Preparation and Application of a Cationic Compound and Its Complex

Abstract

The present invention discloses the preparation and application of a cationic compound and its complex, belonging to the field of pharmaceutical technology, and specifically relates to a cationic compound, which can be added to the original lipid complex. The present invention also discloses a lipid complex as described above, which can be specifically delivered to the lungs, and has the characteristics of low toxicity and good targeting, solves the problem of difficult targeted delivery of nucleic acid drugs, and promotes the development of nucleic acid drugs. The lipid complex prepared by the present invention can effectively target the lungs. It is not possible to achieve lung targeting by adding any cationic lipid with a positive charge to the lipid complex. In the present invention, the tail of the cationic lipid needs to have one or two olefins. The cationic lipid developed by the present invention has lower hemolytic toxicity and better lung targeting compared to the complex formed by the traditional DOTAP lipid material.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the preparation and application of a cationic compound and its complex. Background Art

[0002] As a large class of emerging drug fields, nucleic acid drugs have the characteristics of fast design, wide application, high safety, etc., and are one of the main directions for future drug development. 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 for disease prevention and treatment. Currently, lipid nanoparticles (LNPs) prepared from ionizable cationic lipids are a relatively safe and effective means for 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 will have adverse effects on other normal tissues and organs throughout the body. Therefore, the development of an LNP delivery system with organ targeting has become a research hotspot.

[0003] Researchers have developed selective organ targeting (SORT) technology. This technology introduces SORT lipid molecules with different charges into traditional four-component LNPs, changing the surface properties and biological characteristics of LNPs, thereby achieving the specific delivery of mRNA to specific organs, such as the liver, spleen, or lungs of mice. Although SORT-LNPs can achieve targeted delivery to specific organs, in some cases, their targeting accuracy is not high enough. For example, there may be a situation where part of the drug is delivered to non-target organs, resulting in unnecessary effects on other organs. The added SORT lipid molecules carry a positive charge and may have certain toxicity, which may trigger inflammatory reactions or other immune-related adverse reactions.

[0004] 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 for disease prevention and treatment. Currently, there are few SORT lipid molecules that can effectively achieve organ targeting functions, and a large number of designs and screenings are still needed. Summary of the Invention

[0005] The purpose of the present invention is to provide the preparation and application of a cationic compound and its complex that can be applied to the in vivo and in vitro delivery of therapeutic or prophylactic agents, have high biosafety, and good targeting effects.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0007] A cationic compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0008] Formula (I);

[0009] 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)-, where x is an integer between 0 and 4;

[0010] L2, L3, L5 are each independently selected from unsubstituted C 1-10 alkyl;

[0011] L1, L4 are each independently selected from linear or branched C 1-25 alkyl, C 2-25 alkenyl;

[0012] X1, X2 are each independently selected from an oxygen or sulfur atom;

[0013] R1, R2, R3 are each independently selected from optionally 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, where the substituent groups are 1 or 2 independent OH, SH, C 1-3 hydroxyl, C 1-3 alkoxy or -(C=O)OC 1-3 alkyl.

[0014] Preferably, L4 is or .

[0015] Preferably, the cationic compound or its pharmaceutically acceptable salt is selected from any one of the following:

[0016] Compound 1: ;

[0017] Compound 2: ;

[0018] Compound 3: ;

[0019] Compound 4: ;

[0020] Compound 5: ;

[0021] Compound 6: ;

[0022] Compound 7: ;

[0023] Compound 8: ;

[0024] Compound 9: ;

[0025] Compound 10: ;

[0026] Compound 11: .

[0027] The present invention discloses a lipid complex, comprising: the above-mentioned cationic compound or its pharmaceutically acceptable salt.

[0028] Preferably, the lipid complex further comprises a therapeutic agent or a prophylactic agent.

[0029] 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.

[0030] Preferably, the lipid complex further comprises at least one of phospholipid, structural lipid, and polyethylene glycolated lipid.

[0031] Preferably, the lipid complex is a lipid nanoparticle.

[0032] The present invention discloses a method for preparing a lipid complex, comprising:

[0033] 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 compound, ionizable lipid compound, phospholipid, structural lipid, and polyethylene glycolated lipid;

[0034] Step (2): Mixing an aqueous medium with a buffer solution to obtain an aqueous phase solution;

[0035] Step (3): Mixing the organic phase solution with the aqueous phase solution to obtain a lipid complex.

[0036] Preferably, the aqueous medium comprises a therapeutic agent or a prophylactic agent.

[0037] The present invention discloses the uses of cationic compounds, including at least one of the following 1)-3):

[0038] 1) Encapsulating a therapeutic or prophylactic agent;

[0039] 2) Preparing an in vivo delivery reagent for a therapeutic or prophylactic agent;

[0040] 3) Preparing a transfection kit.

[0041] The present invention provides a lipid complex, comprising a cationic compound.

[0042] Preferably, it further comprises a therapeutic or prophylactic agent.

[0043] Preferably, the therapeutic or prophylactic agent is selected from any at least one of nucleic acid drugs, small molecule drugs, protein drugs, and pharmaceutically active molecules.

[0044] 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.

[0045] Preferably, the protein drug is selected from any at least one of antibodies, enzymes, recombinant proteins, polypeptides, and short peptides.

[0046] 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.

[0047] The structure of 1-126 is shown as follows:

[0048] 1-126.

[0049] More preferably, the ionizable lipid compound is 1-126.

[0050] More preferably, it further comprises any one or more of phospholipids, structural lipids, and polyethylene glycolated lipids.

[0051] More preferably, the phospholipid is selected from any one or at least one of 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-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 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-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine.

[0052] More preferably, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0053] Preferably, the structural lipid is selected from any one or at least one of cholesterol, β-cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, lycopene, ursolic acid, α-tocopherol.

[0054] More preferably, the structural lipid is cholesterol.

[0055] Preferably, the polyethylene glycolylated 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.

[0056] More preferably, the polyethylene glycolylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0057] Preferably, the molar ratio of the ionizable lipid compound, phospholipid, structural lipid, polyethylene glycolylated lipid, and cationic compound is 10-100:0-50:0-50:0-5:10-80.

[0058] Preferably, the molar ratio of the ionizable lipid compound, phospholipid, structural lipid, polyethylene glycolylated lipid, and cationic compound is 10-50:2-20:30-50:0.5-5:10-60.

[0059] More preferably, the molar ratio of the ionizable lipid compound, phospholipid, structural lipid, polyethylene glycolylated lipid, and cationic compound is 15-30:2-10:10-30:0.5-2:30-60.

[0060] Even more preferably, the molar ratio of the ionizable lipid compound, phospholipid, structural lipid, polyethylene glycolylated lipid, and cationic compound is 25:5:19.3:0.8:50.

[0061] Preferably, the complex is a lipid nanoparticle.

[0062] Preferably, the lipid nanoparticle has a particle size of 30 to 600 nm and a Zeta potential of -10 to 40 mV.

[0063] More preferably, the lipid nanoparticle has a particle size of 70 to 500 nm and a Zeta potential of 10 to 40 mV.

