Cationic lipid compound, preparation method and application, and mRNA delivery system

Abstract

The present invention relates to a cationic lipid compound, a preparation method and application thereof, and an mRNA delivery system; wherein, the structure of the cationic lipid compound is shown as general formula (I): #imgabs0# Wherein, in general formula (I): R1 is a saturated group with an ester group or an unsaturated group with an ester group; n is 4-6; R2 is an alkyl group with a carbon atom number greater than or equal to 8; X is O or S. The present invention is mainly used to develop a cationic lipid compound, which is used to prepare an mRNA delivery system (such as, LNP), and has the technical effects of high delivery efficiency, good spleen targeting, and high biosafety.

Description

[0001] This disclosure claims priority to a Chinese patent application filed with the Chinese Patent Office on December 26, 2022, with application number 202211675530.7 and invention name “A Cationic Lipid Compound, Preparation Method and Application, and mRNA Delivery System”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of medical biotechnology, and in particular to a cationic lipid compound, a preparation method and application thereof, and an mRNA delivery system. Background Art

[0003] mRNA (messenger ribonucleic acid) is a type of single-stranded RNA that is polymerized through phosphodiester bonds using one of the double-stranded DNA strands as a template and four ribonucleoside triphosphates (A, U, G, C) as substrates under the catalysis of RNA polymerase.

[0004] mRNA can carry and transmit the genetic information stored in the DNA in the cell nucleus, and plays a key role in the conversion of genetic information into functional proteins. In the cytoplasm, immature mRNA is processed and modified into mature mRNA through steps such as capping, tailing, and intron shearing. Mature mRNA can accurately guide the synthesis of proteins in the cytoplasm. Relatively speaking, because mRNA has a much smaller molecular weight than DNA, it is easy to transfect and there is no risk of integration into host DNA and causing insertion mutations that cause cancer. Therefore, using mRNA as a preventive and therapeutic drug has great advantages and potential in the prevention and treatment of various diseases.

[0005] mRNA nucleic acid drugs use molecular biology methods to introduce the target functional gene or the functional subunit of the target gene into the patient's body in the form of messenger RNA, and express proteins with specific functions through targeted intracellular delivery, late endosomal escape, intracellular translation and post-translational processing and modification, which are used to prevent (functional proteins or subunits activate the host immune system to produce corresponding humoral immunity or cellular immune response) or treat diseases (expressed proteins or subunits have the function of treating diseases or regulating the expression of other genes). Compared with other methods, its advantage is that it can directly activate the body at the molecular level to produce functional antibodies or cellular immune responses against specific pathogens, or repair pathogenic genes or correct the expression of abnormal genes in a targeted manner, thereby achieving the effect of preventing and treating a variety of diseases. mRNA nucleic acid drugs can achieve effects that traditional drugs cannot replace. For example, monoclonal antibody drugs can only act on the cell surface, while mRNA nucleic acid drugs can not only act on extracellular proteins, but also on intracellular proteins, and even in the cell nucleus, and have precise targeting. Among the more than 7,000 diseases faced by humans, about 1 / 3 of the diseases are caused by problems in the expression of functional genes (missing, reducing or overexpressing), such as hemophilia, Duchenne muscular dystrophy (DMD), cystic fibrosis and severe immunodeficiency syndrome (SCID), which are almost incurable in clinical practice, and mRNA nucleic acid drugs are very advantageous for such single gene diseases. In the context of personalized medicine and the popularization of precision medicine. In theory, diseases caused by genetic differences or abnormal gene expression in patients can be accurately and effectively treated with mRNA nucleic acid drugs.

[0006] Although mRNA nucleic acid drugs have great advantages and potential in regulating gene expression and preventing and treating malignant diseases. However, the research and development, preparation and subsequent systemic administration of such drugs face many difficulties, as follows: First, mRNA exists in a single-stranded form, which makes mRNA extremely unstable in vitro and under physiological conditions. It is not only easily degraded by RNA nucleases (RNAase) in the air or blood, but also easily cleared by mononuclear macrophages in tissues and organs such as the liver and spleen; secondly, due to the negative charge of mRNA, it is difficult for it to pass through the cell membrane and enter the cell; thirdly, it is difficult for mRNA to escape from the endosome and enter the cytoplasm to play a role. In addition, the uridine ribonucleoside (U) of mRNA is prone to immunogenicity. In some cases, the generation of immunogenicity may increase the potential toxic side effects of mRNA drugs. Finally, the easy off-target effect is also an important challenge faced by mRNA nucleic acid drugs in the preparation and administration process.

[0007] Therefore, the development of intracellular delivery systems for mRNA nucleic acid drugs is the key to enabling their large-scale clinical application. Summary of the invention

[0008] In view of this, the present invention provides a cationic lipid compound, a preparation method and application, and an mRNA delivery system. The main purpose is to provide a cationic lipid compound, which is used to prepare an mRNA delivery system (such as LNP) and has a technical effect of high delivery efficiency.

[0009] In order to achieve the above object, the present invention mainly provides the following technical solutions:

[0010] In one aspect, an embodiment of the present invention provides a cationic lipid compound, wherein the structure of the cationic lipid compound is shown in general formula (I):

[0011]

[0012] Wherein, in the general formula (I): R1 is a saturated group with an ester group or an unsaturated group with an ester group; n is 4 to 6; R2 is an alkyl group with a carbon number greater than or equal to 8; and X is O or S.

[0013] Preferably, R1 is any one of the following groups:

[0014]

[0015]

[0016] Preferably, the cationic lipid compound is any one of the following compounds:

[0017]

[0018]

[0019] On the other hand, an embodiment of the present invention provides a method for preparing the above-mentioned cationic lipid compound, wherein the preparation formula of the cationic lipid compound is as follows:

[0020]

[0021] Preferably, the preparation steps of the cationic lipid compound are as follows: at room temperature, Tetrabutylammonium fluoride and tetrahydrofuran are mixed and reacted; after the reaction is completed, dilution and washing are performed; then the organic phase is dried and purified to obtain a cationic lipid compound.

[0022] Preferably, when the cationic lipid compound is compound 1, compound 4, compound 5, or compound 9, The preparation formula is as follows:

[0023]

[0024] Preferably, The preparation steps are as follows:

[0025] At room temperature, Potassium carbonate and N,N-dimethylformamide are mixed and heated to 75-85°C for reaction. After the reaction is completed, dilution and washing are performed. The organic phase is then dried and purified to obtain

[0026] Preferably, when the cationic lipid compound is compound 1, compound 5, or compound 9:

[0027] Said All raw materials 1-9 Wherein, the preparation formula of raw material 1-9 is as follows:

[0028]

[0029] Further preferably, the preparation steps of the raw material 1-9 are as follows: reacting the raw material 1-8, dichloromethane solution and trifluoroacetic acid for a set time; spinning the reaction solution to dryness, then diluting and washing, drying the organic phase and purifying it to obtain the raw material 1-9;

[0030] Preferably, the preparation formula of raw material 1-8 is as follows:

[0031]

[0032] Further preferably, the preparation steps of the raw material 1-8 are as follows: sodium hydride and N,N-dimethylformamide are added to a reaction container under ice-water bath conditions, cooled to 0-5°C, and then a tetrahydrofuran solution of the raw material 1-7 is added to the reaction system in batches. After the reaction is carried out for a first set time, the raw material 1-6 is added dropwise to the reaction system for reaction. After the reaction is carried out for a second set time, the reaction temperature is raised to room temperature and the reaction is continued; after the reaction is completed, a quenching treatment is carried out, and then extraction, separation, drying, and purification treatment are carried out to obtain the raw material 1-8;

[0033] Preferably, the preparation formula of raw material 1-6 is as follows:

[0034]

[0035] Further preferably, the preparation steps of raw material 1-6 are as follows: raw material 1-4, linoleic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine, and dichloromethane are reacted at room temperature; after the reaction, extraction and liquid separation are performed, and the organic phase is washed, dried, and purified to obtain raw material 1-6.

