Lipid compounds or their derivatives and pharmaceutical compositions using the same

Abstract

To provide a lipid compound or a derivative thereof, and a pharmaceutical composition using the same.SOLUTION: A lipid compound has a structure represented by Formula (I). (In the formula, Z1 is O or S, Z2 is -Q1-A1-Q2-A2-X1, Z3 and Z2 are the same; Z4 is -Q3-A3-Q4-A4-X2; Q1 and Q3 are each independently a single bond, -O- or -NH-; Q2 and Q4 are each independently a single bond, -O-, -NH-, -S-S- or the like; A1 is a C1-C6 alkylene group; A3 is a single bond or a C1-C6 alkylene group; A2 and A4 are each independently a single bond, a C1-C12 alkylene group or the like; X1 is hydrogen, a C1-C24 alkyl group or the like; and X2 is hydrogen, a C6-C12 aryl group or the like.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] This technical field relates to lipid compounds or their derivatives and pharmaceutical compositions using the same. [Background technology]

[0002] In the biotechnology industry, the success of messenger RNA (mRNA) vaccines, with lipid nanoparticle (LNP) delivery technology—a key element of nucleic acid drugs—being the focus of development, is driving aggressive investment in the nucleic acid drug field. However, ribonucleic acid drugs are easily degraded in the human body and become negatively charged, making them unable to easily pass through cell membranes. Therefore, they require the assistance of a carrier and must be delivered encapsulated within a carrier.

[0003] Ionizable cationic lipids are currently commonly used carriers for lipid nanoparticles. However, when administered in vivo, conventional ionizable cationic lipids can cause significant side effects. Problems already observed include a low percentage of effective delivery to the target, resulting in relatively low or no therapeutic effect. Furthermore, ionizable cationic lipids used as carriers for lipid nanoparticles need to be adjusted to a specific pH value so that activators can be incorporated into the carrier, the activators can be prevented from degradation during administration, and the activators can be released immediately upon reaching their target. [Overview of the project] [Problems that the invention aims to solve]

[0004] Therefore, this industry needs to develop novel lipids that can satisfy the requirements of lipid-nucleic acid delivery systems. [Means for solving the problem]

[0005] According to one embodiment of the present disclosure, the present disclosure provides a lipid compound or a derivative thereof, and the derivative of the lipid compound may be a pharmaceutically acceptable salt of the lipid compound or a solvate of the lipid compound. According to one embodiment of the present disclosure, the lipid compound has a structure represented by formula (I).

[0006]

Chemical formula

[0007] In the formula, Z , 2 , 1 , 2 , 1 , , 2 , , 1 , , 4 , 3 , , 1 , 4 , 3 , , is O or S, and Z 2 is -Q 1 -A 1 -Q 2 -A 2 -X 1 and Z 3 and Z 2 are the same, and Z 4 is -Q 3 -A 3 -Q 4 -A 4 -X 2 and Q 1 and Q 3 each is independently a single bond, -O- or -NH-, and Q 2 and Q 4 each is independently a single bond, -O-, -NH-, -S-S-,

Chemical formula

[0008] According to some embodiments of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the lipid compounds or derivatives thereof as described above in the present disclosure and a helper lipid.

[0009] The following embodiments will be described in detail with reference to the attached drawings. [Brief description of the drawing] The present invention can be better understood by referring to the attached drawings and reading the following detailed description and examples.

[0010] none. [Modes for carrying out the invention]

[0011] In the following detailed description, numerous specific details are provided for illustrative purposes so that the disclosed embodiments may be fully understood. However, it will be apparent that one or more embodiments may be implemented without these specific details. Also, for the sake of brevity, well-known structures and apparatus are shown schematically.

[0012] The following relates to lipid compounds or derivatives thereof as described in this disclosure and pharmaceutical compositions using them. It should be understood that the following description provides many different embodiments or examples for carrying out this disclosure. The specific components and compositions described below are merely illustrative of this disclosure. Naturally, these are merely examples and do not limit this disclosure. In this disclosure, the word “about” means that a particular amount increases or decreases by an amount that would be generally considered reasonable by those skilled in the art.

[0013] This disclosure provides lipid compounds or derivatives thereof. According to one embodiment of this disclosure, the lipid compounds described herein are ionizable cationic phospholipids having a specific structure in which the main core is pentavalent phosphorus and which may contain one to three ionizable tertiary amine groups. The lipid compounds described herein are more readily metabolized in vivo and less toxic. Since the lipid compounds or derivatives described herein can be positively charged in an acidic environment, they can adsorb negatively charged nucleic acids to form lipid nanoparticles (LNPs). In addition, this disclosure also provides pharmaceutical compositions comprising the lipid compounds or derivatives described herein. By having the lipid compounds or derivatives described herein, the pharmaceutical compositions described herein can form lipid nanoparticles (LNPs) that encapsulate ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA), thereby achieving objectives such as the protection of ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA), reducing their degradation rate, and improving the transfection efficiency of cells.

[0014] According to one embodiment of the present disclosure, the lipid compound provided by the present disclosure has a structure represented by formula (I).

[0015]

Chemical formula

[0016] In the formula, Z 1 may be O or S, and Z 2 is -Q 1 -A 1 -Q 2 -A 2 -X 1 and may be, Z 3 and Z 2 are the same, and Z 4 is -Q 3 -A 3 -Q 4 -A 4 -X 2 and may be, and each of Q 1 and Q 3 may each independently be a single bond, -O-, or -NH-. Q 2 and Q 4 may each independently be a single bond, -O-, -NH-, -S-S-,

Chemical formula

Chemical formula

[0017] According to one embodiment of the present disclosure, the derivative of the lipid compound described herein may be a pharmaceutically acceptable salt or solvate of the lipid compound.

