Cationic lipid compounds and compositions for nucleic acid delivery and uses

Abstract

The present invention provides a cationic lipid compound and composition for nucleic acid delivery and uses thereof. The compound is represented by the following formula (I). The present invention further provides the use of the compound as an important component in nucleic acid delivery of nano-lipid particles, including components of the delivery carrier, manufacturing methods, and methods of use. JPEG2025522526000041.jpg38147

Description

[Technical Field]

[0001] The present invention relates to the field of lipid delivery carriers, which are cationic lipid compounds that can be combined with other lipid components to form drug-loadable nanolipid particles and that achieve nucleic acid delivery from the extracellular space to the intracellular space in vitro and in vivo. Specifically, the present invention relates to cationic lipid compounds, compositions, and uses for nucleic acid delivery. [Background technology]

[0002] Nucleic acid drugs are drugs that replace, compensate, block, or modify specific genes by introducing foreign genes into target cells or tissues to achieve the purpose of treating and preventing diseases. Their development and production process is relatively simple, with short development periods, a high success rate in clinical development, and improved flexibility. In recent years, nucleic acid vaccines have proven to have great market potential as one of the main methods for preventing COVID-19.

[0003] However, naked mRNA has a short circulation time in the body, is easily degraded, and is difficult to enter target cells or tissues. Therefore, improving the delivery efficiency of mRNA drugs in the body is an important way to improve the effectiveness of such products.

[0004] Currently, the most widely used nucleic acid drug delivery carrier is lipid nanoparticles, which have the advantages of protecting nucleic acids from rapid degradation in the body, extending cycle time, and enhancing targeted delivery, thereby improving the therapeutic efficacy and targeted delivery of gene drugs. They are composed of two to four lipid components, including a cationic lipid compound, zero to two auxiliary lipids, and zero to one PEG lipid. Among these, cationic lipid compounds play an important role in the encapsulation and release of nucleic acids, making the development of novel, highly efficient, and low-toxicity cationic lipid compounds extremely important. Summary of the Invention

[0005] The present invention provides sulfur-containing cationic lipid compounds, including pharmaceutically acceptable salts thereof and stereoisomers or tautomers thereof, whose primary use is in combination with other lipid components in specific ratios to form lipid nanoparticles for delivery of prophylactic or therapeutic agents (e.g., therapeutic nucleic acids).

[0006] Another object of the present invention is to provide a method for synthesizing the corresponding lipid compounds, which uses readily available raw materials, employs a mild reaction route, has a high product yield, requires little equipment, and is easy to operate.

[0007] In some examples, the therapeutic nucleic acid comprises a plasmid DNA, messenger RNA, antisense oligonucleotide (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, or complementary DNA (cDNA).

[0008] At the same time, the present invention further provides formulation ratios and methods of use when such cationic lipid compounds are used in combination with other lipid components, as well as uses in cells and animal models. In an embodiment of the present invention, a cationic lipid compound having the structure of formula (I) below or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof is used: [ka] wherein L1 and L2 are each independently a divalent linking group or linking bond selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -N(R8)C(=O)-, -C(=O)N(R8)-, -N(R8)C(=O)O-, -OC(=O)N(R8)-, -SC(=O)N(R8)-, -N(R8)C(=O)S-, -C(=S)-, -SC(=S)-, and -C(=S)S-, and R8 is H or C1-C 12 is an alkyl group, R2 and R3 are independently substituted or unsubstituted C1-C 18 Straight chain alkylene group or -R9-L3-R10 wherein R and R 10 are independently substituted or unsubstituted C-C 10 is a linear alkylene group, and L3 is O or S; R4, R5, R6, and R7 are independently hydrogen or substituted or unsubstituted C1-C 30 Aliphatic hydrocarbon group or -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 18 an aliphatic hydrocarbon group, L4 is O or S; R2, R3, R4, R5, R6 and R7 each contain at least one O or S; R1 is H, -R 13 , -OR 13 , -R 13 -OH, -R 13 -OR 14 , -R 13 -OC(=O)R 14 , -R 13 -NHC(=O)-R 14 , -R 13 -OCH3 or -R 13 -N(R 14 )(R 15 ) and R 13 is C1-C 12 is a straight chain alkyl group or a branched chain alkyl group, and R 14 and R 15 are each independently H or C1-C 12 is a straight chain alkyl group, or R 14 and R 15 C3-C together with the N atom attached to them 10 It forms a heterocycloalkyl group.

[0009] In some specific embodiments of the invention, R8 is H or a methyl group.

[0010] In some specific embodiments of the present invention, R is -R 13 -OH or -R 13 -N(R14 )(R 15 ) and R 13 is C1-C 12 is a straight chain alkyl group, R 14 and R 15 are independently C1-C 12 is a straight chain alkyl group, R2 is C1-C 18 is a straight-chain alkylene group, L1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-; R4 and R5 are each independently C1-C 30 is an aliphatic hydrocarbon group, R3 is C1-C 18 is a straight-chain alkylene group, L2 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-; R6 is a methyl group or an ethyl group; R7 is -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 18 is an aliphatic hydrocarbon group, and L4 is O or S.

[0011] In some specific embodiments of the present invention, R 13 is a C1-C8 linear or branched alkyl group, and R 14 and R 15 are each independently H or a C1-C5 straight chain alkyl group, or R 14 and R 15 together with the N atom to which they are attached form a C3-C8 heterocycloalkyl group.

[0012] In some specific embodiments of the present invention, R2 and R3 are independently substituted or unsubstituted C1-C 18 It is a straight chain alkyl group.

[0013] In some specific embodiments of the present invention, R2 and R3 are independently substituted or unsubstituted C1-C 12 It is a straight chain alkyl group.

[0014] In some specific embodiments of the present invention, R, R, R, and R are independently hydrogen or substituted or unsubstituted C-C 18 Aliphatic hydrocarbon group or -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 10 It is an aliphatic hydrocarbon group, L4 is O or S, and R4, R5, R6 and R7 contain at least one O or S and at most two are hydrogen.

[0015] In some specific embodiments of the present invention, the structures of R4, R5, R6, and R7 in the structure of formula (I) are each independently H or the following alkyl chain, or each independently an ether or thioether formed by substituting any carbon atom in the following alkyl chain with O or S: [ka]

[0016] In some specific embodiments of the present invention, R is -R 13 -OH or -R 13 -N(R 14 )(R 15 ) and R 13 is a C1-C5 linear alkyl group, preferably a C2-C4 linear alkyl group, and R 14 and R 15 are independently C1-C 12 are straight-chain alkyl groups, preferably each independently a C1-C3 straight-chain alkyl group; R2 is C2-C 12a straight-chain alkylene group, preferably a C5-C9 straight-chain alkyl group, more preferably a C5-C7 straight-chain alkyl group; L1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-; R4 and R5 are each independently C3-C 13 an aliphatic hydrocarbon group, preferably C6-C 10 is a straight chain alkyl group, more preferably a C6-C8 straight chain alkyl group; R3 is C2-C 10 a straight-chain alkylene group, preferably a C3-C7 straight-chain alkyl group, more preferably a C5-C7 straight-chain alkyl group; L2 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-; R6 is a methyl group or an ethyl group; R7 is -R 11 -L4-R 12 and R 11 is C1-C 10 is an alkyl group, preferably a C1-C3 alkyl group, and R 12 is C3-C 13 alkyl group, preferably C-C 10 L4 is O or S.

[0017] In some specific embodiments of the present invention, R1 is -R 13 -OH and R 13 is C 1-3 is a straight chain alkyl group, R2 is C 5-9 is a straight chain alkyl group, L1 is -OC(=O)- or -C(=O)O-; R4 and R5 each independently represent C 6-10 is a straight chain alkyl group, R3 is C 5-7 is a straight chain alkyl group, L2 is -OC(=O)- or -C(=O)O-; R6 is a methyl group, an ethyl group, or a propyl group; R7 is -R 11 -L4-R 12 and R 11 is a C1-C2 alkyl group, and R 12 is C3-C 13 is an alkyl group, and L4 is O or S.

[0018] In some specific embodiments of the present invention, R1 is -R 13 -OH and R 13 is a C2 straight chain alkyl group, R2 is C 5-7 a straight-chain alkyl group, preferably a C7 straight-chain alkyl group; L1 is -OC(=O)- or -C(=O)O-; R4 and R5 are each independently a C8 linear alkyl group; R3 is C 5-7 a straight-chain alkyl group, preferably a C5 straight-chain alkyl group; L2 is -OC(=O)-, R6 is a methyl group; R7 is -R 11 -L4-R 12 and R 11 is a C1 alkyl group, and R 12 is a C5-C8 alkyl group, and L4 is O or S.

