Lipid molecules containing near-infrared fluorescent indicator and application thereof

Abstract

The invention discloses lipid molecules containing a near-infrared fluorescent tracer agent and application of the lipid molecules, and belongs to the field of fine chemical engineering. According to the series of molecules, near-infrared fluorescent dye with a fluorescent tracing effect is connected with lipid molecules, and the series of molecules belong to a class of novel bifunctional material molecules. The molecule contains a lipid side chain, so that the lipid molecule has the performance that the lipid molecule is combined with RNA / DNA and other base sequences and is self-assembled into nanoparticles, and the lipid molecule becomes a high-quality RNA / DNA delivery carrier material; and the fluorescent dye part shows strong NIR fluorescence, and the emission wavelength can reach 710 nm. Excellent imaging performance can be obtained in three cell line models: Hela, A549 and Raw cells. The whole process change of endosome escape of various cells can be traced with an accurate and good imaging effect, and an effective result associated with mRNA delivery and protein expression can be provided. We predict that we new methods would contribute to the development and optimization of various mRNA delivery vectors.

Description

Technical Field

[0001] The present invention relates to a class of lipid nanomaterials with fluorescent dyes connected to lipid small molecules to achieve a tracing effect, and at the same time capable of delivering nucleic acid drugs such as DNA or RNA to living cells and living bodies, and belongs to the field of fine chemicals. Background Art

[0002] Gene therapy is a method of treating disease by delivering genes into the patient's body as drugs, but this is limited by the fact that gene drugs are easily degraded by biological enzymes. Lipid nanoparticles have attracted attention due to their high delivery efficiency and good biocompatibility. Despite extensive research, more current research focuses on improving delivery efficiency and targeting, resulting in limitations in their effective transport and therapeutic efficacy. Studies have shown that endosomal escape is a key step in the therapeutic efficacy of drug delivery systems and one of the most important obstacles to improving therapeutic efficiency, but we still know little about the mechanisms that control this process.

[0003] Fluorescence tracing technology uses fluorescent substances to label cells and track and visualize their dynamic changes in real time by detecting fluorescent signals. Fluorescent probes are widely used in many fields due to their high sensitivity, specificity, and non-destructive nature. Despite these advantages, there is still a problem of limited penetration depth. Fluorescent signals are easily absorbed and scattered by biological tissues, resulting in limited penetration depth. There is an urgent need to develop near-infrared fluorescent probes (NIR) and to achieve the necessary detection requirements by introducing fluorescent groups on lipid small molecules.

[0004] For these reasons, the development of lipid nanoparticles linked to near-infrared fluorescent dyes for tracer purposes shows great potential. By incorporating tracers into lipid nanoparticles, these nanoparticles enable visualization and tracking of the distribution, metabolism, and mechanism of action of drugs or gene vectors within organisms, providing a deeper understanding of related research. This work is of great significance, potentially resolving many challenges currently associated with improving the delivery efficiency of lipid nanoparticles and possessing high practical implications. Summary of the Invention

[0005] To achieve the fusion of drug delivery and particle tracking, the present invention provides a series of functionalized lipid molecules with high biocompatibility. The technical solution adopted by the present invention is to provide a compound of formula Q having the following general structure:

[0006]

[0007] Among them, R1 is independent of each other. R2 is independent of each other

[0008] R3 is independent of each other Among them, X1 - is an anion, and the total negative charge carried by the anion is equal to the total positive charge carried by the nitrogen-containing groups in the R3 structure.

[0009] In some embodiments, R4 are independently Among them, R5 is independent of each other.

[0010] In some embodiments, R4 is independently Among them, R5 is independent of each other.

[0011] The method for preparing the lipid molecules connected with dye groups has the following reaction formula and reaction steps:

[0012] i) Under argon conditions, compound A, Pd(OAc)2, BINAP, Cs2CO3 and HR1 are heated in an organic solvent to react to obtain compound B.

[0013] ii) removing water with a water separator, and heating the mixture of compound B, TsOH and R6 to react to obtain compound C.

[0014] iii) Compound C was added to an organic solvent at room temperature, and NBS was added in batches. The mixture was stirred for reaction, and compound D was obtained by separation and purification.

