CN116836074A - Ionizable lipid or pharmaceutically acceptable salt thereof, composition and application - Google Patents

Abstract

The invention discloses an ionizable lipid or its pharmaceutically acceptable salt, composition and application. The structure of the ionizable lipid is shown in the general formula I, which is mainly composed of long carbon chains, biodegradable ester bonds, ether bonds and other structures; the ionizable lipid of the present invention or its pharmaceutically acceptable Salts and nucleic acids can be prepared into lipid nanoparticles with a particle size of about 110 nm; the lipid nanoparticles have high in vivo and in vitro transfection efficiency. The present invention is of great significance for enriching the types of ionizable lipids, delivering nucleic acid drugs, and preventing or treating diseases.

Description

An ionizable lipid or its pharmaceutically acceptable salt, composition and application

Technical field

The present invention relates to an ionizable lipid, its composition and application, specifically to an ionizable lipid or its pharmaceutically acceptable salt, composition and application, and belongs to the field of medical technology.

Background technique

Nucleic acid therapeutics face delivery challenges due to low cell permeability and sensitivity to degradation by certain enzymes. Previous studies have demonstrated that cationic lipid-containing compositions, liposomes and lipoplexes serve as transport vehicles to effectively transport nucleic acids into cells and/or intracellular compartments. These compositions generally include one or more "cationic" and/or amino (ionizable) lipids, neutral lipids, structural lipids, and polymeric lipids. Cationic and/or ionizable lipids include amine-containing lipids that are readily protonated. Although a variety of nanoparticle compositions containing such lipids are available, safety, efficacy, and specificity still need to be improved. It is worth noting that the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, which may limit their clinical application. For example, siRNA-encapsulated LNP nanoparticles Preliminary use of steroids and antihistamines is required to eliminate unnecessary immune responses (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, there is a need to develop ionizable lipid compounds that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells for efficient delivery.

Contents of the invention

Purpose of the invention: The first purpose of the present invention is to provide an ionizable lipid or a pharmaceutically acceptable salt thereof for delivering nucleic acid genes. The second purpose of the present invention is to provide an ionizable lipid containing the ionizable lipid. The third object of the present invention is to provide the use of the ionizable lipid or the pharmaceutically acceptable salt thereof or the combination in the preparation of nucleic acid drugs; Lipids enrich the types of ionizable lipids and provide more options for drug delivery, which has important practical significance.

Technical solution: an ionizable lipid or a pharmaceutically acceptable salt thereof according to the present invention, the structure of the ionizable lipid is as shown in Formula I,

Among them, X is a C8-18 branched or linear alkyl group, Y is a C15-18 branched alkyl group, and Z is a methyl or ethyl group.

Further, the X is selected from:

Further, the Y is selected from:

Further, the ionizable lipid is selected from:

The composition of the present invention includes a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or preventive agent, wherein the therapeutic agent or preventive agent is one or more of genetic drugs; the carrier includes One or more of the ionizable lipids of the present invention or pharmaceutically acceptable salts thereof.

Further, the active ingredients of the genetic medicine 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 compounds known in the art. other forms of RNA molecules.

Furthermore, the therapeutic or preventive agent contains at least one mRNA.

Furthermore, the active ingredient is encapsulated in the carrier or adsorbed with the carrier.

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, further preferably 8:1 to 40:1, and more preferably 10 :1～30:1.

Furthermore, the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the carrier is 30% to 70%.

Further, the composition is lipid nanoparticles, and the average particle size of the lipid nanoparticles is 60 nm to 300 nm, preferably 80 nm to 190 nm, and more preferably 90 nm to 120 nm.

Further, the polydispersity index of the lipid nanoparticles is ≤0.30, more preferably ≤0.20.

Further, the carrier also includes structural lipids.

Further, the structural lipids include, but are not limited to, cholesterol, campesterol, stigmasterol, brassisterol, sitosterol, ergosterol, nonsterols, corticosteroids, ursolic acid, tomatine, tomatine and α- One or more tocopherols; structural lipids can well stabilize the structure of the carrier.