[0019] In some specific embodiments of the present invention, L1 and L2 are each independently selected from -OC(=O)-, -C(=O)O-, -C(=O)S-, and -SC(=O)-; R2 and R3 are independently substituted or unsubstituted C1-C 18 is a straight-chain alkylene group, R4, R5, and R6 are independently hydrogen or substituted or unsubstituted C1-C 30 is an aliphatic hydrocarbon group, R7 is -R 11 -L4-R12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 18 an aliphatic hydrocarbon group, L4 is O or S; R1 is H, -R 13 -OH, -R 13 -OCH3 or -R 13 -N(R 14 )(R 15 ) and R 13 is C1-C 12 is a straight chain alkyl group or a branched chain alkyl group, and R 14 and R 15 are each independently H or C1-C 12 is a straight chain alkyl group, or R 14 and R 15 C3-C, along with the N atoms attached to them 10 It forms a heterocycloalkyl group.

[0020] In some specific embodiments of the present invention, L1 and L2 are each independently selected from -OC(=O)-, -C(=O)O-, -C(=O)S-, and -SC(=O)-; R2 and R3 are independently substituted or unsubstituted C3-C 10 is a straight-chain alkylene group, R4, R5, and R6 are independently hydrogen or substituted or unsubstituted C1-C 15 is an aliphatic hydrocarbon group, R7 is -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 18 an aliphatic hydrocarbon group, L4 is O or S; R1 is -R 13 -OH and R 13 is a C1-C6 straight chain alkyl group or a branched chain alkyl group.

[0021] In some specific embodiments of the present invention, L1 and L2 are each independently selected from -OC(=O)- and -C(=O)O-; R2 and R3 are independently substituted or unsubstituted C3-C 10 is a straight-chain alkylene group, R4, R5, and R6 are independently hydrogen or substituted or unsubstituted C1-C 15 is an aliphatic hydrocarbon group, R7 is -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C-C 10 an aliphatic hydrocarbon group, L4 is O or S; R1 is -R 13 -OH and R 13 is a C1-C6 straight chain alkyl group or a branched chain alkyl group.

[0022] In some specific embodiments of the present invention, L1 and L2 are each independently selected from -OC(=O)- and -C(=O)O-; R2 and R3 are independently a substituted or unsubstituted C3-C9 straight chain alkylene group; R4, R5, and R6 are independently hydrogen or substituted or unsubstituted C1-C 12 is an aliphatic hydrocarbon group, R7 is -R 11 -L4-R 12 and R 11 and R 12 is independently in each occurrence a substituted or unsubstituted C1-C9 aliphatic hydrocarbon group, L4 is O or S; R1 is -R 13 -OH and R 13 is a C1-C5 straight chain alkyl group or a branched chain alkyl group.

[0023] In some specific embodiments of the present invention, the cationic lipid compound has one of the structures shown in the table below. [Table 1] JPEG0007770662000004.jpg236170 JPEG0007770662000005.jpg230170 JPEG0007770662000006.jpg228170 JPEG0007770662000007.jpg232170 JPEG0007770662000008.jpg193170

[0024] The present invention further provides liposome formulations comprising one or more cationic lipid compounds of the present invention and a prophylactic or therapeutic nucleic acid for use in the prevention or treatment of a disease.

[0025] The liposome preparation contains one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. The therapeutic agent used in the present invention is a therapeutic nucleic acid, including plasmid DNA, messenger RNA, antisense oligonucleotides (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, and complementary DNA (cDNA). Plasmid DNA, messenger RNA, and antisense oligonucleotides are preferred.

[0026] In some specific embodiments of the present invention, the molar ratio of the nucleic acid to the cationic lipid compound is 20:1 to 1:1.

[0027] In some specific embodiments of the present invention, the molar ratio of the nucleic acid to the cationic lipid compound is 10:1 to 4:1.

[0028] In some specific embodiments of the present invention, the diameter of the liposome preparation is between 50 nm and 300 nm.

[0029] In some specific embodiments of the present invention, the diameter of the liposome preparation is 50 nm to 150 nm, or 150 nm to 200 nm.

[0030] Some specific embodiments of the present invention further comprise one or more other lipid components, including but not limited to neutral lipids, steroids, and polymer-conjugated lipids.

[0031] In some specific embodiments of the present invention, the steroid included is cholesterol.

[0032] In some specific embodiments of the present invention, the molar ratio of cholesterol to cationic lipid compound is (0-1.5):1.

[0033] In some specific embodiments of the present invention, the polymer in the polymer-conjugated lipid is polyethylene glycol (PEG).

[0034] In some specific embodiments of the present invention, the molar ratio of the cationic lipid compound to the polyethylene glycolated lipid is 100:1 to 20:1.

[0035] In some specific embodiments of the invention, the pegylated lipid is PEG-DAG, PEG-PE, PEG-SDAG, PEG-cer, PEG-DMG, or ALC-0159.

[0036] In some specific embodiments of the present invention, the liposome formulation comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0037] In some specific embodiments of the present invention, the neutral lipid is DSPC or DOPE.

[0038] In some specific embodiments of the present invention, the molar ratio of the neutral lipid to the cationic lipid compound is (0-0.5):1.

[0039] In some specific embodiments of the present invention, the liposomal formulation comprises a nucleic acid.

[0040] In some specific embodiments of the invention, the nucleic acid is selected from antisense RNA and / or messenger RNA.

[0041] In some specific embodiments of the invention, the nucleic acid is messenger RNA.

[0042] The present invention further provides the use of the cationic lipid compound of the present invention or the liposome formulation of the present invention in the manufacture of a medicament for inducing protein expression in a subject.

[0043] In some specific embodiments of the invention, the subject is a mammal.

[0044] In some specific embodiments of the invention, the subject is a non-human primate.

[0045] In some specific embodiments of the invention, the subject is a human. Terms used in this specification and claims have the following meanings unless specified to the contrary.

[0046] "Alkyl group" includes substituted or unsubstituted straight or branched chain saturated aliphatic hydrocarbon groups, including, but not limited to, alkyl groups of 1 to 20 carbon atoms, alkyl groups of 1 to 8 carbon atoms, alkyl groups of 1 to 6 carbon atoms, and alkyl groups of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and various branched isomers thereof. Any alkyl group appearing herein shall have a definition consistent with this definition.

[0047] "Alkylene group" includes substituted or unsubstituted straight-chain and branched-chain divalent saturated hydrocarbon groups, -(CH2) v - (v is an integer of 1 to 10). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, and butylene groups. "Aliphatic hydrocarbon group" refers to a saturated or unsaturated, straight or branched chain, linear or cyclic hydrocarbon group. The base The aliphatic hydrocarbon group is selected from alkyl groups, alkenyl groups, alkynyl groups, etc. For example, the term "C1-10 aliphatic hydrocarbon group" includes methyl groups, ethyl groups, n-propyl groups, isopropyl groups, n-butyl groups, isobutyl groups, tert-butyl groups, n-pentyl groups, isopentyl groups, neopentyl groups, n-hexyl groups, n-heptyl groups, vinyl groups, 1-propenyl groups, 2-propenyl groups, 1-methylvinyl groups, 1-butenyl groups, 1-ethylvinyl groups, 1-methyl-2-propenyl groups, and the like. groups, such as 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0048] The term "heterocycloalkyl group" includes, but is not limited to, substituted or unsubstituted saturated heteroatom-containing cyclic hydrocarbon groups containing 3 to 10 atoms, 3 to 8 atoms, and 1 to 3 heteroatoms selected from N, O, and S. The N and S optionally substituted in the heterocycloalkyl ring may be oxidized to various oxidation states. The heterocycloalkyl group may be bonded to a heteroatom or a carbon atom. The heterocycloalkyl group may be bonded to an aromatic or non-aromatic ring. The heterocycloalkyl group may be bonded to a bridged ring or a spiro ring, and non-limiting examples include oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydro-2H-pyranyl, dioxolane, dioxane, pyrrolidinyl, piperidinyl, imidazolidinyl, oxazolidyl, oxazinanyl, morpholinyl, hexahydropyrimidinyl, and piperazinyl.

[0049]

[0013] As described above, the present invention provides cationic lipid compounds and liposome formulations for nucleic acid delivery, and uses thereof.

[0014] The present invention has the following advantages:

[0050] The cationic lipid compounds of the present invention have an ether bond or a thioether bond. The introduction of the ether bond or the thioether bond makes the compounds more easily degradable, increasing the rate of elimination of the lipid compounds in the body, reducing the toxicity of the carriers composed of the compounds, and reducing their residual content in the body. Furthermore, as a result of structural optimization, the screened cationic compounds have better in vivo transfection efficiency than some commercial transfection cationic lipid compounds. In addition, the method for producing the amino lipid compounds has the advantages of easy availability of raw materials, mild reaction conditions, high product yields, low equipment requirements, and simple operation. [Brief explanation of the drawings]

[0051] [Figure 1] 10 shows the relative fluorescence intensity of images of a mouse intramuscularly injected in Example 21. [Figure 2]10 shows the relative fluorescence intensity of live mouse images of pulmonary nebulization delivery in Example 22. [Figure 3] Binding antibody titer of Example 23. [Figure 4] Binding antibody titer of Example 24. [Figure 5] FIG. 10 is a graph showing the evaluation of liver and kidney function in Example 25. [Figure 6] FIG. 10 is a fluorescent image of Example 28. [Figure 7] 13 shows statistics of the fluorescence image results of Example 28. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention will be described in detail below with reference to the drawings and examples, but the present invention is not limited thereto.

