Preparation and application of cationic compound and compound thereof

Abstract

The invention discloses preparation and application of a cationic compound and a compound thereof, belongs to the technical field of medicines, and particularly relates to a cationic compound which can be added into an original lipid compound. The invention also discloses the lipid compound, which can be specifically delivered to the lung, has the characteristics of small toxicity and good targeting property, solves the problem of difficult targeting delivery of nucleic acid drugs, and promotes the development of nucleic acid drugs. The prepared lipid compound can effectively target the lung, lung targeting can be achieved without adding any cationic lipid with positive charges into the lipid compound, and the tail of the cationic lipid needs to be provided with one or two olefins. Compared with a compound formed by a traditional DOTAP lipid material, the cationic lipid developed by the invention has lower hemolytic toxicity and better lung targeting property.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the preparation and application of a cationic compound and a complex thereof. Background Art

[0002] As a major emerging field of medicine, nucleic acid drugs are one of the main directions of future drug development with the characteristics of fast design, wide application and high safety. However, due to the poor cell penetration and easy degradation of nucleic acid drugs themselves, the in vivo application of nucleic acid drugs faces huge challenges. Therefore, it is necessary to develop specific compounds and delivery systems to improve this situation in order to promote nucleic acid drugs as an important means of disease prevention and treatment. At present, lipid nanoparticles (LNPs) prepared from ionizable cationic lipids are a relatively safe and effective means of delivering nucleic acid drugs. In disease treatment, in order to improve the efficacy of drugs and reduce side effects, it is very important to accurately deliver drugs to diseased organs or tissues. Traditional delivery methods often lack specificity and have adverse effects on other normal tissues and organs throughout the body. Therefore, the development of organ-targeted LNP delivery systems has become a hot topic of research.

[0003] Researchers have developed selective organ targeting (SORT) technology. This technology introduces SORT lipid molecules with different charges into traditional four-component LNPs to change the surface properties and biological properties of LNPs, thereby achieving specific delivery of mRNA to specific organs, such as the liver, spleen or lungs of mice. Although SORT-LNP is able to achieve targeted delivery to specific organs, in some cases, its targeting accuracy is not high enough. For example, some drugs may be delivered to non-target organs, resulting in unnecessary effects on other organs. The added SORT lipid molecules are positively charged and have certain toxicity, which may induce inflammatory responses or other immune-related adverse reactions.

[0004] 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. At present, there are few SORT lipid molecules that can effectively achieve organ targeting function, and a lot of design and screening are needed. Summary of the invention

[0005] The purpose of the present invention is to provide a cationic compound and its complex preparation and application which can be applied to the in vivo and in vitro delivery of therapeutic agents or preventive agents, has high biological safety and good targeting effect.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A cationic compound of formula (I) or a pharmaceutically acceptable salt thereof: Formula (I); Wherein, G1, G2, G3, are each independently selected from -(CH2) x -O(C=O)-, -(CH2) x -(C=O)O-, -(CH2) x -(C=O)S-, -(CH2) x -(C=O)NH-, -(CH2) x -O-, -(CH2) x -O(C=O)NH-, -(CH2) x -O(C=O)O- or -(CH2) x NH(C=O)-, wherein x is an integer between 0 and 4; L2, L3, L5 are each independently selected from unsubstituted C 1-10 alkyl; L1 and L4 are each independently selected from a linear or branched C 1-25 Alkyl, C 2-25 alkenyl; X1, 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, C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl or phenyl, wherein the substituent is 1 or 2 independent OH, SH, C 1-3 Hydroxyl, C 1-3 Alkoxy or -(C=O)OC 1-3 alkyl.

[0007] Preferably, L4 is or .

[0008] Preferably, the cationic compound or its pharmaceutically acceptable salt 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: .

[0009] The invention discloses a lipid complex, comprising: the above cationic compound or a pharmaceutical salt thereof.

[0010] Preferably, the lipid complex also includes a therapeutic or prophylactic agent.

[0011] Preferably, the lipid complex further comprises an ionizable lipid compound, and the ionizable lipid compound is at least one of 1-126, D-Lin-MC3-DMA, SM102, ALC-0315, 5A2-SC8, and CKK-E12.

[0012] Preferably, the lipoplex further comprises at least one of phospholipids, structural lipids, and pegylated lipids.

[0013] Preferably, the lipid complex is a lipid nanoparticle.

[0014] The present invention discloses a method for preparing a lipid complex, comprising: Step (1): dissolving a lipid material in an organic solvent to obtain an organic phase solution; the lipid material comprises at least one of the above-mentioned cationic compounds, ionizable lipid compounds, phospholipids, structural lipids, and pegylated lipids; Step (2): mixing the aqueous medium with the buffer to obtain an aqueous solution; Step (3): Mix the organic phase solution with the aqueous phase solution to obtain a lipid complex.

[0015] Preferably, the aqueous medium includes the therapeutic or prophylactic agent.

[0016] The present invention discloses the use of cationic compounds, including at least one of the following 1)-3), 1) Encapsulation of therapeutic or preventive agents; 2) preparing a therapeutic or preventive agent for in vivo delivery; 3) Prepare transfection kit.

[0017] The present invention provides a lipid complex comprising a cationic compound.

[0018] Preferably, a therapeutic or prophylactic agent is also included.

[0019] Preferably, the therapeutic or preventive agent is selected from at least one of nucleic acid drugs, small molecule drugs, protein drugs, and pharmaceutically active molecules.

[0020] Preferably, the nucleic acid drug is selected from any at least one of DNA drugs and RNA drugs; the RNA drug is selected from any at least one of mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA, and lncRNA.

[0021] Preferably, the protein drug is selected from at least one of antibodies, enzymes, recombinant proteins, polypeptides and short peptides.

[0022] Preferably, the ionizable lipid compound is any at least one of 1-126, D-Lin-MC3-DMA, SM102, ALC-0315, 5A2-SC8, and CKK-E12.

[0023] The structure of 1-126 is as follows: 1-126.

[0024] More preferably, the ionizable lipid compound is 1-126.

[0025] More preferably, it further comprises any one or more of phospholipids, structural lipids, and PEGylated lipids.

[0026] More preferably, the phospholipid is selected from distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, Choline, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine Base, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1- any at least one of 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-oleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine.

[0027] More preferably, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0028] Preferably, the structured lipid is selected from any at least one of cholesterol, β-cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, lycopene, ursolic acid, and α-tocopherol.

[0029] More preferably, the structured lipid is cholesterol.

[0030] Preferably, the PEGylated lipid is selected from any at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0031] More preferably, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0032] Preferably, the molar ratio of the ionizable lipid compound, the phospholipid, the structural lipid and the PEGylated lipid, and the cationic compound is 10-100:0-50:0-50:0-5:10-80.