Furthermore, the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the structural lipid is 1:1 to 10:1, preferably 1:1 to 5:1, and further preferably 1: 1 to 4:1, more preferably 1:1 to 2:1.

Further, the carrier also includes neutral lipids.

Further, the neutral lipid compound is any disclosed or undisclosed lipid molecule that exists in an uncharged form or a neutral zwitterionic form within a selected pH value or range.

Further, the neutral lipid is one or more of ceramide, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and their derivatives.

Furthermore, the neutral lipids include but are not limited to 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1 1,2-dioleoyl-sn-glycerol-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))dimethylammonium phosphate)ethyl hydrogen (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), ceramide, sterols and their derivatives.

Furthermore, the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the neutral lipid is 1:2 to 20:1, preferably 1:1 to 10:1, and more preferably 3 :1～6:1.

Further, the carrier also includes polymer conjugated lipids, which mainly include PEG-modified lipid compounds.

Further, the PEG-modified lipid compound is PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine and PEG-modified di- One or more alkylglycerols.

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), and PEG-modified dipalmitoyl phospholipids. Acylcholine (PEG-DPPC), PEG-modified dilauroylphosphatidylethanolamine (PEG-DLPE), PEG-modified distearylphosphatidylethanolamine (PEG-DSPE), PEG-modified cholesterol (Chol-PEG), PEG Modified ceramide (Ceramide-PEG).

Furthermore, the PEG-modified lipid compound is PEG-DMPE or PEG-DMG. Preferably, the relative molecular mass of the PEG is 2,000.

Furthermore, the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the polymer-conjugated lipid is: 10 to 200:1. Preferably it is 10-100:1, More preferably, it is 10-50:1, Still more preferably, it is 25-35:1.

Further, the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt, structural lipid, neutral lipid and polymer conjugated lipid is: (15～60): (15～45) (1～20): (0.5-2), preferably (20～35): (20～35): (1～10): (0.5-1.5).

Further, the carrier also includes one or more other charged lipid compounds.

Furthermore, the charged lipid compounds include but are not limited to 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-di Methylaminomethyl-[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-dioleyl Oxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) Methyl ammonium chloride (DOTAP), 1,2-dimyristoyl-sn-glyceryl-3-ethylcholine phosphate (MOEPC), (R)-5-(dimethylammonium)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).

Furthermore, the composition further includes one or more excipients or diluents commonly used in medicines.

The present invention also includes the use of the ionizable lipid or its pharmaceutically acceptable salt or the composition of the present invention in the preparation of nucleic acid drugs.

Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages:

The present invention provides a brand new ionizable lipid compound, which is non-toxic, biodegradable and has good stability. The ionizable lipid compound enriches the types of lipid compounds, provides more options for delivering nucleic acid drugs, and has important practical significance.

Description of the drawings

Figure 1 is the mass spectrum of compound 1 in Example 1;

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

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

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

Figure 5 is a transmission electron microscope image of H01-mRNA, H02-mRNA, H03-mRNA, H04-mRNA, H05-mRNA and ALC-0315-mRNA in Example 6.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and examples.

Example 1. Synthesis of ionizable lipid compound 1

Compound 1 was synthesized according to the following route:

1. Synthesis of compound 1-1

At 25°C, N,N'-dicyclohexylcarbonimide (DCC) (7.50g, 36.4mmol) was added to 80mL of dichloromethane (DCM) containing 6-bromocaproic acid (5.76g, 29.8mmol). solution and stir the solution at room temperature for 20 minutes. Then, 2-hexyldecanol (6.0 g, 24.8 mmol) and 4-dimethylaminopyridine (DMAP) (170 mg) were added to the above solution at room temperature overnight. The crude product was obtained under reduced pressure and purified by silica gel column chromatography (methanol/dichloromethane=1/20) to obtain the colorless oily product compound 1-1 (2-hexadecyl 6-bromohexanoate, 8.5 g, 20.33mmol, yield 77.3%).

