-1.66(m, 4H), 2.14-2.18(t, 8H), 2.51-2.59(m, 16H), 2.9 0-3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0201] [Example 11] Synthesis of Compound 11 <Synthesis of Intermediate 16> Using 156 mg (0.748 mmol) of intermediate 6 and 49.0 mg (0.340 mmol) of 3-(di-n-propylamino)propylamine, 205 mg of intermediate 16 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0202] [ka]

[0203] <Synthesis of Compound 11> 149 mg of compound 11 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 205 mg (0.366 mmol) of intermediate 16, in the same manner as the synthesis of compound 2 described in Example 2.

[0204] [ka]

[0205] < Compound 11 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 0.98-1.01(m, 12H), 1.17-1.38(m, 40H), 1.59-1.66(m, 4H), 2.14-2.18(t, 8H), 2.51-2.59(m, 14H), 2.9 0-3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0206] [Example 12] Synthesis of Compound 12 <Synthesis of Intermediate 17> Using 156 mg (0.748 mmol) of intermediate 6 and 34.7 mg (0.340 mmol) of (2-aminoethyl)(ethyl)methylamine, 191 mg of intermediate 17 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0207] [ka]

[0208] <Synthesis of Compound 12> 142 mg of compound 12 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 191 mg (0.368 mmol) of intermediate 17, in the same manner as the synthesis of compound 2 described in Example 2.

[0209] [ka]

[0210] < Compound 12 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 0.98-1.01(m, 3H), 1.17-1.38(m, 40H), 1.59-1.66(m, 4H), 2.14-2.18(t, 11H), 2.51-2.59(m, 14H), 2.9 0-3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0211] [Example 13] Synthesis of Compound 13 <Synthesis of Intermediate 18> Using 156 mg (0.748 mmol) of intermediate 6 and 39.5 mg (0.340 mmol) of (2-aminoethyl)(methyl)propylamine, 196 mg of intermediate 18 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0212] [ka]

[0213] <Synthesis of Compound 13> 146 mg of compound 13 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 196 mg (0.368 mmol) of intermediate 18, in the same manner as the synthesis of compound 2 described in Example 2.

[0214] [ka]

[0215] < Compound 13 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 9H), 1.17-1.38(m, 42H), 1.59-1.66(m, 4H), 2.14-2.18(t, 11H), 2.51-2.59(m, 14H), 2.90-3.02(m , 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0216] [Example 14] Synthesis of Compound 14 <Synthesis of Intermediate 19> Using 156 mg (0.748 mmol) of intermediate 6 and 43.6 mg (0.340 mmol) of 2-(2-aminoethyl)-1-methylpyrrolidine, 200 mg of intermediate 19 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0217] [ka]

[0218] <Synthesis of Compound 14> 150 mg of compound 14 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 200 mg (0.367 mmol) of intermediate 19, in the same manner as the synthesis of compound 2 described in Example 2.

[0219] [ka]

[0220] < Compound 14 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.40-1.47(m, 4H), 1.59-1.66 (m, 6H), 2.14-2.18(t, 8H), 2.25-2.30(m, 5H), 2.40-2.46(m, 3H), 2.51 -2.59(m, 8H), 2.90-3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18 -4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0221] [Example 15] Synthesis of Compound 15 <Synthesis of Intermediate 20> Using 156 mg (0.748 mmol) of intermediate 6 and 48.7 mg (0.340 mmol) of 2-(4-methylpiperazin-1-yl)ethane-1-amine, 205 mg of intermediate 20 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0222] [ka]

[0223] <Synthesis of Compound 15> Compound 15 was synthesized in 152 mg in the same manner as the synthesis of Compound 2 described in Example 2, using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 205 mg (0.366 mmol) of intermediate 20.

[0224] [ka]

[0225] < Compound 15 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.59-1.66(m, 4H), 2.14-2.18(m, 11H), 2.25-2.30(m, 8H), 2.40-2.46(m, 4H), 2.51-2.59(m, 8H), 2.90-3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0226] [Example 16] Synthesis of Compound 16 <Synthesis of Intermediate 21> Using 156 mg (0.748 mmol) of intermediate 6 and 53.5 mg (0.340 mmol) of 4-methyl-1-piperazinepropan-1-amine, 210 mg of intermediate 21 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0227] [ka]

[0228] <Synthesis of Compound 16> 155 mg of compound 16 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 210 mg (0.366 mmol) of intermediate 21, in the same manner as the synthesis of compound 2 described in Example 2.

