Ionizable lipid or pharmaceutically acceptable salt thereof, composition and application

Abstract

The present invention discloses an ionizable lipid or a pharmaceutically acceptable salt thereof, a composition, and applications. The structure of the ionizable lipid is shown in Formula I, and is primarily composed of a long carbon chain, biodegradable ester bonds, ether bonds, and other structures. The ionizable lipid or a pharmaceutically acceptable salt thereof and nucleic acids of the present invention can be prepared into lipid nanoparticles with a particle size of approximately 110 nm. These lipid nanoparticles exhibit high in vivo and in vitro transfection efficiency. This invention is of great significance for enriching the variety of ionizable lipids, delivering nucleic acid drugs, and preventing or treating diseases. #imgabs0#

Description

Technical Field

[0001] The present invention relates to an ionizable lipid, a composition and an application thereof, and in particular to an ionizable lipid or a pharmaceutically acceptable salt thereof, a composition and an application thereof, belonging to the field of pharmaceutical technology. Background Art

[0002] Nucleic acid therapeutics face delivery difficulties due to low cell permeability and sensitivity to degradation by certain enzymes. Previous studies have confirmed that compositions containing cationic lipids, liposomes and liposome complexes (lipoplex) are effective as transport vehicles for the delivery of nucleic acids to cells and / or intracellular compartments. These compositions generally contain one or more "cationic" and / or amino (ionizable) lipids, neutral lipids, structural lipids and polymer lipids. Cationic and / or ionizable lipids include amine-containing lipids that are easily protonated. Although there are a variety of nanoparticle compositions containing such lipids, safety, efficacy and specificity still need to be improved. It is worth noting that the increase in the complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, which may limit their clinical application. For example, LNP nanoparticles encapsulating siRNA Steroids and antihistamines are required to eliminate unnecessary immune responses beforehand (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretinamyloidosis, N Engl J Med, 369 (2013) 819-829.). Therefore, it is necessary to develop ionizable lipid compounds that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells to achieve efficient delivery. Summary of the Invention

[0003] Objectives of the invention: The first objective of the present invention is to provide an ionizable lipid or a pharmaceutically acceptable salt thereof for delivering nucleic acid genes. The second objective of the present invention is to provide a composition comprising the ionizable lipid or a pharmaceutically acceptable salt thereof. The third objective of the present invention is to provide the use of the ionizable lipid or a pharmaceutically acceptable salt thereof or the combination in the preparation of nucleic acid drugs. The lipids of the present invention enrich the types of ionizable lipids, provide more options for drug delivery, and have important practical significance.

[0004] Technical solution: The ionizable lipid or a pharmaceutically acceptable salt thereof according to the present invention has a structure as shown in Formula I.

[0005]

[0006] Wherein, X is a C8-18 branched or straight chain alkyl group, Y is a C15-18 branched alkyl group, and Z is a methyl group or an ethyl group.

[0007] Furthermore, X is selected from:

[0008]

[0009] Furthermore, Y is selected from:

[0010]

[0011] Furthermore, the ionizable lipid is selected from:

[0012]

[0013] The composition of the present invention comprises a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or the preventive agent, wherein the therapeutic agent or the preventive agent is one or more gene drugs; and the carrier comprises one or more ionizable lipids or pharmaceutically acceptable salts thereof of the present invention.

[0014] Furthermore, the active ingredients of the gene drug include but are not limited to single-stranded DNA, double-stranded DNA, siRNA, shRNA, miRNA, mRNA, dsRNA, tRNA, locked nucleic acid (LNA), peptide nucleic acid (PNA) and other forms of RNA molecules known in the art.

[0015] Furthermore, the therapeutic or preventive agent comprises at least one mRNA.

[0016] Furthermore, the active ingredient is encapsulated in the carrier or adsorbed to the carrier.

[0017] Furthermore, the mass ratio of the carrier to the therapeutic agent or preventive agent is 1:1 to 100:1, preferably 5:1 to 60:1, more preferably 8:1 to 40:1, and even more preferably 10:1 to 30:1.

[0018] Furthermore, the molar ratio of the ionizable lipid or pharmaceutically acceptable salt thereof to the carrier is 30% to 70%.

[0019] Furthermore, the composition is a lipid nanoparticle, and the average particle size of the lipid nanoparticle is 60nm to 300nm, preferably 80nm to 190nm, and more preferably 90nm to 120nm.

[0020] Furthermore, the polydispersity index of the lipid nanoparticles is ≤0.30, and more preferably ≤0.20.

[0021] Furthermore, the carrier also includes structural lipids.

[0022] Furthermore, the structural lipids include but are not limited to one or more of cholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterols, corticosteroids, ursolic acid, tomatine, tomatine and α-tocopherol; the structural lipids can well stabilize the structure of the carrier.

[0023] Furthermore, the molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof to the structural lipid is 1:1 to 10:1, preferably 1:1 to 5:1, more preferably 1:1 to 4:1, and more preferably 1:1 to 2:1.