[0053] Example 1 Synthesis of compound 2 [ka]

[0054] Step 1: To a solution of compound 2-1 (3.00 g) in tert-butanol (20 mL), 1-decanol (3.23 g) and cesium carbonate (11.1 g) were added sequentially. The solution was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to reveal the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 2-2 (3.93 g, 69% yield).

[0055] Step 2: To a solution of compound 2-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added, and the mixture was stirred at 60 °C for 16 h. TLC showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 2-3 (2.10 g, 95% yield).

[0056] Step 3: Compound 2-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g), and 5-bromo-1-pentanol (1.5 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 2-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to give compound 2-4 (2.8 g, 87% yield) as a colorless oil.

[0057] Step 4: Compound 2-5 (5.0 g) was dissolved in dichloromethane (70 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.48 g), 4-dimethylaminopyridine (DMAP, 3.57 g), and 8-bromooctanoic acid (4.78 g) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 2-5 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and DCM. The mixture was purified (60 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 30 ml / min) to give colorless oily liquid compound 2-6 (8.0 g, 88.9% yield). Compounds 2-6 in Examples 1-6, 8-10, 11-13, and 16-18 below were all synthesized using this method.

[0058] Step 5: Potassium carbonate (7.19 g) was added to a solution of compound 2-6 (8.0 g) and ethanolamine (1.59 g) in acetonitrile (50 mL). The mixture was stirred at 70 °C for 2 h. TLC showed that compound 2-6 had completely disappeared and a single increasingly polar spot had formed. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was added to an appropriate amount of silica gel and DCM and stirred. The mixture was purified (25 g normal-phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 5 min, flow rate 20 mL / min) to give compound 2-7 (4.2 g, 54.9% yield) as a colorless oil.

[0059] Step 6: Compound 2-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 3 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot with a lower polarity than 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 2 (700 mg, 73% yield) as a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.80 (s, 1H), 4.16 - 4.01 (d, J = 3.2 Hz, 2H), 3.75 - 3.45 (m, 6H), 2.78 - 2.68 (dd, J = 8.2, 5.8 Hz, 2H), 2.61 - 2.51 (m, 2H), 2.50 - 2.45 (m, 4H), 2.31 - 2.16 (m, 2H), 1.70 - 1.69 (s, 1H), 1.68 - 1.66 (s, 1H), 1.58 - 1.57 (s, 2H), 1.57 - 1.55 (d, J = 3.4 Hz, 2H), 1.55 - 1.51 (m, 6H), 1.50 - 1.48 (s, 2H), 1.38 - 1.35 (d, J = 1.0 Hz, 4H), 1.35 - 1.30 (m, 20H), 1.30 - 1.27 (m, 20H), 1.19 - 1.17 (m, 3H), 0.91 - 0.88 (m, 9H).

[0060] Example 2 Synthesis of compound 3 [ka]

[0061] Step 1: To a solution of compound 3-1 (3.00 g) in tert-butanol (20 mL), 1-decanol (4.45 g) and cesium carbonate (15.3 g) were added sequentially. The mixture was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to reveal the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 3-2 (3.1 g, 46% yield).

[0062] Step 2: To a solution of compound 3-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added, and the mixture was stirred at 60 °C for 16 h. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 3-3 (2.50 g, 92% yield).

[0063] Step 3: Compound 3-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g), and 5-bromo-1-pentanol (1.5 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 3-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to obtain compound 3-4 (2.6 g, 83% yield) as a colorless oil.

[0064] Step 4: Compound 3-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 3 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot with a lower polarity than 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 3 (750 mg, 80% yield) as a pale yellow oily liquid. 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.14 - 4.02 (d, J = 2.6 Hz, 2H), 3.73 - 3.46 (m, 6H), 2.61 (s, 1H), 2.59 - 2.50 (m, 2H), 2.49 - 2.45 (m, 4H), 2.29 - 2.18 (m, 2H), 1.71 - 1.63 (m, 4H), 1.60 - 1.50 (m, 12H), 1.49 (s, 2H), 1.38 - 1.36 (d, J = 0.6 Hz, 4H), 1.35 - 1.30 (m, 20H), 1.30 - 1.26 (m, 20H), 0.92 (s, 3H), 0.91 - 0.87 (s, 9H).

[0065] Example 3 Synthesis of compound 4 [ka]

[0066] Step 1: To a solution of compound 4-1 (3.00 g) in tert-butanol (20 mL), 1-decanol (4.6 g) and cesium carbonate (16.1 g) were added sequentially. The mixture was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to reveal the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 4-2 (3.0 g, 47.3% yield).

[0067] Step 2: To a solution of compound 4-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added, and the mixture was stirred at 60 °C for 16 h. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 4-3 (2.50 g, 92% yield).

[0068] Step 3: Compound 4-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.1 g), 4-dimethylaminopyridine (DMAP, 1.4 g), and 5-bromo-1-pentanol (1.6 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 3-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to obtain compound 4-4 (2.6 g, 84% yield) as a colorless oil.

[0069] Step 4: Compound 4-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 3 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot with a lower polarity than that of 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% (volume percentage of methanol in DCM / MeOH solution, same below) 10 min, 0-7.5% 20 min, 7.5-7.5% 5 min, flow rate 25 ml / min) and concentrated to give compound 4 (610 mg, 65.7% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.80 - 4.74 (s, 1H), 4.15 - 4.01 (d, J = 2.6 Hz, 2H), 3.73 - 3.44 (m, 6H), 2.56 - 2.50 (m, 3H), 2.49 - 2.45 (m, 4H), 2.29 - 2.18 (m, 2H), 1.71 - 1.47 (m, 18H), 1.41 - 1.26 (m, 46H), 0.96 - 0.92 (m, 3H), 0.99 (s, 9H).

[0070] Example 4 Synthesis of compound 6 [ka]

[0071] Step 1: To a solution of compound 6-1 (19.0 g) in tert-butanol (20 mL), 1-decanol (3.0 g) and cesium carbonate (12.4 g) were added sequentially. The mixture was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to show the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 6-2 (2.1 g, 43% yield).

[0072] Step 2: To a solution of compound 6-2 (2.1 g) in THF (20 mL) and water (10 mL) was added lithium hydroxide (584 mg), and the mixture was stirred at 60 °C for 16 h. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic phases were combined and concentrated to give compound 6-3 (1.6 g, 85% yield).

[0073] Step 3: Compound 6-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g), and 5-bromo-1-pentanol (1.5 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 6-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to obtain compound 6-4 (2.5 g, 82% yield) as a colorless oil.

[0074] Step 4: Compound 6-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 3 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot with a lower polarity than that of 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 6 (742 mg, 75% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.11 (s, 2H), 3.68 - 3.57 (d, J = 5.0 Hz, 4H), 3.55 - 3.46 (s, 2H), 2.61 - 2.43 (m, 8H), 2.28 - 2.17 (s, 2H), 1.73 - 1.63 (d, J = 3.9 Hz, 4H), 1.60 - 1.46 (m, 12H), 1.39 - 1.23 (m, 40H), 0.96 - 0.84 (s, 9H).

[0075] Example 5 Synthesis of compound 7 [ka]

[0076] Step 1: To a solution of compound 2-1 (3.00 g) in tert-butanol (20 mL), octanol (3.1 g) and cesium carbonate (11.0 g) were added sequentially. The solution was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to reveal the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 7-2 (3.6 g, 69% yield).

[0077] Step 2: To a solution of compound 7-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added, and the mixture was stirred at 60 °C for 16 h. TLC showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 7-3 (2.0 g, 94% yield).

[0078] Step 3: Compound 7-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g), and 5-bromo-1-pentanol (1.5 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 7-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and the appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to obtain compound 7-4 (2.5 g, 74% yield) as a colorless oil.

[0079] Step 4: Compound 7-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1 d ammonia water, phosphomolybdic acid). A new spot with a lower polarity than that of 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 7 (750 mg, 75% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.09 (d, J = 2.2 Hz, 2H), 3.78 - 3.37 (m, 6H), 2.75 (s, 1H), 2.55 (d, J = 3.8 Hz, 2H), 2.49 - 2.43 (m, 4H), 2.30 - 2.16 (m, 2H), 1.71 - 1.65 (m, 2H), 1.64 (s, 2H), 1.60 - 1.50 (m, 10H), 1.49 (s, 2H), 1.37(d, J = 0.6 Hz, 4H), 1.35 - 1.30 (m, 20H), 1.30 - 1.26 (m, 16H), 1.22 - 1.14 (m, 3H), 0.99 (s, 9H).

