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, and various carriers and delivery technologies have been developed. Carriers are 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 vulnerable to risks such as non-specific immune responses and have complex production processes, posing numerous challenges for commercialization. Therefore, recent research is focused on addressing these shortcomings by utilizing non-viral carriers. Compared to viral carriers, non-viral carriers offer advantages such as 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 lipid having a structure represented by the following chemical formula 1:

[0007] [Chemical Formula 1]

[0008]

[0009] Here,

[0010] R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0011] R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0012] R3 is an unsubstituted alkylene group,

[0013] R is a hydrogen atom (H) or , wherein R4 is a substituted or unsubstituted alkyl group, alkenyl group, or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group, or alkynylene group, and * represents a point of attachment to a nitrogen atom.

[0014] More specifically, in the chemical formula 1,

[0015] R1 and R4 are each independently substituted or unsubstituted C 1-30 Alkyl group, substituted or unsubstituted C 2-30 Alkenyl group, or substituted or unsubstituted C 2-30 It is an alkynyl group,

[0016] R2 and R5 are each independently substituted or unsubstituted C 1-15 alkylene group, substituted or unsubstituted C 2-15 Alkenylene group, or substituted or unsubstituted C 2-15 It is an alkynylene group,

[0017] R3 is unsubstituted C 2-9 It is an alkylene group.

[0018] More specifically, in the chemical formula 1,

[0019] R1 and R4 are each independently substituted or unsubstituted C 1-20Alkyl group, substituted or unsubstituted C 2-20 Alkenyl group, or substituted or unsubstituted C 2-20 It is an alkynyl group,

[0020] R2 and R5 are each independently substituted or unsubstituted C 1-12 alkylene group, substituted or unsubstituted C 2-12 Alkenylene group, or substituted or unsubstituted C 2-12 It is an alkynylene group,

[0021] R3 is unsubstituted C 2-7 It is an alkylene group.

[0022] More specifically, in the chemical formula 1,

[0023] R1 and R4 are each independently substituted or unsubstituted C 5-20 Alkyl group, substituted or unsubstituted C 5-20 Alkenyl group, or substituted or unsubstituted C 5-20 It is an alkynyl group,

[0024] R2 and R5 are each independently substituted or unsubstituted C 3-12 alkylene group, substituted or unsubstituted C 3-12 Alkenylene group, or substituted or unsubstituted C 3-12 It is an alkynylene group,

[0025] R3 is unsubstituted C 2-5 It is an alkylene group.

[0026] More specifically, the lipid may have any one structure selected from the following chemical formulas A to L:

[0027]

[0028]

[0029] A second aspect of the present invention provides a method for producing a lipid, comprising: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) reacting a compound of formula c with a compound of formula d to obtain a compound of formula e; and (3) reacting a compound of formula e with a compound of formula f to obtain a compound of formula 1-1.

[0030] [chemical formula a]

[0031] HOC(=O)-R2-NH2

[0032] [chemical formula b]

[0033] R1-OH

[0034] [chemical formula c]

[0035] R1-OC(=O)-R2-NH2

[0036] [chemical formula d]

[0037]

[0038] [chemical formula e]

[0039] R1-OC(=O)-R2-NH-C(=O)-CH=CH2

[0040] [chemical formula f]

[0041] tBu-OC(=O)-NH-R3-NH2

[0042] [Chemical Formula 1-1]

[0043] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu

[0044] In the above,

[0045] R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0046] R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0047] R3 is an unsubstituted alkylene group,

[0048] tBu is tert-butyl group,

[0049] X is selected from the group consisting of F, CI, Br and I.

[0050] A third aspect of the present invention provides a method for producing a lipid, comprising: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) reacting a compound of formula c with a compound of formula d to obtain a compound of formula e; and (3) reacting a compound of formula e with a compound of formula f to obtain a compound of formula 1-2.

