Ionizable lipid compound, preparation method therefor and use thereof

Abstract

The present disclosure relates to the technical field of biomedicine, and in particular to an ionizable lipid compound having a structure represented by the following formula (I) or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, or a use thereof, wherein n1, n2, n3 and n4 are each independently an integer from 1 to 20. Further provided is a preparation method for the ionizable lipid compound.

Description

Ionizable lipid compounds, their preparation methods and applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese patent application CN202411854207.5, filed on December 16, 2024; the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of biomedical technology, specifically relating to ionizable lipid compounds, their preparation methods, and applications. Background Technology

[0004] In recent years, significant progress has been made in RNA therapy, represented by mRNA vaccines. However, RNA itself is sensitive to nucleases and has characteristics such as large size and negative charge, making it difficult for RNA to directly cross the cell membrane and enter the cell. The emergence of lipid nanoparticles (LNPs) has solved the problem of RNA delivery. Lipid nanoparticles are typically composed of four components—ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids. Among them, ionizable lipids play an important role in protecting RNA and promoting cytoplasmic transport.

[0005] The unique feature of ionizable lipids is that they are electrically neutral under normal physiological pH conditions, and lipid nanoparticles composed of ionizable lipids exhibit lower toxicity and longer in vivo circulation cycles. Ionizable lipids carry a positive charge at acidic pH, which can cause RNA to aggregate into lipid nanoparticles. After cellular uptake, the lipid nanoparticles are protonated in acidic endosomes and interact with anionic endosome phospholipids to form bilayer-incompatible pyramidal ion pairs. These cationic-anionic lipids facilitate membrane fusion-disruption, endosome escape, and cargo release.

[0006] Some patent documents or academic papers describe methods for preparing ionizable lipid compounds at the milligram level. However, these milligram-level preparation methods often cannot meet the needs of scale-up production. Therefore, it is necessary to develop scale-up synthesis methods for ionizable lipid compounds that meet pharmaceutical purity requirements. Summary of the Invention

[0007] The technical problem this disclosure aims to solve is that current methods for preparing milligram-level ionizable lipid compounds often fail to meet the demands of scale-up production. This disclosure develops a new, safe method for preparing ionizable lipid compounds that satisfies the requirements of scale-up production. Furthermore, it significantly improves production efficiency. This disclosure optimizes the synthetic route for ionizable lipid compounds, enabling scale-up production with good purity.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted in this disclosure is as follows:

[0009] On the one hand, this disclosure provides ionizable lipid compounds having the structure shown in formula (I) or pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers thereof.

[0010] in,

[0011] n1, n2, n3, and n4 are each independent integers from 1 to 20.

[0012] In some preferred embodiments of this disclosure, the ionizable lipid compound, as described above, or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer, wherein n1, n2, n3, and n4 are each independently an integer from 3 to 18; preferably, each n1 is independently an integer from 4 to 15, n2 is an integer from 4 to 10, n3 is an integer from 3 to 10, and n4 is an integer from 4 to 15; preferably, each n1 is independently an integer from 4 to 10, n2 is an integer from 4 to 8, n3 is an integer from 4 to 8, and n4 is an integer from 4 to 12.

[0013] In some preferred embodiments of this disclosure, each n1 is independently 4; in another embodiment, each n1 is independently 5; in another embodiment, each n1 is independently 6; in another embodiment, each n1 is independently 7; in another embodiment, each n1 is independently 8; in another embodiment, each n1 is independently 9; in another embodiment, each n1 is independently 10; in another embodiment, each n1 is independently 11; in another embodiment, each n1 is independently 12; in another embodiment, each n1 is independently 13; in another embodiment, each n1 is independently 14; in another embodiment, each n1 is independently 15.

[0014] In some preferred embodiments of this disclosure, each n1 is an integer from 4 to 15 independently; in another embodiment, each n1 is an integer from 4 to 10 independently; and in yet another embodiment, each n1 is an integer from 4 to 8 independently.

[0015] In some more specific embodiments of this disclosure, each n1 is independently 4, 5, 6, 7, 8, 9 or 10; in another more specific embodiment, each n1 is independently 4, 5, 6, 7 or 8; in yet another more specific embodiment, each n1 is independently 5, 6 or 7; and in yet another more specific embodiment, each n1 is independently 6.

[0016] In some preferred embodiments of this disclosure, n2 is 4; in another embodiment, n2 is 5; in another embodiment, n2 is 6; in another embodiment, n2 is 7; in another embodiment, n2 is 8; in another embodiment, n2 is 9; in another embodiment, n2 is 10; in another embodiment, n2 is 11; in another embodiment, n2 is 12; in another embodiment, n2 is 13; in another embodiment, n2 is 14; in another embodiment, n2 is 15.

[0017] In some preferred embodiments of this disclosure, n2 is an integer from 4 to 10; in another embodiment, n2 is an integer from 4 to 8.

[0018] In some more specific embodiments of this disclosure, n2 is 4, 5, 6, 7, 8 or 9; in another more specific embodiment, n2 is 4, 5, 6, 7 or 8; in yet another more specific embodiment, n2 is 6, 7 or 8; and in yet another more specific embodiment, n2 is 7.

[0019] In some preferred embodiments of this disclosure, n3 is 3; in another embodiment, n3 is 4; in another embodiment, n3 is 5; in another embodiment, n3 is 6; in another embodiment, n3 is 7; in another embodiment, n3 is 8; in another embodiment, n3 is 9; in another embodiment, n3 is 10; in another embodiment, n3 is 11; in another embodiment, n3 is 12; in another embodiment, n3 is 13; in another embodiment, n3 is 14; in another embodiment, n3 is 15.

[0020] In some preferred embodiments of this disclosure, n3 is an integer from 3 to 15; in another embodiment, n3 is an integer from 3 to 10; and in yet another embodiment, n3 is an integer from 4 to 8.

[0021] In some more specific embodiments of this disclosure, n3 is 3, 4, 5, 6, 7, or 8; in another more specific embodiment, n3 is 4, 5, 6, or 7; in yet another more specific embodiment, each n1 is independently 4, 5, or 6; and in yet another more specific embodiment, n3 is 5.

[0022] In some preferred embodiments of this disclosure, n4 is 4; in another embodiment, n4 is 5; in another embodiment, n4 is 6; in another embodiment, n4 is 7; in another embodiment, n4 is 8; in another embodiment, n4 is 9; in another embodiment, n4 is 10; in another embodiment, n4 is 11; in another embodiment, n4 is 12; in another embodiment, n4 is 13; in another embodiment, n4 is 14; in another embodiment, n4 is 15.

[0023] In some preferred embodiments of this disclosure, n4 is an integer from 4 to 15; in another embodiment, n4 is an integer from 4 to 12; in yet another embodiment, n4 is an integer from 4 to 10; and in yet another embodiment, n4 is an integer from 7 to 9.

[0024] In some more specific embodiments of this disclosure, n4 is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; in another more specific embodiment, n4 is 4, 5, 6, 7, 8, 9 or 10; in yet another more specific embodiment, n4 is 7, 8 or 9; and in yet another more specific embodiment, each n1 is independently 8.

[0025] In some preferred embodiments of this disclosure, according to the ionizable lipid compound as described above, or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer, the compound of formula (I) is a compound of the following type:

[0026] On the other hand, this disclosure provides methods for preparing ionizable lipid compounds as described above, or their pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers, including preparation method A or preparation method B:

[0027] The preparation method A includes the following steps: preparing an alcohol (A1M) from a diester of formula (SM-A1) and a haloalkane (SM-A2M) as raw materials.

[0028] Wherein, n1 is as defined in this disclosure;

[0029] X is a halogen.

[0030] In some preferred embodiments of this disclosure, X is selected from any one of fluorine, chlorine, bromine, and iodine.

[0031] The preparation method B includes the following steps: step (B1M) of preparing an alcohol of formula (MTS001-11M) from a ketone of formula (SM-BM) as a raw material.

[0032] Wherein, n1 is as defined in this disclosure.

[0033] Current methods for preparing ionizable lipid compounds at the milligram level often fail to meet the requirements for scale-up production. Therefore, a novel and safe method for the scale-up synthesis of ionizable lipid compounds has been developed, which satisfies these requirements and significantly improves production efficiency.

[0034] In some preferred embodiments of this disclosure, step (A1M) includes the following steps (A11M):

[0035] Wherein, n1 is as defined in this disclosure;

[0036] X is selected from any one of fluorine, chlorine, bromine, and iodine.

[0037] In some preferred embodiments of this disclosure, X is selected from chlorine, bromine, and iodine.

[0038] In some preferred embodiments of this disclosure, X is selected from either bromine or iodine.

[0039] In some preferred embodiments of this disclosure, X is iodine.

[0040] In some preferred embodiments of this disclosure, an alkali is added in step (A11M), wherein the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylamino.

[0041] In some preferred embodiments of this disclosure, the alkali used in step (A11M) is NaH.

[0042] In some preferred embodiments of this disclosure, the base used in step (A11M) is n-BuLi.

[0043] In some preferred embodiments of this disclosure, in step (A11M), the molar ratio of SM-A1 and SM-A2M is 1:2-1:5, preferably 1:2-1:3.

[0044] In some preferred embodiments of this disclosure, in step (A11M), the reaction solvent is selected from any one or a combination of N,N-dimethylformamide, dimethyl sulfoxide, toluene, carbon tetrachloride, chloroform, and tert-butanol; more preferably, in step (A11M), the reaction solvent is N,N-dimethylformamide.

[0045] In some preferred embodiments of this disclosure, the reaction temperature in step (A11M) is -10°C to 70°C; preferably, the reaction temperature in step (A11M) is -10°C to 50°C; more preferably, the reaction temperature in step (A11M) is -5°C to 35°C.

[0046] In some preferred embodiments of this disclosure, step (A1M) includes the following step (A12M):

[0047] Wherein, n1 is as defined in this disclosure.

[0048] In some preferred embodiments of this disclosure, step (A12M) further includes a catalyst, wherein the catalyst is LiCl.

[0049] In some preferred embodiments of this disclosure, in step (A12M), the molar ratio of MTS001-A1M to the catalyst is 1:2 to 1:20; preferably, the molar ratio of MTS001-A1M to the catalyst is 1:5 to 1:15.

[0050] In some preferred embodiments of this disclosure, in step (A12M), the reaction solvent is selected from any one or a combination of N,N-dimethylformamide, dimethyl sulfoxide, toluene, carbon tetrachloride, and chloroform; more preferably, in step (A12M), the reaction solvent is N,N-dimethylformamide.

[0051] In some preferred embodiments of this disclosure, the reaction temperature in step (A12M) is 50°C to 200°C; preferably, the reaction temperature in step (A12M) is 80°C to 180°C; more preferably, the reaction temperature in step (A12M) is 80°C to 150°C.

[0052] In some preferred embodiments of this disclosure, step (A1M) includes the following step (A13M):

[0053] Wherein, n1 is as defined in this disclosure.

[0054] In some preferred embodiments of this disclosure, step (A13M) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, NaBH4, diborane, and aluminum isopropoxide; preferably, in step (A13M), the reducing agent is any one of DIBAL-H and NaBH4; preferably, in step (A13M), the reducing agent is DIBAL-H.