[0080] Example 6 Synthesis of compound 14 [ka]

[0081] Step 1: To a solution of compound 14-1 (3.0 g) and triethylamine (5.3 g) in dichloromethane (50 mL), p-toluenesulfonyl chloride (7.4 g) was added under ice bath conditions. After stirring at room temperature for 3 hours, the reaction mixture was diluted with DCM (30 mL) and washed with dilute hydrochloric acid and brine (100 mL). The organic layers were combined, dried over Na2SO4, and the solvent was removed in vacuo to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: 0-10% EA (volume percent) in n-hexane) to obtain compound 14-2 (6.0 g, 86% yield).

[0082] Step 2: Under ice bath conditions, NaH (680 mg, 60%) was added to a solution of tert-butyldimethylhydroxyethoxysilane (2.0 g) in DMF (20 mL), and the mixture was heated to 0°C. o After stirring at 50°C for half an hour, 14-2 was slowly added to the solution and the solution was heated to 80°C. o The mixture was stirred at RT for 2 h. After the solution cooled to room temperature, it was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic layers were combined and dried over NaSO, and the solvent was removed in vacuo to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with 0-10% EA (volume percent) in n-hexane) to give compound 14-3 (2.5 g, 80% yield).

[0083] Step 3: Under ice bath conditions, a solution of 14-3 (2.5 g) in anhydrous tetrahydrofuran (20 mL) was added with 1M TBAF solution, and the solution was warmed to room temperature and stirred at room temperature for 2 hours. Saturated ammonium chloride solution was added, and the mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined and dried over Na2SO4. The solvent was removed in vacuo to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluting with 0-60% EA (volume percent) in n-hexane) to obtain compound 14-4 (1.4 g, 96% yield).

[0084] Step 4: To a solution of compound 14-4 (1.4 g) in DCM (20 mL), 4-dimethylaminopyridine (DMAP, 1.07 g), 7-bromoheptanoic acid (2.01 g), and 1-ethyl-(3-dimethylaminopropyl) (EDCl, 2.01 g) were added sequentially. The reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with DCM (30 mL) and washed with saturated NaHCO (100 mL) and brine (100 mL). The organic layers were combined, dried over NaSO, and the solvent was removed in vacuo to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with 0-1% EA (volume percent) in n-hexane). The pure fraction was evaporated to give compound 14-5 (2.10 g, 68% yield).

[0085] Step 5: Compound 14-5 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 2-7 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1 d ammonia water, phosphomolybdic acid). A new spot with a lower polarity than that of 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 14 (820 mg, 81% yield) as a pale yellow oily liquid. 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.21 (s, 2H), 3.64 (d, J = 5.0 Hz, 2H), 3.60 (s, 2H), 3.53 (s, 2H), 2.55 (s, 2H), 2.48 (s, 4H), 2.24 (s, 4H),1.71 - 1.65 (m, 4H), 1.58 (d, J = 6.4 Hz, 4H), 1.53 (s, 4H), 1.51 (s, 2H), 1.38 - 1.35 (m, 6H), 1.35 - 1.33 (m, 8H), 1.32 (d, J = 1.0 Hz, 4H), 1.32 - 1.31 (m, 10H), 1.29 (s, 4H), 1.29 (s, 2H), 1.29 - 1.27 (m, 8H), 0.97 - 0.82 (m, 9H).

[0086] Example 7 Synthesis of compound 17 [ka]

[0087] Step 1: To a solution of compound 2-1 (5.00 g) in tert-butanol (40 mL), n-hexanol (2.90 g) and cesium carbonate (27.8 g) were added sequentially. The solution was stirred at room temperature for 4 hours, and then spotted (petroleum ether:ethyl acetate = 10:1) to reveal the formation of a new spot. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to give compound 17-2 (3.14 g, 39.8% yield).

[0088] Step 2: To a solution of compound 17-2 (3.00 g) in THF (20 mL) and water (20 mL), lithium hydroxide (1.03 g) was added, and the mixture was stirred at 60 °C for 16 h. TLC showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 17-3 (1.80 g, 88.7% yield).

[0089] Step 3: Compound 17-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.1 g), 4-dimethylaminopyridine (DMAP, 2.0 g), and 7-bromo-1-heptanol (2.3 g) were weighed sequentially and added in portions to the reaction mixture. The mixture was stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted (PE / EA = 10 / 1, phosphomolybdic acid) against a standard sample of 2-3. A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added. The mixture was stirred and purified (10 g normal-phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to give compound 17-4 (2.8 g, 74% yield) as a colorless oil.

[0090] Step 4: To a solution of compound 44-5 (2.00 g) in DCM (15 mL), 4-dimethylaminopyridine (DMAP, 200 mg) and 7-bromo-1-heptanol (1.51 g) were added sequentially. After stirring the mixture at 25 °C for 5 min, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.62 g) was added, and the reaction mixture was stirred at 25 °C for 1 h. TLC showed the complete disappearance of starting compound 44-5. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-1% EA (volume percent)), and the pure product fraction was evaporated to give compound 44-6 (2.4 g, 74% yield).

[0091] Step 5: Potassium carbonate (1.80 g) was added to a solution of compound 44-6 (2.0 g) and ethanolamine (530 mg) in acetonitrile (50 mL). The mixture was stirred at 70 °C for 3 h. TLC showed that compound 44-6 had completely disappeared and a single increasingly polar spot had appeared. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was added to an appropriate amount of silica gel and DCM, and purified (25 g normal-phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 5 min, flow rate 20 mL / min) to give compound 17-5 (1.0 g, 53.8% yield) as a colorless oil.

[0092] Step 6: Compound 17-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), KCO (527 mg), and compound 17-5 (673 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 2 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot less polar than 17-5 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 17 (725 mg, 70% yield) as a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.15 - 4.03 (m, 4H), 3.56 - 3.45 (m, 6H), 2.75 (s, 1H), 2.55 (d, J = 3.8 Hz, 2H), 2.48 (d, J = 1.0 Hz, 4H), 2.29 (s, 1H), 1.65 (s, 4H), 1.50 - 1.46 (m, 10H), 1.40 - 1.38 (s, 4H), 1.36 (d, J = 0.6 Hz, 2H), 1.34 (d, J = 0.6 Hz, 4H), 1.32 (d, J = 1.0Hz, 10H), 1.32 - 1.30 (m, 10H), 1.28 (d, J = 1.2 Hz, 12H), 1.18 (s, 3H), 0.94 - 0.84 (m, 9H).

[0093] Example 8 Synthesis of compound 28 [ka]

[0094] Step 1: Potassium carbonate (1.2 g) was added to a solution of compound 2-6 (2.0 g) and N,N-diethylethylenediamine (755 mg) in acetonitrile (50 mL). The mixture was stirred at 70 °C for 3 hours. A single spot of increasing polarity was observed. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was stirred with an appropriate amount of silica gel and DCM and purified (25 g normal-phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 mL / min) to give compound 28-1 (900 mg, 30% yield) as a colorless oil.

[0095] Step 2: Compound 28-1 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (146 mg), K2CO3 (406 mg), and compound 7-4 (425 mg) were weighed sequentially and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 2 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1 ml aqueous ammonia, phosphomolybdic acid). A new spot was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and an appropriate amount of DCM and silica gel was added. The mixture was stirred and purified (25 g normal-phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min), and concentrated to give compound 7 (94 mg, 11% yield) as a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.09 (d, J = 2.2 Hz, 2H), 3.73 - 3.60 (m, 4H), 2.75 (s, 1H), 2.69 - 2.57 (m, 8H), 2.51 - 2.40 (m, 4H), 2.31 - 2.17 (m, 2H), 1.72 - 1.65 (m, 4H), 1.60 - 1.50 (m, 10H), 1.48 (s, 2H), 1.36 (d, J = 0.6 Hz, 4H), 1.35 - 1.30 (m, 20H), 1.30 - 1.26 (m, 16H), 1.19 (s, 3H), 1.00 (s, 6H), 0.90 (s, 9H).

[0096] Example 9 Synthesis of compound 29 [ka]

[0097] Step 1: 1-Decanethiol (2.28 g) and KOH (2.20 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Compound 29-1 (2.00 g) was then weighed and added in portions to the reaction mixture, which was then stirred overnight at room temperature. A new spot was observed on TLC (PE / EA = 3 / 1, phosphomolybdic acid). 200 ml of water was added to the reaction mixture, and concentrated HCl was added dropwise to adjust the pH to approximately 3. The mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over Na2SO4 and evaporated under reduced pressure. The mixture was stirred with an appropriate amount of DCM and silica gel, and purified with spot plate monitoring (30 g normal phase column, PE / EA, 0-0% for 10 min, 0-2% for 20 min, 2-2% for 5 min, flow rate 30 ml / min). A portion of the pure product fraction was evaporated to give a white solid 29-2 (950 mg, 30% yield).

[0098] Step 2: Compound 29-2 (950 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg), and 5-bromo-1-pentanol (708 mg) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 29-2 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added, stirred, and purified with spot plate monitoring (10 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 29-3 (1.45 g, 95% yield).