[0015] iv) Under argon protection, compound D was dissolved in an organic solvent, tert-butyl lithium was added, and the mixture was stirred for 0.5 h. The temperature was raised to Dissolve in an organic solvent and add dropwise to the above reaction solution. Warm to room temperature for reaction. Dilute the reaction mixture with saturated NH4Cl and water, and extract with EtOAc. Wash the organic phase with brine, dry and concentrate. Add methanol and R7 to the concentrate and stir at room temperature. Remove the solvent under reduced pressure and purify to obtain compound E.

[0016] The R6 is

[0017] The R7 is HBF4, HCl, HBr, HI, HNO3, H2SO4, HClO4, CH3COOH, CH3SO3H or CF3SO3H.

[0018] The definitions of R1, R2, and R3 are the same as those in the general structural formula.

[0019] The organic solvent in step i is one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dioxane, nitrogen methyl pyrrolidone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphosphoramide, sulfolane, acetonitrile and benzonitrile, and the reaction temperature is 90-180° C.; the reaction temperature in step ii is 130-170° C., and the organic solvent in step iii is one of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, tetrahydrofuran, dioxane, nitrogen methyl pyrrolidone, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, hexamethylphosphoramide, sulfolane, acetonitrile and benzonitrile.

[0020]

[0021] i) The compound was dissolved in tetrahydrofuran, and F1 and NaBH(AcO)3 were added under stirring to react at room temperature. The solvent was evaporated under reduced pressure, and the compound G was obtained by separation and purification.

[0022] ii) Compound G was dissolved in CH2Cl2, triethylamine and DMAP were added, and a solution of p-toluenesulfonyl chloride in dichloromethane was added dropwise with stirring at 0°C. The solvent was evaporated under reduced pressure, and the mixture was redissolved in DMF. NaN3 was added and reacted at 80°C. The mixture was washed and dried, and the solvent was evaporated under reduced pressure. Compound H was isolated and purified.

[0023] The definition of R4 is the same as that in the general structural formula.

[0024]

[0025] i) Under argon protection, compound H, compound E, VcNa and copper sulfate were dissolved in a solvent, stirred at room temperature, washed with water, and the solvent was evaporated under reduced pressure to obtain compound Q through separation and purification.

[0026] The definition of R4 is the same as that in the general structural formula.

[0027] The compound is used in preparing nanomaterials for delivering nucleic acid drugs.

[0028] The compound is used in the preparation of near-infrared fluorescent dyes.

[0029] The compound is used in the preparation of a dual-mode synergistic therapeutic drug for gene therapy and fluorescence tracing.

[0030] The beneficial effects of the present invention are: this series of molecules combines the alkyne group of the near-infrared fluorescent dye with the azide of the lipid molecule through an efficient click reaction, and belongs to a new type of bifunctional material molecule. This series of materials can realize the delivery of gene drugs such as RNA / DNA, and achieve visualization through the luminescence of the fluorescent dye under the excitation of a light source of a specific wavelength. The number of carbons between the drug molecule and the amino core can adjust the overall hydrophilicity of the molecule, which will affect the binding ability of the lipid molecule to the base sequence. The advantages of this series of designs are: on the one hand, the protection and delivery of mRNA can be achieved based on lipid nanoparticles, and it has been verified that drug delivery can be achieved in a variety of target cell lines; on the other hand, the dye connected to the lipid molecule can emit fluorescence under laser irradiation to achieve a tracing effect. This design is intended to provide support for in-depth research on different cell delivery links. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a test chart of the Q1 spectral properties of lipid molecules.

[0032] Figure 2 This is a test chart of the spectral properties of lipid molecules Q1-nanoparticles.

[0033] Figure 3 Confocal imaging of Q1-lipid nanoparticles delivering eGFP mRNA in HeLa cells and monitoring endosomal escape.

[0034] Figure 4 Confocal imaging of Q1-lipid nanoparticles delivering eGFP mRNA in A549 cells and monitoring endosomal escape.

[0035] Figure 5 Confocal imaging of Q1-lipid nanoparticles delivering eGFP mRNA in Raw cells and monitoring endosomal escape. DETAILED DESCRIPTION

[0036] To make the technical solution of the present invention clearer, the following detailed description of the specific embodiments of the present invention is given in conjunction with the technical solution and the accompanying drawings. The present invention is illustrated by the following examples but is not limited thereto. Unless otherwise specified, all parts and percentages are by weight.