[0055] In some preferred embodiments of this disclosure, in step (A13M), the molar ratio of MTS001-A2M to the reducing agent is 1:1-1:5; preferably, the molar ratio of MTS001-A2M to the reducing agent is 1:1.5-1:4; preferably, the molar ratio of MTS001-A2M to the reducing agent is 1:2-1:3.

[0056] In some preferred embodiments of this disclosure, in step (A13M), the reaction solvent is selected from tetrahydrofuran, diethyl ether, 1,4-dioxane, methanol, ethanol, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof; preferably, in step (A13M), the reaction solvent is selected from tetrahydrofuran and diethyl ether, or a combination thereof.

[0057] In some preferred embodiments of this disclosure, the reaction temperature in step (A13M) is -10°C to 80°C; preferably, the reaction temperature in step (A13M) is -5°C to 70°C; more preferably, the reaction temperature in step (A13M) is -5°C to 35°C.

[0058] In some preferred embodiments of this disclosure, preparation method A includes the following steps:

[0059] Wherein, n1 and X are as defined in this disclosure, and the reaction conditions for each step are as described above.

[0060] In some preferred embodiments of this disclosure, step (B1M) includes the following step (B11M):

[0061] Wherein, n1 is as defined in this disclosure.

[0062] In some preferred embodiments of this disclosure, an alkali is added in step (B11M), and the alkali used is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylaminodimethylamine; preferably, in step (B11M), the alkali used is n-BuLi.

[0063] In some preferred embodiments of this disclosure, in step (B11M), the molar ratio of SM-B1M to SM-B2 is 1:1 to 1:5; preferably, the molar ratio of SM-B1M to SM-B2 is 1:1 to 1:3; preferably, the molar ratio of SM-B1M to SM-B2 is 1:1 to 1:2.

[0064] In some preferred embodiments of this disclosure, in step (B11M), the molar ratio of SM-B1M to alkali is 1:1-1:5; preferably, the molar ratio of SM-B1M to alkali is 1:1-1:3; more preferably, the molar ratio of SM-B1M to alkali is 1:1-1:2; and more preferably, the molar ratio of SM-B1M to alkali is 1:1-1:1.5.

[0065] In some preferred embodiments of this disclosure, in step (B11M), the reaction solvent is selected from any one or a combination of tetrahydrofuran, 1,4-dioxane, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide; preferably, in step (B11M), the reaction solvent is selected from tetrahydrofuran.

[0066] In some preferred embodiments of this disclosure, the reaction temperature in step (B11M) is -78°C to 35°C; preferably, the reaction temperature in step (B11M) is -78°C to 25°C; more preferably, the reaction temperature in step (B11M) is -5°C to 25°C.

[0067] In some preferred embodiments of this disclosure, step (B1M) includes the following step (B12M):

[0068] Wherein, n1 is as defined in this disclosure.

[0069] In some preferred embodiments of this disclosure, step (B12M) further includes a catalyst, wherein the catalyst used is HCl.

[0070] In some preferred embodiments of this disclosure, in step (B12M), the molar ratio of MTS001-B1M to the catalyst is 1:1-1:5; preferably, the molar ratio of MTS001-B1M to the catalyst is 1:1-1:3; more preferably, the molar ratio of MTS001-B1M to the catalyst is 1:1.5-1:2.5.

[0071] In some preferred embodiments of this disclosure, in step (B12M), the reaction solvent is selected from tetrahydrofuran, 1,4-dioxane, or a combination thereof.

[0072] In some preferred embodiments of this disclosure, in step (B12M), the reaction temperature is 0°C to 100°C; preferably, the reaction temperature is 20°C to 80°C.

[0073] In some preferred embodiments of this disclosure, step (B1M) includes the following step (B13M):

[0074] Wherein, n1 is as defined in this disclosure.

[0075] In some preferred embodiments of this disclosure, step (B13M) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, and NaBH4; preferably, in step (B13M), the reducing agent is any one of DIBAL-H, LAH, NaBH4, diborane, and aluminum isopropoxide; preferably, in step (B13M), the reducing agent is NaBH4.

[0076] In some preferred embodiments of this disclosure, in step (B13M), the molar ratio of MTS001-B2M to the reducing agent is 1:1-1:5; preferably, the molar ratio of MTS001-B2M to the reducing agent is 1:1-1:3; more preferably, the molar ratio of MTS001-B2M to the reducing agent is 1:1-1:2; even more preferably, the molar ratio of MTS001-B2M to the reducing agent is 1:1-1:1.5.

[0077] In some preferred embodiments of this disclosure, in step (B13M), the reaction solvent is selected from tetrahydrofuran, diethyl ether, 1,4-dioxane, methanol, ethanol, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof; preferably, in step (B13M), the reaction solvent is selected from tetrahydrofuran, diethyl ether, methanol, ethanol, or a combination thereof.

[0078] In some preferred embodiments of this disclosure, in step (B13M), the reaction temperature is -10°C to 50°C; preferably, the reaction temperature is -5°C to 35°C.

[0079] In some preferred embodiments of this disclosure, preparation method A further includes the following esterification step:

[0080] Wherein, n1, n2, and X are as defined in this disclosure.

[0081] In some preferred embodiments of this disclosure, the esterification step may further include a catalyst selected from any one or a combination of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole (HOBt), sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and triethylamine, preferably a combination of EDCI and DMAP.

[0082] In some preferred embodiments of this disclosure, in the esterification step, the molar ratio of MTS001-11M to MTS001-SM3 is 1:1-1:5, preferably 1:1-1:3.

[0083] In some preferred embodiments of this disclosure, in the esterification step, the molar ratio of MTS001-11M to the catalyst is 1:0.1-1:5, preferably 1:0.2-1:3, and more preferably 1:0.2-1:2.

[0084] In some preferred embodiments of this disclosure, in the esterification step, the solvent for the reaction is any one or a combination of dichloromethane, chloroform, chloroform, dimethyl sulfoxide, acetone, benzene, and toluene.

[0085] In some preferred embodiments of this disclosure, the temperature of the reaction in the esterification step is 0–50°C.

[0086] In some preferred embodiments of this disclosure, the temperature of the reaction in the esterification step is 0–35°C.

[0087] In some preferred embodiments of this disclosure, the preparation method further includes the following steps:

[0088] Wherein, n3 and n4 are as defined in this disclosure;

[0089] X is selected from any one of fluorine, chlorine, bromine, and iodine.

[0090] In some preferred embodiments of this disclosure, X is selected from chlorine, bromine, and iodine.

[0091] In some preferred embodiments of this disclosure, X is selected from either bromine or iodine.

[0092] In some preferred embodiments of this disclosure, X is bromine.

[0093] In some preferred embodiments of this disclosure, step (AB3M) further includes a catalyst selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole (HOBt), sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and triethylamine, or combinations thereof.

[0094] In some preferred embodiments of this disclosure, the catalyst in step (AB3M) is a combination of EDCI and DMAP.

[0095] In some preferred embodiments of this disclosure, in step (AB3M), the molar ratio of MTS001-2M to MTS001-2BM is 1:1 to 1:5; preferably, the molar ratio of MTS001-2M to MTS001-2BM is 1:1 to 1:3; more preferably, the molar ratio of MTS001-2M to MTS001-2BM is 1:0.2 to 1:1.5.

[0096] In some preferred embodiments of this disclosure, in step (AB3M), the molar ratio of MTS001-2M to the catalyst is 1:0.1-1:5; preferably, the molar ratio of MTS001-2M to the catalyst is 1:0.2-1:3; more preferably, the molar ratio of MTS001-2M to the catalyst is 1:0.2-1:2.

[0097] In some preferred embodiments of this disclosure, in step (AB3M), the solvent for the reaction is any one or a combination of dichloromethane, chloroform, dimethyl sulfoxide, acetone, benzene, and toluene.

[0098] In some preferred embodiments of this disclosure, in step (AB3M), the reaction temperature is 0–50°C; preferably, the reaction temperature is 0–35°C.

[0099] In some preferred embodiments of this disclosure, the preparation method further includes the following steps:

[0100] Wherein n3, n4, and X are as defined in this disclosure.

[0101] In some preferred embodiments of this disclosure, in step AB4M, the reaction solvent is selected from either anhydrous ethanol or tetrahydrofuran.

[0102] In some preferred embodiments of this disclosure, in step AB4M, the molar ratio of MTS001-3M to ethanolamine is 1:1-20, preferably 1:1-15, and more preferably 1:1-10.

[0103] In some preferred embodiments of this disclosure, a catalyst and / or additive are added in step AB4M; preferably, the catalyst is sodium iodide; preferably, the additive is sodium iodide.

[0104] In some preferred embodiments of this disclosure, in step AB4M, the molar ratio of MTS001-3M to the catalyst is 1:0.05-1:0.3 or 1:0.08-1:0.2.

[0105] In some preferred embodiments of this disclosure, in step AB4M, the molar ratio of MTS001-3M to the additive is 1:0.05-1:0.3 or 1:0.08-1:0.2.

[0106] In some preferred embodiments of this disclosure, the reaction temperature of step AB4M is 0–100°C, preferably 20–80°C, and more preferably 50–80°C.

[0107] In some preferred embodiments of this disclosure, the preparation method further includes the following steps:

[0108] Wherein, n1, n2, n3, n4, and X are as defined in this disclosure.

[0109] In some preferred embodiments of this disclosure, the solvent for step (AB) is selected from any one or a combination of cyclopentyl methyl ether, acetonitrile, tetrahydrofuran, dioxane, and ethanol, preferably one or a combination of cyclopentyl methyl ether and acetonitrile.

[0110] In some preferred embodiments of this disclosure, step (AB) involves adding an alkali selected from any one or a combination of potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium hydroxide, and sodium hydroxide.

[0111] In some preferred embodiments of this disclosure, step (AB) involves adding a catalyst selected from any one of sodium iodide, potassium iodide, sodium bromide, and potassium bromide, preferably any one of sodium iodide and potassium iodide.

[0112] In some preferred embodiments of this disclosure, the reaction temperature of step (AB) is 20–100°C, preferably 20–80°C.

[0113] On the other hand, this disclosure provides methods for preparing ionizable lipid compounds as described above, or their pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers, including preparation method A or preparation method B:

[0114] The preparation method A includes the following steps: step (A1) of preparing an alcohol of formula (MTS001-11) using a diester of formula (SM-A1) and 1-iodoheptane (SM-A2) as raw materials;

[0115] The preparation method B includes the following steps: step (B1) of preparing an alcohol of formula (MTS001-11) from a ketone of formula (SM-B1);

[0116] In some preferred embodiments of this disclosure, step (A1) includes the following step (A11):

[0117] In some preferred embodiments of this disclosure, an alkali is added in step (A11), and the alkali used is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylaminodimethylamine; preferably, the alkali used in step (A11) is NaH.

[0118] In some preferred embodiments of this disclosure, in step (A11), the molar ratio of SM-A1 and SM-A2 is 1:2-1:5, preferably 1:2-1:3.

[0119] In some preferred embodiments of this disclosure, in step (A11), the reaction solvent is selected from any one or a combination of N,N-dimethylformamide, dimethyl sulfoxide, toluene, carbon tetrachloride, chloroform, and tert-butanol; more preferably, in step (A11), the reaction solvent is N,N-dimethylformamide.