[0064] On the other hand, the present invention provides a method for preparing a lipid complex, comprising the following steps:

[0065] (1) Dissolve the ionizable lipid compound and any one or more of phospholipids, structural lipids, and polyethylene glycolylated lipids in an organic solvent to obtain an organic phase solution;

[0066] (2) Mix the therapeutic or prophylactic agent with a buffer solution to obtain an aqueous phase solution;

[0067] (3) Mix the organic phase solution with the aqueous phase solution to obtain a lipid complex.

[0068] Preferably, the organic solvent is at least any one of methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone.

[0069] Preferably, the buffer solution is a citrate buffer solution.

[0070] Preferably, the concentration of the citrate buffer solution is 5-80 mM, and the pH of the citrate buffer solution is 2-6.

[0071] More preferably, the concentration of the citrate buffer solution is 10-50 mM, and the pH of the citrate buffer solution is 3-5.

[0072] Preferably, the N / P of the complex is 1-15. The ratio of N in the ionizable lipid compound and the cationic compound to P of the mRNA as the drug is N / P.

[0073] More preferably, the N / P of the complex is 8.

[0074] The present invention provides the use of a cationic compound for preparing a preparation for improving the delivery efficiency and / or transfection efficiency of a lipid complex.

[0075] The present invention provides the use of a cationic compound for preparing a preparation for reducing the cytotoxicity of a lipid complex.

[0076] The present invention also provides the use of a cationic compound or a pharmaceutically acceptable salt thereof, including at least one of the following 1)-4),

[0077] 1) Encapsulating a therapeutic or prophylactic agent;

[0078] 2) In vitro cell transfection of a therapeutic or prophylactic agent;

[0079] 3) Preparing an in vivo delivery reagent for a therapeutic or prophylactic agent;

[0080] 4) Preparing a transfection kit.

[0081] The lipid complex prepared by the present invention can effectively target the lungs. The lipid complex can achieve lung targeting without adding any cationic lipids with a positive charge. The tail of the cationic lipid needs to have one or two olefins. The present invention solves the problem that traditional lipid nanoparticles will accumulate in the liver after intravenous injection.

[0082] The present invention has simple synthesis, relatively high reaction efficiency and low cost; has relatively low toxicity, low erythrocyte lysis rate in a neutral pH environment, and good safety; has good targeting, and the overall expression in lung tissue in organ distribution can reach more than 70%. The present invention has good reference value for expanding the application scope of LNP and treating lung diseases.

[0083] The present invention has designed and synthesized a brand-new cationic compound from an ionizable lipid compound, capable of preparing a brand-new cationic compound and its complex, which can be applied to the in vivo and in vitro delivery of therapeutic or prophylactic agents, especially nucleic acid drugs, solving the problem of difficult delivery of nucleic acid drugs and promoting the development of nucleic acid drugs; the lipid complex provided by the present invention is a lipid nanoparticle, with uniform nanoparticle size, a particle size of 80 to 500 nm, a Zeta potential of 10 to 40 mV, and an encapsulation efficiency ≥ 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, and is simple to prepare with a short route. Description of the Drawings

[0084] Figure 1 It is a diagram of the hemolysis of lipid nanoparticles at pH 7.4.

[0085] Figure 2 It is a diagram of the hemolysis of lipid nanoparticles at pH 5.5.

[0086] Figure 3 It is a diagram of the imaging of major organs of mice at N / P = 8.

[0087] Figure 4 It is a diagram of the imaging of major organs of mice at N / P = 6.

[0088] Figure 5 It is a diagram of the results of the cytotoxicity test of lipid nanoparticles.

[0089] Figure 6 It is a diagram of the imaging of major organs of mice at 6 h after administration.

[0090] Figure 7 It is a diagram of the fluorescence intensity in the lungs of mice at 6 h after administration.

[0091] Figure 8 It is a diagram of the distribution map of the proportion of lipid nanoparticles in major organs of mice. Detailed Embodiments

[0092] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0093] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and the features in the embodiments in the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0094] It should be noted that the term "alkyl" used in the present invention includes methyl, ethyl, propyl, butyl, pentyl, hexyl, etc. and their possible isomers.

[0095] It should be noted that the term "cycloalkyl" used in the present invention includes cyclopropyl, cyclobutyl, cyclopentyl, etc. and their possible isomers.

[0096] It should be noted that the term "alkoxy" used in the present invention includes methoxy, ethoxy, propoxy, etc. and their possible isomers.

[0097] Example 1: A synthesis method of a cationic compound

[0098] The synthesis route of cationic compound 1 in this example is as follows:

[0099] 。

[0100] Step 1: Synthesis of compound 1-1

[0101] Under zero-degree 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)-octadeca-9,12-dien-1-ol. After stirring for another 2 hours, TLC monitoring showed that the alcohol had 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 under vacuum to obtain a crude product. The crude product was purified by column chromatography, and the pure product fractions were evaporated to obtain a colorless oily compound 1-1 (3.1 g, 93% yield). The column chromatography used a silica gel column, and the eluent was a n-hexane solution containing 5 vol% EA.

[0102] Step 2: Synthesis of compound 1-2

[0103] Dissolve 9.3 mmol of Compound 1-1 in 60 mL of tetrahydrofuran solution, and successively add 46 mmol of paraformaldehyde, 46 mmol of DABCO, and 10 mL of water. After continuing to stir for 12 hours, TLC monitoring shows that Compound 1-1 has completely disappeared. Extract the reaction mixture with 100 mL of ethyl acetate and wash it with 100 mL of water and 100 mL of brine. Combine the organic layers, dry over Na2SO4, and remove the solvent under vacuum to obtain the crude product. Dissolve the crude product in 80 mL of DCM at 0 °C, and add 14 mmol of BoC2O and 0.93 mmol of DMAP. After continuing to stir for 2 hours, TLC monitoring shows that the starting materials have completely disappeared. Remove the solvent under vacuum and purify by column chromatography, and evaporate the pure product fraction to obtain Compound 1-2 as a colorless oil (3.6 g, 84% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 5 vol% EA.

[0104] Step 3: Synthesis of Compound 1-3

[0105] Dissolve 1 mmol of Compound 1-2 in 40 mL of tetrahydrofuran, and successively add 1 mmol of 1,5-pentanediol and 0.1 mmol of DABCO. After stirring at room temperature for 12 hours, TLC monitoring shows that Compound 1-2 has completely disappeared. Remove the solvent under vacuum and purify by column chromatography, and evaporate the pure product fraction to obtain Compound 1-3 as a colorless oil (445 mg, 85% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 10 vol% EA.

[0106] Step 4: Synthesis of Compound 1-4

[0107] Dissolve 0.6 mmol of Compound 1-3 in 25 mL of dichloromethane, and successively add 1.8 mmol of 2-ethylundecanoic acid, 1.8 mmol of EDCI, and 1.8 mmol of DMAP. After stirring at room temperature for 12 hours, TLC monitoring shows that Compound 1-3 has completely disappeared. Remove the solvent under vacuum and purify by column chromatography, and evaporate the pure product fraction to obtain Compound 1-4 as a colorless oil (334 mg, 81% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 5 vol% EA.