[0099] Step 3: Compound 29-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 2-7 (559 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 2 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 2-7. A new spot with a lower polarity than 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified with spot plate monitoring (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A portion of the pure product fraction was evaporated to give compound 29 (850 mg) as a pale yellow oil. 1H NMR (400 MHz, cdcl3) δ 4.86 (p, J = 6.4 Hz, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.56 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 2.61 (dt, J = 11.8, 6.4 Hz, 4H), 2.51 (dd, J = 14.4, 6.8 Hz, 6H), 2.28 (t, J = 7.6 Hz, 2H), 1.70 - 1.55 (m, 6H), 1.50 (dd, J = 16.6, 10.9 Hz, 8H), 1.40 - 1.21 (m, 46H), 0.88 (t, J = 6.8 Hz, 9H).

[0100] Example 10 Synthesis of compound 30 [ka]

[0101] Step 1: To a solution of compound 30-1 (2.00 g) in DMF (15 mL), 1-decanethiol (2.10 g) and sodium hydroxide (1.20 g) were added sequentially, and the reaction mixture was stirred at 70 °C for 3 h. TLC showed the complete disappearance of starting compound 30-1. The reaction mixture was poured into HO (50 mL) and extracted once with EA (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute HCl and extracted three times with EA (20 mL). The organic layers were combined and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to give compound 30-2 (1.70 g, 54% yield).

[0102] Step 2: To a solution of compound 30-2 (1.70 g) in DCM (15 mL), 4-dimethylaminopyridine (DMAP, 160 mg) and 5-bromopentanol (1.31 g) were added sequentially. After stirring the mixture at 25 °C for 5 min, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.56 g) was added, and the reaction mixture was stirred at 25 °C for 2 h. TLC showed that starting compound 30-2 had completely disappeared. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-1% EA (volume percent)), and the pure product fraction was evaporated to give compound 30-3 (1.54 g, 57% yield).

[0103] Step 3: To a solution of compound 30-3 (1.5 g) in ethanol (15 mL), ethanolamine (783 mg) was added, and the mixture was stirred at 70 °C for 12 h. TLC showed that a small amount of starting compound 30-3 remained. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with 1% aqueous ammonia in methanol in dichloromethane containing 0-10% CHOH (volume percent)), and the pure product fraction was evaporated to give compound 30-4 (824 mg, 58% yield).

[0104] Step 4: To a solution of compound 30-4 (724 mg) in DMF (7 mL), heptadecan-9-yl 8-bromooctanoate (1.03 g), NaI (278 mg), and K2CO3 (770 mg) were added sequentially, and the reaction mixture was reacted at 50 °C for 12 h. TLC showed that a small amount of starting compound 30-4 remained. The reaction mixture was poured into HO (50 mL) and extracted three times with EA (20 mL). The organic layers were combined, washed twice with saturated brine (20 mL), and dried over anhydrous sodium sulfate. The mixture was filtered under vacuum and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a solution of 1% aqueous ammonia in methanol in dichloromethane containing 0-10% CH3OH (volume percent)). The pure product fraction was evaporated to give compound 30 (1.02 g, 71% yield). 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (dd, J = 12.4, 6.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.83 (d, J = 7.0 Hz, 1H), 2.73 - 2.38 (m, 12H), 2.27 (t, J = 7.2 Hz, 3H), 1.68 - 1.18 (m, 61H), 0.87 (t, J = 6.8 Hz, 9H).

[0105] Example 11 Synthesis of compound 35 [ka]

[0106] Step 1: To a solution of compound 35-1 (2.00 g) in DMF (15 mL), 1-octyl mercaptan (2.10 g) and sodium hydroxide (1.20 g) were added sequentially, and the reaction mixture was stirred at 70 °C for 3 h. TLC showed the complete disappearance of starting compound 35-1. The reaction mixture was poured into HO (50 mL) and extracted once with EA (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute HCl and extracted three times with EA (20 mL). The organic layers were combined and dried over anhydrous sodium sulfate. The mixture was filtered under vacuum and concentrated to give compound 35-2 (1.80 g, 56.6% yield).

[0107] Step 2: Compound 35-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg), and 5-bromo-1-pentanol (708 mg) were weighed and added in portions to the reaction mixture, followed by stirring at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 35-2 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added and stirred. The mixture was purified with spot plate monitoring (10 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 35-3 (1.30 g, 79.2% yield).

[0108] Step 3: Compound 35-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 2-7 (559 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 35-3. A new spot with a lower polarity than 35-3 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and then stirred with an appropriate amount of DCM and silica gel. The mixture was purified with spot plate monitoring (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A portion of the pure product fraction was evaporated to give compound 35 (830 mg, 56.9% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (dd, J = 12.4, 6.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.83 (d, J = 7.0 Hz, 1H), 2.73 - 2.38 (m, 12H), 2.27 (t, J = 7.2 Hz, 3H), 1.68 - 1.18 (m, 61H), 0.87 (t, J = 6.8 Hz, 9H).

[0109] Example 12 Synthesis of compound 36 [ka]

[0110] Step 1: 1-Octyl mercaptan (2.50 g) and NaOH (2.20 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Compound 36-1 (2.00 g) was then weighed and added in portions to the reaction mixture, which was then stirred overnight at room temperature. TLC (PE / EA = 3 / 1, phosphomolybdic acid) monitored the formation of a new spot. 200 ml of water was added to the reaction mixture, and concentrated HCl was added dropwise to adjust the pH to around 3. The mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over Na2SO4 and evaporated under reduced pressure. The mixture was stirred with an appropriate amount of DCM and silica gel, and purified with spot plate monitoring (30 g normal phase column, PE / EA, 0-0% 10 min, 0-2% 20 min, 2-2% 5 min, flow rate 30 ml / min). A portion of the pure product was evaporated to give a white solid 36-2 (1.09 g, 40.2% yield).

[0111] Step 2: Compound 36-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg), and 5-bromo-1-pentanol (708 mg) were weighed and added in portions to the reaction mixture, followed by stirring at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 36-2 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added and stirred. The mixture was purified with spot plate monitoring (10 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 36-3 (1.20 g, 74.7% yield).

[0112] Step 3: Compound 36-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 2-7 (559 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 2-7. A new spot with a lower polarity than 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and then stirred with an appropriate amount of DCM and silica gel. The mixture was purified with spot plate monitoring (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A portion of the pure product fraction was evaporated to give compound 35 (830 mg, 56.9% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (dd, J = 12.4, 6.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.54 (t, J = 6.0 Hz, 2H), 2.83 (d, J = 7.0 Hz, 1H), 2.73 - 2.38 (m, 14H), 2.27 (t, J = 7.2 Hz, 2H), 1.68 - 1.18 (m, 57H), 0.87 (t, J = 6.8 Hz, 9H).

[0113] Example 13 Synthesis of compound 41 [ka]

[0114] Step 1: To a solution of compound 41-1 (2.00 g) in DMF (15 mL), 1-decanethiol (2.30 g) and sodium hydroxide (1.20 g) were added sequentially, and the reaction mixture was stirred at 40 °C for 4 h. TLC showed the complete disappearance of starting compound 41-1. The reaction mixture was poured into HO (50 mL) and extracted once with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid and extracted three times with ethyl acetate (20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to give compound 41-2 (1.80 g, 56.6% yield).

[0115] Step 2: Compound 41-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.05 g), 4-dimethylaminopyridine (DMAP, 560 mg), and 8-bromooctanoic acid (1.02 g) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 2 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 41-2 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, stirred with an appropriate amount of silica gel and DCM, and purified (15 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min) to give colorless oily liquid compound 41-3 (1.5 g, 77% yield).

[0116] Step 3: Compound 41-3 (700 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 2-7 (559 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 3 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 2-7. A new spot with a lower polarity than 2-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) to give compound 41 (600 mg, 44.8% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (p, J = 6.4 Hz, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.56 (t, J = 5.4 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 2.61 (dt, J = 11.8, 6.4 Hz, 4H), 2.51 (dd, J = 14.4, 6.8 Hz, 6H), 2.28 (t, J = 7.6 Hz, 2H), 1.70 - 1.55 (m, 6H), 1.50 (dd, J = 16.6, 10.8 Hz, 8H), 1.40 - 1.21 (m, 50H), 0.88 (t, J = 6.8 Hz, 9H).

[0117] Example 14 Synthesis of compound 44 [ka]

[0118] Step 1: To a solution of compound 44-1 (2.00 g) in DMF (15 mL), 1-octyl mercaptan (2.63 g) and sodium hydroxide (1.44 g) were added sequentially, and the reaction mixture was stirred at 70 °C for 3 h. TLC showed the complete disappearance of starting compound 44-1. The reaction mixture was poured into HO (50 mL) and extracted once with EA (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute HCl and extracted three times with EA (20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered under vacuum and concentrated to give compound 44-2 (1.63 g, 53% yield).