[0018] According to one embodiment of the present disclosure, a pharmaceutically acceptable salt of a lipid compound may be formed by reacting the lipid compound with a pharmaceutically acceptable acid. Here, pharmaceutically acceptable salts include salts formed from a lipid compound and an inorganic acid, such as hydrochloride, phosphate, diphosphate, hydrobromide, sulfate, sulfinate, or nitrate. According to one embodiment of the present disclosure, pharmaceutically acceptable salts include salts formed from a lipid compound and an organic acid, such as lactate, oxalate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, sulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, trifluoroacetate, amino acid salt, or acetate. In addition, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium.

[0019] According to one embodiment of the present disclosure, the solvate of the lipid compound includes a hydrate of the lipid compound or an alkoxide of the lipid compound.

[0020] According to one embodiment of the present disclosure, a single bond as described herein means that there is no single atom present at the site in question. For example, Z 2 ga-Q 1 -A 1 -Q 2 -A 2 -X 1 In a structure such as Q 2 If Q is a single bond, 2 No single atom exists in the region represented by A 1 and A 2 They are directly connected, Z 2 ga-Q 1 -A 1 -A 2 -X 1 It has this structure.

[0021] According to one embodiment of the present disclosure, the alkyl group described herein may be a linear or branched alkyl group. According to one embodiment of the present disclosure, the alkenyl group described herein may be a linear or branched alkenyl group and may contain at least one carbon-carbon double bond. According to one embodiment of the present disclosure, the alkynyl group described herein may be a linear or branched alkynyl group and may contain at least one carbon-carbon triple bond.

[0022] For example, the C1-C24 alkyl group may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, or an isomer thereof.

[0023] According to one embodiment of the present disclosure, the alkanol group described herein may be a linear or branched alkanol group. According to one embodiment of the present disclosure, the alkenol group described herein may be a linear or branched alkenol group and may contain at least one carbon-carbon double bond. According to one embodiment of the present disclosure, the alkynol group described herein may be a linear or branched alkynyl group and may contain at least one carbon-carbon triple bond. Here, the term "alkanol group" refers to an alkyl group in which at least one hydrogen atom on its carbon is substituted with a hydroxyl group. The term "alkenol group" refers to an alkenyl group in which at least one hydrogen atom on its carbon is substituted with a hydroxyl group. The term "alkynol group" refers to an alkynyl group in which at least one hydrogen atom on its carbon is substituted with a hydroxyl group. In addition, according to one embodiment of the present disclosure, the C6-C12 aryl group described herein may be phenyl, biphenyl, or naphthyl.

[0024] According to one embodiment of this disclosure, the alkylene group described herein may be a linear or branched alkylene group. According to one embodiment of this disclosure, the alkenylene group described herein may be a linear or branched alkenylene group. According to one embodiment of this disclosure, the alkynylene group described herein may be a linear or branched alkynylene group.

[0025] For example, the C1-C24 alkylene group may be a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, or an isomer thereof.

[0026] According to one embodiment of the present disclosure, in a lipid compound having the structure represented by formula (I) described herein, Z 4 is Z 2 Different from (that is, Z 4 is Z 3 (Different from).

[0027] According to one embodiment of the present disclosure, in a lipid compound having the structure represented by formula (I) described herein, Z 2 , Z 3 and Z 4 They are the same.

[0028] According to one embodiment of the present disclosure, in a lipid compound having the structure represented by formula (I) described herein, Q 1 and Q 2 They cannot be single bonds at the same time. In other words, Q 1 When it is a single bond, Q 2 It is not a single bond, and Q 2 When it is a single bond, Q 1 It is not a single bond.

[0029] According to one embodiment of this disclosure, Q 1 It is a single bond, and Q 2 -O-, -NH-, -SS-, [ka] That's fine.

[0030] According to one embodiment of this disclosure, Q 2 It is a single bond, and Q 1 It can be -O- or -NH-.

[0031] According to one embodiment of this disclosure, Q 1 may be -O- or -NH-, and Q 2 -O-, -NH-, -SS-, [ka] That's fine.

[0032] According to one embodiment of the present disclosure, in a lipid compound having the structure represented by formula (I) described herein, Q 3 and Q 4 They cannot be single bonds at the same time. In other words, Q 3 When it is a single bond, Q 4 It is not a single bond, and Q 4 When it is a single bond, Q 3 It is not a single bond.

[0033] According to one embodiment of this disclosure, Q 3 It is a single bond, and Q 4 -O-, -NH-, -SS-, [ka] That's fine.

[0034] According to one embodiment of this disclosure, Q 4 It is a single bond, and Q 3 It can be -O- or -NH-.

[0035] According to one embodiment of this disclosure, Q 3 is -O- or -NH-, Q 4 -O-, -NH-, -SS-, [ka] It is acceptable for it to be, and A 3 It is not a single bond.

[0036] According to one embodiment of the present disclosure, A 2 When is a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkynylene group, Q 2 It is not a single bond.

[0037] According to one embodiment of the present disclosure, A 3 When A is a single bond, 4 This is a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkylylene group.

[0038] According to one embodiment of the present disclosure, A 4 When A is a single bond, 3 It is a C1-C6 alkylene group.