[0051] [chemical formula a]

[0052] HOC(=O)-R2-NH2

[0053] [chemical formula b]

[0054] R1-OH

[0055] [chemical formula c]

[0056] R1-OC(=O)-R2-NH2

[0057] [chemical formula d]

[0058]

[0059] [chemical formula e]

[0060] R1-OC(=O)-R2-NH-C(=O)-CH=CH2

[0061] [chemical formula f]

[0062] tBu-OC(=O)-NH-R3-NH2

[0063] [Chemical Formula 1-2]

[0064] [R1-OC(=O)-R2-NH-C(=O)-CH2-CH2]2-N-R3-NH-C(=O)O-tBu

[0065] In the above,

[0066] R1 is each independently a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0067] R2 is each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0068] R3 is an unsubstituted alkylene group,

[0069] tBu is tert-butyl group,

[0070] X is selected from the group consisting of F, CI, Br and I.

[0071] The fourth aspect of the present invention provides a method for producing a lipid, comprising the step of reacting the compound of formula 1-1 obtained in the second aspect of the present invention with a compound of formula g to obtain a compound of formula 1-3:

[0072] [Chemical Formula 1-1]

[0073] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu

[0074] [chemical formula g]

[0075] R4-OC(=O)-R5-NH-C(=O)-CH=CH2

[0076] [Chemical Formula 1-3]

[0077] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-N(A)-R3-NH-C(=O)O-tBu

[0078] In the above,

[0079] R1 is each independently a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0080] R2 is each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0081] R3 is an unsubstituted alkylene group,

[0082] A is -CH2-CH2-C(=O)-NH-R5-C(=O)O-R4, where R4 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0083] tBu is tert-butyl group,

[0084] X is selected from the group consisting of F, CI, Br and I.

[0085] A fifth aspect of the present invention provides a drug delivery composition comprising a lipid according to the present invention.

[0086] The lipid having a specific structure according to the present invention can easily form a complex with an anionic drug, and by utilizing this complex, the drug can be efficiently delivered into a target biological tissue.

[0087] Figure 1 is a reaction schematic diagram for the lipid synthesis process performed in Example 1.

[0088] Figure 2 is a reaction schematic diagram for the lipid synthesis process performed in Example 2.

[0089] Figure 3 is a reaction schematic diagram for the lipid synthesis process performed in Example 3.

[0090] Figure 4 is a reaction schematic diagram for the lipid synthesis process performed in Example 4.

[0091] Hereinafter, the present invention will be described in more detail.

[0092] The lipid provided according to the first aspect of the present invention has a structure represented by the following chemical formula 1:

[0093] [Chemical Formula 1]

[0094]

[0095] Here,

[0096] R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0097] R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0098] R3 is an unsubstituted alkylene group,

[0099] R is a hydrogen atom (H) or , wherein R4 is a substituted or unsubstituted alkyl group, alkenyl group, or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group, or alkynylene group, and * represents a point of attachment to a nitrogen atom.

[0100] The range of lipids provided according to the first aspect of the present invention includes those having the structure of the above chemical formula 1 as well as their cationic forms.

[0101] 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.

[0102] In this specification, “alkyl”, “alkenyl”, “alkynyl”, “alkylene”, “alkenylene” and “alkynylene” can each independently be branched or unbranched, or cyclic or acyclic.

[0103] According to one specific example of the present invention, in the chemical formula 1,

[0104] R1 and R4 are each independently substituted or unsubstituted C 1-30 Alkyl group, substituted or unsubstituted C 2-30 Alkenyl group, or substituted or unsubstituted C 2-30 It can be an alkynyl group,

[0105] R2 and R5 are each independently substituted or unsubstituted C 1-15 alkylene group, substituted or unsubstituted C 2-15 Alkenylene group, or substituted or unsubstituted C 2-15It may be an alkynylene group,

[0106] R3 is unsubstituted C 2-9 It may be an alkylene group.