Compound 1-1 was analyzed by hydrogen nuclear magnetic spectrum. 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).

2. Synthesis of compound 1-2

Add 2-hexyldecanol (2.42g, 10mmol), zinc chloride (80mg) and two drops of concentrated sulfuric acid to the flask. Epichlorohydrin (1.84g, 20mmol) was added dropwise to the mixture within 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 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 water phase was removed, and the organic phase was dried under reduced pressure to obtain the product compound 1-2(2-(((2-hexyl)oxy)methyl)oxirane, 1.87g, 6.2mmol, yield 74.8% ).

The product compound 1-2 was confirmed by ¹ H NMR and LCMS. 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).

3. Synthesis of compounds 1-3

Methanol (15 ml), methylamine (0.60 g, 13.3 mmol) and 2-(((2-hexadecyl)oxy)methyl)oxirane (1.0 g, 3.34 mmol) were added to the 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 obtained product was compound 1-3 (1-((2-hexadecyl)oxy)-3-(methylamino)propan-2ol, 0.64g, 2.5mmol, 55.6%) as a light yellow oil.

Compound 1-3 of this product was analyzed by LC-TOF, LC-TOF: [M+H] ⁺ 330.32.

4. Synthesis of Compound 1

2-Hexadecyl 6-bromohexanoate (0.836g, 2mmol), K ₂ CO ₃ (447mg, 6.0mmol) and KI (48.6mg, 0.6mmol), 1-((2-hexadecyl) Oxy)-3-(methylamino)propan-2-ol (0.442 g, 1 mmol) was dissolved in CNCH ₃ (15 ml). After the mixed solution was stirred at 90°C for 5 hours, it was filtered and dried under reduced pressure. The resulting residue was redissolved in dichloromethane (50ml) and washed with brine (2x 40ml). 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 is a light yellow oily substance, ionizable lipid compound 1 (0.32g, 0.48mmol, yield 39.0%).

The ionizable lipid compound 1 of this product was confirmed by LC-TOF[M+H]+: 668.66 and ¹ H NMR. ¹ H NMR (600MHz, CDCl ₃ ) δ3.95 (dd, J=21.8, 6.3Hz, 2H), 3.86 (dd, J=9.3, 4.3Hz, 1H), 3.45-3.25 (m, 4H), 2.57- 2.26 (M, 9H), 1.52 (DDD, J=53.8, 38.6, 22.4Hz, 4H), 1.43-1.03 (M, 50H), 0.98-0.77 (M, 12H). The spectrum is shown in Figure 1-2.

Example 2, Synthesis of Ionizable Lipid Compound 2

Compound 2 was synthesized according to the following route:

1. Synthesis of compound 2-1

DCC (7.50 g, 36.4 mmol) was added to 80 mL of a solution of 6-bromocaproic acid (5.76 g, 29.8 mmol) in dichloromethane (DCM) at 25°C, and the solution was stirred at room temperature for 20 minutes. Then, 2-octyldecanol (6.69g, 24.8mmol) and DMAP (170mg) were added to the above solution at room temperature overnight. The crude product was obtained under reduced pressure and purified by silica gel column chromatography (methanol/dichloromethane=1/20) to obtain 8.2 g of the colorless oily product compound 2-1 (2-octadecyl 6-bromocaproate). , 18.4mmol, yield 75.5%).

Compound 2-1 was analyzed by hydrogen nuclear magnetic spectrum. ¹ H NMR (600MHz, CDCl ₃ ): δ3.95 (d, J = 5.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 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).

2. Synthesis of compound 2-2

2-Octyldecanol (2.70, 10 mmol), zinc chloride (80 mg) and two drops of sulfuric acid were added to the flask. Epichlorohydrin (1.84g, 20mmol) was added dropwise to the mixture within 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 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-octadecyl)oxy)methyl)oxirane (1.55g, 4.7mmol, yield 71.4%).