[0039] According to one embodiment of the present disclosure, A 3 It is a single bond, and A 4 When it is a single bond, Q 3 It is -O- or -NH-.

[0040] According to one embodiment of the present disclosure, the lipid compound having the structure represented by formula (I) described herein may be any of the following:

[0041] [ka]

[0042] In the formula, Q 1 Q 2 Q 3 Q 4 , A 1 , A 2 , A 3 , A 4 , X 1 and X 2 The definition is the same as above.

[0043] According to one embodiment of the present disclosure, the disclosure also provides a pharmaceutical composition, for example, a lipid nanoparticle composition. According to one embodiment of the present disclosure, the pharmaceutical composition comprises a lipid compound or a derivative thereof described in the present disclosure and a sterol.

[0044] According to one embodiment of the present disclosure, the pharmaceutical composition contains 100 moles of a lipid compound (and / or its derivative), and the sterol content may be 50 to 300 moles, for example, 60 moles, 80 moles, 100 moles, 120 moles, 150 moles, 180 moles, 200 moles, 230 moles, 250 moles, or 280 moles. According to one embodiment of the present disclosure, the sterol may be cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or a combination thereof.

[0045] According to one embodiment of the present disclosure, the pharmaceutical composition may further contain an auxiliary lipid, which may be 5 to 200 moles, for example, 6 moles, 8 moles, 10 moles, 20 moles, 30 moles, 50 moles, 75 moles, 100 moles, 120 moles, 150 moles, 170 moles, or 190 moles.

[0046] According to one embodiment of the present disclosure, the auxiliary lipids are 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), and 1-stearoyl-2-oleoyl-sn- Lysero-3-phosphocholine (SOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG), sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), La-phosphatidylcholine (EPC), phosphatidylcholine (PC), or combinations thereof.

[0047] According to one embodiment of the present disclosure, the pharmaceutical composition may further contain polyethylene glycol (PEG)-ylated lipids, the amount of which may be 1 to 30 moles, for example, 2 moles, 3 moles, 4 moles, 5 moles, 6 moles, 7 moles, 8 moles, 9 moles, 10 moles, 11 moles, 12 moles, 13 moles, 14 moles, 15 moles, 16 moles, 17 moles, 18 moles, 19 moles, 20 moles, 21 moles, 22 moles, 23 moles, 24 moles, 25 moles, 26 moles, 27 moles, 28 moles, or 29 moles.

[0048] According to one embodiment of the present disclosure, the PEGylated lipid may be polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, polyethylene glycol-modified ceramide, polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, polyethylene glycol-modified dialkylglycerol, polyethylene glycol-modified sterol, polyethylene glycol-modified phospholipid, or a combination thereof.

[0049] According to one embodiment of the present disclosure, the PEGylated lipids are 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-dipalmitoyl-sn-glycerol-3-succinate-polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene Polyethylene glycol (DPPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycerol-3-methoxy-polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-sn-glycerol-methoxy-polyethylene glycol (DPG-PEG), α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159) or a combination thereof.

[0050] According to one embodiment of the present disclosure, the pharmaceutical composition may further contain nucleic acids, the nucleic acid content of which may be 0.1 to 30 mole parts, for example, 0.2 mole parts, 0.5 mole parts, 1 mole part, 1.5 mole parts, 2 mole parts, 3 mole parts, 4 mole parts, 5 mole parts, 6 mole parts, 7 mole parts, 8 mole parts, 9 mole parts, 10 mole parts, 11 mole parts, 12 mole parts, 13 mole parts, 14 mole parts, 15 mole parts, 16 mole parts, 17 mole parts, 18 mole parts, 19 mole parts, 20 mole parts, 21 mole parts, 22 mole parts, 23 mole parts, 24 mole parts, 25 mole parts, 26 mole parts, 27 mole parts, 28 mole parts, or 29 mole parts.

[0051] According to one embodiment of the present disclosure, the nucleic acid may be deoxyribonucleic acid (DNA), plasmid DNA, messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), small activated ribonucleic acid (saRNA), circular ribonucleic acid (circular RNA), or a combination thereof.

[0052] To make the above-described content, other purposes, features, and advantages of this disclosure clearer and easier to understand, exemplary embodiments are shown and described in detail below, but are not limited to these embodiments.

[0053] lipid compounds The lipid compounds described in the examples of this disclosure are listed in Table 1.

[0054] [Table 1] JPEG0007876498000017.jpg199151JPEG0007876498000018.jpg197149JPEG0007876498000019.jpg198150JPEG0007876498000020.jpg198150 JPEG0007876498000021.jpg199151JPEG0007876498000022.jpg198149JPEG0007876498000023.jpg199150JPEG0007876498000024.jpg196148 JPEG0007876498000025.jpg221151JPEG0007876498000026.jpg202152JPEG0007876498000027.jpg199150JPEG0007876498000028.jpg200151 JPEG0007876498000029.jpg197148JPEG0007876498000030.jpg196148JPEG0007876498000031.jpg196149JPEG0007876498000032.jpg196148

[0055] To further explain the methods for producing the lipid compounds described in this disclosure, the production processes for the lipid compounds described in Examples 1-3, 6-15, 18, and 19 are described below.

