Cationic lipid compounds and compositions for nucleic acid delivery and uses thereof

Abstract

The present invention provides cationic lipid compounds, compositions and uses for delivering nucleic acids. The compounds are represented by the following formula (I). The present invention also provides the use of nano-lipid particles with the compounds as key components in nucleic acid delivery, including components of the delivery carrier, preparation methods and usage methods.

Description

Technical Field

[0001] The present invention relates to the field of lipid delivery carriers, which are a class of cationic lipid compounds. After combining with other lipid components, they can form nano-lipid particles capable of carrying drugs, thereby realizing extracellular to intracellular delivery of nucleic acids in vitro and in vivo. Specifically, the present invention relates to cationic lipid compounds, compositions and uses for delivering nucleic acids. Background Art

[0002] Nucleic acid drugs achieve the purpose of treating and preventing diseases by introducing foreign genes into target cells or tissues to replace, compensate, block or correct specific genes. Its R & D and production processes are relatively simple, with advantages such as short R & D cycle, high clinical development success rate, and better improvement plasticity. Nucleic acid vaccines, as one of the main forces in preventing COVID-19 in recent years, have also proven their huge potential in the market.

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

[0004] Currently, the most widely used delivery carrier for nucleic acid drugs is lipid nanoparticles, which have the characteristics of improving the efficacy of gene drugs and targeted delivery, etc. They can protect nucleic acids from being rapidly degraded in vivo, extend the circulation time, and enhance targeted delivery. It consists of 2 to 4 lipid components, including cationic lipid compounds, 0 to 2 kinds of auxiliary lipids, and 0 to 1 kind of PEG lipid. Among them, cationic lipid compounds play a key role in nucleic acid encapsulation and release. Therefore, it is crucial to develop new, efficient, and low-toxic cationic lipid compounds. Summary of the Invention

[0005] The present invention provides a class of sulfur-containing cationic lipid compounds, including pharmaceutically acceptable salts thereof, stereoisomers or tautomers thereof. Its main use is to be used in combination with other lipid components in a specific ratio to form lipid nanoparticles for delivering prophylactic or therapeutic agents (such as therapeutic nucleic acids).

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

[0007] In some examples, therapeutic nucleic acids include plasmid DNA, messenger RNA, antisense oligonucleotides (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, complementary DNA (cDNA).

[0008] Meanwhile, the present invention also provides the formulation ratio and usage method when such cationic lipid compounds are used in combination with other lipid components, as well as their applications in cell and animal models.

[0009] In an embodiment of the present invention, a cationic lipid compound having the following structure of formula (I) is adopted:

[0010]

[0011] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0012] L 1 、L 2 is a linking bond or a divalent linking group, and each of the divalent linking groups is independently selected from -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;

[0013] R 2 and R 3 are independently a substituted or unsubstituted C 1 -C 18 linear alkylene group or -R 9 -L 3 -R 10 -; the R 9 and R 10 are independently a substituted or unsubstituted C 1 -C 10 linear alkylene group, and L 3 is O or S;

[0014] 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 -L4 -R 12 ; said R 11 and R 12 is independently, each time it appears, a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, and L 4 is O or S;

[0015] Among the above R 2 , R 3 , R 4 , R 5 , R 6 and R 7 , at least one of them contains O or S;

[0016] 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 ); R 13 is a C 1 -C 12 linear alkyl or branched alkyl, R 14 and R 15 are each independently H or C 1 -C 12 linear alkyl, or, R 14 and R 15 together with the N atom to which they are attached form a C 3 -C 10 heterocyclic alkyl.

[0017] According to some specific embodiments of the present invention, wherein, R 8 is H or methyl.

[0018] According to some specific embodiments of the present invention, wherein, R 1 is -R 13 -OH or -R 13 -N(R 14 )(R 15 ), R 13 is a C 1 -C 12 linear alkyl; R 14 and R15 Each independently is C 1 -C 12 a linear alkyl group;

[0019] R 2 is C 1 -C 18 a linear alkylene group;

[0020] L 1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-;

[0021] R 4 and R 5 each independently is C 1 -C 30 a hydrocarbyl group;

[0022] R 3 is C 1 -C 18 a linear alkylene group;

[0023] L 2 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-;

[0024] R 6 is methyl or ethyl;

[0025] R 7 is -R 11 -L 4 -R 12 ; said R 11 and R 12 each occurrence is independently a substituted or unsubstituted C 1 -C 18 hydrocarbyl group, and L 4 is O or S.

[0026] According to some specific embodiments of the present invention, wherein, R 13 is C 1 -C 8 a linear alkyl group or a branched alkyl group, R 14 and R 15 each independently is H or C 1 -C 5 a linear alkyl group, or, R 14 and R 15 and the N atom to which it is attached form a C 3 -C 8 heterocycloalkyl group.

[0027] According to some specific embodiments of the present invention, wherein, R 2 and R3 Independently substituted or unsubstituted C 1 -C 18 linear alkyl.

[0028] According to some specific embodiments of the present invention, wherein R 2 and R 3 Independently substituted or unsubstituted C 1 -C 12 linear alkyl.

[0029] According to some specific embodiments of the present invention, wherein R 4 , R 5 , R 6 and R 7 Independently are hydrogen, or substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, or -R 11 -L 4 -R 12 ; said R 11 and R 12 Each occurrence is independently a substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon group, L 4 is O or S; R 4 , R 5 , R 6 and R 7 contain at least one O or S, and at most two are hydrogen.

[0030] According to some specific embodiments of the present invention, wherein R 4 , R 5 , R 6 and R 7 The structures are each independently H or the following alkyl chain, or each independently an ether or thioether formed by replacing any one carbon atom on the following alkyl chain with O or S:

[0031]

[0032] According to some specific embodiments of the present invention, wherein R 1 is -R 13 -OH or -R 13 -N(R 14 )(R 15 ), R 13 is C 1 -C 5 linear alkyl, preferably C 2 -C 4 linear alkyl; R 14 and R 15Each independently is C 1 -C 12 a linear alkyl group, preferably each independently is C 1 -C 3 a linear alkyl group;

[0033] R 2 is C 2 -C 12 a linear alkylene group, preferably is C 5 -C 9 a linear alkyl group, more preferably is C 5 -C 7 a linear alkyl group;

[0034] L 1 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-;

[0035] R 4 and R 5 each independently is C 3 -C 13 a hydrocarbyl group, preferably is C 6 -C 10 a linear alkyl group, more preferably is C 6 -C 8 a linear alkyl group;

[0036] R 3 is C 2 -C 10 a linear alkylene group, preferably is C 3 -C 7 a linear alkyl group, more preferably is C 5 -C 7 a linear alkyl group;

[0037] L 2 is -C(=O)S-, -SC(=O)-, -OC(=O)- or -C(=O)O-;

[0038] R 6 is methyl or ethyl;

[0039] R 7 is -R 11 -L 4 -R 12 ; the R 11 is C 1 -C 10 an alkyl group, preferably is C 1 -C 3 an alkyl group; R 12 is C 3 -C 13 an alkyl group, preferably is C 6 -C10 alkyl, more preferably C 6 -C 8 alkyl; L 4 is O or S.

[0040] According to some specific embodiments of the present invention, wherein,

[0041] R 1 is -R 13 -OH, R 13 is C 1-3 linear alkyl;

[0042] R 2 is C 5-9 linear alkyl;

[0043] L 1 is -OC(=O)- or -C(=O)O-;

[0044] R 4 and R 5 each independently is C 6-10 linear alkyl;

[0045] R 3 is C 5-7 linear alkyl;

[0046] L 2 is -OC(=O)- or -C(=O)O-;

[0047] R 6 is methyl, ethyl or propyl;

[0048] R 7 is -R 11 -L 4 -R 12 ; said R 11 is C 1 -C 2 alkyl; R 12 is C 3 -C 13 alkyl; L 4 is O or S.

[0049] According to some specific embodiments of the present invention, wherein,

[0050] R 1 is -R 13 -OH, R 13 is C 2 linear alkyl;

[0051] R 2 is C 5-7 linear alkyl; preferably C 7 linear alkyl;

[0052] L 1 is -OC(=O)- or -C(=O)O-;

[0053] R 4 and R 5 are each independently C 8 linear alkyl;

[0054] R 3 is C 5-7 linear alkyl; preferably C 5 linear alkyl;

[0055] L 2 is -OC(=O)-;

[0056] R 6 is methyl;

[0057] R 7 is -R 11 -L 4 -R 12 ; said R 11 is C 1 alkyl; R 12 is C 5 -C 8 alkyl; L 4 is O or S.

