CN110283223B

CN110283223B - Cationic lipid molecule and application thereof in nucleic acid delivery

Info

Publication number: CN110283223B
Application number: CN201810225566.2A
Authority: CN
Inventors: 巫林平; 张必良; 宋伟; 王玮
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Guangzhou Ribobio Co ltd
Priority date: 2018-03-19
Filing date: 2018-03-19
Publication date: 2022-01-04
Anticipated expiration: 2038-03-19
Also published as: CN110283223A

Abstract

The invention discloses a cationic lipid molecule and application thereof in nucleic acid delivery. The structural formula of the cationic lipid molecule is shown in a formula i, and the invention also provides a cationic liposome, a lipid complex, a reagent, a kit, a preparation and a pharmaceutical composition on the basis of the cationic lipid molecule. The cationic lipid molecule provided by the invention has simple synthesis process and good stability, and the cationic liposome has high efficiency (can be reflected as high transfection efficiency) and low toxicity; meanwhile, the product is stable and uniform and is easy to prepare; can be used for transfection of various cell lines. Therefore, the siRNA has excellent transmissibility, and can deliver the active substances (such as the exemplified siRNA) to cells (such as the exemplified lung cancer cells), tissues and organs with high efficiency to realize the high-efficiency regulation and control of the active substances. Solves the problems of low toxicity and low transfer efficiency of the cationic liposome in the prior art.

Description

Cationic lipid molecule and application thereof in nucleic acid delivery

Technical Field

The invention relates to a cationic lipid molecule and application thereof in nucleic acid delivery, belonging to the field of nucleic acid delivery systems.

Background

The gene therapy is to introduce exogenous normal gene into target cell to correct or compensate diseases caused by gene deletion or abnormality, so as to achieve the aim of treatment. Gene silencing techniques are representative thereof. To achieve gene silencing, mediating molecules such as siRNA must enter the target cell and act. However, naked siRNA is easily degraded, with a half-life in plasma of less than one hour; and because of its very small size, it is rapidly expelled from the body by the kidneys during circulation; in addition, siRNA cannot cross cell membranes. Therefore, in order to achieve the gene silencing function of siRNA, an effective delivery system is required to protect siRNA from degradation, while increasing cellular uptake, etc.

Cationic liposomes are a very promising class of nucleic acid molecule delivery carrier materials, but conventional liposome synthesis usually requires individual optimization, and the steps are cumbersome, the synthesis cost is relatively difficult and expensive, often resulting in an over-price for the user. In addition, the existing commercial cationic liposome still faces the problems of safety and the challenge of high efficiency. Therefore, the development of novel cationic liposome with high efficiency, low toxicity, simple and feasible preparation and stable property is an urgent problem to be solved.

Disclosure of Invention

The invention aims to provide a novel cationic lipid molecule and application thereof in aspects of nucleic acid delivery and the like, and the provided cationic lipid molecule can be used for efficiently delivering active substances (such as siRNA) into cells (such as exemplified lung cancer cells), tissues and organs so as to realize efficient regulation and control of the active substances.

The structural formula of the cationic lipid molecule provided by the invention is shown as the formula i:

in formula i, A represents C_14-28Acyl or C_14-28An alkoxy group;

b represents

Or not, wherein j is an integer between 1 and 5, m is an integer between 1 and 4, and n is an integer between 2 and 4;

c represents

Wherein k is an integer between 2 and 4;

d represents

Wherein p is an integer between 1 and 4;

e represents

Wherein R is₁And R₂Independently represent C_1-4Alkyl or R₁And R₂Condensed to form an optionally substituted C_5-7Heterocyclyl, X is a chlorine, bromine or iodine atom.

In the formula

Represents a quaternary ammonium salt of a pyridine with substituents in different positions, X is a chlorine, bromine or iodine atom, which comprises

Specifically, the compound of formula i may have the following structure in table 1:

TABLE 1 structural formulas of formulas I-VII

Wherein A is C optionally interrupted by 0, 1 or more double bonds_14-28Acyl or C_25-27Alkoxy radical, C_14-28Acyl specifically includes, but is not limited to: myristoyl (C14: 1, cis-9), palmitoyl (C16: 1, cis-9), oleoyl (C18: 1, cis-9), arachidonyl (C20: 4, cis-5, 8, 11, 14), erucyl (C22: 1, cis-13), alkanoyl (C24: 1, cis-15), myristoyl, palmitoyl, stearoyl, eicosanoyl, docosanoyl, tetracosanoyl, hexacosanoyl or octacosanoyl.

The cationic lipid molecules provided by the present invention are specifically shown in table 2:

TABLE 2 structural formula of each cationic lipid molecule

The cationic lipid molecules provided by the present invention can be prepared by conventional methods in the prior art.

The invention further provides a cationic liposome which is prepared from the cationic lipid molecule or the lipid molecule and auxiliary lipid, wherein the auxiliary lipid is a neutral lipid molecule and/or a PEG lipid molecule;

preferably, the neutral lipid molecule may be DOPE; the PEG lipid molecule may be DSPE-PEG.

The cationic lipid molecules and the neutral lipid molecules may be mixed in an equimolar ratio;

the molar ratio of the cationic lipid molecules to the PEG lipid molecules may be 9: 1 to 2.

The cationic liposome is a nanoparticle with a net positive charge on the surface.

In the cationic liposome, the molar content of the cationic lipid molecules can be 30-70%, preferably 45-50%.

The cationic liposome has at least one of the characteristics (1) and (2):

(1) the particle size is as follows: 80-200 nm, 89-130 nm, 100-200nm, 80-120 nm, 80-100 nm or 80-89 nm;

(2) the surface potential is: 5-70 mv, 30-45mv, 5-45 mv or 5-30 mv.

The cationic liposome is uniform and stable, and can be used as a vector system, such as a gene vector and a transfection reagent.

The preparation method of the cationic liposome of the invention includes but is not limited to an ultrasonic method, a reverse phase evaporation method, an injection method, a freeze drying method, a high pressure homogenization method, a rotary evaporation method, an emulsification method and the like.

Preferably, the emulsification process is also filtered after the clear emulsion is obtained.

The present invention still further provides a lipid complex made of the cationic liposome and a negatively charged active substance (target to be loaded/delivered).

Preferably, the active substance refers to a substance having biological or pharmacological activity, and can be a nucleic acid molecule or a protein molecule;

when the nucleic acid molecule, the cationic liposome is linked to the N: the P ratio can be 20-50: 1.

the nucleic acid may comprise at least one modified nucleic acid analogue or a nucleotide polymer comprising a nucleic acid derivative.

The cationic liposome and the active substance form a lipid complex through electrostatic interaction of positive and negative charges. The lipid complex can form nanoparticles with the particle size of 80-600 nm dispersed in a water phase. The nanoparticles are uniform and stable.

The invention also provides a reagent, a kit, a preparation or a pharmaceutical composition, which comprises the cationic lipid molecule, the cationic liposome or the lipid complex.

The invention also provides application of the cationic lipid molecule, the cationic liposome, the lipid complex, the reagent, the kit, the preparation or the pharmaceutical composition in preparing a product with any one of the following functions 1) to 4):

1) encapsulating an active;

2) delivering an active agent to a cell, tissue or organ;

3) allowing the active substance to exert activity in a cell, tissue or organ;

4) preventing, diagnosing and/or treating diseases.

The cationic lipid molecule provided by the invention has simple synthesis process and good stability, and the cationic liposome has high efficiency (can be reflected as high transfection efficiency) and low toxicity; meanwhile, the product is stable and uniform and is easy to prepare; can be used for transfection of various cell lines. Therefore, the siRNA has excellent transmissibility, and can deliver the active substances (such as the exemplified siRNA) to cells (such as the exemplified lung cancer cells), tissues and organs with high efficiency to realize the high-efficiency regulation and control of the active substances. Solves the problems of low toxicity and low transfer efficiency of the cationic liposome in the prior art.

Drawings

FIG. 1 shows particle size (FIG. 1(A)) and surface potential (FIG. 1(B)) of cationic liposomes prepared by rotary evaporation and corresponding liposome/siRNA complexes.