[0024] Furthermore, the carrier also includes neutral lipids.

[0025] Furthermore, the neutral lipid compound is any lipid molecule, disclosed or undisclosed, that exists in an uncharged form or a neutral zwitterionic form at a selected pH value or range.

[0026] Furthermore, the neutral lipid is one or more of ceramide, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and their derivatives.

[0027] Furthermore, the neutral lipids include but are not limited to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 2-(((2,3-bis(oleoyloxy)propyl))dimethylammoniumphosphate)ethyl hydrogen (DOCP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), ceramide, sterols and their derivatives.

[0028] Furthermore, the molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof to the neutral lipid is 1:2 to 20:1, preferably 1:1 to 10:1, and more preferably 3:1 to 6:1.

[0029] Furthermore, the carrier also includes polymer-conjugated lipids, and the polymer-conjugated lipids mainly include PEG-modified lipid compounds.

[0030] Furthermore, the PEG-modified lipid compound is one or more of PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine and PEG-modified dialkylglycerol.

[0031] Furthermore, polymer conjugated lipids can improve the stability of lipid nanoparticles and reduce protein absorption of lipids. Specifically, the PEG-modified lipid compounds include but are not limited to PEG-modified phosphatidylethanolamine (PEG-DMG), PEG-modified dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-modified dipalmitoylphosphatidylcholine (PEG-DPPC), PEG-modified dilauroylphosphatidylethanolamine (PEG-DLPE), PEG-modified distearoylphosphatidylethanolamine (PEG-DSPE), PEG-modified cholesterol (Chol-PEG), and PEG-modified ceramide (Ceramide-PEG).

[0032] Furthermore, the PEG-modified lipid compound is PEG-DMPE or PEG-DMG, and preferably, the relative molecular mass of the PEG is 2000.

[0033] Furthermore, the molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof to the polymer-conjugated lipid is 10 to 200:1, preferably 10 to 100:1, more preferably 10 to 50:1, and even more preferably 25 to 35:1.

[0034] Furthermore, the molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof, the structural lipid, the neutral lipid and the polymer-conjugated lipid is: (15-60): (15-45): (1-20): (0.5-2), preferably (20-35): (20-35): (1-10): (0.5-1.5).

[0035] Furthermore, the carrier also includes one or more other charged lipid compounds.

[0036] Furthermore, the charged lipid compounds include but are not limited to 1,2-dilinoleoyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylchloro Dimethylaminopropane (DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen).

[0037] Furthermore, the composition further comprises one or more excipients or diluents commonly used in medicines.

[0038] The present invention also includes the use of the ionizable lipid or a pharmaceutically acceptable salt thereof or the composition of the present invention in the preparation of nucleic acid drugs.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0040] The present invention provides a novel ionizable lipid compound that is non-toxic, biodegradable, and stable. The ionizable lipid compound enriches the variety of lipid compounds, provides more options for delivering nucleic acid drugs, and has important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the mass spectrum of compound 1 in Example 1;

[0042] Figure 2 is the NMR spectrum of compound 1 in Example 1;

[0043] Figure 3 is the mass spectrum of compound 2 in Example 2;

[0044] Figure 4 is the NMR spectrum of compound 2 in Example 2;

[0045] Figure 5These are transmission electron micrographs of H01-mRNA, H02-mRNA, H03-mRNA, H04-mRNA, H05-mRNA, and ALC-0315-mRNA in Example 6. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0047] Example 1. Synthesis of ionizable lipid compound 1

[0048] Compound 1 was synthesized according to the following route:

[0049]

[0050] 1. Synthesis of Compound 1-1

[0051] At 25 ℃, N, N'-dicyclohexylcarboximide (DCC) (7.50g, 36.4mmol) is added to 80mL of dichloromethane (DCM) solution containing 6-bromohexanoic acid (5.76g, 29.8mmol), and the solution is stirred at room temperature for 20 minutes. Then, 2-hexyldecanol (6.0g, 24.8mmol) and 4-dimethylaminopyridine (DMAP) (170mg) are added to the above solution at room temperature and spend the night. The crude product is obtained under reduced pressure and purified by silica gel column chromatography (methanol / dichloromethane=1 / 20) to obtain a colorless oily product compound 1-1 (2-hexadecyl 6-bromohexanoate, 8.5g, 20.33mmol, 77.3% yield).

[0052] The compound 1-1 was subjected to H NMR spectrum analysis. 1 H NMR (600MHz, CDCl3): δ3.97(d,J=5.8Hz,2H), 3.40(t,J=6.8Hz,2H), 2.33(t,J=7.4Hz,2H), 1.92–1.82(m,2H ), 1.64(dp,J=15.4,6.4,5.2Hz,3H), 1.48(p,J=7.6,7.1Hz,2H), 1.35-1.08(m,24H), 0.88(t,J=6.9Hz,6H).