[0058] According to some specific embodiments of the present invention, wherein,

[0059] L 1 and L 2 are each independently selected from any one of -OC(=O)-, -C(=O)O-, -C(=O)S-, -SC(=O)-;

[0060] R 2 and R 3 are independently substituted or unsubstituted C 1 -C 18 linear alkylene;

[0061] R 4 , R 5 and R 6 and are independently hydrogen, or substituted or unsubstituted C 1 -C 30 aliphatic hydrocarbon group;

[0062] R 7 is -R 11 -L 4 -R 12 ; said R 11 and R 12Each occurrence is independently a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, L 4 is O or S;

[0063] R 1 is H, -R 13 -OH, -R 13 -OCH 3 or -R 13 -N(R 14 )(R 15 ); R 13 is C 1 -C 12 linear alkyl or branched alkyl, R 14 and R 15 are each independently H or C 1 -C 12 linear alkyl, or, R 14 and R 15 together with the N atom to which it is attached form C 3 -C 10 heterocyclic alkyl.

[0064] According to some specific embodiments of the present invention, wherein,

[0065] L 1 、L 2 are each independently selected from any one of -OC(=O)-, -C(=O)O-, -C(=O)S-, -SC(=O)-;

[0066] R 2 and R 3 are independently substituted or unsubstituted C 3 -C 10 linear alkylene;

[0067] R 4 , R 5 and R 6 are independently hydrogen, or a substituted or unsubstituted C 1 -C 15 aliphatic hydrocarbon group;

[0068] R 7 is -R 11 -L 4 -R 12 ; said R 11 and R 12 each occurrence is independently a substituted or unsubstituted C 1 -C 18 aliphatic hydrocarbon group, L 4 is O or S;

[0069] R1 is -R 13 -OH; R 13 is C 1 -C 6 a straight-chain alkyl or a branched-chain alkyl.

[0070] According to some specific embodiments of the present invention, wherein,

[0071] L 1 、L 2 each independently selected from any one of -OC(=O)-, -C(=O)O-;

[0072] R 2 and R 3 are independently a substituted or unsubstituted C 3 -C 10 straight-chain alkylene;

[0073] R 4 ,R 5 and R 6 and are independently hydrogen, or a substituted or unsubstituted C 1 -C 15 aliphatic hydrocarbon group;

[0074] R 7 is -R 11 -L 4 -R 12 ; said R 11 and R 12 each occurrence is independently a substituted or unsubstituted C 1 -C 10 aliphatic hydrocarbon group, L 4 is O or S;

[0075] R 1 is -R 13 -OH; R 13 is C 1 -C 6 a straight-chain alkyl or a branched-chain alkyl.

[0076] According to some specific embodiments of the present invention, wherein,

[0077] L 1 、L 2 each independently selected from any one of -OC(=O)-, -C(=O)O-;

[0078] R 2 and R 3 are independently a substituted or unsubstituted C 3 -C 9 straight-chain alkylene;

[0079] R4 , R 5 and R 6 and independently are hydrogen, or a substituted or unsubstituted C 1 -C 12 aliphatic hydrocarbon group;

[0080] R 7 is -R 11 -L 4 -R 12 ; said R 11 and R 12 each occurrence independently is a substituted or unsubstituted C 1 -C 9 aliphatic hydrocarbon group, L 4 is O or S;

[0081] R 1 is -R 13 -OH; R 13 is C 1 -C 5 linear alkyl or branched alkyl.

[0082] According to some specific embodiments of the present invention, wherein the cationic lipid compound has one of the structures shown in the following table:

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] The present invention also provides a liposomal formulation comprising one or more cationic lipid compounds of the present invention and a prophylactic or therapeutic nucleic acid, wherein the liposomal formulation is used for preventing or treating a certain disease.

[0090] The liposomal formulation comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. The therapeutic agent used in the present invention is a therapeutic nucleic acid, including plasmid DNA, messenger RNA, antisense oligonucleotide (ASON), microRNA (miRNA), interfering RNA (micRNA), dicer substrate RNA, complementary DNA (cDNA). Preferably, they are plasmid DNA, messenger RNA, and antisense oligonucleotide.

[0091] According to some specific embodiments of the present invention, the molar ratio of the nucleic acid to the cationic lipid compound is from 20:1 to 1:1.

[0092] According to some specific embodiments of the present invention, the molar ratio of the nucleic acid to the cationic lipid compound is from 10:1 to 4:1.

[0093] According to some specific embodiments of the present invention, the diameter of the liposomal formulation is from 50 nm to 300 nm.

[0094] According to some specific embodiments of the present invention, the diameter of the liposomal formulation is from 50 nm to 150 nm, or from 150 nm to 200 nm.

[0095] According to some specific embodiments of the present invention, it further comprises one or more other lipid components, including but not limited to neutral lipids, steroids, and polymer-conjugated lipids.

[0096] According to some specific embodiments of the present invention, the steroid included is cholesterol.

[0097] According to some specific embodiments of the present invention, the molar ratio of the cholesterol to the cationic lipid compound is (0 - 1.5):1.

[0098] According to some specific embodiments of the present invention, the polymer in the polymer-conjugated lipid is polyethylene glycol (PEG).

[0099] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid compound to the polyethylene glycolated lipid is from 100:1 to 20:1.

[0100] According to 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.

[0101] According to some specific embodiments of the present invention, the liposomal formulation comprises one or more neutral lipids selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0102] According to some specific embodiments of the present invention, the neutral lipid is DSPC or DOPE.

[0103] According to some specific embodiments of the present invention, the molar ratio of the neutral lipid to the cationic lipid compound is (0 - 0.5):1.

[0104] According to some specific embodiments of the present invention, the liposomal formulation comprises nucleic acid.

[0105] According to some specific embodiments of the present invention, the nucleic acid is selected from antisense RNA and / or messenger RNA.

[0106] According to some specific embodiments of the present invention, the nucleic acid is messenger RNA.

[0107] The present invention also provides the use of the cationic lipid compound or the liposomal formulation of the present invention in the preparation of a medicament for inducing protein expression in a subject.

[0108] According to some specific embodiments of the present invention, the subject is a mammal.

[0109] According to some specific embodiments of the present invention, the subject is a non-human primate.

[0110] According to some specific embodiments of the present invention, the subject is a human.

[0111] Unless otherwise stated, the terms used in the specification and claims of this application have the following meanings.

[0112] "Alkyl" includes substituted or unsubstituted straight-chain or branched-chain saturated aliphatic hydrocarbon groups, including but not limited to alkyl 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, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and their various branched isomers; the alkyl groups appearing herein have the same definition as this definition.

[0113] "Alkylene" includes substituted or unsubstituted straight-chain and branched-chain divalent saturated hydrocarbon groups, including -(CH 2 ) v -(where v is an integer from 1 to 10), and alkylene examples include but are not limited to methylene, ethylene, propylene and butylene, etc.

[0114] "Aliphatic hydrocarbon group" includes saturated or unsaturated, straight-chain or branched-chain, acyclic or cyclic hydrocarbon groups, aliphatic hydrocarbon groups without heteroatoms and those containing heteroatoms; the heteroatoms refer to nitrogen atom, oxygen atom, fluorine atom, phosphorus atom, sulfur atom, and selenium atom. The types of the aliphatic hydrocarbon group can be selected from alkyl group, alkenyl group, alkynyl group, etc. For example, the term "C1-10 aliphatic hydrocarbon group" includes: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, vinyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 1-ethylethenyl, 1-methyl-2-propenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 1-hexenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 1-methyl-2-propynyl, 3-butynyl, 1-pentynyl, 1-hexynyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, etc.

[0115] "Heterocycloalkyl" includes substituted or unsubstituted saturated cycloalkyl groups containing heteroatoms, including but not limited to those having 3 to 10 atoms, 3 to 8 atoms, containing 1 to 3 heteroatoms selected from N, O or S, and the optionally substituted N and S in the ring of the heterocycloalkyl can be oxidized to various oxidation states. The heterocycloalkyl can be attached to a heteroatom or a carbon atom, the heterocycloalkyl can be attached to an aromatic ring or a non-aromatic ring, and the heterocycloalkyl can be attached with a bridged ring or a spiro ring. Non-limiting examples include epoxyethyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydro-2H-pyranyl, dioxolanyl, dioxanyl, pyrrolidinyl, piperidinyl, imidazolidinyl, oxazolidinyl, oxazinyl, morpholinyl, hexahydropyrimidinyl, piperazinyl.

[0116] In summary, the present invention provides a cationic lipid compound, a liposome preparation for delivering nucleic acids and their uses. The technical solution of the present invention has the following advantages:

[0117] The cationic lipid compound of the present invention has an ether bond or a thioether bond. The introduction of the ether bond or the thioether bond makes the compound more easily degradable, improves the in vivo clearance rate of the lipid compound, makes the carrier composed of the compound less toxic and has less residue in vivo. Moreover, after structural optimization, the in vivo transfection efficiency of the screened cationic compound is better than that of some commercial transfection cationic lipid compounds. And the preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, high product yield, low requirements for instrument equipment and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 For the relative fluorescence intensity of in vivo imaging of intramuscularly injected mice in Example 21;

[0119] Figure 2 Relative fluorescence intensity of in vivo imaging of mice by pulmonary atomization delivery in Example 22;

[0120] Figure 3 Binding antibody titer in Example 23;

[0121] Figure 4 Binding antibody titer in Example 24;

[0122] Figure 5 Graph for evaluating liver and kidney functions in Example 25;

[0123] Figure 6 Fluorescence imaging graph in Example 28;

[0124] Figure 7 Statistical numerical value of fluorescence imaging results in Example 28. Detailed implementation manners

[0125] The technical solutions of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention includes but is not limited to this.

