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, and is a cationic lipid compound that can bind to other lipid components to form drug-carrying nanolipid particles, and can achieve nucleic acid delivery from extracellular to intracellular in vitro and in vivo. Specifically, it relates to cationic lipid compounds and compositions for nucleic acid delivery and their uses.

Background Art

[0002] Nucleic acid pharmaceuticals are those that replace, compensate, block, or modify specific genes so as to achieve the purpose of treating and preventing diseases by introducing foreign genes into target cells or tissues. Its development and production process has advantages such as being relatively simple, having a short development period, a high success rate in clinical development, and better improved plasticity. In recent years, nucleic acid vaccines have proven to have great potential in the market as one of the main forces 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 target tissues. Therefore, improving the in vivo delivery efficiency of mRNA pharmaceuticals is one of the important directions for improving the effectiveness of such products.

[0004] Currently, the most widely applied delivery carrier for nucleic acid pharmaceuticals is lipid nanoparticles, which have characteristics such as improving the therapeutic effect and target delivery effect of gene drugs, protecting nucleic acids from being rapidly degraded in the body, extending the cycle time, and enhancing target delivery. It is composed of 2 to 4 lipid components including a cationic lipid compound, 0 to 2 auxiliary lipids, and 0 to 1 PEG lipid. Among them, the cationic lipid compound plays an important role in the encapsulation and release of nucleic acids. Therefore, developing novel, highly efficient, and low-toxic cationic lipid compounds is very important.

Summary of the Invention

[0005] The present invention provides a sulfur-containing cationic lipid compound including its pharmaceutically acceptable salts, stereoisomers or tautomers. Its main use is to form lipid nanoparticles for the delivery of prophylactic or therapeutic agents (such as therapeutic nucleic acids) by combining with other lipid components in a specific ratio.

[0006] Another object of the present invention is to provide a method for synthesizing the corresponding lipid compound, which uses easily available raw materials, adopts a reaction route with mild conditions, has a high product yield, low equipment requirements, and simple operation.

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

[0008] At the same time, the present invention further provides the formulation ratio and usage method of the preparation when such a cationic lipid compound is used in combination with other lipid components, as well as its use in cells and animal models. In an embodiment of the present invention, a cationic lipid compound having the structure of the following formula (I) or its pharmaceutically acceptable salt, tautomer or stereoisomer is used.

Chemical formula

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

[0010] In some specific embodiments of the present invention, R1 is -R 13 -OH or -R 13 -N(R14 )(R 15 ) and R 13 is a C1-C 12 linear alkyl group, and R 14 and R 15 are each independently a C1-C 12 linear alkyl group, R2 is a C1-C 18 linear alkylene group, L1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-, R4 and R5 are each independently a C1-C 30 aliphatic hydrocarbon group, R3 is a C1-C 18 linear 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 the said R 11 and R 12 are each independently, at each occurrence, a substituted or unsubstituted C1-C 18 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 alkyl group or a branched alkyl group, and R 14 and R 15 are each independently H or a C1-C5 linear 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 a substituted or unsubstituted C1-C 18 linear alkyl group.

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

[0014] In some specific embodiments of the present invention, R4, R5, R6 and R7 are independently hydrogen, or a substituted or unsubstituted C1-C 18 aliphatic hydrocarbon group, or -R 11 -L4-R 12 wherein said R 11 and R 12 are each independently, in each occurrence, a substituted or unsubstituted C1-C 10 aliphatic hydrocarbon group, L4 is O or S, and among R4, R5, R6 and R7, at least one contains 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.

Chemical formula

[0016] In some specific embodiments of the present invention, R1 is -R 13 -OH or -R 13 -N(R 14 )(R 15 ), where R 13 is a C1-C5 linear alkyl group, preferably a C2-C4 linear alkyl group, and R 14 and R 15 are each independently a C1-C 12 linear alkyl group, preferably each independently a C1-C3 linear alkyl group, R2 is a C2-C 12It is a linear alkylene group, preferably a C5-C9 linear alkyl group, more preferably a C5-C7 linear alkyl group, L1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-. R4 and R5 are each independently a C3-C 13 aliphatic hydrocarbon group, preferably a C6-C 10 linear alkyl group, more preferably a C6-C8 linear alkyl group, R3 is a C2-C 10 linear alkylene group, preferably a C3-C7 linear alkyl group, more preferably a C5-C7 linear 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 wherein said R 11 is a C1-C 10 alkyl group, preferably a C1-C3 alkyl group, R 12 is a C3-C 13 alkyl group, preferably a C6-C 10 alkyl group, more preferably a C6-C8 alkyl group, and L4 is O or S.

[0017] In some specific embodiments of the present invention, R1 is -R 13 -OH, R 13 is a C 1-3 linear alkyl group, R2 is a C 5-9 linear alkyl group, L1 is -OC(=O)- or -C(=O)O-. R4 and R5 are each independently a C 6-10 linear alkyl group, R3 is a C 5-7 linear 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 wherein said R 11 is a C1-C2 alkyl group, and R 12 is a C3-C 13 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 linear alkyl group, R2 is a C 5-7 linear alkyl group, preferably a C7 linear alkyl group, L1 is -OC(=O)- or -C(=O)O-, R4 and R5 are each independently a C8 linear alkyl group, R3 is a C 5-7 linear alkyl group, preferably a C5 linear alkyl group, L2 is -OC(=O)-, R6 is a methyl group, R7 is -R 11 -L4-R 12 wherein said 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 any of -OC(=O)-, -C(=O)O-, -C(=O)S-, -SC(=O)-, R2 and R3 are independently a substituted or unsubstituted C1-C 18 linear alkylene group, R4, R5 and R6 are independently hydrogen, or a substituted or unsubstituted C1-C 30 aliphatic hydrocarbon group, R7 is -R 11 -L4-R12 and the R 11 and R 12 is, in each occurrence, independently, a substituted or unsubstituted C1-C 18 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 a C1-C 12 linear alkyl group or a branched alkyl group, R 14 and R 15 are each independently H or a C1-C 12 linear alkyl group, or R 14 and R 15 together with the N atom to which they are attached form a C3-C 10 heterocycloalkyl group.

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

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

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

[0023] In some specific embodiments of the present invention, the cationic lipid compound has one of the structures shown in the following table.

Table 1

[0024] The present invention further provides a liposome preparation containing one or more of the cationic lipid compounds of the present invention and a prophylactic or therapeutic nucleic acid, which is used for the prevention or treatment of a certain disease.

[0025] The liposome preparation contains one or more components selected from neutral lipids, charged lipids, steroids, and polymer complex lipids. The therapeutic agents used in the present invention are therapeutic nucleic acids including plasmid DNA, messenger RNA, antisense oligonucleotides (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, and complementary DNA (cDNA). Preferably, they are plasmid DNA, messenger RNA, and antisense oligonucleotides.

[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 50 nm to 300 nm.