[0053] 2. Synthesis of Compound 1-2

[0054] 2-Hexyldecanol (2.42 g, 10 mmol), zinc chloride (80 mg) and two drops of concentrated sulfuric acid were added to the flask. Epichlorohydrin (1.84 g, 20 mmol) was added dropwise to the mixture over 30 minutes, and then reacted at 120° C. for 2 hours. After the mixture solution was cooled to room temperature, the solid residue was removed by filtration, and the excess epichlorohydrin was removed under reduced pressure. Next, the above solution was mixed with an aqueous sodium hydroxide solution (20%) and stirred at 40° C. for 4 hours. Finally, the aqueous phase was removed and the organic phase was dried under reduced pressure to obtain the product compound 1-2 (2-(((2-hexyldecyl)oxy)methyl)oxirane, 1.87 g, 6.2 mmol, 74.8% yield).

[0055] The product compound 1-2 was 1 H NMR and LCMS confirmed. LCMS: [M+Na] + 321.1. 1 H NMR (600MHz, CDCl3) δ3.96 (p, J=5.5Hz, 1H), 3.78-3.22 (m, 6H), 1.56 (p, J=5.1, 4.0Hz, 1H), 1.37-1.08 (m, 24H), 0.88 (t, J=6.9Hz, 6H).

[0056] 3. Synthesis of Compounds 1-3

[0057] Methanol (15 ml), methylamine (0.60 g, 13.3 mmol), and 2-(((2-hexadecanyl)oxy)methyl)oxirane (1.0 g, 3.34 mmol) were added to a flask and mixed. The mixed solution was stirred at room temperature overnight. The reaction solution was concentrated and further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). The resulting product was a light yellow oil, Compound 1-3 (1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2-ol, 0.64 g, 2.5 mmol, 55.6%).

[0058] The product compound 1-3 was analyzed by LC-TOF, LC-TOF: [M+H] + 330.32.

[0059] 4. Synthesis of Compound 1

[0060] 2-Hexadecyl 6-bromohexanoate (0.836 g, 2 mmol), K2CO3 (447 mg, 6.0 mmol) and KI (48.6 mg, 0.6 mmol), 1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2 alcohol (0.442 g, 1 mmol) were dissolved in CNCH3 (15 ml). The mixed solution was stirred at 90°C for 5 hours, filtered and dried under reduced pressure. The obtained residue was redissolved in dichloromethane (50 ml) and washed with brine (2 x 40 ml). The combined organic layers were concentrated to obtain a crude product, which was further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). Compound 1 was a light yellow oily ionizable lipid compound 1 (0.32 g, 0.48 mmol, yield 39.0%).

[0061] The product can ionize lipid compound 1 by LC-TOF [M+H] +: 668.66 and 1 H NMR confirmed. 1 H NMR (600 MHz, CDCl3) δ 3.95 (dd, J = 21.8, 6.3 Hz, 2H), 3.86 (dd, J = 9.3, 4.3 Hz, 1H), 3.45-3.25 (m, 4H), 2.57-2.26 (M, 9H), 1.52 (DDD, J = 53.8, 38.6, 22.4 Hz, 4H), 1.43-1.03 (M, 50H), 0.98-0.77 (M, 12H). Spectrum see Figure 1-2 .

[0062] Example 2: Synthesis of ionizable lipid compound 2

[0063] Compound 2 was synthesized according to the following route:

[0064]

[0065] 1. Synthesis of Compound 2-1

[0066] Under 25 ℃, DCC (7.50g, 36.4mmol) is joined in the dichloromethane (DCM) solution that 80mL contains 6-bromohexanoic acid (5.76g, 29.8mmol), and at room temperature solution is stirred 20 minutes.Then, at room temperature 2-octyldecanol (6.69g, 24.8mmol) and DMAP (170mg) are added above-mentioned solution and spend the night.Under reduced pressure, obtain crude product, and by silica gel column chromatography (methanol / dichloromethane=1 / 20) purification, obtain colorless oily product compound 2-1 (2-octadecyl 6-bromohexanoate 8.2g, 18.4mmol, productive rate 75.5%).

[0067] The compound 2-1 was subjected to H NMR spectrum analysis.1 H NMR (600MHz, CDCl3): δ3.95(d,J=5.8Hz,2H),3.40(t,J=6.8Hz,2H),2.31(t,J=7.4Hz,2H),1.90–1.81(m,2H ),1.63(dp,J=15.4,6.4,5.2Hz,3H),1.46(p,J=7.6,7.1Hz,2H),1.37-1.06(m,28H),0.88(t,J=6.9Hz,6H).