[0056] Example 1 Compound (1) (2 equivalents) and tetrahydrofuran (THF) were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a solution of lithium bis(trimethylsilyl)amide (LiHMDS) (dissolved in tetrahydrofuran; concentration 1.0 M, LiHMDS 3 equivalents) was added to the reaction flask. After stirring at room temperature for 1 hour, compound (2) (1 equivalent) was added dropwise to the reaction flask. After the reaction was complete, the resulting product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, water was removed with sodium sulfate (Na2SO4) to remove the solvent, and the resulting product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (1). The reaction equation for the above reaction is as follows.

[0057] [ka]

[0058] Next, the lipid compound (1) described in Example 1 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 4.48 (s, 4H), 4.18 (t, J=7.0, 7.5Hz, 2H), 3.40 (s, 4H), 3.09 (m, 9H), 1.68 (s, 9H), 1.36-1.26 (m, 55H), 0.87 (t, J=7.0, 7.0Hz, 12H). Subsequently, lipid compound (1) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 718.7.

[0059] Example 2 Compound (1) (2.5 equivalents) and tetrahydrofuran (THF) were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a solution of lithium bis(trimethylsilyl)amide (LiHMDS) (dissolved in tetrahydrofuran; concentration 1.0 M, LiHMDS 3 equivalents) was added to the reaction flask. After stirring at room temperature for 1 hour, compound (3) (1 equivalent) was added dropwise to the reaction flask. After the reaction was complete, the resulting product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, water was removed with sodium sulfate (Na2SO4) to remove the solvent, and the resulting product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (2). The reaction equation for the above reaction is as follows.

[0060] [ka]

[0061] Next, the lipid compound (2) described in Example 2 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 4.42 (s, 2H), 4.29 (s, 2H), 4.10-4.09 (m, 2H), 3.73 (s, 4H), 3.47-3.43 (m, 2H), 3.32-3.30 (m, 4H), 3.11-3.04 (m, 8H), 1.67 (s, 12H), 1.32-1.25 (m, 77H), 0.88 (t, J=7.5, 7.5Hz, 18H). Subsequently, lipid compound (2) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + =986.1.

[0062] Example 3 Compound (4) (2 equivalents) and tetrahydrofuran (THF) were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a solution of lithium bis(trimethylsilyl)amide (LiHMDS) (dissolved in tetrahydrofuran; concentration 1.0 M, LiHMDS 3 equivalents) was added to the reaction flask. After stirring at room temperature for 1 hour, compound (2) (1 equivalent) was added dropwise to the reaction flask. After the reaction was complete, the resulting product was concentrated and extracted with n-hexane and brine. After collecting the organic phase, water was removed with sodium sulfate (Na2SO4) to remove the solvent, and the resulting product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (3). The reaction equation for the above reaction is as follows.

[0063] [ka]

[0064] Next, the lipid compound (3) described in Example 3 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): 4.13 (t, J=6.5, 7.5Hz, 6H), 3.19 (s, 4H), 3.03-3.02 (m, 8H), 2.17-2.14 (m, 11H), 1.67 (s, 8H), 1.35-1.26 (m, 52H), 0.87 (t, J=7.0, 6.5Hz, 12H). Subsequently, lipid compound (3) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 746.5.

[0065] Example 6 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (6) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (6). The reaction equation for the above reaction is as follows.

[0066] [ka]

[0067] Next, the lipid compound (6) described in Example 6 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 10.62 (s, 1H), 8.73 (s, 1H), 3.60 (s, 3H), 3.22 (s, 3H), 3.09-3.06 (m, 4H), 2.54-2.56 (m, 2H), 2.11-2.13 (m, 4H), 1.63 (s, 12H), 1.32-1.26 (m, 77H), 0.88 (t, J=7.5, 7.5Hz, 18H). Subsequently, lipid compounds (6) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1151.3.

[0068] Example 7 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (7) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (7). The reaction equation for the above reaction is as follows.

[0069] [ka]

[0070] Next, the lipid compound (7) described in Example 7 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 7.98 (s, 3H), 4.49 (s, 7H), 3.21 (s, 6H), 3.10-2.8 (m, 18H), 2.48 (s, 5H), 2.07 (s, 5H), 1.80-1.40 (m, 26H), 1.40-1.10 (m, 78H), 0.84 (t, J=6.0, 7.0Hz, 18H). Subsequently, the lipid compound (7) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1235.5.

[0071] Example 8 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (8) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (8). The reaction equation for the above reaction is as follows.

[0072] [ka]

[0073] Next, the lipid compound (8) described in Example 8 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 11.52 (s, 1H), 5.57 (s, 1H), 3.18 (s, 7H), 2.30 (s, 10H), 2.50 (s, 3H), 2.06 (s, 4H), 1.67-1.65 (m, 11H), 1.34-1.25 (m, 51H), 0.87 (t, J=2.0, 5.5Hz, 9H). Subsequently, the lipid compound (8) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1319.6.

[0074] Example 9 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Next, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (7) (4 equivalents) and compound (9) (1 equivalent) were added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (9). The reaction equation for the above reaction is as follows.

[0075] [ka]

[0076] Next, the lipid compound (9) described in Example 9 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 H-NMR (500 MHz, CDCl3): 1 ¹H-NMR (500MHz, CDCl3): δ 10.84 (s,1H), 8.03 (s,2H), 3.66 (s,2H), 3.33-3.24 (m,6H), 3.04-2.99 (m,16H), 2.81 (s,1H), 2.53 (s,4H), 2.11 (s,4H), 1.76-1.56 (m,12H), 1.33-1.27 (m,38H), 0.88 (t,J=6.5,7.5Hz,9H). Subsequently, the lipid compound (9) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + =955.8.