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

[0030] In some specific embodiments of the present invention, one or more other lipid components including, but not limited to, neutral lipids, steroids, and polymer composite lipids are further included.

[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 the cholesterol to the cationic lipid compound is (0 to 1.5):1.

[0033] In some specific embodiments of the present invention, the polymer in the polymer composite 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 present invention, the polyethylene glycolated 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 includes 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 to 0.5):1.

[0039] In some specific embodiments of the present invention, the liposome formulation contains nucleic acid.

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

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

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

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

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

[0045] In some specific embodiments of the present invention, the subject is a human. The terms used in the specification and claims of the present application have the following meanings unless otherwise stated.

[0046] The "alkyl group" includes, but is not limited to, substituted or unsubstituted linear or branched saturated aliphatic hydrocarbon groups having 1 to 20 carbon atoms, alkyl groups having 1 to 8 carbon atoms, alkyl groups having 1 to 6 carbon atoms, and alkyl groups having 1 to 4 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, neobutyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, and various branched isomers thereof. The alkyl groups appearing in this specification have definitions consistent with this definition.

[0047] The "alkylene group" includes substituted or unsubstituted linear and branched divalent saturated hydrocarbon groups, and -(CH2) v -(where v is an integer from 1 to 10). Examples of alkylene groups include, but are not limited to, methylene group, ethylene group, propylene group, and butylene group. The "aliphatic hydrocarbon group" includes saturated or unsaturated, linear or branched chain or cyclic hydrocarbon groups, which may contain heteroatoms such as nitrogen atoms, oxygen atoms, fluorine atoms, phosphorus atoms, sulfur atoms, and selenium atoms. 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 group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, n-heptyl group, vinyl group, 1-propenyl group, 2-propenyl group, 1-methylvinyl group, 1-butenyl group, 1-ethylvinyl group, 1-methyl-2-propenyl group, 2-butenyl group, 3-butenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-pentenyl group, 1-hexenyl group, ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 1-methyl-2-propynyl group, 3-butynyl group, 1-pentynyl group, 1-hexynyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group, etc.

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

[0049] As described above, the present invention provides a cationic lipid compound, a liposome formulation, and use thereof for nucleic acid delivery. The present invention has the following advantages.

[0050] The cationic lipid compound of the present invention has an ether bond or a thioether bond. By introducing the ether bond or the thioether bond, the compound is more easily decomposed, the in vivo elimination rate of the lipid compound is improved, the toxicity of the carrier composed of the compound is lower, and the residue in the body is less. As a result of structural optimization, the screened cationic compound has better in vivo transfection efficiency than some commercial transfection cationic lipid compounds. In addition, the method for producing the amino lipid compound has the advantages that the raw materials used are easily available, the reaction conditions are mild, the product yield is high, the equipment requirements are low, and the operation is simple.

Brief Description of the Drawings

[0051]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[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

[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 sequentially added. After the solution was stirred at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) indicated the formation of a new spot. The reaction solution was filtered, and the obtained filtrate was concentrated to give a crude product, which was purified by column chromatography (silica gel column, eluted 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 a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic layers were combined and concentrated to obtain compound 2-3 (2.10 g, 95% yield).

[0056] Step 3: Compound 2-3 (2.0 g) was dissolved in DCM (20 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 2-3, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred and purified (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid compound 2-4 (2.8 g, 87% yield).

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

[0058] Step 5: Potassium carbonate (7.19 g) was added to an acetonitrile solution (50 mL) of compound 2-6 (8.0 g) and ethanolamine (1.59 g). The mixture was stirred at 70 °C for 2 h. TLC showed that compound 2-6 completely disappeared and a spot with higher polarity was generated. The reaction solution was filtered, and appropriate amounts of silica gel and DCM were added to the concentrated crude product obtained, stirred, and purified (25 g normal-phase column, PE / EA, 0-0% for 5 min, 0-10% for 20 min, 10-10% for 5 min, flow rate 20 ml / min) to obtain a colorless oily liquid compound 2-7 (4.2 g, 54.9% yield).

[0059] Step 6: Compound 2-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and compound 2-7 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, 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 obtain a pale yellow oily liquid compound 2 (700 mg, 73% yield). 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

Chemical Structure

[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 sequentially added. After the mixture was stirred at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) showed the formation of a new spot. The reaction solution was filtered, and the obtained filtrate was concentrated. The crude product was purified by column chromatography (using a silica gel column and a petroleum ether solution containing 0-10% ethyl acetate (volume percentage) as the eluent) to obtain 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 hours. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic layers were combined and concentrated to obtain compound 3-3 (2.50 g, 92% yield).

[0063] Step 3: Compound 3-3 (2.0 g) was dissolved in DCM (20 ml), stirred at room temperature, and 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with the standard sample of 3-3, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purified (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid compound 3-4 (2.6 g, 83% yield).

[0064] Step 4: Compound 3-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and compound 2-7 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux and stirred at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 d ammonia water, phosphomolybdic acid), and a new spot with lower polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, purified (25 g normal phase column, DCM / MeOH, 0.1% ammonia water, 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 obtain a pale yellow oily liquid compound 3 (750 mg, 80% yield). 11H 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 [Chemical formula]

[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. After the mixture was stirred at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) indicated the formation of a new spot. The reaction solution was filtered, and the obtained filtrate was concentrated. The resulting crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percent)) to obtain 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 a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic layers were combined and concentrated to obtain compound 4-3 (2.50 g, 92% yield).

[0068] Step 3: Compound 4-3 (2.0 g) was dissolved in DCM (20 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with the standard sample of 3-3, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred and purified (10 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 5% for 20 min, 5 - 5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid compound 4-4 (2.6 g, 84% yield).

[0069] Step 4: Compound 4-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and compound 2-7 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, stirred, purified (25 g normal-phase column, DCM / MeOH, 0.1% aqueous ammonia, 0 - 0% (volume percentage of methanol in the DCM / MeOH solution, the same below) for 10 min, 0 - 7.5% for 20 min, 7.5 - 7.5% for 5 min, flow rate 25 ml / min), and concentrated to obtain a pale yellow oily liquid compound 4 (610 mg, 65.7% yield). 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

Chemical Structure

[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 sequentially added. After the mixture was stirred at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) showed the formation of a new spot. The reaction solution was filtered, and the obtained filtrate was concentrated. The crude product was purified by column chromatography (silica gel column, eluent was a petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to obtain 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), lithium hydroxide (584 mg) was added, and the mixture was stirred at 60 °C for 16 hours. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic phases were combined and concentrated to obtain compound 6-3 (1.6 g, 85% yield).

[0073] Step 3: Compound 6-3 (2.0 g) was dissolved in DCM (20 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 6-3, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred and purified (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid compound 6-4 (2.5 g, 82% yield).

