Cycloalkane-based lipid compound for nucleic acid delivery and lipid nanoparticles containing the same

Abstract

The present invention provides a cycloalkane lipid compound represented by the following Chemical Formula 1 and a pharmaceutically acceptable salt thereof. The cycloalkane lipid compound is a compound in which a carbonyl substituent is bound to a central cycloalkane structure. The cycloalkane lipid compound according to the present invention is used as an ionizable lipid, which is one component of lipid nanoparticles for nucleic acid delivery.

Description

[Technical Field]

[0001] The present invention relates to a cycloalkane-based lipid compound for nucleic acid delivery and a lipid nanoparticle containing the same.

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0177848 dated December 19, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference. [Background technology]

[0003] In drug delivery, the drug delivery system (DDS) aims to maximize the efficacy and effectiveness of drugs, deliver the required amount of drugs to the target site, and reduce side effects. The drug delivery system is a high-value-added core technology with a high probability of success, which can generate economic benefits comparable to new drug development, and can contribute to improving the quality of patient treatment by making drug administration more efficient.

[0004] Nucleic acid delivery systems have been extensively studied, particularly for the purpose of controlling desired responses with biological drugs. Nucleic acids, such as antisense RNA, siRNA, and mRNA, are substances that can suppress the expression of specific proteins in vivo and are attracting attention as important tools for the treatment of cancer, genetic diseases, infectious diseases, autoimmune diseases, and other conditions. Some nucleic acids, such as mRNA and plasmids, can induce the expression of specific cellular products, useful for treating diseases associated with protein or enzyme deficiencies. Nucleic acid delivery can increase the levels of existing proteins, replace defective or non-functional versions of proteins, and introduce new proteins and related functionalities into cells or organisms. Nucleic acid-based therapeutics hold great potential, but to realize this potential, more effective delivery of nucleic acids to the appropriate sites within cells or organisms is required.

[0005] Viral delivery vehicles are effective in delivering nucleic acids carrying genetic information, but several drawbacks, such as immunogenicity, limited size of injected DNA, and difficulty in mass production, limit their use. Nonviral delivery vehicles, such as cationic liposomes and polymers, have attracted attention as alternatives. However, these nonviral delivery vehicles can exhibit significantly higher cytotoxicity due to poor biocompatibility and non-biodegradability, and are limited by low transfection efficiency.

[0006] Therefore, as a result of ongoing research into lipid nanoparticles as non-viral delivery vehicles, the present inventors have completed the present invention by synthesizing novel ionizable lipid compounds suitable as components of lipid nanoparticles. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10,166,298 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a cycloalkane-based lipid compound that serves as an ionizable lipid, an important constituent element of lipid nanoparticles for nucleic acid delivery, and that can efficiently deliver nucleic acids from lipid nanoparticles to cells and animals, thereby enabling the prevention or treatment of associated diseases. [Means for solving the problem]

[0009] According to a first aspect of the present invention, The present invention provides a cycloalkane lipid compound represented by the following Chemical Formula 1 and a pharmaceutically acceptable salt thereof:

[0010] [ka]

[0011] According to one embodiment of the present invention, n1 is 1 to 4.

[0012] According to one embodiment of the present invention, n2 is 1-4.

[0013] According to one embodiment of the present invention, when n2 is 2 or 3, the substituents linked to the cycloalkane are each independent.

[0014] According to one embodiment of the present invention, L is [ka] is.

[0015] According to one embodiment of the present invention, the AK1 is alkylene having 1 to 5 carbon atoms.

[0016] According to one embodiment of the present invention, T is alkyl or alkenyl having 6 to 20 carbon atoms, or [ka] is.

[0017] According to one embodiment of the present invention, the AK2 is alkylene having 6 to 10 carbon atoms.

[0018] According to one embodiment of the present invention, AK3 is alkyl or alkenyl having 6 to 10 carbon atoms.

[0019] According to one embodiment of the present invention, when n2 is 2 or 3, the substituents linked to the cycloalkane are linked with a distance such that one or more carbon atoms are located between them.

[0020] According to one embodiment of the present invention, n1 is 2.

[0021] According to one embodiment of the present invention, n2 is 3.

[0022] According to one embodiment of the present invention, the AK1 is alkylene having 3 to 5 carbon atoms.

[0023] According to one embodiment of the present invention, T is alkyl having 8 to 16 carbon atoms.

[0024] According to one embodiment of the present invention, the AK2 is alkylene having 8 to 10 carbon atoms, and the AK3 is alkyl or alkenyl having 8 to 10 carbon atoms.

[0025] According to one embodiment of the present invention, the cycloalkane lipid compound is any of the following compounds 1 to 40.

