A zwitterionic compound, complex and its application

Abstract

This invention discloses an amphoteric compound or its pharmaceutically acceptable salt, complex and its applications, wherein the amphoteric compound has a general structural formula as shown in formula (I); the amphoteric compound or its pharmaceutically acceptable salt of this invention can be used to prepare lipid nanoparticles for drug delivery, and the lipid nanoparticle delivery system has higher transfection efficiency, better delivery efficiency and better mRNA encapsulation rate compared with commercially available delivery systems.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an amphoteric compound, a complex, and its applications. Background Technology

[0002] Nucleic acid drugs, as a large and emerging class of drugs, are characterized by rapid design, wide application, and high safety, making them one of the main directions for future drug development. However, the poor cell penetration and easy degradation of nucleic acid drugs pose significant challenges to their in vivo application. Therefore, it is necessary to develop specific compounds and delivery systems to improve this situation and promote nucleic acid drugs as an important means of disease prevention and treatment.

[0003] Lipid nanoparticles (LNPs) are a class of nanoscale carriers composed of lipid molecules (typically with a particle size of 80-100 nm). They can be used for the efficient delivery of hydrophobic or hydrophilic drugs, especially nucleic acid drugs (such as mRNA, siRNA, etc.). The lipid shell of LNPs encapsulates the mRNA in its core, effectively shielding it from nucleases and preventing its degradation during circulation in vivo.

[0004] Currently, lipid nanoparticles (LNPs) prepared based on ionizable cationic lipids are a relatively safe and effective means of delivering nucleic acid drugs. LNPs typically consist of four components: ionizable lipids, cholesterol, PEGylated lipids, and auxiliary lipids. For example, Chinese patent document CN118845700A discloses a lipid nanoparticle composition for delivering nucleic acid drugs, comprising: a steroid-cationic lipid compound, auxiliary lipids, and polyethylene glycol lipids. Chinese patent document CN118851922A discloses a lipid nanoparticle comprising an organic phase and an aqueous phase, wherein the organic phase includes ionizable lipids, phospholipids, cholesterol, and PEGylated lipids.

[0005] The auxiliary lipids are mainly zwitterionic lipids, which are crucial for regulating the fluidity and phase transition temperature of the lipid bilayer in LNPs. They can promote membrane fusion, disrupt endosomal stability, and improve LNP delivery efficiency. Therefore, the targeted development and introduction of zwitterionic compounds into LNPs can improve the LNP system and increase mRNA delivery efficiency. Summary of the Invention

[0006] This invention provides a method for preparing zwitterionic compounds and their complexes, and their applications. The complexes containing the zwitterionic compounds have high transfection efficiency, good delivery efficiency, and excellent biosafety, and can be used to prepare pharmaceutical compositions or vaccine compositions.

[0007] The technical solution of the present invention is as follows: A zwitterionic compound or a pharmaceutically acceptable salt thereof, said zwitterionic compound having a general structural formula as shown in formula (I): Among them, G1 and G2 are each independently selected from -(CH2). x -O(C=O)-, -(CH2) x -(C=O)O-、-(CH2) x -S(C=O)-, -(CH2) x -(C=O)S-、-(CH2) x -(C=O)NH-, -(CH2) x NH(C=O)-, -(CH2) x -O-、-(CH2) x -O(C=O)NH- or -(CH2) x -O(C=O)O-, where x is an integer from 0 to 4; G3 is selected from C 2-10 alkenyl; L2, L3, L5, and L6 are each independently selected from unsubstituted C. 0-10 alkyl; L1 and L4 are each independently selected from unsubstituted straight chains or branched C-chains. 1-25 Alkyl, C 2-25 alkenyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1, R2, and R3 are each independently selected from any substituted C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl or phenyl, wherein the substituents are one or two independent substituents. , , , C 1-3 Alkyl, C 1-3 Alkyl group or -(C=O)OC 1-3 alkyl.

[0008] Preferably, G1 and G2 are each independently selected from -O(C=O)-, -(C=O)O-, -S(C=O)-, -(C=O)S-, -(C=O)NH-, and -NH(C=O)-; G3 is ; L2 and L3 are each independently selected from unsubstituted C 5-10 Alkyl group; L5 and L6 are each independently selected from unsubstituted C16 groups.0-5 alkyl; L1 and L4 are each independently selected from straight chains or C chains with branches. 5-15 alkyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1, R2, and R3 are each independently selected from any substituted C 1-3 Alkyl groups, wherein the substituents are one or two independent substituents. , , , .

[0009] Preferably, G1 and G2 are each independently selected from -O(C=O)- and -(C=O)O-; -L3-G3-L4 is ; L2 represents unsubstituted C. 5-8 Alkyl group; L5 and L6 are each independently selected from unsubstituted C16 groups. 1-4 alkyl; L1 is a branched C 15-20 alkyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1 and R2 are each independently selected from unsubstituted C 1-3 Alkyl; R3 is selected from , , , .

[0010] Preferably, the zwitterionic compound is: ; L5 and L6 are each independently selected from unsubstituted C. 1-4 alkyl; R1 and R2 are each independently selected from unsubstituted C 1-3 Alkyl; R3 is selected from , , , .

[0011] Preferably, the zwitterionic compound is selected from any one of the following: Compound 1: ; Compound 2: ; Compound 3: ; Compound 4: ; Compound 5: ; Compound 6: ; Compound 7: ; Compound 8: ; Compound 9: ; Compound 10: ; Compound 11: ; Compound 12: ; Compound 13: ; Compound 14: .

[0012] The pharmaceutically usable salt is an acid addition salt or a base addition salt.

[0013] The zwitterionic compounds or their pharmaceutically acceptable salts of the present invention can be used to prepare lipid nanoparticles for drug delivery. The lipid nanoparticle delivery system composed of the zwitterionic compounds or their pharmaceutically acceptable salts of the present invention can be applied to in vivo and in vitro drug delivery. Compared with commercially available delivery systems, this lipid nanoparticle delivery system has higher transfection efficiency, better delivery efficiency and better mRNA encapsulation rate. It can serve as a new method for drug delivery, especially nucleic acid drugs, solving the problem of difficult nucleic acid drug delivery and promoting the development of nucleic acid drugs.

[0014] The present invention also provides a complex comprising the aforementioned zwitterionic compound or a pharmaceutically acceptable salt thereof.

[0015] Preferably, the complex comprises the zwitterionic compound or its pharmaceutically acceptable salt, ionizable lipid, phospholipid and / or structural lipid and / or polyethanolated lipid.

[0016] Preferably, the complex comprises the zwitterionic compound or its pharmaceutically acceptable salt, ionizable lipid, phospholipid, structural lipid, and polyethanolated lipid; the molar ratio of the zwitterionic compound, ionizable lipid, phospholipid, structural lipid, and polyethanolated lipid is 0-20:10-100:0-20:0-50:0-5.

[0017] Further preferably, the molar ratio of zwitterionic compounds, ionizable lipids, phospholipids, structural lipids and polyethanol-modified lipids is 1-10:20-90:0-10:1-50:0.1-5.

[0018] Further preferably, the molar ratio of zwitterionic compounds, ionizable lipids, phospholipids, structural lipids and polyethanol-modified lipids is 1-10:40-60:0-10:30-50:1-5.

[0019] The ionizable lipid is at least one of compounds 1-5, Dlin-MC3-DMA (CAS: 1224606-06-7), SM-102 (CAS: 2089251-47-6), ALC-0315 (CAS: 2036272-55-4), C12-200 (CAS: 1220890-25-4), 5A2-SC8 (1857341-90-2), and CKK-E12 (1432494-65-9).

[0020] Compounds 1-5 have the structure shown in formula (II): .

[0021] The phospholipid is at least one of distearylphosphatidylcholine, 1,2-dioleoyl lecithin, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, and 1,2-distearyl-SN-glycerol-3-phosphoethanolamine.

[0022] The structural lipids mentioned are at least one of cholesterol, β-sitosterol, stigmasterol, and rapeseed sterol.

[0023] The PEGylated lipid is at least one of DMG-PEG 2000 (CAS: 160743-62-4), DSG-PEG 2000 (CAS: 308805-39-2), and DSPE-PEG 2000 (CAS: 474922-77-5).

[0024] The present invention also provides the use of the aforementioned complex in the preparation of lipid nanoparticles for drug delivery.

[0025] The applications include: (1) Dissolve zwitterionic compounds, ionizable lipids, phospholipids, structural lipids and polyethanol-modified lipids in an organic solvent to obtain an organic phase solution; (2) Add the treatment or preventative agent to the buffer solution to obtain an aqueous solution; (3) Mix the organic phase solution and the aqueous phase solution to obtain lipid nanoparticles for drug delivery.