[0074] Step 4: Compound 6-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and compound 2-7 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, 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 obtain a pale yellow oily liquid compound 6 (742 mg, 75% yield). 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

Chemical formula

[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 sequentially added. After the solution was stirred at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) showed the formation of a new spot. The reaction mixture was filtered, and the obtained filtrate was concentrated. The resulting crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-10% ethyl acetate (volume percent)) to obtain 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 hours. TLC showed the formation of a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic layers were combined and concentrated to obtain compound 7-3 (2.0 g, 94% yield).

[0078] Step 3: Compound 7-3 (2.0 g) was dissolved in DCM (20 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, compared with a standard sample of 7-3, and spotted (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred and purified (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid compound 7-4 (2.5 g, 74% yield).

[0079] Step 4: Compound 7-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and compound 2-7 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, 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), concentrated to obtain a pale yellow oily liquid compound 7 (750 mg, 75% yield). 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

Chemical Structure

[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 the mixture 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, the solvent was removed in vacuo to obtain a crude product, and the crude product was purified by column chromatography (silica gel column, eluent: n-hexane solution containing 0 - 10% EA (volume percent)) 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 stirred at 0 o °C for half an hour. Then, 14-2 was gradually added to the solution, and the solution was stirred at 80 o °C for 2 hours. After the solution cooled to room temperature, saturated ammonium chloride solution was added to quench it, and it was extracted with ethyl acetate. The organic layers were combined, dried over Na2SO4, the solvent was removed in vacuo to obtain a crude product.

[0083] Step 3: Under ice bath conditions, 1M TBAF solution was added to a solution of 14-3 (2.5 g) in anhydrous tetrahydrofuran (20 mL). The solution was warmed to room temperature and stirred at room temperature for 2 hours. Saturated ammonium chloride solution was added, diluted with water, and then extracted with ethyl acetate. The organic layers were combined, dried over Na2SO4, the solvent was removed in vacuo to obtain a crude product, and the crude product was purified by column chromatography (silica gel column, eluent: n-hexane solution containing 0 - 60% EA (volume percent)) 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 sequentially added. The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with DCM (30 mL) and washed with saturated NaHCO3 (100 mL) and brine (100 mL). The organic layers were combined, dried over Na2SO4, and the solvent was removed in vacuo to obtain a crude product. The crude product was purified by column chromatography (silica gel column, using an n-hexane solution containing 0 - 1% EA (volume percent) as the eluent), and the pure product fraction was evaporated to obtain 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), K2CO3 (527 mg), and Compound 2-7 (673 mg) were sequentially weighed and added portionwise to the above reaction system, and the mixture was heated to reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, stirred, and purified (25 g normal phase column, DCM / MeOH, 0.1% ammonia water, 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 obtain a pale yellow oily liquid Compound 14 (820 mg, 81% yield). 11H 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

Chemical Structure

[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 sequentially added. After stirring the solution at room temperature for 4 hours, spotting (petroleum ether:ethyl acetate = 10:1) indicated the formation of a new spot. The reaction mixture was filtered, and the obtained filtrate was concentrated. The crude product was purified by column chromatography (using a silica gel column and an eluent of a petroleum ether solution containing 0 - 10% ethyl acetate (volume percent)) to obtain 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) was added lithium hydroxide (1.03 g), and the mixture was stirred at 60 °C for 16 h. TLC indicated the formation of a spot with increased polarity. The reaction mixture was concentrated to remove tetrahydrofuran, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, extracted twice with ethyl acetate (30 mL), the organic layers were combined and concentrated to obtain Compound 17-3 (1.80 g, 88.7% yield).

[0089] Step 3: Compound 17-3 (2.0 g) was dissolved in DCM (20 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with 2-3 standard samples, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred and purified (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) to obtain a colorless oily liquid, Compound 17-4 (2.8 g, 74% yield).

[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 sequentially added. After the mixture was stirred at 25 °C for 5 minutes, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.62 g) was added, and the reaction mixture was stirred at 25 °C for 1 hour. TLC showed that the starting compound 44-5 completely disappeared. The reaction mixture was concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, using a petroleum ether solution containing 0 - 1% EA (volume percent) as the eluent), and the pure product fraction was evaporated to obtain compound 44-6 (2.4 g, 74% yield).

[0091] Step 5: To a solution of compound 44-6 (2.0 g) and ethanolamine (530 mg) in acetonitrile (50 mL), potassium carbonate (1.80 g) was added. The mixture was stirred at 70 °C for 3 hours. TLC showed that compound 44-6 completely disappeared and one spot with increased polarity was generated. The reaction solution was filtered, and an appropriate amount of silica gel and DCM were added to the obtained filtrate and stirred, and purified (25 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 10% for 20 min, 10 - 10% for 5 min, flow rate 20 ml / min) to obtain a colorless oily liquid compound 17-5 (1.0 g, 53.8% yield).

[0092] Step 6: Compound 17-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K2CO3 (527 mg) and Compound 17-5 (673 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 2 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 17-5 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, 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), concentrated to obtain a pale yellow oily liquid compound 17 (725 mg, 70% yield). 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 = 1.0 Hz, 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

Chemical Structure

[0094] Step 1: To a solution of compound 2-6 (2.0 g) and N,N-diethylethylenediamine (755 mg) in acetonitrile (50 mL) was added potassium carbonate (1.2 g). The mixture was stirred at 70 °C for 3 hours. One spot with increased polarity was generated. The reaction solution was filtered, and an appropriate amount of silica gel and DCM were added to the concentrated crude product obtained, followed by stirring and purification (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 obtain a colorless oily liquid compound 28-1 (900 mg, 30% yield).

[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 sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 2 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and it was observed that new spots were generated. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, and an appropriate amount of DCM and silica gel were added, followed by stirring and purification (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 then concentrated to obtain a pale yellow oily liquid compound 7 (94 mg, 11% yield). 11H 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

Chem.

[0097] Step 1: 1-Decanethiol (2.28 g) and KOH (2.20 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Then, Compound 29-1 (2.00 g) was weighed and added portionwise to the above reaction system, and the mixture was stirred overnight at room temperature. New spots were generated 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 about 3. The above mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over Na2SO4 and then evaporated under reduced pressure. Purification (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) was carried out while stirring with an appropriate amount of DCM and silica gel and monitoring by spot plate, and a part of the fraction of the pure product was evaporated to obtain white solid 29-2 (950 mg, 30% yield).