[0077] Example 10 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (10) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (10). The reaction equation for the above reaction is as follows.

[0078] [ka]

[0079] Next, the lipid compound (10) described in Example 10 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J=7.0, 7.5Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J=6.5, 7.0Hz, 9H). Subsequently, lipid compounds (10) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1024.8.

[0080] Example 11 Compound (5) (1 equivalent) and dichloromethane were added to the reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Then, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (6) (4 equivalents) and compound (9) (1 equivalent) were added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (11). The reaction equation for the above reaction is as follows.

[0081] [ka]

[0082] Next, the lipid compound (11) described in Example 11 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J=7.0, 7.5Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J=6.5, 7.0Hz, 9H). Subsequently, lipid compounds (11) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 899.7.

[0083] Example 12 Compound (5) (1 equivalent) and dichloromethane were added to the reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (11) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (12). The reaction equation for the above reaction is as follows.

[0084] [ka]

[0085] Next, the lipid compound (12) described in Example 12 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 8.50 (s, 2H), 6.46 (s, 5H), 4.10-3.90 (m, 3H), 3.90-3.0 (m, 13H), 2.55 (s, 3H), 2.15 (s, 3H), 1.60-1.20 (m, 61H), 0.88 (t, J=7.0, 7.5Hz, 10H). Subsequently, lipid compounds (12) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1499.4.

[0086] Example 13 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (12) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (13). The reaction equation for the above reaction is as follows.

[0087] [ka]

[0088] Next, the lipid compound (13) described in Example 13 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 10.68 (s, 2H), 8.78 (s, 3H), 3.59 (d, J=4.0Hz, 5H), 3.45-3.20 (m, 12H), 3.20-3.0 (m, 11H), 2.60-2.40 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.45 (m, 10H), 1.40-1.20 (m, 32H), 0.88 (t, J=6.5Hz, 15H). Subsequently, the lipid compound (13) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 898.8.

[0089] Example 14 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (13) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (14). The reaction equation for the above reaction is as follows.

[0090] [ka]

[0091] Next, the lipid compound (14) described in Example 14 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 10.67 (s, 2H), 8.75 (s, 2H), 4.25-4.0 (m, 5H), 3.62 (d, J=4.5Hz, 4H), 3.25-3.20 (m, 4H), 3.20-3.0 (m, 9H), 2.65-2.45 (m, 4H), 2.25-2.15 (m, 4H), 1.80-1.60 (m, 9H), 1.45-1.20 (m, 44H), 0.89 (t, J=6.0, 7.5Hz, 12H). Subsequently, lipid compounds (14) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1067.

[0092] Example 15 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (14) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (15). The reaction equation for the above reaction is as follows.

[0093] [ka]

[0094] Next, the lipid compound (15) described in Example 15 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 10.77 (s, 2H), 8.80 (s, 2H), 3.62-3.50 (m, 7H), 3.30-3.20 (m, 5H), 3.20-3.0 (m, 11H), 2.60-2.50 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.50 (m, 11H), 1.50-1.20 (m, 40H), 0.89 (t, J=5.0, 7.0Hz, 14H). Subsequently, the lipid compound (15) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 982.8.

[0095] Example 18 Compound (5) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (15) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (18). The reaction equation for the above reaction is as follows.

[0096] [ka]

[0097] Next, the lipid compound (18) described in Example 18 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 8.66 (s, 2H), 4.60-3.90 (m, 13H), 3.90-3.0 (m, 9H), 2.70-2.40 (m, 4H), 2.30-2.0 (m, 4H), 1.70-1.20 (m, 45H), 0.89 (t, J=6.0, 7.0Hz, 13H). Subsequently, lipid compounds (18) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 864.3.

[0098] Example 19 Compound (5) (1 equivalent) and dichloromethane were added to the reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (16) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (19). The reaction equation for the above reaction is as follows.

[0099] [ka]

[0100] Next, the lipid compound (19) described in Example 19 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown below: 1 ¹H-NMR (500MHz, CDCl3): δ 8.63 (s, 2H), 4.70-3.90 (m, 12H), 3.90-3.0 (m, 17H), 2.70-2.50 (m, 4H), 2.30-2.10 (m, 4H), 1.60-1.20 (m, 48H), 0.88 (t, J=6.5, 7.5Hz, 11H). Subsequently, lipid compounds (19) were analyzed by liquid chromatography-mass spectrometry (LC-MS) and the M / Z ratio was measured: [M+H] + = 1247.1.

[0101] Example 23 Compound (23a) (1 equivalent) and dichloromethane were added to the reaction flask under nitrogen to obtain a solution concentration of 0.05 M. Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (17) (2.5 equivalents) was added dropwise to the reaction flask. After reacting for 12 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (23). The reaction equation for the above reaction is as follows.

[0102] [ka]

[0103] Next, the lipid compound (23) described in Example 23 was analyzed by liquid chromatography-mass spectrometry (LC-MS) and its M / Z ratio was measured: [M+H] + = 1147.6.

[0104] Example 24 Compound (24a) (1 equivalent) and dichloromethane were added to a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Then, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for 10 minutes, compound (18) (2.5 equivalents) was added dropwise to the reaction flask. After reacting for 16 hours, the reaction product was purified by high-performance liquid chromatography (HPLC) (using acetonitrile and aqueous trifluoroacetic acid (TFA) solutions (concentration 0.1%) as eluents) to obtain lipid compound (24). The reaction equation for the above reaction is as follows.