[0036] Preferably, when the cationic lipid compound is compound 4:

[0037] Said It is raw material 4-5; wherein, the preparation formula of raw material 4-5 is as follows:

[0038]

[0039] Further preferably, the preparation steps of raw material 4-5 are as follows: reacting raw material 4-4, dichloromethane solution and trifluoroacetic acid at room temperature; after the reaction, spin-drying the reaction solution, then diluting and washing, drying the organic phase and purifying it to obtain raw material 4-5;

[0040] Preferably, the preparation formula of raw material 4-4 is as follows:

[0041]

[0042] Further preferably, the preparation steps of raw material 4-4 are as follows: sodium hydride and N,N-dimethylformamide are mixed under ice-water bath conditions, and then cooled to 0-5°C, a tetrahydrofuran solution of raw material 1-7 is added to the reaction system in batches, and after reacting for a first set time, raw material 4-3 is added dropwise thereto, and after reacting for a second set time, the temperature is raised to room temperature and the reaction is continued for a third set time; after the reaction is completed, the reaction solution is sequentially quenched, extracted, separated, dried, and purified to obtain raw material 4-4;

[0043] Preferably, the preparation formula of raw material 4-3 is as follows:

[0044]

[0045] Further preferably, the preparation steps of raw material 4-3 are as follows: raw material 4-1, linoleic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane are reacted at room temperature; after the reaction is completed, extraction and separation are performed, and then washing is performed, and the organic phase is dried and purified to obtain raw material 4-3.

[0046] Preferably, when the cationic lipid compound is compound 1:

[0047] Said It is raw material 1-5; wherein, the preparation formula of raw material 1-5 is as follows:

[0048]

[0049] Further preferably, the preparation steps of raw material 1-5 are as follows: reacting raw material 1-3, raw material 1-4, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane at room temperature, performing extraction treatment after the reaction, washing the organic phase, drying and purification treatment to obtain raw material 1-5;

[0050] Preferably, the preparation formula of raw material 1-3 is as follows:

[0051]

[0052] Further preferably, the preparation steps of raw material 1-3 are as follows: at room temperature, raw material 1-2, ethanol, water, and sodium hydroxide are placed in a sealed device, mixed and sealed, and then heated to 105-115°C for reaction. After the reaction is completed, the reaction product is diluted, and then the pH value is adjusted to 4.5-5.5, and then extracted, and the organic phase is dried to obtain raw material 1-3;

[0053] Preferably, the preparation formula of raw material 1-2 is as follows:

[0054]

[0055] Further preferably, the preparation steps of the raw material 1-2 are as follows: after mixing the raw material 1-1, 1-octanol and p-toluenesulfonic acid, the temperature is raised to 100-110° C. and the reaction is carried out for a set time; the reaction system is pressurized and spin-dried, and purification is performed to obtain a colorless oily raw material 1-2.

[0056] When the cationic lipid compound is compound 4:

[0057] Said It is raw material 4-2; wherein, the preparation formula of the raw material 4-2 is as follows:

[0058]

[0059] Preferably, the preparation steps of the raw material 4-2 are as follows: reacting raw material 1-3, raw material 4-1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane at room temperature; after the reaction, performing extraction and liquid separation, washing, drying and purifying the organic phase to obtain raw material 4-2.

[0060] When the cationic lipid compound is compound 5:

[0061] Said It is raw material 5-3; wherein, the preparation formula of the raw material 5-3 is as follows:

[0062]

[0063] Preferably, the preparation steps of raw material 5-3 are as follows: reacting raw material 5-2, 6-bromo-1-hexanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane at room temperature; after the reaction, extracting, washing, drying the organic phase and purifying to obtain raw material 5-3;

[0064] Preferably, the preparation formula of the raw material 5-2 is as follows:

[0065]

[0066] Further preferably, the preparation steps of the raw material 5-2 are as follows: at room temperature, the raw material 5-1, ethanol, water and sodium hydroxide are mixed in a reaction container, the reaction container is sealed, and the temperature is raised to 105-115° C. for reaction; after the reaction is completed, the reaction solution is diluted, and then the pH value is adjusted to 4.5-5.5, and then extraction is performed, and the organic phase is dried to obtain the raw material 5-2;

[0067] Preferably, the preparation formula of raw material 5-1 is as follows:

[0068]

[0069] Further preferably, the preparation steps of raw material 5-1 are as follows: at room temperature, raw material 1-1, hemp alcohol and p-toluenesulfonic acid are mixed evenly, and the temperature is raised to 100-110° C. for reaction; after the reaction, the reaction solution is decompressed and dried by spin drying, and then purified to obtain a colorless oily product raw material 5-1.

[0070] When the cationic lipid compound is compound 9:

[0071] Said It is raw material 9-4; wherein, the preparation formula of raw material 9-4 is as follows:

[0072]

[0073] Preferably, the preparation steps of the raw material 9-4 are as follows: reacting the raw material 9-3, 6-bromo-1-hexanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane at room temperature; after the reaction, extracting and separating the liquids, washing, drying the organic phase and purifying it to obtain the raw material 9-4;

[0074] Preferably, the preparation formula of the raw material 9-3 is as follows:

[0075]

[0076] Further preferably, the preparation steps of raw material 9-3 are as follows: reacting raw material 9-2, ethanol, water and sodium hydroxide at room temperature; after the reaction, adjusting the pH to 6.9-7.1, preferably 7; then extracting and drying the organic phase to obtain raw material 9-3;

[0077] Preferably, the preparation formula of raw material 9-2 is as follows:

[0078]

[0079] Further preferably, raw material 9-1, n-octyl mercaptan, boron trifluoride etherate and dichloromethane are reacted at room temperature; after the reaction is completed, quenching and extraction are performed; the organic phase is washed, dried and purified to obtain raw material 9-2.

[0080] Preferably, when the cationic lipid compound is compound 2, compound 3, compound 6, or compound 7, The preparation formula is as follows:

[0081]

[0082] in, For raw materials 2-2

[0083] Preferably, The preparation steps are as follows:

[0084] At room temperature, the raw materials Br-R1, Potassium carbonate and N,N-dimethylformamide are mixed and heated to 75-85°C for reaction. After the reaction is completed, the reaction solution is diluted and washed, and the organic phase is dried and purified to obtain

[0085] Preferably, the preparation formula of raw material 2-2 is as follows:

[0086]

[0087] Further preferably, the preparation steps of raw material 2-2 are as follows: first, raw material 2-1 and dichloromethane solution are added to a reaction container, and then trifluoroacetic acid is added to react; after the reaction is completed, the reaction solution is spin-dried, diluted, washed, and the organic phase is dried and purified to obtain raw material 2-2;

[0088] Preferably, the preparation formula of raw material 2-1 is as follows:

[0089]

[0090] Further preferably, the preparation steps of raw material 2-1 are as follows: sodium hydride and N,N-dimethylformamide are added to a reaction container under ice-water bath conditions, and after dissolution, they are cooled to 0-5°C; then, a tetrahydrofuran solution of raw material 1-7 is added to the reaction system in batches, and the reaction is carried out for a first set time, and then raw material 1-5 is dropped into the reaction system. After the dropwise addition is completed and the reaction is carried out for a second time, the reaction temperature is raised to room temperature, and the reaction is continued for a third set time; the reaction solution is quenched, extracted, separated, dried, and purified to obtain raw material 2-1.