[0229] [ka]

[0230] <Compound 16 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.68(m, 46H), 2.14-2.18(m, 11H), 2.25-2.30(m, 8H), 2.40-2.46(m, 4H), 2.51-2.59(m, 8H), 2.90 -3.02(m, 8H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0231] [Example 17] Synthesis of Compound 17 <Synthesis of Intermediate 22> Using 156 mg (0.748 mmol) of intermediate 6 and 37.5 mg (0.340 mmol) of 2-(1H-pyrrole-1-yl)ethylamine, 194 mg of intermediate 22 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0232] [ka]

[0233] <Synthesis of Compound 17> Compound 17 was synthesized in 149 mg in the same manner as the synthesis of Compound 2 described in Example 2, using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 194 mg (0.363 mmol) of intermediate 22.

[0234] [ka]

[0235] < Compound 17 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.64-1.68(m, 4H), 2.14-2.18 (m, 8H), 2.50-2.54(m, 8H), 2.59-2.62(m, 2H), 2.90-3.02(m, 8H), 3.69 (s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 4.50-4.55(m, 2H), 5.29 -5.37(m, 4H), 6.12-6.16(m, 2H), 7.00-7.06(m, 4H), 7.27-7.34(m, 6H)

[0236] [Example 18] Synthesis of Compound 18 <Synthesis of Intermediate 23> Using 156 mg (0.748 mmol) of intermediate 6 and 48.4 mg (0.340 mmol) of 2-(1-azepanyl)ethaneamine, 204 mg of intermediate 23 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0237] [ka]

[0238] <Synthesis of Compound 18> 148 mg of compound 18 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 204 mg (0.366 mmol) of intermediate 23, in the same manner as the synthesis of compound 2 described in Example 2.

[0239] [ka]

[0240] <Compound 18 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.59-1.66(m, 8H), 1.75-1.80(m, 4H), 2.14-2.18(m, 8H), 2.25-2.50(m, 12H), 2.90-3.02(m, 8H), 3.07-3.11(m, 4H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0241] [Example 19] Synthesis of Compound 19 <Synthesis of Intermediate 24> Using 156 mg (0.748 mmol) of intermediate 6 and 41.5 mg (0.340 mmol) of 4-(2-aminoethyl)pyridine, 198 mg of intermediate 24 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0242] [ka]

[0243] <Synthesis of Compound 19> Compound 19 was synthesized in 147 mg quantities using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 198 mg (0.368 mmol) of intermediate 24, in the same manner as the synthesis of compound 2 described in Example 2.

[0244] [ka]

[0245] < Compound 19 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.59-1.66(m, 4H), 2.14-2.18(m, 8 H), 2.48-2.54(m, 8H), 2.68-2.73(m, 2H), 2.90-3.02(m, 8H), 3.44-3.46(m, 2H), 3.69(s, 4H), 3.70-3.74(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.20-7.24(m, 2H), 7.27-7.34(m, 4H), 8.53-8.56(m, 2H)

[0246] [Example 20] Synthesis of Compound 20 <Synthesis of Intermediate 25> Using 156 mg (0.748 mmol) of intermediate 6 and 48.4 mg (0.340 mmol) of 1-(3-aminoprop-1-yl)piperidine, 204 mg of intermediate 25 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0247] [ka]

[0248] <Synthesis of Compound 20> 151 mg of compound 20 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 204 mg (0.365 mmol) of intermediate 25, in the same manner as the synthesis of compound 2 described in Example 2.

[0249] [ka]

[0250] < Compound 20 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 42H), 1.47-1.51(m, 4H), 1.55-1.57(m, 2H), 1.59-1.66(m, 4H), 2.14-2.18(m, 8H), 2.25-2.50(m, 1 6H), 2.90-3.02(m, 8H), 3.69(s, 4H), 3.74-3.78(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0251] [Example 21] Synthesis of Compound 21 <Synthesis of Intermediate 26> Using 156 mg (0.748 mmol) of intermediate 6 and 49.0 mg (0.340 mmol) of 1-(2-aminoethyl)pyrrolidine, 205 mg of intermediate 26 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0252] [ka]

[0253] <Synthesis of Compound 21> 153 mg of compound 21 was synthesized using 343 mg (0.823 mmol) of 4-oleoyloxyphenylacetic acid and 205 mg (0.386 mmol) of intermediate 26, in the same manner as the synthesis of compound 2 described in Example 2.