FIG. 2 shows the particle size (FIG. 2(A)) and surface potential (FIG. 2(B)) of the cationic liposome prepared by the emulsion method before and after filtration using a 100nm membrane.

FIG. 3 shows the particle size (FIG. 3(A)) and surface potential (FIG. 3(B)) of long circulating cationic liposomes before and after filtration using a 100nm membrane.

FIG. 4 shows particle size (FIG. 4(A)) and surface potential (FIG. 4(B)) of liposome/siRNA complexes with different nitrogen to phosphorus (N/P) ratios.

FIG. 5 is a fluorescent microscope photograph of liposome/Cy 3-siRNA complex transfected cells.

FIG. 6 shows the expression level of green fluorescent protein in cells after transfection of cells with liposome/siRNA complexes.

FIG. 7 shows the detection of the activity of unloaded liposomes.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Table 3 shows a reference table for English or abbreviated Chinese in the present invention, and Table 4 shows each cell line.

TABLE 3 reference table for English or abbreviated Chinese

TABLE 4 cell lines Table

A549	Human lung adenocarcinoma cell line
		MDA-MB-231	Human breast cancer cell
MCF-7	Human breast cancer cell line
		H1299	Non-small cell lung cancer cell

Example 1 preparation of Compound I-1

(1) Synthesis of Compound 3

Oleic acid 1, HBTU (1.1eq), DIEA (1.2eq) were dissolved in dichloromethane in a single-neck flask. After stirring at room temperature for 2h, diethanolamine 2(1.2eq) was added. After the addition, the mixture was stirred at room temperature for 18 hours. The reaction solution was washed with 1N HCl and saturated Na, respectively₂CO₃The solution was washed with water and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column (petroleum ether: ethyl acetate V: V ═ 1:1) to give 3 as a pale yellow oil in 58.2% yield.¹HNMR(400M，CDCl₃)δppm：(5.305-5.388，m，2H),(3.811-3.836，t，2H)，(3.761-3.787，t，2H)，(3.531-3.556，t，2H)，(3.482-3.508，t，2H)，(2.368-2.406，t，2H)，(2.005-2.019，m，4H)，(1.603-1.638，t，2H)，(1.272-1.308，m，20H)，(0.885-0.901，t，3H)。ESI-MS：m/z 392.93[M+Na]⁺,761.74[2M+Na]⁺。

(2) Synthesis of Compound 5

N-hydroxyethyl morpholine 4 and carbon tetrabromide (1.1eq) were added to a single-neck flask and dissolved in methylene chloride. Triphenylphosphine (1.1eq) was then added slowly. After the addition, the ice bath was removed and the mixture was stirred at room temperature for 30 min. The reaction was concentrated to dryness. Adding 500mL of petroleum ether to separate out a large amount of solid. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column (petroleum ether: ethyl acetate V: V ═ 5:1) to give the intermediate (2- (4-morpholine) ethyl bromide) which was used directly in the next step.

In another two-necked flask, compound 3 and anhydrous tetrahydrofuran were added thereto, and NaH (4.0eq) was slowly added thereto under stirring in an ice bath. After the addition, nitrogen gas is replaced, and the temperature is raised to 60 ℃ for reaction for 1 h. The obtained transparent oily substance was dissolved in anhydrous tetrahydrofuran and added to the reaction solution. After the addition, the reaction was continued at 60 ℃ for 16 hours. The reaction was quenched with water, extracted with ethyl acetate and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to give 5 as a pale yellow oil in 37.3% yield. ESI-MS: m/z 596.94[ M + H ]]⁺，618.81[M+Na]⁺，1213.68[2M+Na]⁺。

(3) Synthesis of Compound 6

In a single neck flask was added 4.0g of compound 5 and methyl iodide (10eq) dissolved in acetonitrile. The temperature is increased to 70 ℃ for reaction for 20 h. The reaction solution was concentrated to dryness to obtain a yellow crude product. Taking the crude product and using C₁₈Column separation gave 6 as a pale yellow solid in 40.6% yield. ESI-MS: m/z 313.08[ M-2I^-]/2，752.47[M-I^-]。¹HNMR(400M，CDCl₃)δppm：(5.321-5.332，d，2H)，(4.066-4.136，m，16H)，(3.562-3.799，m，22H)，(2.309，s，2H)，(1.996-2.003，m，4H)，(1.548，s，2H)，(1.232-1.298，m，20H)，(0.857-0.884，t，3H)。

(4) Synthesis of Compound I-1

Adding chlorine type exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. And adding the compound 6 and absolute ethyl alcohol into another single-mouth bottle, and stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 2 hours at room temperature after the addition is finished. Filtering, concentrating the filtrate to dryness to obtain anhydrous transparent solid I-1 with the yield of 96.0%. ESI-MS: m/z 313.12[ M-2Cl^-]/2，660.57[M-Cl^-]。¹HNMR(400M，CDCl₃)δppm：(5.366，s，2H),(4.079-4.134，m，16H)，(3.552-3.786，m，22H)，(2.322，s，2H)，(2.037，s，4H)，(1.579，s，2H)，(1.297-1.324，m，20H)，(0.909，s，3H)。

Example 2 preparation of Compound I-2

(1) Synthesis of Compound 8

Erucic acid 7 is added into a single-mouth bottle and dissolved in dichloromethane by stirring. HBTU (1.05eq) and DIEA (2eq) were added and stirred at room temperature for 2 h. Diethanolamine (2eq) was added. After the addition, the mixture was stirred at room temperature for 18 hours. The reaction solution was washed with water and dilute hydrochloric acid, and then dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to afford compound 8 in about 83% yield. ESI-MS: m/z 873.22[2M + Na ]]⁺。

(2) Synthesis of Compound 9

N-hydroxyethyl morpholine 4, carbon tetrabromide (1.1eq) and a proper amount of dichloromethane are added into a single-neck flask, and triphenylphosphine (1.1eq) is slowly added after stirring and dissolving. After the addition, the ice bath was removed and the mixture was stirred at room temperature for 30 min. The reaction was concentrated to dryness. Petroleum ether is added to separate out a large amount of solid. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to give the intermediate (2- (4-morpholine) ethyl bromide) which was used directly in the next step.

In a two-necked flask, Compound 8 and anhydrous tetrahydrofuran were added, and NaH (4.0eq) was slowly added at 0 ℃. After the addition, nitrogen gas is replaced, and the temperature is raised to 60 ℃ for reaction for 1 h. Dissolving the intermediate with anhydrous tetrahydrofuran, and adding into the reaction solution. After the addition, the reaction was continued at 60 ℃ for 18 hours. The reaction was quenched by addition of ammonium chloride, extracted with ethyl acetate, washed with saturated brine and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to afford 9 as a brown oil in 47.0% yield. ESI-MS: m/z 652.66[ M + H ]]⁺，1225.50[2M+Na]⁺。

(3) Synthesis of Compound 10

Compound 9 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. Heating to 100 ℃ in a closed manner for reaction at 20 DEG Ch. The reaction solution is concentrated to dryness to obtain a crude product. Crude product of C₁₈The product was obtained in about 10% yield of 48.8% by column separation. ESI-MS: m/z 341.05[ M-2I^-]/2，808.36[M-I^-]。¹HNMR(400M，D₂O)δppm：(5.27，s，2H)，(3.97-4.02，m，12H)，(3.73-3.84，m，6H)，(3.54-3.61，m，14H)，(3.32，s，3H)，(3.30，s，3H)，(2.38，s，2H)，(1.95-1.97，m，4H)，(1.50，s，2H)，(1.22-1.24，m，28H)，(0.81-0.84，t，3H)。

(4) Synthesis of Compound I-2

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. And adding the compound 10 and absolute ethyl alcohol into another single-mouth bottle, and stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtering, and concentrating the filtrate to dryness to obtain the product I-2 with the yield of 89.5%. ESI-MS: m/z 341.05[ M-2Cl^-]/2，716.49[M-Cl^-]。¹HNMR(400M，D₂O)δppm：(5.26-5.29，m，2H)，(3.90-4.70，m，12H)，(3.65-3.73，m，6H)，(3.47-3.60，m，14H)，(3.23，s，6H)，(2.31-2.35，m，2H)，(1.93-1.96，m，4H)，(1.48，s，2H)，(1.22-1.25，m，28H)，(0.81-0.84，t，3H)。