[0098] Step 2: Compound 29-2 (950 mg) was dissolved in DCM (10 ml), stirred at room temperature, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg), and 5-bromo-1-pentanol (708 mg) were sequentially weighed and added to the reaction system in portions, followed by stirring at room temperature for 3 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with the standard sample of 29-2, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added, stirred, and purified (10 g normal-phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) while monitoring with a spot plate. A portion of the fraction of the pure product was evaporated to obtain a 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), K2CO3 (350 mg), and Compound 2-7 (559 mg) were sequentially weighed and added to the above reaction system in portions, followed by heating under reflux with stirring at 85 °C for 2 h. A small amount of the reaction solution was taken, diluted, spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) by comparison with the standard sample of 2-7, and a new spot with lower polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added, 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) while monitoring with a spot plate. A portion of the fraction of the pure product was evaporated to obtain a pale yellow oily liquid, Compound 29 (850 mg). 11H 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 [Chemical formula]

[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 sequentially added, and the reaction mixture was stirred at 70 °C for 3 hours. TLC showed that the starting compound 30-1 had completely disappeared. The reaction solution was poured into H2O (50 mL), EA (20 mL) was added for extraction once, the aqueous phase was adjusted to pH 3 with 2M dilute hydrochloric acid, EA (20 mL) was added for extraction three times, the organic layers were combined, dried over anhydrous sodium sulfate, filtered by suction, and concentrated to obtain 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) were sequentially added 4-dimethylaminopyridine (DMAP, 160 mg) and 5-bromopentanol (1.31 g). After the mixture was stirred at 25 °C for 5 minutes, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCl, 1.56 g) was added, and the reaction mixture was stirred at 25 °C for 2 hours. TLC showed that the starting compound 30-2 had completely disappeared. The reaction mixture was concentrated to obtain a 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 obtain 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) was added ethanolamine (783 mg), and the mixture was stirred at 70 °C for 12 hours. TLC showed that a small amount of the starting compound 30-3 remained. The reaction mixture was concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluting with a dichloromethane solution containing 0 - 10% CH3OH (volume percent) with 1% aqueous ammonia in methanol), and the pure product fraction was evaporated to obtain compound 30-4 (824 mg, 58% yield).

[0104] Step 4: To a solution of compound 30-4 (724 mg) in DMF (7 mL), heptadecane-9-yl 8-bromooctanoate (1.03 g), NaI (278 mg), and K2CO3 (770 mg) were sequentially added, and the reaction mixture was reacted at 50 °C for 12 h. TLC indicated that a small amount of the starting compound 30-4 remained. The reaction solution was poured into H2O (50 mL), EA (20 mL) was added, and the mixture was extracted 3 times. The organic layers were combined, the organic phase was washed twice with saturated brine (20 mL), and the organic phase was dried over anhydrous sodium sulfate. It was filtered by suction and concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, dichloromethane solution containing 0-10% CH3OH (volume percent) with 1% aqueous ammonia in methanol as the eluent), and the pure product fraction was evaporated to obtain 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

Chemical Structure

[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 sequentially added, and the reaction mixture was stirred at 70 °C for 3 hours. TLC showed that the starting compound 35-1 completely disappeared. The reaction solution was poured into H2O (50 mL), EA (20 mL) was added for extraction once, the aqueous phase was adjusted to pH 3 with 2M dilute hydrochloric acid, EA (20 mL) was added for extraction three times, the organic layers were combined, and dried over anhydrous sodium sulfate. Suction filtration was carried out, and the filtrate was concentrated to obtain compound 35-2 (1.80 g, 56.6% yield).

[0107] Step 2: Compound 35-2 (1000 mg) was dissolved in DCM (10 ml), 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 sequentially weighed and added to the reaction system in portions, and stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 35-2, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification was carried out (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) while monitoring with a spot plate. A portion of the fraction of the pure product was evaporated to obtain a 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), K2CO3 (350 mg) and Compound 2-7 (559 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken, diluted, and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) in comparison with a standard sample of 35-3, and a new spot smaller in polarity than 35-3 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, and purification was carried out (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) while monitoring the spot plate, and a part of the fraction of the pure product was evaporated to obtain a pale yellow oily liquid compound 35 (830 mg, 56.9% 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).

[0109] Example 12 Synthesis of Compound 36

Chemical Structure

[0110] Step 1: As a compound, 1-octyl mercaptan (2.50 g) and NaOH (2.20 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Then, compound 36-1 (2.00 g) was weighed and added to the above reaction system in portions, and stirred at room temperature overnight. TLC (PE / EA = 3 / 1, phosphomolybdic acid) monitored that a new spot was generated. 200 ml of water was added to the reaction mixture, and concentrated HCl was added dropwise to adjust the pH to around 3. The above mixture was extracted with 600 ml of ethyl acetate, the organic phase was dried over Na2SO4, and then evaporated under reduced pressure. An appropriate amount of DCM and silica gel were added and stirred, and purification was carried out while monitoring the spot plate (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 fraction of the pure product was evaporated to obtain white solid 36-2 (1.09 g, 40.2% yield).

[0111] Step 2: Compound 36-2 (1000 mg) was dissolved in DCM (10 ml), 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 sequentially weighed and added to the reaction system in portions, and stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) in comparison with the standard sample of 36-2, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification was carried out while monitoring the spot plate (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min). A portion of the fraction of the pure product was evaporated to obtain 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), K2CO3 (350 mg) and Compound 2-7 (559 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken, diluted, spotted (DCM / MeOH = 10 / 1, 1 d aqueous ammonia, phosphomolybdic acid) by comparison with a standard sample of 2-7, and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, and purification was carried out while monitoring the spot plate (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 part of the fraction of the pure product was evaporated to obtain a pale yellow oily liquid compound 35 (830 mg, 56.9% 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, 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

Chemical Structure

[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 sequentially added, and the reaction mixture was stirred at 40 °C for 4 hours. TLC showed that the starting compound 41-1 had completely disappeared. The reaction solution was poured into H2O (50 mL), ethyl acetate (20 mL) was added and extracted once, the aqueous phase was adjusted to pH 3 with 2M dilute hydrochloric acid, ethyl acetate (20 mL) was added and extracted three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered by suction, and concentrated to obtain compound 41-2 (1.80 g, 56.6% yield).

[0115] Step 2: Compound 41-2 (1000 mg) was dissolved in DCM (10 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 2 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 41-2, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification (15 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 5% for 20 min, 5 - 5% for 5 min, flow rate 15 ml / min) gave a 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), K2CO3 (350 mg) and Compound 2-7 (559 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken, diluted, and spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) in comparison with the standard sample of 2-7, and a new spot smaller in polarity than 2-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, and purification was carried out (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 obtain a pale yellow oily liquid, Compound 41 (600 mg, 44.8% yield). 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

Chemical Structure

[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 sequentially added, and the reaction mixture was stirred at 70 °C for 3 hours. TLC showed that the starting compound 44-1 completely disappeared. The reaction solution was poured into H2O (50 mL), EA (20 mL) was added and extracted once, the aqueous phase was adjusted to pH 3 with 2M dilute hydrochloric acid, EA (20 mL) was added and extracted three times, the organic phases were combined and dried over anhydrous sodium sulfate. Filtration was carried out under suction and concentrated to obtain 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 sequentially added. After the mixture was stirred at 25 °C for 5 minutes, 1-ethyl-(3-dimethylaminopropyl) (EDCl, 1.75 g) was added, and the reaction mixture was stirred at 25 °C for 1 hour. TLC showed that the starting compound 44-2 completely disappeared. The reaction mixture was concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, using a petroleum ether solution containing 0-1% EA (volume percent) as the eluent), and the pure product fraction was evaporated to obtain 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 hours. TLC showed that a small amount of the starting compound 44-3 remained. The reaction mixture was concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, using a dichloromethane solution containing 0-10% CH3OH (volume percent) with 1% aqueous ammonia in methanol as the eluent), and the pure product fraction was evaporated to obtain compound 44-4 (800 mg, 56% yield).