[0254] [ka]

[0255] < Compound 21 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.64-1.70(m, 8H), 2.14-2.18(m, 8H), 2.35-2.54(m, 16H), 2.90-3.02(m, 8H), 3.69(s, 4H), 3.74-3.78(m, 4H), 4.18-4.32(m, 8H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0256] [Example 22] Synthesis of Compound 22 Compound 22 was synthesized using the following synthetic route.

[0257] [ka]

[0258] <Synthesis of Intermediate 27> Intermediate 26 was dissolved in 700 mg (1.32 mmol) of dichloromethane, then 404 mg (4.00 mmol) of triethylamine and 387 mg (3.96 mmol) of DSC were added and the mixture was reacted at room temperature for 1 hour. The resulting solution was filtered to obtain a filtrate containing intermediate 27.

[0259] <Synthesis of Intermediate 28> To the filtrate containing intermediate 27, 543 mg (3.96 mmol) of 4-(aminomethyl)phenol and 404 mg (4.00 mmol) of triethylamine were added and the mixture was reacted at room temperature for 4 hours. The resulting solution was washed with 5% by weight aqueous solution of sodium dihydrogen phosphate and 20% by weight aqueous solution of sodium chloride, then dehydrated with sodium sulfate, and the dehydrated filtrate was concentrated using an evaporator. After concentration, the solution was purified by silica gel column using ethanol / chloroform to obtain 776 mg of intermediate 28.

[0260] <Synthesis of Compound 22> Compound 22 was synthesized in 338 mg quantities using 320 mg (0.386 mmol) of intermediate 28 and 240 mg (0.849 mmol) of oleic acid, in the same manner as the synthesis of 4-oleoyloxyphenylacetic acid described in Example 1.

[0261] < Compound 22 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.89(m, 6H), 1.17-1.38(m, 40H), 1.64-1.70(m, 8H), 2.14-2.18(m, 8H), 2.35-2.54(m, 16H), 2.90-3.02(m, 8H), 3.74 -3.78(m, 4H), 3.94-3.98(m, 8H), 4.23(s, 4H), 5.29-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H), 8.17-8.19(m, 2H)

[0262] [Example 23] Synthesis of Compound 23 Compound 23 was synthesized using the following synthetic route.

[0263] [ka]

[0264] <Synthesis of Intermediate 29> Using 5.08 g (18.8 mmol) of n-octanoic anhydride and 2.60 g (17.1 mmol) of 4-hydroxyphenylacetic acid, 3.26 g of intermediate 29 was synthesized in the same manner as the synthesis of 4-oleyloxyphenylacetic acid described in Example 1.

[0265] <Synthesis of Compound 23> Using 485 mg (1.74 mol) of intermediate 29 and 373 mg (0.703 mmol) of intermediate 26, 316 mg of compound 23 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0266] < Compound 23 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.21-1.30(m, 16H), 1.59-1.68(m, 8H), 2.30-2.60(m, 16H), 2.66-2.74(m, 4H), 2.90-3.02(m, 8H), 3.69(s, 4H), 4.18-4.32(m, 8H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0267] [Example 24] Synthesis of Compound 24 Compound 24 was synthesized using the following synthetic route.

[0268] [ka]

[0269] <Synthesis of Intermediate 30> Using 1.96 mg (16.58 mmol) of 1,6-hexanediol and 1.99 mg (15.8 mmol) of acrylic anhydride, 913 mg of intermediate 30 was synthesized in the same manner as the synthesis of intermediate 6 described in Example 2.

[0270] <Synthesis of Intermediate 31> Using 77.7 mg (0.680 mmol) of 1-(2-aminoethyl)pyrrolidine and 258 mg (1.50 mmol) of intermediate 30, 336 mg of intermediate 31 was synthesized in the same manner as the synthesis of intermediate 7 described in Example 2.