[0126] Example 1

[0127] Synthesis of Compound 2

[0128]

[0129] Step 1:

[0130] 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 successively added. After the solution was stirred at room temperature for 4 hours, TLC (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: petroleum ether solution containing 0-10% ethyl acetate (volume percentage)) to obtain Compound 2-2 (3.93 g, 69% yield).

[0131] Step 2:

[0132] 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 hours. TLC showed the formation of a spot with increased polarity. The reaction mixture was concentrated to remove THF, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to obtain Compound 2-3 (2.10 g, 95% yield).

[0133] Step 3:

[0134] Dissolve compound 2-3 (2.0 g) in DCM (20 ml), stir at room temperature. Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g) and 5-bromo-1-pentanol (1.5 g) in turn and add them to the reaction system in batches. Stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the standard sample of 2-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with decreased polarity is observed. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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 colorless oily liquid compound 2-4 (2.8 g, 87% yield).

[0135] Step 4:

[0136] Dissolve compound 2-5 (5.0 g) in dichloromethane (70 ml), stir at room temperature. Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.48 g), 4-dimethylaminopyridine (DMAP, 3.57 g) and 8-bromooctanoic acid (4.78 g) in turn and add them to the reaction system in batches. Stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the standard sample of 2-5 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with decreased polarity is observed. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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 colorless oily liquid compound 2-6 (8.0 g, 88.9% yield). Compounds 2-6 in the following Examples 1-6, 8-10, 11-13, 16-18 are all synthesized by this method.

[0137] Step 5:

[0138] Add potassium carbonate (7.19 g) to the acetonitrile solution (50 mL) of compound 2-6 (8.0 g) and ethanolamine (1.59 g). Stir the mixture at 70 °C for 2 h. TLC shows that compound 2-6 completely disappears and a spot with increased polarity is formed. Filter the reaction solution, concentrate the obtained filtrate to get the crude product, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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 colorless oily liquid compound 2-7 (4.2 g, 54.9% yield).

[0139] Step 6:

[0140] Dissolve compound 2-4 (500 mg) in acetonitrile (10 ml) and stir at room temperature. Then, successively weigh NaI (191 mg), K 2 CO 3 (527 mg) and compound 2-7 (673 mg) and add them to the above reaction system in batches. Heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it, and spot it on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). Observe that there is a new spot with lower polarity than 2-7. After the reaction solution is cooled to room temperature, evaporate it under reduced pressure. Add an appropriate amount of DCM and silica gel to mix the sample, and purify it (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). Concentrate to obtain a pale yellow oily liquid compound 2 (700 mg, 73% yield).

[0141] 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).

[0142] Example 2

[0143] Synthesis of Compound 3

[0144]

[0145]

[0146] Step 1:

[0147] 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 successively added. After the mixture was stirred at room temperature for 4 hours, TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of a new spot. The reaction solution was filtered, and the obtained filtrate was concentrated to give a crude product. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percentage)) to obtain Compound 3-2 (3.1 g, 46% yield).

[0148] Step 2:

[0149] To a solution of Compound 3-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added. 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, and extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to obtain Compound 3-3 (2.50 g, 92% yield).

[0150] Step 3:

[0151] Compound 3-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g) and 5-bromo-1-pentanol (1.5 g) were successively weighed and added to the reaction system in batches, and stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on TLC with a standard sample of 3-3 (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, mixed with an appropriate amount of silica gel and DCM, 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 Compound 3-4 as a colorless oily liquid (2.6 g, 83% yield).

[0152] Step 4:

[0153] Compound 3-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg) and K 2 CO 3(527 mg) and compound 2-7 (673 mg) were added to the above reaction system in batches, 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 on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and new spots with lower polarity than 2-7 were 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 mixed, and then 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). The concentrate was obtained as a pale yellow oily liquid compound 3 (750 mg, 80% yield).

[0154] 1 H 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).

[0155] Example 3

[0156] Synthesis of Compound 4

[0157]

[0158]

[0159] Step 1:

[0160] 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 successively. After the mixture was stirred at room temperature for 4 h, TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of new spots. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percentage)) to obtain compound 4-2 (3.0 g, 47.3% yield).

[0161] Step 2:

[0162] To a solution of Compound 4-2 (3.00 g) in THF (20 mL) and water was added lithium hydroxide (860 mg). 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, and then extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to obtain Compound 4-3 (2.50 g, 92% yield).

[0163] Step 3:

[0164] Compound 4-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.1 g), 4-dimethylaminopyridine (DMAP, 1.4 g) and 5-bromo-1-pentanol (1.6 g) were successively weighed and added to the reaction system in batches, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate for comparison with the standard sample of 3-3 (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 for sample mixing, 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) to obtain Compound 4-4 as a colorless oily liquid (2.6 g, 84% yield).

[0165] Step 4:

[0166] Compound 4-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K 2 CO 3 (527 mg) and Compound 2-7 (673 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of 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 for sample mixing, and purification was carried out (25 g normal-phase column, DCM / MeOH, 0.1% ammonia water, 0 - 0% (volume percentage of methanol in the DCM / MeOH solution, the same hereinafter) 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 Compound 4 as a pale yellow oily liquid (610 mg, 65.7% yield).

[0167] 11H 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).

[0168] Example 4

[0169] Synthesis of Compound 6

[0170]

[0171]

[0172] Step 1:

[0173] To a solution of Compound 6-1 (19.0 g) in tert-butanol (20 mL), 1-decanol (3.0 g) and cesium carbonate (12.4 g) were added successively. After the mixture was stirred at room temperature for 4 hours, TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of a new spot. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percentage)) to obtain Compound 6-2 (2.1 g, 43% yield).

[0174] Step 2:

[0175] To a solution of Compound 6-2 (2.1 g) in THF (20 mL) and water (10 mL), lithium hydroxide (584 mg) was added. 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 THF, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, and extracted twice with ethyl acetate (30 mL). The organic phases were combined and concentrated to obtain Compound 6-3 (1.6 g, 85% yield).

[0176] Step 3:

[0177] Dissolve compound 6-3 (2.0 g) in DCM (20 ml), stir at room temperature. Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g) and 5-bromo-1-pentanol (1.5 g) successively and add them to the reaction system in batches. Stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the standard sample of 6-3 (PE / EA = 10 / 1, phosphomolybdic acid). A new spot with decreased polarity is observed. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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 colorless oily liquid compound 6-4 (2.5 g, 82% yield).

[0178] Step 4:

[0179] Dissolve compound 6-4 (500 mg) in acetonitrile (10 ml), stir at room temperature. Then weigh NaI (191 mg), K 2 CO 3 (527 mg) and compound 2-7 (673 mg) successively and add them to the above reaction system. Heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). A new spot with lower polarity than 2-7 is observed. Cool the reaction solution to room temperature, evaporate it under reduced pressure, add an appropriate amount of DCM and silica gel to mix the sample, purify it (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 concentrate to obtain pale yellow oily liquid compound 6 (742 mg, 75% yield).

[0180] 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).

[0181] Example 5

[0182] Synthesis of Compound 7

[0183]

[0184]

[0185] Step 1:

[0186] To a solution of compound 2-1 (3.00 g) in tert-butanol (20 mL), octanol (3.1 g) and cesium carbonate (11.0 g) were added successively. After the solution was stirred at room temperature for 4 h, TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of a new spot. The reaction mixture was filtered, and the filtrate was concentrated. The crude product obtained was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percentage)) to give compound 7-2 (3.6 g, 69% yield).

[0187] Step 2:

[0188] To a solution of compound 7-2 (3.00 g) in THF (20 mL) and water, lithium hydroxide (860 mg) was added. 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 THF, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, and then extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to give compound 7-3 (2.0 g, 94% yield).

[0189] Step 3:

[0190] Compound 7-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.0 g), 4-dimethylaminopyridine (DMAP, 1.3 g) and 5-bromo-1-pentanol (1.5 g) were weighed successively and added to the reaction system in batches. The mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on TLC with the 7-3 standard sample for comparison (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, appropriate silica gel and DCM were added for sample mixing and purification (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 give compound 7-4 as a colorless oily liquid (2.5 g, 74% yield).

[0191] Step 4:

[0192] Compound 7-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then NaI (191 mg) and K 2 CO 3(527 mg) and compound 2-7 (673 mg) were added batchwise to the above reaction system, 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 on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and new spots with lower polarity than 2-7 were 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 mixed, and then 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). The solution was concentrated to obtain a pale yellow oily liquid compound 7 (750 mg, 75% yield).