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

[0122] Step 5: To a solution of compound 44-4 (800 mg) in DMF (7 mL) were sequentially added 44-6 (1.12 g), NaI (332 mg), and K2CO3 (918 mg), and the reaction mixture was reacted at 50 °C for 12 hours. TLC indicated that a small amount of the starting compound 44-4 remained. The reaction solution was poured into H2O (50 mL), 20 mL of EA was added, and the mixture was extracted 3 times. The organic phases were combined, washed twice with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered by suction, and concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, using a dichloromethane solution containing 0 - 10% CH3OH (volume percent) with 1% aqueous ammonia in methanol as the eluent), and the pure product fraction was evaporated to obtain compound 44 (950 mg, 58% yield). 11H 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

Chemical Structure

[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 portionwise to the above reaction system, and the mixture was stirred at 60 o °C overnight. New spots were formed 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 around 3. The above mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over Na2SO4 and then evaporated under reduced pressure. An appropriate amount of DCM and silica gel were added and stirred, and purification was carried out while monitoring the spot plate (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 fraction of the pure product was evaporated to obtain a colorless oily liquid 46-2 (1.5 g, 62% yield).

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

[0126] Step 3: Compound 46-3 (1.2 g) was dissolved in DCM (10 ml), 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 sequentially weighed and added to the reaction system in portions, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 46-3, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification was carried out (10 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 5% for 20 min, 5 - 5% for 5 min, flow rate 15 ml / min) while monitoring the spot plate. A portion of the fraction of the pure product was evaporated to obtain a colorless oily liquid, Compound 46-4 (1.6 g, 84% yield).

[0127] Step 4: A potassium carbonate (1.52 g) was added to an acetonitrile solution (50 mL) of compound 46-4 (1.6 g) and ethanolamine (447 mg). The mixture was refluxed at 85 °C for 2 hours. TLC showed that compound 46-4 completely disappeared and one spot with increased polarity was generated. The reaction solution was filtered, and an appropriate amount of silica gel and DCM were added to the concentrated crude product obtained, followed by stirring. Purification (25 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 10% for 20 min, 10 - 10% for 5 min, flow rate 20 ml / min) gave a colorless oily liquid compound 46-5 (870 mg, 57% yield).

[0128] Step 5: Compound 46-6 (2.0 g) was dissolved in DCM (10 ml), stirred at room temperature, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.79 g), 4-dimethylaminopyridine (DMAP, 950 mg), and 6-bromo n-hexanol (1.41 mg) were sequentially weighed and added to the reaction system in portions, followed by stirring at room temperature for 3 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) in comparison with a standard sample of 46-6, and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification (25 g normal phase column, PE / EA, 0 - 0% for 5 min, 0 - 5% for 20 min, 5 - 5% for 5 min, flow rate 15 ml / min) was carried out while monitoring with a spot plate. A portion of the fraction of the pure product was evaporated to give a 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), K2CO3 (497 mg) and Compound 46-7 (502 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and a new spot smaller in polarity than 46-7 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and 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 obtain a pale yellow oily liquid Compound 46 (700 mg, 77% yield). 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

Chemical Structure

[0131] Step 1: To an acetonitrile solution (50 mL) of compound 2-6 (8.0 g) and propanolamine (1.9 g), potassium carbonate (7.19 g) was added. The mixture was refluxed at 85 °C for 2 hours. TLC showed that compound 2-6 completely disappeared and one spot with increased polarity was generated. The reaction solution was filtered, and an appropriate amount of silica gel and DCM were added to the concentrated crude product obtained, followed by stirring and purification (40 g normal-phase column, PE / EA, 0-0% for 5 min, 0-10% for 20 min, 10-10% for 5 min, flow rate 20 ml / min) to obtain a colorless oily liquid compound 48-1 (5.0 g, 63.3% yield).

[0132] Step 2: Compound 35-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), K2CO3 (350 mg) and compound 48-1 (620 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, and spotted (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid) in comparison with a standard sample of 48-1, and a new spot smaller in polarity than 48-1 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, and purification was carried out while monitoring the spot plate (25 g normal-phase column, DCM / MeOH, 0.1% ammonia water, 0-0% for 10 min, 0-7.5% for 20 min, 7.5-7.5% for 5 min, flow rate 25 ml / min). A part of the fraction of the pure product was evaporated to obtain a pale yellow oily liquid compound 48 (650 mg, 69% yield). 11H 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 [Chemical Structure]

[0134] Step 1: Potassium carbonate (3.59 g) was added to an acetonitrile solution (50 mL) of Compound 2-6 (4.0 g) and N,N-dimethylethylenediamine (1.53 g). The mixture was stirred and refluxed at 85 °C for 3 hours. TLC showed that Compound 2-6 completely disappeared and one spot with increased polarity was generated. The reaction solution was filtered, and an appropriate amount of silica gel and DCM were added to the obtained concentrated crude product and stirred for purification (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), yielding a colorless oily liquid Compound 55-1 (1.3 g, 32% yield).

[0135] Step 2: To a solution of bromoethanol (2.00 g) in DMF (15 mL), compound 55-2 (1.93 g) and sodium hydroxide (1.20 g) were sequentially added, and the reaction mixture was stirred at 40 °C for 4 hours. TLC indicated that the starting compound 55-2 had completely disappeared. The reaction solution was poured into H2O (50 mL), ethyl acetate (20 mL) was added and extracted once, the aqueous phase was adjusted to pH 3 with 2M dilute hydrochloric acid, ethyl acetate (20 mL) was added and extracted three times, the organic phases were combined, and dried over anhydrous sodium sulfate. Suction filtration was carried out, and concentration gave compound 55-3 (1.56 g, 62.3% yield).

