A triglyceride-derived ionizable lipid compound, preparation method thereof, drug delivery carrier and application thereof

Abstract

The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to an ionizable lipid compound derived from triglycerides, a preparation method thereof, a drug delivery carrier and an application thereof. The chemical structural formula of the ionizable lipid compound derived from triglycerides is shown in formula (I): Formula (I). The ionizable lipid compound has the advantages of high degradability, high safety, and being conducive to the release of the carried nucleic acid drug. The drug delivery carrier containing the compound has excellent properties such as good in vivo targeting, high biological safety, and high mRNA expression efficiency. The target organs and tissues of the drug delivery carrier containing the compound include tumors, heart, liver, spleen, lungs, kidneys, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to an ionizable lipid compound derived from triglyceride, a preparation method thereof, a drug delivery carrier and application thereof. Background Art

[0002] Nucleic acid drugs, including DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA) and mRNA, have attracted extensive attention in recent years due to their great potential in treating cancer, genetic diseases and viral diseases. However, the application of these drugs faces the challenge of unstable chemical properties.

[0003] Nucleic acid drugs are easily degraded into single nucleotides by nucleases in vitro and in vivo, resulting in loss of efficacy. Therefore, the effective delivery of nucleic acid drugs needs to rely on special vector systems. Commonly used delivery vectors include viral vectors and non-viral vectors: viral vectors, such as retroviruses, lentiviruses, and adeno-associated viruses, can efficiently deliver nucleic acid drugs to target cells with their natural cell infection ability. However, the clinical application of viral vectors is limited by their high immunogenicity, limited loading capacity, and complex production processes. In contrast, non-viral vectors have become the focus of research in the field of nucleic acid drug delivery due to their low immunogenicity and high safety. Non-viral vectors mainly encapsulate nucleic acid drugs in the carrier through the electrostatic interaction between cationic materials and the phosphate ions of nucleic acid drugs, thereby protecting the drugs from degradation and changing their cellular entry pathways.

[0004] In non-viral vectors, cationic lipids and cationic polymers are often used as delivery materials. However, due to their strong positive charge, these carriers are easily bound to plasma proteins and then cleared by the reticuloendothelial system (RES), resulting in reduced delivery efficiency. In addition, strong positive charge may also lead to excessive adsorption of cell membranes, affecting the efficiency of vector entry into cells. In order to solve this problem, lipid nanoparticles based on ionizable lipids have received extensive attention in recent years. Ionizable lipids have the ability to adjust the charge state under different environments, making these nanoparticles positively charged in the acidic environment in vitro and able to effectively load nucleic acid drugs. After entering the neutral environment in vivo, they are neutrally charged, reducing the adsorption of plasma proteins and the capture of the reticuloendothelial system, thereby improving the delivery efficiency. Therefore, nanoparticles based on ionizable lipids have broad application prospects in the delivery of nucleic acid drugs. Despite this, the current clinical application of ionizable lipid nanoparticles still faces certain challenges, especially in terms of safety and delivery efficiency. Therefore, the development of more efficient and safe ionizable lipid materials remains a key issue in the widespread application of nucleic acid drugs.

[0005] Triglycerides (TG) are the main form of energy storage in organisms. They exist in adipose tissue and can provide energy when needed by breaking down into glycerol and fatty acids. In addition to energy storage, triglycerides also have the function of heat preservation and protection of internal organs, helping to maintain stable body temperature and reduce damage to organs caused by mechanical impact. In addition, fatty acids in triglycerides are important components of cell membrane phospholipids, and adipose tissue participates in weight regulation and metabolic balance by secreting hormones such as leptin and adiponectin. Triglycerides are also closely related to the absorption and transportation of fat-soluble vitamins (such as vitamins A, D, E, and K), and are essential for a variety of physiological functions of the body.

[0006] Triglycerides (TG) not only play a role in energy storage and supply in the body, but also play an important role in the occurrence, development and metabolism of tumors. In tumor cells, triglycerides are widely used as energy reserves, especially when tumor cells proliferate rapidly. Tumor cells often rely on a large amount of energy to support their high metabolic needs, and triglycerides provide energy support by breaking down into fatty acids and participating in the β-oxidation process. In addition, triglycerides may affect tumor growth, invasion and metastasis by changing lipid metabolism in the tumor microenvironment.