[0033] Preferably, the molar ratio of the ionizable lipid compound, the phospholipid, the structural lipid and the PEGylated lipid, and the cationic compound is 10-50:2-20:30-50:0.5-5:10-60.

[0034] More preferably, the molar ratio of the ionizable lipid compound, the phospholipid, the structural lipid and the PEGylated lipid, and the cationic compound is 15-30:2-10:10-30:0.5-2:30-60.

[0035] More preferably, the molar ratio of the ionizable lipid compound, the phospholipid, the structural lipid and the PEGylated lipid, and the cationic compound is 25:5:19.3:0.8:50.

[0036] Preferably, the complex is a lipid nanoparticle.

[0037] Preferably, the particle size of the lipid nanoparticles is 30 to 600 nm, and the Zeta potential is -10 to 40 mV. More preferably, the particle size of the lipid nanoparticles is 70 to 500 nm, and the Zeta potential of the lipid nanoparticles is 10 to 40 mV.

[0038] In another aspect, the present invention provides a method for preparing a lipid complex, comprising the following steps: (1) dissolving an ionizable lipid compound and any one or more of a phospholipid, a structural lipid, and a pegylated lipid in an organic solvent to obtain an organic phase solution; (2) mixing the therapeutic or preventive agent with a buffer to obtain an aqueous solution; (3) The organic phase solution is mixed with the aqueous phase solution to obtain a lipid complex.

[0039] Preferably, the organic solvent is any at least one of methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone.

[0040] Preferably, the buffer is citrate buffer.

[0041] Preferably, the concentration of the citrate buffer is 5-80 mM, and the pH of the citrate buffer is 2-6.

[0042] More preferably, the concentration of the citrate buffer is 10-50 mM, and the pH of the citrate buffer is 3-5.

[0043] Preferably, the N / P of the complex is 1 to 15. The ratio of N in the ionizable lipid compound and the cationic compound to P in the mRNA as a drug is N / P.

[0044] More preferably, the N / P ratio of the composite is 8.

[0045] The present invention provides a use of a cationic compound for preparing a preparation for improving the delivery efficiency and / or transfection efficiency of a lipid complex.

[0046] The present invention provides a use of a cationic compound for preparing a preparation for reducing the cytotoxicity of a lipid complex.

[0047] The present invention also provides a use of a cationic compound or a pharmaceutically acceptable salt thereof, comprising at least one of the following 1) to 4), 1) Encapsulation of therapeutic or preventive agents; 2) in vitro cell transfection with therapeutic or preventive agents; 3) preparing therapeutic or preventive agent delivery agents in vivo; 4) Prepare transfection kit.

[0048] The lipid complex prepared by the present invention can effectively target the lungs. Lung targeting can be achieved by not adding any cationic lipids with positive charges to the lipid complex. The tail of the cationic lipid needs to have one or two olefins. The present invention solves the problem that traditional lipid nanoparticles accumulate in the liver after intravenous injection.

[0049] The invention has simple synthesis, high reaction efficiency and low cost; it has low toxicity, low red blood cell lysis rate in a neutral pH environment, and good safety; it has good targeting, and the overall expression of lung tissue in organ distribution can reach more than 70%. The invention has good reference value for expanding the application scope of LNP and treating lung diseases.

[0050] The present invention designs and synthesizes a new cationic compound from an ionizable lipid compound, and can prepare a new cationic compound and its complex, which can be applied to the in vivo and in vitro delivery of therapeutic agents or preventive agents, especially nucleic acid drugs, solves the problem of difficult delivery of nucleic acid drugs, and promotes the development of nucleic acid drugs; the lipid complex provided by the present invention is a lipid nanoparticle, and the nanoparticle size is uniform, the particle size is 80 to 500 nm, the Zeta potential is 10 to 40 mV, and the encapsulation rate is ≥97%; the lipid complex provided by the present invention has good biosafety and will not cause obvious hemolysis; the lipid complex provided by the present invention has good delivery efficiency and transfection efficiency, is simple to prepare, and has a short route. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a diagram showing the hemolysis of lipid nanoparticles at pH 7.4.

[0052] Figure 2 This is a diagram showing the hemolysis of lipid nanoparticles at pH 5.5.

[0053] Figure 3 Images of the main organs of mice when N / P is 8.

[0054] Figure 4 Images of the main organs of mice when N / P is 6.

[0055] Figure 5 This is a diagram showing the cytotoxicity test results of lipid nanoparticles.

[0056] Figure 6 The images are of the main organs of mice 6 hours after drug administration.

[0057] Figure 7 The fluorescence intensity diagram in the lungs of mice at 6 hours after drug administration.

[0058] Figure 8 This is the distribution diagram of lipid nanoparticles in the main organs of mice. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0061] It should be noted that the term "alkyl" used in the present invention includes methyl, ethyl, propyl, butyl, pentyl, hexyl and the like and possible isomers thereof.

[0062] It should be noted that the term "cycloalkyl" used in the present invention includes cyclopropyl, cyclobutyl, cyclopentyl and the like and possible isomers thereof.

[0063] It should be noted that the term "alkoxy" used in the present invention includes methoxy, ethoxy, propoxy and the like and possible isomers thereof.

[0064] Example 1: A method for synthesizing a cationic compound The synthesis route of cationic compound 1 in this embodiment is as follows: .

[0065] Step 1: Synthesis of compound 1-1 Under zero temperature conditions, 15 mmol of triethylamine was slowly added to a 60 mL dichloromethane solution containing 10 mmol of acryloyl chloride and 10 mmol of (9Z, 12Z)-octadecane-9,12-dien-1-ol. After stirring for 2 hours, TLC monitoring showed that the alcohol disappeared completely. The reaction mixture was diluted with 100 mL of DCM and washed with 100 mL of water and 100 mL of brine. The combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography and the pure product fraction was evaporated to obtain a colorless oily compound 1-1 (3.1 g, 93% yield). Column chromatography used a silica gel column and the eluent was a n-hexane solution containing 5 vol% EA.

[0066] Step 2: Synthesis of compound 1-2 9.3 mmol of compound 1-1 was dissolved in 60 mL of tetrahydrofuran solution, and 46 mmol of paraformaldehyde, 46 mmol of DABCO and 10 mL of water were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 1-1 completely disappeared. The reaction mixture was extracted with 100 mL of ethyl acetate and washed with 100 mL of water and 100 mL of brine. The combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo to obtain a crude product. The crude product was dissolved in 80 mL of DCM at 0°C, and 14 mmol of BoC2O and 0.93 mmol of DMAP were added. After stirring for 2 hours, TLC monitoring showed that the raw material completely disappeared, the solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oil compound 1-2 (3.6 g, 84% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0067] Step 3: Synthesis of Compound 1-3 1 mmol of compound 1-2 was dissolved in 40 mL of tetrahydrofuran, and 1 mmol of 1,5-pentanediol and 0.1 mmol of DABCO were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 1-2 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 1-3 (445 mg, 85% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 10 vol% EA.