[0026] According to a second aspect of the present invention, The present invention provides lipid nanoparticles comprising the cycloalkane lipid compound, a phospholipid, cholesterol, and a PEG-lipid conjugate.

[0027] According to one embodiment of the present invention, the lipid nanoparticles contain 10 mol % to 40 mol % of a cycloalkane lipid compound based on the total number of moles of the cycloalkane lipid compound, phospholipid, cholesterol, and PEG-lipid conjugate. [Effects of the Invention]

[0028] It is known that ionizable lipids have the greatest impact on efficacy in lipid nanoparticles used for nucleic acid delivery. Specifically, they directly affect nucleic acid delivery efficiency by enabling nucleic acids to escape from endosomes within cells, more so than other compositions. Therefore, novel ionizable lipids with improved delivery efficiency are needed to develop nucleic acid-based gene therapy agents with improved efficacy. In this study, cycloalkane-based lipid compounds were synthesized and their nucleic acid delivery capabilities were evaluated in cell and animal models, confirming their usefulness as lipid nanoparticle compositions for the development of nucleic acid therapeutic agents. [Brief explanation of the drawings]

[0029] [Figure 1]1 is a graph showing the results of particle size (Z-average and polydispersity index (PDI)) measured according to Experimental Example 1. [Figure 2] 1 is a graph showing the results of luminescence intensity (Normalized Luminescence) measured in Experimental Example 1. [Figure 3] 1 is a graph showing the results of particle size (Z-average and polydispersity index (PDI)) measurements of lipid nanoparticles for intramuscular injection according to Experimental Example 2. [Figure 4] 1 is a graph showing the results of luminescence intensity (total flux) measured for lipid nanoparticles for intramuscular injection according to Experimental Example 2. [Figure 5] 1 is a graph showing the results of particle size (Z-average and polydispersity index (PDI)) measurements of lipid nanoparticles for intravenous injection according to Experimental Example 2. [Figure 6] 1 is a graph showing the results of luminescence intensity (total flux) measured for lipid nanoparticles for intravenous injection according to Experimental Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0030] The embodiments provided by the present invention can all be achieved by the following description, which should be understood as describing preferred embodiments of the present invention, and should not be construed as limiting the present invention.

[0031] The present invention provides cycloalkane lipid compounds and lipid nanoparticles containing the same for nucleic acid delivery. The present invention is not limited to the cycloalkane lipid compounds themselves, but also includes isomers such as tautomers or stereoisomers, and pharmaceutically acceptable salts thereof.

[0032] The "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts. The acid addition salt may be, for example, a salt obtained by adding hydrochloric acid, trifluoroacetic acid, formic acid, citric acid, fumaric acid, monosodium fumarate, p-toluenesulfonic acid, stearic acid, disodium citrate, tartaric acid, malic acid, lactic acid, succinic acid, salicylic acid, or the like, but is not particularly limited as long as it is an acid addition salt commonly used in the art. The base addition salt may be, for example, a salt obtained by adding ammonium, sodium, potassium, calcium, magnesium, isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, caffeine, or the like, but is not particularly limited as long as it is a base addition salt commonly used in the art.

[0033] The cycloalkane lipid compound according to the present invention may be an ionizable lipid, which is a lipid whose charge state changes depending on the surrounding pH. Such an ionizable lipid has properties similar to lipids and can play a role in enabling drugs (e.g., anionic drugs and / or nucleic acids) to be encapsulated in lipid nanoparticles with high efficiency through electrostatic interactions with the drugs.

[0034] The term "alkyl" as used herein refers to a straight-chain or branched-chain saturated hydrocarbon containing one radical, which radical acts as a functional group to determine the bonding position, and the bonding position is not particularly limited. Examples of the term "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, and hexyl.

[0035] The term "alkylene" as used herein means a linear or branched saturated hydrocarbon containing two radicals, each of which determines a bonding position as a functional group, and the bonding position is not particularly limited. Examples of the term "alkylene" include, but are not limited to, methylene and ethylene.

[0036] The term "alkenyl" as used herein refers to a straight or branched chain hydrocarbon containing one or more carbon-carbon double bonds, where the one radical determines the bonding position as a functional group, and the bonding position is not particularly limited. Examples of the term "alkenyl" include, but are not limited to, ethenyl and propenyl.

[0037] Hereinafter, embodiments of the cycloalkane lipid compound according to the present invention will be described in detail.

[0038] One embodiment of the present invention provides a cycloalkane lipid compound or a pharmaceutically acceptable salt thereof, wherein "a pharmaceutically acceptable salt" may be an acid addition salt or a base addition salt as described above.