[0007] In view of the above reasons, the present invention is proposed. Summary of the invention

[0008] Based on the above technical background, the main purpose of the present invention is to provide a triglyceride-derived ionizable lipid compound, a preparation method thereof, a drug delivery carrier and application thereof, so as to overcome the deficiencies in the prior art.

[0009] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0010] The first aspect of the present invention is to provide an ionizable lipid compound derived from triglycerides, wherein the chemical structural formula of the ionizable lipid compound derived from triglycerides is as shown in formula (I):

[0011] Formula (I);

[0012] In formula (I), G is (CH2) k , where k is an integer ≥ 1;

[0013] B1 and B2 are each independently selected from (CH2) x One of CH3, wherein x is 0 or an integer greater than 0;

[0014] M1 and M2 are each independently selected from (CH2) n or (CH2) n -SS-(CH2) n , where n is an integer ≥ 1;

[0015] R1 and R2 are each independently selected from a C1-C30 straight-chain or branched alkyl group, a C2-C30 alkene group or a C2-C30 alkyne group.

[0016] The ionizable lipid compound of the present invention has very high specificity and efficiency in nucleic acid drug delivery, especially tumor-targeted mRNA delivery.

[0017] Preferably, in formula (I), G is (CH2) k , where 1≤k≤10 is an integer;

[0018] B1 and B2 are each independently selected from one of CH3, CH2CH3, (CH2)2CH3;

[0019] M1 and M2 are each independently selected from one of (CH2)6, (CH2)4, (CH2)2-SS-(CH2)2, and CH2-SS-CH2;

[0020] R1 and R2 are each independently selected from a C9-C17 straight chain or branched alkyl group, a C9-C17 alkene or a C9-C17 alkyne.

[0021] More preferably, the chemical structural formula of the triglyceride-derived ionizable lipid compound is as shown in any one of S1, S2, S3, S4, S5, S6, S7, S8, C1, C2, C3, C4, C5, C6, C7, and C8:

[0022]

[0023]

[0024]

[0025] .

[0026] The second aspect of the present invention is to provide a method for preparing the triglyceride-derived ionizable lipid compound according to the first aspect of the present invention, the preparation method comprising the following steps:

[0027] Step 1, dissolving an acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stirring for reaction, concentrating and purifying the reaction mixture in vacuo, then dissolving it in a mixed solvent for reaction, and then concentrating and purifying it in vacuo to obtain product I;

[0028] Step 2, dissolving alcohol, diacid, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stirring to react, and then concentrating and purifying in vacuo to obtain product II;

[0029] Step 3: dissolve product I, product II, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stir to react, and then vacuum concentrate and purify to obtain an ionizable lipid compound.

[0030] In step 1,

[0031] Preferably, the acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane and stirred at room temperature for 40 to 50 hours;

[0032] Preferably, the reaction mixture is concentrated and purified in vacuo, dissolved in a mixed solvent, and reacted at room temperature for 10 to 30 minutes.

[0033] Preferably, the equivalent ratio of the acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:1:(1-2):(0.1-0.2).

[0034] In step 2,

[0035] Preferably, the reaction is stirred at room temperature for 40 to 55 hours;

[0036] Preferably, the equivalent ratio of the alcohol, the diacid, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:2:(1-2):(0.1-0.2).

[0037] In step 3,

[0038] Preferably, the reaction is carried out at room temperature for 40 to 55 hours;

[0039] Preferably, the equivalent ratio of the product I, the product II, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:(1-4):(2-4):(0.2-0.4).

[0040] The third aspect of the present invention is to provide a drug delivery carrier, which includes the triglyceride-derived ionizable lipid compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof.

[0041] The fourth aspect of the present invention is to provide a use of the triglyceride-derived ionizable lipid compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof in the preparation of a drug delivery carrier.

[0042] The beneficial effects of the present invention are:

[0043] (1) The core structure of the ionizable lipid compound of the present invention is triglyceride, and contains five ester bonds. Since various esterases are widely present in organisms, this type of compound can be easily degraded. The ionizable lipid compound of the present invention is easily metabolized in the body. Due to the triglyceride contained in the compound, the drug carrier containing the compound can be efficiently taken up by cells with active triglyceride metabolism (such as tumor cells), thereby achieving effective delivery of mRNA.

[0044] (2) Both hydrophobic chains in the ionizable lipid compound of the present invention contain ester bonds, which can improve its degradability.