[0037] The specific embodiments of the present invention are described in detail below in conjunction with the technical solutions:

[0038] Example 1

[0039]

[0040] Under argon, a mixture of compound A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to afford B1 as a pale yellow solid.

[0041] A mixture of B1 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, the solvent removed by rotary evaporation, and purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C1 as a light yellow solid.

[0042] C1 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction solution was diluted with dichloromethane and washed with water. The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D1 as a yellow solid.

[0043] Under argon at -78°C, D1 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain a blue-green solid E1.

[0044] 2 g of the starting compound F (16.9 mmol) was dissolved in 50 mL of tetrahydrofuran, and 13.6 g of compound G1 and NaBH(AcO)3 (84.6 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H1.

[0045] Dissolve 1.5 g of compound H1 in 10 mL of dry CH2Cl2, then add 0.7 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 700 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 300 mg of NaN3. The reaction is continued at 80°C for 5 h. After the reaction, the organic phase, which has been dissolved in a large amount of dichloromethane, is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography to obtain compound K1.

[0046] Under argon, 10 mg of compound K1, 53 mg of compound E1, 10 mg of VcNa, and 10 mg of copper sulfate were dissolved in a mixture of 4 mL of tetrahydrofuran and 2 mL of water. The mixture was stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent removed by rotary evaporation. The target compound was then purified by silica gel column chromatography. The product structure was confirmed by mass spectrometry.

[0047] Example 2

[0048]

[0049] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling with argon (to remove oxygen) for 20 minutes and reacted at 110°C. TLC monitoring was performed until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to obtain a light yellow solid B2.

[0050] A mixture of B2 (0.94 mmol), ethanedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C2 as a light yellow solid.

[0051] C2 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D2 as a yellow solid.

[0052] Under argon at -78°C, D2 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E2.

[0053] 2 g of the starting compound F (16.9 mmol) was dissolved in 50 mL of tetrahydrofuran, and 14 g of compound G2 and NaBH(AcO)3 (84.6 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H2.

[0054] Dissolve 1.5 g of compound H2 in 10 mL of dry CH2Cl2, then add 0.7 mL of triethylamine and a catalytic amount of DMAP. Add dropwise 10 mL of a dichloromethane solution containing 665 mg of p-toluenesulfonyl chloride with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 270 mg of NaN3. The reaction is continued at 80°C for 5 h. After the reaction, the organic phase, which has been dissolved with a large amount of dichloromethane, is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography to obtain compound K2.

[0055] 10 mg of compound K2, 53 mg of compound E2, 10 mg of VcNa and 10 mg of copper sulfate were dissolved in a mixed solvent of 4 mL of tetrahydrofuran and 2 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0056] Example 3

[0057]

[0058] Under argon, a mixture of A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and cyclohexylamine (1.4 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to afford B3 as a pale yellow solid.

[0059] A mixture of B2 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C3 as a light yellow solid.

[0060] C3 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D3 as a yellow solid.

[0061] Under argon at -78°C, D3 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E3.

[0062] 2 g of the starting compound F (16.9 mmol) was dissolved in 50 mL of tetrahydrofuran, and 13.6 g of compound G3 and NaBH(AcO)3 (84.6 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H3.

[0063] Dissolve 1.5 g of compound H3 in 10 mL of dry CH2Cl2, then add 0.7 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 700 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 250 mg of NaN3. The reaction is continued at 80°C for 5 h. After the reaction, the organic phase, which has been dissolved in a large amount of dichloromethane, is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography to obtain compound K3.

[0064] Under argon, 10 mg of compound K3, 53 mg of compound E1, 10 mg of VcNa, and 10 mg of copper sulfate were dissolved in a mixture of 4 mL of tetrahydrofuran and 2 mL of water. The mixture was stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent removed by rotary evaporation. The target compound was then purified by silica gel column chromatography. The product structure was confirmed by mass spectrometry.

[0065] Example 4

[0066]

[0067] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes and reacted at 110°C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B4 as a light yellow solid.

[0068] A mixture of B4 (0.94 mmol), ethanedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The water was then removed. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C4 as a light yellow solid.

[0069] C4 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D4 as a yellow solid.

[0070] Under argon at -78°C, D4 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E4.