[0068] Step 4: Synthesis of Compound 1-4 0.6 mmol of compound 1-3 was dissolved in 25 mL of dichloromethane, and 1.8 mmol of 2-hexyl undecanoic acid, 1.8 mmol of EDCI and 1.8 mmol of DMAP were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 1-3 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain colorless oily compound 1-4 (334 mg, 81% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0069] Step 5: Synthesis of Compound 1-5 0.3 mmol of compound 1-4 was dissolved in a mixed solution of 10 mL DCM and 2 mL MeOH, and 0.9 mmol 3-(dimethylamino)-1-propanethiol and 0.03 mmol TBD were added. After stirring for 24 hours, TLC monitoring showed that compound 1-4 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oil compound 1-5 (202 mg, 83% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0070] Step 6: Synthesis of compound 1 0.2 mmol of compound 1-5 was dissolved in 15 mL of THF solution, and 0.4 mmol of iodomethane was added at zero degrees. After stirring for 2 hours under nitrogen protection, TLC monitoring showed that compound 1-5 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 1 (160 mg, 84% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 1 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.40–5.31 (m, 4H), 4.17–3.98 (m, 4H), 3.72 (dd, J=11.4, 5.5Hz, 2H), 3.61 (ddd, J=23.1, 9.3, 5.5Hz, 2H), 3.44–3 .34 (m, 11H), 2.88–2.60 (m, 7H), 2.38–2.14 (m, 2H), 2.03 (td, J=14.0, 6 .7Hz, 5H), 1.51–1.31 (m, 18H), 1.30–1.20 (m, 34H), 0.90–0.85 (m, 9H).

[0071] Example 2: A method for synthesizing a cationic compound The synthesis route of cationic compound 2 in this embodiment is as follows: .

[0072] Step 1: Synthesis of compound 2-1 0.25 mmol of compound 1-4 was dissolved in a mixed solution of 10 mL DCM and 2 mL MeCN, and 0.75 mmol, 2-dimethylaminoethanethiol and 0.025 mmol TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 1-4 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 2-1 (154 mg, 78% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0073] Step 2: Synthesis of compound 2 0.15 mmol of compound 2-1 was dissolved in 15 mL of THF solution, and 0.3 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 2-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 2 (103 mg, 74% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 2 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.41–5.31 (m, 4H), 4.11 (ddt, J=25.3, 18.5, 6.8Hz, 4H), 3 .97–3.75 (m, 2H), 3.72–3.60 (m, 2H), 3.48 (s, 9H), 3.42 (t, J=6.5Hz, 2H), 3.05–2.86 (m, 5H), 2.77 (t, J=6.7Hz, 2H), 2.38–2.17 (m, 2H), 2.07–2.02 (m, 3H), 1.63 (dd, J=12.7, 6.7 Hz, 5H), 1.39–1.32 (m, 13H), 1.28 (dd, J=10.1, 6.4Hz, 32H), 0.88 (dd, J=4.1, 2.5Hz, 9H).

[0074] Example 3: A method for synthesizing a cationic compound The synthesis route of cationic compound 3 in this embodiment is as follows: .

[0075] Step 1: Synthesis of compound 3-1 0.6 mmol of compound 1-3 was dissolved in 25 mL of dichloromethane, and 1.8 mmol of isostearic acid, 1.8 mmol of EDCI and 1.8 mmol of DMAP were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 1-3 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 3-1 (366 mg, 85% yield). Column chromatography used a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0076] Step 2: Synthesis of compound 3-2 0.2 mmol of compound 3-1 was dissolved in a mixed solution of 10 mL of DCM and 2 mL of MeOH, and 0.5 mmol of 3-(dimethylamino)-1-propanethiol and 0.02 mmol of TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 3-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 11 (120 mg, 83% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0077] Step 3: Synthesis of compound 3 0.1 mmol of compound 3-2 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 3-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 2 (78 mg, 80% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 3 are as follows, 1 H NMR (400MHz, Chloroform-d) δ 5.43–5.27 (m, 4H), 4.17–3.99 (m, 4H), 3.70 (d, J=7.0Hz, 2H), 3.62 (qd, J=9.3, 5.5Hz, 2H), 3.47 (s, 9H), 3.44–3.38 (m, 2H), 2.88–2.74 (m, 5H), 2.73–2.62 (m, 2H), 2.34–2.22 (m, 2H), 2.13– 2.01 (m, 5H), 1.70–1.48 (m, 10H), 1.35–1.19 (m, 44H), 0.93–0.80 (m, 9H).

[0078] Example 4: A method for synthesizing a cationic compound The synthesis route of cationic compound 4 in this embodiment is as follows: .

[0079] Step 1: Synthesis of compound 4-1 At room temperature, 9 mmol EDCI and 9 mmol DMAP were added to a 50 mL dichloromethane solution of 6 mmol 6-bromohexanoic acid and 9 mmol 9-heptadecanol. After stirring for 12 hours, TLC monitoring showed that the alcohol disappeared completely. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 4-1 (2.23 g, 85% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0080] Step 2: Synthesis of compound 4-2 At 60 ° C, 5 mmol of acrylic acid was added to 40 mL of DMF solution of 5 mmol of compound 4-1, and 7.5 mmol of K2CO3 was slowly added. After stirring for 12 hours, TLC monitoring showed that compound 4-1 completely disappeared. The reaction mixture was extracted with 150 mL of ethyl acetate and washed with 100 mL of water and 100 mL of brine. The combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography and the pure product fraction was evaporated to obtain a colorless oily compound 4-2 (1.8 g, 85% yield). Column chromatography used a silica gel column and the eluent was a n-hexane solution containing 5 vol% EA.

[0081] Step 3: Synthesis of compound 4-3 4 mmol of compound 4-2 was dissolved in 50 mL of tetrahydrofuran solution, and 20 mmol of paraformaldehyde, 20 mmol of DABCO and 6 mL of water were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 4-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product. The crude product was dissolved in 80 mL of DCM, and 7.5 mmol of Boc2O and 0.5 mmol of DMAP were added. After stirring for 2 hours, TLC monitoring showed that the raw material completely disappeared, the solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 4-3 (1.7 g, 80% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0082] Step 4: Synthesis of compound 4-4 2 mmol of compound 4-3 was dissolved in 60 mL of tetrahydrofuran, and 2 mmol of 1,6-hexanediol and 0.2 mmol of DABCO were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 4-3 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound, compound 4-4 (362 mg, 92% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 10 vol% EA.