[0026] The treatment or preventive agent mentioned is a nucleic acid drug or nucleic acid vaccine.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention utilizes zwitterionic compounds as shown in structural formula (I) to prepare complexes, thus possessing the following beneficial effects: the complexes provided by the present invention are lipid nanoparticles with uniform nanoparticle size and an encapsulation efficiency ≥85%; the complexes provided by the present invention exhibit excellent biocompatibility, low cytotoxicity, and low hemolysis rate; the complexes provided by the present invention have high transfection efficiency, superior to currently marketed MC3 lipid nanoparticles; the complexes provided by the present invention have good delivery efficiency, with a superior ability to deliver Luciferase mRNA in small animals compared to currently marketed MC3.

[0028] This invention provides a complex with high transfection efficiency, good delivery efficiency and excellent biocompatibility, which can be used for in vivo and in vitro delivery of therapeutic or preventive agents, especially nucleic acid drugs, solving the problem of difficult nucleic acid drug delivery and promoting the development of nucleic acid drugs.

[0029] Attached Figure Description Figure 1 These are in vivo images of mice transfected with Luciferase mRNA using lipid nanoparticles prepared in Examples 15 to 28 of the present invention. Figure 2 Fluorescence intensity thermograms of Luciferase mRNA transfected with lipid nanoparticles in Examples 15 to 28 of the Invention; Figure 3 In vivo imaging images of mice transfected with Luciferase mRNA in Examples 27, 29, 30, 31, and 32 of the invention; Figure 4 The hemolysis conditions of Examples 27 and 29 of the invention at pH 7.4 and pH 5.5; Figure 5 Cell viability diagrams for embodiments 27 and 29 of the invention; Figure 6 These are in vivo imaging images of mice transfected with Luciferase mRNA using lipid nanoparticles, as shown in Examples 33 and 34 of the Invention. Figure 7 These are in vivo imaging images of mice transfected with Luciferase mRNA using lipid nanoparticles, as shown in Examples 35 and 36 of the Invention. Figure 8 These are in vivo imaging images of mice transfected with Luciferase mRNA using lipid nanoparticles, as shown in Examples 37 and 38 of the Invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] Example 1: A method for synthesizing a zwitterionic compound 1, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 1 includes the following steps: Step 1: Synthesis of Compound 1-1 Triethylamine (4.8 mL, 30 mmol, 1.5 equiv.) was slowly added to a solution of acryloyl chloride (1800 mg, 20 mmol, 1 equiv.) and (9Z, 12Z)-octadecane-9,12-dien-1-ol (5320 mg, 20 mmol, 1 equiv.) in dichloromethane (60 mL), and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, dichloromethane (40 mL) was added, and the mixture was washed with saturated sodium chloride aqueous solution (100 mL × 3 times). The organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography with a mobile phase of petroleum ether solution containing 5% ethyl acetate (v / v) to give a colorless oily compound 1-1 (6.0 g), yield: 90%.

[0033] 1 H NMR (400 MHz, CDCl3) δ 6.41 (dd, J = 6.8, 1.6 Hz, 1H), 6.12 (dd, J = 17.6, 10.4 Hz, 1H), 5.80 (dd, J = 10.4, 1.6 Hz, 1H), 5.50 – 5.28 (m, 4H), 4.30 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 2.11 (q, J= 6.8 Hz, 4H),1.75 – 1.56 (m, 2H), 1.50 – 1.18 (m, 16H), 0.81 (t, J = 6.8 Hz, 3H). Step 2: Synthesis of Compounds 1-2 Compound 1-1 (3.1 g, 9.3 mmol, 1 equiv.) was dissolved in tetrahydrofuran (60 mL), followed by the addition of paraformaldehyde (1.84 g, 46 mmol, 5 equiv.), 1,4-diazabicyclo[2.2.2]octane (5.15 g, 46 mmol, 5 equiv.), and 10 mL of water. The mixture was stirred at 60 °C for 12 hours. After the reaction was complete, the mixture was extracted with ethyl acetate (100 mL × 3 times), and the organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by rotary evaporation to obtain a pale yellow oily compound. This compound was dissolved in dichloromethane (80 mL), followed by the addition of di-tert-butyl dicarbonate (3.04 g, 14 mmol, 1.5 equiv.) and 4-dimethylaminopyridine (113 mg, 0.93 mmol, 0.1 equiv.). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography with a mobile phase of petroleum ether solution containing 5% EA (volume percentage) to give a pale yellow oily compound 1-2 (3.6 g) in 84% yield.

[0034] 1 H NMR (400 MHz, CDCl3) δ 6.36 (q, J = 1.2 Hz, 1H), 5.93 (q, J = 1.6Hz, 1H), 5.50 – 5.18 (m, 4H), 4.75 (t, J = 1.2 Hz, 2H), 4.19 (t, J = 6.8 Hz,2H), 2.94 – 2.69 (m, 2H), 2.23 – 2.01 (m, 4H), 1.75 – 1.63 (m, 2H), 1.51 (s,9H), 1.42 – 1.23 (m, 16H), 0.80 (t, J = 6.8 Hz, 3H). Step 3: Synthesis of compounds 1-3 Compounds 1-2 (3.6 g, 8.0 mmol, 1 equiv.) were dissolved in dichloromethane (60 mL), followed by the addition of 1,6-hexanediol (18.9 g, 160.0 mmol, 20 equiv.) and 1,4-diazabicyclo[2.2.2]octane (0.01 g, 0.08 mmol, 0.01 equiv.). The mixture was stirred at room temperature for 8 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using a petroleum ether solution containing 15% ethyl acetate (v / v) as the mobile phase, yielding a pale yellow oily compound 1-3 (3.3 g), in 89% yield.

[0035] 1 H NMR (400 MHz, CDCl3) δ 6.34 (q, J = 1.2 Hz, 1H), 5.90 (q, J = 1.6Hz, 1H), 5.55 – 5.22 (m, 4H), 4.35 – 4.14 (m, 4H), 3.69 (t, J = 6.8 Hz, 2H), 3.52 (t, J = 6.8 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H), 2.10 (q, J = 6.8 Hz, 4H),1.75 – 1.62 (m, 6H), 1.49 – 1.21 (m, 21H), 0.82 (t, J = 6.8 Hz, 3H). Step 4: Synthesis of compounds 1-4 Compounds 1-3 (3.3 g, 7.1 mmol, 1 equiv.) were dissolved in dichloromethane (60 mL), followed by the addition of 2-hexyldecanoic acid (5.5 g, 21.3 mmol, 3 equiv.), 4-dimethylaminopyridine (2.6 g, 21.3 mmol, 3 equiv.), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.0 g, 21.3 mmol, 3 equiv.). The mixture was stirred at room temperature for 8 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using a dichloromethane solution containing 5% methanol (v / v) as the mobile phase, yielding a pale yellow oily compound 1-4 (4.2 g), in 85% yield.

[0036] 1H NMR (400 MHz, CDCl3) δ 6.30 (q, J = 1.6 Hz, 1H), 5.86 (q, J = 1.6Hz, 1H), 5.49 – 5.33 (m, 4H), 4.21 – 4.18 (m, 4H), 4.08 (t, J = 6.8 Hz, 2H), 3.50 (t, J = 6.8 Hz, 2H), 2.80 (t, J = 6.8 Hz, 2H), 2.35 (tt, J = 8.8, 5.2Hz, 1H), 2.07 (q, J = 6.8 Hz, 4H), 1.70 – 1.61 (m, 8H), 1.45 – 1.15 (m, 42H), 1.01 – 0.79 (m, 9H). Step 5: Synthesis of compounds 1-5 Compounds 1-4 (4.2 g, 6.0 mmol, 1 equiv.) were dissolved in dichloromethane (60 mL), followed by the addition of 3-dimethylaminopropanethiol (2.1 g, 18.0 mmol, 3 equiv.), 1,5,7-triazabicyclo[4.4.0]decen-5-ene (2.0 g, 18.0 mmol, 3 equiv.), and acetonitrile (2 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using a dichloromethane solution containing 10% methanol (v / v) as the mobile phase, yielding a pale yellow oily compound 1-5 (4.3 g), in 89% yield.