[0068] 2. Synthesis of Compound 2-2

[0069] 2-Octyldecanol (2.70, 10 mmol), zinc chloride (80 mg) and two drops of sulfuric acid were added to the flask. Epichlorohydrin (1.84 g, 20 mmol) was added dropwise to the mixture over 30 minutes, and then reacted at 120° C. for 2 hours. After the mixture solution was cooled to room temperature, the solid residue was removed by filtration, and the excess epichlorohydrin was removed under reduced pressure. Next, the above solution was mixed with an aqueous sodium hydroxide solution (20%) and stirred at 40° C. for 4 hours. Finally, the aqueous phase was removed and the organic phase was dried under reduced pressure to obtain the product compound 2-2 (2-(((2-octyldecyl)oxy)methyl)oxirane (1.55 g, 4.7 mmol, 71.4% yield).

[0070] The product compound 2-2 was obtained by 1 H NMR and LCMS confirmed. LCMS: [M+Na] + 349.1. 1 H NMR (600MHz, CDCl3) δ3.96 (p, J=5.5Hz, 1H), 3.76-3.24 (m, 6H), 1.56 (p, J=5.1, 4.0Hz, 1H), 1.35-1.06 (m, 28H), 0.88 (t, J=6.9Hz, 6H).

[0071] 3. Synthesis of Compound 2-3

[0072] Methanol (15 ml), methylamine (0.60 g, 13.3 mmol), and 2-(((2-octyldecyl)oxy)methyl)oxirane (1.09 g, 3.34 mmol) were added to a flask and mixed. The mixed solution was stirred at room temperature overnight. The reaction solution was concentrated and further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). The resulting product was a light yellow oil, Compound 2-3 (1-((2-octyldecyl)oxy)-3-(methylamino)propan-2-ol (0.74 g, 2.0 mmol, 61.2%).

[0073] The product compound 2-3 was analyzed by LC-TOF, LC-TOF: [M+H] + 358.36.

[0074] 4. Synthesis of Compound 2

[0075] 2-octadecyl 6-bromohexanoate (0.892 g, 2 mmol), K2CO3 (447 mg, 6.0 mmol) and KI (48.6 mg, 0.6 mmol), 1-((2-octyldecyl)oxy)-3-(methylamino)propan-2 alcohol (0.371 g, 1 mmol) were dissolved in CNCH3 (15 ml). The mixed solution was stirred at 90 ° C for 5 hours, filtered and dried under reduced pressure. The obtained residue was redissolved in dichloromethane (50 ml) and washed with brine (2 x 40 ml). The combined organic layers were concentrated to obtain a crude product, which was further purified by silica gel column chromatography (methanol / dichloromethane=1 / 20). Compound 2 was a light yellow oily ionizable lipid compound 2 (0.45 g, 0.62 mmol, 42.3% yield).

[0076] This product can ionize lipid compound 2 and is tested by LC-TOF [M+H] + :724.57 and 1 H NMR confirmed. 1 H NMR (600 MHz, CDCl3) δ 3.95 (dd, J = 21.8, 6.3 Hz, 2H), 3.86 (dd, J = 9.3, 4.3 Hz, 1H), 3.41-3.23 (m, 4H), 2.55-2.24 (M, 9H), 1.54 (DDD, J = 53.8, 38.6, 22.4 Hz, 4H), 1.47-1.03 (M, 58H), 0.99-0.75 (M, 12H). Spectrum see Figure 3-4 .

[0077] Example 3: Synthesis of ionizable lipid compound 3

[0078] Compound 3 was synthesized according to the following route:

[0079]

[0080] 1. Synthesis of compound 3-1

[0081] At 25°C, N,N'-dicyclohexylcarboximide (DCC) (7.50 g, 36.4 mmol) was added to an 80 mL dichloromethane (DCM) solution containing 6-bromohexanoic acid (5.76 g, 29.8 mmol). The solution was stirred at room temperature for 20 minutes. Heptadecyl-9-ol (6.3 g, 24.8 mmol) and 4-dimethylaminopyridine (DMAP) (170 mg) were then added to the solution overnight at room temperature. The crude product was obtained under reduced pressure and purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20) to afford compound 3-1 (9'-heptadecyl-6-bromohexanoate, 7.4 g, 17.12 mmol, 72.4% yield) as a colorless oil.

[0082] The compound 3-1 was analyzed by nuclear magnetic hydrogen spectrum. 1 H NMR (600MHz, CDCl3) δ4.96–4.78(m,1H),3.40(t,J=6.8Hz,2H),2.31(t,J=7.4Hz,2H),2.00–1 .77(m,2H),1.71–1.62(m,2H),1.57–1.46(m,6H),1.37–1.19(m,24H),0.88(t,J=7.0Hz,6H).

[0083] 2. Synthesis of compound 3-2

[0084] Heptadecyl-9-ol (2.56 g, 10 mmol), zinc chloride (80 mg), and two drops of concentrated sulfuric acid were added to a flask. Epichlorohydrin (1.84 g, 20 mmol) was added dropwise to the mixture over 30 minutes, followed by reaction at 120°C for 2 hours. After the mixture was cooled to room temperature, the solid residue was removed by filtration, and the excess epichlorohydrin was removed under reduced pressure. Next, the solution was mixed with a 20% aqueous sodium hydroxide solution and stirred at 40°C for 4 hours. Finally, the aqueous phase was removed, and the organic phase was dried under reduced pressure to yield the product, compound 3-2 (1.55 g, 4.9 mmol, 68.7% yield).

