Cationic lipid and method for preparing same

Abstract

The present invention relates to a cationic lipid and a method for preparing same and, more specifically, to: a cationic lipid which, due to the specific structure thereof, forms a complex with an anionic drug, and thus is useful for drug delivery; and a method for preparing same.

Description

Cationic lipid and method for producing the same

[0001] The present invention relates to a cationic lipid and a method for preparing the same, and more particularly, to a cationic lipid useful for drug delivery by forming a complex with an anionic drug due to a specific structure, and a method for preparing the same.

[0002] Safe and efficient drug delivery technologies for the treatment of anionic drugs, including nucleic acids, have long been studied, leading to the development of various carriers and technologies. Carriers can be broadly divided into viral carriers, such as those using adenoviruses or retroviruses, and non-viral carriers, such as those using cationic lipids and cationic polymers. Viral carriers are known to be susceptible to risks such as non-specific immune responses and have complex production processes, hindering their commercialization. Therefore, recent research is focused on addressing these shortcomings by utilizing non-viral carriers. Non-viral carriers offer advantages over viral carriers, including fewer side effects in terms of in vivo safety and lower production costs.

[0003] Representative non-viral delivery vehicles used for the delivery of nucleic acid materials include complexes of cationic lipids and nucleic acids (lipoplexes) using cationic lipids, and complexes of polycationic polymers and nucleic acids (polyplexes). These cationic lipids or polycationic polymers have been extensively studied because they stabilize anionic drugs and increase intracellular delivery by forming complexes with them through electrostatic interactions (De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).

[0004] However, polycationic polymers have cytotoxicity due to their multivalent cationic charge, which poses challenges for practical use. Furthermore, nucleic acid-cationic lipid complexes exhibit low stability in the bloodstream, making them difficult to use in vivo. Furthermore, ionic liposomes, which contain cationic lipids, neutral lipids, and fusogenic lipids, have the disadvantages of complex synthesis methods for the cationic lipids used, cytotoxicity, and low intracellular nucleic acid delivery efficiency.

[0005] The purpose of the present invention is to provide a cationic lipid having a specific structure that can easily form a complex with an anionic drug and is useful for drug delivery, and a method for preparing the same.

[0006] A first aspect of the present invention provides a cationic lipid having a structure represented by the following chemical formula 1:

[0007] [Chemical Formula 1]

[0008]

[0009] Here,

[0010] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0011] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0012] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0013] R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0014] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl,

[0015] X - is a monovalent anion.

[0016] More specifically, in the chemical formula 1,

[0017] R1, R2 and R3 are each independently substituted or unsubstituted C 1-6 It is an alkyl group,

[0018] R4 is substituted or unsubstituted divalent C 2-6 It is a hydrocarbon group,

[0019] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent C 2-20 It is a hydrocarbon group,

[0020] R9 and R 10 are independently substituted or unsubstituted saturated or unsaturated monovalent C 2-20 It is a hydrocarbon group,

[0021] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, C 1-13 Alkylene, C 2-13 Alkenylene or C 2-13 Alkynylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 Selected from the group consisting of alkynyl,

[0022] X - is a monovalent anion of an inorganic acid or organic acid.

[0023] More specifically, in the chemical formula 1,

[0024] R1, R2 and R3 are each independently substituted or unsubstituted C 1-3 It is an alkyl group,

[0025] R4 is substituted or unsubstituted C 2-4 It is an alkylene group,

[0026] R5, R6, R7 and R8 are each independently substituted or unsubstituted C 2-13 Alkylene group or C 2-13 It is an alkenylene group,

[0027] R9 and R 10 Each independently substituted or unsubstituted C 6-20 Alkyl group, C 6-20Alkenyl group or C 6-20 It is an alkynyl group,

[0028] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)- and -P(O)(OR')O-, wherein R' is each independently a hydrogen atom and C 1-6 Selected from the group consisting of alkyl,

[0029] X - is F - , Cl - , Br - , I - , nitrate anion, benzoate anion, methanesulfonic acid anion, acetate anion (CH3COO - )(=AcO - ), or trihaloacetate anion (CF3COO - )am.

[0030] More specifically, the cationic lipid may have a structure selected from the following chemical formulas A and B:

[0031]

[0032] A second aspect of the present invention provides a method for preparing a cationic lipid, comprising the steps of: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) protecting a terminal -SH group of the compound of formula c; (3) reacting the compound of formula c with the protected terminal -SH group with a compound of formula d to obtain a compound of formula e; (4) reacting the compound of formula e with a compound of formula f to obtain a compound of formula g; and (5) reacting the compound of formula g with a compound of formula h to obtain a compound of formula 1-1.