[0037] 1 H NMR (400 MHz, CDCl3) δ 5.48 – 5.23 (m, 4H), 4.09 (t, J = 6.8 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 3.70 – 3.59 (m, 2H), 3.38 (td, J = 6.8, 2.0Hz, 2H), 2.79 (m, 5H), 2.50 (t, J = 7.6 Hz, 2H), 2.40 – 2.28 (m, 3H), 2.20(s, 6H), 2.05 (q, J= 6.8 Hz, 4H), 1.72 (p, J = 7.2 Hz, 2H), 1.69 – 1.57 (m,8H), 1.49 – 1.39 (m, 2H), 1.39 – 1.21 (m, 40H), 0.86 (m, 9H). Step 6: Synthesis of Compound 1 Compounds 1-5 (4.3 g, 5.3 mmol, 1 equiv.) were dissolved in anhydrous acetonitrile (60 mL), and 1,3-propanesulfonic acid lactone (1.3 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 25% methanol (v / v) to give compound 1 (2.7 g) as a pale yellow solid, yield: 55%.

[0038] 1 H NMR (400 MHz, CDCl3) δ 5.38 – 5.28 (m, 4H), 4.13 – 4.08 (m, 1H), 4.06 – 4.02 (m, 3H), 3.69 – 3.68 (m, 2H), 3.63 – 3.58 (m, 2H), 3.45 – 3.43(m, 2H), 3.40 – 3.36 (m, 2H), 3.22 (s, 6H), 2.96 – 2.91 (m, 2H), 2.80 – 2.74(m, 5H), 2.67 – 2.58 (m, 2H), 2.31 – 2.21 (m, 3H), 2.06 – 2.01 (m, 6H), 1.62– 1.56 (m, 6H), 1.36 – 1.24 (m, 44H), 0.90 – 0.85 (m, 9H). 13C NMR (101 MHz, CDCl3) δ 176.80, 172.89, 130.34, 130.16, 128.16,128.03, 71.41, 70.45, 65.19, 64.13, 63.71, 63.54, 51.18, 47.79, 46.72, 45.93,32.63, 31.98, 31.82, 31.65, 30.70, 29.82, 29.69, 29.63, 29.57, 29.47, 29.43,29.41, 29.38, 29.35, 29.22, 28.82, 28.76, 27.58, 27.54, 27.36, 27.33, 26.05, 25.94, 25.86, 25.76, 22.79, 22.72, 22.70, 22.63, 14.25, 14.22. Example 2: A method for synthesizing a zwitterionic compound 2, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 2 includes the following steps: Step 1: Synthesis of Compound 2-1 Compound 1-4 (4.2 g, 6.0 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), followed by the addition of 2-dimethylaminoethanethiol (1.9 g, 18.0 mmol, 3 equiv.), 1,5,7-triazabicyclo[4.4.0]decen-5-ene (2.0 g, 18.0 mmol, 3 equiv.), and acetonitrile (2 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using a dichloromethane solution containing 10% methanol (v / v) as the mobile phase, yielding a pale yellow oily compound 2-1 (4.2 g), in 88% yield.

[0039] 1 H NMR (400 MHz, CDCl3) δ 5.44 – 5.27 (m, 4H), 4.08 (m, 4H), 3.70 –3.57 (m, 2H), 3.40 (td, J = 6.6, 1.6 Hz, 2H), 2.91 – 2.71 (m, 5H), 2.69 –2.61 (m, 2H), 2.56 (d, J= 8.0 Hz, 2H), 2.36 – 2.24 (m, 3H), 2.28 (s, 6H), 2.05 (q, J = 6.8 Hz, 4H), 1.62 – 1.32 (m, 8H), 1.29 – 1.21 (m, 40H), 0.88 (m, 9H). Step 2: Synthesis of Compound 2 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and 1,3-propanesulfonic acid lactone (1.3 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 20% ​​methanol (v / v) to give compound 2 (2.5 g) as a pale yellow solid, yield: 52%.

[0040] 1 H NMR (400 MHz, MeOD- d 4) δ 5.39 – 5.32 (m, 4H), 4.21 – 4.15 (m, 1H), 4.11 – 4.06 (m, 3H), 3.68 (d, J = 5.2 Hz, 2H), 3.58 – 3.52 (m, 4H), 3.46 –3.41 (m, 2H), 3.13 (s, 6H), 3.00 – 2.96 (m, 2H), 2.93 – 2.84 (m, 5H), 2.80 –2.77 (t, J = 6.4 Hz, 2H), 2.37 – 2.32 (m, 1H), 2.22 – 2.17 (m, 2H), 2.10 –2.04 (m, 4H), 1.69 – 1.62 (m, 6H), 1.39 – 1.29 (m, 44H), 0.92 – 0.89 (m, 9H). 13C NMR (101 MHz, CDCl3) δ 176.81, 172.99, 130.35, 130.15, 128.17,128.03, 71.44, 70.45, 65.33, 64.14, 50.93, 46.85, 45.92, 32.62, 32.00, 31.84,31.66, 31.64, 31.57, 30.26, 29.85, 29.70, 29.59, 29.49, 29.40, 29.36, 28.84,28.78, 27.59, 27.55, 27.39, 27.34, 26.09, 25.96, 25.88, 25.77, 22.84, 22.81, 22.74, 22.72, 14.28, 14.24. Example 3: A method for synthesizing an amphoteric compound 3, the synthetic route of which is as follows: Step 1: Synthesis of Compound 3 Compounds 1-5 (4.3 g, 5.3 mmol, 1 equiv.) were dissolved in anhydrous acetonitrile (60 mL), and 1,4-butyryl lactone (1.4 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 25% methanol (v / v) to give compound 3 (2.5 g) as a pale yellow solid, yield: 49%.

[0041] 1H NMR (400 MHz, CDCl3) δ 5.37 – 5.28 (m, 4H), 4.13 – 4.10 (m, 1H), 4.05 – 4.02 (m, 3H), 3.63 – 3.52 (m, 4H), 3.44 – 3.36 (m, 4H), 3.20 (s, 6H),2.90 – 2.86 (m, 2H), 2.80 – 2.74 (m, 5H), 2.62 – 2.60 (m, 2H), 2.32 – 2.25(m, 1H), 2.06 – 1.99 (m, 6H), 1.87 – 1.86 (m, 2H), 1.62 – 1.59 (m, 4H), 1.56– 1.51 (m, 4H), 1.33 – 1.24 (m, 44H), 0.89 – 0.84 (m, 9H). 13 C NMR (101 MHz, CDCl3) δ 176.79, 172.87, 130.33, 130.16, 128.15,128.02, 71.40, 70.43, 65.18, 64.35, 64.11, 62.94, 51.13, 50.39, 46.72, 45.92,32.62, 31.98, 31.81, 31.64, 30.73, 29.80, 29.68, 29.62, 29.56, 29.47, 29.41,29.39, 29.37, 29.34, 29.23, 28.81, 28.75, 27.57, 27.53, 27.36, 27.32, 26.03, 25.93, 25.85, 25.75, 22.78, 22.71, 22.69, 22.25, 21.53, 14.24, 14.21. Example 4: A method for synthesizing a zwitterionic compound 4, the synthetic route of which is as follows: Step 1: Synthesis of Compound 4 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and 1,4-butyryl lactone (1.4 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 25% methanol (v / v) to give compound 4 (2.4 g), a pale yellow solid, in 48% yield.

[0042] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.31 (m, 4H), 4.19 – 4.15 (m, 1H), 4.12 – 4.06 (m, 3H), 3.68 (d, J = 5.2 Hz, 2H), 3.57 – 3.53 (m, 2H), 3.46 –3.36 (m, 4H), 3.12 (s, 6H), 3.00 – 2.95 (m, 2H), 2.91 – 2.83 (m, 5H), 2.80 –2.77 (t, J = 6.0 Hz, 2H), 2.36 – 2.32 (m, 1H), 2.10 – 2.05 (m, 4H), 1.88 –1.80 (m, 4H), 1.67 – 1.62 (m, 6H), 1.39 – 1.29 (m, 44H), 0.92 – 0.88 (m, 9H). 13C NMR (101 MHz, CDCl3) δ 176.80, 172.88, 130.35, 130.16, 128.17,128.03, 71.41, 70.42, 65.29, 64.36, 64.13, 63.53, 51.14, 50.36, 46.94, 45.92,32.62, 31.99, 31.83, 31.65, 30.81, 29.83, 29.70, 29.65, 29.58, 29.48, 29.44,29.43, 29.39, 29.35, 28.82, 28.76, 27.58, 27.54, 27.37, 27.33, 26.06, 25.93, 25.86, 25.76, 25.16, 22.79, 22.72, 22.71, 22.21, 21.52, 14.25, 14.22. Example 5: A method for synthesizing an amphoteric compound 5, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 5 includes the following steps: Step 1: Synthesis of Compound 5-1 Compound 1-5 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromoacetate (2.1 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give compound 5-1 (2.4 g), a pale yellow oil, in 44% yield.