[0045] In the compound, M1 and M2 are independently selected from carbon chains (CH2) of different lengths. n or with a disulfide bond in the middle (CH2) n -SS-(CH2) n , where n is an integer not less than 1. The introduction of disulfide bonds enables it to be degraded by reducing substances such as glutathione in the body, which can improve the degradability and safety of ionizable lipid compounds.

[0046] In the compound, R1 and R2 are independently selected from C1-C 30 A straight-chain or branched alkyl group may contain one or more unsaturated bonds in the middle. Compared with a straight-chain alkyl group, a branched structure can further reduce the phase transition temperature of the lipid, which is beneficial to the release of the carried nucleic acid drug. The introduction of unsaturated bonds can also achieve this feature.

[0047] (3) In the ionizable lipid compound, preferably, M1 and M2, R1 and R2 are exactly the same. In this case, the two hydrophobic chain structures are exactly the same, which makes the preparation method of the ionizable lipid compound simpler and more conducive to the synthesis.

[0048] (4) The triglyceride-derived ionizable lipid compound can be used to prepare a drug delivery carrier. The drug carrier containing the compound has excellent properties such as good in vivo targeting, high biosafety, and high mRNA expression efficiency.

[0049] The target organs and tissues of the drug delivery carrier containing the compound include tumors, heart, liver, spleen, lungs, kidneys, etc. The preferred target tissue is tumor, because the abnormal triglyceride metabolism in the tumor is activated, which is more conducive to the delivery of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA prepared by ionizable lipid Dlin-MC3-DMA in different tissues and organs in a mouse pancreatic cancer model are shown;

[0051] Figure 2 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA prepared by SM102 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0052] Figure 3 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of S1 compound prepared in Example 1 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0053] Figure 4 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of S2 compound prepared in Example 2 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0054] Figure 5 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of S3 compound prepared in Example 3 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0055] Figure 6 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of S4 compound prepared in Example 7 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0056] Figure 7 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of the C1 compound prepared in Example 4 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0057] Figure 8 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of C2 compound prepared in Example 5 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0058] Fig. 9 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA of C3 compound prepared in Example 6 in different tissues and organs in a mouse pancreatic cancer model are shown;

[0059] Fig.10 The quantitative results of bioluminescence intensity of lipid nanoparticles loaded with luciferase mRNA and compound C4 prepared in Example 10 in different tissues and organs in a mouse pancreatic cancer model are shown. DETAILED DESCRIPTION

[0060] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0061] The first aspect of the present invention is to provide an ionizable lipid compound derived from triglycerides, wherein the chemical structural formula of the ionizable lipid compound derived from triglycerides is as shown in formula (I):

[0062] Formula (I).

[0063] In formula (I), G is (CH2) k , where k is an integer ≥1.

[0064] B1 and B2 are each independently selected from (CH2) x One of CH3, wherein x is 0 or an integer greater than 0.

[0065] M1 and M2 are each independently selected from (CH2) n or (CH2) n -SS-(CH2) n , where n is an integer ≥1.

[0066] R1 and R2 are each independently selected from a C1-C30 straight-chain or branched alkyl group, a C2-C30 alkene group or a C2-C30 alkyne group.

[0067] Preferably, in formula (I), G is (CH2) k , wherein 1≤k≤10 is an integer, more preferably, G is (CH2)2 or (CH2)3.

[0068] Preferably, B1 and B2 are each independently selected from one of CH3, CH2CH3, and (CH2)2CH3.

[0069] Preferably, M1 and M2 are each independently selected from one of (CH2)6, (CH2)4, (CH2)2-SS-(CH2)2, and CH2-SS-CH2.

[0070] Preferably, R1 and R2 are each independently selected from a C9-C17 straight-chain or branched alkyl group, a C9-C17 alkene group or a C9-C17 alkyne group.

[0071] The ideal ionizable lipid should be electrically neutral under physiological conditions, but positively charged under acidic conditions, and the pH value (pKa) at which the charge transition occurs is about 6.5. When loading nucleic acid drugs, first under acidic conditions, the ionizable lipid is positively charged, and can load the drug through electrostatic interaction with negatively charged nucleic acid drugs, and then the pH is adjusted to physiological conditions (pH=about 7.4), at which time the neutrality can reduce the toxicity of the drug after injection into the body. After the drug is internalized into the cell, under the acidic environment conditions in the lysosome, the ionizable lipid becomes positively charged again, and can interact with the lysosomal membrane, thereby releasing the nucleic acid drug. Therefore, the pKa of the ionizable lipid is crucial to its performance. In the present invention, the pKa of the ionizable lipid is mainly regulated by G and B1, B2 in formula (I). Specifically, the longer G, the longer B1, B2, and the smaller the pKa. Under preferred conditions, B1 and B2 are both CH3 (x=0), and G is (CH2)3 (k=3).