[0271] <Synthesis of Compound 24> Using 336 mg (0.732 mmol) of intermediate 31 and 521 mg (1.87 mol) of intermediate 29, 314 mg of compound 24 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0272] < Compound 24 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.92(t, 6H), 1.21-1.40(m, 24H), 1.48-1.72(m, 16H), 2.31-2.59(m, 16H), 2.6 4-2.73(m, 4H), 3.66(s, 4H), 3.92-4.08(m, 8H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0273] [Example 25] Synthesis of Compound 25 Compound 25 was synthesized using the following synthetic route.

[0274] [ka]

[0275] <Synthesis of Intermediate 32> Using 2.19 g (4.99 mmol) of myristic anhydride and 1.73 g (11.4 mmol) of 4-hydroxyphenylacetic acid, 2.03 g of intermediate 32 was synthesized in the same manner as the synthesis of 4-oleyloxyphenylacetic acid described in Example 1.

[0276] <Synthesis of Compound 25> Compound 25 was synthesized in 260 mg quantities using 215 mg (0.468 mmol) of intermediate 31 and 417 mg (1.15 mmol) of intermediate 32, in the same manner as the synthesis of compound 2 described in Example 2.

[0277] < Compound 25 1 H-NMR (400MHz, DMSO-d6) δ: 0.81-0.92(t, 6H), 1.21-1.44(m, 52H), 1.50-1.71(m, 16H), 1.79-1.93(m, 4H), 2.41-2.51(m, 4H), 2.55-2.77(m, 8H), 3.71(s, 4H), 3.98-4.11(m, 8H), 7.06-7.11(m, 4H), 7.27-7.34(m, 4H)

[0278] [Example 26] Synthesis of Compound 26 Compound 26 was synthesized using the following synthetic route.

[0279] [ka]

[0280] <Synthesis of Intermediate 33> Using 2.07 g (3.75 mmol) of anhydrous stearate and 519 mg (3.41 mmol) of 4-hydroxyphenylacetic acid, 1.50 g of intermediate 33 was synthesized in the same manner as the synthesis of 4-oleyloxyphenylacetic acid described in Example 1.

[0281] <Synthesis of Compound 26> Using 195 mg (0.425 mmol) of intermediate 31 and 402 mg (0.96 mmol) of intermediate 33, 379 mg of compound 26 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0282] < Compound 26 1 H-NMR (400 MHz, chloroform-d) δ: 0.83-0.91(t, 6H), 1.21-1.44(m, 64H), 1.50-1.68(m, 20H), 1.69-1.80(m, 4H), 2.41-2.58(m, 8H), 2.71-2.83(m, 4H), 3.60(s, 4H), 3.98-4.11(m, 8H), 7.00-7.04(m, 4H), 7.21-7.32(m, 4H)

[0283] [Example 27] Synthesis of Compound 27 399 mg of compound 27 was synthesized using 401 mg (0.963 mmol) of 4-oleoyloxyphenylacetic acid and 196 mg (0.427 mmol) of intermediate 31 in the same manner as the synthesis of compound 2 described in Example 2.

[0284] [ka]

[0285] < Compound 27 1H-NMR (600MHz, DMSO-d6) δ: 0.81-0.88(t, 6H), 1.17-1.38(m, 52H), 1.49-1.65(m, 12H), 1.92-2.04(m, 8H), 2.36-2.43(m, 8H), 2.51-2.59(m, 8H), 2.65-2.72(m, 4H), 3.66(s, 4H), 3.98-4.05(m, 8H), 5.28-5.37(m, 4H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0286] [Example 28] Synthesis of Compound 28 Compound 28 was synthesized using the following synthetic route.