[0107] More specifically, in the chemical formula 1,

[0108] R1 and R4 are each independently substituted or unsubstituted C 1-20 Alkyl group, substituted or unsubstituted C 2-20 Alkenyl group, or substituted or unsubstituted C 2-20 It can be an alkynyl group,

[0109] R2 and R5 are each independently substituted or unsubstituted C 1-12 alkylene group, substituted or unsubstituted C 2-12 Alkenylene group, or substituted or unsubstituted C 2-12 It may be an alkynylene group,

[0110] R3 is unsubstituted C 2-7 It may be an alkylene group.

[0111] More specifically, in the chemical formula 1,

[0112] R1 and R4 are each independently substituted or unsubstituted C 5-20 Alkyl group, substituted or unsubstituted C 5-20 Alkenyl group, or substituted or unsubstituted C 5-20 It can be an alkynyl group,

[0113] R2 and R5 are each independently substituted or unsubstituted C 3-12 alkylene group, substituted or unsubstituted C 3-12 Alkenylene group, or substituted or unsubstituted C 3-12 It may be an alkynylene group,

[0114] R3 is unsubstituted C 2-5 It may be an alkylene group.

[0115] More specifically, the lipid may have any one structure selected from the following chemical formulas A to L:

[0116]

[0117]

[0118] A second aspect of the present invention provides a method for producing a lipid, comprising: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) reacting a compound of formula c with a compound of formula d to obtain a compound of formula e; and (3) reacting a compound of formula e with a compound of formula f to obtain a compound of formula 1-1.

[0119] [chemical formula a]

[0120] HOC(=O)-R2-NH2

[0121] [chemical formula b]

[0122] R1-OH

[0123] [chemical formula c]

[0124] R1-OC(=O)-R2-NH2

[0125] [chemical formula d]

[0126]

[0127] [chemical formula e]

[0128] R1-OC(=O)-R2-NH-C(=O)-CH=CH2

[0129] [chemical formula f]

[0130] tBu-OC(=O)-NH-R3-NH2

[0131] [Chemical Formula 1-1]

[0132] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu

[0133] In the above,

[0134] R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0135] R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0136] R3 is an unsubstituted alkylene group,

[0137] tBu is tert-butyl group,

[0138] X is selected from the group consisting of F, CI, Br and I.

[0139] A third aspect of the present invention provides a method for producing a lipid, comprising: (1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; (2) reacting a compound of formula c with a compound of formula d to obtain a compound of formula e; and (3) reacting a compound of formula e with a compound of formula f to obtain a compound of formula 1-2.

[0140] [chemical formula a]

[0141] HOC(=O)-R2-NH2

[0142] [chemical formula b]

[0143] R1-OH

[0144] [chemical formula c]

[0145] R1-OC(=O)-R2-NH2

[0146] [chemical formula d]

[0147]

[0148] [chemical formula e]

[0149] R1-OC(=O)-R2-NH-C(=O)-CH=CH2

[0150] [chemical formula f]

[0151] tBu-OC(=O)-NH-R3-NH2

[0152] [Chemical Formula 1-2]

[0153] [R1-OC(=O)-R2-NH-C(=O)-CH2-CH2]2-N-R3-NH-C(=O)O-tBu

[0154] In the above,

[0155] R1 is each independently a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0156] R2 is each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0157] R3 is an unsubstituted alkylene group,

[0158] tBu is tert-butyl group,

[0159] X is selected from the group consisting of F, CI, Br and I.

[0160] In one specific example of the lipid production method according to the second and third aspects of the present invention, the reaction of step (1) may be performed under reflux in a solvent (e.g., cyclohexane) in the presence of a catalyst (e.g., p-toluenesulfonic acid monohydrate (p-TsOH)), the reaction of step (2) may be performed under conditions of low temperature (e.g., -10°C to 10°C) or room temperature (e.g., 20°C to 30°C) in a solvent (e.g., methylene chloride (MC)) in the presence of a catalyst (e.g., triethylamine (TEA)), and the reaction of step (3) may be performed under reflux in a solvent (e.g., n-butanol (n-BuOH)), but is not limited thereto.