[0072] More preferably, the chemical structural formula of the triglyceride-derived ionizable lipid compound is as shown in any one of S1, S2, S3, S4, S5, S6, S7, S8, C1, C2, C3, C4, C5, C6, C7, and C8:

[0073]

[0074]

[0075]

[0076] .

[0077] The second aspect of the present invention is to provide a method for preparing the triglyceride-derived ionizable lipid compound according to the first aspect of the present invention, the preparation method comprising the following steps:

[0078] Step 1, dissolving an acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stirring for reaction, concentrating and purifying the reaction mixture in vacuo, then dissolving it in a mixed solvent for reaction, and then concentrating and purifying it in vacuo to obtain product I;

[0079] Step 2, dissolving alcohol, diacid, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stirring to react, and then concentrating and purifying in vacuo to obtain product II;

[0080] Step 3: dissolve product I, product II, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stir to react, and then vacuum concentrate and purify to obtain an ionizable lipid compound.

[0081] The above steps are described in detail below.

[0082] In step 1, the acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine are dissolved in anhydrous dichloromethane, and the mixture is stirred at room temperature for 40 to 50 hours, preferably at room temperature for 48 hours.

[0083] The reaction mixture is concentrated and purified in vacuo, dissolved in a mixed solvent, and reacted at room temperature for 10 to 30 minutes, preferably at room temperature for 20 minutes.

[0084] The equivalent ratio of the acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:1:(1-2):(0.1-0.2).

[0085] Preferably, the equivalent ratio of the acid, glycerol acetone acetal, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:1:1.5:0.15.

[0086] The acid is 4-(dimethylamino)butyric acid, 4-(ethylpropylamino)butyric acid or 3-(dimethylamino)propionic acid.

[0087] The mixed solvent is obtained by mixing trifluoroacetic acid, tetrahydrofuran and water. Preferably, the mixed solvent is obtained by mixing trifluoroacetic acid, tetrahydrofuran and water in a volume ratio of 6:8:1.

[0088] In step 2, the reaction is stirred at room temperature for 40 to 55 hours, preferably at room temperature for 48 hours.

[0089] The equivalent ratio of the alcohol, the diacid, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:2:(1-2):(0.1-0.2).

[0090] Preferably, the equivalent ratio of the alcohol, the diacid, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:2:1.5:0.15.

[0091] The alcohol is n-nonanol, 8-methylnonanol, cis-2-nonen-1-ol or 9-heptadecanol.

[0092] The diacid is 3,3'-dithiodipropionic acid or suberic acid.

[0093] In step 3, the reaction is carried out at room temperature for 40 to 55 hours, preferably the reaction is stirred at room temperature for 48 hours.

[0094] The equivalent ratio of the product I, the product II, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:(1-4):(2-4):(0.2-0.4).

[0095] Preferably, the equivalent ratio of the product I, the product II, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:(1.5-3):3:0.3.

[0096] Optionally, the preparation method further comprises step 4, repeating step 2, dissolving the obtained product II, the product obtained in step 3, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine in anhydrous dichloromethane, stirring the reaction at room temperature for 40 to 50 hours, and then vacuum concentrating and purifying to obtain an ionizable lipid compound.

[0097] In step 4, preferably, the reaction is stirred at room temperature for 48 hours.

[0098] The equivalent ratio of product II, the product obtained in step 3, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:(1-2):(2-4):(0.2-0.4).

[0099] Preferably, the equivalent ratio of product II, the product obtained in step 3, 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride and 4-dimethylaminopyridine is 1:1.5:3:0.3.

[0100] The third aspect of the present invention is to provide a drug delivery carrier, which includes the triglyceride-derived ionizable lipid compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof.

[0101] The active ingredient of the drug is selected from at least one of nucleic acid, small molecule drug, macromolecular drug, protein drug, polypeptide and antibody; preferably, the active ingredient of the drug is nucleic acid.