[0120] In some preferred embodiments of this disclosure, the reaction temperature in step (A11) is -10°C to 70°C; preferably, the reaction temperature in step (A11) is -10°C to 50°C; more preferably, the reaction temperature in step (A11) is -5°C to 35°C.

[0121] In some preferred embodiments of this disclosure, step (A1) includes the following step (A12):

[0122] In some preferred embodiments of this disclosure, step (A12) further includes a catalyst, wherein the catalyst used is LiCl.

[0123] In some preferred embodiments of this disclosure, in step (A12), the molar ratio of MTS001-A1 to the catalyst is 1:2 to 1:20; preferably, the molar ratio of MTS001-A1 to the catalyst is 1:5 to 1:15.

[0124] In some preferred embodiments of this disclosure, in step (A12), the reaction solvent is selected from any one or a combination of N,N-dimethylformamide, dimethyl sulfoxide, toluene, carbon tetrachloride, and chloroform; more preferably, in step (A12), the reaction solvent is N,N-dimethylformamide.

[0125] In some preferred embodiments of this disclosure, the reaction temperature in step (A12) is 50°C to 200°C; preferably, the reaction temperature in step (A12) is 80°C to 180°C; more preferably, the reaction temperature in step (A12) is 80°C to 150°C.

[0126] In some preferred embodiments of this disclosure, step (A1) includes the following reduction reaction step (A13):

[0127] In some preferred embodiments of this disclosure, in step (A13), the reducing agent used is any one of Pt / H2, Pb / H2, Ni / H2, diisobutylaluminum hydride (DIBAL-H), lithium aluminum hydride (LAH), NaBH4, LiBH4, and borane; preferably, in step (A13), the reducing agent used is any one of DIBAL-H, LAH, NaBH4, and LiBH4; preferably, in step (A13), the reducing agent used is DIBAL-H.

[0128] In some preferred embodiments of this disclosure, in step (A13), the molar ratio of MTS001-A2 to the reducing agent is 1:1-1:5; preferably, the molar ratio of MTS001-A2 to the reducing agent is 1:1.5-1:4; preferably, the molar ratio of MTS001-A2 to the reducing agent is 1:2-1:3.

[0129] In some preferred embodiments of this disclosure, in step (A13), the reaction solvent is selected from tetrahydrofuran, diethyl ether, 1,4-dioxane, methanol, ethanol, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, N,N-dimethylformamide, or a combination thereof; preferably, in step (A13), the reaction solvent is selected from tetrahydrofuran and diethyl ether, or a combination thereof.

[0130] In some preferred embodiments of this disclosure, the reaction temperature in step (A13) is -10°C to 80°C; preferably, the reaction temperature in step (A13) is -5°C to 70°C; more preferably, the reaction temperature in step (A13) is -5°C to 35°C.

[0131] In some preferred embodiments of this disclosure, step (B1) includes the following step (B11):

[0132] In some preferred embodiments of this disclosure, in step (B11), the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylaminodimethylamine; preferably, in step (B11), the alkali used is n-BuLi.

[0133] In some preferred embodiments of this disclosure, in step (B11), the molar ratio of SM-B1 to SM-B2 is 1:1 to 1:5; preferably, the molar ratio of SM-B1 to SM-B2 is 1:1 to 1:3; preferably, the molar ratio of SM-B1 to SM-B2 is 1:1 to 1:2.

[0134] In some preferred embodiments of this disclosure, in step (B11), the molar ratio of SM-B1 to alkali is 1:1-1:5; preferably, the molar ratio of SM-B1 to alkali is 1:1-1:3; more preferably, the molar ratio of SM-B1 to alkali is 1:1-1:2; and more preferably, the molar ratio of SM-B1 to alkali is 1:1-1:1.5.

[0135] In some preferred embodiments of this disclosure, in step (B11), the reaction solvent is selected from any one or a combination of tetrahydrofuran, 1,4-dioxane, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide; preferably, in step (B11), the reaction solvent is selected from tetrahydrofuran.

[0136] In some preferred embodiments of this disclosure, the reaction temperature in step (B11) is -78°C to 35°C; preferably, the reaction temperature in step (B11) is -78°C to 25°C; more preferably, the reaction temperature in step (B11) is -5°C to 25°C.

[0137] In some preferred embodiments of this disclosure, step (B1) includes the following step (B12):

[0138] In some preferred embodiments of this disclosure, step (B12) further includes a catalyst, wherein the catalyst used is HCl.

[0139] In some preferred embodiments of this disclosure, in step (B12), the molar ratio of MTS001-B1 to the catalyst is 1:1-1:5; preferably, the molar ratio of MTS001-B1 to the catalyst is 1:1-1:3; more preferably, the molar ratio of MTS001-B1 to the catalyst is 1:1.5-1:2.5.

[0140] In some preferred embodiments of this disclosure, in step (B12), the reaction solvent is selected from tetrahydrofuran, 1,4-dioxane, or a combination thereof.

[0141] In some preferred embodiments of this disclosure, in step (B12), the reaction temperature is 0°C to 100°C; preferably, the reaction temperature is 20°C to 80°C.

[0142] In some preferred embodiments of this disclosure, step (B1) includes the following step (B13):

[0143] In some preferred embodiments of this disclosure, step (B13) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, NaBH4, LiBH4, diborane, and aluminum isopropoxide; preferably, in step (B13), the reducing agent is NaBH4.

[0144] In some preferred embodiments of this disclosure, in step (B13), the molar ratio of MTS001-B2 to the reducing agent is 1:1-1:5; preferably, the molar ratio of MTS001-B2 to the reducing agent is 1:1-1:3; more preferably, the molar ratio of MTS001-B2 to the reducing agent is 1:1-1:2; even more preferably, the molar ratio of MTS001-B2 to the reducing agent is 1:1-1:1.5.

[0145] In some preferred embodiments of this disclosure, in step (B13), the reaction solvent is selected from any one or a combination of tetrahydrofuran, diethyl ether, 1,4-dioxane, methanol, ethanol, toluene, carbon tetrachloride, chloroform, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide; preferably, in step (B13), the reaction solvent is selected from any one or a combination of tetrahydrofuran, diethyl ether, methanol, and ethanol.

[0146] In some preferred embodiments of this disclosure, in step (B13), the reaction temperature is -10°C to 50°C; preferably, the reaction temperature is -5°C to 35°C.

[0147] In some preferred embodiments of this disclosure, the preparation method further includes the following steps:

[0148] In some preferred embodiments of this disclosure, step (AB3) further includes a catalyst selected from any one or a combination of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole (HOBt), sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and triethylamine; preferably, the catalyst is a combination of EDCI and DMAP.

[0149] Preferably, in step (AB3), the molar ratio of MTS001-2 to MTS001-20 is 1:1 to 1:5; more preferably, the molar ratio of MTS001-2 to MTS001-20 is 1:1 to 1:3; more preferably, the molar ratio of MTS001-2 to MTS001-20 is 1:0.2 to 1:1.5.

[0150] Preferably, in step (AB3), the molar ratio of MTS001-2 to the catalyst is 1:0.1-1:5; more preferably, the molar ratio of MTS001-2 to the catalyst is 1:0.2-1:3; more preferably, the molar ratio of MTS001-2 to the catalyst is 1:0.2-1:2.

[0151] Preferably, in step (AB3), the solvent for the reaction is any one or a combination of dichloromethane, chloroform, dimethyl sulfoxide, acetone, benzene, and toluene.

[0152] Preferably, in step (AB3), the reaction temperature is 0-50°C; preferably, the reaction temperature is 0-35°C.

[0153] In some preferred embodiments of this disclosure, preparation method A includes the following steps:

[0154] Preparation of (A1X)MTS001-5:

[0155] Dimethyl malonate SM-A1 and 1-iodoheptane SM-A2 react under sodium hydride conditions to obtain MTS001-A1; MTS001-A1 reacts under lithium chloride conditions to obtain MTS001-A2; MTS001-A2 reacts under diisobutylaluminum hydride conditions to obtain MTS001-A11; 8-bromooctanoic acid (SM3) reacts under SOCl2 conditions to obtain the corresponding acyl chloride, and the corresponding acyl chloride reacts with MTS001-A11 under N,N-diisopropylethylamine conditions to obtain MTS001-5;

[0156] Preparation of (A2X)MTS001-4:

[0157] Ethyl isobutyrate (SM1) and N,N-dimethylpropenylurea (DMPU) react with 1,5-dibromopentane in the presence of lithium diisopropylaminoacetate to obtain MTS001-1; MTS001-1 is then reacted with aluminum diisobutylhydride (DIBAL-H) ​​to obtain MTS001-2; MTS001-2 is then reacted with decanoyl chloride under triethylamine conditions to obtain MTS001-3; and MTS001-3 is then reacted with ethanolamine under sodium iodide conditions to obtain MTS001-4.

[0158] Preparation of (A3X)MTS001:

[0159] MTS001-4 and MTS001-5 were reacted under sodium iodide and K2CO3 conditions to obtain MTS001;

[0160] In some preferred embodiments of this disclosure, preparation method B includes the following steps:

[0161] Preparation of (A1Y)MTS001-5:

[0162] (Methoxymethyl)triphenylphosphonium chloride reacts with pentadecane-8-one (SM-B1) under n-butyllithium conditions to give MTS001-B1; MTS001-B1 reacts with HCl conditions to give MTS001-B2; MTS001-B2 reacts with sodium borohydride conditions to give MTS001-11; MTS001-11 reacts with SM3 under the action of 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to give MTS001-5;

[0163] Preparation of (A2Y)MTS001-4:

[0164] Ethyl isobutyrate (SM1) and N,N-dimethylpropenylurea react with 1,5-dibromopentane in the presence of lithium diisopropylaminoacetate to obtain MTS001-1; MTS001-1 is then reacted with aluminum diisobutylhydride (DIBAL-H) ​​to obtain MTS001-2; MTS001-2 is then reacted with n-decanoic acid under EDCI / DMAP conditions to obtain MTS001-3; and MTS001-3 is then reacted with ethanolamine under sodium iodide conditions to obtain MTS001-4.

[0165] Preparation of (A3Y)MTS001:

[0166] MTS001-4 and MTS001-5 were reacted under sodium iodide and potassium carbonate conditions to obtain MTS001;

[0167] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound;

[0168] The purification method for the ionizable lipid compound is liquid chromatography:

[0169] Mobile phase A is an aqueous solution of 0.05% (V / V) trifluoroacetic acid; mobile phase B is an acetonitrile / isopropanol (1:1) solution of 0.05% (V / V) trifluoroacetic acid.

[0170] The eluent of ionizable lipid compounds was collected under the following separation gradient conditions;

[0171] The gradient separation condition is:

[0172] From 0 to 40 minutes, the proportion of mobile phase B increased from 80% to 88%, while the proportion of mobile phase A decreased from 20% to 12%.

[0173] For 40–60 min, the proportion of mobile phase B is 100% and the proportion of mobile phase A is 0%.

[0174] For 60–75 minutes, the proportion of mobile phase B is 80% and the proportion of mobile phase A is 20%.