[0193] 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).

[0194] Example 6

[0195] Synthesis of Compound 14

[0196]

[0197]

[0198] Step 1:

[0199] p-Toluenesulfonyl chloride (7.4 g) was added to a solution of compound 14-1 (3.0 g) and triethylamine (5.3 g) in dichloromethane (50 mL) under ice bath conditions. After the mixture was stirred at room temperature for 3 h, the reaction mixture was diluted with DCM (30 mL) and washed with dilute hydrochloric acid and brine (100 mL). The combined organic layers were dried over Na 2 SO 4 and the solvent was removed under vacuum to obtain a crude product. The crude product was purified by column chromatography (silica gel column, eluent: n-hexane solution containing 0 - 10% EA (volume percentage)) to obtain compound 14-2 (6.0 g, 86% yield).

[0200] Step 2:

[0201] Under ice bath conditions, NaH (680 mg, 60%) was added to a solution of tert-butyldimethylhydroxyethoxysilane (2.0 g) in DMF (20 mL). After stirring the mixture at 0 °C for half an hour, 14-2 was slowly added to the solution, and the solution was stirred at 80 °C for two hours. After the solution was cooled to room temperature, saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: hexane solution containing 0-10% EA (volume percentage)) to obtain compound 14-3 (2.5 g, 80% yield).

[0202] Step 3:

[0203] 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 two hours. Saturated ammonium chloride solution was added, and after dilution with water, the mixture was extracted with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: hexane solution containing 0-60% EA (volume percentage)) to obtain compound 14-4 (1.4 g, 96% yield).

[0204] Step 4:

[0205] To a solution of compound 14-4 (1.4 g) in DCM (20 mL) were successively added 4-dimethylaminopyridine (DMAP, 1.07 g), 7-bromoheptanoic acid (2.01 g) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCl, 2.01 g). The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with DCM (30 mL) and washed with saturated NaHCO 3 (100 mL) and brine (100 mL). The combined organic layers were dried over Na 2 SO 4 and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by column chromatography (silica gel column, eluent: hexane solution containing 0-1% EA (volume percentage)), and the pure product fractions were evaporated to obtain compound 14-5 (2.10 g, 68% yield).

[0206] Step 5:

[0207] Compound 14-5 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg) and K 2 CO3 (527 mg) and Compound 2-7 (673 mg) were added batchwise to the above reaction system, 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 on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and new spots with lower polarity than 2-7 were 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 mixed, and then 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). The concentrate was obtained as a pale yellow oily liquid, Compound 14 (820 mg, 81% yield).

[0208] 1 H 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).

[0209] Example 7

[0210] Synthesis of Compound 17

[0211]

[0212]

[0213] Step 1:

[0214] To a solution of Compound 2-1 (5.00 g) in tert-butanol (40 mL) were successively added n-hexanol (2.90 g) and cesium carbonate (27.8 g). After the solution was stirred at room temperature for 4 h, TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of new spots. The reaction solution was filtered, and the crude product obtained after concentrating the filtrate was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 10% ethyl acetate (volume percentage)) to obtain Compound 17-2 (3.14 g, 39.8% yield).

[0215] Step 2:

[0216] 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). 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 THF, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, and then 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).

[0217] Step 3:

[0218] Compound 17-3 (2.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.1 g), 4-dimethylaminopyridine (DMAP, 2.0 g) and 7-bromo-1-heptanol (2.3 g) were successively weighed and added to the reaction system in batches, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate for comparison with 2-3 standards (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 for sample mixing and purification (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 Compound 17-4 as a colorless oily liquid (2.8 g, 74% yield).

[0219] Step 4:

[0220] To a solution of Compound 44-5 (2.00 g) in DCM (15 mL) were successively added 4-dimethylaminopyridine (DMAP, 200 mg) and 7-bromo-1-heptanol (1.51 g). 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 had completely disappeared. The reaction mixture was concentrated to obtain a crude product, and the crude product was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain Compound 44-6 (2.4 g, 74% yield).

[0221] Step 5:

[0222] To a solution of compound 44-6 (2.0 g) and ethanolamine (530 mg) in acetonitrile (50 mL) was added potassium carbonate (1.80 g). The mixture was stirred at 70 °C for 3 hours. TLC showed that compound 44-6 had completely disappeared and a new spot with increased polarity had formed. The reaction mixture was filtered, and the filtrate was concentrated to obtain a crude product. An appropriate amount of silica gel and DCM were added to the crude product for sample mixing and 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) to obtain compound 17-5 as a colorless oily liquid (1.0 g, 53.8% yield).

[0223] Step 6:

[0224] Compound 17-4 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (191 mg), K 2 CO 3 (527 mg) and compound 17-5 (673 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 2 h. A small amount of the reaction mixture was taken, diluted, spotted on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and a new spot with lower polarity than 17-5 was observed. After the reaction mixture was cooled to room temperature, it was evaporated under reduced pressure. An appropriate amount of DCM and silica gel were added for sample mixing and purification (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 the product was concentrated to obtain compound 17 as a pale yellow oily liquid (725 mg, 70% yield).

[0225] 1 1H 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).

[0226] Example 8

[0227] Synthesis of Compound 28

[0228]

[0229] Step 1:

[0230] Potassium carbonate (1.2 g) was added to a solution of compound 2-6 (2.0 g) and N,N-diethylethylenediamine (755 mg) in acetonitrile (50 mL). The mixture was stirred at 70 °C for 3 hours. A spot with increased polarity was formed. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. An appropriate amount of silica gel and DCM were added to the crude product for sample mixing and purification (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) to obtain a colorless oily liquid compound 28-1 (900 mg, 30% yield).

[0231] Step 2:

[0232] Compound 28-1 (500 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (146 mg), K 2 CO 3 (406 mg) and compound 7-4 (425 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 2 h. A small amount of the reaction solution was taken, diluted, spotted on a plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, 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 for sample mixing and purification (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 then concentrated to obtain a pale yellow oily liquid compound 7 (94 mg, 11% yield).

[0233] 1 1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.09 (d, J = 2.2 Hz, 2H), 3.73 - 3.60 (m, 4H), 2.75 (s, 1H), 2.69 - 2.57 (m, 8H), 2.51 - 2.40 (m, 4H), 2.31 - 2.17 (m, 2H), 1.72 - 1.65 (m, 4H), 1.60 - 1.50 (m, 10H), 1.48 (s, 2H), 1.36 (d, J = 0.6 Hz, 4H), 1.35 - 1.30 (m, 20H), 1.30 - 1.26 (m, 16H), 1.19 (s, 3H), 1.00 (s, 6H), 0.90 (s, 9H).

[0234] Example 9

[0235] Synthesis of Compound 29

[0236]

[0237] Step 1:

[0238] Dissolve 1-decanethiol (2.28 g) and KOH (2.20 g) in ethanol (20 ml), and stir at room temperature. Then weigh compound 29-1 (2.00 g) and add it to the above reaction system in batches, and stir overnight at room temperature. A new spot appears in TLC (PE / EA = 3 / 1, phosphomolybdic acid). Add 200 ml of water to the reaction mixture, and adjust the pH to around 3 by dropping concentrated HCl. Extract the above mixture with 600 ml of ethyl acetate, and dry the organic phase with Na 2 SO 4 After drying, evaporate under reduced pressure. Add an appropriate amount of DCM and silica gel to mix the sample, and purify (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), monitor by TLC, and evaporate the fractions of the pure product to obtain white solid 29-2 (950 mg, 30% yield).

[0239] Step 2:

[0240] Dissolve compound 29-2 (950 mg) in DCM (10 ml), and stir at room temperature. Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg) and 5-bromo-1-pentanol (708 mg) in turn and add them to the reaction system in batches, and stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot it on TLC for comparison with the 29-2 standard sample (PE / EA = 10 / 1, phosphomolybdic acid), and observe a new spot with smaller polarity. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample and purify (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), monitor by TLC, and evaporate the fractions of the pure product to obtain colorless oily liquid compound 29-3 (1.45 g, 95% yield).

[0241] Step 3:

[0242] Dissolve compound 29-3 (750 mg) in acetonitrile (10 ml), and stir at room temperature. Then weigh NaI (170 mg), K 2 CO 3(350 mg) and Compound 2-7 (559 mg) were added batchwise to the above reaction system, and the mixture was heated under reflux with stirring at 85 °C for 2 h. A small amount of the reaction solution was taken, diluted, and spotted on a TLC plate for comparison with the standard sample of 2-7 (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). 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 mixed. Purification was carried out (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). Monitoring by TLC, the fractions of the pure product were evaporated to obtain a pale yellow oily liquid Compound 29 (850 mg).

[0243] 1 H 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).