[0071] 2 g of the starting compound F (16.9 mmol) was dissolved in 50 mL of tetrahydrofuran, and 12.6 g of compound G4 and NaBH(AcO)3 (84.6 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H4.

[0072] Dissolve 1.5 g of compound H4 in 10 mL of dry CH2Cl2, then add 0.7 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 700 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 240 mg of NaN3. The reaction is continued at 80°C for 5 h. After the reaction, the organic phase, which has been dissolved with a large amount of dichloromethane, is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography to obtain compound K4.

[0073] 10 mg of compound K4, 53 mg of compound E4, 10 mg of VcNa and 10 mg of copper sulfate were dissolved in a mixed solvent of 4 mL of tetrahydrofuran and 2 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0074] Example 5

[0075]

[0076] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 minutes and reacted at 110°C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B5 as a light yellow solid.

[0077] A mixture of B5 (0.94 mmol), 3-hydroxypropanethiol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The water was then removed. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C5 as a light yellow solid.

[0078] C5 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D5 as a yellow solid.

[0079] Under argon at -78°C, D5 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The mixture was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E5.

[0080] 1.5 g of the starting compound F was dissolved in 50 mL of tetrahydrofuran, and 16.8 g of compound G5 and NaBH(AcO)3 (63.46 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H5.

[0081] 2.35 g of compound H5 was dissolved in 20 mL of dry CH2Cl2, followed by the addition of 550 mg of triethylamine and a catalytic amount of DMAP. A 10 mL dichloromethane solution containing 778 mg of p-toluenesulfonyl chloride was added dropwise with stirring at 0°C. After the reaction, the solvent was evaporated under reduced pressure, redissolved in 20 mL of DMF, and 353.83 mg of NaN3 was added. The reaction was continued at 80°C for 5 h. After the reaction, the organic phase, which had been dissolved in a large amount of dichloromethane, was washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound K5.

[0082] 10 mg of compound K5, 75 mg of compound E5, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 2 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0083] Example 6

[0084]

[0085] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling argon (to remove oxygen) for 20 minutes and reacted at 110°C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B6 as a light yellow solid.

[0086] A mixture of B6 (0.94 mmol), 2-hydroxyethanethiol (2.1 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C6 as a light yellow solid.

[0087] C6 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D6 as a yellow solid.

[0088] Under argon at -78°C, D6 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E6.

[0089] 1.5 g of the starting compound F was dissolved in 50 mL of tetrahydrofuran, and 16.8 g of compound A8 and NaBH(AcO)3 (63.46 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H6.

[0090] Dissolve 1.5 g of compound H6 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add dropwise 10 mL of a dichloromethane solution containing 496.7 mg of p-toluenesulfonyl chloride with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 226 mg of NaN3. The reaction is continued at 80°C for 5 h. After the reaction, the organic phase, which has been dissolved with a large amount of dichloromethane, is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the solvent. The crude product is purified by silica gel column chromatography to obtain compound K6.

[0091] 10 mg of compound K6, 70 mg of compound E6, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0092] Example 7

[0093]

[0094] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling argon (to deoxygenate) for 20 minutes and reacted at 110°C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B7 as a light yellow solid.

[0095] A mixture of B7 (0.94 mmol), ethanedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The water was then removed. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C7 as a light yellow solid.

[0096] C7 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the NBS, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D7 as a yellow solid.

[0097] Under argon at -78°C, D7 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E7.

[0098] 175.6 mg of the starting compound F was dissolved in 10 mL of tetrahydrofuran solution, 202.76 mg of triethylamine was added, and the mixture was reacted at room temperature for 30 min. 792.45 mg of compound G7 and NaBH(OAc)3 (5.01 mmol) were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H7.

[0099] Dissolve 1.5 g of compound H7 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add dropwise 10 mL of a dichloromethane solution containing 666.1 mg of p-toluenesulfonyl chloride with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 302.8 mg of NaN3. The mixture is allowed to react at 80°C for 5 h. After the reaction, the organic phase is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound K7.

[0100] 10 mg of compound K7, 54.6 mg of compound E7, 5 mg of VcNa and 5 mg of copper sulfate were dissolved in a mixed solvent of 4 mL of tetrahydrofuran and 2 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0101] Example 8

[0102]

[0103] Under argon protection, A (2.97 mmol), Pd (OAc) 2 (0.45 mmol), BINAP (0.32 mmol), Cs 2 CO 3 (8.62 mmol) and The mixture (1.5 mL) was dissolved in toluene bubbling with argon (to remove oxygen) for 20 minutes and reacted at 110°C, monitored by TLC until the reaction was complete. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B8 as a light yellow solid.