[0175] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound;

[0176] The purification of the ionizable lipid compound includes filtration and washing steps:

[0177] The eluent of the ionizable lipid compound is filtered through a hydrophobic PTFE filter, washed with a 1-6% sodium bicarbonate solution, extracted with an organic solvent, separated, and the organic phase is retained. Preferably, the retained organic phase can be repeated with the above filtration, washing, and extraction steps, followed by drying, filtration, and concentration to obtain an ionizable lipid compound with a purity of 95% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 97% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 98% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 99% or higher.

[0178] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound.

[0179] In some preferred embodiments of this disclosure, the purification of the ionizable lipid compound includes filtration and washing steps.

[0180] In some preferred embodiments of this disclosure, the filtration and washing steps include: filtering the eluent of the ionizable lipid compound through a hydrophobic PTFE filter, washing with a 1-3% sodium bicarbonate solution, extracting with an organic solvent, separating, and retaining the organic phase; preferably, the retained organic phase can be repeated with the above filtration, washing, and extraction steps; drying, filtering, and concentrating the obtained organic phase to obtain an ionizable lipid compound with a purity of 95% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 97% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 98% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 99% or higher.

[0181] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound.

[0182] In some preferred embodiments of this disclosure, the purification of the ionizable lipid compound includes filtration and washing steps.

[0183] In some preferred embodiments of this disclosure, the filtration and washing steps include: filtering the eluent of the ionizable lipid compound through a hydrophobic PTFE filter, washing with a 1.8–2.2% sodium bicarbonate solution, extracting with an organic solvent, separating, and retaining the organic phase; preferably, the retained organic phase can be repeated with the above filtration, washing, and extraction steps; drying, filtering, and concentrating the obtained organic phase to obtain an ionizable lipid compound with a purity of 95% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 97% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 98% or higher; preferably, drying, filtering, and concentrating the organic phase to obtain an ionizable lipid compound with a purity of 99% or higher.

[0184] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound, the steps of which are as follows:

[0185] (1) Liquid chromatography purification

[0186] Mobile phase A is an aqueous solution of 0.05% (V / V) trifluoroacetic acid; mobile phase B is a 0.05% (V / V) trifluoroacetic acid solution in acetonitrile / isopropanol (1:1).

[0187] The eluent of ionizable lipid compounds was collected under the following separation gradient conditions;

[0188] The gradient separation condition is:

[0189] From 0 to 40 minutes, the proportion of mobile phase B increased from 80% to 88%, while the proportion of mobile phase A decreased from 20% to 12%.

[0190] For 40–60 min, the proportion of mobile phase B is 100% and the proportion of mobile phase A is 0%.

[0191] For 60–75 min, the proportion of mobile phase B is 80% and the proportion of mobile phase A is 20%.

[0192] (2) Filtration and washing steps:

[0193] The eluent of the ionizable lipid compound is filtered through a hydrophobic PTFE filter, and washed, extracted, separated, and retained using a 1-6% sodium bicarbonate solution, preferably a 1-3% sodium bicarbonate solution, more preferably a 1.8-2.2% sodium bicarbonate solution. Preferably, the retained organic phase can be filtered, washed, and extracted 1-2 times. The obtained organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 95% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 97% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 98% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 99% or higher.

[0194] In some preferred embodiments of this disclosure, the preparation method further includes the purification of the ionizable lipid compound:

[0195] (1) Liquid chromatography purification

[0196] Mobile phase A is an aqueous solution of 0.05% (V / V) trifluoroacetic acid; mobile phase B is a 0.05% (V / V) trifluoroacetic acid solution in acetonitrile / isopropanol (1:1).

[0197] The eluent of ionizable lipid compounds was collected under the following separation gradient conditions;

[0198] The gradient separation condition is:

[0199] From 0 to 40 minutes, the proportion of mobile phase B increased from 80% to 88%, while the proportion of mobile phase A decreased from 20% to 12%.

[0200] For 40–60 min, the proportion of mobile phase B is 100% and the proportion of mobile phase A is 0%.

[0201] For 60–75 min, the proportion of mobile phase B is 80% and the proportion of mobile phase A is 20%.

[0202] (2) Filtration and washing steps:

[0203] The eluent of the ionizable lipid compound is filtered through a hydrophobic PTFE filter, washed and extracted with a 1.8–2.2% sodium bicarbonate solution, retaining the organic phase. Preferably, the retained organic phase can be filtered and washed 1–2 times. The obtained organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 95% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 97% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 98% or higher. Preferably, the organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 99% or higher.

[0204] Preferably, in the above purification step, the organic solvent used for extraction is selected from one or more of n-heptane, hexane, ethyl acetate, dichloromethane, chloroform, petroleum ether, and diethyl ether. Preferably, the organic solvent used for extraction is n-heptane.

[0205] On the other hand, this disclosure provides the application of the ionizable lipid compounds as described above, or pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers thereof, or ionizable lipid compounds as described above, or pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers thereof, prepared by the methods described above, in the preparation of bioactive substance delivery systems.

[0206] In some preferred embodiments of this disclosure, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle, or microbubble; preferably, the delivery system is a liposome or lipid nanoparticle.

[0207] On the other hand, this disclosure provides the use of the preparation method as described above for preparing the ionizable lipid compound described herein or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof.

[0208] On the other hand, the ionizable lipid compound or its pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer prepared by the preparation method described above has a purity of not less than 90%.

[0209] Preferably, its purity is not less than 95%;

[0210] Preferably, its purity is not less than 98%;

[0211] Preferably, its purity is not less than 99%;

[0212] More preferably, its purity is not less than 99.5%.

[0213] On the other hand, this disclosure provides a method for preparing a bioactive substance delivery system, comprising the steps of preparing an ionizable lipid compound as described above or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof, or an ionizable lipid compound or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof prepared by the preparation method described above.

[0214] On the other hand, this disclosure provides ionizable lipid compounds as described above, or pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers thereof, or ionizable lipid compounds or pharmaceutically acceptable salts, solvates, isotopic variants, tautomers, or stereoisomers thereof prepared by the methods described above, for use in the preparation of bioactive substance delivery systems.

[0215] This disclosure has the following advantages:

[0216] (1) This disclosure provides two methods for preparing ionizable lipid compounds, which meet the requirements for scale-up production.

[0217] (2) This disclosure provides a method A and a method B for preparing ionizable lipid compounds, which improves the yield of ionizable lipid compounds.

[0218] (3) This disclosure optimizes the synthetic route of ionizable lipid compounds, realizes scale-up production, and achieves good purity. Detailed Implementation

[0219] Definitions and Explanations

[0220] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used have meanings commonly understood by those skilled in the art. Furthermore, the cell and tissue culture, microbiology-related terms, and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this disclosure, definitions and explanations of relevant terms are provided below. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. It should also be understood that the terminology used in this application is for describing specific embodiments only and is not intended to be limiting.

[0221] Unless otherwise expressly stated, the terms “a,” “an,” and “the” as used in this specification and the appended claims cover one or more types.

[0222] As used in this disclosure, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0223] In the description of this disclosure, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0224] As used in this disclosure and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatically equivalent forms, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0225] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0226] As used in this disclosure, the term "substitution" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are replaced. The terms "optional substitution" or "optionally substituted" mean that substitution is optional, and unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis.

[0227] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0228] In any embodiment, any or all hydrogen atoms present in the compound, or hydrogen atoms in a specific group or portion of the compound, may be replaced by deuterium or tritium. One to a maximum number of hydrogen atoms present in the compound may be replaced by deuterium. One to a maximum number of hydrogen atoms present in any group of the general formula compound or a specific compound may be replaced by deuterium. For example, when a group is described as ethyl, the ethyl group may be C2H5 or a C2H5 in which x (1 to 5) hydrogen atoms are replaced by deuterium, such as C2D. x H 5-x When a group is described as a deuterated ethyl group, the deuterated ethyl group can be a C2H5 with x (1 to 5) hydrogen atoms replaced by deuterium, such as C2D. x H 5-x The stable deuterated derivatives described in this disclosure are preferably stable deuterated isotope derivatives obtained by replacing any deuterated hydrogen atom in each formula with 1 to a maximum number (e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, etc.) of deuterium atoms.

[0229] This disclosure refers to compounds of formula (I), including their isotopic variants, tautomers, stereoisomers, mixtures of stereoisomers, solvates (solvents) or derivatives.

[0230] This disclosure of "compounds" also includes tautomer forms. A tautomer form arises from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton. The terms "tautomer" or "tautomer form" refer to isomers of different functional groups in dynamic equilibrium at room temperature that can rapidly interconvert. It refers to one of two or more structural isomers that exist in equilibrium and readily transform from one isomer form to another. This transformation results in the formal migration of a hydrogen atom, accompanied by the conversion of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomer groups in solution. In solutions where tautomerization is possible, chemical equilibrium of the tautomers will be reached. The exact proportions of the tautomers depend on several factors, including temperature, solvent, and pH conditions. The concept of tautomers that can interconvert through tautomerization is called tautomerism.

[0231] When this specification describes a compound that is readily tautomerizable, but only one of its tautomers is described, it should be understood that all tautomers are included as part of the chemical meaning described. It should be understood that when a compound has tautomeric forms, it is intended to include all tautomeric forms, and the naming of the compound does not exclude any tautomeric form.

[0232] Of the various possible types of tautomerism, two are typically observed. In keto-enol tautomerism, both electrons and hydrogen atoms move simultaneously.

[0233] Common tautomer pairs are: keto-enol, amide-nitrile, lactam-lactam, amide-imine tautomer in heterocycles, imine-enamine, and enamine-enamine.

[0234] The term "isomer" refers to different compounds having the same molecular formula but different atomic arrangements and configurations. Depending on their structure, the compounds of this disclosure can exist in different stereoisomeric forms. These forms include configurational isomers or optical conformational isomers (enantiomers and / or diastereomers, including those that are blocked from rotation). Therefore, this disclosure includes enantiomers, diastereomers, and mixtures thereof. This disclosure further includes all mixtures of the above-described stereoisomers, regardless of proportions, including racemic mixtures.

[0235] Depending on their structure, the compounds disclosed herein can exist in various stable isotopic forms. These forms include those in which one or more hydrogen atoms are replaced by deuterium atoms, those in which one or more nitrogen atoms are replaced by 15N atoms, or those in which one or more carbon, fluorine, chlorine, bromine, sulfur, or oxygen are replaced by stable isotopes of their respective original atoms.

[0236] According to this disclosure, some compounds and salts can exist in different crystalline forms (polymorphs) within the scope of this disclosure.

[0237] The term "alkyl" refers to a chain-like (straight-chain or branched) saturated aliphatic hydrocarbon group. The term "alkyl" can refer to a straight-chain or branched alkyl group containing 1 to 10 carbon atoms. 1-10 Alkyl groups, preferably alkyl groups containing 1 to 6 carbon atoms (C 1-6Alkyl groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof. More preferably are lower alkyl groups containing 1 to 3 carbon atoms (C... 1-3 Alkyl groups, including methyl, ethyl, n-propyl, isopropyl, etc., are used in non-limiting embodiments. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups described in this application.