[0244] Example 10

[0245] Synthesis of Compound 30

[0246]

[0247] Step 1:

[0248] To a solution of Compound 30-1 (2.00 g) in DMF (15 mL) were successively added 1-decanethiol (2.10 g) and sodium hydroxide (1.20 g). The reaction mixture was stirred at 70 °C for 3 h. TLC showed that the starting Compound 30-1 had completely disappeared. The reaction solution was poured into H 2 2O (50 mL), extracted once with EA (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid, and then extracted three times with EA (20 mL). The organic phases were combined, dried over anhydrous sodium sulfate. Filtration was carried out by suction, and the filtrate was concentrated to obtain Compound 30-2 (1.70 g, 54% yield).

[0249] Step 2:

[0250] To a solution of Compound 30-2 (1.70 g) in DCM (15 mL), 4-dimethylaminopyridine (DMAP, 160 mg) and 5-bromopentanol (1.31 g) were added successively. After the mixture was stirred at 25 °C for 5 minutes, 1-ethyl-(3-dimethylaminopropyl) (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, which was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0 - 1% EA (volume percentage)), and the pure product fraction was evaporated to obtain Compound 30-3 (1.54 g, 57% yield).

[0251] Step 3:

[0252] To a solution of Compound 30-3 (1.5 g) in ethanol (15 mL), ethanolamine (783 mg) was added, and the mixture was stirred at 70 °C for 12 hours. TLC showed that a small amount of the starting compound 30-3 remained. The reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, eluent: dichloromethane solution containing 0 - 10% CH 3 OH (volume percentage), 1% ammonia water was added to methanol), and the pure product fraction was evaporated to obtain Compound 30-4 (824 mg, 58% yield).

[0253] Step 4:

[0254] To a solution of Compound 30-4 (724 mg) in DMF (7 mL), octyl 8-bromooctanoate (1.03 g), NaI (278 mg) and K 2 CO 3 (770 mg) were added successively, and the reaction mixture was reacted at 50 °C for 12 hours. TLC showed that a small amount of the starting compound 30-4 remained. The reaction solution was poured into H 2 O (50 mL), extracted 3 times with EA (20 mL), the organic phases were combined, the organic phase was washed 2 times with saturated brine (20 mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration by suction and concentration, a crude product was obtained, which was purified by column chromatography (silica gel column, eluent: dichloromethane solution containing 0 - 10% CH 3 OH (volume percentage), 1% ammonia water was added to methanol), and the pure product fraction was evaporated to obtain Compound 30 (1.02 g, 71% yield).

[0255] 11H 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).

[0256] Example 11

[0257] Synthesis of Compound 35

[0258]

[0259]

[0260] Step 1:

[0261] To a solution of Compound 35-1 (2.00 g) in DMF (15 mL) were successively added 1-octanethiol (2.10 g) and sodium hydroxide (1.20 g). The reaction mixture was stirred at 70 °C for 3 hours. TLC showed that the starting compound 35-1 had completely disappeared. The reaction solution was poured into H 2 2O (50 mL), extracted once with EA (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid, and then extracted three times with EA (20 mL). The combined organic phases were dried over anhydrous sodium sulfate. Filtered by suction and concentrated to obtain Compound 35-2 (1.80 g, 56.6% yield).

[0262] Step 2:

[0263] Compound 35-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg) and 5-bromo-1-pentanol (708 mg) were successively weighed and added to the reaction system in batches, and stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate for comparison with the standard sample of 35-2 (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, mixed with an appropriate amount of silica gel and DCM, 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). Monitored by TLC, and the pure product fractions were evaporated to obtain Compound 35-3 as a colorless oily liquid (1.30 g, 79.2% yield).

[0264] Step 3:

[0265] Dissolve compound 35-3 (750 mg) in acetonitrile (10 ml) and stir at room temperature. Then weigh out NaI (170 mg), K 2 CO 3 (350 mg) and compound 2-7 (559 mg) in batches and add them to the above reaction system. Heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it, and spot it on a TLC plate for comparison with the standard sample of 35-3 (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). Observe that there are new spots with lower polarity than 35-3. After the reaction solution is cooled to room temperature, evaporate it under reduced pressure, add an appropriate amount of DCM and silica gel to mix the sample, and purify it (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). Monitor by TLC plate, evaporate the fractions of the pure product, and obtain compound 35 as a pale yellow oily liquid (830 mg, 56.9% yield).

[0266] 1 1H 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).

[0267] Example 12

[0268] Synthesis of Compound 36

[0269]

[0270] Step 1:

[0271] Dissolve 1-octanethiol (2.50 g) and NaOH (2.20 g) in ethanol (20 ml) and stir at room temperature. Then weigh out compound 36-1 (2.00 g) and add it to the above reaction system in batches. Stir at room temperature overnight. Monitor by TLC (PE / EA = 3 / 1, phosphomolybdic acid) for the formation of new spots. Add 200 ml of water to the reaction mixture, and adjust the pH to around 3 by dropwise addition of concentrated HCl. Extract the above mixture with 600 ml of ethyl acetate. The organic phase is washed with Na 2 SO 4After drying, perform evaporation under reduced pressure. Add an appropriate amount of DCM and silica gel to mix the sample, and purify it (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). Monitor by TLC, evaporate the fractions of the pure product, and obtain white solid 36 - 2 (1.09 g, 40.2% yield).

[0272] Step 2:

[0273] Dissolve compound 36 - 2 (1000 mg) in DCM (10 ml), stir at room temperature. Weigh and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 924 mg), 4-dimethylaminopyridine (DMAP, 95 mg) and 5-bromo-1-pentanol (708 mg) to the reaction system in batches, and stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the 36 - 2 standard sample (PE / EA = 10 / 1, phosphomolybdic acid). Observe a new spot with smaller polarity. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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). Monitor by TLC, evaporate the fractions of the pure product, and obtain colorless oily liquid compound 36 - 3 (1.20 g, 74.7% yield).

[0274] Step 3:

[0275] Dissolve compound 36 - 3 (750 mg) in acetonitrile (10 ml), stir at room temperature. Then weigh and add NaI (170 mg), K 2 CO 3 (350 mg) and compound 2 - 7 (559 mg) to the above reaction system in batches, and heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the 2 - 7 standard sample (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). Observe a new spot with smaller polarity than 2 - 7. Cool the reaction solution to room temperature and evaporate it under reduced pressure, add an appropriate amount of DCM and silica gel to mix the sample, and purify it (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). Monitor by TLC, evaporate the fractions of the pure product, and obtain light yellow oily liquid compound 35 (830 mg, 56.9% yield).

[0276] 11H 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).

[0277] Example 13

[0278] Synthesis of Compound 41

[0279]

[0280]

[0281] Step 1:

[0282] To a solution of Compound 41-1 (2.00 g) in DMF (15 mL) were successively added 1-decanethiol (2.30 g) and sodium hydroxide (1.20 g). 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 H 2 2O (50 mL), extracted once with ethyl acetate (20 mL). The aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid, and then extracted three times with ethyl acetate (20 mL). The combined organic phases were dried over anhydrous sodium sulfate. Filtered by suction and concentrated to obtain Compound 41-2 (1.80 g, 56.6% yield).

[0283] Step 2:

[0284] Compound 41-2 (1000 mg) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.05 g), 4-dimethylaminopyridine (DMAP, 560 mg) and 8-bromooctanoic acid (1.02 g) were successively weighed and added to the reaction system in batches, and stirred at room temperature for 2 h. A small amount of the reaction solution was diluted and spotted on a TLC plate with a standard sample of 41-2 (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, mixed with an appropriate amount of silica gel and DCM, and purified (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) to obtain Compound 41-3 as a colorless oily liquid (1.5 g, 77% yield).

[0285] Step 3:

[0286] Dissolve compound 41-3 (700 mg) in acetonitrile (10 ml) and stir at room temperature. Then successively weigh NaI (170 mg), K 2 CO 3 (350 mg) and compound 2-7 (559 mg) and add them to the above reaction system in batches. Heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it, and spot it on a TLC plate for comparison with the standard sample of 2-7 (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). 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. Add an appropriate amount of DCM and silica gel to mix the sample, and purify it (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) to obtain compound 41 as a pale yellow oily liquid (600 mg, 44.8% yield).

[0287] 1 1H 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).

[0288] Example 14

[0289] Synthesis of Compound 44

[0290]

[0291] Step 1:

[0292] To a solution of compound 44-1 (2.00 g) in DMF (15 mL), successively add 1-octanethiol (2.63 g) and sodium hydroxide (1.44 g). The reaction mixture was stirred at 70 °C for 3 h. TLC showed that the starting compound 44-1 had completely disappeared. Pour the reaction solution into H 2 2O (50 mL), extract once with EA (20 mL), adjust the pH of the aqueous phase to 3 with 2 M dilute hydrochloric acid, extract three times with EA (20 mL), combine the organic phases, and dry over anhydrous sodium sulfate. Filter by suction and concentrate to obtain compound 44-2 (1.63 g, 53% yield).