[0287] [ka]

[0288] <Synthesis of Compound 28> Using 167 mg (0.364 mmol) of intermediate 31 and 433 mg (0.816 mmol) of intermediate 34 synthesized according to the method described in International Publication No. 2024 / 203577, 343 mg of compound 28 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0289] < Compound 28 1 H-NMR (600MHz, DMSO-d6) δ: 0.81-0.86(t, 12H), 1.17-1.38(m, 52H), 1.43-1.65(m, 20H), 1.92-2.0 4(m, 4H), 2.18-2.27(m, 8H), 2.36-2.43(m, 8H), 2.51-2.59(m, 8H), 2.6 5-2.72(m, 4H), 3.66(s, 4H), 3.93-4.05(m, 8H), 4.75-4.82(m, 2H), 5.2 7-5.34(m, 2H), 5.41-5.47(m, 2H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0290] [Example 29] Synthesis of Compound 29 Compound 29 was synthesized using the following synthetic route.

[0291] [ka]

[0292] <Synthesis of Intermediate 35> 3.80 g (22.9 mmol) of 4-(methyldisulfanyl)butanoic acid was dissolved in 38.0 g of chloroform, and then 560 mg (4.58 mmol) of DMAP and 6.58 g (34.4 mmol) of EDC hydrochloride were added and the mixture was reacted at room temperature for 2 hours. The resulting solution was concentrated using an evaporator. After concentration, the solution was purified by silica gel column using ethanol / chloroform to obtain 2.88 g of intermediate 35.

[0293] [ka]

[0294] <Synthesis of Intermediate 36> Using 2.88 g (9.16 mmol) of intermediate 35 and 697 mg (4.58 mmol) of 4-hydroxyphenylacetic acid, 1.10 g of intermediate 36 was synthesized in the same manner as the synthesis of 4-oleyloxyphenylacetic acid described in Example 1.

[0295] <Synthesis of Compound 29> Compound 29 was synthesized in 238 mg quantities using 193 mg (0.364 mmol) of intermediate 26 and 241 mg (0.801 mmol) of intermediate 36, in the same manner as the synthesis of compound 2 described in Example 2.

[0296] < Compound 29 1 H-NMR (600MHz, DMSO-d6) δ: 1.64-1.70(m, 4H), 1.96-2.00(m, 4H), 2.30(s, 6H), 2.35-2.39(m, 4H), 2.47-2.54(m, 16H), 2.90-3. 02(m, 8H), 3.69(s, 4H), 3.74-3.78(m, 4H), 4.18-4.32(m, 8H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0297] [Example 30] Synthesis of Compound 30 Using 167 mg (0.364 mmol) of intermediate 31 and 241 mg (0.801 mmol) of intermediate 36, 216 mg of compound 30 was synthesized in the same manner as the synthesis of compound 2 described in Example 2.

[0298] [ka]

[0299] < Compound 30 1 H-NMR (600MHz, DMSO-d6) δ: 1.41-1.45(m, 8H), 1.58-1.62(m, 8H), 1.64-1.70(m, 4H), 1.96-2.00(m, 4H), 2.30(s, 6H), 2.35-2.39(m, 4H), 2.47-2.54(m, 16H), 3.69(s, 4H), 3.74-3.78(m, 4H), 4.18-4.32(m, 8H), 7.00-7.06(m, 4H), 7.27-7.34(m, 4H)

[0300] [Test Example 1] Measurement of Liposomal pKa Lipid nanoparticles (LNPs) that do not contain nucleic acids were used to evaluate the Liposomal pKa.

[0301] 1. Preparation of LNPs by microfluidic method (1) Preparation of lipid ethanol solution In an Eppendorf tube, ethanol solutions of cationic lipids (5 mM), DOPC (3 mM), Chol (10 mM), and DMG-PEG2k (0.5 mM) were mixed in the desired proportions (catenic lipids:DOPC:Chol:DMG-PEG2k = 52.5:7.5:40:1.5 (molar ratio)) to achieve a total lipid volume of 800 nmol. Ethanol was then added to prepare a lipid ethanol solution (total volume: 1000 μL).

[0302] (2) Preparation of LNPs using microfluidics 1080 μL of acidic malate buffer (20 mM, pH 5.0) containing 30 mM NaCl and 360 μL of lipid ethanol solution were each measured into syringes. Using a high-speed nanopharmaceutical synthesis system, NanoAssemblr (Precision NanoSystems), LNPs were prepared under the following conditions: acidic buffer addition rate: 3 mL / min, lipid ethanol solution addition rate: 1 mL / min, and syringe holder temperature: 25°C. The LNPs were collected in 15 mL tubes. After adding 1000 μL of MES buffer (pH 6.5) to the 15 mL tubes, the resulting mixture was transferred to an Amicon Ultra 4 and concentrated to approximately 100 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 6 min). The obtained concentrate was diluted to 4 mL with PBS and concentrated again under centrifugal conditions (25°C, 1000 g, 6 min) for a total of two times. The obtained concentrate was diluted with PBS to a lipid concentration of 0.5 mM to obtain a dispersion containing LNP.