[0033] [chemical formula a]

[0034] R9-OH

[0035] [chemical formula b]

[0036] HOC(O)-R7-SH

[0037] [chemical formula c]

[0038] R9-OC(O)-R7-SH

[0039] [chemical formula d]

[0040] HS-R5-OH

[0041] [chemical formula e]

[0042] R9-OC(O)-R7-SS-R5-OH

[0043] [chemical formula f]

[0044]

[0045] [chemical formula g]

[0046] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2

[0047] [chemical formula h]

[0048] H2N-R4-N + (R1)(R2)(R3) X -

[0049] [Chemical Formula 1-1]

[0050] [R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2]2-N-R4-N + (R1)(R2)(R3) X -

[0051] In the above,

[0052] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0053] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0054] R5 and R7 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0055] R9 is a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0056] X -is a monovalent anion,

[0057] Hal is selected from the group consisting of F, CI, Br and I.

[0058] A third aspect of the present invention provides a method for producing a cationic lipid, comprising: (1) reacting a compound of formula g obtained in the second aspect of the present invention with a compound of formula h to obtain a compound of formula i; and (2) reacting a compound of formula i with a compound of formula j to obtain a compound of formula 1-2.

[0059] [chemical formula g]

[0060] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2

[0061] [chemical formula h]

[0062] H2N-R4-N + (R1)(R2)(R3) X -

[0063] [chemical formula i]

[0064] R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2-NH-R4-N + (R1)(R2)(R3) X -

[0065] [chemical formula j]

[0066] R 10 -OC(O)-R8-SS-R6-OC(O)-CH=CH2

[0067] [Chemical Formula 1-2]

[0068] R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2-N(A)-R4-N + (R1)(R2)(R3) X -

[0069] In the above,

[0070] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0071] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0072] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0073] R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0074] A is -CH2-CH2-C(O)O-R6-SS-R8-C(O)OR 10 And,

[0075] X - is a monovalent anion.

[0076] A fourth aspect of the present invention provides a drug delivery composition comprising a cationic lipid according to the present invention.

[0077] The cationic lipid according to the present invention can easily form a complex with an anionic drug due to its specific structure, and by utilizing this complex, the drug can be efficiently delivered into a target biological tissue.

[0078] Figure 1 is a reaction schematic diagram for the lipid synthesis process performed in Example 1.

[0079] Figure 2 is a reaction schematic diagram for the lipid synthesis process performed in Example 2.

[0080] Hereinafter, the present invention will be described in more detail.

[0081] The cationic lipid provided according to the first aspect of the present invention has a structure represented by the following chemical formula 1:

[0082] [Chemical Formula 1]

[0083]

[0084] Here,

[0085] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0086] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0087] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0088] R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0089] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl,

[0090] X - is a monovalent anion.

[0091] In this specification, the expression that any group is “substituted or unsubstituted” means that the group is unsubstituted or substituted with —OH, a halogen atom, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 1-6 Halogenated alkyl group, C 1-6 Halogenated alkoxy group, C 3-20 Cycloalkyl group, C 3-20 Heterocycloalkyl group, C 6-20 Aryl group or C 3-20 It means that it is substituted with one or more substituents selected from among heteroaryl groups.

[0092] In this specification, the expression that any group (e.g., heteroaryl, heterocycloalkyl, etc.) is a “hetero” group means that the group has one or more (e.g., 1 to 3) heteroatoms selected from N, O, and S, unless otherwise specified.

[0093] In this specification, “monovalent hydrocarbon group” and “divalent hydrocarbon group” may be branched or unbranched, cyclic or acyclic.

[0094] In this specification, “alkyl”, “alkenyl”, “alkynyl”, “alkylene”, “alkenylene” and “alkynylene” can each independently be branched or unbranched, or cyclic or acyclic.

[0095] According to one specific example of the present invention, in the chemical formula 1,

[0096] R1, R2 and R3 are each independently substituted or unsubstituted C 1-6 It can be an alkyl group,

[0097] R4 is substituted or unsubstituted divalent C 2-6 It may be a hydrocarbon group,

[0098] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent C 2-20 It may be a hydrocarbon group,

[0099] R9 and R 10 are independently substituted or unsubstituted saturated or unsaturated monovalent C 2-20 It may be a hydrocarbon group,

[0100] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, C 1-13 Alkylene, C 2-13 Alkenylene or C 2-13 Alkynylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 Selected from the group consisting of alkynyl,

[0101] X -It can be a monovalent anion of an inorganic acid or an organic acid.

[0102] More specifically, in the chemical formula 1,

[0103] R1, R2 and R3 are each independently substituted or unsubstituted C 1-3 It can be an alkyl group,

[0104] R4 is substituted or unsubstituted C 2-4 It may be an alkylene group,

[0105] R5, R6, R7 and R8 are each independently substituted or unsubstituted C 2-13 Alkylene group or C 2-13 It may be an alkenylene group,

[0106] R9 and R 10 Each independently substituted or unsubstituted C 6-20 Alkyl group, C 6-20 Alkenyl group or C 6-20 It can be an alkynyl group,

[0107] L1, L2, L3 and L4 can each be independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)- and -P(O)(OR')O-, wherein R' is each independently a hydrogen atom and C 1-6 may be selected from the group consisting of alkyl,

[0108] X - is F - , Cl - , Br - , I - , nitrate anion, benzoate anion, methanesulfonic acid anion, acetate anion (CH3COO - )(=AcO - ), or trihaloacetate anion (CF3COO - ) may be.