[0238] In one embodiment, the substituent is independently selected from oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight-chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, -C(=O)NH (substituted or unsubstituted alkyl, or substituted or unsubstituted). -Phenyl), -C(=O)N(H or alkyl)2, -OC(=O)N(substituted or unsubstituted alkyl)2, NHC(=O)NH(substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl), -NHC(=O)alkyl, -N(substituted or unsubstituted alkyl)C(=O)(substituted or unsubstituted alkyl), -NHC(=O)(substituted or unsubstituted alkyl), -C(OH)(substituted or unsubstituted alkyl)2 and -C(NH2)(substituted or unsubstituted alkyl)2. In another embodiment, for example, the optional substituents are selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, -OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and C(=O)OH. In yet another embodiment, the substituents are independently selected from C 1-6 Alkyl, -OH, C 1-6 Alkoxy, halogen, amino, acetamino, oxo, and nitro groups. In yet another embodiment, the substituents are independently selected from C10.1-6 Alkyl, C 1-6 Alkyl, halogen, acetamino, and nitro groups. As used in this disclosure, when the substituent is alkyl or alkoxy, the carbon chain can be branched, linear, or cyclic.

[0239] The term "alkylene" refers to a divalent group formed by removing one hydrogen atom from an alkyl group, which may be substituted or unsubstituted. The "alkylene" is preferably a divalent group of a straight-chain or branched saturated aliphatic hydrocarbon containing 1 to 8 carbon atoms, more preferably a divalent alkyl group containing 1 to 6 carbon atoms (C1 to C2). 1-6 Alkylenes, examples of which include, but are not limited to, -CH2-, -CH(CH3)-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH2CH2- or -(CH2)4- and their stereoisomers.

[0240] The term "heteroatom" is selected from nitrogen, oxygen, or sulfur. Nitrogen may optionally be substituted; sulfur may also optionally be substituted, for example, by oxidation, thus forming S(O). t3 (where t3 is an integer from 0 to 2).

[0241] The term "aryl" refers to phenyl or naphthyl, or phenyl or naphthyl substituted with the following groups: halogen, C 1-8 Alkyl, hydroxyl, nitro, trifluoromethyl, etc. Phenyl or monosubstituted phenyl groups are preferred; phenyl is the most preferred.

[0242] The terms "solvent" or "solvent compound" as used in this disclosure refer to complexes formed by the compounds of this disclosure with solvents. These complexes either react in the solvent or precipitate or crystallize from the solvent. For example, a complex formed with water is called a "hydrate". Solvents of the compounds represented by formula (I) of this disclosure are within the scope of this disclosure.

[0243] As used in this disclosure, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of the present invention.

[0244] Pharmaceutically acceptable base addition salts are those formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include sodium, potassium, magnesium, and calcium. Suitable amines include N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine.

[0245] The base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the required base in a conventional manner to form a salt. The free acid can be regenerated by contacting the salt form with an acid in a conventional manner and then separating the free acid. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents; however, for the purposes of this invention, the salts are equivalent to their respective free acids.

[0246] Salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, and iodides prepared from inorganic acids, such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthate, methanesulfonate, gluconate, lactobionate, laurylsulfonate, and hydroxyethanesulfonate. Salts can also be prepared from organic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, alkyl diacids, aromatic acids, and aliphatic and aromatic sulfonic acids. Representative salts include acetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinates, caprylates, sebacic acid salts, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, naphthates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, and methanesulfonates. Pharmaceutically acceptable salts may include alkali metal and alkaline earth metal-based cations, such as sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Salts of amino acids are also included, such as arginine salts, gluconates, and galacturons.

[0247] This disclosure includes prodrugs of the aforementioned compounds. The prodrugs include known amino and carboxyl protecting groups, which are released under physiological conditions by hydrolysis or via enzymatic reactions to yield the parent compound.

[0248] As used in this disclosure, the term "hydroxyl" refers to -OH.

[0249] As used in this disclosure, the term "oxo" refers to =O.

[0250] As used in this disclosure, the term "carboxyl group" refers to -C(=O)OH.

[0251] Example

[0252] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below in conjunction with the chemical reaction formulas in the embodiments of this disclosure. Obviously, the described embodiments are some embodiments of this disclosure, but not all embodiments. The following is merely a further description of this disclosure, and the protection scope of this disclosure is not limited thereto.

[0253] In the specific embodiments of this disclosure, the technical means or methods not specifically described are conventional technical means or methods in the art. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available. Table 1 below lists the abbreviations of common chemical substances used in the embodiments and comparative examples of this disclosure.

[0254] Table 1. Abbreviations of common chemical substances

[0255] Example 1

[0256] Preparation method A of compound MTS001

[0257] (1) Preparation of MTS001-A1

[0258] Dimethyl malonate SM-A1 (10 g, 75 mmol) was dissolved in N,N-dimethylformamide (200 mL, 20 V). The solution was cooled to 0 °C, and 1-iodoheptane SM-A2 (43 g, 189 mmol, 2.5 eq.) was added in a single batch. Maintaining the internal temperature below 20 °C, sodium hydride (60% mineral oil suspension, 7.6 g, 189 mmol, 2.5 eq.) was slowly added in portions. The reaction system was heated to room temperature and maintained for 6 hours. The reaction was monitored by TLC. Water (400 mL) was slowly added to the mixture for quenching, followed by extraction with diisopropyl ether (200 mL × 2). The organic phases were combined, washed with water (200 mL), and then concentrated under vacuum. The concentrated residue was purified by silica gel column chromatography using PE:EA (100:1–20:1) as the eluent. A pale yellow oil, MTS001-A1 (23 g, 92% yield), was obtained. 1 H NMR (CDCl3) δ: 0.77-0.93 (m, 6H), 1.05-1.19 (m, 4H), 1.20-1.34 (m, 16H), 1.79-1.93 (m, 4H), 3.70 (s, 6H).

[0259] (2) Preparation of MTS001-A2

[0260] MTS001-A1 (23 g, 70 mmol) was dissolved in N,N-dimethylformamide (345 ml), and lithium chloride (30 g, 700 mmol, 10 eq.) was added while stirring. The solution was heated to 120 °C and stirred for 12 hours under nitrogen protection. The reaction was monitored by TLC. Water (690 ml) and ethyl acetate (345 ml) were added to the mixture, and the mixture was stirred for 1 hour. Phase separation was performed; the organic phase was washed with water (230 ml × 3) and concentrated under vacuum. The concentrated residue was purified by silica gel column chromatography using PE:EA (100:1–50:1) as the eluent. A pale yellow oil, MTS001-A2 (15 g, 79% yield), was obtained.

[0261] (3) Preparation of MTS001-11

[0262] Dissolve 15 g (56 mmol) of MTS001-A2 in 345 mL of N,N-dimethylformamide. Cool the solution to 0 °C and slowly add diisobutylaluminum hydride (1 M hexane solution, 140 mL, 140 mmol, 2.5 eq.) under nitrogen protection. Monitor the reaction by TLC. Slowly add 200 mL of 10% citric acid solution to quench the reaction and stir the mixture until both phases are clear. Perform phase separation, wash the organic phase with 150 mL of water and concentrate under vacuum. Purify the concentrated residue by silica gel column chromatography with PE:EA (50:1–20:1) as eluent to obtain a pale yellow oily substance, MTS001-11 (12 g, yield 81%), with a GC purity of 98.0% and a maximum single impurity of 1.3%.

[0263] (4) Preparation of MTS001-5

[0264] To a solution of SM3 (13.3 g, 59 mmol, 1.2 eq.) in dichloromethane (120 mL, 10 V), SOCl2 (10.8 mL, 149 mmol, 3 eq.) was added dropwise at 0 °C. The solution was stirred at 0 °C for 4 hours and concentrated under vacuum to obtain the corresponding acyl chloride. MTS001-11 (12 g, 49 mmol) was dissolved in dichloromethane (120 mL, 10 V). The solution was cooled to 0 °C, and N,N-diisopropylethylamine (26 mL, 149 mmol, 3 eq.) was added. The previously obtained acyl chloride was then slowly added to this solution, and the mixture was stirred at 0 °C for 4 hours. The reaction was monitored by TLC. The mixture was quenched with saturated NaHCO3 (60 mL, 5 V), the organic phase was separated, and the solution was dissolved in 1 M HCl (120 mL, 10 V) and water (120 mL, 10 V). The organic phase was concentrated under vacuum, and the residue was purified by silica gel column chromatography with PE:EA (100:1 to 50:1) as the eluent. MTS001-5 (19g, yield 86%) was obtained as a pale yellow liquid with a purity of 94.3%.

[0265] (5) Preparation of MTS001-1

[0266] Under a nitrogen atmosphere, ethyl isobutyrate (65 g, 560 mmol, 1 eq.) and N,N-dimethylpropenylurea (172.2 g, 1340 mmol, 2.4 eq.) were dissolved in tetrahydrofuran (520 mL, 8 V). The mixture was then stirred for 2 hours at -50 °C under a nitrogen atmosphere, followed by the addition of lithium diisopropylamino (336 mL, 672 mmol, 1.2 eq.) (reaction 1). In another reaction (reaction 2), 1,5-dibromopentane (140.4 g, 616 mmol, 1.1 eq.) was dissolved in tetrahydrofuran (260 mL, 4 V) and then cooled to -50 °C under a nitrogen atmosphere. The solution from reaction 1 was slowly added to the solution from reaction 2 at -50 °C under a nitrogen atmosphere, and the mixture was reacted at -50 °C for 1 hour. The reaction was monitored using LC-MS. The mixture was quenched with 1N HCl (10V, 65ml) and stirred at 10–20°C for 10 minutes. The mixture was extracted with ethyl acetate, the organic phase was concentrated, and the residue was purified by silica gel column chromatography with PE:EA (10:1) as the eluent, thus obtaining MTS001-1 (70g, yield 47%) as a pale yellow liquid with a purity of 98.8%.

[0267] (6) Preparation of MTS001-2

[0268] MTS001-1 was dissolved in tetrahydrofuran, and the reaction temperature was lowered to -10°C. Diisobutylaluminum hydride (609 mL, 609 mmol, 2.30 eq.) was added to the reaction vessel from -10°C to 0°C. The reaction was carried out under a nitrogen atmosphere at 0°C for 1 hour, followed by a reaction at 25–30°C for 4 hours. The reaction was quenched by adding 1N HCl (12V, 840 mL), and the organic phase was extracted with ethyl acetate. The organic phase was concentrated, and the residue was purified by silica gel column chromatography using PE:EA (10:1) as the eluent. This yielded MTS001-2 (53 g, 90% yield) as a pale yellow liquid with a purity of 99.4%.

[0269] (7) Preparation of MTS001-3

[0270] MTS001-2 was added to dichloromethane (795 mL, 15°C), and the reaction was cooled to 0°C. Triethylamine (72.3 g, 716 mmol, 3 eq.) was added to the reaction mixture, and the mixture was stirred at 0°C for 10 minutes. Decanoyl chloride (49.9 g, 262.5 mmol, 1.1 eq.) was added at 0°C, and the reaction was stirred at 25°C for 3 hours. After the reaction was complete, it was quenched with 5% NaHCO3 (25 ± 5°C, stirring for at least 10 minutes). The aqueous phase was extracted with dichloromethane (530 mL), and the organic phase was washed with citric acid (25 ± 5°C, stirring for at least 10 minutes), followed by washing with 5% NaCl (25 ± 5°C, stirring for at least 10 minutes). The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with PE:EA (100:1) as the eluent. MTS001-3 (70g, yield 78%) was obtained as a pale yellow liquid with a purity of 97.1%.