[0039] The cycloalkane lipid compound according to one embodiment of the present invention may be a compound having a structure as shown in Chemical Formula 1 below.

[0040] [ka]

[0041] As can be seen from Chemical Formula 1, the ionizable lipid compound according to the present invention has a central structure of cycloalkane, and is therefore named a "cycloalkane-based lipid compound."

[0042] In the chemical formula 1, n1 specifies the number of carbon atoms forming the ring of the cycloalkane. The n1 is defined as a natural number with no decimal points, and according to one embodiment of the present invention, n1 is 1 to 4. When n1 is 1, the cycloalkane becomes pentagonal cyclopentane; when n1 is 2, the cycloalkane becomes hexagonal cyclohexane. When n1 is 3, the cycloalkane becomes heptane; and when n1 is 4, the cycloalkane becomes octagonal cyclooctane. When n1 is 1 to 4, the bond angle with the adjacent carbon atom is appropriate, allowing for the formation of a more stable ring structure. The cycloalkane lipid compound according to one embodiment of the present invention may be a compound in the chemical formula 1 where n1 is 1 to 4, 1 to 3, 1 to 2, or 2.

[0043] In Chemical Formula 1, n2 specifies the number of carbonyl-based substituents linked to the cycloalkane. The portion not linked to the carbonyl-based substituent may be unsubstituted, or may be replaced by an alkyl group having 1 to 4 carbon atoms, as commonly introduced in the art, within the range of structural similarity. n2 is defined as a natural number without decimal points. According to one embodiment of the present invention, n2 is 1 to 4. When n2 is 2 to 4, multiple substituents are freely linked to carbon atoms forming the cycloalkane ring, and each substituent is independently defined. According to one embodiment of the present invention, when n2 is 2 to 4, the substituents linked to the cycloalkane are spaced apart so that one or more carbon atoms are located between them. Spaced apart multiple substituents reduce steric hindrance between the substituents, resulting in improved functionality due to more dispersed substituent placement. A cycloalkane lipid compound according to one embodiment of the present invention may be a compound in Chemical Formula 1 where n2 is 1 to 4, 2 to 4, 2 to 3, or 3.

[0044] The carbonyl-based substituent linked to the cycloalkane has a structure in which a linking group (L) and a terminal group (T) are linked to the carbonyl. According to one embodiment of the present invention, the linking group (L) is [ka] The linker (L) is represented in the same positional relationship as in Chemical Formula 1, with the left-side linking point being linked to the carbon of the carbonyl, and the right-side linking point being linked to the terminal group (T). When the right-side nitrogen has two linking points, they are all linked to the terminal group (T). In this case, the two terminal groups (T) linked to the right-side nitrogen are independently defined. A cycloalkane lipid compound according to one embodiment of the present invention is a cycloalkane lipid compound represented by Chemical Formula 1, wherein the linking group (L) is [ka] may be.

[0045] According to one embodiment of the present invention, AK1 in the linking group (L) is alkylene having 1 to 5 carbon atoms. In the AK1, the bonding position is not limited to the terminal carbon, so when it is bonded to a carbon other than the terminal, it may form a branched alkyl substituent. In the cycloalkane lipid compound according to one embodiment of the present invention, AK1 in the linking group (L) may be alkylene having 2 to 5 or 3 to 5 carbon atoms.

[0046] The terminal group (T) of the carbonyl-based substituent is connected to the right nitrogen of the linking group (L) to form one terminal of the compound. The terminal group (T) may be alkyl or alkenyl, or an ester group may be introduced into the alkyl or alkenyl chain. According to one embodiment of the present invention, the terminal group (T) is alkyl or alkenyl having 6 to 20 carbon atoms, [ka] When the terminal group (T) is an alkyl or alkenyl having 6 to 20 carbon atoms, the bonding position of the alkyl or alkenyl is not limited to the terminal carbon, and therefore when bonding to a carbon atom other than the terminal, a branched alkyl substituent may be formed. In one embodiment of the cycloalkane lipid compound of the present invention, the terminal group (T) may be an alkyl or alkenyl having 8 to 20, 8 to 18, or 8 to 16 carbon atoms.

[0047] According to one embodiment of the present invention, the terminal group (T) is [ka] In this case, AK2 may be alkylene having 6 to 10 carbon atoms or 8 to 10 carbon atoms, and AK3 may be alkyl or alkenyl having 6 to 10 carbon atoms or 8 to 10 carbon atoms. In AK2 or AK3, the bonding position is not limited to the terminal carbon, so when bonding to a carbon atom other than the terminal, a branched alkyl substituent may be formed. In a cycloalkane lipid compound according to one embodiment of the present invention, AK2 in the terminal group (T) may be alkylene having 8 carbon atoms, and AK3 may be alkyl or alkenyl having 8 or 9 carbon atoms.