Example 3 preparation of Compound I-3

(1) Synthesis of Compound 11

A single-necked flask was taken, and compound 5 and methanol were added thereto. After the catalyst is dissolved, Pd/C is added, hydrogen is replaced, and then the mixture is stirred for 18 hours at room temperature. Filtration through celite and concentration of the filtrate to dryness afforded compound 11, which was used directly in the next step. ESI-MS: m/z 598.52[ M + H ]]⁺，620.50[M+Na]⁺。¹HNMR(CD₃OD,ppm)：δ(3.69-3.73，m，8H),(3.61-3.66，m，12H)，(2.53-2.65，m，12H)，(2.45，t，2H)，(1.61，t，2H)，(1.31-1.35，m，28H)，(0.92，t，3H)。

(2) Synthesis of Compound 12

Compound 11 and DMF were added to a single vial, stirred to dissolve, and methyl iodide (10eq) was added. Heating to 100 ℃ under a sealed condition and reacting for 20 h. The reaction solution is concentrated to dryness and washed with ethyl acetate to obtain a crude product. Crude product of C₁₈Column separation gave 12 as a pale yellow solid in 63.5% yield. ESI-MS: m/z 314.17[ M-2I-]/2，754.37[M-I-]。¹HNMR(400M，D₂O)δppm：(3.98-4.05，m，12H)，(3.74-3.85，m，6H)，(3.55-3.68，m，14H)，(3.33，s，3H)，(3.31，s，3H)，(2.39，s，2H)，(1.52，s，2H)，(1.24，s，28H)，(0.82-0.86，t，3H)。

(3) Synthesis of Compound I-3

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. And adding the compound 12, absolute ethyl alcohol and water into another single-mouth bottle, and stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtering, and washing the filter cake with a small amount of anhydrous ethanol. The filtrate was concentrated to dryness to give a colorless transparent solid (I-3) in a yield of 94.0%. ESI-MS: m/z 313.95[ M-2Cl-]/2，662.35[M-Cl-]。¹HNMR(400M，D₂O)δppm：(3.92-4.04，m，12H),(3.67-3.74，m，6H)，(3.47-3.62，m，14H)，(3.27，s，3H)，(3.24，s，3H)，(2.33-2.36，t，2H)，(1.49-1.51，m，2H)，(1.24，s，28H),(0.83-0.86，t，3H)。

Example 4 preparation of Compound II-1

(1) Synthesis of Compound 14

Compound 13 and triethylamine (1.1eq) were added to a two-port flask and dissolved in THF with stirring, and MsCl (1.05eq) was added at 0 ℃. The reaction was maintained at 0 ℃ for 1 h. Filtering with diatomite, and washing the filter cake with a small amount of THF to obtain an intermediate filtrate for later use.

Dissolving compound 3 in THF in another double-mouth bottle, and adding at 0 deg.CNaH (3 eq). After the addition, nitrogen gas is replaced, and the temperature is raised to 60 ℃ for reaction for 1 h. The intermediate filtrate (ca. 3eq) was added by syringe. After the addition, the reaction was continued at 60 ℃ for 2 hours. The reaction was quenched with water, extracted with ethyl acetate, washed with saturated brine and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was isolated by column chromatography to afford compound 14, which was used directly in the next step. ESI-MS: m/z 1125.20[2M + Na ]]⁺。

(2) Synthesis of Compound 15

Compound 14 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. The temperature is raised to 100 ℃ in a sealed way for reaction for 20 h. The reaction solution is concentrated to dryness to obtain a crude product. Crude product is processed by C₁₈And (5) performing column chromatography separation to obtain a product 15. ESI-MS: m/z 290.95[ M-2I^-]/2，708.17[M-I^-]。¹HNMR(400M，D₂O)δppm：(8.76-8.78，d，2H)，(8.69-8.71，d，2H)，(7.91-7.93，d，4H)，(5.22-5.24，m，2H)，(4.87，s，2H)，(4.80，s，2H)，(4.30，s，3H)，(4.28，s，3H)，(3.63-3.81，m，8H)，(2.42-2.45，m，2H)，(1.89-1.91，m，4H)，(1.51，s，2H)，(1.16-1.21，m，20H)，(0.76-0.79，m，3H)。

(3) Synthesis of Compound II-1

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. Another single-necked bottle was taken, and compound 15 and absolute ethanol were added thereto, followed by stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtration and concentration of the filtrate to dryness gave the product (II-1) in about 90.9% yield. ESI-MS: m/z 290.91[ M-2Cl^-]/2，615.99[M-Cl^-]。¹HNMR(400M，D₂O)δppm：(8.69-8.76，m，4H)，(7.89-7.91，m，4H)，(5.23-5.24，m，2H)，(4.70-4.85，m，2H)，(4.32，s，3H)，(4.30，s，3H)，(3.64-3.81，m，8H)，(2.42-2.46，m，2H)，(1.90-1.91，m，4H)，(1.51-1.54，m，2H)，(1.17-1.22，m，20H)，(0.77-0.80，t，3H)。

Example 5 preparation of Compound II-2

(1) Synthesis of Compound 16

Compound 13 and triethylamine (1.1eq) were added to a two-port flask and dissolved in THF with stirring, and MsCl (1.05eq) was added at 0 ℃. The reaction was maintained at 0 ℃ for 1 h. Celite was added for filtration and the filter cake was washed with a small amount of THF to give the intermediate filtrate which was used directly in the next step.

Compound 8, anhydrous tetrahydrofuran, was added to a two-necked flask and NaH (3.0eq) was added slowly with stirring in an ice bath. After the addition, nitrogen gas is replaced, and the temperature is raised to 60 ℃ for reaction for 1 h. The intermediate filtrate was added by syringe. After the addition, the reaction was continued at 60 ℃ for 2 hours. The reaction was quenched with water, extracted with ethyl acetate and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product is separated by column chromatography and then passes through a column to obtain the compound 16, and the yield is 53.9 percent. ESI-MS: m/z 1237.33[2M + Na ]]⁺。¹HNMR(CDCl₃,ppm)：δ(8.35-8.60，m，4H)，(7.19-7.28，m，4H)，(5.34-5.36，m，2H)，(4.50-4.63，m，4H)，(3.61-3.78，m，8H),(2.38-2.42，m，2H)，(1.99-2.04，m，4H)，(1.62-1.65，m，2H)，(1.27-1.35，m，28H),(0.88，t，3H)。

(2) Synthesis of Compound 17

Compound 16 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. The temperature is raised to 100 ℃ in a sealed way for reaction for 20 h. The reaction solution is concentrated to dryness and washed with petroleum ether to obtain a crude product. Crude product of C₁₈Column chromatography separation to obtain light yellow product 17 with 31.1% yield. ESI-MS: m/z 318.96[ M-2I^-]/2，764.21[M-I^-]。¹HNMR(D₂O,ppm)：δ(8.81-8.83，d，2H)，(8.72-8.74，d，2H),(7.94-7.97，dd，4H)，(5.26，s，2H)，(4.90，s，2H)，(4.83，s，2H)，(4.34，s，3H),(4.31，s，3H)，(3.66-3.85，m，8H)，(2.46，s，2H)，(1.92-1.96，m，4H)，(1.54，s，2H)，(1.21-1.28，m，28H),(0.82，t，3H)。

(3) Synthesis of Compound II-2

Adding anion exchange into single-mouth bottleAnd (5) washing impurities of the resin by using absolute methanol, and removing the methanol for later use. Another single-necked flask was taken, and compound 17 and anhydrous methanol were added thereto and stirred at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtering, and concentrating the filtrate to dryness to obtain a light brown product II-2 with the yield of 94.3%. ESI-MS: m/z 318.97[ M-2Cl^-]/2，672.08[M-Cl^-]。¹HNMR(D₂O，ppm)：δ(8.69-8.78，m，4H)，(7.89-7.92，dd，4H)，(5.23-5.26，m，2H)，(4.85，s，2H)，(4.79，s，2H)，(4.33，s，3H)，(4.30，s，3H)，(3.63-3.82，m，8H)，(2.44，t，2H)，(1.90-1.93，m，4H)，(1.53，s，2H)，(1.19-1.22，m，28H)，(0.80，t，3H)。

Example 6 preparation of Compound III-1

(1) Synthesis of Compound 20

Compound 18, DMF and dichloromethane were added to a single-neck flask and suspended with stirring. Oxalyl chloride (4.5eq) was added at 0 ℃ and after addition, the temperature was raised to 50 ℃ to react for 1h and then dissolved clearly. The reaction was continued at 50 ℃ for 3 h. And concentrating the reaction solution to be dry to obtain a yellow solid, and adding dichloromethane to obtain an acyl chloride solution of the yellow solid for later use.