[0293] Step 2:

[0294] To a solution of compound 44-2 (1.63 g) in DCM (15 mL) were successively added 4-dimethylaminopyridine (DMAP, 172 mg) and 5-bromopentanol (1.41 g). 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 had completely disappeared. The reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain compound 44-3 (1.65 g, 62% yield).

[0295] Step 3:

[0296] To a solution of compound 44-3 (1.5 g) in acetonitrile (15 mL) were added ethanolamine (960 mg) and potassium carbonate (1.15 g), and the solution was stirred at 70 °C for 12 hours. TLC showed a small amount of the starting compound 44-3 remaining. The reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, eluent: dichloromethane solution containing 0-10% CH 3 OH (volume percentage), with 1% ammonia water added to methanol), and the pure product fraction was evaporated to obtain compound 44-4 (800 mg, 56% yield).

[0297] Step 4:

[0298] To a solution of compound 44-5 (2.00 g) in DCM (15 mL) were successively added 4-dimethylaminopyridine (DMAP, 200 mg) and 7-bromo-1-heptanol (1.51 g). 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 had completely disappeared. The reaction mixture was concentrated to obtain a crude product, which was purified by column chromatography (silica gel column, eluent: petroleum ether solution containing 0-1% EA (volume percentage)), and the pure product fraction was evaporated to obtain compound 44-6 (2.40 g, 74% yield).

[0299] Step 5:

[0300] To a solution of compound 44-4 (800 mg) in DMF (7 mL) were successively added 44-6 (1.12 g), NaI (332 mg) and K 2 CO 3 (918 mg), and the reaction mixture was reacted at 50 °C for 12 hours. TLC showed a small amount of the starting compound 44-4 remaining. The reaction solution was poured into H2 O (50 mL), add EA (20 mL) and extract 3 times. Combine the organic phases. Wash the organic phases with saturated sodium chloride (20 mL) 2 times. Dry the organic phases with anhydrous sodium sulfate. Filter by suction and concentrate to obtain the crude product. Purify the crude product by column chromatography (silica gel column, eluent: dichloromethane solution containing 0 - 10% CH 3 OH (volume percentage), add 1% ammonia water to methanol), and evaporate the pure product fraction to obtain compound 44 (950 mg, 58% yield).

[0301] 1 1H NMR (400 MHz, Chloroform - d) δ 4.78 (s, 1H), 4.09 (d, J = 3.2 Hz, 2H), 3.72 - 3.55 (m, 2H), 3.05 - 2.86 (m, 2H), 2.84 (s, 1H), 2.59 - 2.41 (m, 8H), 2.31 - 2.17 (m, 2H), 1.72 - 1.64 (m, 4H), 1.62 - 1.60 (s, 2H), 1.59 - 1.51 (m, 8H), 1.55 (s, 2H), 1.39 - 1.35 (m, 6H), 1.33 (d, J = 3.0 Hz, 6H), 1.31 (d, J = 3.0 Hz, 12H), 1.31 - 1.28 (m, 4H), 1.30 - 1.26 (m, 12H), 1.24 (s, 3H), 0.94 - 0.82 (m, 9H).

[0302] Example 15

[0303] Synthesis of Compound 46

[0304]

[0305] Step 1:

[0306] Dissolve 1 - heptanethiol (2.1 g) and NaOH (1.0 g) in DMF (20 ml) and stir at room temperature. Then weigh compound 46 - 1 (2.00 g) and add it to the above reaction system in batches. Stir overnight at 60 °C. TLC (PE / EA = 3 / 1, phosphomolybdic acid) shows the formation of a new spot. Add 200 ml of water to the reaction mixture, and adjust the pH to around 3 by dropping concentrated HCl. Extract the above mixture with 600 ml of ethyl acetate. After drying the organic phase with Na 2 SO 4 dry and evaporate under reduced pressure. Add an appropriate amount of DCM and silica gel to mix the sample, and purify (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). Monitor by TLC plate, and evaporate the pure product fraction to obtain a colorless oily liquid 46 - 2 (1.5 g, 62% yield).

[0307] Step 2:

[0308] 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 showed the formation of a spot with increased polarity. The reaction mixture was concentrated to remove THF, diluted with water, extracted once with ethyl acetate (30 mL), the aqueous phase was adjusted to pH = 2 with dilute hydrochloric acid, and then extracted twice with ethyl acetate (30 mL). The organic layers were combined and concentrated to obtain compound 46-3 (1.2 g, 88% yield).

[0309] Step 3:

[0310] Compound 46-3 (1.2 g) was dissolved in DCM (10 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.01 g), 4-dimethylaminopyridine (DMAP, 534 mg) and 6-bromohexanol (792 mg) were successively weighed and added to the reaction system in batches, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate for comparison with the standard sample of 46-3 (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with decreased polarity was observed. The reaction solution was evaporated under reduced pressure, mixed with an appropriate amount of silica gel and DCM, 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). Monitored by TLC, the pure product fraction was evaporated to obtain compound 46-4 as a colorless oily liquid (1.6 g, 84% yield).

[0311] Step 4:

[0312] Potassium carbonate (1.52 g) was added to a solution of compound 46-4 (1.6 g) and ethanolamine (447 mg) in acetonitrile (50 mL). The mixture was refluxed at 85 °C for 2 h. TLC showed the complete disappearance of compound 46-4 and the formation of a spot with increased polarity. The reaction solution was filtered, and the resulting filtrate was concentrated to obtain a crude product. The crude product was mixed with an appropriate amount of silica gel and DCM, 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 compound 46-5 as a colorless oily liquid (870 mg, 57% yield).

[0313] Step 5:

[0314] Compound 46-6 (2.0 g) was dissolved in DCM (10 ml), and the mixture was stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.79 g), 4-dimethylaminopyridine (DMAP, 950 mg) and 6-bromohexanol (1.41 mg) were successively weighed and added to the reaction system in batches, and the mixture was stirred at room temperature for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate together with the standard sample of 46-6 (PE / EA = 10 / 1, phosphomolybdic acid), and a new spot with a smaller polarity was observed. The reaction solution was evaporated under reduced pressure, and an appropriate amount of silica gel and DCM were added for sample mixing 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). The TLC plate was monitored, and the pure product fraction was evaporated to obtain colorless oily liquid compound 46-7 (2.8 g, 86% yield).

[0315] Step 6:

[0316] Compound 46-5 (500 mg) was dissolved in acetonitrile (10 ml), and the mixture was stirred at room temperature. Then, NaI (180 mg), K 2 CO 3 (497 mg) and compound 46-7 (502 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and a new spot with a smaller polarity than 46-7 was observed. 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 for sample mixing and purification (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). The mixture was concentrated to obtain pale yellow oily liquid compound 46 (700 mg, 77% yield).

[0317] 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).

[0318] Example 16

[0319] Synthesis of Compound 48

[0320]

[0321]

[0322] Step 1:

[0323] Potassium carbonate (7.19 g) was added to a solution of compound 2-6 (8.0 g) and propanolamine (1.9 g) in acetonitrile (50 mL). The mixture was refluxed at 85 °C for 2 hours. TLC showed that compound 2-6 completely disappeared and a new spot with increased polarity was formed. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. An appropriate amount of silica gel and DCM were added for sample mixing 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 colorless oily liquid compound 48-1 (5.0 g, 63.3% yield).

[0324] Step 2:

[0325] Compound 35-3 (750 mg) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then, NaI (170 mg), K 2 CO 3 (350 mg) and compound 48-1 (620 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 3 h. A small amount of the reaction solution was diluted and spotted on a TLC plate for comparison with the 48-1 standard sample (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and a new spot with lower 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 for sample mixing and purification (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). The TLC plate was monitored, and the pure product fractions were evaporated to obtain pale yellow oily liquid compound 48 (650 mg, 69% yield).

[0326] 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).

[0327] Example 17

[0328] Synthesis of Compound 55

[0329]

[0330]

[0331] Step 1:

[0332] To a solution of compound 2 - 6 (4.0 g) and N,N - dimethylethylenediamine (1.53 g) in acetonitrile (50 mL) was added potassium carbonate (3.59 g). The mixture was stirred and refluxed at 85 °C for 3 hours. TLC showed that compound 2 - 6 completely disappeared and a new spot with increased polarity was formed. The reaction mixture was filtered, and the filtrate was concentrated to obtain a crude product. An appropriate amount of silica gel and DCM were added to the crude product for sample mixing and purification (25 g normal phase column, 0.1% NH 3 H 2 O, MeOH / DCM, 0 - 0% for 5 min, 0 - 10% for 20 min, 10 - 10% for 5 min, flow rate 20 ml / min) to obtain colorless oily liquid compound 55 - 1 (1.3 g, 32% yield).