The product compound 2-2 was confirmed by ¹ H NMR and LCMS. 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).

3. Synthesis of compound 2-3

Methanol (15 ml), methylamine (0.60 g, 13.3 mmol) and 2-(((2-octadecyl)oxy)methyl)oxirane (1.09 g, 3.34 mmol) were added to the 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 obtained product was a light yellow oil compound 2-3(1-((2-octyl)oxy)-3-(methylamino)propan-2ol, (0.74g, 2.0mmol, 61.2%).

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

4. Synthesis of compound 2

2-Octadecyl 6-bromohexanoate (0.892g, 2mmol), K ₂ CO ₃ (447mg, 6.0mmol) and KI (48.6mg, 0.6mmol), 1-((2-octadecanyl)oxy) -3-(Methylamino)propan-2-ol (0.371 g, 1 mmol) was dissolved in CNCH ₃ (15 ml). After the mixed solution was stirred at 90°C for 5 hours, it was filtered and dried under reduced pressure. The resulting residue was redissolved in dichloromethane (50ml) and washed with brine (2x 40ml). 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 is a light yellow oily substance, ionizable lipid compound 2 (0.45g, 0.62mmol, yield 42.3%).

The ionizable lipid compound 2 of this product was confirmed by LC-TOF [M+H] ⁺ : 724.57 and ¹ H NMR. ¹ H NMR (600MHz, CDCl ₃ ) δ3.95 (dd, J=21.8, 6.3Hz, 2H), 3.86 (dd, J=9.3, 4.3Hz, 1H), 3.41-3.23 (m, 4H), 2.55- 2.24 (M, 9H), 1.54 (DDD, J=53.8, 38.6, 22.4Hz, 4H), 1.47-1.03 (M, 58H), 0.99-0.75 (M, 12H). The spectrum is shown in Figure 3-4.

Example 3. Synthesis of ionizable lipid compound 3

Compound 3 was synthesized according to the following route:

1. Synthesis of compound 3-1

At 25°C, N,N'-dicyclohexylcarbonimide (DCC) (7.50g, 36.4mmol) was added to 80mL of dichloromethane (DCM) containing 6-bromocaproic acid (5.76g, 29.8mmol). solution and stir the solution at room temperature for 20 minutes. Then, heptadecyl-alcohol-9 (6.3 g, 24.8 mmol) and 4-dimethylaminopyridine (DMAP) (170 mg) were added to the above solution at room temperature overnight. The crude product was obtained under reduced pressure and purified by silica gel column chromatography (methanol/dichloromethane=1/20) to obtain the colorless oily product compound 3-1 (9'-heptadecyl-6-bromocaproate). , 7.4g, 17.12mmol, yield 72.4%).

Compound 3-1 was analyzed by hydrogen nuclear magnetic spectrum. ¹ H NMR (600MHz, CDCl ₃ ) δ4.96–4.78 (m, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.31 (t, J = 7.4 Hz,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).

2. Synthesis of compound 3-2

Add heptadecyl-alcohol-9 (2.56g, 10mmol), zinc chloride (80mg) and two drops of concentrated sulfuric acid to the flask. Epichlorohydrin (1.84g, 20mmol) was added dropwise to the mixture within 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 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 product compound 3-2, 1.55 g, 4.9 mmol, yield 68.7%).

The product compound 3-2 was confirmed by ¹ H NMR and LCMS. 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).

3. Synthesis of compound 3-3

Methanol (15 ml), methylamine (0.60 g, 13.3 mmol) and compound 3-2 (1.0 g, 3.32 mmol) were added to the 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 obtained product was light yellow oil compound 3-3, 0.72g, 2.1mmol, 59.6%).