[0119] Step 2: To a solution of compound 44-2 (1.63 g) in DCM (15 mL), 4-dimethylaminopyridine (DMAP, 172 mg) and 5-bromopentanol (1.41 g) were added sequentially. After stirring the mixture at 25 °C for 5 min, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.75 g) was added, and the reaction mixture was stirred at 25 °C for 1 h. TLC showed that starting compound 44-2 had completely disappeared. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-1% EA (volume percent)), and the pure product fraction was evaporated to give compound 44-3 (1.65 g, 62% yield).

[0120] Step 3: To a solution of compound 44-3 (1.5 g) in acetonitrile (15 mL), ethanolamine (960 mg) and potassium carbonate (1.15 g) were added, and the solution was stirred at 70 °C for 12 h. TLC showed that a small amount of starting compound 44-3 remained. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with 1% aqueous ammonia in methanol in dichloromethane containing 0-10% CHOH (volume percent)), and the pure product fraction was evaporated to give compound 44-4 (800 mg, 56% yield).

[0121] Step 4: To a solution of compound 44-5 (2.00 g) in DCM (15 mL), 4-dimethylaminopyridine (DMAP, 200 mg) and 7-bromo-1-heptanol (1.51 g) were added sequentially. After stirring the mixture at 25 °C for 5 min, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.62 g) was added, and the reaction mixture was stirred at 25 °C for 1 h. TLC showed the complete disappearance of starting compound 44-5. The reaction mixture was concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a petroleum ether solution containing 0-1% EA (volume percent)), and the pure product fraction was evaporated to give compound 44-6 (2.40 g, 74% yield).

[0122] Step 5: To a solution of compound 44-4 (800 mg) in DMF (7 mL), 44-6 (1.12 g), NaI (332 mg), and K2CO3 (918 mg) were added sequentially, and the reaction mixture was reacted at 50 °C for 12 h. TLC showed that a small amount of starting compound 44-4 remained. The reaction mixture was poured into H2O (50 mL) and extracted three times with EA (20 mL). The organic phases were combined, washed twice with saturated brine (20 mL), and dried over anhydrous sodium sulfate. The mixture was suction filtered and concentrated to give the crude product. The crude product was purified by column chromatography (silica gel column, eluting with a solution of 1% aqueous ammonia in methanol in dichloromethane containing 0-10% CH3OH (volume percent)). The pure product fraction was evaporated to give compound 44 (950 mg, 58% yield). 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.09 (d, J = 3.2 Hz, 2H), 3.72 - 3.55 (m, 2H), 3.05 - 2.86 (m, 2H), 2.84 (s, 1H), 2.59 - 2.41 (m, 8H), 2.31 - 2.17 (m, 2H), 1.72 - 1.64 (m, 4H), 1.62 - 1.60 (s, 2H), 1.59 - 1.51 (m, 8H), 1.55(s, 2H), 1.39 - 1.35 (m, 6H), 1.33 (d, J = 3.0 Hz, 6H), 1.31 (d, J = 3.0 Hz, 12H), 1.31 - 1.28 (m, 4H), 1.30-1.26(m, 12H), 1.24 (s, 3H), 0.94 - 0.82 (m, 9H).

[0123] Example 15 Synthesis of compound 46 [ka]

[0124] Step 1: 1-Heptanethiol (2.1 g) and NaOH (1.0 g) were dissolved in DMF (20 ml) and stirred at room temperature. Then, compound 46-1 (2.00 g) was weighed and added in portions to the reaction system. o The mixture was stirred overnight at 3°C. TLC (PE / EA = 3 / 1, phosphomolybdic acid) showed the formation of a new spot. 200 mL of water was added to the reaction mixture, and concentrated HCl was added dropwise to adjust the pH to approximately 3. The mixture was extracted with 600 mL of ethyl acetate, and the organic phase was dried over Na2SO4 and evaporated under reduced pressure. An appropriate amount of DCM and silica gel were added, and the mixture was stirred. Purification was performed with spot plate monitoring (30 g normal-phase column, PE / EA, 0-0% 10 min, 0-2% 20 min, 2-2% 5 min, flow rate 30 mL / min). A portion of the pure product fraction was evaporated to give a colorless oily liquid 46-2 (1.5 g, 62% yield).

[0125] Step 2: To a solution of compound 46-2 (1.5 g) in THF (20 mL) and water, lithium hydroxide (360 mg) was added, and the mixture was stirred at 60 °C for 16 h. TLC showed the formation of an increasingly polar spot. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, and extracted once with ethyl acetate (30 mL). The aqueous phase was adjusted to pH 2 with dilute hydrochloric acid and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 46-3 (1.2 g, 88% yield).

[0126] Step 3: Compound 46-3 (1.2 g) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.01 g), 4-dimethylaminopyridine (DMAP, 534 mg), and 6-bromo-n-hexanol (792 mg) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 46-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added and stirred. The mixture was purified with spot plate monitoring (10 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 46-4 (1.6 g, 84% yield).

[0127] Step 4: Potassium carbonate (1.52 g) was added to a solution of compound 46-4 (1.6 g) and ethanolamine (447 mg) in acetonitrile (50 mL). The mixture was refluxed at 85 °C for 2 h. TLC showed that compound 46-4 had completely disappeared and one increasingly polar spot had appeared. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was stirred with an appropriate amount of silica gel and DCM and purified (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 5 min, flow rate 20 mL / min) to give compound 46-5 (870 mg, 57% yield) as a colorless oil.

[0128] Step 5: Compound 46-6 (2.0 g) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.79 g), 4-dimethylaminopyridine (DMAP, 950 mg), and 6-bromo-n-hexanol (1.41 mg) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 46-6 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added and stirred. The mixture was purified with spot plate monitoring (25 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 46-7 (2.8 g, 86% yield).

[0129] Step 6: Compound 46-5 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (180 mg), KCO (497 mg), and compound 46-7 (502 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid). A new spot with a lower polarity than 46-7 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 46 (700 mg, 77% yield) as a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.10 (m, 4H), 3.64 (m, 3H), 2.61 - 2.42 (m, 8H), 2.29 (s, 1H), 1.98 - 1.90 (m, 2H), 1.67 - 1.61 (m, 6H), 1.59 - 1.45 (m, 8H), 1.43 (s, 4H), 1.41 - 1.37 (m, 4H), 1.37 - 1.33 (m, 10H), 1.33 - 1.30 (m, 14H), 1.30 - 1.26 (m, 12H), 0.95 - 0.83 (m, 12H).

[0130] Example 16 Synthesis of compound 48 [ka]

[0131] Step 1: Potassium carbonate (7.19 g) was added to a solution of compound 2-6 (8.0 g) and propanolamine (1.9 g) in acetonitrile (50 mL). The mixture was refluxed at 85 °C for 2 h. TLC showed that compound 2-6 had completely disappeared and a single increasingly polar spot had formed. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was stirred with an appropriate amount of silica gel and DCM and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 5 min, flow rate 20 mL / min) to give compound 48-1 (5.0 g, 63.3% yield) as a colorless oil.

[0132] Step 2: Compound 35-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 48-1 (620 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 48-1. A new spot with a lower polarity than 48-1 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and then stirred with an appropriate amount of DCM and silica gel. The mixture was purified with spot plate monitoring (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A portion of the pure product fraction was evaporated to give compound 48 (650 mg, 69% yield) as a pale yellow oily liquid. 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.09 (d, J = 3.2 Hz, 2H), 3.65 - 3.56 (m, 2H), 3.00 - 2.88 (m, 3H), 2.56 - 2.40 (m, 8H), 2.31 - 2.17 (m, 2H), 1.73 - 1.63 (m, 6H), 1.62 (d, J = 1.0 Hz, 2H), 1.59 - 1.50 (m, 8H), 1.58 (s, 2H), 1.40 - 1.35 (m, 6H), 1.34 - 1.30 (m, 18H), 1.30 - 1.27 (m, 16H), 1.24 (s, 3H), 0.95 - 0.82 (m, 9H).

[0133] Example 17 Synthesis of compound 55 [ka]

[0134] Step 1: Potassium carbonate (3.59 g) was added to a solution of compound 2-6 (4.0 g) and N,N-dimethylethylenediamine (1.53 g) in acetonitrile (50 mL). The mixture was stirred at 85 °C for 3 h. TLC showed that compound 2-6 had completely disappeared and a single increasingly polar spot had formed. The reaction mixture was filtered, and the resulting filtrate was concentrated. The crude product was stirred with an appropriate amount of silica gel and DCM and purified (25 g normal-phase column, 0.1% NH3H2O, MeOH / DCM, 0-0% for 5 min, 0-10% for 20 min, 10-10% for 5 min, flow rate 20 mL / min) to give compound 55-1 (1.3 g, 32% yield) as a colorless oil.