[0085] The product compound 3-2 was 1 H NMR and LCMS confirmed. LCMS: [M+Na] + 335.3. 1 H NMR (600MHz, CDCl3) δ3.62-3.12 (m, 3H), 2.62-2.35 (m, 3H), 1.39-1.14 (m, 28H), 0.88 (t, J=6.9Hz, 6H).

[0086] 3. Synthesis of Compound 3-3

[0087] Methanol (15 ml), methylamine (0.60 g, 13.3 mmol), and compound 3-2 (1.0 g, 3.32 mmol) were added to a flask and mixed. The mixed solution was stirred at room temperature overnight. The reaction solution was concentrated and further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). The product was obtained as a light yellow oil (compound 3-3, 0.72 g, 2.1 mmol, 59.6%).

[0088] The product compound 3-3 was analyzed by LC-TOF, LC-TOF: [M+H] + 344.50.

[0089] 4. Synthesis of Compound 3

[0090] Compound 3-1 (0.872 g, 2 mmol), K2CO3 (447 mg, 6.0 mmol) and KI (48.6 mg, 0.6 mmol) and compound 3-3 (0.442 g, 1 mmol) were dissolved in CNCH3 (15 ml). The mixed solution was stirred at 90 ° C for 5 hours, filtered and dried under reduced pressure. The obtained residue was redissolved in dichloromethane (50 ml) and washed with brine (2x40 ml). The combined organic layers were concentrated to obtain a crude product, which was further purified by silica gel column chromatography (methanol / dichloromethane=1 / 20). Compound 3 is a light yellow oily ionizable lipid compound 3 (0.42 g, 0.60 mmol, yield 45.3%).

[0091] The product can ionize lipid compound 3 by LC-TOF [M+H] +: 696.68 and 1 H NMR confirmed. 1 HNMR (600MHz, CDCl3) δ 4.96-4.78 (m, 1H), 3.62-3.15 (m, 4H), 2.45-2.24 (M, 9H), 1.47-1.01 (M, 63H), 0.99-0.74 (M, 12H).

[0092] Example 4. Synthesis of ionizable lipid compound 4

[0093] Compound 4 was synthesized according to the following route:

[0094]

[0095] 1. Synthesis of Compound 1-1

[0096] Compound 1-1 was prepared in the same manner as in Example 1 to obtain a colorless oily product, compound 1-1, namely 2-hexadecyl 6-bromohexanoate.

[0097] 2. Synthesis of compound 4-2

[0098] Add n-tetradecanol (2.14 g, 10 mmol), zinc chloride (80 mg) and two drops of concentrated sulfuric acid to the flask. Add epichlorohydrin (1.84 g, 20 mmol) dropwise to the mixture over 30 minutes, and then react at 120° C. for 2 hours. After the mixture solution is cooled to room temperature, the solid residue is removed by filtration, and the excess epichlorohydrin is removed under reduced pressure. Next, the above solution is mixed with an aqueous sodium hydroxide solution (20%) and stirred at 40° C. for 4 hours. Finally, the aqueous phase is removed and the organic phase is dried under reduced pressure to obtain the product compound 4-2 (2-(((2-tetradecyl)oxy)methyl)oxirane, 2.07 g, 7.7 mmol, 81.8% yield).

[0099] The product compound 4-2 was 1 H NMR and LCMS confirmed. LCMS: [M+Na] + 293.3. 1 H NMR (600MHz, CDCl3) δ3.94 (p, J=5.5Hz, 1H), 3.82-3.20 (m, 6H), 1.54 (p, J=5.1, 4.0Hz, 1H), 1.35-1.06 (m, 23H), 0.88 (t, J=6.9Hz, 3H).

[0100] 3. Synthesis of compound 4-3

[0101] Methanol (15 ml), methylamine (0.60 g, 13.3 mmol), and 2-(((2-hexadecanyl)oxy)methyl)oxirane (0.90 g, 3.34 mmol) were added to a flask and mixed. The mixed solution was stirred at room temperature overnight. The reaction solution was concentrated and further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). The resulting product was a light yellow oil, compound 4-3 (1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2-ol, 0.78 g, 2.6 mmol, 67.7%).

[0102] The product compound 4-3 was analyzed by LC-TOF, LC-TOF: [M+H] + 302.30.

[0103] 4. Synthesis of Compound 4

[0104] 2-Hexadecyl 6-bromohexanoate (0.836 g, 2 mmol), K2CO3 (447 mg, 6.0 mmol) and KI (48.6 mg, 0.6 mmol), 1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2 alcohol (0.301 g, 1 mmol) were dissolved in CNCH3 (15 ml). The mixed solution was stirred at 90°C for 5 hours, filtered and dried under reduced pressure. The obtained residue was redissolved in dichloromethane (50 ml) and washed with brine (2 x 40 ml). The combined organic layers were concentrated to obtain a crude product, which was further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). Compound 4 was a light yellow oily ionizable lipid compound 4 (0.44 g, 0.69 mmol, 52.3% yield).