[0083] Step 5: Synthesis of compound 4-5 0.5 mmol of compound 4-4 was dissolved in 30 mL of dichloromethane, and 0.5 mmol of 2-hexyl undecanoic acid, 1 mmol of EDCI and 1 mmol of DMAP were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 4-4 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 4-5 (328 mg, 83% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0084] Step 6: Synthesis of Compound 4-6 0.2 mmol of compound 4-5 was dissolved in a mixed solution of 10 mL DCM and 2 mL MeCN, and 0.5 mmol 3-(dimethylamino)-1-propanethiol and 0.02 mmol TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 4-5 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 4-6 (149 mg, 82% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0085] Step 7: Synthesis of compound 4 0.1mmol of compound 4-6 was dissolved in 10mL of THF solution, and 0.2mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 4-6 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 4 (89mg, 85% yield). The column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10vol% MeOH. The NMR data of compound 4 are as follows, 1H NMR (400MHz, Chloroform-d) δ4.85 (p, J=6.2Hz, 1H), 4.08 (dt, J=23.2, 6.6Hz, 4H), 3. 75 (dd, J=10.6, 6.0Hz, 2H), 3.62 (ddd, J=21.4, 9.3, 5.5Hz, 2H), 3.48 (s, 9H), 3.41 (t, J =6.5Hz, 2H), 2.82 (q, J=5.8Hz, 3H), 2.68 (dd, J=12.6, 6.4Hz, 2H), 2.30 (t, J=7.3Hz, 3H ), 2.21–2.07 (m, 2H), 1.73–1.41 (m, 18H), 1.32–1.20 (m, 48H), 0.88 (t, J=6.7Hz, 12H).

[0086] Example 5: A method for synthesizing a cationic compound The synthesis route of cationic compound 5 in this embodiment is as follows: .

[0087] Step 1: Synthesis of compound 5-1 6 mmol of compound 4-1 was dissolved in 50 mL of ultra-dry DMF solution, and 2 mmol of 3,5-dihydroxybenzyl alcohol, 20 mmol of anhydrous potassium carbonate and 0.2 mmol of potassium iodide were added, and the mixture was reacted at 80°C for 24 hours. TLC monitoring showed that compound 4-1 completely disappeared, and the reaction mixture was washed with ethyl acetate, and the filtered solution was extracted with ethyl acetate and saturated brine, and finally extracted with saturated lithium chloride. After 4-5 extractions, the organic phase layers were combined and dried over anhydrous Na2SO4. After the solvent was removed in vacuo, it was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 5-1 (380 mg, 45% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 15 vol% EA.

[0088] Step 2: Synthesis of compound 5-2 0.3 mmol of compound 5-1 was dissolved in 20 mL of DCM solution, and 0.6 mmol of N, N-dimethyl-β-alanine and 0.5 mmol of EDCI were added in sequence. 0.9 mmol of triethylamine was added during stirring. Stirring was continued for 12 hours. TLC monitored that compound 5-1 was completely reacted. The solvent was removed in vacuo and purified by column chromatography. The pure product fraction was evaporated to obtain a colorless oily compound 5-2 (151 mg, 53% yield). Column chromatography used a silica gel column, and the eluent was a DCM solution containing 10 vol% MeOH.

[0089] Step 3: Synthesis of compound 5 0.1 mmol of compound 5-2 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 5-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 4 (85 mg, 78% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 5 are as follows, 1 H NMR (400MHz, Chloroform-d) δ6.37 (d, J=2.1Hz, 2H), 6.32 (t, J=2.1Hz, 1H), 4.97 (s, 2H), 4.7 9 (p, J=6.3Hz, 2H), 3.86 (t, J=6.4Hz, 4H), 3.74–3.63 (m, 2H), 3.34 (s, 9H), 2.54 (t, J=6.4Hz, 2H), 2.26 (dd, J=16.4, 9.0Hz, 4H), 2.03 (d, J=6.3Hz, 2H), 1.77–1.68 (m, 4H), 1.62 (dt, J=14. 5, 7.1Hz, 4H), 1.43 (d, J=6.3Hz, 8H), 1.19 (d, J=13.6Hz, 53H), 0.80 (dd, J=8.6, 5.1Hz, 12H).

[0090] Example 6: A method for synthesizing a cationic compound The synthesis route of cationic compound 6 in this embodiment is as follows: .

[0091] Step 1: Synthesis of compound 6-1 0.5 mmol of compound 1-3 was dissolved in 20 mL of dichloromethane, and 1 mmol of 1-decanoic acid, 1 mmol of EDCI and 1 mmol of DMAP were added in sequence. After stirring at room temperature for 12 hours, TLC monitoring showed that compound 1-3 completely disappeared. The solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 6-1 (254 mg, 84% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0092] Step 2: Synthesis of compound 6-2 0.2 mmol of compound 6-1 was dissolved in a mixed solution of 8 mL of DCM and 2 mL of MeCN, and 0.5 mmol of 3-(dimethylamino)-1-propanethiol was added. After stirring for 2 hours, TLC monitoring showed that compound 8-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 6-2 (117 mg, 81% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0093] Step 3: Synthesis of compound 6 0.1 mmol of compound 6-2 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 5-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 6 (72 mg, 84% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 6 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.45–5.25 (m, 4H), 4.17–3.99 (m, 4H), 3.81–3.70 (m, 2H), 3.61 (qd, J=9.2, 5.4Hz, 2H), 3.46 (s, 9H), 3.40 (t, J=6.5Hz, 2H), 2.88–2.59 (m, 7H), 2.33–2.24 (m, 2H), 2.15–1.98 (m, 6H), 1.56–1.49 (m, 2H), 1.37–1.22 (m, 38H), 0.90–0.83 (m, 6H).

[0094] Example 7: A method for synthesizing a cationic compound The synthesis route of cationic compound 7 in this embodiment is as follows: .

[0095] Step 1: Synthesis of compound 7-1 0.3 mmol of compound 1-2 was dissolved in 20 mL of THF solution, and 0.6 mmol of (9Z, 12Z)-octadecane-9,12-dien-1-ol and 0.03 mmol of DABCO were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 1-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 7-1 (122 mg, 79% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 3 vol% EA.