[0043] 1H NMR (400 MHz, CDCl3) δ 5.32 – 5.17 (m, 4H), 4.17 – 4.13 (m, 1H), 4.10 – 4.06 (m, 3H), 3.94 (s, 2H), 3.68 – 3.51 (m, 4H), 3.40 – 3.28 (m, 2H),3.19 (s, 6H), 2.90 – 2.85 (m, 1H), 2.81 – 2.75 (m, 4H), 2.56 – 2.50 (m, 2H),2.24 – 2.15 (m, 1H), 1.97 – 1.84 (m, 6H), 1.61 – 1.46 (m, 6H), 1.44 (s, 9H),1.40 – 1.11 (m, 44H), 0.91 – 0.73 (m, 9H). Step 2: Synthesis of Compound 5 Compound 5-1 (2.4 g, 2.3 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 5 (1.9 g) as a pale yellow solid, yield: 85%.

[0044] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.29 (m, 4H), 4.17 – 4.13 (m, 1H), 4.10 – 4.06 (m, 3H), 3.84 (s, 2H), 3.69 – 3.65 (m, 4H), 3.46 – 3.39 (m, 2H), 3.26 (s, 6H), 2.90 – 2.85 (m, 1H), 2.81 – 2.75 (m, 4H), 2.63 – 2.60 (m, 2H), 2.38 – 2.30 (m, 1H), 2.08 – 2.03 (m, 6H), 1.67 – 1.62 (m, 6H), 1.39 – 1.29(m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.16, 174.56, 168.73, 130.96, 130.87,129.14, 129.05, 72.16, 71.59, 65.93, 65.25, 64.94, 64.56, 51.81, 47.89,47.11, 33.81, 33.06, 32.91, 32.71, 31.08, 30.81, 30.65, 30.62, 30.52, 30.41,30.38, 30.34, 29.85, 29.81, 29.77, 28.61, 28.59, 28.23, 27.10, 26.99, 26.94, 26.61, 23.77, 23.72, 23.69, 23.68, 14.56, 14.53. Example 6: A method for synthesizing a zwitterionic compound 6, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 6 includes the following steps: Step 1: Synthesis of Compound 6-1 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromoacetate (2.1 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give a pale yellow oily compound 6-1 (2.5 g), yield: 47%.

[0045] 1H NMR (400 MHz, CDCl3) δ 5.43 – 5.28 (m, 4H), 4.18 – 3.95 (m, 4H), 3.79 (s, 2H), 3.70 – 3.65 (m, 2H), 3.59 – 3.54 (m, 2H), 3.35 – 3.30 (m, 2H),3.10 (s, 6H), 2.97 – 2.62 (m, 7H), 2.36 – 2.18 (m, 1H), 2.06 – 2.01 (m, 4H),1.62 – 1.47 (m, 6H), 1.44 (s, 9H), 1.42 – 1.20 (m, 44H), 0.93 – 0.79 (m, 9H). Step 2: Synthesis of Compound 6 Compound 6-1 (2.5 g, 2.5 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 6 (1.8 g) as a pale yellow solid, yield: 76%.

[0046] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.31 (m, 4H), 4.18 – 4.14 (m, 1H), 4.11 – 4.06 (m, 3H), 3.87 (s, 2H), 3.85 – 3.79 (m, 2H), 3.67 – 3.66 (m, 2H), 3.45 – 3.41 (m, 2H), 3.26 (s, 6H), 2.95 – 2.77 (m, 7H), 2.36 – 2.32 (m, 1H), 2.10 – 2.04 (m, 4H), 1.67 – 1.62 (m, 6H), 1.41 – 1.29 (m, 44H), 0.93 – 0.89(m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.29, 174.36, 168.59, 130.96, 130.87,129.14, 129.06, 72.19, 71.47, 68.86, 66.01, 65.28, 64.93, 64.44, 54.82,51.76, 47.97, 47.15, 33.82, 33.05, 32.91, 32.71, 31.23, 30.80, 30.62, 30.51,30.40, 30.37, 30.33, 29.84, 29.82, 28.60, 28.59, 28.21, 27.10, 26.98, 26.94, 26.59, 26.50, 25.49, 23.76, 23.68, 14.50, 14.47. Example 7: A method for synthesizing a zwitterionic compound 7, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 7 includes the following steps: Step 1: Synthesis of Compound 7-1 Compounds 1-5 (4.3 g, 5.3 mmol, 1 equiv.) were dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromobutyrate (2.4 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give compound 7-1 (2.7 g), a pale yellow oil, in 50% yield.

[0047] 1 H NMR (400 MHz, CDCl3) δ 5.41 – 5.25 (m, 4H), 4.15 – 3.99 (m, 4H), 3.70 – 3.52 (m, 2H), 3.42 – 3.32 (m, 6H), 3.06 (s, 6H), 2.87 – 2.64 (m, 5H),2.58 – 2.55 (m, 2H), 2.38 (t, J= 6.8 Hz, 2H), 2.23 – 2.15 (m, 1H), 2.04 –1.99 (m, 8H), 1.66 – 1.47 (m, 6H), 1.41 (s, 9H), 1.39 – 1.20 (m, 44H), 0.85(m, 9H). Step 2: Synthesis of Compound 7 Compound 7-1 (2.7 g, 2.7 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 7 (2.0 g) as a pale yellow solid, yield: 77%.

[0048] 1 H NMR (400 MHz, MeOD- d 4) δ 5.39 – 5.32 (m, 4H), 4.18 – 4.12 (m, 1H), 4.10 – 4.06 (m, 3H), 3.66 – 3.65 (m, 2H), 3.46 – 3.37 (m, 6H), 3.13 (s, 6H), 2.91 – 2.86 (m, 1H), 2.83 – 2.77 (m, 4H), 2.67 – 2.62 (m, 2H), 2.45 (t, J =6.8 Hz, 2H), 2.38 – 2.31 (m, 1H), 2.11 – 2.02 (m, 8H), 1.67 – 1.60 (m, 6H), 1.40 – 1.29 (m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.31, 175.32, 174.62, 130.96, 130.86,129.15, 129.05, 72.18, 71.62, 65.98, 65.26, 64.23, 63.99, 54.81, 51.67,49.85, 47.95, 47.14, 33.81, 33.08, 33.05, 32.90, 32.70, 31.93, 31.14, 30.88,30.79, 30.76, 30.70, 30.62, 30.60, 30.51, 30.48, 30.39, 30.35, 30.32, 30.13, 29.83, 29.81, 29.66, 28.59, 28.58, 28.20, 27.09, 26.96, 26.93, 26.58, 23.75, 23.67, 23.43, 18.98, 14.49, 14.46. Example 8: A method for synthesizing a zwitterionic compound 8, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 8 includes the following steps: Step 1: Synthesis of Compound 8-1 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromobutyrate (2.36 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give a pale yellow oily compound 8-1 (2.8 g), yield: 52%.

[0049] 1 H NMR (400 MHz, CDCl3) δ 5.41 – 5.25 (m, 4H), 4.16 – 3.98 (m, 4H), 3.58 – 3.53 (m, 2H), 3.50 – 3.36 (m, 6H), 3.14 (s, 6H), 3.05 – 2.71 (m, 7H),2.42 (t, J= 6.4 Hz, 2H), 2.30 – 2.21 (m, 1H), 2.04 – 1.95 (m, 6H), 1.66 –1.47 (m, 6H), 1.41 (s, 9H), 1.39 – 1.20 (m, 44H), 0.91 – 0.81 (m, 9H). Step 2: Synthesis of Compound 8 Compound 8-1 (2.8 g, 2.8 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 8 (2.1 g) as a pale yellow solid, yield: 78%.

[0050] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.30 (m, 4H), 4.19 – 4.15 (m, 1H), 4.10 – 4.06 (m, 3H), 3.68 – 3.67 (m, 2H), 3.59 – 3.55 (m, 2H), 3.46 – 3.35(m, 4H), 3.14 (s, 6H), 3.01 –2.97 (m, 2H), 2.93 –2.87 (m, 2H), 2.85 –2.77 (m,3H), 2.45 (t, J = 6.8 Hz, 2H), 2.36 – 2.32 (m, 1H), 2.09 – 2.01 (m, 6H), 1.67 – 1.62 (m, 6H), 1.39 – 1.29 (m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.31, 175.39, 174.41, 130.96, 130.86,129.15, 129.05, 72.22, 71.45, 66.07, 65.25, 64.37, 64.18, 51.47, 48.09,47.14, 33.82, 33.09, 33.05, 32.90, 32.70, 31.82, 31.27, 30.79, 30.77, 30.72,30.62, 30.60, 30.51, 30.49, 30.44, 30.40, 30.36, 30.34, 30.32, 29.82, 29.81, 28.60, 28.58, 28.20, 27.09, 26.95, 26.91, 26.58, 25.17, 23.76, 23.67, 18.99, 14.50, 14.47. Example 9: A method for synthesizing a zwitterionic compound 9, the synthetic route of which is as follows: A method for synthesizing a zwitterionic compound 9 includes the following steps: Step 1: Synthesis of Compound 9-1 Compound 1-5 (4.3 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromopentanoate (2.5 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give a pale yellow oily compound 9-1 (2.5 g), yield: 45%.