[0108] Step 5: Synthesis of Compound 1-5

[0109] Dissolve 0.3 mmol of compound 1-4 in a mixed solution of 10 mL of DCM and 2 mL of MeOH, add 0.9 mmol of 3-(dimethylamino)-1-propanethiol and 0.03 mmol of TBD. After continuing to stir for 24 hours, TLC monitoring showed that compound 1-4 completely disappeared. The solvent was removed under vacuum, and the crude product was obtained and purified by column chromatography. The pure product fraction was evaporated to obtain compound 1-5 as a colorless oil (202 mg, 83% yield). Column chromatography was performed using a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0110] Step 6: Synthesis of compound 1

[0111] Dissolve 0.2 mmol of compound 1-5 in 15 mL of THF solution. At zero degree condition, add 0.4 mmol of methyl iodide. After continuing to stir for 2 hours under nitrogen protection, TLC monitoring showed that compound 1-5 completely disappeared. The solvent was removed under vacuum, and the crude product was obtained and purified by column chromatography. The pure product fraction was evaporated to obtain compound 1 as a pale yellow oil (160 mg, 84% yield). Column chromatography was performed using 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 (400 MHz, Chloroform-d) δ 5.40–5.31 (m, 4H), 4.17–3.98 (m, 4H), 3.72 (dd, J = 11.4, 5.5 Hz, 2H), 3.61 (ddd, J = 23.1, 9.3, 5.5 Hz, 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, 6.7 Hz, 5H), 1.51–1.31 (m, 18H), 1.30–1.20 (m, 34H), 0.90–0.85 (m, 9H).

[0112] Example 2: A method for synthesizing a cationic compound

[0113] The synthesis route of the cationic compound 2 in this example is as follows:

[0114] .

[0115] Step 1: Synthesis of compound 2-1

[0116] Dissolve 0.25 mmol of Compound 1-4 in a mixed solution of 10 mL of DCM and 2 mL of MeCN. Add 0.75 mmol of 2-dimethylaminoethanethiol and 0.025 mmol of TBD. After continuing to stir for 2 hours, TLC monitoring shows that Compound 1-4 has completely disappeared. Remove the solvent under vacuum to obtain the crude product, which is purified by column chromatography. The pure product fraction is evaporated to obtain Compound 2-1 as a colorless oil (154 mg, 78% yield). Column chromatography is performed using a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH.

[0117] Step 2: Synthesis of Compound 2

[0118] Dissolve 0.15 mmol of Compound 2-1 in 15 mL of THF solution. At 0 °C, add 0.3 mmol of methyl iodide. After continuing to stir for 24 hours under nitrogen protection, TLC monitoring shows that Compound 2-1 has completely disappeared. Remove the solvent under vacuum to obtain the crude product, which is purified by column chromatography. The pure product fraction is evaporated to obtain Compound 2 as a pale yellow oil (103 mg, 74% yield). Column chromatography is performed using a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH. The NMR data of Compound 2 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 5.41–5.31 (m, 4H), 4.11 (ddt, J = 25.3, 18.5, 6.8 Hz, 4H), 3.97–3.75 (m, 2H), 3.72–3.60 (m, 2H), 3.48 (s, 9H), 3.42 (t, J = 6.5 Hz, 2H), 3.05–2.86 (m, 5H), 2.77 (t, J = 6.7 Hz, 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, 6.4 Hz, 32H), 0.88 (dd, J = 4.1, 2.5 Hz, 9H).

[0119] Example 3: A method for synthesizing a cationic compound

[0120] The synthetic route of the cationic Compound 3 in this example is as follows:

[0121] 。

[0122] Step 1: Synthesis of Compound 3-1

[0123] Dissolve 0.6 mmol of Compound 1-3 in 25 mL of dichloromethane, and successively add 1.8 mmol of isostearic acid, 1.8 mmol of EDCI, and 1.8 mmol of DMAP. After stirring at room temperature for 12 hours, TLC monitoring showed that Compound 1-3 completely disappeared. The solvent was removed under vacuum and purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 3-1 as a colorless oil (366 mg, 85% yield). Column chromatography was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0124] Step 2: Synthesis of Compound 3-2

[0125] Dissolve 0.2 mmol of Compound 3-1 in a mixed solution of 10 mL of DCM and 2 mL of MeOH, add 0.5 mmol of 3-(dimethylamino)-1-propanethiol and 0.02 mmol of TBD. After continuing to stir for 2 hours, TLC monitoring showed that Compound 3-1 completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 11 as a colorless oil (120 mg, 83% yield). Column chromatography was performed using a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0126] Step 3: Synthesis of Compound 3

[0127] Dissolve 0.1 mmol of Compound 3-2 in 10 mL of THF solution. Under ice-cooled conditions, add 0.2 mmol of methyl iodide. After continuing to stir for 24 hours under nitrogen protection, TLC monitoring showed that Compound 3-2 completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 2 as a pale yellow oil (78 mg, 80% yield). Column chromatography was performed using 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 (400 MHz, Chloroform-d) δ 5.43–5.27 (m, 4H), 4.17–3.99 (m, 4H), 3.70 (d, J = 7.0 Hz, 2H), 3.62 (qd, J = 9.3, 5.5 Hz, 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).

[0128] Example 4: Synthesis Method of a Cationic Compound

[0129] The synthesis route of the cationic compound 4 in this example is as follows:

[0130] 。

[0131] Step 1: Synthesis of Compound 4-1

[0132] At room temperature, 9 mmol of EDCI and 9 mmol of DMAP were added to a 50 mL dichloromethane solution of 6 mmol of 6-bromohexanoic acid and 9 mmol of 9-heptadecanol. After stirring for another 12 hours, TLC monitoring showed that the alcohol had completely disappeared. The solvent was removed under vacuum 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 was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0133] Step 2: Synthesis of Compound 4-2

[0134] At 60 °C, 5 mmol of acrylic acid was added to a 40 mL DMF solution of 5 mmol of compound 4-1, and then 7.5 mmol of K2CO3 was slowly added. After stirring for another 12 hours, TLC monitoring showed that compound 4-1 had 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 under vacuum 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 was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0135] Step 3: Synthesis of Compound 4-3

[0136] 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 another 12 hours, TLC monitoring showed that compound 4-2 had completely disappeared. The solvent was removed under vacuum 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 materials had completely disappeared. The solvent was removed under vacuum 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 was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0137] Step 4: Synthesis of Compound 4-4

[0138] Dissolve 2 mmol of Compound 4-3 in 60 mL of tetrahydrofuran, and successively add 2 mmol of 1,6-hexanediol and 0.2 mmol of DABCO. After stirring at room temperature for 12 hours, TLC monitoring shows that Compound 4-3 has completely disappeared. Remove the solvent under vacuum and purify by column chromatography, and evaporate the pure product fraction to obtain a colorless oily compound, Compound 4-4 (362 mg, 92% yield). Column chromatography uses a silica gel column, and the eluent is a n-hexane solution containing 10 vol% EA.