[0105] [ka]

[0106] Next, the lipid compound (24) described in Example 24 was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the M / Z ratio was measured: [M+H] + = 1175.7.

[0107] Preparation of composition

[0108] Example 35 Luciferase mRNA (product number R1018, purchased from APExBIO) was dissolved in an acidic buffer (product number J63669, purchased from Alfa Aesar) (pH value 4.5) to obtain an aqueous nucleic acid solution (concentration: 0.1 mg / mL).

[0109] The lipid compound (1) prepared in Example 1, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and DMG-PEG lipid (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol) (product number: DMG-PEG-2K, purchased from Nippon Fine Chemical) were dissolved in ethanol to obtain a lipid solution (concentration: 10-15 mg / mL). The content of lipid compound (1), cholesterol, DSPC, and DMG-PEG lipid is shown in Table 2.

[0110] Next, the nucleic acid aqueous solution and the lipid solution were mixed using a microfluidic system (NanoAssemblr Ignite system, purchased from Precision NanoSystem Inc.), and the volumes of the nucleic acid aqueous solution and the lipid solution were adjusted so that the ratio of nitrogen atoms (N) of the lipid compound (1) to phosphorus atoms (P) derived from luciferase mRNA (N / P) was 12. Subsequently, after ultrafiltration or dialysis, the resulting composition was self-assembled to form nucleic acid-lipid nanoparticle carriers (1).

[0111] Examples 36-43 Nucleic acid-lipid nanoparticle carriers (2) to (9) were obtained by the same method as in Example 35, but the components and / or content in the lipid solution and the N / P value (adjusted by the volume ratio of the nucleic acid aqueous solution to the lipid solution) were changed based on Table 1.

[0112] Comparative Example 1 A nucleic acid-lipid nanoparticle carrier (10) was obtained by the same method as in Example 35, but the lipid compound (1) was replaced with (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA), and the N / P value was adjusted (by adjusting the volume ratio of the nucleic acid aqueous solution to the lipid solution).

[0113] Comparative Example 2 A nucleic acid-lipid nanoparticle carrier (11) was obtained by the same method as in Example 35, but the lipid compound (1) was replaced with ptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid (SM-102), and the N / P value was adjusted (by adjusting the volume ratio of the nucleic acid aqueous solution to the lipid solution).

[0114] [Table 2]

[0115] Examples 44-52 Nucleic acid-lipid nanoparticle carriers (12) to (20) were obtained by the same method as in Example 35, but the components and / or content in the lipid solution and the N / P value (adjusted by the volume ratio of the nucleic acid aqueous solution to the lipid solution) were changed based on Table 3.

[0116] [Table 3]

[0117] Examples 53-61 Nucleic acid-lipid nanoparticle carriers (21) to (29) were obtained by the same method as in Example 35, but the components and / or content in the lipid solution and the N / P value (adjusted by the volume ratio of the nucleic acid aqueous solution to the lipid solution) were changed based on Table 4.

[0118] [Table 4]

[0119] Examples 62-70 Nucleic acid-lipid nanoparticle carriers (30) to (38) were obtained by the same method as in Example 35, but the components and / or content in the lipid solution and the N / P value (adjusted by the volume ratio of the nucleic acid aqueous solution to the lipid solution) were changed based on Table 5.

[0120] [Table 5]

[0121] Characterization of pharmaceutical compositions The particle size, polydispersity index (PdI), zeta potential, ribonucleic acid recovery rate (mRNA recovery), ribonucleic acid encapsulation efficiency (EE), and acid dissociation constant (pKa) of nucleic acid-lipid nanoparticle carriers (1) to (9) obtained in Examples 35 to 43, and nucleic acid-lipid nanoparticle carriers (10) and (11) obtained in Comparative Examples 1 and 2 were evaluated. The results are shown in Table 6.

[0122] The particle size, polydispersity index (PdI), and zeta potential of nucleic acid-lipid nanoparticle carriers were evaluated as follows: 20 μL of sample was taken, added to 330 μL of Dulbecco's phosphate-buffered saline (DPBS), and after uniform shaking, the liposome particle size and polydispersity index (PdI) were measured using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical). Furthermore, 20 μL of sample was taken, added to 680 μL of sodium chloride aqueous solution (concentration: 10 mM), and after uniform shaking, the liposome zeta potential was measured using a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical).

[0123] The ribonucleic acid recovery rate (mRNA recovery) was evaluated as follows: After sterile filtering of the nucleic acid lipid nanoparticle carrier, 100 μL of the sample was taken and added to 25 μL of 5% Triton X-100. Then, after incubation at 37°C for 10 minutes, 100 μL of chloroform was added. After 10 minutes of high-speed shaking (rotation speed: 2000 rpm) and 30 minutes of centrifugation (centrifugal force: 14000 × g), 80 μL of the supernatant was taken and its absorbance was analyzed using a UV-Vis spectrophotometer (UV7, Mettler Toledo). Subsequently, the ribonucleic acid concentration was calculated using the formula (1) shown below, and the recovery rate was calculated using the formula (2) shown below. In the formulas, C is the ribonucleic acid concentration, A 260nmVf is the absorbance value of ribonucleic acid at a wavelength of 260 nm, Vf is the volume of the supernatant (μL), and W is the initial weight of ribonucleic acid (ng).