[0051] 1 H NMR (400 MHz, CDCl3) δ 5.41 – 5.23 (m, 4H), 4.14 – 3.98 (m, 4H), 3.68 – 3.51 (m, 2H), 3.45 – 3.34 (m, 6H), 3.10 (s, 6H), 2.95 – 2.78 (m, 5H),2.67 (m, 2H), 2.43 (t, J= 7.2 Hz, 2H), 2.27 – 2.21 (m, 1H), 2.17 – 1.91 (m,6H), 1.88 – 1.76 (m, 2H), 1.70 – 1.51 (m, 8H), 1.40 (s, 9H), 1.50 – 1.20 (m,44H), 0.90 – 0.80 (m, 9H). Step 2: Synthesis of Compound 9 Compound 9-1 (2.5 g, 2.4 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 9 (1.9 g) as a pale yellow solid, yield: 82%.

[0052] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.30 (m, 4H), 4.20 – 4.14 (m, 1H), 4.11 – 4.06 (m, 4H), 3.67 – 3.65 (m, 2H), 3.46 – 3.37 (m, 5H), 3.10 (s, 6H), 2.91 –2.85 (m, 1H), 2.83 – 2.77 (m, 4H), 2.66 – 2.62 (m, 2H), 2.42 (t, J =7.2 Hz, 2H), 2.36 – 2.32 (m, 1H), 2.09 – 2.04 (m, 6H), 1.85 – 1.79 (m, 2H), 1.68 – 1.60 (m, 8H), 1.36 – 1.24 (m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.33, 176.80, 174.61, 130.96, 130.86,129.14, 129.04, 72.18, 71.61, 66.00, 65.26, 65.03, 64.17, 54.81, 51.45,47.98, 47.14, 34.02, 33.81, 33.04, 32.89, 32.70, 31.93, 31.81, 31.19, 30.78,30.70, 30.61, 30.60, 30.50, 30.48, 30.39, 30.34, 30.31, 29.82, 29.80, 29.71, 28.59, 28.58, 28.20, 27.09, 26.95, 26.92, 26.58, 23.75, 23.67, 23.53, 23.00, 22.71, 20.87, 14.49, 14.46. Example 10: A method for synthesizing an amphoteric compound 10, the synthetic route of which is as follows: A method for synthesizing an amphoteric compound 10 includes the following steps: Step 1: Synthesis of Compound 10-1 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and tert-butyl bromopentanoate (2.5 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give a pale yellow oily compound 10-1 (3.1 g), yield: 56%.

[0053] 1H NMR (400 MHz, CDCl3) δ 5.44 – 5.27 (m, 4H), 4.18 – 4.01 (m, 4H), 3.68 – 3.50 (m, 2H), 3.40 – 3.27 (m, 6H), 3.08 (s, 6H), 2.95 – 2.73 (m, 7H),2.37 – 2.25 (m, 2H), 2.20 – 2.15 (m, 1H), 2.11 – 2.01 (m, 4H), 1.84 – 1.68(m, 2H), 1.61 – 1.48 (m, 8H), 1.40 (s, 9H), 1.45 – 1.23 (m, 44H), 0.93 – 0.81 (m, 9H). Step 2: Synthesis of Compound 10 Compound 10-1 (3.1 g, 3.0 mmol, 1 equiv.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography using a dichloromethane solution containing 20% ​​methanol (v / v) as the mobile phase, to give compound 10 (2.2 g) as a pale yellow solid, yield: 76%.

[0054] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.30 (m, 4H), 4.19 – 4.15 (m, 1H), 4.11 – 4.06 (m, 3H), 3.68 – 3.67 (m, 2H), 3.57 – 3.53 (m, 2H), 3.46 – 3.35(m, 4H), 3.12 (s, 6H), 2.99 –2.94 (m, 2H), 2.92 –2.83 (m, 3H), 2.80 –2.77 (m,2H), 2.42 (t, J = 7.2 Hz, 2H), 2.36 – 2.32 (m, 1H), 2.09 – 2.04 (m, 4H), 1.86 – 1.80 (m, 2H), 1.68 – 1.62 (m, 8H), 1.39 – 1.29 (m, 44H), 0.93 – 0.89 (m,9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.30, 176.55, 174.42, 130.96, 130.86,129.15, 129.05, 72.22, 71.47, 66.09, 65.25, 65.07, 64.67, 51.26, 48.12,47.14, 33.86, 33.81, 33.05, 32.90, 32.70, 31.94, 31.28, 30.79, 30.71, 30.62,30.60, 30.51, 30.40, 30.36, 30.34, 30.32, 29.82, 28.59, 28.58, 28.20, 27.09, 26.95, 26.91, 26.58, 25.33, 23.76, 23.67, 23.02, 22.65, 14.50, 14.47. Example 11: A method for synthesizing an amphoteric compound 11, the synthetic route of which is as follows: Step 1: Synthesis of Compound 11 Compounds 1-5 (4.3 g, 5.3 mmol, 1 equiv.) were dissolved in anhydrous acetonitrile (60 mL), and di-tert-butylchloromethyl phosphate (2.7 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give compound 11 (2.4 g), a pale yellow oil, in 43% yield.

[0055] 1 H NMR (400 MHz, CDCl3) δ 5.35 – 5.28 (m, 4H), 5.13 – 5.11 (d, J= 8.0Hz, 2H), 4.10 – 4.06 (m, 1H), 4.04 – 3.99 (m, 3H), 3.62 – 3.54 (m, 2H), 3.47 – 3.43 (m, 2H), 3.37 – 3.33 (m, 2H), 3.18 (s, 6H), 2.78 –2.71 (m, 5H), 2.59 –2.55 (m, 2H), 2.30 – 2.23 (m, 1H), 2.03 – 1.98 (m, 6H), 1.61 – 1.56 (m, 6H),1.40 (m, 9H), 1.29 – 1.21 (m, 44H), 0.86 – 0.81 (m, 9H). 13 C NMR (101 MHz, CDCl3) δ 176.80, 172.90, 130.31, 130.15, 128.11,128.00, 84.00, 83.92, 71.36, 70.43, 65.10, 64.12, 53.55, 47.95, 47.89, 46.59,45.91, 32.62, 31.96, 31.80, 31.62, 31.60, 30.61, 30.22, 30.00, 29.96, 29.78,29.66, 29.59, 29.54, 29.45, 29.39, 29.37, 29.35, 29.32, 29.13, 28.78, 28.72, 27.56, 27.51, 27.33, 27.29, 25.99, 25.92, 25.83, 25.72, 22.76, 22.69, 22.67, 22.46, 14.22, 14.19. 31 P NMR (162 MHz, CDCl3) δ -7.13. Example 12: A method for synthesizing an amphoteric compound 12, the synthetic route of which is as follows: Step 1: Synthesis of Compound 12 Compound 2-1 (4.2 g, 5.3 mmol, 1 equiv.) was dissolved in anhydrous acetonitrile (60 mL), and di-tert-butylchloromethyl phosphate (2.7 g, 10.6 mmol, 2 equiv.) and anhydrous potassium carbonate (5.8 g, 42.4 mmol, 8 equiv.) were added. The mixture was stirred at 60 °C for 96 hours. After the reaction was complete, the mixture was filtered, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 10% methanol (v / v) to give compound 12 (2.6 g), a pale yellow oil, in 47% yield.