[0105] The product can ionize lipid compound 4 by LC-TOF [M+H] +: 640.40 and 1 H NMR confirmed. 1 HNMR (600MHz, CDCl3) δ3.95 (dd, J=21.8, 6.3Hz, 2H), 3.88 (dd, J=9.3, 4.3Hz, 1H), 3.47-3.23 (m, 4H ), 2.59-2.24 (M, 9H), 1.53 (DDD, J=53.8, 38.6, 22.4Hz, 4H), 1.43-1.03 (M, 52H), 0.99-0.79 (M, 9H).

[0106] Example 5 Synthesis of ionizable lipid compound 5

[0107] Compound 5 was synthesized according to the following route:

[0108]

[0109] 1. Synthesis of Compound 2-1

[0110] Compound 2-1 was prepared in the same manner as in Example 2 to obtain a colorless oily product, compound 2-1, ie, 2-octadecyl 6-bromohexanoate.

[0111] 2. Synthesis of Compound 1-2

[0112] Compound 1-2 was prepared in the same manner as in Example 1 to obtain a colorless oily product, compound 1-2, namely 1-2(2-(((2-hexadecanyl)oxy)methyl)oxirane.

[0113] 3. Synthesis of Compounds 1-3

[0114] Compound 1-3 was prepared in the same manner as in Example 1 to obtain a colorless oily product, compound 1-3, namely 1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2-ol.

[0115] 4. Synthesis of Compound 5

[0116] 2-octadecyl 6-bromohexanoate (0.932 g, 2 mmol), K2CO3 (447 mg, 6.0 mmol) and KI (48.6 mg, 0.6 mmol), 1-((2-hexadecanyl)oxy)-3-(methylamino)propan-2 alcohol (0.442 g, 1 mmol) were dissolved in CNCH3 (15 ml). The mixed solution was stirred at 90°C for 5 hours, filtered and dried under reduced pressure. The obtained residue was redissolved in dichloromethane (50 ml) and washed with brine (2 x 40 ml). The combined organic layers were concentrated to obtain a crude product, which was further purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20). Compound 5 was a light yellow oily ionizable lipid compound 5 (0.44 g, 0.63 mmol, yield 51.40%).

[0117] The product can ionize lipid compound 5 by LC-TOF [M+H] +: 696.68 and 1 H NMR confirmed. 1 HNMR (600MHz, CDCl3) δ3.96 (dd, J=21.8, 6.3Hz, 2H), 3.84 (dd, J=9.3, 4.3Hz, 1H), 3.48-3.22 (m, 4H) , 2.55-2.23 (M, 9H), 1.53 (DDD, J=53.8, 38.6, 22.4Hz, 4H), 1.45-1.01 (M, 54H), 0.98-0.75 (M, 12H).

[0118] Example 6 Particle size, polydispersity coefficient, encapsulation efficiency and transmission electron microscopy of lipid nanoparticle preparations

[0119] 1. Preparation of the composition

[0120] The ionizable lipid compound 1 in Example 1, the ionizable lipid compound 2 in Example 2, the ionizable lipid compound 3 in Example 3, the ionizable lipid compound 4 in Example 4, the ionizable lipid compound 5 in Example 5 and the commercially available ionizable lipid ALC-0315 were dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution.

[0121] Briefly, a 200 μL solution of firefly luciferase mRNA (Fluc mRNA) encoding firefly luciferase (at a concentration of 0.5 μg / μL) was dissolved in 700 μL of 20 mM citrate buffer (pH 4) as the aqueous phase. Ionizable lipid compound 1 (1.242 mg), DSPC (0.294 mg), cholesterol (0.553 mg), and DMG-PEG 2000 (0.141 mg) were dissolved in 300 μL of ethanol at a molar ratio of 50:10:38.5:1.5 as the organic phase. The aqueous and organic phases were mixed using a microfluidic mixer (N / P = 6:1) to prepare a LNP-mRNA solution. The prepared LNP-mRNA solution was quickly added to an ultrafiltration tube containing 10 volumes of PBS (pH 7.4) standard solution and centrifuged at 5000 rpm for 30 minutes to remove the ethanol and citrate buffer. An Amicon Ultra centrifugal filter was used to obtain a lipid nanoparticle formulation solution of the desired concentration, designated H01-Fluc mRNA. The preparation procedures for ionizable lipid compound 2 in Example 2, ionizable lipid compound 3 in Example 3, ionizable lipid compound 4 in Example 4, ionizable lipid compound 5 in Example 5, and the commercially available ionizable lipid ALC-0315 lipid nanoparticles were similar to those described above, resulting in lipid nanoparticle formulation solutions designated H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA. Sucrose was added to each of the six lipid nanoparticle formulation solutions at a 5% sucrose concentration for later use.