[0102] The delivery methods of the drug delivery carrier include intravenous injection, intraperitoneal injection, subcutaneous injection, intratumoral injection, etc. Preferably, the injection method is intraperitoneal injection.

[0103] In order to achieve effective drug loading, the ionizable lipid compound or its pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug described in the present invention needs to be combined with other lipids, including phospholipids, cholesterol, pegylated lipids, etc.

[0104] The drug delivery carrier can be used in tumor targeting drugs.

[0105] The fourth aspect of the present invention is to provide a use of the triglyceride-derived ionizable lipid compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof in the preparation of a drug delivery carrier.

[0106] Example

[0107] The present invention is further described below by specific examples, which are limited to illustrating the present invention but not to limiting the scope of the present invention. The raw materials used in the examples of the present invention were all purchased.

[0108] Example 1

[0109] Synthesis of compound S1:

[0110] Synthesis of compound A3: A1 (4-(dimethylamino)butyric acid, 1 equivalent), A2 (glycerol acetone acetal, 1 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride (EDC, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP, 0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. After the reaction mixture was concentrated in vacuo, the residue was purified by silica gel column chromatography. The obtained product was dissolved in a mixed solvent of trifluoroacetic acid, tetrahydrofuran and water (volume ratio of 6:8:1) and reacted at room temperature for 20 minutes. After vacuum concentration, the residue was purified by silica gel column chromatography to obtain A3.

[0111] Synthesis of A6: A4 (1 equivalent of n-nonanol), A5 (2 equivalents of 3,3'-dithiodipropionic acid), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0112] Synthesis of S1: A3 (1 equivalent), A6 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S1. The synthesis route of S1 is shown in formula (1). Its structural characterization results are: 1H NMR (500 MHz, Methanol-d4) δ 5.37 – 5.08 (m, 1H), 4.45 – 4.13 (m, 4H), 4.10 (t, J = 6.6 Hz, 4H), 2.94 (tq, J = 5.8, 3.2 Hz, 8H), 2.83 – 2.68 0.90 (t, J = 6.8 Hz, 6H). FTMS(m / z): [M+H] + calculated for C 39 H 71 NO 10 S4, 842.40 found: 842.4.

[0113] Formula (1).

[0114] Example 2

[0115] Synthesis of compound S2:

[0116] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0117] Synthesis of A8: A7 (8-methylnonanol, 1 equivalent), A5 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0118] Synthesis of S2: A3 (1 equivalent), A8 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S2. The synthetic route of S2 is shown in formula (2). Its structural characterization results are: 1H NMR (500 MHz, Methanol-d4) δ 5.34 – 5.08 (m, 1H), 4.43 – 4.33 (m, 2H), 4.22 (ddd, J = 19.8,12.1, 6.2 Hz, 2H), 4.17 – 4.06 (m, 4H), 2.94 (tq, J = 6.2, 3.4 Hz, 8H), 2.77(ddt, J = 19.6, 10.9, 5.1 Hz, 8H), 2.43 – 2.31 (m, 4H), 2.25 (d, J = 2.9 Hz,6H), 1.83 – 1.74 (m, 2H), 1.72 – 1.55 (m, 4H), 1.46 – 1.21 (m, 6H), 1.21 –1.08 (m, 16H), 0.95 – 0.76 (m, 12H). FTMS(m / z): [M+H] + calculated for C 41 H 75 NO 10 S4, 870.43 found: 870.5.

[0119] Formula (2).

[0120] Example 3

[0121] Synthesis of compound S3:

[0122] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0123] Synthesis of A10: A9 (cis-2-nonen-1-ol, 1 equivalent), A5 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0124] Synthesis of S3: A3 (1 equivalent), A10 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S3. The synthetic route of S3 is shown in formula (3). Its structural characterization results are: 1H NMR (500 MHz, Methanol-d4) δ 5.73 – 5.63 (m, 2H), 5.56 (dt, J = 11.7, 7.1 Hz, 2H), 5.37 –5.10 (m, 1H), 4.68 (d, J = 7.0 Hz, 4H), 4.45 – 4.02 (m, 4H), 2.97 (q, J = 6.2Hz, 8H), 2.78 (dt, J = 23.6, 7.0 Hz, 8H), 2.39 (ddd, J = 15.8, 8.1, 4.1 Hz,4H), 2.30 (d, J = 1.5 Hz, 6H), 2.15 (q, J = 7.4 Hz, 4H), 1.89 – 1.74 (m, 2H), 1.50 – 1.26 (m, 18H), 0.93 (t, J = 6.5 Hz, 6H). FTMS(m / z): [M+H] + calculated for C 39 H 67 NO 10 S4, 838.36 found: 838.37360.