In another single-neck flask, compound 19(2.1eq), anhydrous pyridine and dichloromethane were added and dissolved by stirring. The above acid chloride solution was added dropwise at 0 ℃. After the addition, the reaction is carried out for 2 hours at room temperature, and a large amount of solid is separated out in the stirring process. And pouring out the mother liquor to obtain a residual crude product. The crude product was separated by column chromatography to give a thick oil 20 in the form of a reddish brown oil which was used directly in the next step. ESI-MS: m/z 464.48[ M + H ]]⁺。

(2) Synthesis of Compound 21

Compound 20 was added to a two-necked flask and dissolved in methanol with stirring. Pd/C is added, and hydrogen balloons are respectively sleeved on the two bottle mouths to replace hydrogen. The reaction was carried out at room temperature for 16h, and two hydrogen balloons were replaced again. The reaction was continued at room temperature until the reaction was complete. Spreading diatomite, filtering, and concentrating the filtrate to dryness to obtain crude product. Subjecting the crude product to column chromatographySeparation gave 21 as a light brown viscous oil in 95.2% yield. ESI-MS: m/z 434.44[ M + H ]]⁺，889.47[2M+Na]⁺。¹HNMR(400M，DMSO-d6)δppm：(8.447，s，2H)，(7.464，s，1H)，(7.110，s，2H)，(5.417，s，2H)，(3.256-3.271，m，8H)，(3.168，s，4H)，(2.419，s，12H)，(1.682-1.715，m，4H)。

(3) Synthesis of Compound 22

Compound 1, DMF and dichloromethane were added to a single-neck flask. After stirring to dissolve, oxalyl chloride (2.5eq) was added at 0 ℃. After the addition, the temperature is raised to 50 ℃ and the mixture is refluxed for 2 hours. Concentrating the reaction liquid to dryness, and adding dichloromethane to obtain an acyl chloride solution for later use.

Another single-neck flask was charged with compound 21(0.8eq), anhydrous pyridine (3.0eq) and DMF. After stirring and dissolving, the acyl chloride solution is dripped into the ice bath. After the addition, the reaction was carried out at room temperature for 2 hours. Concentrating to remove the solvent to obtain a crude product, and performing column chromatography separation on the crude product to obtain a product 22 with the yield of 65.6%. ESI-MS: m/z 698.78, [ M + H ]]⁺，1395.66，[2M+H]⁺。¹HNMR(CDCl₃,ppm)：δ(9.314，s，1H)，(8.357，s，2H)，(8.165，s，2H)，(8.018，s，1H)，(5.313-5.356，m，2H),(3.817，s，8H)，(3.482，s，4H)，(2.946，s，12H)，(2.433，s，2H)，(2.002-2.016，m，8H)，(1.674，s，2H)，(1.270-1.304，m，20H)，(0.891，t，3H)。

(4) Synthesis of Compound 23

Compound 22 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. And sealing and raising the temperature to 100 ℃ for reaction for 24 hours. Concentrating the reaction liquid to dryness, and washing with a petroleum ether-ethyl acetate system to obtain a crude product. The crude product was isolated by C18 column to give 23 as a pale yellow solid in 21.3% yield. ESI-MS: m/z 364.60[ M-2I^-]/2，854.59[M-I^-]。¹HNMR(CDCl₃,ppm)：δ(8.090，s，2H)，(7.943，s，1H)，(5.335，s，2H)，(4.016，s，8H)，(3.513-3.644，m，16H),(3.232，s，6H)，(2.454，s，2H)，(2.152，s，4H)，(2.007，s，4H)，(1.641，s，2H)，(1.273，s，20H),(0.883，s，3H)。

(5) Synthesis of Compound III-1

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. Another single-necked flask was taken, and compound 23, absolute ethanol and water were added thereto and stirred at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 20 hours at room temperature after the addition is finished. Filtration and concentration of the filtrate to dryness gave III-1 as a pale yellow solid in about 88% yield. ESI-MS: m/z364.85, [ M-2Cl ]^-]/2，763.19，[M-Cl^-]。¹HNMR(CDCl₃,ppm)：δ(7.97，s，2H)，(7.85，s，1H)，(5.23-5.24，d，2H)，(3.91，t，8H)，(3.39-3.51，m，16H),(3.12，s，6H)，(2.33，s，2H)，(2.05，s，4H)，(1.91，s，4H)，(1.54，s，2H)，(1.17-1.20，m，20H),(0.79，t，3H)。

Example 7 preparation of Compound III-2

(1) Synthesis of Compound 24

A single-neck flask was taken, compound 22 was added thereto, and methanol was added thereto with stirring. After the catalyst is dissolved, Pd/C is added, hydrogen is replaced, and then the mixture is stirred for 18 hours at room temperature. After filtration through celite, the filtrate was concentrated to dryness to obtain the compound 24 as a solid in 90.6% yield. ESI-MS: m/z 700.55[ M + H ]]⁺。

(2) Synthesis of Compound 25

Compound 24 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. And sealing and raising the temperature to 100 ℃ for reaction for 24 hours. Concentrating the reaction liquid to dryness, and washing with a petroleum ether-ethyl acetate system to obtain a crude product. Half of the crude product is separated by C18 column chromatography to obtain the product 25 with the yield of 25.8%. ESI-MS: m/z364.85 [ M-2I^-]/2，856.48[M-I^-]。¹HNMR(D₂O,ppm)：δ(8.00，s，2H)，(7.86，s，1H)，(3.93，s，8H)，(3.43-3.57，m，16H)，(3.15，s，6H)，(2.37，s，2H)，(2.07，s，4H)，(1.56，s，2H)，(1.21，s，28H)，(0.82，s，3H)。

(3) Synthesis of Compound III-2

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. Adding compound 25, anhydrous ethanol, and water into another single-mouth bottle, and stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtration and concentration of the filtrate to dryness gave product III-2 in about 92% yield. ESI-MS: m/z 364.94, [ M-2Cl ]^-]/2，764.40[M-Cl^-]。¹HNMR(D₂O，ppm)：δ(7.96，s，2H)，(7.85，s，1H)，(3.89-3.92，t，8H)，(3.39-3.49，m，16H)，(3.12，s，6H)，(2.34，s，2H)，(2.04，s，4H)，(1.54，s，2H)，(1.19，s，28H)，(0.80，t，3H)。

Example 8 preparation of Compound IV-1

(1) Synthesis of Compound 27

Compound 1, DMF and dichloromethane were added to a single-neck flask. Adding thionyl chloride (2.0eq) at 0 ℃, heating to room temperature and reacting for 2h after the addition is finished. Concentrating the reaction liquid to dryness, and adding dichloromethane to obtain an acyl chloride solution for later use.