[0091] Preferably, when the cationic lipid compound is compound 2: the Br-R1 is raw material 2-4; wherein the preparation formula of raw material 2-4 is as follows:

[0092]

[0093] Preferably, the preparation steps of the raw material 2-4 are as follows: 6-bromo-1-hexanol, oleic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane are reacted at room temperature; after the reaction is completed, extraction and separation are performed, and then the organic phase is washed, dried and purified to obtain raw material 2-4.

[0094] When the cationic lipid compound is compound 3: the Br-R1 is raw material 3-2; wherein the preparation formula of the raw material 3-2 is as follows:

[0095]

[0096] Preferably, the preparation steps of the raw material 3-2 are as follows: 6-bromohexanoic acid, linoleyl alcohol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane are reacted at room temperature; after the reaction is completed, extraction and separation are performed, and the organic phase is washed, dried and purified to obtain raw material 3-2.

[0097] When the cationic lipid compound is compound 6: the Br-R1 is raw material 6-2; wherein the preparation formula of raw material 6-2 is as follows:

[0098]

[0099] Preferably, the preparation steps of the raw material 6-2 are as follows: 6-bromohexanoic acid, raw material 6-1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane are reacted at room temperature; after the reaction is completed, extraction and separation are performed, and then the organic phase is washed, dried and purified to obtain raw material 6-2;

[0100] When the cationic lipid compound is compound 7: the Br-R1 is raw material 7-2; wherein the preparation formula of raw material 7-2 is as follows:

[0101]

[0102] Preferably, the preparation steps of raw material 7-2 are as follows: 6-bromohexanoic acid, raw material 7-1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane (200 mL) are reacted at room temperature; after the reaction is completed, extraction and separation are performed, and the organic phase is washed, dried and purified to obtain raw material 7-2.

[0103] Preferably, the preparation formula of compound 8 is as follows:

[0104]

[0105] The preparation steps of the compound 8 are as follows: at room temperature, raw material 8-4, tetrabutylammonium fluoride and tetrahydrofuran solution are mixed and reacted; after the reaction is completed, the reaction solution is diluted and washed; the organic phase is dried and purified to obtain compound 8;

[0106] Preferably, the preparation formula of raw material 8-4 is as follows:

[0107]

[0108] Further preferably, the preparation steps of the raw material 8-4 are as follows: at room temperature, the raw material 8-3, octanethiol and benzoin dimethyl ether are mixed to obtain a reaction system; the reaction system is irradiated with ultraviolet light to react; after the reaction is completed, purification is performed to obtain the raw material 8-4;

[0109] Preferably, the preparation formula of the raw material 8-3 is as follows:

[0110]

[0111] Further preferably, the preparation steps of the raw material 8-3 are as follows: at room temperature, raw material 1-9, raw material 8-2, potassium carbonate and N,N-dimethylformamide are mixed, and the temperature is raised to 75-85° C. for reaction; after the reaction is completed, the reaction solution is diluted and washed, and then the organic phase is purified to obtain raw material 8-3;

[0112] Preferably, the preparation formula of raw material 8-2 is as follows:

[0113]

[0114] Further preferably, the preparation steps of the raw material 8-2 are as follows: 8-bromooctanoic acid, propargyl alcohol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, triethylamine and dichloromethane are reacted at room temperature; after the reaction is completed, extraction and separation are performed; then, the organic phase is washed, dried and purified to obtain the raw material 8-2.

[0115] Preferably, the cationic lipid compound described in any one of the above items is used to prepare an mRNA delivery system; preferably, the mRNA delivery system is a LNP composition.

[0116] Preferably, any of the above cationic lipid compounds is used in the preparation of nucleic acid drugs.

[0117] On the other hand, an embodiment of the present invention further provides an mRNA delivery system, wherein the mRNA delivery system comprises any of the cationic lipid compounds described above; preferably, the mRNA delivery system is a LNP composition.

[0118] Preferably, the above-mentioned mRNA delivery system is used in the preparation of nucleic acid drugs.

[0119] Compared with the prior art, the cationic lipid compound, preparation method and application, and mRNA delivery system of the present invention have at least the following beneficial effects:

[0120] On the one hand, the embodiments of the present invention develop a new cationic lipid compound, whose structure is shown in the general formula (I). The cationic lipid compound is used to prepare an mRNA delivery system (LNP composition), which has the characteristics of high delivery efficiency, good spleen targeting, and high biological safety.

[0121] On the other hand, an embodiment of the present invention provides an mRNA delivery system (LNP). Since the mRNA delivery system includes the above-mentioned cationic lipid compound, the mRNA delivery system (LNP) has the characteristics of high delivery efficiency, good spleen targeting and high biosafety.

[0122] In addition, the above-mentioned cationic lipid compounds and mRNA delivery systems (LNP) are mainly used for the preparation of nucleic acid drugs.

[0123] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 It is a graph of cell transfection efficiency of the lipid nanoparticle preparation prepared by compound 1 and the control preparations (MC3, SM-102 lipid nanoparticle preparations) in Example 1 of the present invention; wherein, (a) is a quantitative graph of luciferase activity after the lipid nanoparticle preparation prepared by compound 1 and the control preparation were transfected into cells; (b) is a multiple comparison graph of the transfection efficiency of compound 1 and SM102 lipid nanoparticle preparation relative to MC3 lipid nanoparticle preparation.

[0125] Figure 2 It is a diagram of the in vivo transfection efficiency of the lipid nanoparticle preparation prepared by compound 1 and the control MC3 in Example 3 of the present invention; wherein, (a) is a live imaging diagram 6 hours after intravenous administration of the lipid nanoparticle preparation; (b) is a diagram showing the change trend of the total amount of photons of the luciferase protein expressed by mice over time within 48 hours; (c) is a diagram showing the relative weight change of mice within 7 days.

[0126] Figure 3 This is a comparison chart of the delivery efficiency of the lipid nanoparticle preparation prepared from compounds 1-9 of the present invention and the control in the liver in Example 5 of the present invention.

[0127] Figure 4 This is a comparison chart of the delivery efficiency of the lipid nanoparticle preparation prepared from compounds 1-9 of the present invention and the control in the spleen in Example 5 of the present invention.

[0128] Figure 5 This is a comparison chart of spleen targeting of lipid nanoparticle preparations prepared from compounds 1-9 of the present invention and a control in Example 5 of the present invention. DETAILED DESCRIPTION

[0129] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention application are described in detail below in conjunction with the accompanying drawings and preferred embodiments. In the following description, different "one embodiment" or "embodiment" does not necessarily refer to the same embodiment. In addition, specific features, structures, or characteristics in one or more embodiments may be combined in any suitable form.

[0130] The present invention develops a novel cationic lipid compound, which is used to prepare an mRNA delivery system, and has a technical effect of high delivery efficiency. Here, the structure of the cationic lipid compound is shown in general formula (I):

[0131]

[0132] Wherein, in the general formula (I), R1 is a saturated group with an ester group or an unsaturated group with an ester group; n is 4 to 6; R2 is an alkyl group with a carbon number greater than or equal to 8; and X is O or S.

[0133] Preferably, the cationic lipid compound of the present invention is any one of Compounds 1 to 9. The preparation method of Compounds 1 to 9 is described in detail below.

[0134] 1. Compound 1

[0135]

[0136] The preparation steps of compound 1 are as follows:

[0137]

[0138] At room temperature, in a 100 mL three-necked flask, raw material 1-1 (10.0 g, 63.7 mmol), 1-octanol (24.8 g, 191 mmol), p-toluenesulfonic acid (0.55 g, 3.2 mmol) were added, mixed evenly, heated to 100-110°C, reacted for 72 hours, cooled, and TLC plate showed that the raw material reaction was complete. The reaction system was pressurized and dried, and then silica gel was added for mixing, and a colorless oily product compound 1-2 (raw material 1-2) was obtained by column chromatography.