[0124] C=(A 260nm ×40) / (100 / 125) Equation (1) Recovery rate (%) = [(C × Vf) / W] × 100% Equation (2)

[0125] The encapsulation efficiency (EE) of ribonucleic acid was measured using the Quant-iT RiboGreen RNA quantitative detection kit (RNA Assay kit) (purchased from Invitrogen). Specifically, the concentration of mRNA in a dispersion containing nucleic acid-lipid particles was quantified in the presence and absence of 0.2% Triton X-100 surfactant, and the encapsulation efficiency (EE) of ribonucleic acid was calculated using the formula (3) shown below. In the formula, EE is the encapsulation efficiency of ribonucleic acid, and C Free This is the concentration of unencapsulated ribonucleic acid, C All This represents the total ribonucleic acid concentration.

[0126] EE(%)=(1-C Free / C All )×100% Equation (3)

[0127] To determine the acid dissociation constant (pKa), a titration method based on 6-p-toluidino-2-naphthalenesulfonic acid (TNS) was used. The fluorescence intensity titration curves of 18 samples in buffer solutions were analyzed using a microdisk fluorescence analyzer (Infinite F200 Pro, Tecan, excitation wavelength: 320 nm, emission wavelength: 448 nm) at pH values ​​ranging from 2.5 to 11.0. The titration curves were fitted using a sigmoid titration, and the calculated acid dissociation constant (pKa) was determined at the pH value at which the nucleic acid-lipid nanoparticle carrier reached half of its maximum fluorescence intensity.

[0128] [Table 6]

[0129] The particle size, polydispersity index (PdI), zeta potential, ribonucleic acid recovery rate (mRNA recovery), ribonucleic acid encapsulation efficiency (EE), and acid dissociation constant (pKa) of the nucleic acid-lipid nanoparticle carriers (12) to (20) obtained in Examples 44 to 52 were evaluated. The results are shown in Table 7.

[0130] [Table 7]

[0131] The particle size, polydispersity index (PdI), zeta potential, ribonucleic acid recovery rate (mRNA recovery), ribonucleic acid encapsulation efficiency (EE), and acid dissociation constant (pKa) of the nucleic acid-lipid nanoparticle carriers (21) to (29) obtained in Examples 53 to 61 were evaluated. The results are shown in Table 8.

[0132] [Table 8]

[0133] The particle size, polydispersity index (PdI), zeta potential, ribonucleic acid recovery rate (mRNA recovery), ribonucleic acid encapsulation efficiency (EE), and acid dissociation constant (pKa) of the nucleic acid-lipid nanoparticle carriers (30) to (38) obtained in Examples 62 to 70 were evaluated. The results are shown in Table 9.

[0134] [Table 9]

[0135] Tables 6 to 9 show that the particle size of nucleic acid-lipid nanoparticle carriers formed by self-assembly of the pharmaceutical compositions of this disclosure ranged from approximately 50 nm to 250 nm, and the polydispersity index could be between 0.03 and 0.33. In some examples, the ribonucleic acid recovery rate (%) of nucleic acid-lipid nanoparticle carriers formed by self-assembly of some of the pharmaceutical compositions of this disclosure was higher than approximately 80%, and the acid dissociation constant (pKa) could be in the range of 5 to 8.5.

[0136] Evaluation of transfection efficiency and cell viability: HEK293 cells were cultured in 5 × 10⁶ well plates. 4 Cells were seeded in wells and cultured for 24 hours. Next, 97 μL of Opti-MEM medium and 3 μL of lipofectamine reagent (purchased from Invitrogen) were homogeneously mixed and reacted at room temperature for 10 minutes to obtain the first solution. 1 μL of luciferase mRNA (product number: R1018, purchased from APExBIO) (concentration: 1 mg / mL) and 99 μL of Opti-MEM® medium were homogeneously mixed to obtain the second solution. Then, the second solution was added to the first solution, homogeneously mixed, and reacted at room temperature for 5 minutes to obtain the transfection solution. Next, the lipid nanoparticle (LNP) solutions prepared in Examples 35-43 were diluted with 1×PBS to obtain diluted lipid nanoparticle solutions (volume: 200 μL, concentration: 5 μg / mL). Next, 800 μL of cell medium (product number: MT-10-009-CVS, purchased from Corning) was added to each diluted lipid nanoparticle solution and mixed with the transfection solution to obtain each cell co-culture solution (luciferase mRNA concentration: 0.1 μg / 100 μL). Subsequently, 100 μL of each cell co-culture solution was added to each 96-well culture plate and co-cultured with cells (0.1 μg mRNA / well). After 24 hours of incubation, the supernatant was obtained. TakeNext, it was added to 20 μL of the cell viability assay reagent (GF-AFC). After reacting at 37 °C for 35 to 40 minutes, the fluorescence value of the reaction product was measured with a fluorometer (Ex: 380 nm; Em: 540 nm). By subtracting the fluorescence detection intensity of the sample and the fluorescence value of the untreated negative control group, the cell viability in the presence of different lipid nanoparticle (LNP) solutions was calculated. The results are shown in Table 10.

[0137]

Table 10

[0138] As can be seen from the above, it is understood that the lipid compounds described in the present disclosure have little effect on cell viability when performing in vivo cell experimental analysis.

[0139] Next, 100 μL of ONE-Glo Reagent was added to each well of the culture plate, and its luminescence value was measured within 3 minutes to evaluate the transfection ability of different lipid nanoparticle (LNP) solutions. The results are shown in Table 11.