Another single-neck flask was charged with Compound 26(0.73eq), triethylamine and dichloromethane. The acyl chloride solution is dripped into the mixture under ice bath stirring. After the addition, the reaction was carried out at room temperature for 1 hour. Washing with water, dilute sodium hydroxide and dilute hydrochloric acid respectively. Dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product is separated by column chromatography to obtain the product 27 with the yield of 73.5 percent. ESI-MS: m/z 969.15[2M + Na ]]⁺。

(2) Synthesis of Compound 28

Compound 27, tetrahydrofuran and water were added to a single-neck flask. Anhydrous lithium hydroxide (4.0eq) was added with stirring, and after the addition was completed, the reaction was carried out at room temperature for 18 hours. The tetrahydrofuran was removed by concentration and the residue was adjusted to pH 2 with 4N HCl to precipitate a large amount of solid. Filtration and concentration of the filter cake to dryness yielded product 28 as an off-white solid. ESI-MS: m/z 889.74[2M-H ]]^-。

(3) Synthesis of Compound 30

Compound 28, HBTU (2.2eq), DIEA (3.0eq) and DMF were added to a single neck flask. Stir at room temperature for 1 h. Compound 29(2.2eq) was added. After the addition, the mixture was stirred at room temperature for 2 hours. The reaction was quenched with water and extracted with dichloromethane. The organic phase was washed with water 3 times and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dryness to obtain crude product. The crude product was passed through the column using a dichloromethane-methanol system to afford 30 as a pale orange solid in about 92% yield. ESI-MS: m/z 1173.33[2M + Na ]]⁺。

(4) Synthesis of Compound 31

Compound 30 and anhydrous methanol were added to a single-neck flask, and DMF and methyl iodide were added with stirring. And sealing and raising the temperature to 60 ℃ for reaction for 24 hours. Concentrating the reaction liquid to dryness, and washing with a petroleum ether-ethyl acetate system to obtain a crude product. The crude product is separated by C18 column chromatography to obtain a light yellow solid product 31 with the yield of 24.8 percent. ESI-MS: m/z 327.95[ M-2I^-]/2，782.17[M-I^-]。¹HNMR(D₂O，ppm)：δ(8.56-8.58，t，4H)，(8.03，s，2H)，(7.95，s，1H)，(7.84-7.85，d，4H)，(5.24，s，2H)，(4.64，s，4H)，(4.17，s，6H)，(2.38，s，2H)，(1.92，s，4H)，(1.56，s，2H)，(1.18-1.21，m，20H)，(0.78-0.79，t，3H)。

(5) Synthesis of Compound IV-1

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. Adding compound 31, anhydrous ethanol, and water into another single-mouth bottle, and stirring at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtering, and concentrating the filtrate to be dry to obtain the product IV-1 with the yield of about 93 percent. ESI-MS: m/z 327.94[ M-2Cl^-]/2，690.23[M-Cl^-]。¹HNMR(D₂O，ppm)：δ(8.56-8.58，m，4H)，(7.81-8.02，m，7H)，(5.22，s，2H)，(4.66-4.71，m，4H)，(4.18-4.20，m，6H)，(2.34，s，2H)，(1.90，s，4H)，(1.55，s，2H)，(1.16-1.23，m，20H)，(0.76-0.79，t，3H)。

Example 9 preparation of Compound V-1

(1) Synthesis of Compound 33

Compound 32, DMF and dichloromethane were added to a single-neck flask and dissolved with stirring. Oxalyl chloride (2.0eq) was added at 0 ℃ and after addition, the temperature was raised to 50 ℃ and refluxed for 2 h. Concentrating the reaction liquid to dryness, and adding dichloromethane to obtain an acyl chloride solution for later use.

Another single-neck flask was charged with compound 21(0.95eq), anhydrous pyridine (3.0eq), and DMF, and dissolved by stirring. The acyl chloride solution is dripped into the mixture at the temperature of zero ℃. After the addition, the reaction was carried out at room temperature for 2 hours. The solvent was removed by concentration, and the residue was separated by silica gel column chromatography to give 33 as a pale yellow oil with a yield of 71.7%. ESI-MS: m/z 576.64[ M + H ]]⁺，1151.60[2M+H]⁺，1173.55[2M+Na]⁺。

(2) Synthesis of Compound 34

Compound 33, tetrahydrofuran and water were added to a single-neck flask and dissolved with stirring. Anhydrous lithium hydroxide (4.0eq) was added and the reaction was carried out at room temperature for 20 h. The tetrahydrofuran was removed by concentration, water was added, washing was with dichloromethane and the aqueous phase was adjusted to pH 6 with 6N HCl. The aqueous phase was concentrated to dryness to afford crude inorganic salt-containing product 34 as an oil which was used directly in the next step. ESI-MS: m/z 562.51[ M + H ]]⁺，568.59[M+Li]⁺。

(3) Synthesis of Compound 36

Weighing the compound 35 into a single-mouth bottle, and adding dichloromethane to dissolve for later use. Another single-neck flask was charged with Compound 34 and anhydrous DMF, and a solution of HOBT (1.5eq), DIEA (3eq), and Compound 35 in dichloromethane was added with stirring. After the addition, the mixture was stirred at room temperature for half an hour. HBTU (1.5eq) was added and the reaction was carried out at room temperature for 16 hours. The reaction solution was concentrated to dryness, and methylene chloride was added to dissolve the residue, which was then washed with water and dried over anhydrous sodium sulfate. Filtered and the filtrate concentrated to dryness. Passing through a silica gel column to obtain a light yellow oily substance 36 with the yield of 47.7 percent. ESI-MS: m/z 1106.14[ M + H ]]⁺，1128.07[M+Na]⁺。¹HNMR(400M，CDCl₃)δppm：(9.581，s，1H)，(8.294-8.297，d，2H)，(8.033-8.058，t，2H)，(7.969，s，1H)，(6.948，s，1H)，(5.305-5.326，m，1H)，(3.681-3.704，t，8H)，(3.616-3.645，m，12H)，(3.512-3.562，m，6H)，(3.442-3.469，t，2H)，(3.106-3.211，m，1H)，(2.483-2.541，m，14H)，(2.248-2.385，m，4H)，(1.725-2.015，m，12H)，(0.995-1.582，m，22H)，(0.970，s，3H)，(0.898-0.915，d，3H)，(0.850-0.871，dd，6H)，(0.664，s，3H)。

(4) Synthesis of Compound 37

Compound 36 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. And sealing and raising the temperature to 100 ℃ for reaction for 24 hours. And (4) supplementing methyl iodide, and continuing the closed reaction at 100 ℃ for 24 hours. The reaction solution is concentrated to be dry and is washed by a petroleum ether/ethyl acetate system to obtain a crude product. Crude product is passed through C₁₈Column, affording 23 as a pale yellow solid in 25.5% yield. ESI-MS: m/z 568.36[ M-2I^-]/2，1261.99[M-I^-]。

(5) Synthesis of Compound V-1

Adding anion exchange resin into the single-mouth bottle, washing impurities with absolute ethyl alcohol, and removing ethyl alcohol for later use. Another single-necked flask was charged with compound 37 and absolute ethanol, and stirred at room temperature. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filtering, and washing the filter cake with a small amount of anhydrous ethanol. The filtrate was concentrated to dryness to give a colorless transparent solid 18 with a yield of 98.0%. ESI-MS: m/z 568.17[ M-2Cl^-]/2，1169.87[M-Cl^-]。¹HNMR(400M，D₂O)δppm：(7.93，s，2H)，(7.85，s，1H)，(5.47，s，1H)，(4.01，s，8H)，(3.37-3.69，m，34H)，(3.21，s，6H)，(0.73-2.36，m，54H)。

Example 10 preparation of Compound VI-1

(1) Synthesis of Compound 40

In a single-neck flask were added benzyl alcohol 38 and triethylamine (1.1 e)q) dissolved in dichloromethane with stirring. Phenyl chloroformate 39(1.0eq) was weighed and added dropwise slowly at 0 ℃. After the addition, the mixture was stirred at room temperature for 18 hours. The reaction solution is washed with water and dilute hydrochloric acid, respectively, and with NaHCO₃The pH was adjusted to 8. The organic phase was dried with anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to give a clear oil 40 in about 82% yield.¹HNMR(400M，CDCl₃)δppm：(7.38-7.49，m，7H)，(7.20-7.32，m，3H)，(5.30，s，2H)。