[0161] The fourth aspect of the present invention provides a method for producing a lipid, comprising the step of reacting the compound of formula 1-1 obtained in the second aspect of the present invention with a compound of formula g to obtain a compound of formula 1-3:

[0162] [Chemical Formula 1-1]

[0163] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu

[0164] [chemical formula g]

[0165] R4-OC(=O)-R5-NH-C(=O)-CH=CH2

[0166] [Chemical Formula 1-3]

[0167] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-N(A)-R3-NH-C(=O)O-tBu

[0168] In the above,

[0169] R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group,

[0170] R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0171] R3 is an unsubstituted alkylene group,

[0172] A is -CH2-CH2-C(=O)-NH-R5-C(=O)O-R4, where R4 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0173] tBu is tert-butyl group,

[0174] X is selected from the group consisting of F, CI, Br and I.

[0175] In one specific example of the lipid production method according to the fourth aspect of the present invention, the reaction may be performed under reflux in a solvent (e.g., n-butanol (n-BuOH)), but is not limited thereto.

[0176] The lipid having a specific structure according to the present invention can easily form a complex with an anionic drug, making it useful for drug delivery.

[0177] Accordingly, according to the fifth aspect of the present invention, a drug delivery composition comprising the lipid of the present invention is provided.

[0178] In one embodiment, the drug may be selected from a nucleic acid, a polypeptide, a virus or a combination thereof, and preferably may be a nucleic acid.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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).

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] [Example]

[0191] Example 1

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

[0193] [Chemical Formula A]

[0194]

[0195] 1-2. Synthesis of 2-octyldodecyl 6-acrylamidohexanoate

[0196] 6-aminohexanoic acid (1.05 g, 8.04 mmol, 1.20 eq), 2-octyldodecan-1-ol (2.00 g, 6.70 mmol, 1.00 eq), p-toluenesulfonic acid monohydrate (p-TsOH) (2.29 g, 12.06 mmol, 1.80 eq), and cyclohexane (100 mL) were added to a 250 mL 3-neck round-bottom flask (RBF), and stirred and refluxed using a Dean-Stark trap and a condenser. After 24 hours, the mixture was cooled to room temperature, concentrated in vacuo, extracted with methylene chloride (or dichloromethane, MC) and aqueous NaOH solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo to obtain low-purity 2-octyldodecyl 6-aminohexanoate. Without further purification, the previously obtained 2-octyldodecyl 6-aminohexanoate, methylene chloride (33 mL), and triethylamine (TEA) (1.49 g, 14.74 mmol, 2.20 eq) were added to a 100 mL 3-neck RBF, cooled to 0°C, and acryloyl chloride (0.67 g, 7.37 mmol, 1.10 eq) was added dropwise. The temperature of the reactor was raised to room temperature (20-25℃) and stirred. After 18 hours, the mixture in the reactor was extracted with aqueous HCl solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane = 1:2 to obtain 2-octyldodecyl 6-acrylamidohexanoate (1928.20 mg, yield: 62%).

[0197] 1H-NMR (400 MHz, CDCl3) δ 6.29 (d, 1H), 6.10 (m, 1H), 5.64 (d, 1H), 3.97 (d, 2H), 3.37 (t, 2H), 2.14 (t, 2H), 1.68 - 1.26 (m, 41H), 0.90 (t, 6H)

[0198] 1-3. Synthesis of the compound of chemical formula A

[0199] In a 100 mL 3-neck RBF, 2-octyldodecyl 6-acrylamidohexanoate (120.00 mg, 2.58 mmol, 2.40 eq), tert-butyl N-(2-aminoethyl)carbamate (171.99 mg, 1.07 mmol, 1.00 eq), and n-butanol (n-BuOH) (10 mL) were added, stirred, and refluxed. After 48 h, the mixture was concentrated in vacuo at 80°C, and purified using a silica column with methylene chloride:methanol:ammonium hydroxide = 20:1:0.1 to obtain the compound of formula A.