[0048] The cycloalkane lipid compound according to one embodiment of the present invention may be a compound in which three carbonyl-based substituents are connected to cyclohexane, one carbon atom is located between the substituents, and the carbonyl-based substituents are uniformly dispersed. The three carbonyl-based substituents may have the same structure.

[0049] According to one embodiment of the present invention, the cycloalkane lipid compound is any of compounds 1 to 40 in Table 1 below.

[0050] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0051] Hereinafter, embodiments of lipid nanoparticles containing the aforementioned cycloalkane-based lipid compounds will be described in detail.

[0052] One embodiment of the present invention provides lipid nanoparticles comprising a cycloalkane-based lipid compound, a phospholipid, cholesterol, and a PEG-lipid conjugate, wherein the cycloalkane-based lipid compound is as described above.

[0053] The phospholipids act to encapsulate and protect the core formed by the interaction of the cycloalkane lipid compound and the drug within the lipid nanoparticle. Furthermore, the phospholipids bind to the phospholipid bilayer of target cells, facilitating cell membrane passage and endosomal escape during intracellular delivery of the drug. The phospholipids may be any substance commonly used in the art that has the above functionality. According to one embodiment of the present invention, the phospholipid is distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (distearoylphosphatidylethanolamine), or the like. DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,The phospholipid may be selected from the group consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dioctadecanoyl-sn-glycero-3-phosphoserine (DSPS), and combinations thereof. The phospholipid may be selected based on the type of nucleic acid to be delivered.

[0054] The cholesterol provides morphological robustness to the lipid packing within the lipid nanoparticles and is dispersed in the core and surface of the nanoparticles, thereby improving the stability of the nanoparticles.

[0055] The PEG-lipid conjugate refers to a lipid having a hydrophilic polymer, polyethyleneglycol (PEG), attached to one end of the lipid. The PEG-lipid conjugate not only contributes to the serum particle stability of lipid nanoparticles and prevents aggregation within the nanoparticles, but also protects nucleic acids from degradative enzymes during in vivo delivery, enhancing the in vivo stability of nucleic acids and increasing the half-life of drugs encapsulated in the nanoparticles. In the PEG-lipid conjugate, PEG and lipid can be conjugated according to methods commonly used in the art, and PEG can be conjugated to the lipid via a linker, if desired. According to one embodiment of the present invention, in the PEG-lipid conjugate, the lipid is selected from the group consisting of ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), cholesterol, and combinations thereof.

[0056] The lipid nanoparticles are prepared by mixing a cycloalkane lipid compound, a phospholipid, cholesterol, and a PEG-lipid conjugate to form a lipid mixture, and then mixing the mixture with nucleic acids such as antisense RNA, siRNA, and mRNA. Therefore, the lipid nanoparticles may further contain nucleic acids. The specific preparation method is not particularly limited as long as it is a method commonly used in the art.

[0057] In the lipid mixture, the cycloalkane lipid compound, phospholipid, cholesterol and PEG-lipid conjugate are mixed in an appropriate ratio taking into account the functionality of each component.

[0058] According to one embodiment of the present invention, the cycloalkane lipid compound is contained in the lipid mixture at 10 mol% to 40 mol% based on the total number of moles of the cycloalkane lipid compound, phospholipid, cholesterol and PEG-lipid complex. Specifically, the cycloalkane lipid compound is contained in the lipid mixture at 10 mol% or more, 11 mol% or more, 12 mol% or more, 13 mol% or more, 14 mol% or more, 15 mol% or more based on the total number of moles of the cycloalkane lipid compound, phospholipid, cholesterol and PEG-lipid complex, and at 40 mol% or less, 39 mol% or less, 38 mol% or less, 37 mol% or less, 36 mol% or less, 35 mol% or less, 34 mol% or less, 33 mol% or less, 32 mol% or less, 31 mol% or less, 30 mol% or less.

[0059] According to one embodiment of the present invention, the phospholipid is contained in the lipid mixture at 10 mol% to 50 mol% based on the total number of moles of the cycloalkane lipid compound, phospholipid, cholesterol, and PEG-lipid conjugate. Specifically, the phospholipid is contained in the lipid mixture at 10 mol% or more, 11 mol% or more, 12 mol% or more, 13 mol% or more, 14 mol% or more, 15 mol% or more, 16 mol% or more, 17 mol% or more, 18 mol% or more, 19 mol% or more, or 20 mol% or more based on the total number of moles of the cycloalkane lipid compound, phospholipid, cholesterol, and PEG-lipid conjugate. It may be contained in the lipid mixture at 50 mol% or less, 49 mol% or less, 48 ​​mol% or less, 47 mol% or less, 46 mol% or less, 45 mol% or less, 44 mol% or less, 43 mol% or less, 42 mol% or less, 41 mol% or less, or 40 mol% or less.