[0333] Step 2:

[0334] To a solution of bromoethanol (2.00 g) in DMF (15 mL) were successively added compound 55 - 2 (1.93 g) and sodium hydroxide (1.20 g). The reaction mixture was stirred at 40 °C for 4 hours. TLC showed that the starting compound 55 - 2 completely disappeared. The reaction mixture was poured into H 2 O (50 mL), extracted once with ethyl acetate (20 mL), the aqueous phase was adjusted to pH 3 with 2 M dilute hydrochloric acid, and then extracted three times with ethyl acetate (20 mL). The combined organic phases were dried over anhydrous sodium sulfate. Filtration was carried out by suction, and the filtrate was concentrated to obtain compound 55 - 3 (1.56 g, 62.3% yield).

[0335] Step 3:

[0336] Dissolve compound 55-3 (872 mg) in DCM (10 mL), stir at room temperature. Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.05 g), 4-dimethylaminopyridine (DMAP, 560 mg) and 8-bromooctanoic acid (1.02 g) in turn and add them to the reaction system in batches, stir at room temperature for 2 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate for comparison with the standard sample of 55-3 (PE / EA = 10 / 1, phosphomolybdic acid), and observe a new spot with smaller polarity. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample, and purify it (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) to obtain colorless oily liquid compound 55-4 (1.27 g, 70% yield).

[0337] Step 4:

[0338] Dissolve compound 55-1 (500 mg) in acetonitrile (10 ml), stir at room temperature. Then weigh NaI (146 mg), K 2 CO 3 (406 mg) and compound 55-4 (393 mg) in turn and add them to the above reaction system, heat and reflux with stirring at 85 °C for 3 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and observe the formation of a new spot. After the reaction solution is cooled to room temperature, evaporate it under reduced pressure, add an appropriate amount of DCM and silica gel to mix the sample, purify it (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 concentrate to obtain pale yellow oily liquid compound 55 (108 mg, 14% yield).

[0339] 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).

[0340] Example 18

[0341] Synthesis of Compound 57

[0342]

[0343]

[0344] Step 1:

[0345] Dissolve Compound 55-1 (500 mg) in acetonitrile (10 ml) and stir at room temperature. Then successively weigh NaI (146 mg), K 2 CO 3 (406 mg) and Compound 29-3 (422 mg) were added to the above reaction system in batches, and the mixture was heated under reflux and stirred at 85 °C for 2 h. Take a small amount of the reaction solution, dilute it and spot it on a TLC plate (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid), and observe the formation of a new spot. 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 mixed, 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 57 (103 mg, 12% yield).

[0346] 1 1H 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).

[0347] Example 19

[0348] Synthesis of Compound 51

[0349]

[0350]

[0351] Step 1:

[0352] Dissolve compound 1 - hexanethiol (2.10 g) and NaOH (2.40 g) in ethanol (20 ml), and stir at room temperature. Then weigh compound 51 - 1 (2.00 g) and add it to the above reaction system in batches, and stir at room temperature overnight. TLC (PE / EA = 3 / 1, phosphomolybdic acid) monitoring shows the formation of a new spot. Add 200 ml of water to the reaction mixture, and adjust the pH to around 3 by dropping concentrated hydrochloric acid. Extract the above mixture with 600 ml of ethyl acetate, and the organic phase is dried over anhydrous Na 2 SO 4 After drying, evaporate under reduced pressure. Add an appropriate amount of DCM and silica gel to mix the sample, and purify (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). Monitor by TLC spotting, and evaporate the fractions of the pure product to obtain white solid 51 - 2 (2.10 g, 85.8% yield).

[0353] Step 2:

[0354] Dissolve compound 51 - 2 (1000 mg) in DCM (10 ml), and stir at room temperature. Weigh 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 986 mg), 4 - dimethylaminopyridine (DMAP, 105 mg) and 7 - bromo - 1 - heptanol (1050 mg) in turn and add them to the reaction system in batches, and stir at room temperature for 3 h. Take a small amount of the reaction solution, dilute it and spot - plate it with the 51 - 2 standard sample for comparison (PE / EA = 10 / 1, phosphomolybdic acid), and observe a new spot with a smaller polarity. Evaporate the reaction solution under reduced pressure, add an appropriate amount of silica gel and DCM to mix the sample and purify (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). Monitor by TLC spotting, and evaporate the fractions of the pure product to obtain colorless oily liquid compound 51 - 3 (1.40 g, 75.0% yield).

[0355] Step 3:

[0356] Dissolve compound 51 - 3 (750 mg) in acetonitrile (10 ml), and stir at room temperature. Then weigh NaI (170 mg), K 2 CO 3(350 mg) and Compound 2-7 (559 mg) were added batchwise to the above reaction system, 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 on a TLC plate for comparison with the 51-3 standard sample (DCM / MeOH = 10 / 1, 1 drop of ammonia water, phosphomolybdic acid). A new spot with lower 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 mixed, and then 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). TLC plate monitoring was carried out, and the fractions of the pure product were evaporated to obtain a pale yellow oily liquid compound 51 (740 mg, 50.7% yield).

[0357] 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).

[0358] Example 20

[0359] The luciferase mRNA was diluted in a 10 - 100 mM citric acid buffer at pH 4.0; each lipid component (the cationic lipid shown in the present invention: DSPC: cholesterol: PEG lipid (DMG-PEG2000)) was dissolved in ethanol according to a molar ratio of 50:10:38.5:1.5.

[0360] 3 mL of the mRNA buffer and 1 mL of the lipid solution were respectively loaded into two 5 mL syringes, which were installed on a microfluidic injection pump. The chip was connected to the syringes, the flow rate of the injection pump was set, and the start button of the injection pump was clicked to inject into the chip at a flow rate ratio of 3:1. Observe the color of the product at the outlet of the chip. After discarding the first 5 drops of milky white droplets (about 100 μL), the subsequent samples were collected into an EP tube. The collected product was placed in a dialysis bag and dialyzed against 10 mM PBS (pH 7.4) for 6 hours (cut-off molecular weight: 100 kDa), and then ultrafiltered and concentrated to the desired concentration. Then the lipid nanoparticles were filtered through a 0.22 μm sterile filter and stored at 4 °C.

[0361] According to the instructions of the Ribogreen kit, the encapsulation efficiency of the product was tested and calculated; the particle size, polydispersity index (PDI), and Zeta potential analysis were performed using standard detection methods on the Zetasizer nano instrument of Malvern Corporation.

[0362] The test results of the particle size, PDI, and encapsulation efficiency of the mRNA-loaded LNP prepared in this example are shown in Table 1. The results show that the nanoparticles formed by the lipids and mRNA under this formulation have a high encapsulation efficiency and a uniform particle size of about 100 nm, meeting the basic characteristics of nucleic acid delivery vectors.

[0363] Table 1. Characterization results of nanoparticles prepared with different cationic lipids

[0364]

[0365]

[0366] *DLin-MC3-DMA is the cationic lipid of the commercial nucleic acid delivery system Onpattro.

[0367] **Lipid M in Table 1 is from "Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines" Mol Ther Nucleic Acids 2019 Vol.15 Pages 1 - 11 and is a lipid similar to SM-102.

[0368] Example 21 Determination of the in vivo expression effect of luciferase mRNA delivered by tail vein injection of a nano-lipid particle composition

[0369] BALB / c mice at 6 - 8 weeks of age were injected with LUC-mRNA-lipid nanoparticles containing 5 μg mRNA through the tail vein (the nucleotide sequence corresponding to LUC-mRNA can be found in SEQ ID NO:1 of Patent Publication CN114380724A), and the preparation method was the same as that in Example 19. At specific time points, 100 μg of D-Luciferin Potassium Salt was injected into the tail vein of the mice, and a PerkinElmer small animal imaging system was used for detection. Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability, and it 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 replacement of pseudouridine. The detection results are as Figure 1, wherein the level of delivery of mRNA to the liver by the nano-lipid particle composition designed by the present invention is mostly equivalent to or better than that of DLin-MC3-DMA; some compounds are better than Lipid M.

[0370] Example 22 Determination of the in vivo expression effect of luciferase mRNA delivered by pulmonary atomization of nano-lipid particle composition

[0371] 6-8 week-old BALB / c mice were delivered with LUC-mRNA-lipid nanoparticles containing 5 μg mRNA by pulmonary atomization. The preparation method was the same as that in Example 19. At specific time points, 100 μg of D-Luciferin Potassium Salt was injected into the tail vein of the mice, and a PerkinElmer small animal imaging system was used for detection. The detection results are as Figure 2 , wherein the fluorescence level expressed by the nano-lipid particle composition composed of Compounds 7 and 35 is better than that of Lipid M and SM-102.

[0372] Example 23 Delivery of SARS-CoV-2 mRNA vaccine using nano-lipid particle composition

[0373] 6-week-old BALB / c mice were immunized by intramuscular injection with mRNA COVID-19 vaccines (Omicron antigen mRNA, the corresponding nucleotide sequence is shown in SEQ ID NO:6 of Patent Publication CN114380724A) delivered by different nano-lipid particle compositions on days 0 and 14. Blood was collected on day 28 (14 days after the second immunization), and the neutralizing antibody titer was measured by enzyme-linked immunosorbent assay to evaluate the protective effect of the mRNA COVID-19 vaccines delivered by different nano-lipid particle compositions against SARS-CoV-2 virus strain infection. The results are as Figure 3 shown. From the results, it can be seen that the binding antibody titer induced by the nano-lipid particle composition composed of Compound 35 for delivering COVID-19 mRNA is 1.4 million, while that of Lipid M is about 0.93 million.