[0139]

[0140] At room temperature, in a 100mL sealed tube, add raw material 1-2 (5.0g, 15.4mmol), ethanol (15mL), water (15mL) and sodium hydroxide (1.8g, 46.2mmol), mix well and seal, then heat to 105-115°C and react for 16 hours. Cool, TLC spot plate, show that the raw material reaction is complete. Then add ethyl acetate (50mL) to dilute, use 1mol / L hydrochloric acid to neutralize the system to pH 4.5-5.5. Extract twice with ethyl acetate (50mL), dry and spin-dry after the organic phase is combined. The obtained crude product 1-3 (raw material 1-3) is not further purified and is directly used in the next step.

[0141]

[0142] At room temperature, in a 250 mL single-mouth bottle, raw material 1-3 (4.0 g, 11.6 mmol), raw material 1-4 (2.1 g, 11.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.7 g, 13.9 mmol), 4-dimethylaminopyridine (0.15 g, 1.2 mmol), triethylamine (2.3 g, 23.2 mmol) and dichloromethane (100 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (100 mL) and water (100 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and raw material 1-5 was obtained by column chromatography.

[0143]

[0144] At room temperature, in a 500 mL single-mouth bottle, raw material 1-4 (10.0 g, 55.6 mmol), linoleic acid (15.6 g, 55.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.8 g, 66.7 mmol), 4-dimethylaminopyridine (0.68 g, 5.6 mmol), triethylamine (11.2 g, 111 mmol) and dichloromethane (200 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials reacted completely, and then ethyl acetate (400 mL) and water (400 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and raw material 1-6 was obtained by column chromatography.

[0145]

[0146] Under ice-water bath conditions, sodium hydride (60% content, 18.5mmol, 0.74g) and N,N-dimethylformamide (50mL) were added to a 250mL three-necked flask with a thermometer inserted, dissolved, cooled to 0-5°C, and the reaction system was slowly added with a tetrahydrofuran solution (50mL) of raw material 1-7 (4.0g, 13.2mmol) in batches. After reacting for 30 minutes, raw material 1-6 (5.8g, 13.2mmol) was added to the above system through a constant pressure dropping funnel. After the addition was completed, the reaction was heated to room temperature after 1 hour and the reaction was continued for 5 hours. TLC monitoring showed that most of the raw materials reacted completely. Saturated ammonium chloride solution was slowly added to the reaction solution for quenching, and then ethyl acetate (100mL) was added for extraction and separation.

[0147] After drying and spin drying, silica gel is added to mix the sample, and raw material 1-8 is obtained by column chromatography.

[0148]

[0149] At room temperature, in a 100 mL single-mouth bottle, raw material 1-8 (2.0 g, 3.0 mmol) and dichloromethane solution (20 mL) were added, followed by trifluoroacetic acid (5 mL), and the reaction was continued for 1 hour. After TLC monitoring, most of the raw materials were reacted completely. The reaction solution was spin-dried, and then ethyl acetate (100 mL) was added, and washed 2-3 times with saturated sodium bicarbonate (10 mL). The organic phase was dried and spin-dried, and then silica gel was added to mix the sample, and raw material 1-9 was obtained by column chromatography.

[0150]

[0151] At room temperature, in a 100 mL flask, add raw material 1-9 (1.0 g, 1.7 mmol), raw material 1-5 (0.90 g, 1.7 mmol), potassium carbonate (0.47 g, 3.4 mmol) and N, N-dimethylformamide (20 mL), respectively, mix well, heat to 80 ° C, react for 16 hours, cool, and TLC plate, showing that the raw material reaction is complete. Add ethyl acetate (50 mL) to dilute, and wash with water (50 mL). After the organic phase is dried, add silica gel to mix, and obtain raw material 1-10 by column chromatography.

[0152]

[0153] At room temperature, in a 50mL flask, add raw material 1-10 (0.8g, 1.7mmol), tetrabutylammonium fluoride (0.89g, 3.4mmol) and tetrahydrofuran solution (20mL). Mix well and react for 4 hours. TLC spot plate shows that the raw material reaction is complete. Add ethyl acetate (50mL) to dilute and wash with water (50mL). After the organic phase is dried, add silica gel to mix the sample and obtain the product compound 1 by column chromatography. Among them, the NMR data of compound 1 are as follows:

[0154] 1H NMR (400MHz, CDCl3) δ5.32(m,4H),4.46(t,J=5.6Hz,1H),4.02(t,J=6.7Hz,1H),3.58–3.48(m,5H),3.38(m,1H),2.74(t,J=6.4Hz,-1H),2.44–2 .31(m,2H),2.26(t,J=7.5Hz,1H),2.02(q,J=6.9Hz,5H),1.90(q,J=7.0Hz,2H),1.59(m,3H),1.40–1.18(m,24H),0.86(m,9H).C54H103NO7Exact Mass:877.7735,found[M+H] + :878.77845.

[0155] 2. Compound 2

[0156]

[0157] The preparation steps of compound 2 are as follows:

[0158]

[0159] Under ice-water bath conditions, sodium hydride (60% content, 18.5mmol, 0.74g) and N,N-dimethylformamide (50mL) were added to a 250mL three-necked flask with a thermometer inserted, dissolved, cooled to 0-5°C, and the reaction system was slowly added in batches with a tetrahydrofuran solution (20mL) of raw material 1-7 (4.0g, 13.2mmol). After reacting for 30 minutes, raw material 1-5 (6.7g, 13.2mmol) was added to the above system through a constant pressure dropping funnel. After the addition was complete, the reaction was heated to room temperature after 1 hour and the reaction was continued for 5 hours. TLC monitoring showed that most of the raw materials reacted completely. Saturated ammonium chloride solution was slowly added to the reaction solution for quenching, and then ethyl acetate (50mL) was added for extraction and separation.

[0160] After drying and spin drying, silica gel is added to mix the sample, and the raw material 2-1 is obtained by column chromatography.

[0161]

[0162] At room temperature, in a 100mL single-mouth bottle, add raw material 2-1 (3.0g, 4.1mmol) and dichloromethane solution (40mL), then add trifluoroacetic acid (8mL), react for 1 hour, TLC monitoring, most of the raw materials reacted completely, the reaction solution was spin-dried, then ethyl acetate (200mL) was added, and washed 2-3 times with saturated sodium bicarbonate (50mL), the organic phase was dried, spin-dried, and then silica gel was added to mix the sample, and the raw material 2-2 was obtained by column chromatography.

[0163]

[0164] At room temperature, in a 500 mL single-necked bottle, 6-bromo-1-hexanol (10.0 g, 55.6 mmol), oleic acid (2-3, 15.7 g, 55.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.8 g, 66.7 mmol), 4-dimethylaminopyridine (0.68 g, 5.6 mmol), triethylamine (11.2 g, 111 mmol) and dichloromethane (200 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (400 mL) and water (400 mL) were added for extraction and separation. The organic phase was then washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 2-4 was obtained by column chromatography.

[0165]

[0166] At room temperature, in a 100 mL flask, raw material 2-4 (1.0 g, 2.2 mmol), raw material 2-2 (1.38 g, 2.2 mmol), potassium carbonate (0.61 g, 4.4 mmol) and N, N-dimethylformamide (20 mL) were added respectively, mixed evenly, heated to 80 ° C for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute and washed with water (50 mL). After the organic phase was dried, silica gel was added and mixed, and raw material 2-5 was obtained by column chromatography.