[0109] More specifically, the cationic lipid may have a structure selected from the following chemical formulas A and B:

[0110]

[0111] A second aspect of the present invention provides a method for preparing a cationic lipid, comprising the steps of: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) protecting a terminal -SH group of the compound of formula c; (3) reacting the compound of formula c with the protected terminal -SH group with a compound of formula d to obtain a compound of formula e; (4) reacting the compound of formula e with a compound of formula f to obtain a compound of formula g; and (5) reacting the compound of formula g with a compound of formula h to obtain a compound of formula 1-1.

[0112] [chemical formula a]

[0113] R9-OH

[0114] [chemical formula b]

[0115] HOC(O)-R7-SH

[0116] [chemical formula c]

[0117] R9-OC(O)-R7-SH

[0118] [chemical formula d]

[0119] HS-R5-OH

[0120] [chemical formula e]

[0121] R9-OC(O)-R7-SS-R5-OH

[0122] [chemical formula f]

[0123]

[0124] [chemical formula g]

[0125] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2

[0126] [chemical formula h]

[0127] H2N-R4-N + (R1)(R2)(R3) X -

[0128] [Chemical Formula 1-1]

[0129] [R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2]2-N-R4-N + (R1)(R2)(R3) X -

[0130] In the above,

[0131] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0132] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0133] R5 and R7 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0134] R9 is a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0135] X - is a monovalent anion,

[0136] Hal is selected from the group consisting of F, CI, Br and I.

[0137] In one specific example of the cationic lipid production method according to the second aspect of the present invention, the reaction of step (1) may be carried out under reflux in the presence of a catalyst (e.g., p-toluenesulfonic acid monohydrate (p-TsOH)) in a solvent (e.g., toluene), the protection of step (2) may be carried out in the presence of a catalyst (e.g., acetic acid) in a solvent (e.g., ethanol) using an appropriate mercapto protecting agent (e.g., 2,2-dipyridyldisulfide), the reaction of step (3) may be carried out in the presence of a catalyst (e.g., acetic acid) in a solvent (e.g., ethanol), and the reaction of step (4) may be carried out in the presence of a catalyst (e.g., triethylamine (TEA)) in a solvent (e.g., methylene chloride (MC)) at a low temperature (e.g., -10°C to 10°C) or room temperature (e.g., The reaction can be performed under conditions of 20°C to 30°C, and the reaction of step (5) can be performed at elevated temperature (e.g., 80°C to 150°C) in the presence of a catalyst (e.g., triethylamine (TEA)) in a solvent (e.g., isopropyl alcohol (IPA)), but is not limited thereto.

[0138] A third aspect of the present invention provides a method for producing a cationic lipid, comprising: (1) reacting a compound of formula g obtained in the second aspect of the present invention with a compound of formula h to obtain a compound of formula i; and (2) reacting a compound of formula i with a compound of formula j to obtain a compound of formula 1-2.

[0139] [chemical formula g]

[0140] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2

[0141] [chemical formula h]

[0142] H2N-R4-N + (R1)(R2)(R3) X -

[0143] [chemical formula i]

[0144] R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2-NH-R4-N + (R1)(R2)(R3) X -

[0145] [chemical formula j]

[0146] R 10 -OC(O)-R8-SS-R6-OC(O)-CH=CH2

[0147] [Chemical Formula 1-2]

[0148] R9-OC(O)-R7-SS-R5-OC(O)-CH2-CH2-N(A)-R4-N + (R1)(R2)(R3) X -

[0149] In the above,

[0150] R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group,

[0151] R4 is a substituted or unsubstituted divalent hydrocarbon group,

[0152] R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group,

[0153] R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0154] A is -CH2-CH2-C(O)O-R6-SS-R8-C(O)OR 10 And,

[0155] X - is a monovalent anion.

[0156] In one specific example of a method for producing a cationic lipid according to the third aspect of the present invention, the reactions of steps (1) and (2) may be performed in a solvent (e.g., isopropyl alcohol (IPA)) in the presence of a catalyst (e.g., triethylamine (TEA)) at an elevated temperature (e.g., 80°C to 150°C), but are not limited thereto.

[0157] The cationic lipid according to the present invention can easily form a complex with an anionic drug, making it useful for drug delivery.

[0158] Accordingly, according to a fourth aspect of the present invention, a drug delivery composition comprising the cationic lipid of the present invention is provided.

[0159] In one embodiment, the drug may be selected from a nucleic acid, a polypeptide, a virus, or a combination thereof.