[0104] A mixture of B8 (0.94 mmol), ethanedithiol (2.2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C8 as a light yellow solid.

[0105] C8 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D8 as a yellow solid.

[0106] Under argon at -78°C, D8 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E8.

[0107] 70.5 mg of the starting compound F was dissolved in 5 mL of tetrahydrofuran solution, 81.4 mg of triethylamine was added, and the mixture was reacted at room temperature for 30 min. 515.62 mg of compound G8 and NaBH(OAc)3 (2.01 mmol) were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H8.

[0108] Dissolve 1.5 g of compound H8 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 471 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 215 mg of NaN3. The mixture reacts at 80°C for 5 h. After the reaction, the organic phase is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound K8.

[0109] 10 mg of compound K8, 73 mg of compound E8, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0110] Example 9

[0111]

[0112] Under argon, a mixture of compound A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and pyrrolidine (1.2 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to afford B9 as a pale yellow solid.

[0113] A mixture of B9 (0.94 mmol), ethylene glycol (2 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C9 as a light yellow solid.

[0114] C9 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between additions. After the addition of all the NBS, the reaction mixture was allowed to react for 60 minutes. The reaction solution was diluted with dichloromethane and washed with water. The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D9 as a yellow solid.

[0115] Under argon at -78°C, D9 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to give a blue-green solid E9.

[0116] 70.5 mg of compound F was dissolved in 5 mL of tetrahydrofuran solution, 81.4 mg of triethylamine was added, and the mixture was reacted at room temperature for 30 min. 515.62 mg of compound G9 and NaBH(OAc)3 (2.01 mmol) were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H9.

[0117] Dissolve 1.5 g of compound H9 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 471 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 215 mg of NaN3. The mixture reacts at 80°C for 5 h. After the reaction, the organic phase is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound K9.

[0118] 10 mg of compound K9, 77 mg of compound E9, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0119] Example 10

[0120]

[0121] Under argon, a mixture of A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and methylamine (1.5 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to afford B10 as a pale yellow solid.

[0122] A mixture of B10 (0.94 mmol), ethylene glycol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The mixture was then separated. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and C10 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0123] C10 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the NBS, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D10 as a yellow solid.

[0124] Under argon at -78°C, D10 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E10.

[0125] 1.5 g of the starting compound F (14.4 mmol) was dissolved in 50 mL of tetrahydrofuran, and 20.48 g of the compound G10 and NaBH(AcO)3 (72.01 mmol) were added with stirring. The mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H10.

[0126] 1.26 g of compound H10 was dissolved in 10 mL of dry CH2Cl2, followed by the addition of 280.39 mg of triethylamine and a catalytic amount of DMAP. A 10 mL dichloromethane solution containing 396.17 mg of p-toluenesulfonyl chloride was added dropwise with stirring at 0°C. After the reaction, the solvent was evaporated under reduced pressure, redissolved in 10 mL of DMF, and 180.13 mg of NaN3 was added. The reaction was continued at 80°C for 5 h. After the reaction, the organic phase was washed with multiple small 20 mL portions of saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound K10.

[0127] 10 mg of compound K10, 73 mg of compound E10, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0128] Example 11

[0129]

[0130] Under argon, a mixture of A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and thiomorpholine (1.5 mL) was dissolved in toluene bubbled with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and purification by column chromatography (ethyl acetate:petroleum ether = 1:25) afforded B11 as a pale yellow solid.

[0131] A mixture of B11 (0.94 mmol), propylene glycol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The water was then removed. When TLC indicated the complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and the mixture was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford C11 as a light yellow solid.

[0132] C11 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the ingredients, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D11 as a yellow solid.

[0133] Under argon at -78°C, D11 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E11.

[0134] 220 mg of compound F was dissolved in 10 mL of tetrahydrofuran, and an appropriate amount of triethylamine was added to adjust the pH of the solution to neutral. The mixture was reacted at room temperature for 30 min. 1.86 g of compound G11 and NaBH(AcO)3 (6.28 mmol) were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H11.