[0271] (8) Preparation of MTS001-4

[0272] MTS001-3 (65 g, 173 mmol, 1 eq.), ethanolamine (53.2 g, 865 mmol, 5 eq.), and sodium iodide (2.6 g, 17 mmol, 0.1 eq.) were added to ethanol (325 mL), and the solution was stirred at 70 °C for 3 hours. The reaction solution was cooled to 25 °C, and water (650 mL) was added. The mixture was extracted with dichloromethane (10 V × 2, 650 mL), the organic phases were combined and washed with water (325 mL), the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with DCM:MeOH (50:1–20:1) as the eluent. MTS001-4 (41.9 g, yield 68%) was obtained as a pale yellow liquid with a purity of 93.1%.

[0273] (9) Preparation of MTS001

[0274] MTS001-4 (11.1 g, 31 mmol, 1 eq.) and MTS001-5 (18 g, 40.3 mmol, 1.30 eq.) were dissolved in cyclopentyl methyl ether (111 mL) and acetonitrile (33.3 mL). Sodium iodide (5.6 g, 37.4 mmol, 1.2 eq.) and K2CO3 (12.9 g, 93 mmol, 3 eq.) were added, and the mixture was heated at 70 °C for 48 hours. Dichloromethane (143 mL) and water (143 mL) were added to the mixture for phase separation. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (143 mL). The organic phases were combined and washed with water (143 mL). The organic phases were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by silica gel column chromatography with n-heptane:ethyl acetate (10:1 to 5:1) as the eluent, thus yielding 10 g of MTS001 in a yield of 45%, as a pale yellow liquid with a purity of 93.1% (HPLC-CAD).

[0275] Example 2

[0276] Preparation method B of compound MTS001

[0277] (1) Preparation of MTS001-B1

[0278] (Methoxymethyl)triphenylphosphonium chloride (SM-B2, 2.76 kg, 8.04 mol, 1.4 eq.) was suspended in THF (13 L). The solution was cooled to -20 °C, and n-butyllithium (2.5 M hexane solution, 3.0 L, 7.46 mol, 1.3 eq.) was slowly added under nitrogen. The mixture was stirred for 2 hours while maintaining the temperature below 0 °C. Pentadecane-8-one (SM-B1, 1.3 kg, 5.74 mol) in THF (5.2 L) was slowly added to the reaction mixture. The mixture was brought to room temperature and stirred for another 18 hours. GC monitoring was performed until the reaction was complete. The reaction was quenched by slowly adding cold water (13 L). The mixture was extracted with n-heptane (13 L) and stirred for 30 minutes. The aqueous phase was discarded, and the organic phase was washed with water (13 L) and replaced with heptane (13 L) under vacuum. The mixture was filtered to remove insoluble solids. The residue was concentrated under vacuum and purified by silica gel column chromatography (PE elution). This yielded a light yellow oily substance, MTS001-B1 (1170g, yield 80%), with a purity of 90.8%.

[0279] (2) Preparation of MTS001-B2

[0280] MTS001-B1 (1170 g, 4.6 mol) was dissolved in tetrahydrofuran (5.8 L), and 4M HCl solution (2340 mL, 9.2 mol, 2 eq.) was added while stirring. The solution was heated to 70 °C and stirred for 18 hours. The reaction was monitored by GC. The mixture was cooled to 25 °C, and n-heptane (11.7 L) was added to the mixture. The mixture was stirred for 10 minutes, and the organic phase was separated. The mixture was washed with water (11.7 L) and 5% NaHCO3 (5.8 L). The organic phase was concentrated under vacuum. The crude product MTS001-B2 obtained was used directly in the next step without further purification.

[0281] (3) Preparation of MTS001-11

[0282] MTS001-B2 (crude product, 4.6 mol) was dissolved in methanol (11.7 L, 10 V). The solution was cooled to 0 °C, and sodium borohydride (174 g, 4.6 mol, 1 eq.) was slowly added in several batches while stirring. The solution was stirred at 0 °C for 2 hours, and the reaction was monitored by GC. The mixture was quenched by slowly adding water (23.4 L), and the mixture was extracted with ethyl acetate (11.7 L). The organic phase was washed with water (11.7 L), and the mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography (PE:EA = 50:1 to 20:1 elution) to obtain a light yellow oily substance MTS001-11 (890 g, 2-step yield 80%) with a purity of 99.4%.

[0283] (4) Preparation of MTS001-5

[0284] MTS001-11 (300 g, 1.24 mol, 1 eq.) was added to dichloromethane (3.0 L). SM3 (331 g, 1.48 mol, 1.2 eq.) and 4-dimethylaminopyridine (45 g, 0.37 mol, 0.3 eq.) were added to the reaction mixture at 25 °C. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (285 g, 1.48 mol, 1.2 eq.) was then added to the reaction mixture. The mixture was stirred at 25 °C for 2 hours, and the reaction was monitored by HPLC. The reaction was quenched with water (1.5 L). The lower organic phase was separated and concentrated under vacuum. The residue was dissolved in methanol (2.25 L), and 0.5 M... HCl solution (750 mL) and n-heptane (3.0 L) were added and stirred for 1 hour. The mixture was separated, and the lower aqueous phase was discarded. Methanol / water (3:1, 3 L) was added and stirred for 1 hour. The lower aqueous phase was discarded. The n-heptane solution was washed with 5% NaHCO3 solution (1.5 L). The upper organic phase was separated and concentrated under vacuum to obtain a light yellow oily substance MTS001-5 (510 g, yield 92%) with a purity of 95.1%.

[0285] (5) Preparation of MTS001-1

[0286] Under a nitrogen atmosphere, ethyl isobutyrate (1100 g, 9.48 mol, 1 eq.) and N,N-dimethylpropenylurea (2915 g, 22.74 mol, 2.4 eq.) were dissolved in tetrahydrofuran (8.8 L). Under a nitrogen atmosphere, lithium diisopropylamino (2M tetrahydrofuran / n-hexane solution, 5.7 L, 11.4 mol, 1.2 eq.) was added at -50 °C, and the mixture was stirred at -50 °C for 2 hours (reaction vessel 1). In another reactor (reaction vessel 2), 1,5... Dibromopentane (3240 g, 14.2 mol, 1.5 eq.) was dissolved in tetrahydrofuran (4.4 L) and then cooled to -50 °C under a nitrogen atmosphere. Under a nitrogen atmosphere, the solution in container 1 was slowly transferred to the solvent in container 2 at -50 °C. The resulting mixture was stirred at -50 °C for 1 hour, then raised to room temperature and reacted for 12 hours. The reaction was monitored by GC. 1 M hydrochloric acid solution (8.8 L) was added dropwise to the reaction system while maintaining the temperature at 10-20 °C and stirring for 10 minutes. Methyl tert-butyl ether (8.8 L ml) was added to the mixture for extraction. The reaction was stirred at 10-20 °C for 10 minutes, and the organic phase was separated and collected. Add anhydrous sodium sulfate (2.0 kg, 2.0 w / w) and dry, then evaporate to dryness. The residue is purified by silica gel column chromatography (PE:EA = 40:1 to 10:1 elution) to give a light yellow oily substance MTS001-1 (1350 g, yield 54%) with a purity of 95.4%.

[0287] (6) Preparation of MTS001-2

[0288] MTS001-1 (1300 g, 4.92 mol, 1 eq.) was dissolved in tetrahydrofuran (6.5 L). The temperature of the reactants was lowered to 5 ± 5 °C. Diisobutylaluminum hydride (1 M toluene solution, 11.3 L, 11.3 mol, 2.3 eq.) was added to the reactor at 5 ± 5 °C. The reaction was carried out at 0 °C for 1 hour under a nitrogen atmosphere, followed by a reaction at 25-30 °C for 12 hours. 12V (15.6 L) EA and 12V 1N HCl (15.6 L) were added to the reactor. The reaction solution was added in batches with stirring. The reaction was stirred at 10-20 °C for 10 minutes. The mixture was separated, the organic phase was concentrated, and the residue was purified by silica gel column chromatography (PE:EA = 40:1 to 10:1 elution) to obtain a light yellow oily substance MTS001-2 (980 g, yield 90%) with a purity of 96.2%.

[0289] (7) Preparation of MTS001-3

[0290] Add dichloromethane (18.62 kg) to the reactor, control the temperature at 15 ± 5 °C, add MTS001-2 (700.0 g, 1.0 eq), add N,N-dimethylaminopyridine (119.0 g, 0.3 eq) and n-decanoic acid (651.0 g, 1.2 eq), start stirring, and cool the reaction system to 15 ± 5 °C. At 15 ± 5 °C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (728.0 g, 1.2 eq.) to the reaction system. After the addition is complete, maintain the reaction system at 15 ± 5 °C and stir for at least 30 minutes. Warm the reaction system back to 25 ± 5 °C and stir for at least 12 hours. After the reaction is complete as monitored by GC, transfer the reaction solution to a rotary evaporator for concentration. Add n-heptane (1.43 kg, 3.0 V) to the concentrate and concentrate again until no fraction remains. Add methanol (3.32 kg, 6.0 V) and water (1.40 kg, 2.0 V) to the concentrate, stir at room temperature, then add citric acid monohydrate (280.0 g, 0.4 w / w) and stir until a clear solution is obtained. Add methanol (1.66 kg, 3.0 V) and water (700.0 g, 1.0 V) again, stir at room temperature, then add citric acid monohydrate (140.0 g, 0.2 w / w) and stir until a clear solution is obtained. Add n-heptane (7.14 kg, 15.0 V), stir, let stand, separate the layers, discard the lower aqueous phase, and retain the upper organic phase in the reactor. Add methanol (4.98 kg, 9.0 V) and water (2.10 kg, 3.0 V), stir, let stand, separate the layers, discard the lower aqueous phase, add sodium bicarbonate solution (7.00 kg water and 140.0 g sodium bicarbonate) to the upper organic phase, stir until the solution is clear, let stand, separate the layers, collect the upper organic phase in a container, and add anhydrous sodium sulfate (2.10 kg) to dry. Filter with a 200-mesh filter cloth, collect the filtrate, and wash the filter cake with n-heptane. Concentrate the filtrate and eluent under reduced pressure until no fraction remains, to obtain product MTS001-3, net weight 1.355 kg.