[0056] 1 H NMR (400 MHz, MeOD- d 4) δ 5.39 – 5.32 (m, 4H), 5.13 – 5.11 (d, J =5.2 Hz, 2H), 4.20 – 4.14 (m, 1H), 4.10 – 4.06 (m, 3H), 3.68 – 3.67 (d, J =5.2 Hz, 2H), 3.60 – 3.56 (m, 2H), 3.46 – 3.41 (m, 2H), 3.12 (s, 6H), 3.10 –2.97 (m, 2H), 2.93 –2.83 (m, 3H), 2.80 –2.77 (m, 2H), 2.36 – 2.32 (m, 1H), 2.10 – 2.05 (m, 4H), 1.67 – 1.62 (m, 4H), 1.47 (m, 9H), 1.39 – 1.29 (m, 44H), 0.86 – 0.81 (m, 9H). 13 C NMR (101 MHz, MeOD- d4) δ 178.15, 174.34, 130.95, 130.85, 129.14,129.04, 85.79, 72.18, 71.51, 65.98, 65.23, 61.83, 48.01, 47.11, 33.81, 33.05,32.90, 32.85, 32.70, 31.97, 31.22, 30.80, 30.74, 30.63, 30.61, 30.57, 30.51,30.46, 30.41, 30.38, 30.33, 30.23, 30.19, 29.85, 29.82, 28.60, 28.58, 28.22, 27.10, 26.98, 26.94, 26.59, 25.12, 23.76, 23.68, 23.67, 14.54, 14.51. 31 P NMR (162 MHz, MeOD- d 4) δ -7.06. Example 13: A method for synthesizing an amphoteric compound 13, the synthetic route of which is as follows: Step 1: Synthesis of Compound 13 Compound 11 (2.4 g, 2.3 mmol, 1 equiv.) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 25% methanol (v / v) to give compound 13 (1.3 g) as a pale yellow oil, yield: 56%.

[0057] 1 H NMR (400 MHz, MeOD- d 4) δ 5.40 – 5.29 (m, 4H), 4.85 (d, J = 8.0 Hz,2H), 4.19 – 4.13 (m, 1H), 4.12 – 4.06 (m, 3H), 3.66 – 3.65 (m, 2H), 3.46 –3.40 (m, 4H), 3.08 (s, 6H), 2.90 – 2.86 (m, 1H), 2.81 – 2.76 (m, 4H), 2.63(td, J= 6.8, 2.4 Hz, 2H), 2.38 – 2.31 (m, 1H), 2.12 – 2.04 (m, 6H), 1.69 –1.62 (m, 4H), 1.39 – 1.29 (m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, CDCl3) δ 176.75, 172.98, 130.31, 130.13, 128.14,128.01, 114.19, 71.37, 70.54, 65.11, 64.13, 53.56, 50.78, 47.66, 46.64,45.90, 32.61, 31.98, 31.82, 31.65, 31.62, 30.24, 29.84, 29.82, 29.69, 29.60,29.57, 29.47, 29.45, 29.39, 29.35, 28.84, 28.77, 27.57, 27.53, 27.37, 27.31, 26.07, 25.97, 25.88, 25.75, 22.82, 22.79, 22.72, 22.70, 14.25, 14.21. 31 P NMR (162 MHz, CDCl3) δ -2.01. Example 14: A method for synthesizing an amphoteric compound 14, the synthetic route of which is as follows: Step 1: Synthesis of Compound 14 Compound 12 (2.6 g, 2.5 mmol, 1 equiv.) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the solution was purified by silica gel column chromatography with a mobile phase of dichloromethane containing 25% methanol (v / v) to give compound 14 (1.2 g) as a pale yellow oil, yield: 60%.

[0058] 1 H NMR (400 MHz, MeOD- d 4) δ 5.37 – 5.32 (m, 4H), 4.80 – 4.781 (d, J=7.6 Hz, 2H), 4.18 – 4.14 (m, 1H), 4.10 – 4.06 (m, 3H), 3.68 – 3.67 (d, J =5.6 Hz, 2H), 3.56 – 3.52 (m, 2H), 3.46 – 3.41 (m, 2H), 3.07 (s, 6H), 3.00 –2.95 (m, 2H), 2.92 –2.83 (m, 3H), 2.80 –2.77 (m, 2H), 2.37 – 2.32 (m, 1H), 2.10 – 2.04 (m, 4H), 1.67 – 1.62 (m, 4H), 1.38 – 1.29 (m, 44H), 0.93 – 0.89 (m, 9H). 13 C NMR (101 MHz, MeOD- d 4) δ 178.31, 174.47, 130.95, 130.87, 129.13,129.05, 86.59, 72.19, 71.53, 65.99, 65.29, 61.68, 47.99, 47.15, 33.82, 33.05,32.90, 32.70, 31.82, 31.22, 30.80, 30.76, 30.62, 30.60, 30.51, 30.48, 30.41,30.39, 30.37, 30.32, 29.84, 29.82, 28.59, 28.38, 28.21, 27.39, 27.10, 26.99, 26.94, 26.58, 25.19, 23.76, 23.67, 14.49, 14.46. 31 P NMR (162 MHz, MeOD- d 4) δ -1.86. In this invention, ionizable lipid compounds 1-5 were also used to prepare lipid nanoparticles. The structures of ionizable lipid compounds 1-5 are described above.

[0059] Example 15: A method for preparing lipid nanoparticles i. Preparation of organic phase solutions: Compounds 1-5, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-PEG were prepared at concentrations of 90 mg / mL, 23.5 mg / mL, 15 mg / mL, 25 mg / mL, and 30 mg / mL, respectively. 2000And ethanol solutions of zwitterionic compounds. DSPC, cholesterol, and DMG-PEG 2000 were purchased from Iveto (Shanghai) Pharmaceutical Technology Co., Ltd. 2000 An organic phase solution was prepared by mixing (polyethylene glycol-modified lipids): DSPC (phospholipids): zwitterionic compound in a molar ratio of 50: 38.5: 1.5: 5: 5. The zwitterionic compound was compound 1 prepared in Example 1.

[0060] ii. Preparation of aqueous solution: Firefly luciferase mRNA was added to 10 mM citrate buffer to prepare an aqueous solution; Luciferase mRNA was purchased from APExBIO, the pH of the citrate buffer was 4.5, the concentration of Luciferase mRNA was 1 mg / mL, and Luciferase mRNA and citrate buffer were mixed at a volume ratio of 41:900.

[0061] iii. Preparation of mRNA-LNPs based on microfluidic method: After cleaning and installing the microfluidic chip, the organic phase and the aqueous phase were rapidly mixed in the microfluidic chip according to the ratio of N-containing lipids in the organic phase to phosphate-containing mRNA in the aqueous phase (N / P) of 6, the volume ratio of organic phase to aqueous phase of 2:1, and the flow rate of 20 mL / min to prepare mRNA-LNPs. iv. Dialysis: Dialyze the mRNA-LNPs prepared in step iii in 100-fold volume of DPBS buffer at room temperature for 2-3 h. v. Ultrafiltration: Using a 100 kDa ultrafiltration tube, the mRNA-LNPs obtained from dialysis in step iv are centrifuged at 4000 rpm for 20 min. This process is repeated 3 times to concentrate the volume to about one-tenth of the original volume, thus obtaining the finished mRNA-LNPs product.

[0062] Example 16: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 2 obtained in Example 2.

[0063] Everything else is the same as in Example 15.

[0064] Example 17: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 3 obtained in Example 3.

[0065] Everything else is the same as in Example 15.

[0066] Example 18: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 4 obtained in Example 4.

[0067] Everything else is the same as in Example 15.

[0068] Example 19: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 5 obtained in Example 5.

[0069] Everything else is the same as in Example 15.

[0070] Example 20: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 6 obtained in Example 6.

[0071] Everything else is the same as in Example 15.

[0072] Example 21: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound is compound 7 obtained in Example 7.

[0073] Everything else is the same as in Example 15.

[0074] Example 22: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound is compound 8 obtained in Example 8.

[0075] Everything else is the same as in Example 15.

[0076] Example 23: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 9 obtained in Example 9.

[0077] Everything else is the same as in Example 15.

[0078] Example 24: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 10 prepared in Example 10.

[0079] Everything else is the same as in Example 15.

[0080] Example 25: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 11 prepared in Example 11.

[0081] Everything else is the same as in Example 15.

[0082] Example 26: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 12 prepared in Example 12.

[0083] Everything else is the same as in Example 15.

[0084] Example 27: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 13 obtained in Example 13.

[0085] Everything else is the same as in Example 15.

[0086] Example 28: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 14 prepared in Example 14.

[0087] Everything else is the same as in Example 15.

[0088] Example 29: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound is DSPC.

[0089] Everything else is the same as in Example 15.

[0090] Example 30: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 13 obtained in Example 13; the mixing ratio was compound 1-5: cholesterol: DMG-PEG. 2000 DSPC: zwitterionic compound = 50: 38.5: 1.5: 7.5: 2.5 (molar ratio).

[0091] Everything else is the same as in Example 15.

[0092] Example 31: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 13 obtained in Example 13; the mixing ratio was compound 1-5: cholesterol: DMG-PEG. 2000 DSPC: zwitterionic compound = 50: 38.5: 1.5: 2.5: 7.5 (molar ratio).