[0139] Step 5: Synthesis of Compound 4-5

[0140] Dissolve 0.5 mmol of Compound 4-4 in 30 mL of dichloromethane, and successively add 0.5 mmol of 2-ethylundecanoic acid, 1 mmol of EDCI and 1 mmol of DMAP. After stirring at room temperature for 12 hours, TLC monitoring shows that Compound 4-4 has completely disappeared. Remove the solvent under vacuum and purify by column chromatography, and evaporate the pure product fraction to obtain a colorless oily compound, Compound 4-5 (328 mg, 83% yield). Column chromatography uses a silica gel column, and the eluent is a n-hexane solution containing 5 vol% EA.

[0141] Step 6: Synthesis of Compound 4-6

[0142] Dissolve 0.2 mmol of Compound 4-5 in a mixed solution of 10 mL of DCM and 2 mL of MeCN, add 0.5 mmol of 3-(dimethylamino)-1-propanethiol and 0.02 mmol of TBD. After continuing to stir for 2 hours, TLC monitoring shows that Compound 4-5 has completely disappeared. Remove the solvent under vacuum to obtain a crude product and purify by column chromatography, and evaporate the pure product fraction to obtain a colorless oily compound, Compound 4-6 (149 mg, 82% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH.

[0143] Step 7: Synthesis of Compound 4

[0144] Dissolve 0.1 mmol of Compound 4-6 in 10 mL of THF solution, and add 0.2 mmol of methyl iodide under zero-degree conditions. After continuing to stir for 24 hours under nitrogen protection, TLC monitoring shows that Compound 4-6 has completely disappeared. Remove the solvent under vacuum to obtain a crude product and purify by column chromatography, and evaporate the pure product fraction to obtain a pale yellow oily compound, Compound 4 (89 mg, 85% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH. The NMR data of Compound 4 are as follows, 11H NMR (400 MHz, Chloroform-d) δ 4.85 (p, J = 6.2 Hz, 1H), 4.08 (dt, J = 23.2, 6.6 Hz, 4H), 3.75 (dd, J = 10.6, 6.0 Hz, 2H), 3.62 (ddd, J = 21.4, 9.3, 5.5 Hz, 2H), 3.48 (s, 9H), 3.41 (t, J = 6.5 Hz, 2H), 2.82 (q, J = 5.8 Hz, 3H), 2.68 (dd, J = 12.6, 6.4 Hz, 2H), 2.30 (t, J = 7.3 Hz, 3H), 2.21–2.07 (m, 2H), 1.73–1.41 (m, 18H), 1.32–1.20 (m, 48H), 0.88 (t, J = 6.7 Hz, 12H).

[0145] Example 5: Synthesis Method of a Cationic Compound

[0146] The synthesis route of the cationic compound 5 in this example is as follows:

[0147] .

[0148] Step 1: Synthesis of Compound 5-1

[0149] Dissolve 6 mmol of compound 4-1 in 50 mL of ultradry DMF solution, add 2 mmol of 3,5-dihydroxybenzyl alcohol, 20 mmol of anhydrous potassium carbonate, and 0.2 mmol of potassium iodide, and react at 80 °C for 24 hours. TLC monitoring shows that compound 4-1 has completely disappeared. Wash the reaction mixture with ethyl acetate, filter the obtained solution, extract it with ethyl acetate and saturated brine, and finally extract it with saturated lithium chloride, 4-5 times. Combine the organic phase layers, dry with anhydrous Na2SO4. After removing the solvent under vacuum, purify by column chromatography, and evaporate the pure product fraction to obtain colorless oily compound 5-1 (380 mg, 45% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 15 vol% EA.

[0150] Step 2: Synthesis of Compound 5-2

[0151] Dissolve 0.3 mmol of compound 5-1 in 20 mL of DCM solution. Sequentially add 0.6 mmol of N,N-dimethyl-β-alanine and 0.5 mmol of EDCI. Add 0.9 mmol of triethylamine during stirring. Continue stirring for 12 hours. Monitor the reaction by TLC until compound 5-1 is completely reacted. Remove the solvent under vacuum and purify by column chromatography. Evaporate the pure product fraction to obtain compound 5-2 as a colorless oil (151 mg, 53% yield). Column chromatography is performed using a silica gel column, and the eluent is a DCM solution containing 10 vol% MeOH.

[0152] Step 3: Synthesis of compound 5

[0153] Dissolve 0.1 mmol of compound 5-2 in 10 mL of THF solution. At 0 °C, add 0.2 mmol of methyl iodide. Continue stirring under nitrogen protection for 24 hours. Monitor the reaction by TLC until compound 5-2 completely disappears. Remove the solvent under vacuum to obtain the crude product and purify by column chromatography. Evaporate the pure product fraction to obtain compound 4 as a pale yellow oil (85 mg, 78% yield). Column chromatography is performed using a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 5 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 6.37 (d, J = 2.1 Hz, 2H), 6.32 (t, J = 2.1 Hz, 1H), 4.97 (s, 2H), 4.79 (p, J = 6.3 Hz, 2H), 3.86 (t, J = 6.4 Hz, 4H), 3.74–3.63 (m, 2H), 3.34 (s, 9H), 2.54 (t, J = 6.4 Hz, 2H), 2.26 (dd, J = 16.4, 9.0 Hz, 4H), 2.03 (d, J = 6.3 Hz, 2H), 1.77–1.68 (m, 4H), 1.62 (dt, J = 14.5, 7.1 Hz, 4H), 1.43 (d, J = 6.3 Hz, 8H), 1.19 (d, J = 13.6 Hz, 53H), 0.80 (dd, J = 8.6, 5.1 Hz, 12H).

[0154] Example 6: A method for synthesizing a cationic compound

[0155] The synthesis route of the cationic compound 6 in this example is as follows:

[0156] 。

[0157] Step 1: Synthesis of compound 6-1

[0158] Dissolve 0.5 mmol of Compound 1-3 in 20 mL of dichloromethane, and successively add 1 mmol of 1-decanoic acid, 1 mmol of EDCI, and 1 mmol of DMAP. After stirring at room temperature for 12 hours, TLC monitoring showed that Compound 1-3 completely disappeared. The solvent was removed under vacuum and purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 6-1 as a colorless oil (254 mg, 84% yield). Column chromatography was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA.

[0159] Step 2: Synthesis of Compound 6-2

[0160] Dissolve 0.2 mmol of Compound 6-1 in a mixed solution of 8 mL of DCM and 2 mL of MeCN, and add 0.5 mmol of 3-(dimethylamino)-1-propanethiol. After continuing to stir for 2 hours, TLC monitoring showed that Compound 8-2 completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 6-2 as a colorless oil (117 mg, 81% yield). Column chromatography was performed using a silica gel column, and the eluent was a dichloromethane solution containing 10 vol% MeOH.