[0140]

Table 11

[0141] As can be seen from Table 11, it is understood that the lipid compounds described in the present disclosure can actually improve the transfection efficiency of HEK293 cells.

[0142] It will be apparent to those skilled in the art that various modifications and changes can be made to the disclosed embodiments. The detailed descriptions and examples are intended to be regarded merely as illustrative, and the true scope of the present disclosure is shown by the following claims and their equivalents.

Claims

1. A lipid compound or a derivative thereof, wherein the lipid compound has a structure represented by formula (I). 【Chemistry 1】 (wherein, Z １ is O or S, and Z ２ is -Q １ -A １ -Q ２ -A ２ -X １ and Z ３ is the same as Z ２ and Z ４ is -Q ３ -A ３ -Q ４ -A ４ -X ２ and Q １ is a single bond, Q ３ is a single bond, -O- or -NH-, and Q ２ is -O-, -NH-, -S-S-, or a group of the following formula 【Chemistry 2】 Q ４ These are single bonds, -O-, -NH-, -S-S-, or the following groups 【Transformation 3】 A １ is a C1-C6 alkylene group, A ３ is a C1-C6 alkylene group, A ２ and A ４ Each of them is independently a single bond, a C1-C12 alkylene group, a C2-C12 alkenylene group, or a C2-C12 alkylylene group, X １ Ha-NR １ R ２ X ２ is hydrogen, C6-C12 aryl group, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group, -NR ７ R ８ , or the basis of the following formula 【Chemistry 4】 And R １ is a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, or -A ９ - Q ７ -X ５ And R ２ is a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, or -A １０ - Q ８ -X ６ And R ９ , R １０ , R １１ and R １２ Each of them is independently hydrogen or a C1-C6 alkyl group, R ７ is hydrogen, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group or -A １１ - Q ９ -X ７ And R ８ is hydrogen, C1-C24 alkyl group, C2-C24 alkenyl group, C2-C24 alkynyl group, C1-C24 alkanol group, C2-C24 alkenol group, C2-C24 alkynol group or -A １２ - Q １０ -X ８ A ５ A ６ A ７ A ８ A ９ A １０ A １１ and A １２ Each of them is independently a C1-C12 alkylene group, Q ５ Q ６ Q ７ Q ８ Q ９ and Q １０ Each of these independently corresponds to -O-, -NH-, -S-S-, or the base given by the following equations. 【Transformation 5】 and X ３ , X ４ , X ５ , X ６ , X ７ and X ８ Each of these is independently a C1-C24 alkyl group, and here, the derivative of the lipid compound is a pharmaceutically acceptable salt of the lipid compound or a solvate of the lipid compound, and the solvate of the lipid compound is a hydrate of the lipid compound or an alkoxide of the lipid compound.

2. Q ３ Q is a single bond, ４ The base is -O-, -NH-, -S-S-, or the base of the following equations. 【Transformation 6】 The lipid compound or derivative thereof according to claim 1.

3. Q ４ It is a single bond, and Q ３ The lipid compound or derivative thereof according to claim 1, wherein is -O- or -NH-.

4. A ４ It is a single bond, A ３ The lipid compound or derivative thereof according to claim 1, wherein is a C1-C6 alkylene group.

5. Z ４ and Z ２ A lipid compound or derivative thereof according to claim 1, which is different from the above.

6. Z ２ Z ３ and Z ４ The lipid compound or derivative thereof according to claim 1, wherein the same properties are present.

7. The lipid compound or a derivative thereof according to claim 1, wherein the lipid compound is any of the following compounds. 【Transformation 7】 (wherein, A １ , A ２ , A ３ , A ４ , Q １ , Q ２ , Q ３ , Q ４ , X １ and X ２ are defined the same as in claim 1.)

8. The lipid compound or its derivative according to claim 1, Sterols and A pharmaceutical composition containing the following:

9. The pharmaceutical composition according to claim 8, wherein the sterol is cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or a combination thereof.

10. It further contains auxiliary lipids, the auxiliary lipids being 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-stearoyl-2-oleoyl-sn-glycero-3- The pharmaceutical composition according to claim 8, comprising phosphocholine (SOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG), sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), L-a-phosphatidylcholine (EPC), phosphatidylcholine (PC), or a combination thereof.

11. The pharmaceutical composition according to claim 8, further comprising polyethylene glycol (PEG)-ylated lipids, wherein the polyethylene glycol (PEG)-ylated lipids are polyethylene glycol-modified phosphatidylethanolamine, polyethylene glycol-modified phosphatidic acid, polyethylene glycol-modified ceramide, polyethylene glycol-modified dialkylamine, polyethylene glycol-modified diacylglycerol, polyethylene glycol-modified dialkylglycerol, polyethylene glycol-modified sterol, polyethylene glycol-modified phospholipid, or a combination thereof.

12. The polyethylene glycol (PEG) lipids include 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-dipalmitoyl-sn-glycerol-3-succinate-polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol. The pharmaceutical composition according to claim 11, wherein the glycol is (DPPE-PEG), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycerol-3-methoxy-polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-sn-glycerol-methoxy-polyethylene glycol (DPG-PEG), α-[2-(ditetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethanediyl) or a combination thereof.

13. The pharmaceutical composition according to claim 8, further comprising a nucleic acid, wherein the nucleic acid is deoxyribonucleic acid (DNA), plasmid DNA, messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), small activated ribonucleic acid (saRNA), circular ribonucleic acid (circular RNA), or a combination thereof.

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