[0093] Everything else is the same as in Example 15.

[0094] Example 32: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the zwitterionic compound was compound 13 obtained in Example 13; the mixing ratio was compound 1-5: cholesterol: DMG-PEG. 2000 DSPC: zwitterionic compound = 50: 38.5: 1.5: 0:10 (molar ratio).

[0095] Everything else is the same as in Example 15.

[0096] Example 33: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid was SM102 (CAS: 2089251-47-6), and the zwitterionic compound was DSPC.

[0097] Everything else is the same as in Example 15.

[0098] Example 34: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid was SM102, and the zwitterionic compound was compound 13 prepared in Example 13.

[0099] Everything else is the same as in Example 15.

[0100] Example 35: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid is ALC-0315 (CAS: 2036272-55-4), and the zwitterionic compound is DSPC.

[0101] Everything else is the same as in Example 15.

[0102] Example 36: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid was ALC-0315, and the zwitterionic compound was compound 13 prepared in Example 13.

[0103] Everything else is the same as in Example 15.

[0104] Example 37: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid was DLin-MC3-DMA (CAS: 1224606-06-7), and the zwitterionic compound was DSPC.

[0105] Everything else is the same as in Example 15.

[0106] Example 38: A method for preparing lipid nanoparticles The difference between this embodiment and embodiment 15 lies in the following steps: In the preparation of the ethanol phase solution, the ionizable lipid was DLin-MC3-DMA, and the zwitterionic compound was compound 13 prepared in Example 13.

[0107] Everything else is the same as in Example 15.

[0108] Experimental example: This invention uses a Malvern Zetasizer Nano ZSZEN3600 (MalvernUK) to determine the size and polydispersity index of the lipid nanoparticles prepared in Examples 15-38 by dynamic light scattering, and to measure their zeta potential; Quant-i™ RiboGreen is used. ® The encapsulation efficiency of the lipid nanoparticles prepared in Examples 15-38 was determined using an RNA quantification assay kit (Thermo Fisher Scientific, UK). The test results are shown in Table 1.

[0109] Table 1. Physicochemical properties characterization of lipid nanoparticles As can be seen from Table 1, the lipid nanoparticles of the present invention have a particle size of 80-300 nm, a zeta potential of -5-15 mV, and an encapsulation efficiency of >85%.

[0110] This invention tested the apparent pKa of the lipid nanoparticles prepared in Examples 15-38. The mRNA escape is mainly achieved by the pH-sensitive lipid nanoparticles disrupting the endosome membrane in an acidic intracellular environment (pH=3-5.5). mRNA-LNPs with ideal apparent pKa carry almost no charge at physiological pH, preventing non-specific binding to proteins or cells in vivo and causing toxicity; under acidic conditions, they acquire a positive charge, promoting endosome escape and releasing mRNA, thereby exerting a therapeutic effect. This invention uses the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence method to determine the apparent pKa of mRNA-LNPs. The specific method is as follows: lipid nanoparticles were incubated with pKa buffers at pH values ​​of 3, 4, 5, 6, 6.5, 7, 7.5, 8, 9, 10, and 11, respectively. Then, TNS dye was added to measure the fluorescence value of the samples at wavelengths of 321 / 445 nm. The apparent pKa was obtained by plotting and fitting the results, which are shown in Table 2.

[0111] Table 2 Apparent pKa of lipid nanoparticles This invention conducted animal experiments on lipid nanoparticles prepared from zwitterionic compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, specifically Examples 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. After dialysis, ultrafiltration, and quantification, the lipid nanoparticles were delivered to 6-8 week old female Babl / c mice via tail vein injection. These lipid nanoparticles, encapsulating Firefly Luciferase mRNA, were administered in vivo at a dose of 1 μg per mouse. Small animal in vivo fluorescence imaging (IVIS) was performed 6 hours after administration. Lumina III (PE Corporation), and then the mice were immediately euthanized. The classic formulation of lipid nanoparticles in Example 29 was used as a control group and compared with the experimental group containing the zwitterionic compound of the present invention from the same batch. The test results are as follows. Figure 1 As shown. 10% DSPC represents the lipid nanoparticles prepared in Example 29 (classic lipid nanoparticle formulation) in each experimental batch; DSPC+compound 1 represents the lipid nanoparticles prepared in Example 15; DSPC+compound 2 represents the lipid nanoparticles prepared in Example 16; DSPC+compound 3 represents the lipid nanoparticles prepared in Example 17; DSPC+compound 4 represents the lipid nanoparticles prepared in Example 18; DSPC+compound 5 represents the lipid nanoparticles prepared in Example 19; DSPC+compound 6 represents the lipid nanoparticles prepared in Example 20; DSPC+compound 7 represents the lipid nanoparticles prepared in Example 21; DSPC+compound 8 represents the lipid nanoparticles prepared in Example 22; DSPC+compound 9 represents the lipid nanoparticles prepared in Example 23; DSPC+compound 10 represents the lipid nanoparticles prepared in Example 24; DSPC+compound 11 represents the lipid nanoparticles prepared in Example 25; DSPC+compound 12 represents the lipid nanoparticles prepared in Example 26; DSPC+compound 13 represents the lipid nanoparticles prepared in Example 27; and DSPC+compound 14 represents the lipid nanoparticles prepared in Example 28. Compared with classic formulations of lipid nanoparticles from the same batch, Examples 15, 17, 23, and 27 all showed stronger mRNA expression levels, with Example 27 showing the strongest mRNA expression level, indicating that zwitterionic compound 13 helps to improve the in vivo mRNA delivery level of lipid nanoparticles.

[0112] Will Figure 1The results were quantitatively processed. The results of each formulation were normalized according to the bioluminescence intensity of the classic lipid nanoparticle formulation group within the same experimental batch. A heatmap was used to display the multiples of bioluminescence intensity of different experimental groups (Examples 15-28) compared to the classic lipid nanoparticle formulation group. Figure 2 As shown. Example 27 exhibited the strongest mRNA expression level, indicating that zwitterionic compound 13 helps to improve the in vivo delivery level of mRNA from lipid nanoparticles.

[0113] This invention conducted animal experiments on the lipid nanoparticles of Examples 27, 29, 30, 31, and 32 prepared from zwitterionic compound 13. After dialysis, ultrafiltration, and quantification, the lipid nanoparticles were delivered to 6-8 week old female Babl / c mice via tail vein injection at a dose of 1 μg / mouse. Small animal in vivo fluorescence imaging (IVIS Lumina III, PE) was performed 6 hours after administration, followed by immediate euthanasia. The test results are as follows: Figure 3 As shown. Among them, Figure 3 In this formulation, 10% DSPC represents the lipid nanoparticles prepared in Example 29, 2.5% Compound 13 represents the lipid nanoparticles prepared in Example 30, 5% Compound 13 represents the lipid nanoparticles prepared in Example 27, 7.5% Compound 13 represents the lipid nanoparticles prepared in Example 31, and 10% Compound 13 represents the lipid nanoparticles prepared in Example 32. Compared with the lipid nanoparticles prepared in Example 29 (classic formulation), lipid nanoparticles prepared with different mixing ratios of Compound 13 all showed varying degrees of increased mRNA expression levels, indicating that the zwitterionic compound 13 helps improve the in vivo mRNA delivery level of lipid nanoparticles. The lipid nanoparticles prepared in Example 27 showed the highest degree of improvement in mRNA expression levels.

[0114] In summary, the lipid nanoparticles prepared by zwitterionic compounds 1, 3, 9, and 13 in this invention have a stronger in vivo mRNA delivery capability compared to Example 29 (classic formulation); among all zwitterionic compounds, compound 13 has the best mRNA delivery effect, and at a mixing ratio of 5%, it has the highest degree of improvement in mRNA expression level.