[0200] 1 H-NMR (400 MHz, CDCl3) δ 7.11 (s, 1H), 4.93 (s, 1H), 3.96 (d, 2H), 3.24 (q, 4H), 2.89 (t, 2H), 2.75 (t, 2H), 2.30-2.36 (m, 4H), 1.62-1.68 (m, 3H), 1.44-1.56 (m, 2H), 1.47 (s, 9H), 1.20-1.39 (m, 35H), 0.90 (t, 6H)

[0201] Example 2

[0202] 2-1. A compound of the following chemical formula B was prepared according to the synthetic outline shown in Fig. 2.

[0203] [Chemical Formula B]

[0204]

[0205] 2-2. Synthesis of 2-hexyloctyl 6-acrylamidohexanoate

[0206] A 500 mL 3-neck RBF was charged with 6-aminohexanoic acid (1.84 g, 13.99 mmol, 1.20 eq), 2-hexyl-1-n-octanol (2.50 g, 11.66 mmol, 1.00 eq), p-toluenesulfonic acid monohydrate (3.99 g, 20.99 mmol, 1.80 eq), and cyclohexane (120 mL), and a Dean-Stark trap and a condenser were installed, followed by stirring and refluxing. After 24 h, the mixture was cooled to room temperature, concentrated in vacuo, extracted with methylene chloride and an aqueous NaOH solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo to obtain low-purity 2-hexyloctyl 6-aminohexanoate. Without further purification, the previously obtained 2-hexyloctyl 6-aminohexanoate, methylene chloride (60 mL), and triethylamine (2.60 g, 25.65 mmol, 2.20 eq) were added to a 250 mL 3-neck RBF, cooled to 0°C, and acryloyl chloride (1.16 g, 12.83 mmol, 1.10 eq) was added dropwise. The temperature of the reactor was raised to room temperature (20–25°C) and stirred. After 18 h, the mixture in the reactor was extracted with aqueous HCl solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane = 1:2 to obtain 2-hexyloctyl 6-acrylamidohexanoate (3.63 g, yield: 82%).

[0207] 1H-NMR (400 MHz, CDCl3) δ 6.29 (d, 1H), 6.10 (m, 1H), 5.64 (d, 1H), 3.97 (d, 2H), 2.33 (t, 2H), 1.68 - 1.54 (m, 5H), 1.41 (m, 2H), 1.30 (m, 20H), 0.90 (t, 6H)

[0208] 2-3. Synthesis of the compound of chemical formula B

[0209] In a 100 mL 3-neck RBF, 2-hexyloctyl 6-acrylamidohexanoate (1429.07 mg, 3.74 mmol, 2.40 eq), tert-butyl N-(2-aminoethyl)carbamate (250.00 mg, 1.56 mmol, 1.00 eq), and n-butanol (10 mL) were added, stirred, and refluxed. After 48 h, the mixture was concentrated in vacuo at 80°C and purified using a silica column with methylene chloride:methanol:ammonium hydroxide = 15:1:0.1 to obtain the compound of formula B.

[0210] 1 H-NMR (400 MHz, CDCl3) δ 7.09 (s, 1H), 4.96 (s, 1H), 3.96 (d, 2H), 3.24 (q, 4H), 2.90 (t, 2H), 2.76 (t, 2H), 2.37 (t, 2H), 2.31 (t, 2H), 1.60-1.80 (m, 3H), 1.47-1.57 (m, 2H), 1.51 (s, 9H), 1.20-1.39 (m, 24H), 0.90 (t, 6H)

[0211] Example 3

[0212] 3-1. A compound of the following chemical formula C was prepared according to the synthetic outline shown in Figure 3.