[0125] Formula (3).

[0126] Example 4

[0127] Synthesis of compound C1:

[0128] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0129] Synthesis of A12: A4 (1 equivalent), A11 (suberic acid, 2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0130] Synthesis of C1: A3 (1 equivalent), A12 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound C1. The synthetic route of C1 is shown in formula (4). Its structural characterization results are: 1H NMR (600 MHz, Methanol-d4) δ 5.31 – 5.04 (m, 1H), 4.42 – 4.30 (m, 2H), 4.19 – 4.09 (m, 2H), 4.06 (t, J = 6.6 Hz, 4H), 2.43 – 2.28 (m, 18H), 1.84 – 1.76 (m, 2H), 1.62(pd, J = 7.1, 3.1 Hz, 12H), 1.37 – 1.27 (m, 32H), 0.90 (t, J = 6.9 Hz, 6H).FTMS(m / z): [M+H] + calculated for C 43 H 79 NO 10 , 770.57 found: 770.6.

[0131] Formula (4).

[0132] Example 5

[0133] Synthesis of compound C2:

[0134] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0135] Synthesis of A13: A7 (1 equivalent), A11 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0136] Synthesis of C2: A3 (1 equivalent), A13 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound C2. The synthetic route of C2 is shown in formula (5). Its structural characterization results are: 1H NMR (500 MHz, Chloroform-d) δ 5.26 (ddd, J = 10.2, 5.9, 4.4 Hz, 1H), 4.30 (dt, J = 11.9, 4.8 Hz, 2H), 4.19 – 3.77 (m, 6H), 2.40 (dt, J = 17.8, 7.4 Hz, 4H), 2.35 –2.18 (m, 14H), 1.85 (p, J = 7.4 Hz, 2H), 1.62 (qq, J = 7.1, 3.6, 2.9 Hz,14H), 1.49 – 1.01 (m, 28H), 0.96 – 0.68 (m, 12H). FTMS(m / z): [M+H] + calculated for C 45 H 83 NO 10 , 798.60 found: 798.6.

[0137] Formula (5).

[0138] Example 6

[0139] Synthesis of compound C3:

[0140] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0141] Synthesis of A14: A9 (1 equivalent), A11 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0142] Synthesis of C3: A3 (1 equivalent), A14 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound C3. The synthetic route of C3 is shown in formula (6). Its structural characterization results are: 1H NMR (500 MHz, Methanol-d4) δ 5.64 (dt, J = 10.7, 7.6 Hz, 2H), 5.57 – 5.48 (m, 2H), 5.29 –5.03 (m, 1H), 4.62 (d, J = 6.9 Hz, 4H), 4.21 – 3.95 (m, 4H), 2.33 (dtd, J =21.4, 7.5, 3.4 Hz, 12H), 2.25 (s, 6H), 2.12 (qd, J = 7.4, 1.5 Hz, 4H), 1.79(p, J = 7.4 Hz, 2H), 1.62 (pd, J = 6.9, 4.3 Hz, 8H), 1.49 – 1.20 (m, 24H),0.95 – 0.83 (m, 6H). FTMS(m / z): [M+H] + calculated for C 43 H 75 NO 10 , 766.54 found:766.54895.

[0143] Formula (6).

[0144] Example 7

[0145] Synthesis of compound S4:

[0146] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0147] Synthesis of A16: A15 (9-heptadecanol, 1 equivalent), A5 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0148] Synthesis of S4: A3 (1 equivalent), A16 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S4, whose structural formula is shown in Formula (7).

[0149] Formula (7).

[0150] Example 8

[0151] Synthesis of compound S5:

[0152] Synthesis of compound A18: A17 (4-(ethylpropylamino)butyric acid, 1 equivalent), A2 (glycerol acetone acetal, 1 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride (EDC, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP, 0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography. The obtained product was dissolved in a mixed solvent of trifluoroacetic acid, tetrahydrofuran and water (volume ratio of 6:8:1) and reacted at room temperature for 20 minutes. After vacuum concentration, the residue was purified by silica gel column chromatography to obtain A18.

[0153] Synthesis of A6: A6 was synthesized according to the same procedure as in Example 1.