[0115] This invention tested the safety and endosome escape ability of the lipid nanoparticles prepared in Examples 27 and 29. The mRNA escape is mainly achieved by the pH-sensitive lipid nanoparticles disrupting the endosome membrane in an acidic intracellular environment (pH=3-5.5). This invention simulates the interaction between lipid nanoparticles and the cell membrane in a neutral pH environment; and the interaction between lipid nanoparticles and the endosome membrane in an acidic pH environment of intracellular endosomes; thereby verifying the safety and endosome escape ability of lipid nanoparticles prepared from ionizable lipid compounds. This invention verifies the safety and endosome escape ability of lipid nanoparticles through an in vitro hemolysis experiment. The specific operation is as follows: free mRNA or lipid nanoparticles with a final mRNA concentration of 5 μg / ml were co-incubated with mouse red blood cell solution (final volume percentage equal to 4%) at 37°C for 1 hour, then the supernatant was collected by centrifugation, and the UV absorbance of the supernatant at 575 nm was measured to demonstrate hemolysis. The test results are as follows. Figure 4 As shown, 10% DSPC represents the lipid nanoparticles prepared in Example 29 (classic formulation), and DSPC + compound 13 represents the lipid nanoparticles prepared in Example 27. In an environment of pH 7.4, the erythrocyte lysis rate is very low, indicating that the lipid nanoparticles have minimal disruptive effect on the cell membrane and do not cause hemolysis. This demonstrates that the lipid nanoparticles of Example 27 prepared with zwitterionic compound 13 exhibit excellent biocompatibility. The hemolysis rate is approximately 5%, consistent with the lipid nanoparticles prepared in Example 29 (classic formulation), demonstrating excellent safety. In an environment of pH 5.5, the erythrocyte lysis rate of the lipid nanoparticles of Example 27 prepared with zwitterionic compound 13 is higher than that of the lipid nanoparticles prepared in Example 29 (classic formulation), indicating that the lipid nanoparticles with zwitterionic compound 13 have stronger endosome escape ability.

[0116] The lipid nanoparticles prepared in Examples 27 and 29 of this invention were subjected to cytotoxicity tests. The cytotoxicity was assessed using a commercially available cell proliferation assay kit (MTS, Promega). 40,000 293T cells were seeded per well in a 96-well plate and cultured overnight. 293T cells were then transfected with 0.2 μg mRNA of Luciferase mRNA lipid nanoparticles per well. After 24 hours of transfection, the old culture medium was removed and replaced with fresh medium containing MTS. The cells were incubated for approximately 2 hours, and the absorbance at 490 nm was measured using a TACAN (SPARK) microplate reader. The test results are as follows: Figure 5 As shown. The lipid nanoparticles prepared in Example 29 (classic formulation) and the lipid nanoparticles prepared in Example 27 from zwitterionic compound 13 had no significant effect on cell viability, indicating that they did not have significant cytotoxicity.

[0117] Among all zwitterionic compounds, zwitterionic compound 13 showed the best transfection efficiency. Therefore, we selected zwitterionic compound 13 in combination with commercially available ionizable lipid compounds SM102, ALC-0315, and MC3, and compared them with lipid nanoparticles without zwitterionic compound 13. Lipid nanoparticles encapsulating Firefly Luciferase mRNA (Examples 33, 34, 35, 36, 37, and 38) were delivered to 6-8 week old female Babl / c mice via tail vein injection at a dose of 1 μg / mouse. In vivo fluorescence imaging (IVIS Lumina III, PE) was performed on the mice 6 hours after administration, followed by immediate euthanasia. The test results for Examples 33 and 34 are as follows: Figure 6 As shown, 10% DSPC represents the lipid nanoparticles prepared in Example 33, and DSPC + compound 13 represents the lipid nanoparticles prepared in Example 34. (The last sentence appears to be incomplete and possibly refers to a different context.) Figure 6 It can be seen that when preparing lipid nanoparticles using the commercially available ionizable lipid SM102, the addition of zwitterionic compound 13 resulted in a decrease in fluorescence signal in mice, failing to improve the in vivo mRNA delivery level of the lipid nanoparticles. The test results of Examples 35 and 36 are as follows... Figure 7 As shown, 10% DSPC represents the lipid nanoparticles prepared in Example 35, and DSPC+compound 13 represents the lipid nanoparticles prepared in Example 36. (The last sentence appears to be incomplete and possibly refers to a different context.) Figure 7 It can be seen that when preparing lipid nanoparticles using the commercially available ionizable lipid ALC-0315, the addition of zwitterionic compound 13 did not significantly change the fluorescence signal in mice, indicating minimal impact on the in vivo mRNA delivery level of the lipid nanoparticles. The test results of Examples 37 and 38 are as follows... Figure 8 As shown, 10% DSPC represents the lipid nanoparticles prepared in Example 37, and DSPC+compound 13 represents the lipid nanoparticles prepared in Example 38. (The last sentence appears to be incomplete and possibly refers to a different context.) Figure 8 It can be seen that when lipid nanoparticles were prepared using commercially available ionizable lipid MC3, the addition of zwitterionic compound 13 increased the fluorescence signal in mice, significantly improving the in vivo mRNA delivery level of the lipid nanoparticles.

[0118] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A zwitterionic compound or a pharmaceutically acceptable salt thereof, characterized in that, The zwitterionic compound has the general structural formula as shown in formula (I): G1, G2are each independently selected from -(CH2) x -(C=O)NH-, -(CH2) x -O(C=O)-, -(CH2) x -(C=O)S-, -(CH2) x -S(C=O)-, -(CH2) x -(C=O)NH-, -(CH2) x NH(C=O)-, -(CH2) x -O-, -(CH2) x -O(C=O)NH- or -(CH2) x -O(C=O)O-, wherein x is an integer from 0 to 4; G3 is selected from C 2-10 alkenyl; L2, L3, L5, and L6 are each independently selected from unsubstituted C. 0-10 alkyl; L1 and L4 are each independently selected from unsubstituted straight chains or branched C-chains. 1-25 Alkyl, C 2-25 alkenyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1, R2, and R3 are each independently selected from any substituted C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-8 cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl or phenyl, wherein the substituents are one or two independent substituents. , , , C 1-3 Alkyl, C 1-3 Alkyl group or -(C=O)OC 1-3 alkyl.

2. The zwitterionic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, G1 and G2 are each independently selected from -O(C=O)-, -(C=O)O-, -S(C=O)-, -(C=O)S-, -(C=O)NH-, and -NH(C=O)-; G3 is ; L2 and L3 are each independently selected from unsubstituted C 5-10 Alkyl group; L5 and L6 are each independently selected from unsubstituted C16 groups. 0-5 alkyl; L1 and L4 are each independently selected from straight chains or C chains with branches. 5-15 alkyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1, R2, and R3 are each independently selected from any substituted C 1-3 Alkyl groups, wherein the substituents are one or two independent substituents. , , , .

3. The zwitterionic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, G1 and G2 are each independently selected from -O(C=O)- and -(C=O)O-; -L3-G3-L4 is ; L2 represents unsubstituted C. 5-8 Alkyl group; L5 and L6 are each independently selected from unsubstituted C16 groups. 0-4 alkyl; L1 is a branched C 15-20 alkyl; X1 and X2 are each independently selected from oxygen or sulfur atoms; R1 and R2 are each independently selected from unsubstituted C 1-3 Alkyl; R3 is selected from , , , .

4. The zwitterionic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The zwitterionic compound is: ； L5 and L6 are each independently selected from unsubstituted C. 1-4 alkyl; R1 and R2 are each independently selected from unsubstituted C 1-3 Alkyl; R3 is selected from , , , .

5. The zwitterionic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The zwitterionic compound is selected from any one of the following: Compound 1: ; Compound 2: ; Compound 3: ; Compound 4: ; Compound 5: ; Compound 6: ; Compound 7: ; Compound 8: ; Compound 9: ; Compound 10: ; Compound 11: ; Compound 12: ; Compound 13: ; Compound 14: .

6. The zwitterionic compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The pharmaceutically usable salt is an acid addition salt or a base addition salt.

7. A complex, characterized in that, It includes the zwitterionic compound or its pharmaceutically acceptable salt as described in any one of claims 1-6.

8. The complex according to claim 7, characterized in that, The complex comprises the zwitterionic compound of any one of claims 1-6 or its pharmaceutically acceptable salt, ionizable lipid, phospholipid, structural lipid and polyethanolated lipid; the molar ratio of the zwitterionic compound, ionizable lipid, phospholipid, structural lipid and polyethanolated lipid is 0-20:10-100:0-20:0-50:0-5.

9. The complex according to claim 7, characterized in that, The ionizable lipid is at least one of compounds 1-5, Dlin-MC3-DMA, SM-102, ALC-0315, C12-200, 5A2-SC8, and CKK-E12; compounds 1-5 have the structure shown in formula (II): ； The phospholipid is at least one of distearylphosphatidylcholine, 1,2-dioleoyl lecithin, 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, and 1,2-distearyl-SN-glycerol-3-phosphoethanolamine. The structural lipids mentioned are at least one of cholesterol, β-sitosterol, stigmasterol, and campesterol; The PEGylated lipid is at least one of DMG-PEG 2000, DSG-PEG 2000, and DSPE-PEG 2000.

10. Use of the complex according to any one of claims 7-9 in the preparation of lipid nanoparticles for drug delivery.

Images