[0096] Step 2: Synthesis of compound 7-2 0.25 mmol of compound 7-1 was dissolved in a mixed solution of 8 mL of DCM and 2 mL of MeCN, and 0.75 mmol of 3-(dimethylamino)-1-propanethiol and 0.025 mmol of TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 7-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 7-2 (131 mg, 73% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0097] Step 3: Synthesis of compound 7 0.1 mmol of compound 7-2 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 7-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 7 (67 mg, 78% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 7 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.41–5.29 (m, 8H), 4.10 (qt, J=10.8, 6.8Hz, 2H), 3.83–3.71 (m, 2H), 3.61 (qd, J=9.2, 5.4Hz, 2H), 3 .49–3.35 (m, 11H), 2.88–2.60 (m, 9H), 2.13–1.97 (m, 10H), 1.63 (dd, J=14.0, 7.0Hz, 3H), 1.34–1.26 (m, 32H), 0.90–0.87 (m, 6H).

[0098] Example 8: A method for synthesizing a cationic compound The synthesis route of cationic compound 8 in this embodiment is as follows: .

[0099] Step 1: Synthesis of compound 8-1 0.5 mmol of compound 1-2 was dissolved in 20 mL of THF solution, and 1 mmol of 1-octadecanol and 0.05 mmol of DABCO were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 1-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 8-1 (186 mg, 72% yield). Column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 3 vol% EA.

[0100] Step 2: Synthesis of compound 8-2 0.2 mmol of compound 8-1 was dissolved in a mixed solution of 8 mL DCM and 2 mL MeCN, and 0.6 mmol 3-(dimethylamino)-1-propanethiol and 0.02 mmol TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 8-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 8-2 (98 mg, 68% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0101] Step 3: Synthesis of compound 8 0.1 mmol of compound 8-2 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 8-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 8 (60 mg, 70% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 8 are as follows, 1H NMR (400MHz, Chloroform-d) δ5.42–5.31 (m, 4H), 4.18–4.02 (m, 2H), 3.76 (dd, J=9.1, 6.5Hz, 2H), 3.61 (ddd, J=19.1, 9.3, 5.5Hz, 2H), 3.46 (s, 9H), 3.39 ( t, J=6.7Hz, 2H), 2.86–2.61 (m, 7H), 2.07 (dt, J=13.9, 7.5Hz, 6H), 1.67–1.60 (m, 2H), 1.54–1.49 (m, 2H), 1.30–1.23 (m, 41H), 0.88 (dt, J=7.0, 3.3Hz, 6H).

[0102] Example 9: A method for synthesizing a cationic compound The synthesis route of cationic compound 9 in this embodiment is as follows: .

[0103] Step 1: Synthesis of compound 9-1 0.2 mmol of compound 8-1 was dissolved in a mixed solution of 8 mL DCM and 2 mL MeCN, and 0.6 mmol 2-dimethylaminoethanethiol and 0.02 mmol TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 8-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 9-1 (78 mg, 55% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0104] Step 2: Synthesis of compound 9 0.1 mmol of compound 9-1 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 9-1 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 9 (69 mg, 82% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 9 are as follows, 1H NMR (400MHz, Chloroform-d) δ5.42–5.31 (m, 4H), 4.19–4.03 (m, 2H), 3.97–3.77 (m, 2H), 3.68–3.62 (m, 2H), 3.50 (d, J=3.8Hz, 9H), 3.40 (t, J=6.7Hz, 2H) , 2.93 (tt, J=21.0, 10.3Hz, 5H), 2.77 (t, J=6.6Hz, 2H), 2.08–1.99 (m, 4H), 1 .67–1.61 (m, 2H), 1.54–1.49 (m, 2H), 1.33–1.23 (m, 46H), 0.92–0.86 (m, 6H).

[0105] Example 10: A method for synthesizing a cationic compound The synthesis route of the cationic compound 10 in this embodiment is as follows: .

[0106] Step 1: Synthesis of compound 10 0.1 mmol of compound MC3 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 9-4 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 8 (60 mg, 76% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 10 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.43–5.28 (m, 8H), 4.84 (p, J=6.2Hz, 1H), 3.81–3.70 (m, 2H), 3.46 (s, 9H), 2.77 (t, J =6.6Hz, 4H), 2.52 (t, J=6.5Hz, 2H), 2.11–1.98 (m, 10H), 1.55–1.47 (m, 4H), 1.33–1.20 (m, 36H), 0.91–0.86 (m, 6H).

[0107] Example 11: A method for synthesizing a cationic compound The synthesis route of the cationic compound 11 in this embodiment is as follows: .

[0108] Step 1: Synthesis of compound 11-1 Under zero degree conditions, 10.5 mmol of triethylamine was slowly added to a 40 mL dichloromethane solution of 7 mmol of acryloyl chloride and 7 mmol of oleyl alcohol. After stirring for 2 hours, TLC monitoring showed that oleyl alcohol completely disappeared. The reaction mixture was diluted with 100 mL of DCM and washed with 100 mL of water and 100 mL of brine. The combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography and the pure product fraction was evaporated to obtain a colorless oily compound 11-1 (1.9 g, 88% yield). Column chromatography used a silica gel column and the eluent was a n-hexane solution containing 5 vol% EA. The NMR data of compound 11-1 are as follows, 1 H NMR (400MHz, Chloroform-d) δ6.40 (dd, J=17.3, 1.5Hz, 1H), 6.12 (dd, J=17.3, 10.4Hz, 1H), 5.81 (dd, J=10.4, 1.5Hz, 1H) , 5.44–5.26 (m, 2H), 4.15 (t, J=6.7Hz, 2H), 2.15–1.90 (m, 4H), 1.71–1.60 (m, 2H), 1.34–1.23 (m, 22H), 0.90–0.87 (m, 3H).

[0109] Step 2: Synthesis of compound 11-2 6 mmol of compound 11-1 was dissolved in 30 mL of tetrahydrofuran solution, and 12 mmol of paraformaldehyde, 12 mmol of DABCO and 2 mL of water were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 11-1 completely disappeared. The reaction mixture was extracted with 100 mL of ethyl acetate and washed with 100 mL of water and 100 mL of brine. The combined organic layers were dried over Na2SO4, and the solvent was removed in vacuo to obtain a crude product. The crude product was dissolved in 40 mL of DCM at 0°C, and 7.5 mmol of Boc2O and 0.05 mmol of DMAP were added. After stirring for 2 hours, TLC monitoring showed that the raw material completely disappeared, the solvent was removed in vacuo and purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 11-2 (1.2 g, 53% yield). The column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA. The NMR data of compound 11-2 are as follows, 1H NMR (400MHz, Chloroform-d) δ6.37 (dd, J=3.8, 1.3Hz, 1H), 5.86 (dd, J=2.5, 1.4Hz, 1H), 5.42–5.32 (m, 2H), 4.80 (t, J=1.2Hz, 2H) , 4.16 (t, J=6.7Hz, 2H), 2.05–1.94 (m, 4H), 1.69–1.64 (m, 2H), 1.49 (d, J=2.5Hz, 9H), 1.30–1.25 (m, 22H), 0.85 (d, J=0.7Hz, 3H).