[0135] Step 2: Compound 55-2 (1.93 g) and sodium hydroxide (1.20 g) were added sequentially to a solution of bromoethanol (2.00 g) in DMF (15 mL), and the reaction mixture was stirred at 40 °C for 4 h. TLC showed the complete disappearance of starting compound 55-2. The reaction mixture was poured into HO (50 mL) and extracted once with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid and extracted three times with ethyl acetate (20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was vacuum filtered and concentrated to give compound 55-3 (1.56 g, 62.3% yield).

[0136] Step 3: Compound 55-3 (872 mg) was dissolved in DCM (10 mL) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.05 g), 4-dimethylaminopyridine (DMAP, 560 mg), and 8-bromooctanoic acid (1.02 g) were weighed and added in portions to the reaction mixture, followed by stirring at room temperature for 2 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 55-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, stirred with an appropriate amount of silica gel and DCM, and purified (15 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 mL / min) to give colorless oily liquid compound 55-4 (1.27 g, 70% yield).

[0137] Step 4: Compound 55-1 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (146 mg), K2CO3 (406 mg), and compound 55-4 (393 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1 ml aqueous ammonia, phosphomolybdic acid). A new spot was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and an appropriate amount of DCM and silica gel was added and stirred. The mixture was purified (25 g normal-phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 55 (108 mg, 14% yield) as a pale yellow oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.23 (s, 2H), 2.80 (s, 2H), 2.60 (s, 2H), 2.57 (s, 2H), 2.52 (s, 2H), 2.45 (s, 4H), 2.30 (s, 6H), 2.27 (s, 2H), 2.24 (s, 2H), 1.71 - 1.66 (m, 2H), 1.62 (s, 2H), 1.58 - 1.57 (d, J = 6.8 Hz, 4H), 1.54 (d, J = 6.8 Hz, 2H), 1.53 - 1.50 (m, 4H), 1.37 (d, J = 1.0 Hz, 6H), 1.35 - 1.30 (m, 24H), 1.30 - 1.25 (m, 16H), 0.96 - 0.81 (m, 9H).

[0138] Example 18 Synthesis of compound 57 [ka]

[0139] Step 1: Compound 55-1 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (146 mg), KCO (406 mg), and compound 29-3 (422 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85°C for 2 hours. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1 d ammonia water, phosphomolybdic acid). The formation of a new spot was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min) and concentrated to give compound 57 (103 mg, 12% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.12 (s, 2H), 2.96 - 2.77 (m, 2H), 2.62 - 2.56 (m, 6H), 2.50 (s, 2H), 2.45 (d, J = 2.2 Hz, 4H), 2.30 (s, 6H), 2.24 (s, 2H), 1.71 - 1.66 (m, 2H), 1.65 (s, 2H), 1.62 (s, 2H), 1.58 (s, 2H), 1.57 - 1.50 (m, 6H), 1.49 (s, 2H), 1.38 (d, J = 0.8 Hz, 6H), 1.34 - 1.30 (m, 18H), 1.30 - 1.26 (m, 20H), 0.94 - 0.82 (m, 9H).

[0140] Example 19 Synthesis of compound 51 [ka]

[0141] Step 1: 1-Hexanethiol (2.10 g) and NaOH (2.40 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Compound 51-1 (2.00 g) was then weighed and added in portions to the reaction mixture, which was then stirred overnight at room temperature. A new spot was observed on TLC (PE / EA = 3 / 1, phosphomolybdic acid). 200 ml of water was added to the reaction mixture, and concentrated hydrochloric acid was added dropwise to adjust the pH to approximately 3. The mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The mixture was stirred with an appropriate amount of DCM and silica gel, and purified with spot plate monitoring (30 g normal phase column, PE / EA, 0-0% 10 min, 0-2% 20 min, 2-2% 5 min, flow rate 30 ml / min). A portion of the pure product was evaporated to give a white solid 51-2 (2.10 g, 85.8% yield).

[0142] Step 2: Compound 51-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 986 mg), 4-dimethylaminopyridine (DMAP, 105 mg), and 7-bromo-1-heptanol (1050 mg) were weighed and added in portions to the reaction mixture, which was then stirred at room temperature for 3 h. A small amount of the reaction mixture was diluted and spotted against a standard sample of 51-2 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added and stirred. The mixture was purified with spot plate monitoring (10 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 ml / min). A portion of the pure product fraction was evaporated to give colorless oily liquid compound 51-3 (1.40 g, 75.0% yield).

[0143] Step 3: Compound 51-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), KCO (350 mg), and compound 2-7 (559 mg) were weighed and added in portions to the reaction mixture. The mixture was heated to reflux and stirred at 85 °C for 3 h. A small amount of the reaction mixture was diluted and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) against a standard sample of 51-3. A new spot with a lower polarity than 51-3 was observed. The reaction mixture was cooled to room temperature, evaporated under reduced pressure, and stirred with an appropriate amount of DCM and silica gel. The mixture was purified by TLC monitoring (25 g normal phase column, DCM / MeOH, 0.1% aqueous ammonia, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A portion of the pure product fraction was evaporated to give compound 51 (740 mg, 50.7% yield) as a pale yellow oily liquid. 1 H NMR (400 MHz, Chloroform-d) δ 4.86 (p, J = 6.3 Hz, 1H), 4.09 (t, J = 6.6 Hz, 2H), 3.51 (t, J = 5.4 Hz, 2H), 2.82 (dd, J = 12.7, 7.0 Hz, 1H), 2.65 (h, J = 6.9 Hz, 1H), 2.58 - 2.54 (m, 3H), 2.53 - 2.47 (m, 2H), 2.43 (q, J = 7.0 Hz, 4H), 2.27 (t, J = 7.5 Hz, 2H), 1.82 - 1.18 (m, 53H), 0.87 (t, J = 6.6 Hz, 7H).

[0144] Example 20 Luciferase mRNA was diluted in 10-100 mM citrate buffer, pH 4.0, and each lipid component (cationic lipid shown in the present invention: DSPC: cholesterol: PEG lipid (DMG-PEG2000)) was dissolved in ethanol at a molar ratio of 50:10:38.5:1.5.

[0145] 3 mL of mRNA buffer and 1 mL of lipid solution were placed in a 5 mL syringe and attached to a microfluidic syringe pump. The chip was connected to the syringe, the flow rate of the syringe pump was set, and the start button was clicked to inject the solution into the chip at a flow rate ratio of 3:1. The color of the product at the chip outlet was observed, and the first 5 milky white droplets (approximately 100 μL) were discarded. The remaining samples were collected in an EP tube. The collected material was placed in a dialysis bag and dialyzed against 10 mM PBS (pH 7.4) for 6 hours (molecular weight cutoff: 100 kDa). The collected material was then concentrated to the desired concentration by ultrafiltration. The lipid nanoparticles were then filtered through a 0.22 μm sterile filter and stored at 4°C.

[0146] The product encapsulation rate was tested and calculated according to the Ribogreen kit instructions, and particle size and polydispersity index (PDI) detection, Zeta potential analysis were performed using standard detection methods on a Malvern Zetasizer nano instrument. The particle size, PDI, and encapsulation rate of the mRNA-loaded LNPs prepared in this example are shown in Table 1. As can be seen from the results, the nanoparticles formed by this combination of lipids and mRNA had a high encapsulation rate and a uniform particle size of approximately 100 nm, satisfying the basic properties of a nucleic acid delivery carrier.

[0147] [Table 2]

[0148] Example 21 Measurement of the in vivo expression effect of luciferase mRNA delivered by tail vein injection of nanolipid particle composition Six- to eight-week-old BALB / c mice were injected via the tail vein with LUC-mRNA-lipid nanoparticles containing 5 μg of mRNA prepared as in Example 19 (see SEQ ID NO: 1 in Patent Publication CN114380724A for the nucleotide sequence corresponding to LUC-mRNA). At specific time points, mice were injected with 100 μg of D-luciferin potassium salt via the tail vein and detected using a PerkinElmer small animal imaging system. Fluc is commonly used in mammalian cell cultures to measure gene expression and cellular activity and emits biological fluorescence in the presence of the substrate luciferin. The mRNA used had a basic ARCA cap structure, a polyA tail length of 100-120 nt, and was fully substituted with pseudouracil. As shown in Figure 1, nanolipid particle compositions composed of compounds designed in this invention delivered mRNA to the liver at levels comparable to or better than DLin-MC3-DMA, with some compounds even superior to Lipid M.

[0149] Example 22 Measurement of the in vivo expression efficacy of luciferase mRNA delivered by pulmonary nebulization of nanolipid particle composition LUC-mRNA-lipid nanoparticles containing 5 μg of mRNA prepared as in Example 19 were delivered to the lungs of 6-8 week-old BALB / c mice by aerosol. At specific time points, the mice were injected with 100 μg of D-luciferin potassium salt via the tail vein, and the results were detected using a PerkinElmer small animal imaging system. As shown in Figure 2, the nanolipid particle composition consisting of compounds 7 and 35 showed superior fluorescence levels of the delivered mRNA compared to Lipid M and SM-102.