[0160] The above “nucleic acid” may be, for example, but is not limited to, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof.

[0161] The above “polypeptide” may mean a protein that is active in the body, such as an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a polypeptide sequence of an antigen, an analogue or precursor thereof, which can be recognized as an antigen through a series of processes in the body.

[0162] In one specific embodiment, the lipid of the present invention forms a complex with a drug, and the complex can be encapsulated within a nanoparticle structure formed by an amphiphilic block copolymer.

[0163] In one specific example, the amphiphilic block copolymer may be an AB type block copolymer including a hydrophilic A block and a hydrophobic B block. The AB type block copolymer forms core-shell type polymer nanoparticles in an aqueous solution, in which the hydrophobic B block forms a core (inner wall) and the hydrophilic A block forms a shell (outer wall).

[0164] In one specific example, the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof.

[0165] More specifically, the hydrophilic A block may be at least one selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0166] In addition, if necessary, a functional group, ligand, or functional group capable of reaching a specific tissue or cell, or a functional group capable of promoting intracellular delivery may be chemically bonded to the terminal of the hydrophilic A block to control the distribution of the nanoparticle carrier in the body or increase the efficiency of delivery of the nanoparticle carrier into a cell. In one specific example, the functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies to cell surface receptors. More specifically, the functional group or ligand may be at least one selected from the group consisting of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, antibodies to transferrin receptors, etc.

[0167] The above hydrophobic B block is a biocompatible biodegradable polymer, and in one specific example, it may be at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine.

[0168] More specifically, the hydrophobic B block may be at least one selected from the group consisting of polylactide (PLA), polyglycolide, polycaprolactone, polydioxan-2-one, a copolymer of polylactide and glycolide, a copolymer of polylactide and polydioxan-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

[0169] Additionally, in one specific example, the hydrophobic B block may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to a hydroxyl group at the end of the hydrophobic B block to increase the hydrophobicity of the hydrophobic B block and thereby improve the stability of the nanoparticle.

[0170] Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for explaining the present invention and the scope of the present invention is not limited in any way by these examples.

[0171] [Example]

[0172] Example 1

[0173] 1-1. A compound of the following chemical formula A was prepared according to the synthetic outline shown in Figure 1.

[0174] [Chemical Formula A]

[0175]

[0176] 1-2. Synthesis of 2-hexyldecyl 3-mercaptopropanoate

[0177] 2-hexyl-1-decanol (15.00 g, 61.87 mmol, 1.0 eq), 3-mercaptopropionic acid (7.88 g, 74.25 mmol, 1.2 eq), p-toluenesulfonic acid monohydrate (p-TsOH) (21.18 g, 111.37 mmol, 1.8 eq), and toluene (300 mL) were added to a 500 mL 3-neck round-bottom flask (RBF), and stirred and refluxed using a Dean-Stark trap and a condenser. After 7 hours, the mixture was concentrated in vacuo, extracted with methylene chloride (or dichloromethane, MC) and a saturated aqueous solution of sodium bicarbonate, and the methylene chloride layer was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:30) to obtain 2-hexyldecyl 3-mercaptopropanoate (14.5 g, yield: 70.9%).

[0178] 1 H-NMR (400 MHz, CDCl3)δ4.02 (d, 2H), 2.80-2.76 (m, 2H), 2.65 (t, 2H), 1.64-1.62 (m, 2H), 1.31-1.21 (m, 24H), 0.88 (t, 6H)

[0179] 1-3. Synthesis of 2-hexyldecyl 3-(pyridin-2-yldisulfaneyl)propanoate

[0180] 2,2-dipyridyldisulfide (12 g, 54.45 mmol, 1.5 eq) and ethanol (228 mL) were added to a 500 mL 1-neck RBF and stirred to completely dissolve. 2-hexyldecyl 3-mercaptopropanoate (12 g, 36.30 mmol, 1 eq) and acetic acid (4.36 mL, 76.23 mmol, 2.1 eq) were sequentially added and stirred. After 4 h, the mixture was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:6) to obtain 2-hexyldecyl 3-(pyridin-2-yldisulfanyl)propanoate (10.8 g, yield: 67.7%).

[0181] 1 H-NMR (400 MHz, CDCl3)δ8.48-8.46 (m, 1H), 7.70-7.68 (m, 1H), 7.66-7.62 (m, 1H), 7.11-7.08 (m, 1H), 4.00 (d, 2H), 3.05 (t, 2H), 2.77 (t, 2H), 1.61-1.59 (m, 1H), 1.33-1.26 (m, 24H), 0.88 (t, 6H)

[0182] 1-4. Synthesis of 2-hexyldecyl 3-((2-hydroxyethyl)disulfaneyl)propanoate

[0183] 2-Hexyldecyl 3-(pyridin-2-yldisulfanyl)propanoate (9 g, 20.47 mmol, 1.0 eq) and ethanol (100 mL) were added to a 250 mL 1-neck RBF and stirred to completely dissolve. 2-mercaptoethanol (2.40 g, 30.70 mmol, 1.5 eq) and acetic acid (2.46 mL, 42.98 mmol, 2.1 eq) were sequentially added and stirred. After 5 h, the mixture was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:4) to obtain 2-hexyldecyl 3-((2-hydroxyethyl)disulfanyl)propanoate (6.2 g, yield: 74.5%).