[0110] Step 3: Synthesis of compound 11-3 0.5 mmol of compound 11-2 was dissolved in 20 mL of THF solution, and 1 mmol of 1-octadecanol and 0.05 mmol of DABCO were added in sequence. After stirring for 12 hours, TLC monitoring showed that compound 11-2 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 11-3 (247 mg, 82% yield). The column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 3 vol% EA. The NMR data of compound 11-3 are as follows, 1 H NMR (400MHz, Chloroform-d) δ6.33–6.23 (m, 1H), 5.93–5.80 (m, 1H), 5.41–5.27 (m, 2H), 4.22–4.09 (m, 4H), 3. 46 (dd, J=21.2, 14.5Hz, 2H), 2.10–1.93 (m, 4H), 1.71–1.55 (m, 5H), 1.52–1.12 (m, 51H), 0.88 (t, J=6.8Hz, 6H).

[0111] Step 4: Synthesis of compound 11-4 0.2 mmol of compound 11-3 was dissolved in a mixed solution of 8 mL DCM and 2 mL MeCN, and 0.6 mmol 3-(dimethylamino)-1-propanethiol and 0.02 mmol TBD were added. After stirring for 2 hours, TLC monitoring showed that compound 11-3 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a colorless oily compound 11-4 (111 mg, 77% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 11-4 are as follows, 1H NMR (400MHz, Chloroform-d) δ 5.39–5.29 (m, 2H), 4.15–4.04 (m, 2H), 3.63 (qd, J=9.3, 5. 9Hz, 2H), 3.44–3.34 (m, 2H), 2.91–2.69 (m, 3H), 2.55 (t, J=7.4Hz, 2H), 2.34 (dd, J=16.3, 9.0Hz, 2H), 2.21 (s, 6H), 1.99 (dd, J=13.4, 6.7Hz, 4H), 1.79–1.68 (m, 2H), 1.61 (dt, J=15 .6, 7.9Hz, 2H), 1.51 (dd, J=13.1, 6.4Hz, 2H), 1.32–1.19 (m, 52H), 0.87 (t, J=6.7Hz, 6H).

[0112] Step 5: Synthesis of compound 11 0.1 mmol of compound 11-4 was dissolved in 10 mL of THF solution, and 0.2 mmol of iodomethane was added at zero degrees. After stirring for 24 hours under nitrogen protection, TLC monitoring showed that compound 11-4 completely disappeared. The solvent was removed in vacuo to obtain a crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain a light yellow oily compound 11 (63 mg, 73% yield). Column chromatography used a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 11 are as follows, 1 H NMR (400MHz, Chloroform-d) δ5.39–5.32 (m, 2H), 4.17–4.03 (m, 2H), 3.77 (dd, J=9 .3, 6.7Hz, 2H), 3.61 (qd, J=9.2, 5.4Hz, 2H), 3.46 (s, 9H), 3.39 (t, J=6.7Hz, 2H), 2. 87–2.77 (m, 3H), 2.68 (dtd, J=20.3, 13.7, 6.8Hz, 2H), 2.18–1.96 (m, 6H), 1.63 (dd, J=13.7, 6.6Hz, 2H), 1.54–1.49 (m, 2H), 1.33–1.23 (m, 52H), 0.88 (t, J=6.8Hz, 6H).

[0113] The present invention also uses an ionizable lipid compound 1-126, and the structure of the ionizable lipid compound 1-126 is shown below: 1-126.

[0114] Example 12: A method for preparing lipid nanoparticles Preparation of ethanol phase solution: Dissolve ionizable lipid compound, DSPC, cholesterol, DMG-PEG 2000, SORT lipid molecule and cationic compound in ethanol to obtain ethanol phase solution. The ionizable lipid compound is compound 1-126, DSPC is purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., cholesterol is purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd. and DMG-PEG 2000 is purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., the ionizable lipid compound, DSPC, cholesterol, DMG-PEG 2000 and SORT lipid molecule are mixed in a molar ratio of 25:5:19.3:0.8:50, and the concentration of ionizable lipid compound in ethanol phase solution is 30 mg / mL. The cationic compound is compound 1 prepared in Example 1, and the amount of cationic compound used is determined by the N / P ratio in liposome nanoparticles.

[0115] Preparation of aqueous solution: Luciferase mRNA was added to 50 mM citrate buffer to obtain 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.

[0116] Preparation of liposome nanoparticles: The ethanol phase solution and the aqueous phase solution are mixed by a microfluidic mixing method to form lipid particles, and then dialyzed with PBS, and finally the obtained product is filtered through a 0.2 μm sterile filter to obtain lipid nanoparticles. The ethanol phase solution and the aqueous phase solution are mixed in a volume ratio of 1:2. The ethanol phase solution contains cationic compounds, and the ratio of N in the ionizable lipid compound and the cationic compound in the ethanol phase solution to P of Luciferase mRNA in the aqueous phase solution is the N / P ratio, and the N / P ratio in this embodiment is 6.

[0117] Example 13: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is the compound 2 prepared in Example 2.

[0118] The rest is the same as Example 12.

[0119] Example 14: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is compound 3 prepared in Example 3.

[0120] The rest is the same as Example 12.

[0121] Example 15: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is compound 4 prepared in Example 4.

[0122] The rest is the same as Example 12.

[0123] Example 16: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is compound 5 prepared in Example 5.

[0124] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0125] The rest is the same as Example 12.

[0126] Example 17: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is compound 6 prepared in Example 6.

[0127] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0128] The rest is the same as Example 12.

[0129] Example 18: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is compound 7 prepared in Example 7.

[0130] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0131] The rest is the same as Example 12.

[0132] Example 19: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that the following steps are performed: In the preparation of the ethanol phase solution, the cationic compound is Compound 8 prepared in Example 8.

[0133] The rest is the same as Example 12.

[0134] Example 20: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, the cationic compound is Compound 8 prepared in Example 8.

[0135] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0136] The rest is the same as Example 12.

[0137] Example 21: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, the cationic compound is the compound 9 prepared in Example 9.

[0138] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0139] The rest is the same as Example 12.

[0140] Example 22: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, the cationic compound is the compound 10 prepared in Example 10.

[0141] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0142] The rest is the same as Example 12.

[0143] Example 23: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, the cationic compound is the compound 11 prepared in Example 11.

[0144] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0145] The rest is the same as Example 12.