[0154] Synthesis of S5: A18 (1 equivalent), A6 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S5, whose structural formula is shown in Formula (8).

[0155] Formula (8).

[0156] Example 9

[0157] Synthesis of compound S6:

[0158] Synthesis of compound A20: A19 (3-(dimethylamino)propionic acid, 1 equivalent), A2 (glycerol acetone acetal, 1 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbamate hydrochloride (EDC, 1.5 equivalents) and 4-dimethylaminopyridine (DMAP, 0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. After the reaction mixture was concentrated in vacuo, the residue was purified by silica gel column chromatography. The obtained product was dissolved in a mixed solvent of trifluoroacetic acid, tetrahydrofuran and water (volume ratio of 6:8:1) and reacted at room temperature for 20 minutes. After vacuum concentration, the residue was purified by silica gel column chromatography to obtain A20.

[0159] Synthesis of A6: A6 was synthesized according to the same procedure as in Example 1.

[0160] Synthesis of S6: A20 (1 equivalent), A6 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S6, whose structural formula is shown in Formula (9).

[0161] Formula (9).

[0162] Example 10

[0163] Synthesis of compound C4:

[0164] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0165] Synthesis of A21: A15 (9-heptadecanol, 1 equivalent), A11 (2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0166] Synthesis of C4: A3 (1 equivalent), A21 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound C4, whose structural formula is shown in Formula (10).

[0167] Formula (10).

[0168] Embodiment 11

[0169] Synthesis of compound C5:

[0170] Synthesis of compound A18: A18 was synthesized according to the same procedure as Example 8.

[0171] Synthesis of A12: A12 was synthesized according to the same procedure as Example 4.

[0172] Synthesis of C5: A18 (1 equivalent), A12 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product, compound C5, whose structural formula is shown in formula (11).

[0173] Formula (11).

[0174] Example 12

[0175] Synthesis of compound C6:

[0176] Synthesis of compound A20: A20 was synthesized according to the same procedure as Example 9.

[0177] Synthesis of A12: A12 was synthesized according to the same procedure as Example 4.

[0178] Synthesis of C6: A20 (1 equivalent), A12 (3 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound C6, whose structural formula is shown in Formula (12).

[0179] Formula (12).

[0180] Embodiment 13

[0181] Synthesis of compound S7:

[0182] Synthesis of compound A3: A3 was synthesized according to the same procedure as in Example 1.

[0183] Synthesis of A6: A6 was synthesized according to the same procedure as in Example 1.

[0184] Synthesis of A22: A3 (1 equivalent), A6 (1.5 equivalents), EDC (3 equivalents) and DMAP (0.3 equivalents) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product, compound A22.

[0185] Synthesis of A24: A15 (9-heptadecanol, 1 equivalent), A23 (2,2'-dithiodiacetic acid, 2 equivalents), EDC (1.5 equivalents) and DMAP (0.15 equivalents) were dissolved in anhydrous dichloromethane and stirred at room temperature for 48 hours. The reaction solution was concentrated in vacuo and the residue was purified by silica gel column chromatography.

[0186] Synthesis of compound S7: A22 (1 equivalent), A24 (1.5 equivalent), EDC (3 equivalents) and DMAP (0.3 equivalent) were dissolved in anhydrous dichloromethane and reacted at room temperature for 48 hours. After the reaction solution was concentrated in vacuo, the residue was purified by silica gel column chromatography to obtain the final product compound S7. The synthesis route of S7 is shown in formula (13).

[0187] Formula (13).

[0188] Experimental example

[0189] Experimental Example 1 Preparation loaded with mRNA

[0190] Eight ionizable lipid compounds, including compounds S1-S4 prepared in Examples 1-3 and 7, and compounds C1-C4 prepared in Examples 4-6 and 10, as well as two commercially available ionizable lipids Dlin-MC3-DMA and SM102, were loaded with mRNA together with other substances to prepare lipid nanoparticles loaded with mRNA.

[0191] The preparation steps of lipid nanoparticles loaded with mRNA are as follows: lipids are mixed according to the molar ratio of ionized lipid: DSPC: cholesterol: DMG-PEG2000 of 50:10:38.5:1.5, and the lipids are dissolved in ethanol at a concentration of 20 mM to prepare a lipid solution. mRNA is dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL to obtain an mRNA solution. Then, the lipid solution and the mRNA solution are quickly mixed at a volume ratio of 3:1, and the total mass ratio of RNA to lipid is 15 μg / 1 μmol. The mixture is incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles are dialyzed in PBS for 1 hour and then diluted with PBS to a final mRNA concentration of 0.01 mg / mL. All lipids can effectively load mRNA, with an encapsulation efficiency of more than 80% and a size of about 100 nm.