[0184] 1 H-NMR (400 MHz, CDCl3)δ4.03 (d, 2H), 3.89 (t, 2H), 2.96 (t, 2H), 2.87 (t, 2H), 2.76 (t, 2H), 1.63 (br, 1H), 1.58 (br, 1H), 1.32-1.25 (m, 24H), 0.88 (t, 6H)

[0185] 1-5. Synthesis of 2-((3-((2-hexyldecyl)oxy)-3-oxopropyl)disulfaneyl)ethyl acrylate

[0186] 2-Hexyldecyl 3-((2-hydroxyethyl)disulfanyl)propanoate (5 g, 12.29 mmol, 1 eq), triethylamine (TEA) (1.89 mL, 13.52 mmol, 1.1 eq), and methylene chloride (120 mL) were added to a 250 mL 1-neck RBF, cooled to 0°C, and acryloyl chloride (1.22 g, 13.52 mmol, 1.1 eq) was added dropwise. The temperature of the reactor was raised to room temperature and stirred. After 3 hours, the mixture in the reactor was extracted with a saturated aqueous solution of sodium bicarbonate, and the methylene chloride layer was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:6) to obtain 2-((3-((2-hexyldecyl)oxy)-3-oxopropyl)disulfanyl)ethyl acrylate (4.4 g, yield: 77.7%).

[0187] 1 H-NMR (400 MHz, CDCl3)δ6.43 (dd, 1H), 6.13 (dd, 1H), 5.86 (dd, 1H), 4.42 (t, 2H), 4.01 (d, 2H), 2.97-2.94 (m, 4H), 2.75 (t, 2H), 1.63 (br, 1H), 1.31-1.27 (m, 24H), 0.88 (t, 6H)

[0188] 1-6. Synthesis of the compound of chemical formula A

[0189] In a 25 mL 1-neck RBF, (2-aminoethyl)trimethylammonium chloride hydrochloride (0.17 g, 0.94 mmol, 1 eq), triethylamine (0.13 mL, 0.94 mmol, 1 eq), and isopropyl alcohol (10 mL) were added and stirred to dissolve. Then, 2-((3-((2-hexyldecyl)oxy)-3-oxopropyl)disulfanyl)ethyl acrylate (1.09 g, 2.36 mmol, 2.5 eq) was added, and the temperature of the reactor was set to 110°C and stirred. After stirring for 6 days, the mixture in the reactor was concentrated in vacuo and separated through a silica column with methylene chloride:methanol:ammonium hydroxide (8:1:0.1), and dried under vacuum. After this, hexane was added to the mixture and filtered to obtain a compound of chemical formula A (0.105 g, 10.9%).

[0190] 1 H-NMR (400 MHz, CDCl3) δ 4.34 (t, 4H), 4.00 (d, 4H), 3.96 (br, 2H), 3.44 (s, 9H), 3.07 (br, 2H), 2.96-2.88 (m, 12H), 2.74 (t, 4H), 2.56 (br, 4H), 1.63 (br, 2H), 1.31-1.15 (m, 48H), 0.88 (t, 6H)

[0191] Example 2

[0192] 2-1. A compound of the following chemical formula B was prepared according to the synthetic outline shown in Fig. 2.

[0193] [Chemical Formula B]

[0194]

[0195] 2-2. Synthesis of 2-nonyl 3-mercaptopropanoate

[0196] Nonanol (15.00 g, 103.98 mmol, 1.0 eq), 3-mercaptopropionic acid (13.24 g, 124.77 mmol, 1.2 eq), p-toluenesulfonic acid monohydrate (35.60 g, 187.16 mmol, 1.8 eq), and toluene (380 mL) were added to a 500 mL 3-neck RBF, and a Dean-Stark trap and a condenser were installed, followed by stirring and refluxing. After 7 hours, the mixture was concentrated in vacuo, extracted with methylene chloride and a saturated aqueous sodium bicarbonate solution, and the methylene chloride layer was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:30) to obtain 2-nonyl 3-mercaptopropanoate (16.8 g, yield: 69.5%).