[0150] Example 23: Delivery of a novel coronavirus mRNA vaccine by nanolipid particle compositions Six-week-old BALB / c mice were immunized with mRNA novel coronavirus pneumonia vaccines (Omicron antigen mRNA; the corresponding nucleotide sequence is shown in Patent Publication CN114380724A) delivered via different nanolipid particle compositions via intramuscular injection on days 0 and 14. Blood samples were collected on day 28 (14 days after the secondary immunization) and neutralizing antibody titers were measured by enzyme-linked immunosorbent assay to evaluate the protective effect of the mRNA novel coronavirus pneumonia vaccines delivered via different nanolipid particle compositions against SARS-CoV-2 virus strain infection. As shown in Figure 3, the nanolipid particle composition consisting of compound 35 induced a binding antibody titer of 1.4 million, with a Lipid M titer of approximately 930,000, due to the delivered novel coronavirus mRNA. Example 24: Delivery of a novel coronavirus mRNA vaccine to the lungs using a nanolipid particle composition According to Example 20, more lipid nanoparticles were prepared and the immune effect of delivering a novel coronavirus mRNA vaccine to the lungs was examined. Eight-week-old BALB / c mice were immunized on day 0 with 2 μg of mRNA novel coronavirus pneumonia vaccine (Omicron antigen mRNA; the corresponding nucleotide sequence is SEQ ID NO: 6 in Patent Publication CN114380724A) delivered using different nanolipid particle compositions via pulmonary aerosolization. Blood samples were collected on day 14, and binding antibody titers were measured using enzyme-linked immunosorbent assay to evaluate the protective effect of mRNA novel coronavirus pneumonia vaccines delivered using different nanolipid particle compositions against SARS-CoV-2 virus strain infection (results are shown in Figure 4). The results show that the binding antibody titers of the compounds of the present invention were significantly improved compared to Lipid 5.

[0151] Example 25: Properties of nanolipid particle compositions with different lipid components and ratios To investigate the formulation potential of compound 35 with different co-lipids and lipid ratios, an experimental design was conducted. Nanoparticles were prepared by mixing lipid mixtures with different structured lipids (DOPE, DSPC), different lipid ratios (cation 45-55%, PEG lipid 1.5-2.5%, structured lipid 8-22%, cholesterol 20.5-45.5%), different nitrogen / phosphorus ratios (5-10), and Luc mRNA using a microfluidic control method (as in Example 19). The results are shown in Table 2.

[0152] [Table 3]

[0153] As can be seen from the results in Table 2, nanoparticles formed using a lipid mixture and mRNA within the above ranges had a uniform particle size, a high encapsulation rate, and good formulation suitability.

[0154] Example 26: Study of the in vivo expression effect of luciferase mRNA delivered by different cations The effect of luciferase mRNA delivered by more cationic lipids was examined according to the methods of Example 20 and Example 21. Table 3 below shows the characteristic results.

[0155] [Table 4]

[0156] Note that, because the experimental lots were different, the data for encapsulation rate and particle size DPI in Table 3 differ from those in Table 1.

[0157] As can be seen from the above table, the above compound has a high encapsulation rate, uniform particle size, and a much higher efficiency of delivering Luciferase mRNA into the body than the commercially available lipid DLin-MC3-DMA.

[0158] Example 27: Study of expression efficiency using cationic lipid proteins In this example, the in vivo transfection efficacy of erythropoietin (EPO) with different cationic lipids was compared. Nanoparticles were prepared as in Example 20. The nucleotide sequence corresponding to EPO mRNA was CN114380724B, SEQ ID NO:2. After preparation, 20 μg of EPO-mRNA-lipid nanoparticles were injected via the tail vein of 6-8 week-old female Balb / c mice. Six hours later, blood was collected from the eye margins of the mice. The serum was centrifuged and the expression level of EPO protein was measured by ELISA. The mRNA used was characterized by an ARCA cap structure, a 100-120 nt polyA tail, and fully substituted pseudouracil residues. The preparation and protein detection results are shown in Table 4.

[0159] [Table 5]

[0160] Note: Because the experimental lots were different, the data on the encapsulation rate and particle size DPI in Table 4 differ from those in Tables 1 and 3.

[0161] As can be seen from the above table, the above compound has a high encapsulation rate, uniform particle size, and the protein translation efficiency of EPO mRNA delivery into the body is much higher than that of the commercially available lipid DLin-MC3-DMA.

[0162] Example 28: Study of abnormal toxicity caused by cationic lipids In this experiment, the nanoparticles prepared in Example 27 were injected via the tail vein of female SD rats weighing 200-250 g at a dose of 5 mg / kg, and control mice were injected with a corresponding volume of saline. Mice in the DLin-MC3-DMA group died within 18 h after injection. No abnormalities were observed in the weight, feeding, or activity of the other mice during the observation period. This indicates that DLin-MC3-DMA lipids exhibited stronger toxicity. Blood samples were taken 120 h after injection, and glutamic acid pyruvate transferase (ALT), glutamic acid oxaloacetate transferase (AST), urea nitrogen (BUN), and creatinine (SCRI) were analyzed using an automated biochemical analyzer to assess liver and kidney function. As can be seen from the results in Figure 5, lipids No. 35 (compound 35) and No. 51 (compound 51) showed a slight decrease in BUN values ​​(although both values ​​were within the normal range for healthy individuals), but other lipid nanoparticles showed no significant changes in other indicators. Therefore, such cationic lipids do not affect liver and kidney function, have low toxicity, and are suitable for use in protein replacement pipelines.

[0163] Example 29 Comparison of the effects of intratumoral injection of nanolipid particle compositions In the preparation of Example 20, lipid nanoparticles encapsulating different types of luciferase mRNA were prepared and injected into the right flank of 6-8 week old female C57BL6 mice at a dose of 5 × 10 5 B16F10 cells (derived from the Cell Bank of the Chinese Academy of Sciences, CSTR: 19375.09.3101MOUTCM36) were subcutaneously injected per mouse, and tumor growth was observed and recorded periodically. On the 8th day after tumor inoculation, the tumor length (L, mm) and width (D, mm) were measured with a caliper and calculated using the formula V = (L × D 2 ) / 2, the tumor volume (V) was calculated. 3Mice were randomly divided into groups and injected intratumorally with 25 μL / 2.5 μg of lipid nanoparticles loaded with different types of luciferase mRNA. 24 hours later, autopsy imaging was performed to detect the fluorescence intensity of the mouse liver and tumor (shown in Figures 6 and 7). The results showed that compounds 35 and 51 had higher fluorescence intensity in the tumor and lower fluorescence intensity in the liver. Their superior tumor in situ expression effect and lower liver metastasis indicated their application in the encapsulation and delivery of several tumor-therapeutic nucleic acid drugs.

Claims

1. A cationic lipid compound for nucleic acid delivery, having the structure of the following structural formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: 【Chemistry 1】 where: R 1 is —R 13 —OH, where R 13 is a C 2 straight chain alkyl group; R 2 is a C 5-7 linear alkylene group; L 1 is —OC(═O)— or —C(═O)O—; R 4 and R 5 are each independently a C 8 linear alkyl group; R 3 is a C 5-7 linear alkylene group; L 2 is -OC(=O)-; R 6 is a methyl group; R 7 is —R 11 -L 4 —R 12 , wherein R 11 is a C 1 alkyl group, R 12 is a C 5 -C 8 alkyl group, and L 4 is O or S.

2. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the cationic lipid compound has one of the structures shown in the table below. Table 1

3. A liposome preparation comprising the cationic lipid compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and a prophylactic or therapeutic nucleic acid, for use in the prevention or treatment of a disease.

4. 4. The liposome formulation according to claim 3, wherein the molar ratio of the nucleic acid to the cationic lipid compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is 20:1 to 1:

1.

5. The liposome preparation according to claim 3, wherein the diameter of the liposome preparation is 50 nm to 300 nm.

6. The liposome preparation according to claim 3, further comprising one or more other lipid components including neutral lipids, steroids, and polymer-conjugated lipids.

7. The liposome formulation of claim 6, wherein the steroid contained therein is cholesterol.

8. 8. The liposome preparation according to claim 7, wherein the molar ratio of the cholesterol to the cationic lipid compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is (0-1.5):

1.

9. The liposome formulation of claim 6, wherein the polymer in the polymer-conjugated lipid is polyethylene glycol (PEG).

10. 10. The liposome preparation according to claim 9, wherein the molar ratio of the cationic lipid compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, to the polyethylene glycol-conjugated lipid is 100:1 to 20:

1.

11. The liposome preparation according to claim 6, wherein the neutral lipid is one or a combination of two or more selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

12. 12. The liposome formulation according to claim 11, wherein the molar ratio of the neutral lipid to the cationic lipid compound, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is (0-0.5):

1.

13. The liposome formulation according to claim 3 , wherein the nucleic acid is selected from antisense RNA and / or messenger RNA.

14. A pharmaceutical for inducing protein expression in a subject, comprising the cationic lipid compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

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