[0167]

[0168] At room temperature, in a 50 mL flask, add raw material 2-5 (0.6 g, 1.3 mmol), tetrabutylammonium fluoride (0.68 g, 2.6 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC spot plate shows that the raw material reaction is complete. Add ethyl acetate (30 mL) to dilute and wash with water (30 mL). After the organic phase is dried, add silica gel and mix the sample, and obtain the product compound 2 by column chromatography.

[0169] 3. Compound 3

[0170]

[0171] The preparation steps of compound 3 are as follows:

[0172]

[0173] At room temperature, in a 500 mL single-necked bottle, 6-bromohexanoic acid (10.0 g, 51.5 mmol), linoleyl alcohol (raw material 3-1, 13.7 g, 51.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.8 g, 61.8 mmol), 4-dimethylaminopyridine (0.63 g, 5.2 mmol), triethylamine (10.4 g, 103 mmol) and dichloromethane (200 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (400 mL) and water (400 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 3-2 was obtained by column chromatography.

[0174]

[0175] At room temperature, in a 100 mL flask, raw material 3-2 (1.0 g, 2.2 mmol), raw material 2-2 (1.4 g, 2.2 mmol), potassium carbonate (0.61 g, 4.4 mmol) and N, N-dimethylformamide (20 mL) were added respectively, mixed evenly, heated to 80 ° C for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute and washed with water (50 mL). After the organic phase was dried, silica gel was added to mix the sample, and raw material 3-3 was obtained by column chromatography.

[0176]

[0177] At room temperature, in a 50 mL flask, add raw material 3-3 (1.0 g, 1.0 mmol), tetrabutylammonium fluoride (0.53 g, 2.0 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material is completely reacted. Add ethyl acetate (30 mL) to dilute and wash with water (30 mL). After the organic phase is dried, add silica gel to mix the sample and obtain the product compound 3 by column chromatography.

[0178] 4. Compound 4

[0179]

[0180] The preparation steps of compound 4 are as follows:

[0181]

[0182] At room temperature, in a 250 mL single-mouth bottle, raw material 1-3 (4.0 g, 11.6 mmol), raw material 4-1 (1.8 g, 11.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.7 g, 13.9 mmol), 4-dimethylaminopyridine (0.15 g, 1.2 mmol), triethylamine (2.3 g, 23.2 mmol) and dichloromethane (50 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (50 mL) and water (50 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the product 4-2 was obtained by column chromatography.

[0183]

[0184] At room temperature, in a 500mL single-necked bottle, raw material 4-1 (10.0g, 65.8mmol), linoleic acid (18.4g, 65.8mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12.6g, 65.8mmol), 4-dimethylaminopyridine (0.81g, 6.6mmol), triethylamine (13.3g, 132mmol) and dichloromethane (250mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (500mL) and water (500mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 4-3 was obtained by column chromatography.

[0185]

[0186] Under ice-water bath conditions, sodium hydride (60% content, 18.5mmol, 0.74g) and N,N-dimethylformamide (50mL) were added to a 250mL three-necked flask with a thermometer inserted, dissolved, cooled to 0-5°C, and the reaction system was slowly added in batches to a tetrahydrofuran solution (50mL) of raw material 1-7 (4.0g, 13.2mmol). After reacting for 30 minutes, raw material 4-3 (5.5g, 13.2mmol) was added to the above system through a constant pressure dropping funnel. After the addition was complete, the reaction was heated to room temperature after 1 hour and the reaction was continued for 5 hours. TLC monitoring showed that most of the raw materials reacted completely. Saturated ammonium chloride solution was slowly added to the reaction solution for quenching, and then ethyl acetate (100mL) was added for extraction and separation.

[0187] After drying and spin drying, silica gel is added to mix the sample, and the raw material 4-4 is obtained by column chromatography.

[0188]

[0189] At room temperature, in a 100mL single-mouth bottle, raw material 4-4 (2.0g, 3.1mmol) and dichloromethane solution (20mL) were added, and then trifluoroacetic acid (5mL) was added. The reaction was carried out for 1 hour. Under TLC monitoring, most of the raw materials were reacted completely. The reaction solution was spin-dried, and then ethyl acetate (100mL) was added, and washed with saturated sodium bicarbonate (50mL) 2-3 times. The organic phase was dried and spin-dried, and then silica gel was added to mix the sample, and the raw material 4-5 was obtained by column chromatography.

[0190]

[0191] At room temperature, in a 100 mL flask, raw material 4-5 (1.0 g, 1.9 mmol), raw material 4-2 (0.89 g, 1.9 mmol), potassium carbonate (0.52 g, 3.8 mmol) and N, N-dimethylformamide (20 mL) were added, mixed evenly, heated to 80 ° C, reacted for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute, and washed with water (50 mL).

[0192] After the organic phase is dried, silica gel is added to mix the sample, and raw material 4-6 is obtained by column chromatography.

[0193]

[0194] At room temperature, in a 50 mL flask, add raw material 4-6 (0.8 g, 0.9 mmol), tetrabutylammonium fluoride (0.47 g, 1.8 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material reaction is complete. Add ethyl acetate (50 mL) to dilute and wash with water (30 mL). After the organic phase is dried, add silica gel to mix the sample and obtain the product compound 4 by column chromatography.

[0195] 5. Compound 5

[0196]

[0197] The preparation steps of compound 5 are as follows:

[0198]

[0199] At room temperature, in a 250mL three-necked flask, raw material 1-1 (10.0g, 63.7mmol), n-decyl alcohol (30.1g, 19mmol), p-toluenesulfonic acid (0.55g, 3.2mmol) were added respectively, mixed evenly, heated to 105°C for 72 hours, cooled, and TLC plate showed that the raw material reaction was complete. The reaction system was dried under reduced pressure, and then silica gel was added for mixing, and a colorless oily product raw material 5-1 was obtained by column chromatography.

[0200]

[0201] At room temperature, in a 100mL sealed tube, add raw material 5-1 (5.0g, 13.1mmol), ethanol (15mL), water (15mL) and sodium hydroxide (1.6g, 39.3mmol), mix well and seal, then heat to 110°C and react for 16 hours. Cool, TLC plate, show that the raw material reaction is complete. Then add ethyl acetate (50mL) to dilute, use 1mol / L hydrochloric acid to neutralize the system to pH = 5.0. Extract twice with ethyl acetate (50mL), dry and spin dry after combining the organic phases. The obtained crude product 5-2 is not further purified and used directly in the next step.

[0202]

[0203] At room temperature, in a 250 mL single-necked bottle, raw material 5-2 (4.0 g, 10.0 mmol), 6-bromo-1-hexanol (1.8 g, 10.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12.0 mmol), 4-dimethylaminopyridine (0.12 g, 1.0 mmol), triethylamine (2.2 g, 20.0 mmol) and dichloromethane (100 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials reacted completely, and then ethyl acetate (100 mL) and water (100 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 5-3 was obtained by column chromatography.

[0204]

[0205] At room temperature, in a 100 mL flask, raw material 5-3 (1.0 g, 1.8 mmol), raw material 1-9 (1.2 g, 1.8 mmol), potassium carbonate (0.5 g, 3.6 mmol) and N, N-dimethylformamide (20 mL) were added respectively, mixed evenly, heated to 80 ° C for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute and washed with water (50 mL). After the organic phase was dried, silica gel was added and mixed, and raw material 5-4 was obtained by column chromatography.

[0206]

[0207] At room temperature, in a 50 mL flask, add raw material 5-4 (1.0 g, 1.0 mmol), tetrabutylammonium fluoride (0.5 g, 2.0 mmol) and tetrahydrofuran solution (20 mL). Mix well and react for 4 hours. TLC plate shows that the raw material reaction is complete. Add ethyl acetate (50 mL) to dilute and wash with water (50 mL). After the organic phase is dried, add silica gel to mix the sample and obtain the product compound 5 by column chromatography.