[0197] 1 H-NMR (400 MHz, CDCl3)δ4.09 (t, 2H), 2.80-2.73 (m, 2H), 2.66 (t, 2H), 1.66-1.60 (m, 3H), 1.36-1.27 (m, 12H), 0.88 (t, 3H)

[0198] 2-3. Synthesis of nonyl 3-(pyridin-2-yldisulfaneyl)propanoate

[0199] 2,2-Dipyridyldisulfide (19.91 g, 90.37 mmol, 1.5 eq) and ethanol (180 mL) were added to a 500 mL 1-neck RBF and stirred to completely dissolve. 2-Nonyl 3-mercaptopropanoate (14 g, 60.25 mmol, 1 eq) and acetic acid (7.24 mL, 126.52 mmol, 2.1 eq) were sequentially added and stirred. After 4 h, the mixture was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:7) to obtain nonyl 3-(pyridin-2-yldisulfanyl)propanoate (13.5 g, yield: 65.6%).

[0200] 1H-NMR (400 MHz, CDCl3)δ8.47-8.46 (m, 1H), 7.70-7.66 (m, 1H), 7.65-7.62 (m, 1H), 7.11-7.08 (m, 1H), 4.08 (t, 2H), 3.05 (t, 2H), 2.76 (t, 2H), 1.64-1.58 (m, 2H), 1.34-1.27 (m, 12H), 0.88 (t, 3H)

[0201] 2-4. Synthesis of nonyl 3-((2-hydroxyethyl)disulfaneyl)propanoate

[0202] Nonyl 3-(pyridin-2-yldisulfanyl)propanoate (12 g, 35.14 mmol, 1.0 eq) and ethanol (140 mL) were added to a 250 mL 1-neck RBF and stirred to completely dissolve. 2-mercaptoethanol (4.12 g, 52.70 mmol, 1.5 eq) and acetic acid (4.22 mL, 73.79 mmol, 2.1 eq) were sequentially added and stirred. After 4 h, the mixture was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:7) to obtain nonyl 3-((2-hydroxyethyl)disulfanyl)propanoate (8.1 g, yield: 74.7%).

[0203] 1 H-NMR (400 MHz, CDCl3)δ4.08 (t, 2H), 3.89 (t, 2H), 2.96 (t, 2H), 2.88 (t, 2H), 2.75 (t, 2H), 2.10 (br, 1H), 1.66-1.60 (m, 2H), 1.37-1.27 (m, 12H), 0.88 (t, 3H)

[0204] 2-5. Synthesis of 2-((3-(nonyloxy)-3-oxopropyl)disulfaneyl)ethyl acrylate

[0205] Nonyl 3-((2-hydroxyethyl)disulfanyl)propanoate (6.5 g, 21.07 mmol, 1 eq), triethylamine (3.23 mL, 23.18 mmol, 1.1 eq), and methylene chloride (100 mL) were added to a 250 mL 1-neck RBF and cooled to 0°C. Acryloyl chloride (2.10 g, 23.18 mmol, 1.1 eq) was added dropwise. The reactor was heated to room temperature and stirred. After 3 hours, the mixture in the reactor was extracted with a saturated aqueous solution of sodium bicarbonate, and the methylene chloride layer was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane (1:4) to obtain 2-((3-(nonyloxy)-3-oxopropyl)disulfanyl)ethyl acrylate (5.1 g, yield: 66.8%).

[0206] 1 H-NMR (400 MHz, CDCl3)δ6.43 (dd, 1H), 6.13 (dd, 1H), 5.86 (dd, 1H), 4.42 (t, 2H), 4.10 (t, 2H), 2.97-2.91 (m, 4H), 2.74 (t, 2H), 1.66-1.60 (m, 2H), 1.35-1.27 (m, 12H), 0.88 (t, 3H)

[0207] 2-6. Synthesis of the compound of chemical formula B

[0208] In a 25 mL 1-neck RBF, (2-aminoethyl)trimethylammonium chloride hydrochloride (0.22 g, 1.26 mmol, 1 eq), triethylamine (0.18 mL, 1.26 mmol, 1 eq), and isopropyl alcohol (5 mL) were added and stirred to dissolve. 2-((3-(nonyloxy)-3-oxopropyl)disulfanyl)ethyl acrylate (1.05 g, 2.89 mmol, 2.3 eq) was added, and the temperature of the reactor was set to 110°C and stirred. After stirring for 7 days, the mixture in the reactor was concentrated in vacuo, separated through a silica column with methylene chloride:methanol:ammonium hydroxide (8:1:0.1), and dried in vacuo. Thereafter, ether was added to the mixture, and filtration was performed to obtain a compound of chemical formula B (0.082 g, 7.9%).

[0209] 1 H-NMR (400 MHz, CDCl3) δ 4.33 (t, 4H), 4.09 (t, 4H), 3.88 (t, 2H), 3.43 (s, 9H), 3.00 (br, 2H), 2.96-2.90 (m, 8H), 2.83 (t, 4H), 2.73 (t, 4H), 2.51 (t, 4H), 1.66-1.60 (m, 4H), 1.35-1.28 (m, 24H), 0.88 (m, 3H)

[0210] [Example of manufacturing a composition for drug delivery]

[0211] (1) Manufacturing Example 1

[0212] 1. Preparation of raw materials

[0213] As shown in the table below, the materials required for formulation preparation were dissolved in each dilution solvent to prepare the required concentration. When dissolving, the materials were warmed to room temperature, then the solvent was added and dissolved.