[0146] Example 24: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, no cationic compound was used.

[0147] In the preparation of liposome nanoparticles, the amount of ethanol solution was adjusted to make the N / P ratio 6.

[0148] The rest is the same as Example 12.

[0149] Example 25: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, no cationic compound was used.

[0150] In the preparation of liposome nanoparticles, the amount of ethanol solution was adjusted to make the N / P ratio 8.

[0151] The rest is the same as Example 12.

[0152] Example 26: A method for preparing lipid nanoparticles Compared with Example 12, this embodiment is different in that: In the preparation of the ethanol phase solution, the cationic compound is DOTAP.

[0153] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0154] The rest is the same as Example 12.

[0155] Example 27: A method for preparing lipid nanoparticles Compared with Example 23, this embodiment is different in that: In the preparation of the ethanol phase solution, the ionizable lipid compound is MC3.

[0156] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0157] The rest is the same as Example 23.

[0158] Example 28: A method for preparing lipid nanoparticles Compared with Example 23, this embodiment is different in that: In the preparation of the ethanol phase solution, the ionizable lipid compound is MC3 and the cationic compound is DOTAP.

[0159] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0160] The rest is the same as Example 23.

[0161] Example 29: A method for preparing lipid nanoparticles Compared with Example 23, this embodiment is different in that: In the preparation of the ethanol phase solution, the ionizable lipid compound is SM102.

[0162] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0163] The rest is the same as Example 23.

[0164] Example 30: A method for preparing lipid nanoparticles Compared with Example 23, this embodiment is different in that: In the preparation of the ethanol phase solution, the ionizable lipid compound is SM102 and the cationic compound is DOTAP.

[0165] In the preparation of liposome nanoparticles, the N / P ratio was 8.

[0166] The rest is the same as Example 23.

[0167] Test example: The present invention uses Malvern Zetasizer Nano ZS ZEN 3600 (Malvern UK) to measure the size and polydispersity index of the lipid nanoparticles prepared in Examples 12-30 by dynamic light scattering, and determines the Zeta potential thereof; uses Quant-it Ribo green RNA quantitative determination kit (Thermo Fisher Scientific, UK) to determine the encapsulation efficiency of the lipid nanoparticles prepared in Examples 12-30, and the test results are shown in Table 1.

[0168] Table 1 Physicochemical properties of lipid nanoparticles

[0169] Example 22 is a lipid nanoparticle prepared from cationic compound 10. After dialysis, obvious particle aggregation can be observed in the dialysis bag, and the quantitative recovery mass is 0, so subsequent testing is impossible.

[0170] As can be seen from Table 1, the particle size of the lipid nanoparticles of the present invention is 80-500 nm, the Zeta potential is 15-35 mV, and the encapsulation efficiency is >97%.

[0171] The present invention tests the safety and endosomal escape ability of the lipid nanoparticles prepared in Examples 12-30. The mRNA escape is mainly achieved by the destruction of the endosomal membrane by pH-sensitive lipid nanoparticles in the intracellular acidic environment (pH=3-5.5). The present invention simulates the interaction between lipid nanoparticles and cell membranes in a neutral pH environment; and the interaction between lipid nanoparticles and endosomal membranes in the acidic pH environment of intracellular endosomes; thereby verifying the safety and endosomal escape ability of lipid nanoparticles prepared by ionizable lipid compounds. The present invention verifies the hemolysis experiment of lipid nanoparticles in vitro, and the specific operation is as follows: free mRNA or lipid nanoparticles with a final mRNA concentration of 5μg / ml are incubated with a mouse red blood cell solution (final volume percentage equal to 4%) at 37°C for 1 hour, and then the supernatant is collected by centrifugation, and the ultraviolet absorption of the supernatant at 575 nm is measured to prove the hemolysis.

[0172] The results for pH 7.4 are as follows Figure 1 As shown, wherein 1 represents the lipid nanoparticles prepared in Example 12, 2 represents the lipid nanoparticles prepared in Example 13, 3 represents the lipid nanoparticles prepared in Example 14, 4 represents the lipid nanoparticles prepared in Example 15, 5 represents the lipid nanoparticles prepared in Example 16, 6 represents the lipid nanoparticles prepared in Example 17, 7 represents the lipid nanoparticles prepared in Example 18, 8 (N / P=6) represents the lipid nanoparticles prepared in Example 19, 8 (N / P=8) represents the lipid nanoparticles prepared in Example 20, 9 represents the lipid nanoparticles prepared in Example 21, 10 represents the lipid nanoparticles prepared in Example 22, 11 represents the lipid nanoparticles prepared in Example 23, and 1-126 represent the lipid nanoparticles prepared in Example 25. In an environment with a pH of 7.4, the red blood cell lysis rate is very low, and the lipid nanoparticles have a very low destructive effect on the cell membrane and will not cause hemolysis. This shows that the lipid nanoparticles of Examples 17, 20, 21, and 23 prepared from cationic compounds 6, 8, 9, and 11 have excellent biosafety and a hemolysis rate of about 10%, and good safety, that is, the use of cationic compounds 6, 8, 9, and 11 at N / P=8 has excellent biosafety, and other cationic compounds show higher hemolysis due to more tail branches or more olefins in the tail. In the present invention, cationic compounds 6, 8, 9, and 11 also have excellent biosafety at other N / P values.

[0173] The results for pH 5.5 are as follows Figure 2As shown, wherein 1 represents the lipid nanoparticles prepared in Example 12, 2 represents the lipid nanoparticles prepared in Example 13, 3 represents the lipid nanoparticles prepared in Example 14, 4 represents the lipid nanoparticles prepared in Example 15, 5 represents the lipid nanoparticles prepared in Example 16, 6 represents the lipid nanoparticles prepared in Example 17, 7 represents the lipid nanoparticles prepared in Example 18, 8 (N / P=6) represents the lipid nanoparticles prepared in Example 19, 8 (N / P=8) represents the lipid nanoparticles prepared in Example 20, 9 represents the lipid nanoparticles prepared in Example 21, 10 represents the lipid nanoparticles prepared in Example 22, 11 represents the lipid nanoparticles prepared in Example 23, and 1-126 represent the lipid nanoparticles prepared in Example 25. In an environment of pH 5.5, the lipid nanoparticles of Examples 17, 20, 21, and 23 prepared from cationic compounds 6, 8, 9, and 11 have comparable erythrocyte lysis rates in an acidic pH environment, with no significant differences, meaning that cationic compounds 6, 8, 9, and 11 have excellent biosafety when used at N / P=8, and cationic compounds 6, 8, 9, and 11 also have excellent biosafety at other N / P values.