[0208] 6. Compound 6

[0209]

[0210] The preparation steps of compound 6 are as follows:

[0211]

[0212] At room temperature, in a 500mL single-mouth bottle, 6-bromohexanoic acid (10.0g, 51.0mmol), raw material 6-1 (9.9g, 51.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.8g, 51.0mmol), 4-dimethylaminopyridine (0.62g, 5.1mmol), triethylamine (10.3g, 102mmol) and dichloromethane (200mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials reacted completely, and then ethyl acetate (400mL) and water (400mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 6-2 was obtained by column chromatography.

[0213]

[0214] At room temperature, in a 100 mL flask, raw material 6-2 (1.0 g, 2.7 mmol), raw material 2-2 (1.7 g, 2.7 mmol), potassium carbonate (0.75 g, 5.4 mmol) and N, N-dimethylformamide (20 mL) were added respectively, mixed evenly, heated to 80 ° C, reacted for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute and washed with water (50 mL). After the organic phase was dried, silica gel was added to mix the sample, and raw material 6-3 was obtained by column chromatography.

[0215]

[0216] At room temperature, in a 50 mL flask, add raw material 6-3 (1.0 g, 1.1 mmol), tetrabutylammonium fluoride (0.57 g, 2.2 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material is completely reacted. Add ethyl acetate (30 mL) to dilute and wash with water (30 mL). After the organic phase is dried, add silica gel and mix the sample, and obtain the product compound 6 by column chromatography.

[0217] 7. Compound 7

[0218]

[0219] The preparation steps of compound 7 are as follows:

[0220]

[0221] At room temperature, in a 500mL single-mouth bottle, 6-bromohexanoic acid (10.0g, 43.9mmol), raw material 7-1 (8.5g, 43.9mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.4g, 43.9mmol), 4-dimethylaminopyridine (0.54g, 4.4mmol), triethylamine (8.9g, 87.8mmol) and dichloromethane (200mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (400mL) and water (400mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 7-2 was obtained by column chromatography.

[0222]

[0223] At room temperature, in a 100 mL flask, raw material 7-2 (1.0 g, 2.5 mmol), raw material 2-2 (1.6 g, 2.5 mmol), potassium carbonate (0.69 g, 5.0 mmol) and N, N-dimethylformamide (20 mL) were added respectively, mixed evenly, heated to 80 ° C for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (50 mL) was added to dilute and washed with water (50 mL). After the organic phase was dried, silica gel was added and mixed, and raw material 7-3 was obtained by column chromatography.

[0224]

[0225] At room temperature, in a 50 mL flask, add raw material 7-3 (1.0 g, 1.0 mmol), tetrabutylammonium fluoride (0.53 g, 2.0 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material is completely reacted. Add ethyl acetate (30 mL) to dilute and wash with water (30 mL). After the organic phase is dried, add silica gel to mix the sample and obtain the product compound 7 by column chromatography.

[0226] 8. Compound 8

[0227]

[0228] The preparation steps of compound 8 are as follows:

[0229]

[0230] At room temperature, in a 500mL single-mouth bottle, add 8-bromooctanoic acid (raw material 8-1, 10.0g, 45.0mmol), propargyl alcohol (2.5g, 45.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.6g, 45.0mmol), 4-dimethylaminopyridine (0.55g, 4.5mmol), triethylamine (9.1g, 90.0mmol) and dichloromethane (200mL). After reacting overnight, TLC monitoring showed that most of the raw materials reacted completely, and then ethyl acetate (400mL) and water (400mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried, spin-dried, and then silica gel was added for mixing, and the raw material 8-2 was obtained by column chromatography.

[0231]

[0232] At room temperature, in a 100 mL flask, raw material 1-9 (2.0 g, 3.6 mmol), raw material 8-2 (0.92 g, 3.6 mmol), potassium carbonate (1.0 g, 7.2 mmol) and N, N-dimethylformamide (50 mL) were added respectively, mixed evenly, heated to 80 ° C for 16 hours, cooled, and TLC plate showed that the raw material reaction was complete. Ethyl acetate (100 mL) was added to dilute and washed with water (100 mL). After the organic phase was dried, silica gel was added and mixed, and raw material 8-3 was obtained by column chromatography.

[0233]

[0234] At room temperature, raw material 8-3 (0.5 g, 0.67 mmol), octanol (0.55 g, 3.8 mmol) and benzoin dimethyl ether (20 mg, 4% wt / wt) were added to a 20 mL reaction bottle and mixed well. The reaction system was heated to 10 mW / cm 2 The reaction mixture was irradiated with ultraviolet light for 3 minutes, and TLC showed that the reaction of the raw material was complete. Silica gel was then added to mix the sample, and the raw material 8-4 was obtained by column chromatography.

[0235]

[0236] At room temperature, in a 25 mL flask, add raw material 8-4 (0.5 g, 0.5 mmol), tetrabutylammonium fluoride (0.26 g, 1.0 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material is completely reacted. Add ethyl acetate (50 mL) to dilute and wash with water (50 mL). After the organic phase is dried, add silica gel and mix the sample, and obtain the product compound 8 by column chromatography.

[0237] 9. Compound 9

[0238]

[0239] The preparation steps of compound 9 are as follows:

[0240]

[0241] At room temperature, in a 250 mL single-mouth bottle, add raw material 9-1 (2.0 g, 15.4 mmol), n-octyl mercaptan (5.8 g, 40 mmol), boron trifluoride etherate (2.2 g, 15.4 mmol) and dichloromethane (100 mL). The reaction was carried out at room temperature for 45 minutes. TLC monitoring showed that most of the raw materials reacted completely, and then water (100 mL) was added for quenching. Dichloromethane (50 mL) was extracted twice, and the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried, spin-dried, and then silica gel was added for mixing, and raw material 9-2 was obtained by column chromatography.

[0242]

[0243] At room temperature, in a 100mL flask, add raw material 9-2 (1.0g, 15.4mmol), ethanol (15mL), water (15mL) and sodium hydroxide (1.8g, 46.2mmol), mix well, and react at room temperature for 4 hours. Cool, TLC plate, show that the raw material reaction is complete. Use 1mol / L hydrochloric acid to neutralize the system to pH = 7.0. Extract twice with dichloromethane (50mL), dry and spin dry after combining the organic phases. The obtained crude product 9-3 is not further purified and is directly used in the next step.

[0244]

[0245] At room temperature, in a 50 mL single-mouth bottle, raw material 9-3 (1.5 g, 4.0 mmol), 6-bromo-1-hexanol (0.72 g, 4.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.77 g, 4.0 mmol), 4-dimethylaminopyridine (49 mg, 0.4 mmol), triethylamine (0.81 g, 8.0 mmol) and dichloromethane (20 mL) were added. After reacting overnight, TLC monitoring showed that most of the raw materials were reacted completely, and then ethyl acetate (50 mL) and water (50 mL) were added for extraction and separation, and then the organic phase was washed with saturated ammonium chloride 2-3 times, the organic phase was dried and spin-dried, and then silica gel was added for mixing, and the raw material 9-4 was obtained by column chromatography.

[0246]

[0247] At room temperature, in a 100 mL flask, add raw material 9-4 (1.0 g, 1.9 mmol), raw material 1-9 (1.0 g, 1.9 mmol), potassium carbonate (0.51 g, 3.7 mmol) and N, N-dimethylformamide (20 mL), mix well, heat to 80 ° C, react for 16 hours, cool, and TLC plate, showing that the raw material reaction is complete. Add ethyl acetate (50 mL) to dilute and wash with water (50 mL). After the organic phase is dried, add silica gel to mix and obtain raw material 9-5 by column chromatography.