[0214]

[0215] 2. Mixing raw materials

[0216] The required amounts of raw materials were taken according to the ratio of each compound of chemical formula A and B:DOPE:cholesterol:DMG-PEG=50:10:38.5:1.5 to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6 and then mixed. Ethanol was added to the ethanol layer so that the sum of all raw materials was within 12.5 mM, and the aqueous phase and the ethanol phase were mixed while maintaining a volume ratio of 3:1. To lower the total ethanol content after mixing, buffer exchange was performed as follows: The mixed solution was concentrated by centrifugation at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume: 0.5 mL or 4 mL or 15 mL), then diluted with PBS and centrifuged to concentrate. This process was repeated to perform buffer exchange.

[0217] The specific process sequence is as follows.

[0218] 1) Two autoclaved tubes were prepared (Tube (A), (B)).

[0219] 2) DOPE and compounds of chemical formulas A and B, cholesterol, and DMG-PEG in molar numbers calculated according to the experimental conditions were sequentially added to Tube (A), and mixed by vortexing after addition.

[0220] 3) Ethanol was added as needed to ensure that the sum of all raw materials in the ethanol phase was within 6.25-12.5 mM.

[0221] 4) In Tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) (= prepared by diluting 3 M sodium acetate buffer to 20 mM and titrating to pH 4.6 with 1 M HCl) were mixed. The ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase and added.

[0222] 5) Mixing of Tube (A) and Tube (B) was performed using a microfluidics device (Ignite, Precision Nanosystem). Microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min.

[0223] 6) The resulting mixture from step 5 was concentrated by centrifugation at 4,000 rpm using an Amicon-Ultra tube filter (50K), then diluted with PBS and centrifuged to remove excess ethanol by repeating the process, and then concentrated to a final x mg / mL (theoretical concentration).

[0224] 3. Evaluation of physical properties of the formulation

[0225] 1) For the manufactured formulation, particle characteristics (i.e., zeta-average particle size, polydispersity index (PDI), and zeta-potential) were confirmed using a particle size analyzer (Dynamic Light Scattering, DLS), and the results are shown in Table 1 below.

[0226] 2) For the manufactured formulation, mRNA encapsulation efficiency was confirmed through Ribo-green assay, and the results are shown in Table 1 below.

[0227] [Table 1]

[0228]

[0229] (2) Manufacturing Example 2

[0230] 1. Preparation of raw materials

[0231] As shown in the table below, the materials required for formulation preparation were dissolved in each dilution solvent to prepare the required concentration. When dissolving, the materials were warmed to room temperature, then the solvent was added and dissolved.

[0232]

[0233] 2. Mixing raw materials

[0234] The required amounts of raw materials were taken according to the ratio of MC3: each compound of chemical formula A and B: cholesterol: DMG-PEG = 50:10:38.5:1.5 and then mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5. Ethanol was added to the ethanol layer so that the sum of all raw materials was within 12.5 mM, and the aqueous phase and the ethanol phase were mixed while maintaining a volume ratio of 3:1. To lower the total ethanol content after mixing, buffer exchange was performed as follows: The mixed solution was concentrated by centrifugation at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume: 0.5 mL or 4 mL or 15 mL), then diluted with PBS and centrifuged to concentrate. This process was repeated to perform buffer exchange.

[0235] The specific process sequence is as follows.

[0236] 1) Two autoclaved tubes were prepared (Tube (A), (B)).

[0237] 2) MC3 and compounds of chemical formulas A and B, cholesterol, and DMG-PEG in molar quantities calculated according to the experimental conditions were sequentially added to Tube (A), and mixed by vortexing after addition.

[0238] 3) Ethanol was added as needed to ensure that the sum of all raw materials in the ethanol phase was within 6.25-12.5 mM.

[0239] 4) In Tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) (= prepared by diluting 3 M sodium acetate buffer to 20 mM and titrating to pH 4.6 with 1 M HCl) were mixed. The ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase and added.

[0240] 5) Mixing of Tube (A) and Tube (B) was performed using a microfluidics device (Ignite, Precision Nanosystem). Microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min.

[0241] 6) The resulting mixture from step 5 was concentrated by centrifugation at 4,000 rpm using an Amicon-Ultra tube filter (50K), then diluted with PBS and centrifuged to remove excess ethanol by repeating the process, and then concentrated to a final x mg / mL (theoretical concentration).

[0242] 3. Evaluation of physical properties of the formulation

[0243] 1) For the manufactured formulation, particle characteristics (i.e., zeta-average particle size, polydispersity index (PDI), and zeta-potential) were confirmed using a particle size analyzer (Dynamic Light Scattering, DLS), and the results are shown in Table 2 below.