[0174] The present invention conducted an animal experiment on the lipid nanoparticles of Examples 16, 17, 18, 20, 21, 23, and 25 prepared from cationic compounds 5, 6, 7, 8, 9, 11, and 1-126 at N / P=8. After dialyzing, ultrafiltration, and quantification of the lipid nanoparticles, the lipid nanoparticles of Examples 16, 17, 18, 20, 21, 23, and 25 encapsulating Luciferase mRNA were delivered to 6-8 week old female Babl / c mice by tail vein injection at a dose of 10 μg / mouse. Small animal in vivo fluorescence imaging (IVISLuminaIII, PE Company) was performed 6 hours after administration, and then the mice were immediately euthanized. The heart, liver, spleen, lung, kidney and other major organs of the mice were imaged. The test results are as follows: Figure 3 shown.

[0175] The present invention conducted an animal experiment on the lipid nanoparticles of Examples 12, 13, 14, 15, 19, and 24 prepared from cationic compounds 1, 2, 3, 4, 8, and 1-126 at N / P=6. After dialyzing, ultrafiltration, and quantification of the lipid nanoparticles, the lipid nanoparticles of Examples 12, 13, 14, 15, 19, and 24 encapsulating Luciferase mRNA were delivered to 6-8 week old female Babl / c mice by tail vein injection at a dose of 2 μg / mouse. Small animal in vivo fluorescence imaging (IVISLuminaIII, PE Company) was performed 6 hours after administration, and then the mice were immediately euthanized, and the heart, liver, spleen, lung, kidney and other major organs of the mice were imaged. The test results are as follows: Figure 4 shown.

[0176] In summary, the lipid nanoparticles prepared from cationic compounds 8, 9, and 11 of the present invention have obvious lung targeting ability; from a structural point of view, the two tail structures of cationic compounds 8 and 9 are the same, and the number of carbons on the chain containing N at the head is different. The two tail structures of cationic compounds 8 and 11 are similar, with only one olefin difference, and the number of carbons on the chain containing N at the head is the same. Compounds with other structures have high hemolysis due to the large number of branches in the tail, and the large number of olefins in the tail, and basically have no targeting effect. Among all cationic compounds, cationic compound 11 has the best targeting effect.

[0177] Cationic compound 11 has the best targeting effect among all cationic compounds, so we chose cationic compound 11 to compare with commercial ionizable lipid compounds SM102, MC3 and commercial cationic compound DOTAP. The lipid nanoparticles of Examples 23, 26, 27, 27, 29 and 30 encapsulating Luciferase mRNA were delivered to 6-8 week old female Babl / c mice by tail vein injection at a dose of 10 μg / mouse. The safety test and cytotoxicity test of the lipid nanoparticles were carried out. Toxicity experiments were performed using a commercially available cell proliferation assay kit (MTS, Promega) to evaluate the cytotoxicity of lipid nanoparticles. 40,000 293T cells / well were seeded in a 96-well plate and cultured overnight. 293T cells were transfected with Luciferase mRNA lipid nanoparticles at a dose of 0.2 μg mRNA per well. 24 hours after transfection, the old medium was removed and replaced with a new medium containing MTS. The cells were incubated in an incubator for about 2 hours. The absorbance at 490 nm was detected using a TACAN (SPARK) microplate reader. The test results are shown in Figure 5 , and small animal in vivo fluorescence imaging (IVISLuminaIII, PE company) was performed 6 hours after administration, and then the mice were euthanized immediately, and the main organs of the mice, such as the heart, liver, spleen, lung, and kidney, were imaged. The test results are as follows Figure 6 As shown, the fluorescence intensity in the lung is expressed as Figure 7 The distribution of lipid nanoparticles in various organs in Examples 23, 26, 27, 28, 29, and 30 is shown in Figure 8 .pass Figure 5 It can be seen that the hemolysis of cationic compound 11 at pH 7.4 is lower than that of DOTAP when paired with ionizable lipid compounds 1-126, MC3, and SM102, showing good safety, no significant difference in cytotoxicity, and low toxicity. Figure 6 , 7, 8 It can be seen that cationic compound 11 exhibits better lung targeting effect when paired with MC3, which is equivalent to the targeting effect when paired with SM102, and cationic compound 11 exhibits better lung share.

[0178] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0179] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A cationic compound of formula (I) or a pharmaceutically acceptable salt thereof: Formula (I); in, G1, G2, G3 are each independently selected from -(CH2) x -O(C=O)-, -(CH2) x -(C=O)O-, -(CH2) x -(C=O)S-, -(CH2) x -(C=O)NH-, -(CH2) x -O-, -(CH2) x -、-(CH2) x -O(C=O)NH-, -(CH2) x -O(C=O)O- or -(CH2) x NH(C=O)-, wherein x is an integer between 0 and 4; L1, L2, L3, L4 are each independently selected from a linear or branched C 1-25 Alkyl, cis C 2-25 alkenyl; L5 is selected from unsubstituted C 1-10 alkyl; X1, 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, C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, C 3-8 Cycloalkynyl or phenyl, wherein the substituent is 1 or 2 independent OH, SH, C 1-3 Hydroxyl, C 1-3 Alkoxy or -(C=O)OC 1-3 alkyl.

2. The cationic compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The L4 is or .

3. The cationic compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The cationic compound or its pharmaceutically acceptable salt 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: .

4. A lipid complex comprising: A cationic compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

5. A lipid complex according to claim 4, characterized in that: The lipid complex also includes a therapeutic or prophylactic agent.

6. A lipid complex according to claim 4, characterized in that: The lipid complex also includes an ionizable lipid compound, and the ionizable lipid compound is at least one of 1-126, D-Lin-MC3-DMA, SM102, ALC-0315, 5A2-SC8, and CKK-E12.

7. A lipid complex according to claim 4, characterized in that: The lipid complex further includes at least one of phospholipids, structural lipids, and PEGylated lipids.

8. A method for preparing a lipid complex according to any one of claims 4 to 7, comprising: Step (1): dissolving the lipid material in an organic solvent to obtain an organic phase solution; The lipid material comprises at least one of the cationic compound, ionizable lipid compound, phospholipid, structured lipid, and pegylated lipid according to any one of claims 1 to 2; Step (2): mixing the aqueous medium with the buffer to obtain an aqueous solution; Step (3): Mix the organic phase solution with the aqueous phase solution to obtain a lipid complex.

9. The method for preparing a lipid complex according to claim 8, characterized in that: The aqueous medium includes a therapeutic or prophylactic agent.

10. The use of the cationic compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, comprising at least one of the following 1) to 3), 1) Encapsulation of therapeutic or preventive agents; 2) preparing a therapeutic or preventive agent for in vivo delivery; 3) Prepare transfection kit.

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