[0122] 2. Lipid nanoparticle size, polydispersity coefficient, encapsulation efficiency and transmission electron microscopy detection

[0123] The particle size and polydispersity index (PDI) of the lipid nanoparticles H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA obtained above were measured using a Malvern particle size analyzer (Malvern UK). The results are shown in Table 1.

[0124] The mRNA encapsulation efficiency (EE%) was determined using RiboGreen reagent (Thermo Fisher Scientific, Cat No. 5, R11491). First, to determine the free mRNA in the lipid nanoparticle formulations, 10 μL of each of the H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA lipid nanoparticle formulations containing 5% sucrose was placed in a centrifuge tube and diluted with 990 μL of 1× TE buffer (purchased from Nanjing Novozymes Biotechnology Co., Ltd.). 100 μL of the diluted solution was then added to a 96-well plate. Then, 100 μL of RiboGreen dye was added to the wells and incubated for 5 min. The free mRNA concentration was determined using a spectrophotometer (Thermo Fisher Scientific, USA) with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. To determine the total mRNA content in the lipid nanoparticle formulation, 10 μL of the lipid nanoparticle formulation solution containing 5% sucrose, H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA, was taken into a centrifuge tube and diluted with 990 μL of 2% TE-Triton buffer (purchased from Nanjing Novozymes Biotechnology Co., Ltd.). 100 μL of the diluted solution was added to a 96-well plate. Then, 100 μL of RiboGreen dye was added to the plate wells and incubated for 5 minutes. The total mRNA concentration was measured using a spectrophotometer (Thermo Fisher Scientific, USA) with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The calculation formula for EE (%) is as follows:

[0125] EE (%) = (total mRNA concentration - free mRNA concentration) / total mRNA concentration × 100%

[0126] The results are shown in Table 1.

[0127] Table 1 Physicochemical properties of H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles

[0128]

[0129]

[0130] As can be seen from Table 1, the particle size range of the H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles prepared by the present invention is 98-115nm, and the PDI value is all less than 0.2. This shows that the six kinds of lipid nanoparticles all have excellent physicochemical properties. Simultaneously, the H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles prepared by the present invention all have higher mRNA encapsulation efficiency, and the encapsulation efficiency of the H01-Fluc mRNA and H03-Fluc mRNA lipid nanoparticles is higher than that of the ALC-0315-Fluc mRNA lipid nanoparticles.

[0131] The surface morphology of lipid nanoparticles H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA was observed using a transmission electron microscope (TEM, JEOL, Japan). The prepared lipid nanoparticles were dropped onto different 200-mesh carbon-coated copper grids. After air-drying, 2% (w / v) phosphotungstic acid was added dropwise to the copper grids for approximately 1-2 minutes. The treated samples were photographed at an accelerating voltage of 200 kV. The results are shown in FIG. Figure 5 As shown, Figure 5 The transmission electron micrographs of H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles in Example 6 are shown in FIG. Figure 5Transmission electron microscopy images show that the lipid nanoparticles prepared in this example, H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA, are all spherical, and their sizes are consistent with the results measured by the Malvern particle size analyzer described above. There are no significant morphological differences between the lipid nanoparticles containing H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, and H05-Fluc mRNA and the lipid nanoparticles containing ALC-0315-Fluc mRNA.

[0132] Example 7 Cell transfection experiment

[0133] In order to study the in vitro mRNA delivery of ionizable lipid compounds 1, 2, 3, 4, 5 and commercially available ALC-0315, enhanced green fluorescent protein mRNA (eGFP mRNA) was encapsulated in lipid nanoparticles to obtain H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, H05-eGFP mRNA and ALC-0315-eGFP mRNA lipid nanoparticle formulation solutions. The preparation process was the same as in Example 6. HEK293 cells or DC2.4 cells (2×10 4 Cells (cells / well, sourced from the ATCC Biological Standards Resource Center, USA) were seeded in 6-well plates and cultured in 2 mL of DMEM medium until 80% confluency was achieved. The cells were then washed three times with a PBS standard solution, and the medium was replaced with 2 mL of Opti-MEM serum-free medium. The prepared lipid nanoparticle formulations containing 5% sucrose (mRNA concentration of 1.5 μg / ml) containing H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, H05-eGFP mRNA, and ALC-0315-eGFP mRNA were added. After 24 hours of incubation, the cells were harvested and the positive rate of cellular eGFP protein was determined by flow cytometry (Accui C6, USA). The results are shown in Table 2.

[0134] Table 2 In vitro transfection data of H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, H05-eGFP mRNA and ALC-0315-eGFP mRNA lipid nanoparticles

[0135]

[0136] As can be seen from Table 2, the lipid nanoparticle preparations H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, and H05-eGFP mRNA mediated by ionizable lipid compounds 1, 2, 3, 4, and 5 can be effectively transfected into HEK293 cells or DC2.4 cells, and the transfection effect of the lipid nanoparticle preparations mediated by compounds 1 and 2 is better than that of the commercially available lipid ALC-0315, indicating good application prospects.