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

4. Synthesis of compound 3

Compound 3-1 (0.872g, 2mmol), K ₂ CO ₃ (447mg, 6.0mmol), KI (48.6mg, 0.6mmol), and compound 3-3 (0.442g, 1mmol) were dissolved in CNCH ₃ (15ml). After the mixed solution was stirred at 90°C for 5 hours, it was filtered and dried under reduced pressure. The resulting 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 substance and ionizable lipid compound 3 (0.42g, 0.60mmol, yield 45.3%).

The ionizable lipid compound 3 of this product was confirmed by LC-TOF[M+H]+: 696.68 and ^1H NMR. ¹ HNMR (600MHz, CDCl ₃ ) δ4.96-4.78 (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).

Example 4. Synthesis of ionizable lipid compound 4

Compound 4 was synthesized according to the following route:

1. Synthesis of compound 1-1

Compound 1-1 was prepared in the same manner as in Example 1, and a colorless oily product, compound 1-1, that is, 2-hexadecyl 6-bromohexanoate, was obtained.

2. Synthesis of compound 4-2

Add n-tetradecanol (2.14g, 10mmol), zinc chloride (80mg) and two drops of concentrated sulfuric acid to the flask. Epichlorohydrin (1.84g, 20mmol) was added dropwise to the mixture within 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 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 water phase was removed, and the organic phase was dried under reduced pressure to obtain the product compound 4-2(2-((2-tetradecyl)oxy)methyl)oxirane, 2.07g, 7.7mmol, yield 81.8 %).

The product compound 4-2 was confirmed by ¹ H NMR and LCMS. 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).

3. Synthesis of compound 4-3

Methanol (15 ml), methylamine (0.60 g, 13.3 mmol) and 2-(((2-hexyl)oxy)methyl)oxirane (0.90 g, 3.34 mmol) were added to the 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 obtained product was compound 4-3 (1-((2-hexadecyl)oxy)-3-(methylamino)propan-2ol, 0.78g, 2.6mmol, 67.7%) as a light yellow oil.

Compound 4-3 of this product was analyzed by LC-TOF, LC-TOF: [M+H] ⁺ 302.30.

4. Synthesis of compound 4

2-Hexadecyl 6-bromohexanoate (0.836g, 2mmol), K ₂ CO ₃ (447mg, 6.0mmol) and KI (48.6mg, 0.6mmol), 1-((2-hexadecyl) Oxy)-3-(methylamino)propan-2-ol (0.301 g, 1 mmol) was dissolved in CNCH ₃ (15 ml). After the mixed solution was stirred at 90°C for 5 hours, it was filtered and dried under reduced pressure. The resulting residue was redissolved in dichloromethane (50ml) and washed with brine (2x 40ml). 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 is a light yellow oily substance and ionizable lipid compound 4 (0.44g, 0.69mmol, yield 52.3%).

The ionizable lipid compound 4 of this product was confirmed by LC-TOF [M+H]+: 640.40 and ¹ H NMR. ¹ HNMR (600MHz, CDCl ₃ ) 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).

Example 5 Synthesis of Ionizable Lipid Compound 5

Compound 5 was synthesized according to the following route:

1. Synthesis of compound 2-1

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

2. Synthesis of compound 1-2

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

3. Synthesis of compounds 1-3

Compound 1-3 was prepared in the same manner as in Example 1, and a colorless oily product, compound 1-3, namely 1-((2-hexyl)oxy)-3-(methylamino)propan-2ol, was obtained.

4. Synthesis of compound 5

2-Octadecyl 6-bromohexanoate (0.932g, 2mmol), K ₂ CO ₃ (447mg, 6.0mmol) and KI (48.6mg, 0.6mmol), 1-((2-hexadecyl) Oxy)-3-(methylamino)propan-2-ol (0.442 g, 1 mmol) was dissolved in CNCH ₃ (15 ml). After the mixed solution was stirred at 90°C for 5 hours, it was filtered and dried under reduced pressure. The resulting residue was redissolved in dichloromethane (50ml) and washed with brine (2x 40ml). 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 is a light yellow oily substance and ionizable lipid compound 5 (0.44g, 0.63mmol, yield 51.40%).