[0161] Step 3: Synthesis of Compound 6

[0162] Dissolve 0.1 mmol of Compound 6-2 in 10 mL of THF solution. At zero degree condition, add 0.2 mmol of methyl iodide. After continuing to stir for 24 hours under nitrogen protection, TLC monitoring showed that Compound 5-2 completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography, and the pure product fraction was evaporated to obtain Compound 6 as a pale yellow oil (72 mg, 84% yield). Column chromatography was performed using 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 (400 MHz, Chloroform-d) δ 5.45–5.25 (m, 4H), 4.17–3.99 (m, 4H), 3.81–3.70 (m, 2H), 3.61 (qd, J = 9.2, 5.4 Hz, 2H), 3.46 (s, 9H), 3.40 (t, J = 6.5 Hz, 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).

[0163] Example 7: A synthesis method of a cationic compound

[0164] The synthetic route of the cationic compound 7 in this example is as follows:

[0165] 。

[0166] Step 1: Synthesis of compound 7-1

[0167] Dissolve 0.3 mmol of compound 1-2 in 20 mL of THF solution, and successively add 0.6 mmol of (9Z,12Z)-octadeca-9,12-dien-1-ol and 0.03 mmol of DABCO. After continuing to stir for 12 hours, TLC monitoring shows that compound 1-2 has completely disappeared. The solvent is removed under vacuum, and the crude product is purified by column chromatography, and the pure product fraction is evaporated to obtain colorless oily compound 7-1 (122 mg, 79% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 3 vol% EA.

[0168] Step 2: Synthesis of compound 7-2

[0169] Dissolve 0.25 mmol of compound 7-1 in a mixed solution of 8 mL of DCM and 2 mL of MeCN, and add 0.75 mmol of 3-(dimethylamino)-1-propanethiol and 0.025 mmol of TBD. After continuing to stir for 2 hours, TLC monitoring shows that compound 7-1 has completely disappeared. The solvent is removed under vacuum, and the crude product is purified by column chromatography, and the pure product fraction is evaporated to obtain colorless oily compound 7-2 (131 mg, 73% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH.

[0170] Step 3: Synthesis of compound 7

[0171] Dissolve 0.1 mmol of compound 7-2 in 10 mL of THF solution, and add 0.2 mmol of methyl iodide under zero-degree conditions. After continuing to stir for 24 hours under nitrogen protection, TLC monitoring shows that compound 7-2 has completely disappeared. The solvent is removed under vacuum, and the crude product is purified by column chromatography, and the pure product fraction is evaporated to obtain light yellow oily compound 7 (67 mg, 78% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 7 is as follows, 11H NMR (400 MHz, Chloroform-d) δ 5.41–5.29 (m, 8H), 4.10 (qt, J = 10.8, 6.8 Hz, 2H), 3.83–3.71 (m, 2H), 3.61 (qd, J = 9.2, 5.4 Hz, 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, 7.0 Hz, 3H), 1.34–1.26 (m, 32H), 0.90–0.87 (m, 6H).

[0172] Example 8: A method for synthesizing a cationic compound

[0173] The synthesis route of the cationic compound 8 in this example is as follows:

[0174] 。

[0175] Step 1: Synthesis of compound 8-1

[0176] Dissolve 0.5 mmol of compound 1-2 in 20 mL of THF solution, and sequentially add 1 mmol of 1-octadecanol and 0.05 mmol of DABCO. After continuing to stir for 12 hours, TLC monitoring shows that compound 1-2 has completely disappeared. The solvent is removed under vacuum, and the crude product is obtained and purified by column chromatography. The pure product fraction is evaporated to obtain a colorless oily compound 8-1 (186 mg, 72% yield). Column chromatography uses a silica gel column, and the eluent is a hexane solution containing 3 vol% EA.

[0177] Step 2: Synthesis of compound 8-2

[0178] Dissolve 0.2 mmol of compound 8-1 in a mixed solution of 8 mL of DCM and 2 mL of MeCN, and add 0.6 mmol of 3-(dimethylamino)-1-propanethiol and 0.02 mmol of TBD. After continuing to stir for 2 hours, TLC monitoring shows that compound 8-1 has completely disappeared. The solvent is removed under vacuum, and the crude product is obtained and purified by column chromatography. The pure product fraction is evaporated to obtain a colorless oily compound 8-2 (98 mg, 68% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH.

[0179] Step 3: Synthesis of compound 8

[0180] Dissolve 0.1 mmol of compound 8-2 in 10 mL of THF solution. At 0 °C, add 0.2 mmol of methyl iodide. After stirring for another 24 hours under nitrogen protection, TLC monitoring shows that compound 8-1 has completely disappeared. Remove the solvent under vacuum to obtain the crude product, which is purified by column chromatography. The pure product fraction is evaporated to obtain compound 8 as a pale yellow oil (60 mg, 70% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 8 are as follows, 1 H NMR (400 MHz, Chloroform-d) δ 5.42–5.31 (m, 4H), 4.18–4.02 (m, 2H), 3.76 (dd, J = 9.1, 6.5 Hz, 2H), 3.61 (ddd, J = 19.1, 9.3, 5.5 Hz, 2H), 3.46 (s, 9H), 3.39 (t, J = 6.7 Hz, 2H), 2.86–2.61 (m, 7H), 2.07 (dt, J = 13.9, 7.5 Hz, 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, 3.3 Hz, 6H).

[0181] Example 9: A method for synthesizing a cationic compound

[0182] The synthetic route of the cationic compound 9 in this example is as follows:

[0183] 。

[0184] Step 1: Synthesis of compound 9-1

[0185] Dissolve 0.2 mmol of compound 8-1 in a mixed solution of 8 mL of DCM and 2 mL of MeCN, add 0.6 mmol of 2-dimethylaminoethanethiol and 0.02 mmol of TBD. After stirring for 2 hours, TLC monitoring shows that compound 8-1 has completely disappeared. Remove the solvent under vacuum to obtain the crude product, which is purified by column chromatography. The pure product fraction is evaporated to obtain compound 9-1 as a colorless oil (78 mg, 55% yield). Column chromatography uses a silica gel column, and the eluent is a dichloromethane solution containing 10 vol% MeOH.

[0186] Step 2: Synthesis of compound 9

[0187] Dissolve 0.1 mmol of compound 9-1 in 10 mL of THF solution. At 0 °C, add 0.2 mmol of methyl iodide. After stirring for an additional 24 hours under nitrogen protection, TLC monitoring showed that compound 9-1 had completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography. The pure product fraction was evaporated to obtain compound 9 as a pale yellow oil (69 mg, 82% yield). Column chromatography was performed using a silica gel column, and the eluent was dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 9 are as follows, 1 H NMR (400 MHz, Chloroform-d) δ 5.42–5.31 (m, 4H), 4.19–4.03 (m, 2H), 3.97–3.77 (m, 2H), 3.68–3.62 (m, 2H), 3.50 (d, J = 3.8 Hz, 9H), 3.40 (t, J = 6.7 Hz, 2H), 2.93 (tt, J = 21.0, 10.3 Hz, 5H), 2.77 (t, J = 6.6 Hz, 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).