[0248]

[0249] At room temperature, in a 50 mL flask, add raw material 9-5 (0.8 g, 0.8 mmol), tetrabutylammonium fluoride (0.41 g, 1.6 mmol) and tetrahydrofuran solution (10 mL). Mix well and react for 4 hours. TLC plate shows that the raw material is completely reacted. Add ethyl acetate (50 mL) to dilute and wash with water (50 mL). After the organic phase is dried, add silica gel and mix the sample, and obtain the product compound 9 by column chromatography.

[0250] The following uses Compound 1 as an example to illustrate the technical effect of the cationic lipid compound of the present invention applied in the mRNA delivery system LNP.

[0251] Example 1

[0252] Preparation and testing of lipid nanoparticle formulations.

[0253] 1. Preparation steps:

[0254] The compound 1 of the present invention was dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution. In addition, luciferase (Fluc) mRNA was diluted with 50 mM citrate buffer (pH=4) to obtain an mRNA aqueous solution.

[0255] Lipid nanoparticles were prepared by mixing ethanol lipid solution and mRNA aqueous solution in a volume ratio of 1:3 through a microfluidic device, and then dialyzed against 1X PBS for 18 hours to remove ethanol and complete the citrate buffer exchange process. Finally, the lipid nanoparticle solution was filtered through 0.2 μm sterile filtration and ultrafiltration concentration to obtain the compound 1 lipid nanoparticle preparation encapsulating luciferase mRNA.

[0256] In addition, MC3 lipid nanoparticles were prepared using the same method as a control.

[0257] 2. Detection steps

[0258] Using Litesizer TM The lipid nanoparticle size, polydispersity index (PDI) and potential (Zeta) were measured by 500 (Anton Paar, Austria). The particle size and potential were measured in 0.1% PBS. The encapsulation efficiency of the lipid nanoparticle preparation was detected by the RiboGreen method. The test results are shown in Table 1:

[0259] Table 1

[0260] Preparation name Particle size PDI (%) Zeta EE(%) MC3-Luc 95.53±6.11 20.2±2.35 17.0±6.56 95.6±3.07 Compound 1-Luc 112.16±0.66 21.8±9.69 23.1±3.04 89.7±1.56

[0261] As can be seen from Table 1, compound 1 of the present invention as an ionizable lipid can form lipid nanoparticles.

[0262] Example 2

[0263] Lipid nanoparticle formulations and cell transfection.

[0264] The lipid nanoparticle preparation of Compound 1 encapsulating Fluc mRNA in Example 1 was prepared. Meanwhile, lipid nanoparticle preparations of MC3 and SM-102 were prepared as controls.

[0265] The above three preparations were diluted to 160 ng / mL with culture medium and added to Hek293 cells. After 24 hours, the culture medium was removed and washed with PBS, then the cell lysis solution was added and incubated for 10 minutes. After lysis, luciferase substrate was added to complete the detection. The test results are as follows Figure 1 shown.

[0266] from Figure 1 As shown in Figures (a) and (b), the cell transfection ability of the lipid nanoparticle preparation of compound 1 of the present invention is enhanced by more than ten times compared with the MC3 lipid nanoparticle preparation, and is comparable to the transfection ability of the lipid nanoparticle preparation of SM-102. It can be seen that the compound of the present invention has excellent delivery efficiency when applied to the mRNA delivery system.

[0267] Example 3

[0268] Lipid nanoparticle formulations for in vivo transfection.

[0269] BALB / c mice were divided into two groups, 3 mice in each group, and each group was injected intravenously with a single dose of 5 μg / mouse of MC3 or compound 1 lipid nanoparticle preparation. Luciferase substrate was injected intraperitoneally at 0, 6, 10, 24, and 48 hours, and imaging and quantification were performed using a live imaging instrument after waiting for 10 minutes. In addition, to examine the biosafety of the compound 1 lipid nanoparticle preparation, the MC3 lipid nanoparticle preparation was used as a control, and the changes in mouse body weight were recorded before administration, 6h, 1 day, 2 days, 4 days, and 7 days after administration.

[0270] 6h in vivo imaging results (see Figure 2 (a) in Figure 3 shows that the in vivo transfection efficiency of the lipid nanoparticle preparation of compound 1 of the present invention is higher than that of the lipid nanoparticle preparation of MC3 and the lipid nanoparticle preparation of compound 1 can achieve sustained high expression of Fluc in vivo (see Figure 2 In addition, the lipid nanoparticle formulation of compound 1 did not cause a significant decrease in body weight (see Figure 2 (c) in the figure), indicating that it has good biosafety.

[0271] In summary, the ionizable lipid compound 1 of the present invention, when used for mRNA delivery in vivo and in vitro, has the technical effects of high delivery efficiency, good spleen targeting, and high biosafety.

[0272] The following uses Compound 1-9 as an example to further illustrate the technical effect of the cationic lipid compound of the present invention in the mRNA delivery system LNP.

[0273] Example 4

[0274] Preparation and testing of lipid nanoparticle formulations.

[0275] 1. Preparation steps:

[0276] The above compound of the present invention was dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:15:34.5:0.5 to prepare ethanol lipid solutions.

[0277] The N1-methyl-pseudouridine-modified luciferase mRNA was diluted with 50 mM citrate buffer (pH=4.0) to obtain an mRNA aqueous solution.

[0278] Lipid nanoparticles were prepared by mixing ethanol lipid solution and mRNA aqueous solution in a volume ratio of 1:3 by microfluidic device, and dialyzed against 1X PBS for 18 hours to remove ethanol and complete the citrate buffer exchange process. Finally, the lipid nanoparticle solution was sterile filtered (0.2 μm) and ultrafiltration concentrated to obtain lipid nanoparticle preparations encapsulating luciferase mRNA (respectively referred to as: compound 1 LNP, compound 2 LNP, compound 3 LNP, compound 4 LNP, compound 5 LNP, compound 6 LNP, compound 7 LNP, compound 8 LNP, compound 9 LNP).

[0279] In addition, MC3 and SM102 lipid nanoparticle preparations were prepared by the same method as controls.

[0280] 2. Detection steps

[0281] Using Litesizer TM The particle size, polydispersity index (PDI) and potential (Zeta) of lipid nanoparticles were measured by 500 (Anton Paar, Austria). The particle size and potential were measured in 0.1% PBS. The encapsulation efficiency of lipid nanoparticle preparations was detected by RiboGreen method, and the test results are shown in Table 2:

[0282] Table 2

[0283] Preparation name Particle size (nm) PDI (%) Zeta(mV) EE(%) MC3 LNP 148.37 15.3 10.8 92.86 SM102 LNP 131.04 7.3 1.4 86.08 Compound 1 LNP 155.66 13.3 8.7 86.55 Compound 2 LNP 190.53 7.1 10.6 87.79 Compound 3 LNP 167.82 14.9 5.9 90.16 Compound 4 LNP 196.87 6.2 4.7 88.99 Compound 5 LNP 150.34 5.2 1.8 90.77 Compound 6 LNP 214.60 17.3 9.5 56.36 Compound 7 LNP 165.38 3.6 4.3 92.30 Compound 8 LNP 127.27 18.6 3.6 92.33 Compound 9 LNP 175.53 10.9 6.2 93.23

[0284] As can be seen from Table 2, compounds 1-9 of the present invention as cationic lipids can form lipid nanoparticles.

[0285] Example 5

[0286] Lipid nanoparticle formulations for in vivo transfection.