(2) Synthesis of Compound 42

Compound 40 and dichloromethane were added to a single-neck flask and dissolved with stirring. The compound 41(1.0eq) was weighed, added dropwise to the reaction mixture, and reacted at room temperature for 40 hours after the addition. Washing the reaction liquid with water and dilute sodium hydroxide, concentrating the organic phase to near dryness, adding hydrochloric acid to form salt, and recrystallizing with ethanol to obtain white solid 42 hydrochloride. Sodium carbonate was used for liberation to give off-white solid 42 in 53.0% yield. ESI-MS: m/z 372.09[ M + H ]]⁺。¹HNMR(400M，CD₃OD)δppm：(7.34-7.37，m，10H)，(5.12，s，4H)，(3.45-3.48，m，4H)，(3.18-3.21，m，4H)。

(3) Synthesis of Compound 43

Compound 32(1.0eq), HBTU (1.1eq), DIEA (2.0eq) were added to a single-neck flask and dissolved with stirring in dichloromethane. The reaction was stirred at room temperature for 1 h. Compound 42(0.95eq) was added and the reaction stirred at room temperature for 20 h. The reaction solution was washed with water, dilute hydrochloric acid and sodium hydrogencarbonate, respectively, and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product was passed through a silica gel column to give oil 43 in 91.6% yield.¹HNMR(CDCl₃,ppm)：δ(7.34-7.36，m，10H)，(5.08-5.12，d，4H)，(3.66，s，3H)，(3.29-3.48，m，8H)，(2.29-2.37，m，4H)，(1.60-1.65，m，4H)。

(4) Synthesis of Compound 44

Compound 43 was added to a single-neck flask and dissolved in tetrahydrofuran with stirring. Anhydrous lithium hydroxide (4.0eq) was weighed and dissolved in water and added to the reaction solution, after which the reaction was carried out at room temperature for 6 hours. Concentrating to remove tetrahydrofuran, and extracting with petroleum ether-ethyl acetateThe system was washed twice and the aqueous phase was adjusted to pH 2 with 4N HCl. Extracted with dichloromethane and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dryness to obtain the compound 8 with the yield of about 82.3 percent. ESI-MS: m/z 498.09[ M-H]^-。

(5) Synthesis of Compound 45

Compound 44(1.0eq), anhydrous DMF, dichloromethane were added to a single-neck flask and dissolved with stirring. Slowly adding thionyl chloride (2.0eq), heating to 40 ℃ and refluxing for 2h after the addition is finished. Concentrating the reaction liquid to dryness, and adding dichloromethane to obtain an acyl chloride solution for later use.

In another single-neck flask, compound 26(0.9eq) and triethylamine (2.0eq) were added to dichloromethane and dissolved with stirring. The above acid chloride solution was added dropwise at 0 ℃. After the addition, the reaction was carried out at room temperature for 2 hours. The reaction mixture was washed with 1M sodium hydroxide, 1M hydrochloric acid and saturated sodium hydrogencarbonate, and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product is passed through a silica gel column to obtain a white solid 45 with the yield of 80.6 percent. ESI-MS: m/z 713.32[ M + Na ]]⁺，1402.80[2M+Na]⁺。¹HNMR(400M，CDCl₃)δppm：(8.48-8.49，d，2H)，(8.31-8.32，t，1H)，(7.27-7.33，m，10H)，(5.04-5.08，d，4H)，(3.93，s，6H)，(3.42-3.47，m，4H)，(3.27-3.33，m，4H)，(2.37-2.42，m，4H)，(1.64-1.70，m，4H)。

(6) Synthesis of Compound 46

Compound 45, methanol and ethyl acetate were added to a two-necked flask and dissolved with stirring. After Pd/C is added, hydrogen balloons are sleeved on both bottle mouths to replace hydrogen. The reaction was carried out at room temperature for 16h, and two hydrogen balloons were replaced again. The reaction was continued for 4h at room temperature, filtered over celite and the filtrate was concentrated to dryness to give 46 as a clear oil in 70.3% yield. ESI-MS: m/z 421.38[ M-H]^-。

(7) Synthesis of Compound 47

Compound 1, DMF, and dichloromethane were added to a single-neck flask, and dissolved with stirring. Thionyl chloride (2.0eq) was added under ice-cooling and after addition, the temperature was raised to 40 ℃ for 2h reaction. Concentrating the reaction liquid to dryness, and adding dichloromethane to obtain an acyl chloride solution for later use.

In another single-neck flask, compound 46(0.45eq) and triethylamine (2.0eq) were added and dissolved in dichloromethane. The acyl chloride solution is dripped into the mixture under ice bath stirring. After the addition, the reaction was carried out at room temperature for 1 hour.

The reaction solution is washed by water, diluted sodium hydroxide, diluted hydrochloric acid and saturated sodium chloride solution respectively, and then is dried by adding anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness. After methylene chloride was dissolved, column chromatography was performed to obtain about 17.4g of a product. ESI-MS: m/z 973.77[ M + Na ]]⁺，¹HNMR(CD₃OD,ppm)：δ(8.51-8.52，d，2H)，(8.34，t，1H)，(5.29-5.37，m，4H)，(3.95，s，6H)，(3.48-3.51，m，4H)，(3.33-3.39，m，4H)，(2.45-2.50，m，4H)，(2.14-2.22，m，4H)，(1.99-2.05，m，8H)，(1.71-1.78，m，4H)，(1.55-1.61，m，4H)，(1.27-1.33，m，40H)，(0.89-0.92，m，6H)。

(8) Synthesis of Compound 48

Compound 47 was added to a single-necked flask and dissolved in tetrahydrofuran. Anhydrous lithium hydroxide (8.0eq) was weighed, dissolved in water and added to the reaction mixture, and after the addition was completed, the reaction was carried out at room temperature for 18 hours. The tetrahydrofuran was removed by concentration and the aqueous phase was adjusted to pH 2 with 4N HCl. Extracted with dichloromethane and dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate to dryness to obtain the product 48 with the yield of about 95.0%. ESI-MS: m/z 922.03[ M-H]^-。

(9) Synthesis of Compound 49

Compound 48, HBTU (2.4eq), DIEA (3.0eq) were dissolved in dichloromethane in a single vial and stirred at room temperature for 1 h. Compound 16(2.4eq) was added. After the addition, the mixture was stirred at room temperature for 3 hours. The reaction mixture was washed with water and 1N sodium hydroxide, and dried over anhydrous sodium sulfate. Filtering, and concentrating the filtrate to dryness to obtain crude product. The crude product is subjected to column chromatography by using a dichloromethane-methanol system to obtain a light yellow oily substance 49 with the yield of 46.6 percent. ESI-MS: m/z 1175.92[ M + H ]]⁺。¹HNMR(CDCl₃，ppm)：δ(10.59，d，1H)，(8.70，s，2H)，(7.94-8.07，m，4H)，(7.53，s，1H)，(5.31-5.38，m，4H)，(3.70-3.72，m，8H)，(3.49-3.63，m，12H)，(2.41-2.56，m，15H)，(2.22-2.26，m，2H)，(1.95-2.10，m，11H)，(1.48-1.84，m，12H)，(1.08-1.36，m，40H)，(0.87-0.91，m，6H)。

(10) Synthesis of Compound 50

Compound 49 and anhydrous DMF were added to a single-neck flask and methyl iodide was added with stirring. The temperature is raised to 100 ℃ in a sealed way to react for 18 h. The reaction solution is concentrated to dryness and washed with petroleum ether to obtain a crude product. Crude product of C₁₈Column chromatography separation is carried out to obtain 50 percent of light yellow solid product with the yield of 50.2 percent. ESI-MS: m/z 602.67[ M-2I^-]/2，1331.73[M-I^-]。¹HNMR(D₂O，ppm)：δ(8.29-8.30，d，2H)，(8.21-8.22，m，1H)，(5.34-5.37，m，4H)，(4.02-4.05，m，8H)，(3.68-3.71，m，4H)，(3.48-3.66，m，16H)，(3.34-3.40，m，4H)，(3.28，s，6H)，(2.47-2.52，m，4H)，(2.15-2.26，m，8H)，(2.01-2.08，m，8H)，(1.70-1.81，m，4H)，(1.59-1.63，m，4H)，(1.30-1.34，m，40H)，(0.90-0.94，m，6H)。