[0060] According to one embodiment of the present invention, the cholesterol is contained in the lipid mixture at 20 mol% to 60 mol% based on the total number of moles of cycloalkane lipid compounds, phospholipids, cholesterol and PEG-lipid conjugates. Specifically, the cholesterol is contained in the lipid mixture at 20 mol% or more, 21 mol% or more, 22 mol% or more, 23 mol% or more, 24 mol% or more, 25 mol% or more, 26 mol% or more, 27 mol% or more, 28 mol% or more, 29 mol% or more, 30 mol% or more based on the total number of moles of cycloalkane lipid compounds, phospholipids, cholesterol and PEG-lipid conjugates, and at 60 mol% or less, 59 mol% or less, 58 mol% or less, 57 mol% or less, 56 mol% or less, 55 mol% or less, 54 mol% or less, 53 mol% or less, 52 mol% or less, 51 mol% or less, 50 mol% or less.

[0061] According to one embodiment of the present invention, the PEG-lipid conjugate is contained in the lipid mixture at 0.2 mol% to 3.0 mol% based on the total molar amount of the cycloalkane lipid compound, phospholipid, cholesterol, and PEG-lipid conjugate. Specifically, the PEG-lipid conjugate is contained in the lipid mixture at 0.2 mol% or more, 0.25 mol% or more, 0.3 mol% or more, 0.35 mol% or more, 0.4 mol% or more, 0.45 mol% or more, or 0.5 mol% or more, based on the total molar amount of the cycloalkane lipid compound, phospholipid, cholesterol, and PEG-lipid conjugate, and at 3.0 mol% or less, 2.5 mol% or less, 2 mol% or less, 1.5 mol% or less, or 1 mol% or less.

[0062] According to one embodiment of the present invention, the nucleic acid contained in the lipid nanoparticles is selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), SAM (self-amplifying RNA), circular ribonucleic acid, aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, DNAzyme, and sgRNA for gene correction, and combinations thereof. [Example]

[0063] Compound preparation: Specific methods for producing compounds 1 to 40 in Table 1 above will be described below.

[0064] Compounds 1 to 40 according to one embodiment of the present invention are basically prepared according to the following reaction scheme 1.

[0065] [ka]

[0066] Compound 45 finally produced in Reaction Scheme 1 can be specified as Compounds 1 to 40 depending on the type of R. In Reaction Scheme 1, the amine of Compound 44 reacts with an aldehyde (RCHO) to introduce an R group into the amine. When the R group contains an ester group, such as Compound 6, it can be obtained according to Reaction Scheme 2 below.

[0067] [ka]

[0068] Specifically, compounds 1–40 were prepared by adding one drop of dimethylformamide (DMF) to a solution of cis,cis-1,3,5-cyclohexanetricarboxylic acid (compound 41) (3.2 mmol, 691.8 mg, 1.0 equiv.) in SOCl (8 mL) at room temperature. The reaction mixture was heated to 90 °C and stirred for 3 h. Excess SOCl was removed under reduced pressure, and the residue was co-evaporated with toluene to give the cis,cis-1,3,5-tricarbonyl chloride compound (compound 42) in quantitative yield. The cis,cis-1,3,5-tricarbonyl chloride compound (compound 42) was dissolved in anhydrous dichloromethane (DCM) (6 mL) and added dropwise to a solution of amine (compound 54) or alcohol (compounds 55-57) (3.15 equiv., 10.0 mmol) and triethylamine (6.0 equiv., d = 0.728, 19.2 mmol, 2.67 mL) in DCM (6 mL), where amine (compound 54) or alcohol (compounds 55-57) is the following compound:

[0069] [ka]

[0070] The reaction mixture was stirred at 0 °C for 1 h and then at room temperature overnight. The reaction mixture was then diluted with DCM (50 mL), washed with 1 M HCl (2 × 15 mL), dried over NaSO, and concentrated in vacuo to give a solid. The residue was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution from 100% hexane to hexane / EA (50 / 50, by volume) to give compounds 58–61.