The ionizable lipid compound 5 of this product was confirmed by LC-TOF[M+H]+: 696.68 and ^1H NMR. ¹ HNMR (600MHz, CDCl ₃ ) 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).

Example 6 Detection of particle size, polydispersity coefficient, encapsulation efficiency and transmission electron microscope of lipid nanoparticle preparation

1. Preparation of composition

The ionizable lipid compound 1 in Example 1, the ionizable lipid compound 2 in Example 2, the ionizable lipid compound 3 in Example 3, and the ionizable lipid compound in Example 4 are Compound 4, ionizable lipid compound 5 in Example 5 and 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 respectively. Prepare ethanolic lipid solution.

Briefly, a volume of 200 μL of firefly luciferase mRNA (FlucmRNA) solution encoding firefly luciferase (concentration of 0.5 μg/μL) was dissolved in 700 μL of 20 mM citrate buffer at pH=4 as the aqueous phase. Ionizable lipid compound 1 (1.242mg), DSPC (0.294mg), cholesterol (0.553mg) and DMG-PEG2000 (0.141mg) were dissolved in 300 μL ethanol at a molar ratio of 50:10:38.5:1.5 as organic Mutually. Mix the above aqueous phase and organic phase through a microfluidic mixer (N/P=6:1) to prepare an LNP-mRNA solution. Quickly add the prepared LNP-mRNA solution into an ultrafiltration tube containing 10 times the volume of PBS (pH=7.4) standard solution, and centrifuge at 5000 r/min for 30 minutes to remove the ethanol and citrate buffer. Use Amicon Ultra centrifugal filters to obtain the desired concentration of lipid nanoparticle formulation solution, designated as H01-Fluc mRNA. Ionizable lipid compound 2 in Example 2, Ionizable lipid compound 3 in Example 3, Ionizable lipid compound 4 in Example 4, Ionizable lipid compound in Example 5 5. The preparation process of commercially available ionizable lipid ALC-0315 lipid nanoparticles is the same as described above. The obtained lipid nanoparticle preparation solutions are recorded as H02-Fluc mRNA, H03-FlucmRNA, H04-Fluc mRNA, and H05-Fluc mRNA. and ALC-0315-Fluc mRNA. Add sucrose to the six obtained lipid nanoparticle preparation solutions based on 5% sucrose and set aside for use.

2. Lipid nanoparticle size, polydispersity coefficient, encapsulation efficiency and transmission electron microscope detection

The particle size 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 was detected using a Malvern particle size analyzer (Malvern UK). diameter, polydispersity coefficient (PDI), and the results are shown in Table 1.

RiboGreen reagent (Thermo Fisher Scientific, Cat No. 5, R11491) was used to detect the mRNA encapsulation efficiency (EE%). First, in order to determine the free mRNA in the lipid nanoparticle preparation, the above-mentioned H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315 containing 5% sucrose were taken. -10 μL of the Fluc mRNA lipid nanoparticle preparation solution was placed in a centrifuge tube, and 990 μL of 1×TE buffer (purchased from Nanjing Novezan Biotechnology Co., Ltd.) was added to dilute it, and 100 μL of the diluted solution was added to a 96-well plate. . Then, add 100 μL of RiboGreen dye to the plate well and incubate for 5 min. The concentration of free mRNA was measured using a spectrophotometer (Thermo Fisher Scientific, USA), with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. In order to determine the total mRNA content in the lipid nanoparticle preparation, take the above-mentioned lipid nanoparticle preparation solutions containing 5% sucrose H01-FlucmRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mRNA and 10 μL of the ALC-0315-FlucmRNA lipid nanoparticle preparation solution was placed in a centrifuge tube, and 990 μL of 2% TE-Triton buffer (purchased from Nanjing Novezan Biotechnology Co., Ltd.) was added to dilute it, and 100 μL of the diluted solution was added to in 96-well plates. Then, add 100 μL of RiboGreen dye to the plate well and incubate for 5 min. The total mRNA concentration value 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 of EE(%) is as follows:

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

The results are shown in Table 1.