[0136] Step 3: Compound 55-3 (872 mg) was dissolved in DCM (10 mL), 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 sequentially weighed and added to the reaction system in portions, and stirred at room temperature for 2 h. A small amount of the reaction solution was taken, diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) by comparison with a standard sample of 55-3, and a new spot with lower polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification (15 g normal phase column, PE / EA, 0-0% 5 min, 0-5% 20 min, 5-5% 5 min, flow rate 15 mL / min) gave a 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 sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and it was observed that a new spot was formed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and 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 obtain a pale yellow oily liquid compound 55 (108 mg, 14% yield). 1 1H 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

Chemical Structure

[0139] Step 1: 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 29-3 (422 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 2 h. A small amount of the reaction solution was taken and diluted, spotted (DCM / MeOH = 10 / 1, 1 drop of aqueous ammonia, phosphomolybdic acid), and it was observed that a new spot was formed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and 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 obtain a pale yellow oily liquid Compound 57 (103 mg, 12% yield). 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

Chemical Structure

[0141] Step 1: As a compound, 1-hexanethiol (2.10 g) and NaOH (2.40 g) were dissolved in ethanol (20 ml) and stirred at room temperature. Then, compound 51-1 (2.00 g) was weighed and added portionwise to the above reaction system, and stirred at room temperature overnight. It was monitored by TLC (PE / EA = 3 / 1, phosphomolybdic acid) that new spots were generated. 200 ml of water was added to the reaction mixture, and concentrated hydrochloric acid was added dropwise to adjust the pH to around 3. The above mixture was extracted with 600 ml of ethyl acetate, and the organic phase was dried over anhydrous Na2SO4 and then evaporated under reduced pressure. An appropriate amount of DCM and silica gel were added and stirred, and purification was carried out (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) while monitoring the spot plate, and a part of the fraction of the pure product was evaporated to obtain 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 sequentially and added portionwise to the reaction system, and stirred at room temperature for 3 h. A small amount of the reaction solution was taken and diluted, spotted (PE / EA = 10 / 1, phosphomolybdic acid) in comparison with the standard sample of 51-2, and a new spot with a smaller polarity was observed. The reaction solution was evaporated under reduced pressure, an appropriate amount of silica gel and DCM were added and stirred, and purification was carried out (10 g normal phase column, PE / EA, 0-0% for 5 min, 0-5% for 20 min, 5-5% for 5 min, flow rate 15 ml / min) while monitoring the spot plate, and a part of the fraction of the pure product was evaporated to obtain 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), K2CO3 (350 mg) and Compound 2-7 (559 mg) were sequentially weighed and added to the above reaction system in portions, and the mixture was heated under reflux with stirring at 85 °C for 3 h. A small amount of the reaction solution was taken, diluted, spotted (DCM / MeOH = 10 / 1, 1d aqueous ammonia, phosphomolybdic acid) by comparison with a standard sample of 51-3, and a new spot smaller in polarity than 51-3 was observed. After the reaction solution was cooled to room temperature, it was evaporated under reduced pressure, an appropriate amount of DCM and silica gel were added and stirred, and purification was carried out (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) while monitoring with a TLC plate, and a part of the fraction of the pure product was evaporated to obtain a pale yellow oily liquid compound 51 (740 mg, 50.7% yield). 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 a citric acid buffer at 10 - 100 mM and 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 respectively placed into 5 mL syringes, 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 of the syringe pump was clicked to inject into the chip in a manner with a flow rate ratio of 3:1. The product color at the chip outlet was observed. After discarding the first 5 drops (about 100 μL) of milky white droplets, the subsequent samples were collected into EP tubes. The collected samples were placed into a dialysis bag and dialyzed through 10 mM PBS (pH 7.4) for 6 hours (molecular weight cut-off: 100 KDa), and then concentrated to a preferred concentration by ultrafiltration. Furthermore, the lipid nanoparticles were filtered through a 0.22 μm sterile filter and stored at 4°C.

[0146] According to the description of the Ribogreen kit, the encapsulation efficiency of the product was tested and calculated. With a Malvern Zetasizer nano instrument, the particle size, polydispersity index (PDI), and Zeta potential analysis were performed by standard detection methods. The detection results of the particle size, PDI, and encapsulation efficiency of the mRNA-loaded LNP produced in this example are shown in Table 1. As can be seen from the results, the nanoparticles formed by the combination of lipid and mRNA have a high encapsulation efficiency, a uniform particle size of about 100 nm, and meet the basic characteristics 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 the nano-lipid particle composition 6 - 8-week-old BALB / c mice were injected with 5 μg of LUC-mRNA-lipid nanoparticles containing mRNA adjusted in the same manner as in Example 19 (the nucleotide sequence corresponding to LUC-mRNA refers to SEQ ID NO:1 in Patent Publication CN114380724A) via the tail vein. At specific time points, 100 μg of D-Luciferin Potassium Salt was injected into the mice via the tail vein and detected using a PerkinElmer small animal imaging system. Fluc is generally used in mammalian cell cultures to measure gene expression and cell activity and emits bioluminescence in the presence of the substrate luciferin. The basic characteristics of the mRNA used were an ARCA cap structure, a polyA tail length of 100 - 120 nt, and complete substitution with pseudouridine. The results, as shown in Figure 1, indicate that the nano-lipid particle composition consisting of the compounds designed in the present invention delivered mRNA to the liver at levels almost equivalent to or higher than those of DLin-MC3-DMA, and some compounds were superior to Lipid M.

[0149] Example 22 Measurement of the in vivo expression effect of luciferase mRNA delivered by pulmonary atomization of the nano-lipid particle composition 6 - 8-week-old BALB / c mice were delivered with LUC-mRNA-lipid nanoparticles containing 5 μg of mRNA adjusted in the same manner as in Example 19 to the lungs by spraying. At specific time points, 100 μg of D-Luciferin Potassium Salt was injected into the mice via the tail vein and detected using a PerkinElmer small animal imaging system. The results, as shown in Figure 2, indicate that the nano-lipid particle composition consisting of Compounds 7 and 35 was superior to Lipid M and SM-102 in terms of the fluorescence levels expressed by the delivered mRNA.

[0150] Example 23 Delivery of the novel coronavirus mRNA vaccine by the nano-lipid particle composition Six-week-old BALB / c mice were immunized by intramuscular injection on days 0 and 14 with an mRNA COVID-19 vaccine (Omicron antigen mRNA, the corresponding nucleotide sequence refers to SEQ ID NO:6 of Patent Publication CN114380724A) delivered by different nanolipid particle compositions. Blood was collected on day 28 (14 days after the secondary immunization), and the neutralizing antibody titer was measured by enzyme-linked immunosorbent assay to evaluate the protective effect of the mRNA COVID-19 vaccine delivered by different nanolipid particle compositions against SARS-CoV-2 virus strain infection. The results, as shown in Figure 3, were that the nanolipid particle composition consisting of Compound 35 had a binding antibody titer of 1.4 million for the delivered COVID-19 mRNA, and Lipid M was about 0.93 million. Example 24 Delivery of the novel coronavirus mRNA vaccine to the lungs by the nano-lipid particle composition According to Example 20, more lipid nanoparticles were produced to verify the immune effect by delivering the COVID-19 mRNA vaccine to the lungs. Eight-week-old BALB / c mice were immunized by nebulizing 2 μg of an mRNA COVID-19 vaccine (Omicron antigen mRNA, the corresponding nucleotide sequence refers to SEQ ID NO:6 of Patent Publication CN114380724A) delivered by different nanolipid particle compositions to the lungs on day 0. Blood was collected on day 14, and the binding antibody titer was measured by enzyme-linked immunosorbent assay to evaluate the protective effect (the results were shown in Figure 4) of the mRNA COVID-19 vaccine delivered by different nanolipid particle compositions against SARS-CoV-2 virus strain infection. As can be seen from the results, the binding antibody titer by the compound of the present invention was improved to be superior to Lipid 5.