[0303] 2. Measurement of Liposomal pKa 20 mM citrate buffer, sodium phosphate buffer, and Tris-HCl buffer containing 150 mM NaCl were prepared at various pH levels in the range of pH 3.0 to 10.0. The TNS (Sigma) solution was diluted with ultrapure water to a concentration of 0.6 mM. 2 μL of the TNS solution, 12 μL of the dispersion containing LNP prepared in [Test Example 1] 1., and 186 μL of the buffers adjusted to various pH levels 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 relative fluorescence intensity was calculated as a percentage, with the maximum fluorescence intensity at each LNP set to 100% and the minimum to 0%. The pH at which the relative fluorescence intensity was 50% was defined as the Liposomal pKa. The Liposomal pKas of the cationic lipids and LNPs used are shown in Table 3.

[0304] [Table 3]

[0305] 3.Results As shown in Table 3, the liposomal pKa of LNPs containing compound 1, compound 3, compound 6, compound 7, compound 27, or the comparative compound was within a favorable range for endosomal escape (4.5–9.5).

[0306] [Test Example 2] Preparation and physical property evaluation of mRNA-encapsulated particles 1. Preparation of LNPs by microfluidic method (1) Preparation of lipid ethanol solution In an Eppendorf tube, ethanol solutions of cationic lipids (5 mM), DOPC (5 mM), Chol (10 mM), and DMG-PEG2k (0.5 mM) were mixed in the desired ratio (catenic lipids:DOPC:Chol:DMG-PEG2k = 50:10:40:1 (molar ratio)) to achieve a total lipid volume of 800 nmol. Then, ethanol was added to prepare a lipid ethanol solution (total volume: 3067 μL).

[0307] (2) Preparation of acidic buffer solutions for nucleic acids A 0.6 mg / mL mRNA solution was weighed into a 5 mL tube to a value of 4.0 μg. Acidic malate buffer (20 mM, pH 3.0) was added to this to prepare an acidic buffer solution of nucleic acids (total volume: 7480 μL).

[0308] (3) Preparation of LNPs using microfluidics Acidic buffer solution of nucleic acid and ethanol solution of lipid were weighed into syringes. Using a high-speed nanopharmaceutical synthesis system, NanoAssemblr (Precision NanoSystems), LNPs were prepared under the following conditions: addition rate of acidic buffer solution of nucleic acid: 3 mL / min, addition rate of ethanol solution of lipid: 1 mL / min, and syringe holder temperature: 25°C. The LNPs were collected in a 15 mL tube. 3000 μL of MES buffer (pH 6.5) was added to the 15 mL tube, and the resulting mixture was transferred to an Amicon Ultra 4 and concentrated to approximately 500 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 6 min). The obtained concentrate was diluted to 4 mL with PBS and concentrated again under centrifugal conditions (25°C, 1000 g, 6 min) for a total of two times. The obtained concentrate was diluted with PBS to a lipid concentration of 2 mM to obtain a dispersion containing LNPs.

[0309] 2. Measurement of particle size, PdI, and zeta potential of mRNA-encapsulated LNPs The particle size, PdI (Polydispersity Index), and zeta potential of mRNA-encapsulated LNPs prepared by the method described in 1. above were measured using dynamic light scattering (Zetasizer Nano; Malvern). The cationic lipids used and the results are shown in Table 4.

[0310] [Table 4]

[0311] 3.Results As shown in Table 4, the particle size of mRNA-encapsulated LNPs containing compound 1, compound 3, compound 6, compound 7, or the comparative compound was within a more preferable range (30 nm to 200 nm), and the potential at physiological pH (zeta potential) was also within a preferable range (-15 mV to +15 mV).