[0135] Dissolve 1.5 g of compound H11 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 450 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 200 mg of NaN3. The mixture reacts at 80°C for 5 h. After the reaction, the organic phase is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound K11.

[0136] 10 mg of compound K11, 73 mg of compound E11, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0137] Example 12

[0138]

[0139] Under argon, a mixture of A (2.97 mmol), Pd(OAc)2 (0.45 mmol), BINAP (0.32 mmol), Cs2CO3 (8.62 mmol), and thiomorpholine dioxide (1.5 mL) was dissolved in toluene bubbling with argon (to deoxygenate) for 20 min. The reaction was allowed to proceed at 110°C and monitored by TLC until completion. After cooling to room temperature, the solid was removed by filtration, the solvent was removed by rotary evaporation, and the product was purified by column chromatography (ethyl acetate:petroleum ether = 1:25) to afford B12 as a pale yellow solid.

[0140] A mixture of B12 (0.94 mmol), heptanediol (2.3 mL), and TsOH (0.29 mmol) was dissolved in toluene and reacted at 110°C for 4 h and then at 150°C for 12 h. The water was then removed. When TLC indicated complete disappearance of the starting material, the mixture was washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and C12 was purified by column chromatography (ethyl acetate / petroleum ether = 20 / 1) to afford a light yellow solid.

[0141] C12 (0.91 mmol) was dissolved in N,N-dimethylformamide at room temperature and stirred until completely dissolved. NBS (1.82 mmol) was added to the reaction mixture in five portions, with 5-minute intervals between each addition. After the addition of all the NBS, the mixture was allowed to react for 60 minutes. The reaction mixture was diluted with dichloromethane, washed with water, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. Purification by column chromatography (dichloromethane:petroleum ether = 2:1) afforded D12 as a yellow solid.

[0142] Under argon at -78°C, D12 (0.2 g, 0.38 mmol) was dissolved in ultra-dry tetrahydrofuran (30 mL). Tert-butyl lithium (1.3 M) (1.6 mL, 1.71 mmol) was added dropwise, and the reaction was stirred at -78°C for 30 min. The reaction mixture was cooled to -20°C, and W (0.24 g, 0.95 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred for 12 h. The product was quenched with saturated ammonium chloride solution, extracted with dichloromethane (30 mL x 3), washed with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The remaining solid was dissolved in methanol (3 mL), and glacial acetic acid (100 uL) was added. After stirring at room temperature for 10 min, the reaction solution was purified by column chromatography (dichloromethane:methanol = 10:1) to yield a blue-green solid (120 mg, 56%). The blue-green solid (35 mg, 0.06 mmol) was dissolved in 4 mL of methanol, potassium hydroxide (20 mg) was added and stirred at room temperature overnight. The reaction solution was evaporated to remove the solvent and purified by column chromatography (dichloromethane: methanol = 10:1) to obtain E12.

[0143] 220 mg of compound F was dissolved in 10 mL of tetrahydrofuran, and an appropriate amount of triethylamine was added to adjust the pH of the solution to neutral. The mixture was reacted at room temperature for 30 min. 1.86 g of compound G12 and NaBH(AcO)3 (6.28 mmol) were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound H12.

[0144] Dissolve 1.5 g of compound H12 in 10 mL of dry CH2Cl2, then add 0.5 mL of triethylamine and a catalytic amount of DMAP. Add 10 mL of a dichloromethane solution containing 450 mg of p-toluenesulfonyl chloride dropwise with stirring at 0°C. After the reaction, evaporate the solvent under reduced pressure, redissolve the mixture in 10 mL of DMF, and then add 200 mg of NaN3. The mixture reacts at 80°C for 5 h. After the reaction, the organic phase is washed with multiple small amounts of 20 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound K12.

[0145] 10 mg of compound K12, 73 mg of compound E12, 15 mg of VcNa and 15 mg of copper sulfate were dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 4 mL of water under argon protection, stirred at room temperature for 30 min, washed with water, extracted with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The target compound was separated and purified by silica gel chromatography.