[0188] Example 10: A method for synthesizing a cationic compound

[0189] The synthetic route of the cationic compound 10 in this example is as follows:

[0190] .

[0191] Step 1: Synthesis of compound 10

[0192] Dissolve 0.1 mmol of compound MC3 in 10 mL of THF solution. At 0 °C, add 0.2 mmol of methyl iodide. After stirring for an additional 24 hours under nitrogen protection, TLC monitoring showed that compound 9-4 had completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography. The pure product fraction was evaporated to obtain compound 8 as a pale yellow oil (60 mg, 76% yield). Column chromatography was performed using a silica gel column, and the eluent was dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 10 are as follows, 11H NMR (400 MHz, Chloroform-d) δ 5.43–5.28 (m, 8H), 4.84 (p, J = 6.2 Hz, 1H), 3.81–3.70 (m, 2H), 3.46 (s, 9H), 2.77 (t, J = 6.6 Hz, 4H), 2.52 (t, J = 6.5 Hz, 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).

[0193] Example 11: Synthesis Method of a Cationic Compound

[0194] The synthesis route of the cationic compound 11 in this example is as follows:

[0195] 。

[0196] Step 1: Synthesis of Compound 11-1

[0197] 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 another 2 hours, TLC monitoring showed that the oleyl alcohol had 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 under vacuum to obtain a crude product. The crude product was purified by column chromatography, and the pure product fractions were evaporated to obtain a colorless oily compound 11-1 (1.9 g, 88% yield). Column chromatography was performed using a silica gel column, and the eluent was a hexane solution containing 5 vol% EA. The NMR data of compound 11-1 are as follows, 1 1H NMR (400 MHz, Chloroform-d) δ 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.5 Hz, 1H), 5.44–5.26 (m, 2H), 4.15 (t, J = 6.7 Hz, 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).

[0198] Step 2: Synthesis of Compound 11-2

[0199] Dissolve 6 mmol of compound 11-1 in 30 mL of tetrahydrofuran solution, and successively add 12 mmol of paraformaldehyde, 12 mmol of DABCO, and 2 mL of water. After stirring for an additional 12 hours, TLC monitoring showed that compound 11-1 had 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 the 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 an additional 2 hours, TLC monitoring showed that the starting material had completely disappeared. The solvent was removed in vacuo and the product was purified by column chromatography, and the pure product fractions were evaporated to give compound 11-2 as a colorless oil (1.2 g, 53% yield). Column chromatography was performed using a silica gel column with a hexane solution containing 5 vol% EA as the eluent. The NMR data of compound 11-2 are as follows, 1 H NMR (400 MHz, Chloroform-d) δ 6.37 (dd, J = 3.8, 1.3 Hz, 1H), 5.86 (dd, J = 2.5, 1.4 Hz, 1H), 5.42–5.32 (m, 2H), 4.80 (t, J = 1.2 Hz, 2H), 4.16 (t, J = 6.7 Hz, 2H), 2.05–1.94 (m, 4H), 1.69–1.64 (m, 2H), 1.49 (d, J = 2.5 Hz, 9H), 1.30–1.25 (m, 22H), 0.85 (d, J = 0.7 Hz, 3H).

[0200] Step 3: Synthesis of compound 11-3

[0201] Dissolve 0.5 mmol of compound 11-2 in 20 mL of THF solution, and successively add 1 mmol of 1-octadecanol and 0.05 mmol of DABCO. After stirring for an additional 12 hours, TLC monitoring showed that compound 11-2 had completely disappeared. The solvent was removed in vacuo to obtain the crude product, which was purified by column chromatography, and the pure product fractions were evaporated to give compound 11-3 as a colorless oil (247 mg, 82% yield). Column chromatography was performed using a silica gel column with a hexane solution containing 3 vol% EA as the eluent. The NMR data of compound 11-3 are as follows, 11H NMR (400 MHz, Chloroform-d) δ 6.33–6.23 (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, 14.5 Hz, 2H), 2.10–1.93 (m, 4H), 1.71–1.55 (m, 5H), 1.52–1.12 (m, 51H), 0.88 (t, J = 6.8 Hz, 6H).

[0202] Step 4: Synthesis of Compound 11-4

[0203] Dissolve 0.2 mmol of Compound 11-3 in a mixed solution of 8 mL of DCM and 2 mL of MeCN, add 0.6 mmol of 3-(dimethylamino)-1-propanethiol and 0.02 mmol of TBD. After stirring for another 2 hours, TLC monitoring showed that Compound 11-3 had completely disappeared. The solvent was removed under vacuum to obtain the crude product, which was purified by column chromatography. The pure product fraction was evaporated to obtain Compound 11-4 as a colorless oil (111 mg, 77% yield). Column chromatography was performed using 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: 1 1H NMR (400 MHz, Chloroform-d) δ 5.39–5.29 (m, 2H), 4.15–4.04 (m, 2H), 3.63 (qd, J = 9.3, 5.9 Hz, 2H), 3.44–3.34 (m, 2H), 2.91–2.69 (m, 3H), 2.55 (t, J = 7.4 Hz, 2H), 2.34 (dd, J = 16.3, 9.0 Hz, 2H), 2.21 (s, 6H), 1.99 (dd, J = 13.4, 6.7 Hz, 4H), 1.79–1.68 (m, 2H), 1.61 (dt, J = 15.6, 7.9 Hz, 2H), 1.51 (dd, J = 13.1, 6.4 Hz, 2H), 1.32–1.19 (m, 52H), 0.87 (t, J = 6.7 Hz, 6H).

[0204] Step 5: Synthesis of Compound 11

[0205] Dissolve 0.1 mmol of compound 11-4 in 10 mL of THF solution. At zero degree Celsius, add 0.2 mmol of methyl iodide. After continuously stirring for 24 hours under nitrogen protection, TLC monitoring shows that compound 11-4 completely disappears. Remove the solvent under vacuum to obtain the crude product, which is purified by column chromatography. Evaporate the pure product fraction to obtain compound 11 as a pale yellow oil (63 mg, 73% yield). Column chromatography uses a silica gel column, and the eluent is dichloromethane solution containing 10 vol% MeOH. The NMR data of compound 11 is as follows, 1 H NMR (400 MHz, Chloroform-d) δ 5.39–5.32 (m, 2H), 4.17–4.03 (m, 2H), 3.77 (dd, J = 9.3, 6.7 Hz, 2H), 3.61 (qd, J = 9.2, 5.4 Hz, 2H), 3.46 (s, 9H), 3.39 (t, J = 6.7 Hz, 2H), 2.87–2.77 (m, 3H), 2.68 (dtd, J = 20.3, 13.7, 6.8 Hz, 2H), 2.18–1.96 (m, 6H), 1.63 (dd, J = 13.7, 6.6 Hz, 2H), 1.54–1.49 (m, 2H), 1.33–1.23 (m, 52H), 0.88 (t, J = 6.8 Hz, 6H).

[0206] The ionizable lipid compound 1-126 is also used in the present invention. The structure of the ionizable lipid compound 1-126 is shown as follows:

[0207] 1-126.