[0291] (8) Preparation of MTS001-4

[0292] Add MTS001-3 (1.355 kg, 1.0 eq) and anhydrous ethanol (4.28 kg) to the reaction vessel, then add ethanolamine (2.20 kg, 10.0 eq) and sodium iodide (54.2 g, 0.1 eq). Adjust the temperature to 70 ± 5 °C and stir for at least 3 hours. Monitor the reaction with HPLC until complete. Cool the reaction system to 25 ± 5 °C, add water (20.33 kg) and ethyl acetate (18.29 kg) to the reaction vessel, stir, let stand, separate the layers, and extract the lower aqueous phase again with ethyl acetate (18.29 kg). Combine the organic phases in the reaction vessel, add a lemon juice solution (13.55 kg water and 677.5 g citric acid monohydrate), stir, let stand, and separate the layers. The lower aqueous phase was discarded, and saturated sodium chloride aqueous solution was added to the upper organic phase. The mixture was stirred, allowed to stand, and separated. The upper organic phase was collected, and anhydrous sodium sulfate (4.07 kg, 3.0 w / w) was added to the organic phase. The mixture was stirred, filtered, and the filtrate was collected. The filter cake was washed with ethyl acetate (4.88 kg). The filtrate and eluent were transferred to rotary evaporator for concentration. The mixture was purified by silica gel column chromatography with dichloromethane as eluent A and methanol as eluent B. The elution ratios were 100 / 0; 100 / 1; 90 / 1; 80 / 1; 70 / 1; 60 / 1; 50 / 1; 40 / 1; 30 / 1; 20 / 1; and 10 / 1, respectively. The eluent was collected, filtered through a 0.22 μm hydrophobic PTFE filter, and concentrated until no fraction was obtained, yielding MTS001-4 745.5 g with an HPLC purity of 97.2%.

[0293] (9) Preparation of crude MTS001

[0294] Add cyclopentyl methyl ether (CpMe, 3.10 kg) and acetonitrile (1.05 kg) to the reactor, start stirring, and set the temperature to 25 ± 5 °C. Add MTS001-4 (450.0 g, 1.0 eq), anhydrous potassium carbonate (522.0 g, 3.0 eq), and sodium iodide (225.0 g, 1.2 eq) to the reactor. Add MTS001-5 (675.0 g, 1.2 eq) to the reactor. Raise the temperature of the reaction system to 70 ± 5 °C and stir for at least 48 hours. Monitor the reaction by HPLC. After the reaction was completed, the system temperature was cooled to 20-30℃, and the reaction solution was filtered through a 200-mesh filter cloth. The filter cake was washed with cyclopentyl methyl ether (774.0 g), and the filtrate was collected. The filtrate was concentrated by rotary evaporation and purified by silica gel column chromatography. The eluent A was dichloromethane, and the eluent B was methanol. The elution ratios were 100 / 0; 100 / 1; 90 / 1; 80 / 1; 70 / 1; 60 / 1; 50 / 1; 40 / 1; 30 / 1; 20 / 1; 10 / 1. The eluents were combined and concentrated by rotary evaporation to obtain 593.9 g of crude MTS001, with a yield of 65.2%.

[0295] (10) Purification of MTS001

[0296] Prepare mobile phase A: a 0.05% (v / v) aqueous solution of trifluoroacetic acid. Add water (1.0 wt) to a clean container, start stirring, add trifluoroacetic acid (0.00077 wt), and stir thoroughly until homogeneous. Set aside for use. Mobile phase A can be prepared multiple times according to requirements.

[0297] Prepare mobile phase B: a 0.05% (v / v) trifluoroacetic acid solution in acetonitrile / isopropanol (1:1). Add acetonitrile (1.0 wt) to a clean container, then add isopropanol (1 wt, the amount of isopropanol to be added is calculated based on the amount of acetonitrile) and trifluoroacetic acid (0.00197 wt, the amount of trifluoroacetic acid to be added is calculated based on the amount of acetonitrile) with stirring until homogeneous. Set aside for use. Mobile phase B can be prepared multiple times according to requirements.

[0298] To prepare a 2% sodium bicarbonate solution: Add water (1.0 wt) to a clean container, turn on the stirrer, add sodium bicarbonate (0.02 wt, the amount of sodium bicarbonate added should be calculated based on the amount of sterile water for injection), and stir until dissolved. The sodium bicarbonate solution can be prepared multiple times according to requirements.

[0299] To prepare a 2% sodium chloride solution: Add water (1.0 wt) to a clean container, turn on the stirrer, add sodium chloride (0.02 wt), and stir until dissolved. Set aside for later use. The sodium chloride solution can be prepared multiple times according to requirements.

[0300] The solution obtained by dissolving crude MTS001 (568.7 g) in acetonitrile and isopropanol (the weight ratio of acetonitrile and isopropanol was 1:1) g was filtered through a 0.22 μm hydrophobic PTFE filter for later use.

[0301] Chromatographic column: 250*200mm reversed-phase column (5kg AQ-C18 packing).

[0302] Separation gradient: B% = 80%-88%, time 40 minutes; B% = 100%-100%, time 20 minutes; B% = 80%-80%, time 15 minutes. Collect the pure fraction (start timing from the beginning of elution, collect the eluent of the first main peak after 25 minutes, the collection time can be adjusted according to the actual situation). The combined preparation solution was transferred to the reactor through a 0.22 μm hydrophilic PTFE filter. Stirring was started, and 0.5 times the weight of the eluent (2% sodium bicarbonate solution) and 0.3 times the weight of the eluent (n-heptane) were added. The mixture was stirred for at least 10 minutes and allowed to stand for at least 10 minutes. The liquid was separated, and the lower aqueous phase was discarded. The upper organic phase was retained in the reactor. 0.2 times the weight of the organic phase (2% sodium chloride solution) was added to the reactor. The mixture was stirred, allowed to stand for separation, and the aqueous phase was discarded. 0.05 times the weight of the organic phase (anhydrous sodium sulfate) was added to the organic phase. The mixture was stirred, filtered through a 200-mesh filter cloth, and the filtrate was collected. The reactor and filter cake were rinsed with n-heptane. The combined filtrates were filtered through a 0.22 μm hydrophobic PTFE filter and concentrated to obtain 429.1 g of MTS001 with a purity of 99.7%.

[0303] Comparative Example 1

[0304] Milligram-scale preparation method of compound MTS001

[0305] Under an inert nitrogen atmosphere, sodium hydride (2.0 g, 50.64 mmol, 1.6 eq.) was added to a tetrahydrofuran (50.0 mL) solution of nonanoic acid SM2 (5.0 g, 31.65 mmol, 1.0 eq.). The resulting mixture was stirred at 0 °C for 10 min. Lithium diisopropylamide (2 mol / L, 28.50 mL, 56.97 mmol, 1.8 equivalence) was added to the mixture at 0 °C. The resulting mixture was stirred at 0 °C for 10 min. 1-Iodoheptane (10.7 g, 47.48 mmol, 1.5 eq.) was added to the mixture. The resulting mixture was stirred at 40 °C for 12 h, the reaction was quenched with saturated ammonium chloride solution (10.0 mL), diluted with 500 mL of water and extracted with 3 x 500 mL of dichloromethane. The combined organic layers were washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (3:1). 5 g (62% yield) of MTS001-10 was obtained as a yellow oil. 1 H NMR(300MHz, CDCl3)δ:0.85-0.89(m,6H),1.25-1.32(m,19H),1.41-1.67-2.27(m,4H),2.29-2.33(m,1H),10.29(s,1H)

[0306] At 0 °C, a borane-tetrahydrofuran complex solution (1 mol / L, 23.4 mL, 23.43 mmol, 3.0 eq.) was added to a stirred solution of MTS001-10 (2.0 g, 7.81 mmol, 1.0 eq.) in tetrahydrofuran (5.0 mL). The resulting solution was heated to 75 °C and stirred for 3 hours. The mixture was cooled to room temperature and the reaction was quenched with methanol (10.0 mL). The mixture was diluted with 100 mL of water, extracted with 3 × 200 mL of dichloromethane, and the organic layers were combined, washed with 3–200 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (20:1). 1.6 g (yield 85%) of MTS001-11 was given as a colorless oil with a purity of 98%.

[0307] Under an inert nitrogen atmosphere, 8-bromooctanoic acid (1.11 g, 4.96 mmol, 1.2 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.19 g, 6.20 mmol, 1.5 eq.), and 4-dimethylaminopyridine (756.0 mg, 6.20 mmol, 1.5 mol) were added to a solution of MTS001-11 (1.0 g, 4.13 mmol, 1.0 eq.) in dichloromethane (10.0 mL). The resulting mixture was stirred at 25 °C for 6 hours, diluted with 100 mL of water, extracted with 3 x 300 mL of dichloromethane, and the organic layers were combined. The mixture was washed with 3 x 100 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (50:1). 1g (56% yield) of MTS001-5 was obtained as a yellow oily substance;

[0308] At 0 °C, lithium diisopropylamide (43.0 mL, 86.00 mmol, 2.0 eq.) was added to a stirred solution of methyl isobutyrate (4.4 g, 43.0 mmol, 1.0 eq.) in tetrahydrofuran (100.0 mL). The resulting mixture was stirred at 0 °C for 30 min. 1,5-Dibromo-pentane (20.0 g, 86.0 mmol, 1.0 eq.) was added to the above solution at 0 °C. The resulting mixture was stirred at 25 °C for 5 h, the reaction was quenched with saturated ammonium chloride solution (1.0 mL), diluted with 300 mL of water, extracted with 3 x 500 mL dichloromethane, the organic layers were combined, washed with 3 x 500 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (50:1). 10g (yield 46%) of MTS001-1 was obtained, which was a light yellow oily substance;

[0309] At 0°C, a borane-tetrahydrofuran complex solution (100.0 mL) was added to a stirred solution of MTS001-1 (10.0 g, 40.0 mmol, 1.0 eq.) in 20.0 mL of tetrahydrofuran. The resulting solution was stirred at 75°C for 3 hours. The mixture was cooled to 25°C, diluted with 100 mL of water, and extracted with 3 x 500 mL of dichloromethane. The organic layers were combined, washed with 3 x 500 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 8 g (90%) of MTS001-2 was obtained as a colorless oil.

[0310] At 0 °C, triethylamine (13.5 g, 134.00 mmol, 1.0 eq.) and decanoyl chloride (11.0 g, 58.0 mmol, 1.3 eq.) were added to a stirred solution of MTS001-2 (10.0 g, 44.00 mmol, 1.0 eq.) in 100.0 mL of dichloromethane. The resulting mixture was stirred at 25 °C for 3 hours, diluted with 100 mL of water, and extracted with 3 x 300 mL of dichloromethane. The organic layers were combined, washed with 3 x 300 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (10:1). 10 g (60% yield) of MTS001-3 was obtained as a colorless oil.

[0311] Under an inert nitrogen atmosphere, potassium carbonate (1.5 g, 11.1 mmol, 3.0 eq.) and MTS001-3 (1.4 g, 3.70 mmol, 1.0 eq.) were added to a stirred solution of ethanolamine (2.3 g, 37.2 mmol, 10.0 eq.) in acetonitrile (15.0 mL). The resulting solution was stirred at 70 °C for 3 hours. The mixture was cooled to 25 °C, diluted with 10 mL of water, extracted with 3 x 50 mL dichloromethane, and the organic layers were combined, washed with 3 x 50 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with dichloromethane / methanol (10:1). 1 g (76% yield) of MTS001-4 was given as a colorless oil. 1 H NMR(300MHz,CD3Cl)δ:0.85-0.89(m,9H),1.20-1.26(m,21H),1.59-1.62(m,4H) ),2.29-2.34(m,2H),2.73-2.76(m,2H),2.87-2.91(m,2H),3.72-3.77(m,3H);

[0312] Under a nitrogen inert atmosphere, potassium carbonate (161.5 mg, 1.17 mmol, 3.0 eq.), sodium iodide (141.5 mg, 0.95 mmol, 2.5 eq.), and MTS001-4 (135.0 mg, 0.39 mmol, 1.0 eq.) were added to a solution of MTS001-5 (202.8 mg, 0.45 mmol, 1.2 eq.) in N,N-dimethylformamide (2.0 mL). The reaction mixture was stirred at 70 °C for 6 h. The mixture was cooled to 25 °C, diluted with 10 mL of water, extracted with 3 x 50 mL dichloromethane, and the combined organic layers were washed with 3 x 50 mL saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography: Xselect CSH F-phenyl OBD column 19 x 250 mm, 5 μm; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 20 mL / min; gradient: from 75% B to 95% B over 9 min, yielding 83 mg MTS001, 30% yield, as a yellow oil with a purity of 98.8%.