[0146] Example 13

[0147]

[0148] Example 14

[0149]

[0150] Example 15

[0151] Q1 spectral property test of lipid molecules:

[0152] The photophysical properties of compound Q1 were characterized by HP 8435-UV 3100 UV-visible spectrophotometer and PTI-700 fluorescence spectrophotometer. The absorption and emission spectra of Q1 in different solvents are shown in Figure 2. Figure 1 As shown, the sample concentration was 5 μmol / L.

[0153] like Figure 1 As shown, the absorption range of Q1 is 600-720nm, with an absorption peak at around 675nm, and the emission range is 660-800nm, with an emission peak at around 710nm. It has good absorption and emission intensities in the near-infrared region and can be used as a near-infrared fluorescent dye with good properties in subsequent experimental research.

[0154] In addition to studying the photophysical properties of Q1, the photophysical properties of Q1-constructed nanoparticles in aqueous solutions at varying pH values were further investigated to explore their potential mechanisms in different physiological environments. The preparation method was as follows: 2 mg / mL G1 lipid molecules, 2 mg / mL phospholipids, 2 mg / mL cholesterol, and 0.4 mg / mL polyethylene glycol were mixed in an ethanolic solution at a molar ratio of 20:30:40:0.75 to form an ethanol phase and thoroughly mixed. Calf thymus DNA (Ct-DNA) was dissolved in 10 mmol / L sodium citrate buffer (pH 3) to prepare a DNA premix at a DNA concentration of 0.004 μg / μL. The DNA premix was added to the ethanol phase (1:1 volume ratio) and thoroughly mixed. The Q1-lipid nanoparticle stock solution was then diluted with an equal volume of phosphate buffer to obtain the Q1-lipid nanoparticle stock solution.

[0155] like Figure 2 As shown, the Q1-lipid nanoparticles exhibited a complete and regular absorption peak pattern in PBS buffer solution, producing stable emission light without aggregated particles that would otherwise cause fluorescence quenching. This suggests that in the acidic environment of a simulated lysosome, the lipid nanoparticles exhibited absorption and emission similar to those of a monomeric structure, and that they dissociated within the lysosomal membrane to release the material, thereby exerting their corresponding function.

[0156] Example 16

[0157] Assays to monitor Q1-lipid nanoparticle delivery of eGFP mRNA and track endosomal escape:

[0158] An ethanol phase was prepared by mixing 2 mg / mL Q1 lipid molecules, 2 mg / mL phospholipids, 2 mg / mL cholesterol, and 0.4 mg / mL polyethylene glycol in an ethanolic solution at a molar ratio of 20:30:40:0.75. The mixture was then thoroughly mixed. A green fluorescent protein messenger RNA (eGFP mRNA) premix was prepared by diluting it with 10 mmol / L sodium citrate buffer (pH 3) to a concentration of 0.004 μg / μL. The mRNA premix was added to the ethanol phase (v / v = 1:1) and thoroughly mixed. The resulting solution was then diluted with an equal volume of phosphate buffered saline (PBS) to obtain a Q1 lipid nanoparticle stock solution.

[0159] 24 hours before treatment, Hela, A549, and Raw cells were cultured at 8×10 4 The cells were cultured at a density of 1 mL in confocal culture dishes, with the volume of MEM medium for Hela cells being 1 mL. This medium contained 10% (V / V) FBS and 1% penicillin / streptomycin. The volume of DMEM medium for Raw and A549 cells was 1 mL. This medium contained 10% (V / V) FBS and 1% penicillin / streptomycin. All cells were cultured in a cell culture incubator at 37°C and 5% CO2. Three types of cells (passages between 2 and 30, passaged 1 to 4 times after recovery) were cultured on the cell culture dishes for 24 hours before use. The cells were stimulated with Q1 lipid nanoparticles encapsulating 2.5 μg eGFP mRNA for 1, 3, 6, and 18 hours, respectively, for a total of 12 groups with four different culture time periods. The culture medium was not removed and no washing was required. Green fluorescent protein expression was directly detected using a two-photon laser confocal microscope (Olympus FV1000), with the eGFP excitation wavelength set to 488 nm, the RAL excitation wavelength set to 635 nm, and the detector wavelengths set to 500–550 nm and 680–760 nm. Images were processed using FV10-ASW Viewer software.