[0208] Example 12: A method for preparing lipid nanoparticles

[0209] Preparation of the ethanol phase solution: Dissolve the ionizable lipid compound, DSPC, cholesterol, DMG-PEG 2000, SORT lipid molecule, and cationic compound in ethanol to obtain the ethanol phase solution. The ionizable lipid compound is compound 1-126. DSPC is purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., cholesterol is purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., and DMG-PEG 2000 is purchased from AVT (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. The concentration of the ionizable lipid compound in the ethanol phase solution is 30 mg / mL. The cationic compound is compound 1 prepared in Example 1, and the usage amount of the cationic compound is determined by the N / P ratio in the liposome nanoparticles.

[0210] 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 the citrate buffer were mixed at a volume ratio of 41:900.

[0211] Preparation of lipid nanoparticles: The ethanol solution and the aqueous solution were mixed by a microfluidic mixing method to form lipid particles, then dialyzed with PBS, and finally the obtained product was filtered through a 0.2 μm sterile filter to obtain lipid nanoparticles. The ethanol solution and the aqueous solution were mixed at a volume ratio of 1:2. The ethanol solution contained a cationic compound. The ratio of N in the ionizable lipid compound and the cationic compound in the ethanol solution to P of Luciferase mRNA in the aqueous solution was the N / P ratio. The N / P ratio in this example was 6.

[0212] Example 13: A method for preparing lipid nanoparticles

[0213] This example is different from Example 12 in the following steps:

[0214] In the preparation of the ethanol solution, the cationic compound was Compound 2 prepared in Example 2.

[0215] Others were the same as in Example 12.

[0216] Example 14: A method for preparing lipid nanoparticles

[0217] This example is different from Example 12 in the following steps:

[0218] In the preparation of the ethanol solution, the cationic compound was Compound 3 prepared in Example 3.

[0219] Others were the same as in Example 12.

[0220] Example 15: A method for preparing lipid nanoparticles

[0221] This example is different from Example 12 in the following steps:

[0222] In the preparation of the ethanol solution, the cationic compound was Compound 4 prepared in Example 4.

[0223] Others were the same as in Example 12.

[0224] Example 16: A method for preparing lipid nanoparticles

[0225] This example is different from Example 12 in the following steps:

[0226] In the preparation of the ethanol phase solution, the cationic compound is Compound 5 prepared in Example 5.

[0227] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0228] Others are the same as in Example 12.

[0229] Example 17: A method for preparing lipid nanoparticles

[0230] This example is different from Example 12 in the following steps:

[0231] In the preparation of the ethanol phase solution, the cationic compound is Compound 6 prepared in Example 6.

[0232] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0233] Others are the same as in Example 12.

[0234] Example 18: A method for preparing lipid nanoparticles

[0235] This example is different from Example 12 in the following steps:

[0236] In the preparation of the ethanol phase solution, the cationic compound is Compound 7 prepared in Example 7.

[0237] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0238] Others are the same as in Example 12.

[0239] Example 19: A method for preparing lipid nanoparticles

[0240] This example is different from Example 12 in the following steps:

[0241] In the preparation of the ethanol phase solution, the cationic compound is Compound 8 prepared in Example 8.

[0242] Others are the same as in Example 12.

[0243] Example 20: A method for preparing lipid nanoparticles

[0244] This example is different from Example 12 in the following steps:

[0245] In the preparation of the ethanol phase solution, the cationic compound is Compound 8 prepared in Example 8.

[0246] In the preparation of liposomal nanoparticles, the N / P ratio was 8.

[0247] The others were the same as in Example 12.

[0248] Example 21: A method for preparing lipid nanoparticles

[0249] Compared with Example 12, the differences in this example were as follows:

[0250] In the preparation of the ethanol-phase solution, the cationic compound was Compound 9 prepared in Example 9.

[0251] In the preparation of liposomal nanoparticles, the N / P ratio was 8.

[0252] The others were the same as in Example 12.

[0253] Example 22: A method for preparing lipid nanoparticles

[0254] Compared with Example 12, the differences in this example were as follows:

[0255] In the preparation of the ethanol-phase solution, the cationic compound was Compound 10 prepared in Example 10.

[0256] In the preparation of liposomal nanoparticles, the N / P ratio was 8.

[0257] The others were the same as in Example 12.

[0258] Example 23: A method for preparing lipid nanoparticles

[0259] Compared with Example 12, the differences in this example were as follows:

[0260] In the preparation of the ethanol-phase solution, the cationic compound was Compound 11 prepared in Example 11.

[0261] In the preparation of liposomal nanoparticles, the N / P ratio was 8.

[0262] The others were the same as in Example 12.

[0263] Example 24: A method for preparing lipid nanoparticles

[0264] Compared with Example 12, the differences in this example were as follows:

[0265] In the preparation of the ethanol-phase solution, no cationic compound was used.

[0266] In the preparation of liposomal nanoparticles, the amount of ethanol solution was adjusted to make the N / P ratio 6.

[0267] The others were the same as in Example 12.

[0268] Example 25: A method for preparing lipid nanoparticles

[0269] This example is different from Example 12 in the following steps:

[0270] In the preparation of the ethanol-phase solution, no cationic compound was used.

[0271] In the preparation of the liposome nanoparticles, the amount of the ethanol solution was adjusted to make the N / P ratio 8.

[0272] Others are the same as in Example 12.

[0273] Example 26: A method for preparing lipid nanoparticles

[0274] This example is different from Example 12 in the following steps:

[0275] In the preparation of the ethanol-phase solution, the cationic compound is DOTAP.

[0276] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0277] Others are the same as in Example 12.

[0278] Example 27: A method for preparing lipid nanoparticles

[0279] This example is different from Example 23 in the following steps:

[0280] In the preparation of the ethanol-phase solution, the ionizable lipid compound is MC3.

[0281] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0282] Others are the same as in Example 23.

[0283] Example 28: A method for preparing lipid nanoparticles

[0284] This example is different from Example 23 in the following steps:

[0285] In the preparation of the ethanol-phase solution, the ionizable lipid compound is MC3 and the cationic compound is DOTAP.

[0286] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0287] Others are the same as in Example 23.

[0288] Example 29: A method for preparing lipid nanoparticles

[0289] This example is different from Example 23 in the following steps:

[0290] In the preparation of the ethanolic phase solution, the ionizable lipid compound is SM102.

[0291] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0292] Others are the same as in Example 23.

[0293] Example 30: A method for preparing lipid nanoparticles

[0294] This example is different from Example 23 in the following steps:

[0295] In the preparation of the ethanolic phase solution, the ionizable lipid compound is SM102 and the cationic compound is DOTAP.

[0296] In the preparation of the liposome nanoparticles, the N / P ratio is 8.

[0297] Others are the same as in Example 23.

[0298] Test example:

[0299] In the present invention, the size and polydispersity index of the lipid nanoparticles prepared in Examples 12 - 30 were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS ZEN3600 (Malvern, UK), and their Zeta potential was measured; the encapsulation efficiency of the lipid nanoparticles prepared in Examples 12 - 30 was measured using a Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK). The test results are shown in Table 1.