[0287] BALB / c mice were divided into 11 groups, three mice in each group. Each group was injected with MC3 or SM102 or compound 1 or compound 2 or compound 3 or compound 4 or compound 5 or compound 6 or compound 7 or compound 8 or compound 9 lipid nanoparticle formulations once via tail vein (0.3 mg / kg dose). Specifically, the first group was injected with a control MC3 lipid nanoparticle formulation (MC3LNP), the second group was injected with a control SM102 lipid nanoparticle formulation (SM102 LNP), the third group was injected with a compound 1 lipid nanoparticle formulation (Compound 1LNP), the fourth group was injected with a compound 2 lipid nanoparticle formulation (Compound 2LNP), the fifth group was injected with a compound 3 lipid nanoparticle formulation (Compound 3LNP), the sixth group was injected with a compound 4 lipid nanoparticle formulation (Compound 4LNP), the seventh group was injected with a compound 5 lipid nanoparticle formulation (Compound 5LNP), the eighth group was injected with a compound 6 lipid nanoparticle formulation (Compound 6LNP), the ninth group was injected with a compound 7 lipid nanoparticle formulation (Compound 7LNP), the tenth group was injected with a compound 8 lipid nanoparticle formulation (Compound 8LNP), and the eleventh group was injected with a compound 9 lipid nanoparticle formulation (Compound 9LNP).

[0288] After 6 hours, the luciferase substrate was injected intraperitoneally. After waiting for 10 minutes, the liver, spleen, lungs, kidneys and other organs were dissected and removed. The fluorescence signal was observed and quantified using a small animal in vivo imaging system.

[0289] According to the tail vein injection results, it can be seen that the delivery efficiency of the lipid nanoparticle preparations of compounds 1-9 of the present invention in the liver is significantly lower than that of the control SM102 lipid nanoparticle preparation (see Figure 3The delivery efficiency of the lipid nanoparticle preparations of compounds 1-9 in the spleen is equivalent to or higher than that of the SM102 lipid nanoparticle preparations, and is significantly higher than that of the MC3 lipid nanoparticle preparations (see Figure 4 The spleen targeting of the lipid nanoparticle preparations of the compounds 1-9 of the present invention is significantly higher than that of the lipid nanoparticle preparations of SM102 and MC3 (see Figure 5 shown).

[0290] In summary, the ionizable lipid compound 1-9 developed by the present invention has the technical effects of high delivery efficiency and good spleen targeting in in vivo mRNA delivery.

[0291] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A cationic lipid compound, characterized in that The cationic lipid compound is any one of the following compounds:

2. The method for preparing the cationic lipid compound according to claim 1, characterized in that: The preparation formula of the cationic lipid compound is as follows:

3. The method for preparing a cationic lipid compound according to claim 2, characterized in that: The preparation steps of the cationic lipid compound are as follows: at room temperature, Tetrabutylammonium fluoride and tetrahydrofuran are mixed and reacted; after the reaction is completed, dilution and washing are performed; then the organic phase is dried and purified to obtain a cationic lipid compound.

4. The method for preparing a cationic lipid compound according to claim 2, characterized in that: If the cationic lipid compound is compound 4, compound 5, or compound 9, The preparation formula is as follows:

5. The method for preparing a cationic lipid compound according to claim 4, characterized in that: The preparation steps are as follows: At room temperature, Potassium carbonate and N,N-dimethylformamide are mixed and heated to 75-85°C for reaction. After the reaction is completed, dilution and washing are performed. The organic phase is then dried and purified to obtain 6. The method for preparing a cationic lipid compound according to claim 4, characterized in that: When the cationic lipid compound is compound 5 or compound 9: Said All raw materials 1-9 Wherein, the preparation formula of raw material 1-9 is as follows:

7. The method for preparing a cationic lipid compound according to claim 6, characterized in that: The preparation formula of raw material 1-8 is as follows:

8. The method for preparing a cationic lipid compound according to claim 7, characterized in that: The preparation formula of raw material 1-6 is as follows:

9. The method for preparing a cationic lipid compound according to claim 4, characterized in that: When the cationic lipid compound is compound 4: Said It is raw material 4-5; wherein, the preparation formula of raw material 4-5 is as follows:

10. The method for preparing a cationic lipid compound according to claim 9, characterized in that: The preparation formula of raw material 4-4 is as follows:

11. The method for preparing a cationic lipid compound according to claim 10, characterized in that: The preparation formula of raw material 4-3 is as follows:

12. The method for preparing a cationic lipid compound according to claim 4, characterized in that: When the cationic lipid compound is compound 4: Said It is raw material 4-2; wherein, the preparation formula of the raw material 4-2 is as follows:

13. The method for preparing a cationic lipid compound according to claim 4, characterized in that: When the cationic lipid compound is compound 5: Said It is raw material 5-3; wherein, the preparation formula of the raw material 5-3 is as follows:

14. The method for preparing a cationic lipid compound according to claim 13, characterized in that: The preparation formula of the raw material 5-2 is as follows:

15. The method for preparing a cationic lipid compound according to claim 14, characterized in that: The preparation formula of raw material 5-1 is as follows:

16. The method for preparing a cationic lipid compound according to claim 4, characterized in that: When the cationic lipid compound is compound 9: Said It is raw material 9-4; wherein, the preparation formula of raw material 9-4 is as follows:

17. The method for preparing a cationic lipid compound according to claim 16, characterized in that: The preparation formula of the raw material 9-3 is as follows:

18. The method for preparing a cationic lipid compound according to claim 17, characterized in that: The preparation formula of raw material 9-2 is as follows:

19. The method for preparing a cationic lipid compound according to claim 2, characterized in that: If the cationic lipid compound is compound 2, compound 3, compound 6, or compound 7, The preparation formula is as follows: in, For raw materials 2-2 20. The method for preparing a cationic lipid compound according to claim 19, characterized in that: The preparation steps are as follows: At room temperature, the raw materials Br-R1, Potassium carbonate and N,N-dimethylformamide are mixed and heated to 75-85°C for reaction. After the reaction is completed, the reaction solution is diluted and washed, and the organic phase is dried and purified to obtain 21. The method for preparing a cationic lipid compound according to claim 20, characterized in that: The preparation formula of raw material 2-2 is as follows:

22. The method for preparing a cationic lipid compound according to claim 21, characterized in that 5, the preparation formula of raw material 2-1 is as follows:

23. The method for preparing a cationic lipid compound according to claim 19, characterized in that: When the cationic lipid compound is compound 2: The Br-R1 is raw material 2-4; wherein the preparation formula of raw material 2-4 is as follows:

24. The method for preparing a cationic lipid compound according to claim 19, characterized in that: When the cationic lipid compound is compound 3: The Br-R1 is raw material 3-2; wherein the preparation formula of the raw material 3-2 is as follows:

25. The method for preparing a cationic lipid compound according to claim 19, characterized in that: When the cationic lipid compound is compound 6: The Br-R1 is raw material 6-2; wherein the preparation formula of raw material 6-2 is as follows:

26. The method for preparing a cationic lipid compound according to claim 19, characterized in that: When the cationic lipid compound is compound 7: The Br-R1 is raw material 7-2; wherein the preparation formula of raw material 7-2 is as follows:

27. Use of the cationic lipid compound according to claim 1 in preparing an mRNA delivery system.

28. Use of the cationic lipid compound according to claim 27 in preparing an mRNA delivery system, characterized in that: The mRNA delivery system is a LNP composition.

29. Use of the cationic lipid compound according to claim 1 in the preparation of nucleic acid drugs.

30. An mRNA delivery system, characterized in that The mRNA delivery system comprises the cationic lipid compound of claim 1.

31. The mRNA delivery system according to claim 30, characterized in that The mRNA delivery system is a LNP composition.

32. Use of the mRNA delivery system according to claim 30 or 31 in the preparation of nucleic acid drugs.

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