[0071] [ka] 1 H NMR and MS results: 11H NMR (300 MHz, DMSO-d) δ 10.50 (broad singlet, 3H), 8.10 (broad singlet, 3H), 7.41 - 7.32 (multiplet, 45H), 3.15 - 2.97 (multiplet, 6H), 2.82 - 2.61 (multiplet, 6H), 2.07 - 1.93 (multiplet, 9H), 1.42 (doublet, J = 11.2 Hz, 3H), 1.26 - 1.17 (multiplet, 3H). MS (ESI, m / z): [M+H] + Calculated for C 75 H 79 N6O3, 1111.6; found: 1111.7.

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[0075] To a solution of the core amine compounds (compounds 54–57) (0.1 mmol, 1.0 equiv.) in DCM (1.5 mL) was added trifluoroacetic acid (TFA) (0.5 mL). After stirring the mixture at room temperature for 2 h, the solvent and excess TFA were evaporated, and the residue was dissolved in methanol (MeOH) and concentrated three times. To a suspension of the residue from the previous step in 10 mL of anhydrous tetrahydrofuran, the aldehyde (0.8 mmol, 8.0 equiv.) was added, followed by NaBH(OAc)3 (170 mg, 0.8 mmol, 8.0 equiv.) and triethylamine (TEA) (42 μL, 0.3 mmol, 3.0 equiv.), and the resulting reaction mixture was stirred at room temperature for 48 h. Here, the aldehyde was an alkyl aldehyde containing 8, 10, 12, 14, or 16 carbon atoms, or the aldehydes of compounds 49–53. The reaction was quenched with saturated aqueous NaHCO3 and extracted with DCM (3 × 25 mL). The combined organic phases were washed with 60 mL of 1 M NaHCO3 and dried over anhydrous Na2SO4. The solution was filtered, and the solvent was removed under reduced pressure. The residue was purified by column chromatography using a CombiFlash RF system with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution from 100% CHCl2 to CHCl2 / MeOH / NH4OH (75 / 22 / 3 by volume) to give compounds 1–40.

[0076] [ka] 1 H NMR results: 1 H NMR (300 MHz, Chloroform-d) δ 7.26 (s, 3H), 3.43 - 3.08 (m, 6H), 2.44 (dt, J = 27.4, 7.2 Hz, 21H), 2.04 (d, J = 11.8 Hz, 3H), 1.62 (p, J = 6.6 Hz, 9H), 1.41 (t, J = 7.5 Hz, 12H), 1.35 - 1.15 (m, 60H), 0.87 (t, J = 6.6 Hz, 18H).

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[0108] [Chemistry] 1 Results of 1H NMR: 1 1H NMR (300 MHz, Chloroform-d) δ 4.84 (q, J = 6.0 Hz, 3H), 2.98 - 2.27 (m, 20H), 2.28 - 2.10 (m, 3H), 1.83 - 1.62 (m, 6H), 1.62 - 1.36 (m, 18H), 1.35 - 1.13 (m, 111H), 0.87 (t, J = 6.6 Hz, 27H).

[0109] [Chemistry] 1 Results of 1H NMR: 1 1H NMR (300 MHz, Chloroform-d) δ 4.85 (p, J = 6.2, 5.8 Hz, 3H), 2.44 (s, 21H), 2.23 (d, J = 12.0 Hz, 3H), 1.85 - 1.62 (m, 6H), 1.62 - 1.52 (m, 6H), 1.35 - 1.15 (m, 12H), 1.25 (s, 135H), 0.93 - 0.80 (m, 27H).

[0110] [Chemistry] 1 Results of 1H NMR: 1 1H NMR (300 MHz, Chloroform-d) δ 5.01 - 4.69 (m, 3H), 2.91 - 2.29 (m, 21H), 2.25 (d, J = 12.2 Hz, 3H), 1.95 - 1.69 (m, 6H), 1.68 - 1.45 (m, 18H), 1.45 - 1.05 (m, 159H), 0.97 - 0.81 (m, 27H).

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Chem.

[0116] Experimental example Experimental Example 1: Preparation of lipid nanoparticles for in vitro studies Lipid nanoparticles (LNPs) encapsulating Firefly luciferase-encoding mRNA (UTR-optimized luciferase mRNA N1-methylpseudouridine (m1Ψ)) were prepared using a 20:30:40:0.75 molar ratio of ionizable lipid, phospholipid, cholesterol, and PEG-lipid conjugate. The ionizable lipids were compounds 4, 5, 9, 10, 19, 20, 28, 29, 30, and 40 obtained in the previous examples. The phospholipid was 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids Inc., Cat: 850275P). The cholesterol was purchased from Sigma-Aldrich, and the PEG-lipid conjugate was 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (NOF America Corporation).