[0192] Experimental Example 2 Specificity and effectiveness test of delivery

[0193] A mouse orthotopic pancreatic cancer model was constructed. The lipid nanoparticles loaded with luciferase mRNA prepared in Experimental Example 1 were injected into the mouse via intraperitoneal injection at a dose of 0.1 mg / kg mRNA. 3 hours after injection, the mouse was injected with luciferase substrate. The mouse was killed 2 minutes later, and the tumor and major organs were removed. Bioluminescence imaging was performed, and the luminescence intensity of each tissue and organ was quantitatively tested. The test results are shown in Figures 1 to 10 .

[0194] from Figure 1 It can be seen that Dlin-MC3-DMA has higher luminescence intensity in the liver and tumor, and the luminescence intensity in the liver is higher than that in the tumor.

[0195] Figure 2 Among them, the luminescence intensity of SM102 was the highest in the liver, followed by the luminescence intensity in the tumor.

[0196] Figure 3 Among them, the luminescence intensity of S1 in tumor was the highest, which was higher than that in spleen.

[0197] Figure 4 Among them, S2 has the highest luminescence intensity in tumors and has almost no expression in other organs.

[0198] Figure 5 Among them, the luminescence intensity of S3 was the highest in the tumor, and the luminescence intensity in the spleen was significantly lower than that in the tumor, and there was basically no expression in other organs.

[0199] Figure 6 Among them, S4 has a certain luminescence intensity in the tumor, liver and spleen, and the luminescence intensity in the tumor is the highest.

[0200] Figure 7 Among them, C1 has a certain luminescence intensity in various organs and tumors, and its luminescence intensity in tumors is the highest.

[0201] Figure 8 Among them, C2 has a certain luminescence intensity in the lung, liver, spleen and tumor, and its luminescence intensity is the highest in the tumor.

[0202] Fig. 9 Among them, C3 has a certain luminescence intensity in the liver, spleen and tumor, and the luminescence intensity in the tumor is the highest.

[0203] Fig.10 Among them, C4 has a certain luminescence intensity in the liver, spleen and tumor, and the luminescence intensity in the tumor is the highest.

[0204] In summary, the tested ionizable lipids can all achieve efficient expression of mRNA in the tumor site, but compared with the commercial lipids Dlin-MC3-DMA and SM102, the expression levels of the eight ionizable lipids prepared in the present invention in the liver, spleen and other organs of mice are greatly reduced, especially the C2 ionizable lipid, which shows a higher tumor luminescence intensity than the two commercial lipids, and the luminescence intensity in each major organ is greatly reduced, indicating the high specificity and effectiveness of the ionizable lipid compounds described in the present invention in tumor-targeted mRNA delivery.

[0205] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. An ionizable lipid compound derived from triglycerides, characterized in that The chemical structural formula of the triglyceride-derived ionizable lipid compound is shown in formula (I): Formula (I); In formula (I), G is (CH2) k , where 1≤k≤10 is an integer; B1 and B2 are each independently selected from one of CH3, CH2 CH3, and (CH2)2 CH3; M1 and M2 are each independently selected from (CH2)6 and (CH2)4; R1 and R2 are each independently selected from a C9-C17 straight chain or branched alkyl group, Or, M1 and M2 are selected from one of (CH2)2-SS-(CH2)2 and CH2-SS-CH2; R1 and R2 are each independently selected from a C9-C17 straight chain or branched alkyl group, optionally containing one or more unsaturated bonds in the middle.

2. The triglyceride-derived ionizable lipid compound according to claim 1, characterized in that The chemical structural formula of the triglyceride-derived ionizable lipid compound is shown in any one of S1, S2, S3, S4, S5, S6, S7, S8, C1, C2, C4, C5, C6, C7, and C8: 。 3. A drug delivery carrier, characterized in that: The drug delivery carrier comprises the triglyceride-derived ionizable lipid compound according to claim 1 or 2.

4. Use of the triglyceride-derived ionizable lipid compound according to claim 1 or 2 in the preparation of a drug delivery carrier.

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