[0160] The results of Hela cell experiments are as follows Figure 3As shown. Using the eGFP green channel, a clear green fluorescence signal can be observed inside the cells of the Q1-lipid nanoparticle-treated group, with a distinct green diffuse fluorescence distributed throughout the cytoplasm at 6 and 18 hours. This experimental result demonstrates that lipid nanoparticles can deliver eGFP mRNA into living cells and express a certain amount of enhanced green fluorescent protein (EGFP). Using the RAL red channel, after 1 and 3 hours of incubation, the internalized particles are primarily localized in endosomes, with a predominantly spherical shape and a small number of thin stripes. As the co-incubation time increases, the proportion of thin stripes increases, becoming predominantly thin stripes with a small number of spherical shapes. After 18 hours of incubation with cells, the particles escape from the endosomes, with the vast majority of thin stripes visible in confocal images, indicating that the particles facilitate the release of mRNA into the cytoplasm. This experimental result demonstrates that compound Q1-lipid nanoparticles can accurately monitor the distribution of mRNA within living cells, using dye luminescence as a tracer. By analyzing the two channels together, we can draw a preliminary conclusion: when the degree of endosomal escape increases, the protein expression of the cell increases accordingly.

[0161] The results of the A549 cell experiment are as follows Figure 4 As shown. Through the EGFP green channel, it can be seen that green fluorescence signals can be observed inside the cells of the particle-treated group, but there is no green diffuse fluorescence distribution in the cell cavity. This experimental result shows that lipid-based nanoparticles can deliver eGFP mRNA into living cells, but do not express a certain amount of enhanced green fluorescent protein. Then observe the RAL red channel. It can be seen that the red channel intensity is weak at 1 hour and increases at 3 hours. As the culture time increases to 6 hours, the intensity increases significantly. The distribution is mainly spherical, including a small number of non-spherical filaments. Because A549 cells are small, the distribution of organelles under the confocal microscope is not clear. After the incubation time is extended to 18 hours, the proportion of filamentous distribution increases. This experimental result shows that Q1-lipid nanoparticles can monitor the distribution in living cells with a certain accuracy and play a tracing role through the luminescence of the dye.

[0162] Raw cell experiment results are as follows Figure 5 As shown in the figure. Using the eGFP green channel, green fluorescence can be observed within the cells of the particle-treated group, but there is no diffuse green fluorescence distribution within the cell lumen. This experimental result indicates that the lipid nanoparticles based on Compound Q1 can deliver low levels of eGFP mRNA into living cells, but do not express a certain amount of enhanced green fluorescent protein. Observing the red channel, the red channel intensity is stronger at 1 hour and is stronger than that of the two cells mentioned above. The intensity continues to increase with increasing culture time.

[0163] In summary, Q1 lipid nanoparticles can protect mRNA, improve transfection efficiency, and link dyes to act as tracers.

Claims

1. A compound of formula Q or a salt thereof: in, R1 is independent of each other R2 is independent of each other R3 is independent of each other Among them, X1 - is an anion, the total negative charge of the anion is equal to the total positive charge of the nitrogen-containing groups in the R3 structure; R4 is independent of each other C9-C 12 Saturated alkane or C 16 -C 18 wherein R5 is independently a C6-C8 saturated alkane group or a C8-C9 unsaturated alkane group, and p is independently an integer of 1-18.

2. The compound or salt thereof according to claim 1, characterized in that: R1 is independent of each other 3. The compound or salt thereof according to claim 2, characterized in that: R2 is independent of each other 4. The compound or salt thereof according to claim 3, characterized in that: R3 is independent of each other Among them, X1 - It is an anion, and the total negative charge carried by the anion is equal to the total positive charge carried by the nitrogen-containing groups in the R3 structure.

5. The compound or salt thereof according to claim 4, characterized in that: R4 each independent Among them, R5 is independent of each other.

6. The compound or salt thereof according to claim 5, characterized in that: R4 is independent of each other Among them, R5 is independent of each other.

7. A pharmaceutical composition, characterized in that The invention comprises the compound or salt thereof according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 8, characterized in that It also includes carriers and / or pharmaceutical excipients.

9. Use of the compound according to any one of claims 1 to 6 in the preparation of nanomaterials for delivering nucleic acids.

10. Use of the compound according to any one of claims 1 to 6 in the preparation of materials containing fluorescent tracer effects.

11. Use of the compound according to any one of claims 1 to 6 in dual-mode synergistic therapeutic drugs for drug delivery and fluorescence tracing.

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