[0213] [Chemical Formula C]

[0214]

[0215] 3-2. Synthesis of 2-hexyloctyl 8-acrylamidooctanoate

[0216] 8-Aminooctanoic acid (1.78 g, 11.19 mmol, 1.20 eq), 2-hexyloctan-1-ol (2.00 g, 9.33 mmol, 1.00 eq), p-toluenesulfonic acid monohydrate (3.19 g, 16.79 mmol, 1.80 eq), and cyclohexane (100 mL) were added to a 250 mL 3-neck RBF, and a Dean-Stark trap and a condenser were installed, followed by stirring and refluxing. After 24 h, the mixture was cooled to room temperature, concentrated in vacuo, extracted with methylene chloride and an aqueous NaOH solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo to obtain low-purity 2-hexyloctyl 8-aminooctanoate. Without further purification, the previously obtained 2-hexyloctyl 8-aminooctanoate, methylene chloride (100 mL), and triethylamine (2.08 g, 20.52 mmol, 2.20 eq) were added to a 250 mL 3-neck RBF, cooled to 0°C, and acryloyl chloride (0.93 g, 10.26 mmol, 1.10 eq) was added dropwise. The temperature of the reactor was raised to room temperature (20–25°C) and stirred. After 18 h, the mixture in the reactor was extracted with aqueous HCl solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane = 1:1 to obtain 2-hexyloctyl 8-acrylamidooctanoate (2838.70 mg, yield: 74%).

[0217] 1 H-NMR (400 MHz, CDCl3)δ6.29 (d, 1H), 6.10 (m, 1H), 5.63 (d, 1H), 3.97 (d, 2H), 3.35 (q, 2H), 2.31 (t, 2H), 1.65 - 1.51 (m, 4H), 1.35 - 1.27 (m, 27H), 0.90 (t, 6H)

[0218] 3-3. Synthesis of the compound of chemical formula C

[0219] A 100 mL 3-neck RBF was charged with 2-hexyloctyl 8-acrylamidooctanoate (1130.14 mg, 2.76 mmol, 2.60 eq), tert-butyl N-(2-aminoethyl)carbamate (170.00 mg, 1.06 mmol, 1.00 eq), and n-butanol (11 mL), stirred, and refluxed. After 48 h, the mixture was concentrated in vacuo at 80°C, and purified using a silica column with methylene chloride:methanol:ammonium hydroxide = 15:1:0.1 to obtain the compound of formula C.

[0220] 1 H-NMR (400 MHz, CDCl3) δ 7.00 (s, 1H), 4.86 (s, 1H), 3.96 (d, 2H), 3.23 (q, 4H), 2.88 (t, 2H), 2.74 (t, 2H), 2.34 (t, 2H), 2.29 (t, 2H), 1.60-1.67 (m, 6H), 1.44-1.57 (m, 9H), 1.27-1.32 (m, 27H), 0.90 (t, 6H)

[0221] Example 4

[0222] 4-1. A compound of the following chemical formula D was prepared according to the synthetic outline shown in Fig. 4.

[0223] [Chemical Formula D]

[0224]

[0225] 4-2. Synthesis of 2-hexyldecyl 6-acrylamidohexanoate

[0226] A 500 mL 3-neck RBF was added 6-aminohexanoic acid (3.25 g, 24.74 mmol, 1.20 eq), 2-hexyldecan-1-ol (5.00 g, 20.62 mmol, 1.00 eq), p-toluenesulfonic acid monohydrate (7.06 g, 37.12 mmol, 1.80 eq), and cyclohexane (150 mL), and a Dean-Stark trap and a condenser were installed, followed by stirring and refluxing. After 24 h, the mixture was cooled to room temperature, concentrated in vacuo, extracted with methylene chloride and an aqueous NaOH solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo to obtain low-purity 2-hexyldecyl 6-aminohexanoate. Without further purification, the previously obtained 2-hexyldecyl 6-aminohexanoate, methylene chloride (100 mL), and triethylamine (4.59 g, 45.36 mmol, 2.20 eq) were added to a 250 mL 3-neck RBF, cooled to 0°C, and acryloyl chloride (2.05 g, 22.68 mmol, 1.10 eq) was added dropwise. The temperature of the reactor was raised to room temperature (20–25°C) and stirred. After 18 h, the mixture in the reactor was extracted with 3% aqueous HCl solution, and the organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo and purified using a silica column with ethyl acetate:hexane = 1:1 to obtain 2-hexyldecyl 6-acrylamidohexanoate (6.01 g, yield: 71%).