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

As can be seen from Table 1, the particle size range of 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, PDI values are less than 0.2. This indicates that all six lipid nanoparticles have excellent physicochemical properties. At the same time, 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 The encapsulation rate of H01-Fluc mRNA and H03-Fluc mRNA lipid nanoparticles is higher than that of ALC-0315-Fluc mRNA lipid nanoparticles.

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 were observed by transmission electron microscopy (TEM, JEOL, Japan) ; The lipid nanoparticles prepared above were dropped on different 200 mesh carbon film copper grids; after natural air drying, 2% (w/v) phosphotungstic acid was dropped into the above copper grids, about 1-2 minutes; the treated sample was photographed at an accelerating voltage of 200kV; the results are shown in Figure 5. Figure 5 shows H01-Fluc mRNA, H02-Fluc mRNA, H03-FlucmRNA, and H04-Fluc in Example 6 Transmission electron microscopy images of lipid nanoparticles of mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA. The transmission electron microscopy image of Figure 5 shows that the lipid nanoparticles H01-Fluc mRNA, H02-Fluc mRNA and H03 prepared in this example -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 above-mentioned Malvern particle size analyzer. There is no big difference in the morphology of H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-FlucmRNA, H05-Fluc mRNA lipid nanoparticles and ALC-0315-Fluc mRNA lipid nanoparticles.

Example 7 Cell Transfection Experiment

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 The preparation process of H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, H05-eGFP mRNA and ALC-0315-eGFP mRNA lipid nanoparticle preparation solutions is the same as in Example 6. HEK293 cells or DC2.4 cells (2×10 ⁴ cells/well, cells are from the ATCC Biological Standards Resource Center of the United States) were seeded in a 6-well plate, and 2 mL DMEM medium was used to culture the cells until 80% confluence was reached; then, Wash the cells three times with PBS standard solution, replace the medium with 2 mL of Opti-MEM serum-free medium, and add the prepared H01-eGFP mRNA, H02-eGFP mRNA, H03-eGFP mRNA, and H04-eGFPmRNA containing 5% sucrose respectively. , H05-eGFP mRNA and ALC-0315-eGFP mRNA lipid nanoparticle preparation solution (mRNA concentration is 1.5 μg/ml). After incubation for 24 hours, the cells were collected, and the positive rate of eGFP protein in the cells was detected by flow cytometry (Accui C6, USA). The results are shown in Table 2.

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

As can be seen from Table 2, the lipid nanoparticle formulations H01-eGFPmRNA, H02-eGFP mRNA, H03-eGFP mRNA, H04-eGFP mRNA, and H05-eGFP mediated by ionizable lipid compounds 1, 2, 3, 4, and 5 The mRNA can be efficiently transfected into HEK293 cells or DC2.4 cells, and the transfection effect of the lipid nanoparticle preparation mediated by compounds 1 and 2 is better than that of the commercially available lipid ALC-0315, which has good application prospects.

Example 8 Cytotoxicity Experiment

HEK293 cells or DC2.4 cells were seeded in a 96-well plate (2000 cells/well) and cultured overnight; after the cells adhered, the H01-Fluc mRNA and H02-containing 5% sucrose prepared in Example 6 were Fluc mRNA, H03-FlucmRNA, H04-Fluc mRNA, H05-Fluc mRNA and ALC-0315-Fluc mRNA lipid nanoparticle preparation solutions were added to the 96-well plate, and the mRNA concentration was controlled to 2 μg/ml; after 24 hours of incubation, use Cell Counting Kit-8 (CCK-8, APExBIO, USA) was used to evaluate cytotoxicity; the OD value at 450 nm was measured by a microplate reader. The cell survival rate was calculated based on the OD value, and the results are shown in Table 3.

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

The results in Table 3 show that the six lipid nanoformulations have very little cytotoxicity and have good in vivo application prospects.