[0312] [Test Example 3] Preparation and physical property evaluation of mRNA-encapsulated particles 1. Preparation of LNPs by microfluidic method (1) Preparation of lipid ethanol solution In an Eppendorf tube, ethanol solutions of cationic lipids (5 mM), DOPC (5 mM), Chol (10 mM), and DMG-PEG2k (0.5 mM) were mixed in the desired ratio (catenic lipids:DOPC:Chol:DMG-PEG2k = 40:10:48:2 (molar ratio)) to achieve a total lipid volume of 800 nmol. Then, ethanol was added to prepare a lipid ethanol solution (total volume: 3200 μL).

[0313] (2) Preparation of acidic buffer solutions for nucleic acids A 0.6 mg / mL mRNA solution was weighed into a 5 mL tube to a value of 4.0 μg. Acidic citrate buffer (20 mM, pH 5.0) was added to this to prepare an acidic buffer solution of nucleic acid (total volume: 15.6 mL).

[0314] (3) Preparation of LNPs using microfluidics Acidic buffer solution of nucleic acid and ethanol solution of lipid were weighed into syringes. Using a high-speed nanopharmaceutical synthesis system, NanoAssemblr (Precision NanoSystems), LNPs were prepared under the following conditions: addition rate of acidic buffer solution of nucleic acid: 3 mL / min, addition rate of ethanol solution of lipid: 1 mL / min, and syringe holder temperature: 25°C. The LNPs were collected in 15 mL tubes. 3000 μL of Tris-buffered saline (pH 7.6) was added to the 15 mL tubes, and the resulting mixture was transferred to an Amicon Ultra 4 and concentrated to approximately 500 μL by ultrafiltration under centrifugal conditions (25°C, 1000 g, 6 min). The obtained concentrate was diluted to 4 mL with Tris-buffered saline and concentrated again under centrifugal conditions (25°C, 1000 g, 6 min), a total of two times. The obtained concentrate was diluted with Tris-buffered saline to a lipid concentration of 2 mM to obtain a dispersion containing LNP.

[0315] 2. Measurement of particle size, PdI, and zeta potential of mRNA-encapsulated LNPs The particle size, PdI (Polydispersity Index), and zeta potential of mRNA-encapsulated LNPs prepared by the method described in 1. above were measured using dynamic light scattering (Zetasizer Nano; Malvern). The cationic lipids used and the results are shown in Table 5.

[0316] [Table 5]

[0317] 3.Results As shown in Table 5, the particle size of mRNA-encapsulated LNPs prepared using compound 27 as the cationic lipid was within a preferred range (30 nm to 300 nm), and the potential at physiological pH (zeta potential) was also within a preferred range (-15 mV to +15 mV).

[0318] [Test Example 4] Evaluation of gene expression in HeLa cells in vitro 1. Preparation of mRNA-encapsulated LNPs LNPs containing luciferase-expressing mRNA were prepared using compound 3, compound 7, or a comparative compound, according to the method described in [Test Example 2]1. Additionally, LNPs containing luciferase-expressing mRNA were prepared using compound 27, according to the method described in [Test Example 3]1.

[0319] 2. Time-course evaluation of gene expression in HeLa cells 24 hours before transfection, human cervical cancer cells, HeLa cells, were transfected into 5.0 × 10⁶ cells. 4 Cells were seeded in a 3.5 cm dish at a concentration of 2 mL / dish. After 24 hours, the culture medium was replaced with D-MEM containing 0.1 mM D-luciferin. The prepared mRNA-encapsulated LNPs were diluted with PBS or Tris-buffered saline to an mRNA concentration of approximately 1 μg / mL. The diluted mRNA-encapsulated LNP solution (approximately 200 μL, mRNA: 0.2 μg) was added to a 3.5 cm dish and placed in a KronosDio incubator-type luminometer. The luciferase luminescence intensity was measured for 2 minutes every hour. The cumulative luciferase luminescence intensity over 24 hours was calculated from the obtained time-dependent expression changes. Table 6 shows the cationic lipid used, the cumulative luciferase luminescence intensity over 24 hours, and the relative luminescence intensity (= cumulative luciferase luminescence intensity over 24 hours when using the cationic lipid in the example / cumulative luciferase luminescence intensity over 24 hours when using the comparative compound). Note that "E+0a" (a: integer) listed in Table 6 is equivalent to "10 a This represents "5.19E+08". For example, "5.19E+08" is equivalent to "5.19×10 8 This represents ".