[0117] The mRNAs were prepared as follows: DNA templates were synthesized by PCR amplification of the corresponding plasmids using forward and reverse primers containing 120T at the 5' end. The DNA templates were purified with a QIAquick PCR Purification Kit (Qiagen) and examined by agarose gel electrophoresis. All mRNAs were synthesized by in vitro transcription using an AmpliScribe T7-Flash Transcription Kit (Lucigen) with 100% N-methyl-pseudouridine-5'-triphosphate (TriLink)-substituted UTP for in vivo studies, and wild-type unconverted UTP purified with RNA Clean & Concentrator (Zymo) according to the manufacturer's instructions for in vitro studies. The mRNA cap-1 construct was synthesized using the Vaccinia Capping System (NEB) and Cap 2'-O-methyltransferase (NEB), followed by further purification with RNA Clean & Concentrator (Zymo). After measuring the concentration using a NanoDrop 2000 Spectrophotometer (Thermo), all mRNAs were diluted to the desired concentration with 1x TE, aliquoted, and stored at -80°C for future use. The synthesized mRNA was analyzed for quality using a TapeStation RNA BioAnalyzer. The mRNA concentration was fixed at 0.01 mg / mL and prepared in a final volume of 100 μL. The mass ratio of ionizable lipid to mRNA was fixed at 10. All lipid materials were dissolved in ethanol to the desired concentration, and mRNA was dissolved in 10 mM citrate buffer (pH 3.0). Of the final volume of 100 μL, 50 μL was 1x PBS, 25 μL was the aqueous phase, and 25 μL was the ethanol phase. In vitro mRNA-encapsulated LNPs were prepared by rapid pipetting.

[0118] Cells were previously plated overnight in white 96-well plates (Corning 3917) at a density of 20,000 cells / 100 μL / well. For in vitro studies, 5 μL of freshly prepared mRNA-encapsulated LNPs was added to at least three wells within 1 hour of preparation. After 18 hours of treatment, cells were treated with 100 μL of Bright-Glo Luciferase substrate (Promega E2650). After 5 minutes of shaking incubation in the dark, luminescence intensity was measured using a Cytation 5 (Biotek, US). Particle size was measured using a Malvern Zetasizer NanoZS (Malvern, UK).

[0119] Additionally, unless otherwise noted, all chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). DOPE (Avanti Polar Lipids Inc., Cat: 850275P), DSPC (Avanti Polar Lipids Inc., Cat: 850365), DMG-PEG2000 (NOF America Corporation), and the human liver epithelial cell line, Hep3B, were purchased from ATCC (HB-8064) and cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% FBS. The mouse myoblast cell line, C2C12, was purchased from ATCC (CRL-1772) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS. All cells were cultured in a humidified 37°C incubator containing 5% CO2.

[0120] The particle size (Z-average and polydispersity index (PDI)) measured based on the above content is shown in Figure 1, and the luminescence intensity (Normalized Luminescence) is shown in Figure 2. In Figures 1 and 2, "Lipid N" refers to lipid nanoparticles containing an ionizable lipid, Compound N. For comparison, Figures 1 and 2 also show the results of the measurement of the particle size (Z-average and polydispersity index (PDI)) of known lipid nanoparticles, ALC-0315 LNP (BioNTech's Company). TM ) results are also presented.

[0121] As can be seen from FIG. 1, the lipid nanoparticles prepared using a compound according to one embodiment of the present invention as an ionizable lipid have a Z-average of 200 nm or less and a PDI of 0.3 or less, indicating that they have an appropriate particle size for lipid nanoparticles.

[0122] 2, lipid nanoparticles incorporating a compound according to an embodiment of the present invention as an ionizable lipid exhibited similar delivery efficacy to ALC-0315 in both Hep3B and C2C12 cell lines. This study demonstrated the potential of lipid nanoparticles for delivery to liver and muscle cells.

[0123] Example 2: Formulation of lipid nanoparticles for in vivo administration Lipid nanoparticles administered for in vivo analysis were prepared using a Precision NanoSystems Nanoassemblyr microfluidic device (Vancouver, BC, Canada). Unless otherwise specified, lipids were dissolved in ethanol. mRNA was diluted with 10 mM citrate buffer (pH 3.0) and mixed with 1X PBS (pH 7.4). The overall flow rate was fixed at 12 mL / min, and the ethanol to aqueous phase ratio was 1:3. For in vivo studies, mRNA-encapsulated LNPs were dialyzed in 1X PBS (pH 7.4 buffer) for 90 minutes using Slide-A-Lyzer Dialysis Cassettes (MWCO 3.5K) purchased from Life Technologies (Grand Island, NY, USA) prior to administration. The total volume change after dialysis (if any) was recorded, and the injection volume was adjusted appropriately to match the target mRNA dose. Prepared lipid nanoparticles were stored at 4°C for up to 18 hours before administration. As in Experimental Example 1, the particle size of the lipid nanoparticles was measured using a Malvern Zetasizer NanoZS (Malvern, UK).