(11) Synthesis of Compound VI-1

And (3) adding anion exchange resin into the single-mouth bottle, washing impurities with acetonitrile, and removing the acetonitrile for later use. And adding the compound 50 and the acetonitrile into another single-mouth bottle, and stirring at room temperature in water. After dissolving, adding the mixture into the resin, and stirring the mixture for 18 hours at room temperature after the addition is finished. Filter and wash the filter cake with a small amount of acetonitrile. The filtrate is concentrated to be dry, and a small amount of toluene is added for spin drying to obtain a product VI-1, wherein the yield is about 73 percent. ESI-MS: m/z 602.66[ M-2Cl^-]/2，1239.86[M-Cl^-]。¹HNMR(D₂O，ppm)：δ(8.29-8.29，d，2H)，(8.23-8.23，t，1H)，(5.34-5.37，m，4H)，(4.02-4.04，m，8H)，(3.62-3.67，m，4H)，(3.47-3.60，m，16H)，(3.34-3.39，m，4H)，(3.26，s，6H)，(2.47-2.51，m，4H)，(2.15-2.24，m，8H)，(2.01-2.10，m，8H)，(1.70-1.81，m，4H)，(1.57-1.63，m，4H)，(1.30-1.41，m，40H)，(0.90-0.94，m，6H)。

Examples 11,

The cationic compound of the I-3, III-1 and III-2 liposome synthesized by the invention and DOPE (molar ratio: 1:1) are dissolved in a chloroform/methanol mixed system (10mL, 1:1, v/v), poured into a round-bottom flask of a rotary evaporator, and evaporated for 2 hours under the vacuum condition at the constant temperature of 50 ℃ in a water bath. Then dried in vacuum for 4h at 40 ℃ in a vacuum drying oven to completely remove the organic solvent. Adding physiological saline (6mL), shaking at constant temperature in water bath of 50 deg.C under normal pressure for 1h to obtain white emulsion, transferring the white emulsion into a small glass bottle, and performing ultrasonic treatment for 30min until the emulsion is substantially transparent. Finally the emulsion was sterilized on a 220 μm filter (Millipore Corp.) filter head, transferred to a new sample vial and stored at 4 ℃. Particle size, polydispersity and surface charge measurements were performed using a Zetasizer Nano particle sizer (fig. 1).

Examples 12,

Absolute ethyl alcohol is used for preparing cationic compounds I-1, I-2, I-3, II-1, II-2, III-1, III-2, VI-1, VI-2, V-1 and DOPE solution with the concentration of 0.01mol/L respectively. Adding a mixed solution of cationic compound and DOPE (50%: 50%, mol/mol) in ethanol at an equal molar ratio into 0.2M sodium acetate buffer aqueous solution with pH of 5, uniformly swirling, stirring by ventilation and magnetic force until ethanol in the mixed system is volatilized, and adding equal amount of distilled water to obtain transparent emulsion. A portion of the emulsion was filtered through a 100nm filter membrane using a liposome extrusion apparatus (Mini-Extruder, Avanti Lipids Polar, Inc, USA), and the particle size was compared with the particle size before and after filtration and the surface potential change was performed using a Zetasizer Nano particle sizer (FIG. 2).

Examples 13,

Cationic compound I-1, DOPE (dioleoylphosphatidylethanolamine) and DSPE-PEG (distearoylphosphatidylethanolamine-polyethylene glycol) are respectively in a molar ratio of 45%: 45 percent; 5% and 40%: 40%: preparing an ethanol solution with the concentration of 10 percent, adding a 0.2M sodium acetate buffer aqueous solution with the pH value of 5, uniformly swirling, ventilating and magnetically stirring until ethanol in a mixed system is volatilized, and adding equal amount of distilled water to obtain transparent emulsion. A part of the emulsion was filtered through a 100nm filtration membrane using a liposome extruder, and the particle size and surface potential changes before and after filtration were compared using a Zetasizer Nano particle sizer (FIG. 3).

As can be seen from examples 11, 12 and 13, the cationic compound synthesized by the present invention can be prepared into sterile cationic liposomes by a rotary evaporation method and an emulsification method. In fact, various conventional liposome preparation methods, such as an ultrasonic method, a reverse phase evaporation method, an injection method, a freeze drying method, a high pressure homogenization method and the like, are suitable for preparing the nanoliposome by using the cationic lipid molecule.

FIG. 1 shows the measurement results of the particle size and surface potential of the cationic liposome prepared by the rotary evaporation method using a Zetasizer Nano particle size analyzer. The particle size range of the obtained liposome is 89-130 nm, and the surface potential range is about 30-45 mV. FIG. 2 shows the results of particle size and surface potential measurements before and after filtration of cationic liposomes prepared by emulsification with a 100nm membrane using a liposome extrusion apparatus. The liposome directly prepared by the emulsification method has the particle size of 100-200nm and the surface charge of 30-70 mV before the filtration of a 100nm membrane by a liposome extruder is not adopted. After filtering through a 100nm membrane of a liposome extrusion instrument, the particle sizes of all the liposomes become more uniform within the range of 80-120 nm, the surface potential of the liposomes is correspondingly reduced, and the phenomenon that individual samples are sharply reduced within the range of 5-45 mV exists. FIG. 3 shows the results of particle size and surface potential measurements before and after filtration with a DSPE-PEG extruder (100nm membrane) in which auxiliary lipid molecules were added to liposomes containing I-1 and DOPE as the main components. The addition of DSPE-PEG can increase the surface hydrophilicity of the liposome and increase the circulation time in vivo. The increase in DSPE-PEG slightly increased the liposome particle size (fig. 3 (a)). However, the surface potential decreased with increasing DSPE-PEG content, from 53.6 + -1.6 mV down to 20.0 + -0.6 mV for 10% DSPE-PEG (FIG. 3 (B)).

From the above results, it can be seen that the nano-liposome solution with uniform and stable particle size can be obtained by adopting different liposome preparation methods and adding different auxiliary lipid molecules, and the surface potentials are all positive, thus proving that the liposome surface has net charges.

Examples 14,

The preparation and characterization of the I-1/DOPE liposome and siRNA compound comprises the following specific steps: after 10ul of the sample was diluted in 1ml of ultrapure water, particle size and surface charge were measured by Zetasizer Nano.

mu.L of the liposome solutions I-1/DOPE, I-3/DOPE, III-1/DOPE, and III-2/DOPE prepared in example 12 were added to 20. mu.L of Opti-MEM, and mixed to give solution 1. mu.L of siRNA (concentration 20. mu.M) was added to 24. mu.L of Opti-MEM medium as solution 2. And dropwise adding the solution 2 into the solution 1, uniformly mixing, standing at room temperature for 30min to obtain the I-1/DOPE, I-3/DOPE, III-1/DOPE, III-2/DOPE liposome and siRNA nano-composite. Then, the particle size, polydispersity and surface potential of the nanocomposite were measured using a Zetasizer Nano particle sizer (I-3/DOPE, III-1/DOPE, III-2/DOPE liposome and siRNA nanocomposite measurement results are shown in FIG. 1, and I-1/DOPE liposome and siRNA nanocomposite measurement results are shown in FIG. 4.)

FIG. 4 shows the results of particle size and surface potential measurements of I-1 liposomes and siRNA complexes at different nitrogen to phosphorus (N/P) ratios. The cationic liposome and the siRNA nucleic acid form composite nanoparticles through electrostatic interaction. It can be observed from FIG. 4 that the surface charge of the complex increases with increasing N/P ratio. When the N/P ratio of the cationic liposome I-1 to the siRNA is 10:1, the particle size is 581 +/-44 nm, and the surface potential is-18.7 +/-5.6 mV, which is a negative value. Indicating that at this N/P ratio, the liposomes did not compress the siRNA well and formed good complexes. When the N/P ratio is 20:1, the particle size is 218 + -61 nm, and the surface potential is 13.3 + -2.4 mV. The amount of charge carried by the surface of the liposome and siRNA complex is an important factor affecting transfection efficiency. The surface potential is a physical quantity used for measuring the strength of attraction or repulsion between materials, and is too low, so that nanoparticles are more prone to agglomeration, the stability of a system is influenced, the capability of wrapping nucleic acid molecules is weaker, and the transfection efficiency is reduced. And because the cell membrane is negatively charged, the complex formed by the carrier material and the nucleic acid is electropositive, which can help the nano-complex to enter the cell more easily. Therefore, for liposomal siRNA complexes, appropriate surface potentials will help ensure higher transfection efficiency of the complexes, as well as stable presence in body fluid transport.