[0227] 1 H-NMR (400 MHz, CDCl3)δ6.29 (d, 1H), 6.10 (m, 1H), 5.64 (d, 1H), 3.97 (d, 2H), 3.36 (q, 2H), 2.33 (t, 2H), 1.68 - 1.54 (m, 5H), 1.41 - 1.30 (m, 28H), 0.90 (m, 6H)

[0228] 4-3. Synthesis of the compound of chemical formula D

[0229] 2-Hexyldecyl 6-acrylamidohexanoate (2500 mg, 6.10 mmol, 3 eq), tert-butyl N-(2-aminoethyl)carbamate (325.92 mg, 2.03 mmol, 1.00 eq), and n-butanol (20 mL) were added to a 100 mL 3-neck RBF, stirred, and refluxed. After 96 h, the mixture was concentrated in vacuo at 80°C and purified using a silica column with methylene chloride:methanol:ammonium hydroxide = 15:1:0.1 to obtain the compound of formula D.

[0230] 1 H-NMR (400 MHz, CDCl3)δ6.52 (t, 2H), 3.97 (d, 4H), 3.25 (q, 4H), 3.17 (br, 2H), 2.71 (t, 4H), 2.48 (t, 2H), 2.29 (m, 8H), 1.62 - 1.26 (m, 64H), 1.47 (s, 9H), 0.89 (t, 12H)

[0231] [Example of manufacturing a composition for drug delivery]

[0232] 1. Preparation of raw materials

[0233] 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.

[0234]

[0235] 2. Mixing raw materials

[0236] The required amount of raw materials was taken according to the ratio of compound of chemical formula A:DOPE:cholesterol:DMG-PEG=50:10:38.5:1.5 and mixed, with the NP ratio (amine group of lipid: phosphate group of mRNA) set to 6. 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 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.

[0237] The specific process sequence is as follows.

[0238] 1) Two autoclaved tubes were prepared (Tube (A), (B)).

[0239] 2) Each compound of chemical formula A to C and DOPE, cholesterol, and DMG-PEG in the molar number calculated according to the experimental conditions were sequentially added to Tube (A) and mixed by vortexing.

[0240] 3) Ethanol was added to the ethanol phase as needed so that the sum of all raw materials was within 12.5 mM.

[0241] 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.

[0242] 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.

[0243] 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).

[0244] 3. Evaluation of physical properties of the formulation

[0245] 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.

[0246] 2) For the manufactured formulation, mRNA encapsulation efficiency was confirmed through Ribo-green assay, and the results are shown in Table 1 below.

[0247] [Table 1]

[0248]

Claims

1. A lipid having a structure represented by the following chemical formula 1: [Chemical Formula 1] Here, R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R3 is an unsubstituted alkylene group, R is a hydrogen atom (H) or , wherein R4 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, and * represents a point of attachment to a nitrogen atom.

2. In paragraph 1, R1 and R4 are each independently substituted or unsubstituted C 1-30 Alkyl group, substituted or unsubstituted C 2-30 Alkenyl group, or substituted or unsubstituted C 2-30 It is an alkynyl group, R2 and R5 are each independently substituted or unsubstituted C 1-15 alkylene group, substituted or unsubstituted C 2-15 Alkenylene group, or substituted or unsubstituted C 2-15 It is an alkynylene group, R3 is unsubstituted C 2-9 Alkylene group, lipid.