[0300] Table 1 Physicochemical properties of lipid nanoparticles

[0301]

[0302] Example 22 was the lipid nanoparticles prepared with cationic compound 10. After dialysis, obvious particle aggregation could be observed in the dialysis bag, and the quantitatively recovered mass was 0, making subsequent testing impossible.

[0303] 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 > 97%.

[0304] The present invention tested the safety and endosomal escape ability of the lipid nanoparticles prepared in Examples 12 - 30. The realization of mRNA escape is mainly because the pH-sensitive lipid nanoparticles disrupt the endosomal membrane in the intracellular acidic environment (pH = 3 - 5.5). The present invention simulated the interaction of lipid nanoparticles with the cell membrane in a neutral pH environment; and the interaction of lipid nanoparticles with the endosomal membrane in the acidic pH environment of intracellular endosomes; thereby verifying the safety and endosomal escape ability of the lipid nanoparticles prepared from the ionizable lipid compounds. The present invention verified this through 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 5 μg / ml were co-incubated with mouse erythrocyte solution (the final volume percentage is equal to 4%) at 37°C for 1 hour, then the supernatant was collected by centrifugation, and the ultraviolet absorption of the supernatant at 575 nm was measured to prove the hemolysis situation.

[0305] The results at pH 7.4 are as Figure 1 shown, where 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 represents the lipid nanoparticles prepared in Example 25. The erythrocyte lysis rate is very low in an environment with pH 7.4, and the lipid nanoparticles have a very low destructive effect on the cell membrane and do not cause hemolysis. This indicates that the lipid nanoparticles of Examples 17, 20, 21, and 23 prepared from cationic compounds 6, 8, 9, and 11 have excellent biosafety. The hemolysis rate is about 10%, and the safety is good, that is, the use of cationic compounds 6, 8, 9, and 11 at N / P = 8 has excellent biosafety. Other cationic compounds show higher hemolytic properties due to more branched tails or more olefins in the tails. In the present invention, cationic compounds 6, 8, 9, and 11 also have excellent biosafety at other N / P values.

[0306] The results at pH 5.5 are as Figure 2As shown in the figure, where 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 represents the lipid nanoparticles prepared in Example 25. In an environment with a pH of 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 and show no significant differences, that is, the use of cationic compounds 6, 8, 9, and 11 at N / P = 8 has excellent biosafety, and cationic compounds 6, 8, 9, and 11 also have excellent biosafety at other N / P values.

[0307] The present invention conducted animal experiments 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 dialysis, 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 at a dose of 10 μg / mouse by tail vein injection, and small animal in - vivo fluorescence imaging (IVIS Lumina III, PE company) was performed 6 hours after administration. Then the mice were immediately euthanized, and the main organs of the mice such as the heart, liver, spleen, lung, and kidney were imaged. The test results are as Figure 3 shown.

[0308] The present invention conducted animal experiments 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 dialysis, 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 at a dose of 2 μg / mouse by tail vein injection, and small animal in - vivo fluorescence imaging (IVIS Lumina III, PE company) was performed 6 hours after administration. Then the mice were immediately euthanized, and the main organs of the mice such as the heart, liver, spleen, lung, and kidney were imaged. The test results are as Figure 4 shown.

[0309] Based on the above analysis, the lipid nanoparticles prepared from cationic compounds 8, 9, and 11 of the present invention have obvious lung targeting ability; in terms of structure, the two tails of cationic compounds 8 and 9 have the same structure, but the number of carbons on the N-containing chain of the head is different. The two tails of cationic compounds 8 and 11 have similar structures, differing only by one alkene, and the number of carbons on the N-containing chain of the head is the same. However, compounds with other structures have high hemolytic activity due to more branches in the tail and a large number of alkenes in the tail, and basically have no targeting effect. Among all cationic compounds, the best targeting effect is shown by cationic compound 11.

[0310] Among all cationic compounds, the best targeting effect is shown by cationic compound 11. Therefore, we chose to use cationic compound 11 and compare it with the commercial ionizable lipid compound SM102, MC3, and the commercial cationic compound DOTAP. 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 at a dose of 10 μg / mouse by tail vein injection for safety testing and cytotoxicity testing of the lipid nanoparticles. The cytotoxicity experiment evaluated the cytotoxicity of the lipid nanoparticles through a commercially available cell proliferation detection kit (MTS, Promega). 40,000 293T cells / well were seeded in a 96-well plate and cultured overnight. The 293T cells were transfected with Luciferase mRNA lipid nanoparticles at a dose of 0.2 μg mRNA per well. After 24 hours of transfection, the old medium was removed and replaced with a new medium containing MTS. After incubating in the incubator for about 2 hours, the absorbance at 490 nm was measured using a TACAN (SPARK) microplate reader. The test results are shown in Figure 5 , and small animal in vivo fluorescence imaging (IVIS Lumina III, PE company) was performed 6 hours after administration. Then the mice were immediately euthanized, and the main organs such as the heart, liver, spleen, lungs, and kidneys of the mice were imaged. The test results are as shown in Figure 6 , the fluorescence intensity expression in the lungs is as shown in Figure 7 , and the proportion distribution of the lipid nanoparticles of Examples 23, 26, 27, 28, 29, and 30 in each organ is shown in Figure 8 . By Figure 5 , it can be seen that when cationic compound 11 is paired with any one of the ionizable lipid compounds 1-126, MC3, and SM102, the hemolysis at pH 7.4 is lower than that of the pairing of DOTAP and the ionizable lipid compound, showing better safety. There is no significant difference in cytotoxicity and the toxicity is low. By Figure 6 , 7As can be seen from 8, the cationic compound 11 exhibits better lung targeting effect when paired with MC3, comparable targeting effect when paired with SM102, and the cationic compound 11 shows a better lung occupancy ratio.

[0311] The above-described embodiments and / or implementation manners are merely used to illustrate the preferred embodiments and / or implementation manners for implementing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0312] Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of language expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, embellishments or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, embellishments, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A cationic compound, Select any one of the following: Compound 6: ; Compound 8: ; Compound 9: ; Compound 11: .

2. A lipid complex comprising: The cationic compound according to any one of claims 1, The lipid complex also includes an ionizable lipid compound, which is 1-126 , D-Lin-MC3-DMA, SM102, ALC-0315, 5A2-SC8, CKK-E12; The lipid complex further includes at least one of phospholipids, structural lipids, and PEGylated lipids.

3. A lipid complex according to claim 2, characterized in that: The lipid complex also includes a therapeutic or prophylactic agent.

4. A method for preparing a lipid complex according to any one of claims 2 to 3, comprising: Step (1): dissolving a lipid material in an organic solvent to obtain an organic phase solution; the lipid material comprises the cationic compound according to claim 1, an ionizable lipid compound, and at least one of a phospholipid, a structural lipid, and a pegylated lipid; 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.

5. The method for preparing a lipid complex according to claim 4, characterized in that: The aqueous medium includes a therapeutic or prophylactic agent.

6. The use of the cationic compound according to claim 1, selected from 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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