As can be seen from this example 14, the cationic liposome of the present invention can spontaneously form a liposome/nucleic acid complex with nucleic acid by positive and negative charge interaction. In fact, various conventional methods for preparing liposome-nucleic acid complexes, such as liposome membrane and nucleic acid solution hydration, lyophilization-rehydration method, etc., are suitable for the preparation of liposome-nucleic acid complexes described in the present invention. Similarly, the cationic liposome of the present invention is also suitable for various nucleic acid encapsulation complexing experiments other than siRNA, including but not limited to DNA, messenger mRNA, microRNA, etc., which are effective in forming small-particle, uniform and stable liposome/nucleic acid complexes.

Examples 15,

A549 cells are transfected by the I-1, I-3, III-1, III-2, V-1, VI-1 and VI-2 liposome and Cy3 fluorescence labeled siRNA (Cy3-siRNA) compound. A549 cells at 2X 10⁵The density of each cell/well is paved on a 24-well plate, and a DMEM culture solution containing 10% calf serum is added for culturing for 24 hours until the cell density reaches about 70%. The culture medium was removed and washed twice with PBS. The liposome/siRNA complexes prepared in example 14 were diluted to 500. mu.L of Opti-MEM medium and added to 24-well plates. 37 ℃ and 5% CO₂Incubate under conditions for 4 h. The culture medium containing the liposome/siRNA complex was removed and incubated for 24 hours with DMEM medium containing 10% calf serum. The transfection of Cy3-siRNA was then observed with a fluorescence microscope with excitation light at 550nm and emission light at 570 nm. FIG. 5 shows fluorescence microscopy pictures of transfection results of liposome/Cy 3-siRNA complexes, red fluorescence being Cy3-siRNA transfected into cells by liposomes. The commercially available lipofamamine 2000(lipo2000) transfection reagent can deliver Cy3-siRNA into cells, and the liposomes of the invention can similarly transfect Cy3-siRNA into the cytoplasm. In contrast, some of the liposomes of the invention transfected cells had a much stronger red fluorescence intensity than lipo2000, indicating that some of the liposomes of the invention could more efficiently transfect Cy3-siRNA, such as III-1, V-1 and VI-1 liposomes.

Examples 16,

A549 cells expressing Green Fluorescent Protein (GFP) at 2X 10⁵The density of each cell/well is paved on a 24-well plate, and DMEM culture solution containing 10% calf serum is added for culturing for 24 hours until the cell density reaches about 70%. The culture medium was removed and washed twice with PBS. The liposome/siRNA complexes prepared by the method of example 14 were diluted to 500. mu.L of Opti-MEM medium and added to 24-well plates. 37 ℃ and 5% CO₂Incubator incubation under conditions 4h. The culture medium containing the liposome/siRNA complex was removed and incubated for 48h with DMEM medium containing 10% calf serum. Removing culture solution, washing with PBS twice, digesting cells with pancreatin, centrifuging at 4 deg.C and 1000rpm for 5min to obtain cell precipitate, washing cell precipitate with PBS, centrifuging at 4 deg.C and 1000rpm for 5min, and discarding supernatant. The cell pellet was resuspended in serum-free DMEM medium and assayed at 1X 10 intervals using a flow cytometer (BD, FACScalibur, BD Biosciences)⁴Relative fluorescence intensity of the cells.

From example 16, the novel cationic liposome/siRNA complexes of the present invention can achieve delivery of siRNA into various cell types including, but not limited to, A549, MDA-MB-231, MCF-7 cells, etc., by similar cell transfection procedures as in example 15 and example 16. In fact, for certain cells, specific liposomes in the cationic liposome combinatorial library of the present invention can deliver siRNA into these cells with high efficiency. FIG. 6 shows the expression level of cellular green fluorescent protein after transfection of cells with exemplary liposome/siRNA complexes. The liposome/siRNA compound prepared by the liposome can effectively reduce the expression of genes corresponding to siRNA when cells are transfected. With the adjustment of the N/P ratio of the liposome and the siRNA, the down regulation of the target gene by the liposome/siRNA complex of the invention is equivalent to or even superior to the marker post reagent Lipo2000 of the transfection reagent on the market.

Examples 17,

A549 cells were seeded in 96-well plates (about 1.5X 10)⁴One cell/well), 100 μ L of fresh cell suspension (medium containing 10% FBS) was added to each well at 37 deg.C, 5% CO₂Is incubated in the environment of (1). After 24 hours, the medium was discarded and washed twice with PBS, and 100 μ L of the liposome particles prepared in example 12 of the present invention were added to each well, and 3 parallel groups were set for each sample. After the cells are incubated at 37 ℃ for 24 hours again, 20 microliter of CCK-8 reagent is supplemented, the cells are cultured in an incubator for 2 hours, and the absorbance at the wavelength of 450nm is read by a microplate reader for cytotoxicosis detection. The cell viability was determined as 100% by taking the absorbance of unstimulated cells at a wavelength of 450nm as the cell viabilityCell viability under plastid stimulation.

As can be seen from example 17, the toxicity of the empty cationic liposomes of the present invention can be tested and evaluated in various cell lines including, but not limited to, A549, MDA-MB-231, MCF-7 and H1299 cells by performing cytotoxicity assays similar to those described in example 17. Figure 7 shows methanol of the present invention that is cytotoxic to a549 cells using a variety of empty cationic liposomes. At equivalent levels, the unloaded cations listed have very little toxicity to the cell line, and some liposomes may even have a cell growth promoting effect. Compared with the Lipo2000 transfection reagent on the market, the cationic liposome of the invention shows better biocompatibility.

While the preferred embodiments of the present invention have been described, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and the scope of the invention is to be determined by the appended claims.

Claims

1. A compound of formula I, formula II, formula III, formula IV, formula V or formula VI:

in each formula, A represents C containing 0-3 double bonds_14-28An acyl group;

said C is_14-28Acyl is myristoyl, palmitoyl, oleoyl, arachidonoyl, erucyl, nervoyl, myristoyl, palmitoyl, stearoyl, eicosanoyl, docosanoyl, tetracosanoyl, hexacosanoyl or octacosanoyl;

x is chlorine, bromine or iodine atom.

k is an integer between 2 and 4; p is an integer between 1 and 4; j is an integer of 1-5; m is an integer between 1 and 4; n is an integer of 2-4.

2. A cationic liposome, which is 1) or 2) below:

1) prepared from the compound of claim 1;

2) made from a compound of claim 1 and a co-lipid;

the auxiliary lipid is neutral lipid molecule and/or PEG lipid molecule.

3. A lipid complex made from the cationic liposome of claim 2 and a negatively charged active substance;

the active substance with negative charge is a nucleic acid molecule or a protein molecule.

4. An agent comprising a compound of claim 1, a cationic liposome of claim 2, or a lipid complex of claim 3.

5. A kit comprising a compound of claim 1, a cationic liposome of claim 2, or a lipid complex of claim 3.

6. A formulation comprising a compound of claim 1, a cationic liposome of claim 2, or a lipid complex of claim 3.

7. A pharmaceutical composition comprising a compound of claim 1, a cationic liposome of claim 2, or a lipid complex of claim 3.

8. Use of a compound according to claim 1, a cationic liposome according to claim 2, a lipid complex according to claim 3, a reagent according to claim 4, a kit according to claim 5, a formulation according to claim 6 or a pharmaceutical composition according to claim 7 for the preparation of a product having any one of the following functions 1) to 3):

1) encapsulating an active;

2) delivering an active agent to a cell, tissue or organ;

3) the active substance is activated in cells, tissues or organs.