Example 9. Fluorescence detection in vivo

In order to study 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. , three in each group. H01-Fluc mRNA, H02-Fluc mRNA, H03-FlucmRNA, H04-FlucmRNA, H05-FlucmRNA and ALC-0315-Fluc containing 5% sucrose prepared in Example 6 were injected into the muscles of mice in each group. 100 μL of the mRNA lipid nanoparticle preparation solution encapsulates 20 μg of Fluc mRNA. 3, 6, 24, and 48 hours after completion of injection, 100 μL of luciferase substrate (concentration: 30 mg/mL) was intraperitoneally injected into the mice and allowed to react for 5 min. The bioluminescence signal image was obtained by a mouse imager (PerkinElmer), and the results are shown in Table 4.

Table 4 Mouse in vivo imaging experimental data of H01-Fluc mRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-FlucmRNA and ALC-0315-Fluc mRNA lipid nanoparticles

As shown in Table 4, the lipid nanoparticle preparations H01-FlucmRNA, H02-Fluc mRNA, H03-Fluc mRNA, H04-Fluc mRNA, H05-Fluc mediated by ionizable lipid compounds 1, 2, 3, 4 and 5 mRNA lipid nanoparticles can effectively deliver mRNA in vivo and complete expression, and the in vivo delivery effect of lipid nanoparticle preparations mediated by compounds 1, 2, and 3 is better than that of commercially available lipid ALC-0315, with good Application prospects.

Example 10. Stability testing

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

Table 5 Mouse in vivo imaging in vivo experimental data (stability test)

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

Claims (10)

1. An ionizable lipid or a pharmaceutically acceptable salt thereof, characterized in that the structure of the ionizable lipid is as shown in Formula I, Among them, X is a C8-18 branched or linear alkyl group, Y is a C15-18 branched alkyl group, and Z is a methyl or ethyl group. 2. The ionizable lipid or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, the X is selected from: The Y is selected from: 3. The ionizable lipid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the ionizable lipid is selected from: 4. A composition, characterized in that the composition includes a therapeutic agent or a preventive agent and a carrier for delivering the therapeutic agent or preventive agent, wherein the therapeutic agent or preventive agent is siRNA, shRNA, or mRNA. One or more; the carrier includes one or more of the ionizable lipids or pharmaceutically acceptable salts thereof according to any one of claims 1-3; the carrier and a therapeutic or preventive agent The mass ratio is: 1~100:1. 5. The composition according to claim 4, characterized in that the composition is lipid nanoparticles, and the average particle size of the lipid nanoparticles is 60 nm to 300 nm; the polydispersity of the lipid nanoparticles Index ≤ 0.30. 6. The composition according to claim 4, wherein the carrier further comprises a structural lipid, and the structural lipid is cholesterol, campesterol, stigmasterol, brassinosterol, sitosterol, and ergosterol. , one or more of non-sterols, corticosteroids, ursolic acid, tomatine, tomatine and alpha-tocopherol; The molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the structural lipid is 1 to 10:1. 7. The composition according to claim 6, wherein the carrier further includes neutral lipids, and the neutral lipids are ceramide, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and derivatives thereof One or more substances; the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt to the neutral lipid is 1:2 to 20:1. 8. The composition according to claim 7, wherein the carrier further comprises a polymer conjugated lipid, and the polymer conjugated lipid is PEG-modified phosphatidylethanolamine, PEG-modified ceramide, One or more of PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine and PEG-modified dialkylglycerol; the ionizable lipid or its pharmaceutically acceptable The molar ratio of salt to polymer conjugated lipid is 10 to 200:1. 9. The composition according to claim 8, characterized in that the molar ratio of the ionizable lipid or its pharmaceutically acceptable salt, structural lipid, neutral lipid and polymer conjugated lipid It is (15～60): (15～45): (1～20): (0.5-2). 10. Use of the ionizable lipid according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof or the composition according to any one of claims 4 to 9 in the preparation of nucleic acid drugs.