3. In paragraph 1, R1 and R4 are each independently substituted or unsubstituted C 1-20 Alkyl group, substituted or unsubstituted C 2-20 Alkenyl group, or substituted or unsubstituted C 2-20 It is an alkynyl group, R2 and R5 are each independently substituted or unsubstituted C 1-12 alkylene group, substituted or unsubstituted C 2-12 Alkenylene group, or substituted or unsubstituted C 2-12 It is an alkynylene group, R3 is unsubstituted C 2-7 Alkylene group, lipid.

4. In paragraph 1, R1 and R4 are each independently substituted or unsubstituted C 5-20 Alkyl group, substituted or unsubstituted C 5-20 Alkenyl group, or substituted or unsubstituted C 5-20 It is an alkynyl group, R2 and R5 are each independently substituted or unsubstituted C 3-12 alkylene group, substituted or unsubstituted C 3-12 Alkenylene group, or substituted or unsubstituted C 3-12 It is an alkynylene group, R3 is unsubstituted C 2-5 Alkylene group, lipid.

5. In paragraph 1, a lipid having any one structure selected from the following chemical formulas A to L: 6.(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 reacting a compound of chemical formula c with a compound of chemical formula d to obtain a compound of chemical formula e; and (3) A method for producing a lipid, comprising: a step of reacting a compound of chemical formula e with a compound of chemical formula f to obtain a compound of chemical formula 1-1; [chemical formula a] HOC(=O)-R2-NH2 [chemical formula b] R1-OH [chemical formula c] R1-OC(=O)-R2-NH2 [chemical formula d] [chemical formula e] R1-OC(=O)-R2-NH-C(=O)-CH=CH2 [chemical formula f] tBu-OC(=O)-NH-R3-NH2 [Chemical Formula 1-1] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu In the above, R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R3 is an unsubstituted alkylene group, tBu is tert-butyl group, X is selected from the group consisting of F, CI, Br and I. 7.(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 reacting a compound of chemical formula c with a compound of chemical formula d to obtain a compound of chemical formula e; and (3) A method for producing a lipid, comprising: a step of reacting a compound of chemical formula e with a compound of chemical formula f to obtain a compound of chemical formula 1-2; [chemical formula a] HOC(=O)-R2-NH2 [chemical formula b] R1-OH [chemical formula c] R1-OC(=O)-R2-NH2 [chemical formula d] [chemical formula e] R1-OC(=O)-R2-NH-C(=O)-CH=CH2 [chemical formula f] tBu-OC(=O)-NH-R3-NH2 [Chemical Formula 1-2] [R1-OC(=O)-R2-NH-C(=O)-CH2-CH2]2-N-R3-NH-C(=O)O-tBu In the above, R1 is each independently a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R2 is each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R3 is an unsubstituted alkylene group, tBu is tert-butyl group, X is selected from the group consisting of F, CI, Br and I.

8. A method for producing a lipid, comprising: a step of reacting the compound of chemical formula 1-1 obtained in paragraph 6 with a compound of chemical formula g to obtain a compound of chemical formula 1-3; [Chemical Formula 1-1] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-NH-R3-NH-C(=O)O-tBu [chemical formula g] R4-OC(=O)-R5-NH-C(=O)-CH=CH2 [Chemical Formula 1-3] R1-OC(=O)-R2-NH-C(=O)-CH2-CH2-N(A)-R3-NH-C(=O)O-tBu In the above, R1 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R2 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R3 is an unsubstituted alkylene group, A is -CH2-CH2-C(=O)-NH-R5-C(=O)O-R4, where R4 is a substituted or unsubstituted alkyl group, alkenyl group or alkynyl group, R5 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, tBu is tert-butyl group, X is selected from the group consisting of F, CI, Br and I.

9. A drug delivery composition comprising the lipid of any one of claims 1 to 5.

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