[0313] 1 H NMR(300MHz, CDCl3)δ:0.80-0.90(m,15H),1.13-1.72(m,62H),2.22-2.27(m,4H),2.64-2.79(m,4H),3.71(s,2H),3.88-3.90(m,2H); MS m / z[M+H] + (ESI): 724.80.

[0314] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

An ionizable lipid compound having the structure shown in formula (I) or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, in, n1, n2, n3, and n4 are each independent integers from 1 to 20. The ionizable lipid compound according to claim 1, or its pharmaceutically acceptable salt, isomer, solvate, or prodrug, is characterized in that, n1, n2, n3, and n4 are each independently an integer from 3 to 18; preferably, each n1 is independently an integer from 4 to 15, n2 is an integer from 4 to 10, n3 is an integer from 3 to 10, and n4 is an integer from 4 to 15; preferably, each n1 is independently an integer from 4 to 10, n2 is an integer from 4 to 8, n3 is an integer from 4 to 8, and n4 is an integer from 4 to 12. The ionizable lipid compound according to claim 1 or 2, or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer, is characterized in that, Formula (I) refers to the following compounds: The method for preparing an ionizable lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer according to any one of claims 1-3 is characterized in that, This includes preparation method A or preparation method B; The preparation method A includes the following steps: preparing an alcohol (A1M) with the formula (MTS001-11M) using a diester (SM-A1M) and a haloalkane (SM-A2M) as raw materials; Wherein, n1 is defined as in claims 1 to 2; Wherein, X is a halogen; preferably, X is selected from any one of fluorine, chlorine, bromine, and iodine; The preparation method B includes the following steps: step (B1M) of preparing an alcohol of formula (MTS001-11M) from a ketone of formula (SM-B1M); Wherein, n1 is as defined in claims 1 to 2. The preparation method according to claim 4 is characterized in that, The step (A1M) includes the following steps (A11M): Wherein, n1 and X are defined as in claims 1 to 2; Preferably, an alkali is added in step (A11M), wherein the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylamino. The preparation method according to claim 4 or 5 is characterized in that, The step (A1M) includes the following step (A12M): Wherein, n1 is defined as in claims 1 to 2; Preferably, the step (A12M) further includes a catalyst, wherein the catalyst is LiCl. The preparation method according to claim 4, 5 or 6 is characterized in that, The step (A1M) includes the following step (A13M): Wherein, n1 is defined as in claims 1 to 2; Preferably, the step (A13M) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, NaBH4, diborane, and aluminum isopropoxide. The preparation method according to claim 4 is characterized in that, The step (B1M) includes the following steps (B11M): Wherein, n1 is defined as in claims 1 to 2; Preferably, an alkali is added in step (B11M), wherein the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylamino. The preparation method according to claim 4 or 8 is characterized in that, The step (B1M) includes the following step (B12M): Wherein, n1 is defined as in claims 1 to 2; Preferably, step (B12M) further includes a catalyst, wherein the catalyst is HCl. The preparation method according to claim 4, 8 or 9 is characterized in that, The step (B1M) includes the following step (B13M): Wherein, n1 is defined as in claims 1 to 2; Preferably, the step (B13M) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, NaBH4, diborane, and aluminum isopropoxide. The preparation method according to any one of claims 4 to 10 is characterized in that, It also includes the following steps: Wherein, n3 and n4 are as defined in claims 1 to 2; X is selected from halogens; preferably, X is selected from any one of fluorine, chlorine, bromine, and iodine; Preferably, the step (AB3M) further includes a catalyst selected from any one or a combination of DCC, DIC, EDCI, DMAP, DBU, HOBt, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, DIPEA (N,N-diisopropylethylamine) and triethylamine. The method for preparing the ionizable lipid compound or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer according to claim 3 is characterized in that, This includes preparation method A or preparation method B; The preparation method A includes the following steps: step (A1) of preparing an alcohol of formula (MTS001-11) using a diester of formula (SM-A1) and 1-iodoheptane (SM-A2) as raw materials; The preparation method B includes the following steps: step (B1) of preparing an alcohol of formula (MTS001-11) from a ketone of formula (SM-B1); The preparation method according to claim 12 is characterized in that, Step (A1) includes the following steps (A11): Preferably, an alkali is added in step (A11), wherein the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylamino. The preparation method according to claim 12 or 13 is characterized in that, Step (A1) includes the following step (A12): Preferably, step (A12) further includes a catalyst, wherein the catalyst is LiCl. The preparation method according to claim 14 is characterized in that, The step (A1) includes the following reduction reaction step (A13): Preferably, in step (A13), the reducing agent used is any one of Pt / H2, Pb / H2, Ni / H2, DIBAL-H, LAH, NaBH4, LiBH4, and borane. The preparation method according to claim 12 is characterized in that, Step (B1) includes the following step (B11): Preferably, in step (B11), the alkali is any one of NaH, n-BuLi, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and lithium diisopropylamino. The preparation method according to claim 16 is characterized in that, Step (B1) includes the following step (B12): Preferably, step (B12) further includes a catalyst, wherein the catalyst is HCl. The preparation method according to claim 17 is characterized in that, In step (B1), the following reduction reaction step (B13) is included: Preferably, step (B13) further includes a reducing agent, which is any one of Pt / H2, Pb / H2, Ni / H2, H2O2, DIBAL-H, LAH, NaBH4, LiBH4, diborane, and aluminum isopropoxide. The preparation method according to any one of claims 12 to 18 is characterized in that, It also includes the following steps: Preferably, step (AB3) further includes a catalyst selected from any one or a combination of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1-hydroxybenzotriazole (HOBt), sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, DIPEA (N,N-diisopropylethylamine), and triethylamine. The preparation method according to any one of claims 4 to 19 is characterized in that, Preparation method A includes the following steps: Preparation of (A1X)MTS001-5: Dimethyl malonate SM-A1 and 1-iodoheptane SM-A2 react under sodium hydride conditions to obtain MTS001-A1; MTS001-A1 reacts under lithium chloride conditions to obtain MTS001-A2; MTS001-A2 reacts under diisobutylaluminum hydride conditions to obtain MTS001-11; 8-bromooctanoic acid (SM3) SM3 reacts under SOCl2 conditions to obtain the corresponding acyl chloride, and the corresponding acyl chloride reacts with MTS001-11 under N,N-diisopropylethylamine conditions to obtain MTS001-5; Preparation of (A2X)MTS001-4: Ethyl isobutyrate and N,N-dimethylpropenylurea react with 1,5-dibromopentane in the presence of lithium diisopropylaminoacetate to obtain MTS001-1; MTS001-1 is then reacted with diisobutylaluminum hydride (DIBAL-H) ​​to obtain MTS001-2; MTS001-2 is then reacted with decanoyl chloride under triethylamine conditions to obtain MTS001-3; and MTS001-3 is then reacted with ethanolamine under sodium iodide conditions to obtain MTS001-4. Preparation of (A3X)MTS001: MTS001-4 and MTS001-5 were reacted under sodium iodide and K2CO3 conditions to obtain MTS001; The preparation method according to any one of claims 4 to 19 is characterized in that, Preparation method B includes the following steps: Preparation of (A1Y)MTS001-5: (Methoxymethyl)triphenylphosphonium chloride reacts with pentadecane-8-one (SM-B1) under n-butyllithium conditions to give MTS001-B1; MTS001-B1 reacts with HCl conditions to give MTS001-B2; MTS001-B2 reacts with sodium borohydride conditions to give MTS001-11; MTS001-11 reacts with SM3 under the action of 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to give MTS001-5; Preparation of (A2Y)MTS001-4: Ethyl isobutyrate and N,N-dimethylpropenylurea react with 1,5-dibromopentane in the presence of lithium diisopropylaminoacetate to obtain MTS001-1; MTS001-1 is then reacted with diisobutylaluminum hydride to obtain MTS001-2; MTS001-2 is then reacted with n-decanoic acid under EDCI / DMAP conditions to obtain MTS001-3; and MTS001-3 is then reacted with ethanolamine under sodium iodide conditions to obtain MTS001-4. Preparation of (A3Y)MTS001: MTS001-4 and MTS001-5 were reacted under sodium iodide and potassium carbonate conditions to obtain MTS001; The preparation method according to any one of claims 4 to 21 is characterized in that, It also includes the purification of ionizable lipid compounds, wherein the purification method is liquid chromatography: Mobile phase A is an aqueous solution of 0.05% (V / V) trifluoroacetic acid; mobile phase B is a 0.05% (V / V) trifluoroacetic acid solution in acetonitrile / isopropanol (1:1). The eluent of ionizable lipid compounds was collected under the following separation gradient conditions; The gradient separation condition is: From 0 to 40 minutes, the proportion of mobile phase B increased from 80% to 88%, while the proportion of mobile phase A decreased from 20% to 12%. For 40–60 min, the proportion of mobile phase B is 100% and the proportion of mobile phase A is 0%. For 60–75 min, the proportion of mobile phase B is 80% and the proportion of mobile phase A is 20%. Preferably, the purification further includes filtration and washing steps; Preferably, the filtration and washing steps include the following steps: filtering the eluent of the ionizable lipid compound, washing with 1-6% sodium bicarbonate solution, extracting with an organic solvent, separating, and retaining the organic phase. Preferably, the filtration and washing steps can be repeated. The obtained organic phase is dried, filtered, and concentrated to obtain an ionizable lipid compound with a purity of 95% or higher. The application of the ionizable lipid compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof, or the ionizable lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer, or stereoisomer thereof prepared by the preparation method according to any one of claims 4 to 22, in the preparation of a bioactive substance delivery system; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle, or microbubble; preferably, the delivery system is a liposome or lipid nanoparticle. The preparation method according to any one of claims 4 to 22 is used for preparing the ionizable lipid compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof. The ionizable lipid compound or its pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer obtained by the preparation method according to any one of claims 4 to 22 is characterized in that, Its purity is not less than 90%; Preferably, its purity is not less than 95%; Preferably, its purity is not less than 98%; Preferably, its purity is not less than 99%; More preferably, its purity is not less than 99.5%. A method for preparing a bioactive substance delivery system, comprising the steps of preparing an ionizable lipid compound as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof, or an ionizable lipid compound or a pharmaceutically acceptable salt, solvate, isotope variant, tautomer or stereoisomer thereof prepared by the method described in any one of claims 4 to 22. The ionizable lipid compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof, or the ionizable lipid compound or a pharmaceutically acceptable salt, solvate, isotopic variant, tautomer or stereoisomer thereof prepared by the preparation method according to any one of claims 4 to 22, is used to prepare a bioactive substance delivery system.

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