[0244] 2) For the manufactured formulation, mRNA encapsulation efficiency was confirmed through Ribo-green assay, and the results are shown in Table 2 below.

[0245] [Table 2]

[0246]

Claims

1. A cationic lipid having a structure represented by the following chemical formula 1: [Chemical Formula 1] Here, R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group, R4 is a substituted or unsubstituted divalent hydrocarbon group, R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group, R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl, X - is a monovalent anion.

2. In paragraph 1, R1, R2 and R3 are each independently substituted or unsubstituted C 1-6 It is an alkyl group, R4 is substituted or unsubstituted divalent C 2-6 It is a hydrocarbon group, R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent C 2-20 It is a hydrocarbon group, R9 and R 10 are independently substituted or unsubstituted saturated or unsaturated monovalent C 2-20 It is a hydrocarbon group, L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O- and -S(O)2-, wherein L' is a direct bond, C 1-13 Alkylene, C 2-13 Alkenylene or C 2-13 Alkynylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 Selected from the group consisting of alkynyl, X - is a monovalent anion of an inorganic acid or organic acid, Cationic lipids.

3. In paragraph 1, R1, R2 and R3 are each independently substituted or unsubstituted C 1-3 It is an alkyl group, R4 is substituted or unsubstituted C 2-4 It is an alkylene group, R5, R6, R7 and R8 are each independently substituted or unsubstituted C 2-13 Alkylene group or C 2-13 It is an alkenylene group, R9 and R 10 Each independently substituted or unsubstituted C 6-20 Alkyl group, C 6-20 Alkenyl group or C 6-20 It is an alkynyl group, L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)- and -P(O)(OR')O-, wherein R' is each independently a hydrogen atom and C 1-6 Selected from the group consisting of alkyl, X - is F - , Cl - , Br - , I - , nitrate anion, benzoate anion, methanesulfonic acid anion, acetate anion (CH3COO - ), or trihaloacetate anion (CF3COO - )person, Cationic lipids.

4. In the first paragraph, a cationic lipid having a structure selected from the following chemical formulas A and B: 5.(1) A step of reacting a compound of chemical formula a with a compound of chemical formula b to obtain a compound of chemical formula c; (2) A step of protecting the terminal -SH group of the compound of chemical formula c; (3) A step of reacting a compound of formula c having a protected terminal -SH group with a compound of formula d to obtain a compound of formula e; (4) a step of reacting a compound of chemical formula e with a compound of chemical formula f to obtain a compound of chemical formula g; and (5) a step of reacting a compound of chemical formula g with a compound of chemical formula h to obtain a compound of chemical formula 1-1; Method for preparing cationic lipids: [chemical formula a] R9-OH [chemical formula b] HOC(O)-R7-SH [chemical formula c] R9-OC(O)-R7-SH [chemical formula d] HS-R5-OH [chemical formula e] R9-OC(O)-R7-SS-R5-OH [chemical formula f] [chemical formula g] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2 [chemical formula h] H2N-R4-N + (R1)(R2)(R3) X - [Chemical Formula 1-1] [R9-OC(O)-R7-S-S-R5-OC(O)-CH2-CH2]2-N-R4-N + (R1)(R2)(R3) X - In the above, R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group, R4 is a substituted or unsubstituted divalent hydrocarbon group, R5 and R7 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group, R9 is a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, X - is a monovalent anion, Hal is selected from the group consisting of F, CI, Br and I. 6.(1) A step of reacting the compound of chemical formula g obtained in paragraph 5 with the compound of chemical formula h to obtain a compound of chemical formula i; and (2) a step of reacting a compound of chemical formula i with a compound of chemical formula j to obtain a compound of chemical formula 1-2; Method for preparing cationic lipids: [chemical formula g] R9-OC(O)-R7-SS-R5-OC(O)-CH=CH2 [chemical formula h] H2N-R4-N + (R1)(R2)(R3) X - [chemical formula i] R9-OC(O)-R7-S-S-R5-OC(O)-CH2-CH2-NH-R4-N + (R1)(R2)(R3) X - [chemical formula j] R 10 -OC(O)-R8-S-S-R6-OC(O)-CH=CH2 [Chemical Formula 1-2] R9-OC(O)-R7-S-S-R5-OC(O)-CH2-CH2-N(A)-R4-N + (R1)(R2)(R3) X - In the above, R1, R2 and R3 are each independently a substituted or unsubstituted alkyl group, R4 is a substituted or unsubstituted divalent hydrocarbon group, R5, R6, R7 and R8 are each independently a substituted or unsubstituted saturated or unsaturated divalent hydrocarbon group, R9 and R 10 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, A is -CH2-CH2-C(O)O-R6-SS-R8-C(O)OR 10 And, X - is a monovalent anion.

7. A drug delivery composition comprising a cationic lipid according to any one of claims 1 to 4.

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