[0374] Example 24 Delivery of SARS-CoV-2 mRNA vaccine using nano-lipid particle composition by pulmonary route

[0375] We prepared more lipid nanoparticles according to Example 20 to verify their immune effects in delivering the mRNA vaccine against the novel coronavirus via the lungs. Using 8-week-old BALB / c mice, on day 0, the mice were immunized by nebulizing 2 μg of the mRNA COVID-19 vaccine (Omicron antigen mRNA, the corresponding nucleotide sequence is shown in SEQ ID NO:6 of Patent Publication CN114380724A) delivered by different nano-lipid particle compositions into the lungs. Blood was collected on day 14, and the binding antibody titer was measured by enzyme-linked immunosorbent assay to evaluate the protective effect of the mRNA COVID-19 vaccine delivered by different nano-lipid particle compositions against the infection of the SARS-CoV-2 virus strain (the results are as Figure 4 shown). The results showed that the increase in the binding antibody titer caused by the compound of this patent was better than that of Lipid 5.

[0376] Characterization of nano-lipid particle compositions with different lipid components and ratios in Example 25

[0377] To study the drug-likeness under different helper lipids and lipid ratios, 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 microfluidics (the same method as in Example 19) to prepare nanoparticles. The results are shown in Table 2.

[0378] Table 2. Characterization results of nanoparticles prepared with different lipid ratios (molar ratio)

[0379]

[0380] From the results in Table 2, it can be seen that the nanoparticles formed by the lipid mixtures and mRNA within this range have uniform particle sizes, high encapsulation rates, and good drug-likeness.

[0381] Study on the in vivo expression effect of different cations delivering luciferase mRNA in Example 26

[0382] We verified the effects of more cationic lipids delivering luciferase mRNA according to the methods of Example 20 and Example 21. The following Table 3 shows the characterization results.

[0383] Table 3

[0384] Number Cationic lipid Entrapment efficiency (%) Particle size (nm) PDI Relative fluorescence brightness (%) 1 Compound 7 96.8 82.9 0.15 754±32 2 Compound 16 95.6 90.9 0.20 574±102 3 Compound 17 97.6 68.2 0.11 686±45 4 Compound 23 97.0 75.4 0.10 764±39 5 Compound 35 96.2 80.4 0.14 962±78 6 Compound 44 97.1 71.1 0.10 843±52 7 Compound 45 96.8 80.9 0.15 559±33 8 Compound 51 98.0 91.0 0.11 734±51 9 DLin-MC3-DMA 0.2 101.5 0.16 100

[0385] Note: Due to different experimental batches, the encapsulation rate and particle size DPI data in this Table 3 are different from those in Table 1.

[0386] As can be seen from the above table, the above compounds have a high encapsulation efficiency, uniform particle size, and the efficiency of delivering Luciferase mRNA in vivo is much higher than that of the commercial lipid DLin-MC3-DMA.

[0387] Study on the protein expression efficiency of different cationic lipids in Example 27

[0388] In this example, the effects of different cationic lipids on in vivo transfection of erythropoietin (EPO) were compared. The preparation method of the nanoparticles was the same as that in Example 20. The nucleotide sequence corresponding to EPO mRNA can be found in CN114380724B, SEQ ID NO: 2. After preparation, female Balb / c mice aged 6-8 weeks were injected with 20 μg of EPO-mRNA-lipid nanoparticles via the tail vein. After 6 hours, blood was collected from the orbital cavity of the mice. After centrifuging to separate the serum, the expression level of EPO protein was measured by ELISA. The basic characteristics of the mRNA used were the ARCA cap structure, the polyA tail length was 100-120 nt, and pseudouridine was completely replaced. The preparation and protein detection results are shown in Table 4.

[0389] Table 4

[0390]

[0391]

[0392] Note: Due to different experimental batches, the encapsulation efficiency and particle size DPI data in Table 4 are different from those in Table 1 and Table 3.

[0393] As can be seen from the above table, the above compounds have a high encapsulation efficiency, uniform particle size, and the protein translation efficiency of delivering EPO mRNA in vivo is much higher than that of the commercial lipid DLin-MC3-DMA.

[0394] Study on the abnormal toxicity of different cationic lipids in Example 28

[0395] In this experiment, the nanoparticles prepared 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. The control group of mice was injected with the corresponding volume of normal saline. The mice in the DLin-MC3-DMA group died within 18 h after injection, and the body weight, food intake, and activity status of the remaining mice showed no abnormalities during the observation period. This shows that the DLin-MC3-DMA lipid has stronger toxic and side effects. Blood was collected 120 h after injection, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (SCR) were detected using an automatic biochemical analyzer as indicators for evaluating liver and kidney function. Figure 5The results showed that, except for a slight decrease in the BUN values of lipids No. 35 (Compound 35) and No. 51 (Compound 51) (but the values were all within the healthy normal range), there were no significant fluctuations in the other indicators of the other lipid nanoparticles, indicating that this type of cationic lipid did not affect liver and kidney functions, had low toxicity and side effects, and was suitable for use in protein replacement pipelines.

[0396] Comparison of the intratumoral injection effects of different nano-lipid particle compositions in Example 29

[0397] We prepared different types of lipid nanoparticles encapsulating luciferase mRNA according to the preparation method of Example 20. Using 6-8 week-old female C57BL6 mice, we performed a right abdominal subcutaneous injection with 5x10 5 B16F10 cells (from the Cell Bank of the Chinese Academy of Sciences, CSTR: 19375.09.3101MOUTCM36) / per mouse, regularly observed and recorded tumor growth. On the 8th day after tumor inoculation, the tumor length (L, mm) and width (D, mm) were measured with a vernier caliper, and its tumor volume (V) was calculated. The calculation formula was V = (L x D 2 ) / 2. Mice with a tumor volume of 80-120 mm 3 were randomly grouped and intratumorally injected with different types of lipid nanoparticles encapsulating luciferase mRNA. The injection volume was 25 μL / 2.5 μg. After 24 h, anatomical imaging was performed to detect the fluorescence brightness of the mouse liver and tumor (as shown in Figure 6 and Figure 7 ). The results showed that the brightness at the tumor site of Compounds 35 and 51 was higher and the brightness of the liver was lower. This indicates that their in-situ tumor expression effect is better and the liver metastasis is lower, making them suitable for the encapsulation and delivery of some nucleic acid drugs related to tumor treatment.

Claims

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

2. The cationic lipid compound according to claim 1, wherein, the cationic lipid compound has one of the structures shown in the following table:

3. A liposomal formulation comprising the cationic lipid compound according to any one of claims 1-2 and a prophylactic or therapeutic nucleic acid, wherein, the formulation is for preventing or treating a certain disease.

4. The liposomal formulation according to claim 3, wherein, the molar ratio of the nucleic acid to the cationic lipid compound is from 20:1 to 1:

1.

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

1.

6. The liposomal formulation according to claim 3, wherein, the diameter of the liposomal formulation is from 50 nm to 300 nm.

7. The liposomal formulation according to claim 6, wherein, the diameter of the liposomal formulation is from 50 nm to 150 nm, or from 150 nm to 200 nm.

8. The liposomal formulation according to claim 3, wherein, it further comprises one or more other lipid components, including neutral lipids, steroids and polymer-conjugated lipids.

9. The liposomal formulation according to claim 8, wherein, the steroid comprised is cholesterol.

10. The liposomal formulation according to claim 9, wherein, the molar ratio of the cholesterol to the cationic lipid compound is (0-1.5):

1.

11. The liposomal formulation according to claim 8, wherein, the polymer in the polymer-conjugated lipid is polyethylene glycol (PEG).

12. The liposomal formulation according to claim 11, wherein, the molar ratio of the cationic lipid compound to the PEG-conjugated lipid is from 100:1 to 20:

1.

13. The liposomal formulation according to claim 11, wherein, the PEG-conjugated lipid is PEG-DAG, PEG-PE, PEG-SDAG, PEG-cer, PEG-DMG or ALC-0159.

14. The liposomal formulation according to claim 8, wherein, the neutral lipid is selected from one or more combinations of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

15. The liposomal formulation according to claim 14, wherein, the molar ratio of the neutral lipid to the cationic lipid compound is (0-0.5):

1.

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

17. Use of the cationic lipid compound according to any one of claims 1-2 or the liposomal formulation according to any one of claims 3-16 in the preparation of a medicament for inducing protein expression in a subject.

18. The use according to claim 17, wherein, the subject is a mammal.

19. The use according to claim 17, wherein, the subject is a non-human primate or a human.

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