[0320] [Table 6]

[0321] 3.Results A higher cumulative luciferase emission intensity over 24 hours, i.e., a higher total luciferase activity, indicates higher gene expression. As shown in Table 6, LNPs containing compound 3, compound 7, or compound 27 showed superior gene expression activity compared to LNPs containing the comparison compounds. Therefore, it has become clear that the LNPs containing the cationic lipids of the present invention are beneficial as LNPs with good nucleic acid delivery efficiency. [Industrial applicability]

[0322] The cationic lipids of the present invention are useful in nucleic acid drugs, gene therapy, biochemical experiments, and the like.

Claims

1. Formula (1): 【Chemistry 1】 (In the formula, R １ This represents an alkyl group having 1 to 4 carbon atoms and having a nitrogen-containing heterocyclic group, a tertiary amino group, or a hydroxyl group as a substituent. R ２ａ and R ２ｂ Each of these independently represents an alkylene group having 1 to 16 carbon atoms and containing at least one degradable bond. R ３ａ and R ３ｂ Each of these independently represents an alkoxy group or halogen atom having 1 to 4 carbon atoms. ma and mb each independently represent integers from 0 to 4. R ４ａ and R ４ｂ Each of them is independent of R ５ -CO-O-* (where * represents the bond position) and R ５ is a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms which may contain at least one dissociable bond, or R ６ -CO-(CH ２ ) ｐ -* (In the above formula, * represents the bonding position, and R ６ represents the residue of a fat-soluble vitamin having a hydroxyl group or the residue of a sterol derivative having a hydroxyl group, and p represents an integer of 1 to 8.) represents.) Cationic lipids represented by [the symbol].

2. The nitrogen-containing heterocyclic group is a pyrrolidinyl group, morpholinyl group, piperazinyl group, piperidyl group, azepanyl group, pyrrolyl group, pyrazolyl group, pyridyl group, or indolyl group. The tertiary amino group is a di(alkyl)amino group, and The cationic lipid according to claim 1, wherein the number of carbon atoms of each of the two alkyl groups in the di(alkyl)amino group is independently 1 to 4.

3. R ２ａ and R ２ｂ However, each independently, equation (2): **-(CH ２ ) ａ -COO-O-(CH ２ ) ｂ -(S-S) ｅ -(CH) ２ ) ｃ -O-CO-(NH) ｆ -(CH) ２ ) ｄ -*** (2) (In the formula, ** represents the bonding position with the nitrogen atom in formula (1), *** represents the bond position of the benzene ring to the carbon atom in formula (1), a to d each independently represent an integer from 1 to 4, and e and f each independently represent either 0 or 1. The cationic lipid according to claim 1, which represents a divalent group indicated by .

4. The cationic lipid according to claim 1, wherein ma and mb are both 0.

5. R ５ The cationic lipid according to claim 1, wherein the cationic lipid is a monovalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, which may contain at least one degradable bond.

6. A lipid membrane structure comprising a cationic lipid as described in any one of claims 1 to 5 as a constituent lipid of the membrane.

7. Furthermore, the lipid membrane structure according to claim 6, further comprising nucleic acids.

8. A nucleic acid delivery agent comprising a cationic lipid according to any one of claims 1 to 5.

9. Furthermore, the nucleic acid introduction agent according to claim 8, which further comprises nucleic acid.

10. A pharmaceutical composition comprising a cationic lipid according to any one of claims 1 to 5.

11. The pharmaceutical composition according to claim 10, further comprising nucleic acid.

12. A method for introducing nucleic acids contained in a nucleic acid delivery agent into cells, comprising contacting the nucleic acid delivery agent described in claim 9 with cells in vitro.

13. A method for introducing nucleic acids contained in a nucleic acid delivery agent into target cells in a living organism, comprising administering the nucleic acid delivery agent described in claim 9 to a living organism.

14. A method for producing a cell pharmaceutical product containing cells expressing the gene in the nucleic acid, comprising contacting the nucleic acid delivery agent described in claim 9 with cells to introduce the nucleic acid contained in the nucleic acid delivery agent into the cells.

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