[0137] Example 8 Cytotoxicity Experiment

[0138] HEK293 cells or DC2.4 cells were seeded in 96-well plates (2000 cells / well) and cultured overnight. After the cells adhered, the lipid nanoparticle formulations containing H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA prepared in Example 6 and containing 5% sucrose were added to the 96-well plates to control the mRNA concentration to 2 μg / ml. After 24 hours of incubation, cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8, APExBIO, USA). OD values ​​at 450 nm were measured using a microplate reader. Cell viability was calculated based on the OD values, as shown in Table 3.

[0139] Table 3 In vitro toxicity data of H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles

[0140]

[0141]

[0142] The results in Table 3 show that the cytotoxicity of the six lipid nanoformulations is very low and they have good prospects for in vivo application.

[0143] Example 9: In vivo fluorescence detection

[0144] To investigate the in vivo mRNA delivery of ionizable lipid compounds 1, 2, 3, 4, 5, and commercially available ALC-0315, 18 female BALB / c mice weighing 16-18 g were randomly divided into six groups of three mice each. Each group of mice received an intramuscular injection of 100 μL of a lipid nanoparticle solution containing 20 μg of Fluc mRNA, encapsulated with 5% sucrose, H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA, prepared in Example 6. Three, six, 24, and 48 hours after the injection, 100 μL of luciferase substrate (30 mg / mL) was injected intraperitoneally into the mice and allowed to react for 5 minutes. Bioluminescence signals were acquired using a mouse imager (PerkinElmer). The results are shown in Table 4.

[0145] Table 4 In vivo imaging experimental data of mice using H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticles

[0146]

[0147] Table 4 shows that the lipid nanoparticle formulations H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, and H05-Fluc mRNA mediated by ionizable lipid compounds 1, 2, 3, 4, and 5 can effectively deliver mRNA in vivo and complete expression. In addition, the in vivo delivery effect of the lipid nanoparticle formulations mediated by compounds 1, 2, and 3 is better than that of the commercially available lipid ALC-0315, and has good application prospects.

[0148] Example 10: Stability test

[0149] Eighteen female BALB / c mice weighing 16-18 g were randomly divided into six groups of three mice each. Every ten days, 100 μl of the lipid nanoformulation containing 5% sucrose, prepared in Example 6, containing H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA, and ALC-0315-Fluc mRNA, was injected intramuscularly into the mice. The bioluminescence signal was measured in the mice using the same experimental procedures as in Example 9. The results are shown in Table 5.

[0150] Table 5 In vivo imaging experimental data of mice (stability test)

[0151]

[0152] Table 5 shows that the lipid nanoparticle formulations H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, and H05-Fluc mRNA mediated by ionizable lipid compounds 1, 2, 3, 4, and 5 have longer-lasting stability than the commercially available ALC-0315-mediated formulation ALC-0315-Fluc mRNA.

Claims

1. An ionizable lipid or a pharmaceutically acceptable salt thereof, characterized in that: The ionizable lipids are selected from: or .

2. A composition, characterized in that The composition includes a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or the preventive agent, wherein the therapeutic agent or the preventive agent is one or more of siRNA, shRNA, and mRNA; the carrier includes one or more of the ionizable lipids or pharmaceutically acceptable salts thereof according to claim 1; and the mass ratio of the carrier to the therapeutic agent or the preventive agent is: 1 to 100:

1.

3. The composition according to claim 2, characterized in that The composition is lipid nanoparticles, the average particle size of the lipid nanoparticles is 60nm-300nm, and the polydispersity index of the lipid nanoparticles is ≤0.

30.

4. The composition according to claim 2, characterized in that The carrier further comprises a structural lipid, wherein the structural lipid is one or more of cholesterol, campesterol, stigmasterol, brassicasterol, sitosterol, ergosterol, non-sterols, corticosteroids, ursolic acid, tomatine, tomatine and α-tocopherol; The molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof to the structural lipid is 1 to 10:

1.

5. The composition according to claim 4, characterized in that The carrier also includes a neutral lipid, which is one or more of ceramide, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and their derivatives; the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the neutral lipid is 1:2~20:

1.

6. The composition according to claim 5, characterized in that The carrier also includes a polymer-conjugated lipid, which is one or more of PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine, and PEG-modified dialkylglycerol; the molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof to the polymer-conjugated lipid is 10-200:

1.

7. The composition according to claim 6, characterized in that The molar ratio of the ionizable lipid or a pharmaceutically acceptable salt thereof, the structural lipid, the neutral lipid and the polymer-conjugated lipid is (15-60): (15-45): (1-20): (0.5-2).

8. Use of the ionizable lipid or pharmaceutically acceptable salt thereof according to claim 1 or the composition according to any one of claims 2 to 7 in the preparation of a nucleic acid drug.

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