[0151] Example 25 Characteristics of nano-lipid particle compositions with different lipid components and ratios To study the formulation properties at different ratios of auxiliary lipids and lipids, an experimental design was carried out for Compound 35. Different structural lipids (DOPE, DSPC), different lipid ratios (cationic 45 - 55%, PEG lipid 1.5 - 2.5%, structural lipid 8 - 22%, cholesterol 20.5 - 45.5%), lipid mixtures with different nitrogen / phosphorus ratios (5 - 10), and Luc mRNA were mixed by the microfluidic control method (similar to Example 19) to produce nanoparticles. The results are shown in Table 2.

[0152]

Table 3

[0153] As can be seen from the results in Table 2, the nanoparticles formed by the lipid mixtures and mRNA within this range had uniform particle sizes, high encapsulation rates, and good formulation properties.

[0154] Example 26 Study on the in vivo expression effect of luciferase mRNA delivered by different cations According to the methods of Example 20 and Example 21, the effect of luciferase mRNA delivered by more cationic lipids was verified. The following Table 3 shows the characteristic results.

[0155]

Table 4

[0156] Note that due to different experimental lots, the data of the encapsulation rate and particle size DPI in Table 3 are different 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 the delivery efficiency of Luciferase mRNA into the body is much higher than that of the commercially available lipid DLin - MC3 - DMA.

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

[0159]

Table 5

[0160] Note: Due to different experimental lots, the data of encapsulation efficiency and particle size DPI in Table 4 are different from those in Table 1 and Table 3.

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

[0162] Example 28 Study on the abnormal toxicity by cationic lipids In this experiment, the nanoparticles produced in Example 27 were used. Female SD rats weighing 200 - 250 g were injected via the tail vein at a dose of 5 mg / kg, and the mice in the control group were injected with the corresponding volume of physiological saline. The mice in the DLin - MC3 - DMA group died within 18 h after injection. For the other mice, there were no abnormal manifestations in terms of body weight, food intake, and activity status during the observation period. This indicated that the DLin - MC3 - DMA lipid had stronger toxicity. Blood was collected 120 h after injection, and alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (SCR) were detected using an automatic biochemical analyzer as liver and kidney function evaluation indicators. As can be seen from the results in Figure 5, for the lipids of No. 35 (Compound 35) and No. 51 (Compound 51), the BUN value decreased slightly (however, all values were within the healthy normal index range), and for the other lipid nanoparticles, the other indicators did not show obvious fluctuations. Therefore, such cationic lipids do not affect liver and kidney functions, have low toxicity, and are applicable for use in the protein replacement pipeline.

[0163] Example 29 Comparison of the intratumoral injection effect by the nano-lipid particle composition In the production of Example 20, lipid nanoparticles encapsulating different types of luciferase mRNA were produced. On the right flank of 6 - 8 - week - old female C57BL6 mice, 5 × 10 5 cells of B16F10 (derived from the Cell Bank of Chinese Academy of Sciences, CSTR:19375.09.3101MOUTCM36) / mouse were subcutaneously injected, and tumor growth was regularly observed and recorded. On the 8th day after tumor inoculation, the tumor length (L, mm) and width (D, mm) were measured with calipers, and the tumor volume (V) was calculated according to the formula V = (L × D 2 ) / 2. When the tumor volume was 80 - 120 mm 3Mice were randomly grouped, and 24 hours after injecting 25 μL / 2.5 μg of lipid nanoparticles encapsulating different types of luciferase mRNA into the tumor, the fluorescence intensities of the mouse liver and tumor (shown in Figures 6 and 7) were detected by anatomical imaging. As can be seen from the results, the intensities in the tumors of Compounds 35 and 51 were higher and those in the liver were lower. It was shown that it was applicable to the encapsulation and delivery of some nucleic acid drugs related to tumor treatment because of its better in situ expression effect in the tumor and lower liver metastasis.

Claims

1. A cationic lipid compound for nucleic acid delivery, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, having the structure of the following structural formula (I). 【Chemical 1】 Here, L 1 and L 2 are each independently a divalent linking group or linking bond selected from any of -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -S-S-, -C(=O)S-, -SC(=O)-, -N(R 8 )C(=O)-, -C(=O)N(R 8 )-, -N(R 8 )C(=O)O-, -OC(=O)N(R 8 )-, -SC(=O)N(R 8 )-, -N(R 8 )C(=O)S-, -C(=S)-, -SC(=S)-, and -C(=S)S-, and the R 8 is H or C 1 -C 12 alkyl group R 2 and R 3 each independently represents a substituted or unsubstituted C 1 -C 18 linear alkylene group or -R 9 -L 3 -R 10 -, wherein said R 9 and R 10 each independently represents a substituted or unsubstituted C 1 -C 10 linear alkylene group, and L 3 is O or S, R 4 、R 5 、R 6 and R 7 are, independently, hydrogen, or a substituted or unsubstituted C 1 -C 30 aliphatic hydrocarbon group, or -R 11 -L 4 -R 12 wherein said R 11 and R 12 are, independently at each occurrence, a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, and L 4 is O or S, The above R 2 、R 3 、R 4 、R 5 、R 6 and R 7 include at least one O or S, R 1 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 -OCH 3 or -R 13 -N(R 14 )(R 15 ), and R 13 is a C 1 -C 12 linear alkyl group or a branched alkyl group, and R 14 and R 15 are each independently H or a C 1 -C 12 linear alkyl group, or R 14 and R 15 together with the N atom to which they are attached form a C 3 -C 10 heterocycloalkyl group.

2. R 8 The cationic lipid compound according to claim 1, wherein R is H or a methyl group.

3. R 13 is a C 1 -C 8 linear alkyl group or branched alkyl group, and R 14 and R 15 are each independently H or C 1 -C 5 linear alkyl group, or R 14 and R 15 together with the N atom to which they are attached form a C 3 -C 8 heterocycloalkyl group, the cationic lipid compound according to claim 2.

4. R 2 and R 3 each independently is a substituted or unsubstituted C 1 -C 18 linear alkyl group, the cationic lipid compound according to claim 3.

5. R 2 and R 3 each independently is a substituted or unsubstituted C 1 -C 12 linear alkyl group, the cationic lipid compound according to claim 4.

6. R 4 、R 5 、R 6 and R 7 are, independently, hydrogen, or a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, or -R 11 -L 4 -R 12 wherein said R 11 and R 12 are, independently in each occurrence, a substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon group, L 4 is O or S, and R 4 、R 5 、R 6 and R 7 contain at least one O or S and at most two are hydrogen. The cationic lipid compound according to claim 5.