[0124] All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Ohio State University IACUC (Approval ID: 2014A00000106-R2). Wild-type C57BL / 6 female mice, 8 to 10 weeks old, were used for all experiments. Three mice were used per group. For intramuscular injections, each mouse was injected into both the left and right posterior flanks. LNP was injected intramuscularly into the posterior flanks of the mice or intravenously via the tail vein. For intramuscular injections, the target dose was 0.8 μg per injection for a dose of 0.04 mg / kg for a 20 g mouse. LNP was formulated to a concentration of 0.02 mg / mL mRNA. For intravenous injections, the target dose was 6 μg mRNA per tail vein injection for a dose of 0.3 mg / kg. LNP was formulated to a concentration of 0.03 mg / mL mRNA. Approximately 6, 28, and 54 hours after injection, the luminescence intensity (total flux) in the liver of the mice was measured using IVIS Lumina II (Perkin Elmer, Inc.).

[0125] Experimental Example 2 was performed under the same conditions as Experimental Example 1 unless otherwise specified. Experiments for intramuscular injection (IM) and intravenous injection (IV) were performed using lipid nanoparticles prepared separately. Lipid nanoparticles containing compounds 4, 5, 9, 10, and 40 were prepared as lipid nanoparticles for intramuscular injection, and lipid nanoparticles containing compounds 4, 9, and 10 were prepared as lipid nanoparticles for intravenous injection. The particle size (Z-average and polydispersity index (PDI)) measured for the lipid nanoparticles for intramuscular injection according to the above procedure is shown in FIG. 3, and the luminescence intensity (total flux) measured for the lipid nanoparticles for intravenous injection according to the above procedure is shown in FIG. 5, and the luminescence intensity (total flux) measured for the lipid nanoparticles for intravenous injection according to the above procedure is shown in FIG. 6.

[0126] 3 and 5, it can be seen that lipid nanoparticles prepared using a compound according to one embodiment of the present invention as an ionizable lipid have a Z-average of 200 nm or less and a PDI of 0.3 or less when prepared using a microfluidic device for in vivo administration, and thus have particle sizes at appropriate levels for lipid nanoparticles.

[0127] 4 and 6, it can be seen that lipid nanoparticles prepared using a compound according to one embodiment of the present invention as an ionizable lipid can effectively deliver mRNA to the liver and muscle in vivo, even by intramuscular or intravenous injection.

[0128] While the present invention has been described primarily with reference to the specific embodiments set forth above, it should be understood that those of ordinary skill in the art can make various modifications and variations to the present invention which are also within the scope of the present invention as defined by the claims appended below.

Claims

1. Cycloalkane lipid compounds of the following formula 1 and pharmaceutically acceptable salts thereof: 【Chemistry 1】 In the above Chemical Formula 1, The n 1 is 1 to 4, The n 2 is 1 to 4, The n 2 is 2 or 3, each substituent attached to the cycloalkane is independently The L is 【Chemistry 2】 and Said AK 1 is an alkylene having 1 to 5 carbon atoms, T is an alkyl or alkenyl having 6 to 20 carbon atoms; or 【Transformation 3】 and Said AK 2 is an alkylene having 6 to 10 carbon atoms, Said AK 3 is alkyl or alkenyl having 6 to 10 carbon atoms.

2. The n 2 is 2 or 3, the substituents linked to the cycloalkane are linked at a distance such that there is one or more carbon atoms between them. The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1.

3. The n 1 The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1, wherein R is 2.

4. The n 2 The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1, wherein R is 3.

5. Said AK 1 The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1, wherein is an alkylene having 3 to 5 carbon atoms.

6. 2. The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1, wherein T is alkyl having 8 to 16 carbon atoms.

7. Said AK 2 is an alkylene having 8 to 10 carbon atoms, Said AK 3 The cycloalkane lipid compound and its pharmaceutically acceptable salt according to claim 1, wherein is alkyl or alkenyl having 8 to 10 carbon atoms.

8. The cycloalkane lipid compound according to claim 1, characterized in that the cycloalkane lipid compound is any one of the following compounds 1 to 40, or a pharmaceutically acceptable salt thereof: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 [Chemistry 4-5] [Chemistry 4-6] 【Chemistry 4-7】 【Chemistry 4-8】 【Chemistry 4-9】 【Chemistry 4-10】

9. A lipid nanoparticle comprising the cycloalkane lipid compound of claim 1, a phospholipid, cholesterol, and a PEG-lipid conjugate.

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