7. R in the structure of the above formula 4 , R 5 ,R 6 and R 7 The structures of are each independently H or the following alkyl chain, or are each independently an ether or thioether formed by substituting any carbon atom in the following alkyl chain with O or S. The cationic lipid compound according to claim 6. 【Chemical Formula 2】

8. R 1 is -R 13 -OH, and R 13 is a C 1-3 linear alkyl group R 2 is a C 5-9 linear alkyl group, and L 1 is -OC(=O)- or -C(=O)O-, R 4 and R 5 each independently is a C 6-10 linear alkyl group, R 3 is C 5-7 a linear alkyl group, L 2 is -OC(=O)- or -C(=O)O- and R 6 is a methyl group, an ethyl group or a propyl group, R 7 is -R 11 -L 4 -R 12 and the R 11 is C 1 -C 2 an alkyl group, and R 12 is C 3 -C 13 an alkyl group, and L 4 is O or S. The cationic lipid compound according to claim 1.

9. R 1 is -R 13 -OH, and R 13 is a C 2 linear alkyl group R 2 is a C 5-7 linear alkyl group, and L 1 is -OC(=O)- or -C(=O)O- and R 4 and R 5 each independently is a C 8 linear alkyl group, R 3 is C 5-7 a straight-chain alkyl group, L 2 is -OC(=O)-, R 6 is a methyl group, R 7 is -R 11 -L 4 -R 12 and the said R 11 is a C 1 alkyl group, and R 12 is a C 5 -C 8 alkyl group, and L 4 is O or S. The cationic lipid compound according to claim 8.

10. L 1 and L 2 are each independently selected from any of -OC(=O)-, -C(=O)O-, -C(=O)S-, -SC(=O)- R 2 and R 3 each independently is a substituted or unsubstituted C 1 -C 18 linear alkylene group, R 4 、R 5 and R 6 each independently represents hydrogen, or a substituted or unsubstituted C 1 -C 30 aliphatic hydrocarbon group, R 7 is -R 11 -L 4 -R 12 wherein said R 11 and R 12 are, independently at each occurrence, a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, and L 4 is O or S, R 1 is H, -R 13 -OH, -R 13 -OCH 3 or -R 13 -N(R 14 )(R 15 ) and R 13 is a C 1 -C 12 linear alkyl group or a branched alkyl group, and R 14 and R 15 are each independently H or a C 1 -C 12 linear alkyl group, or R 14 and R 15 together with the N atom to which they are attached form a C 3 -C 10 heterocycloalkyl group, the cationic lipid compound according to claim 1.

11. L 1 and L 2 are each independently selected from among -OC(=O)-, -C(=O)O-, -C(=O)S-, -SC(=O)- R 2 and R 3 each independently is a substituted or unsubstituted C 3 -C 10 linear alkylene group, R 4 、 R 5 and R 6 each independently is hydrogen or a substituted or unsubstituted C 1 -C 15 aliphatic hydrocarbon group, R 7 is -R 11 -L 4 -R 12 wherein said R 11 and R 12 are, independently at each occurrence, a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, and L 4 is O or S, R 1 is -R 13 -OH, and R 13 is C 1 -C 6 The cationic lipid compound according to claim 10, which is a linear alkyl group or a branched alkyl group.

12. L 1 and L 2 are each independently selected from either -OC(=O)- or -C(=O)O- R 2 and R 3 each independently is a substituted or unsubstituted C 3 -C 10 linear alkylene group, R 4 、 R 5 and R 6 each independently represents hydrogen, or a substituted or unsubstituted C 1 -C 15 aliphatic hydrocarbon group, R 7 is -R 11 -L 4 -R 12 and the said R 11 and R 12 are, independently at each occurrence, a substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon group, and L 4 is O or S, R 1 is -R 13 -OH, and R 13 is C 1 -C 6 The cationic lipid compound according to claim 11, which is a linear alkyl group or a branched alkyl group.

13. L 1 and L 2 are each independently selected from either -OC(=O)- or -C(=O)O- R 2 and R 3 each independently is a substituted or unsubstituted C 3 -C 9 linear alkylene group, R 4 、R 5 and R 6 each independently is hydrogen or a substituted or unsubstituted C 1 -C 12 aliphatic hydrocarbon group, R 7 is -R 11 -L 4 -R 12 and the said R 11 and R 12 are, independently at each occurrence, a substituted or unsubstituted C 1 -C 9 aliphatic hydrocarbon group, and L 4 is O or S, R 1 is -R 13 -OH, and R 13 is C 1 -C 5 The cationic lipid compound according to claim 12, which is a linear alkyl group or a branched alkyl group.

14. The cationic lipid compound according to claim 5, having one of the structures shown in the following table. 【Table 1】

15. A liposomal formulation comprising the cationic lipid compound according to any one of claims 1 to 14 and a prophylactic or therapeutic nucleic acid, which is used for the prevention or treatment of a certain disease.

16. The liposomal formulation according to claim 15, wherein the molar ratio of the nucleic acid to the cationic lipid compound is 20:1 to 1:

1.

17. The liposomal formulation according to claim 16, wherein the molar ratio of the nucleic acid to the cationic lipid compound is 10:1 to 4:

1.

18. The liposomal formulation according to claim 15, wherein the diameter of the liposomal formulation is 50 nm to 300 nm.

19. The liposomal formulation according to claim 18, wherein the diameter of the liposomal formulation is 50 nm to 150 nm or 150 nm to 200 nm.

20. The liposomal formulation according to claim 15, further comprising one or more other lipid components including neutral lipids, steroids and polymer composite lipids.

21. The liposomal formulation according to claim 20, wherein the steroid contained is cholesterol.

22. The liposomal formulation according to claim 21, wherein the molar ratio of the cholesterol to the cationic lipid compound is (0 to 1.5):

1.

23. The liposomal formulation according to claim 15, wherein the polymer in the polymer composite lipid is polyethylene glycol (PEG).

24. The liposomal formulation according to claim 23, wherein the molar ratio of the cationic lipid compound to the polyethylene glycol composite lipid is 100:1 to 20:

1.

25. The liposomal formulation according to claim 23, wherein the polyethylene glycol composite lipid is PEG-DAG, PEG-PE, PEG-SDAG, PEG-cer, PEG-DMG or ALC-0159.

26. The liposome formulation according to claim 20, wherein the neutral lipid is one or a combination of more than one selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

27. The liposome formulation according to claim 26, wherein the molar ratio of the neutral lipid to the cationic lipid compound is (0 to 0.5):

1.

28. The liposome formulation according to claim 15, wherein the nucleic acid is selected from antisense RNA and / or messenger RNA.

29. Use of the cationic lipid compound according to any one of claims 1 to 14 or the liposome formulation according to any one of claims 15 to 28 in the manufacture of a drug for inducing protein expression in a subject.

30. The use according to claim 29, wherein the subject is a mammal.

31. The use according to claim 29, wherein the subject is a non-human primate or a human.

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