Lipid composition

Abstract

The present invention relates to mucosal drug delivery systems, in particular to lipid compositions comprising therapeutic and / or prophylactic agents, such as RNA, which can be used for mucosal administration of therapeutic and / or prophylactic agents to treat or prevent diseases, such as infectious diseases.

Description

A lipid composition

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202211381081.5 filed on November 6, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a drug delivery system, and in particular to a lipid composition for mucosal administration and related products and their use in treating or preventing infectious diseases. Background Art

[0004] Nanoparticle compositions, liposomes, and liposome complexes containing lipids are used as transport vehicles to effectively deliver bioactive substances such as small molecule drugs, proteins, and nucleic acids to cells and / or intracellular compartments. These lipid compositions generally include cationic lipids, structural lipids, helper lipids, and / or surfactants.

[0005] Existing lipid-based drug delivery systems, such as liposomes and lipid nanoparticle (LNP) drug delivery systems, have been widely used in recent years. However, in actual use, these drug delivery systems have been found to have problems when delivering bioactive substances such as nucleic acids to the mucosa, such as low expression of mRNA in the mucosa and the resulting weak immunogenicity.

[0006] At present, researchers have found that the application of disclosed high molecular polymers, positively charged liposomes, viral vectors or some commercial transfection reagents for mucosal administration such as nasal administration will cause a large amount of protein expression in the lungs. Researchers have also compared the three-component LNP drug delivery system comprising different lipid combinations such as cationic lipids, auxiliary phospholipids and PEG, and found that the immunogenicity and expression of the same nucleic acid delivered by different routes of administration (intramuscular route, intradermal route, nasal administration) in vivo are different, and the effect of nasal administration immunity is significantly weaker than intramuscular and subcutaneous administration, and after nasal administration, the nucleic acid delivered can be distributed in a large number in mouse throat and stomach (G.Anderluzzi, et al.The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency, Journal of Controlled Release 342 (2022) 388-399.).

[0007] There is a need in the art for a drug delivery system that, after mucosal administration, can be efficiently expressed only in the mucosa to stimulate mucosal immunity and better protect the body.

[0008] Summary of the Invention

[0009] In one aspect, the present invention provides a lipid composition for mucosal administration, comprising a therapeutic agent or prophylactic agent and a lipid encapsulating the therapeutic agent or prophylactic agent, wherein the lipid encapsulating the therapeutic agent or prophylactic agent comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer associates with the therapeutic agent or prophylactic agent to form a complex, and the cationic polymer is co-encapsulated in the lipid to form a lipid multimer complex. In one embodiment, the lipid composition comprises 2.5-20 mol% of a polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

[0010] In one embodiment, the therapeutic or prophylactic agent is a nucleic acid, such as RNA, particularly mRNA.

[0011] In one embodiment, the cationic lipid comprises a lipid compound of formula (I), (II), (III), (IV) or a pharmaceutically acceptable salt thereof, as defined herein. Preferably, the cationic lipid is M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2.

[0012] In one embodiment, the cationic lipid does not include T5'.

[0013] In one embodiment, the lipid composition comprises 30-60 mol% of a cationic lipid, 5-40 mol% of a phospholipid, 10-70 mol% of a steroid, and 2.5-20 mol% of a polyethylene glycol-modified lipid.

[0014] In one embodiment, the lipid composition comprises 30-60 mol% of a cationic lipid, 5-40 mol% of a phospholipid, 10-60 mol% of a steroid, and 2.5-20 mol% of a polyethylene glycol-modified lipid.

[0015] In one embodiment, the lipid composition comprises 35-50 mol% of a cationic lipid, 10-35 mol% of a phospholipid, 15-50 mol% of a steroid, and 2.5-20 mol% of a polyethylene glycol-modified lipid.

[0016] In one embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of a phospholipid, 15-30 mol% of a steroid, and 2.5-20 mol% of a polyethylene glycol-modified lipid.

[0017] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 2.5-20 mol% of DMG-PEG.

[0018] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 3.75-10 mol% of DMG-PEG.

[0019] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 3.75-5 mol% of DMG-PEG.

[0020] In a particularly preferred embodiment, the lipid composition comprises 42.5 mol% of a cationic lipid, 35 mol% of DOPE, 18.75 mol% of cholesterol, and 3.75 mol% of DMG-PEG.

[0021] In one aspect, the present invention also provides a pharmaceutical composition comprising the lipid composition of the present invention, and optionally a pharmaceutically acceptable excipient.

[0022] In another aspect, the lipid composition or pharmaceutical composition of the present invention is used for administration to the nasal cavity, oral cavity, conjunctiva, rectum, or vaginal mucosa.

[0023] In a preferred embodiment, the lipid composition or pharmaceutical composition is for nasal administration.

[0024] In a preferred embodiment, the nasal administration includes nasal instillation, nasal spray administration or nasal inhalation.

[0025] In one embodiment, the nasal spray administration is performed via an aerosol delivery device.

[0026] In another aspect, the present invention also provides a method for treating or preventing a disease or condition, comprising administering a therapeutic or prophylactic agent to a subject in a multiple-dose regimen, wherein at least one dose is administered by a mucosal route.

[0027] In another aspect, the present invention further provides a nasal drop or nasal spray comprising the lipid composition of the present invention and a pharmaceutically acceptable excipient.

[0028] In another aspect, the present invention also provides use of the lipid composition of the present invention, the pharmaceutical composition of the present invention, or the nasal drops or nasal spray of the present invention in preparing a medicament for treating or preventing a disease or condition in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figures 1A and 1B show the in vivo expression of LPP preparations prepared with different formulations in the third round of screening. Figure 1A is an in vivo imaging image of mice; Figure 1B is a statistical graph of luciferase expression, represented by the area under the curve (AUC).

[0030] Figures 2A-2D show the immunogenicity test results of LPP preparations containing COVID-19 mRNA using different immunization schedules. Figure 2A shows the immunization schedule; Figure 2B shows the ELISpot test results of splenocytes in each group of mice immunized using different immunization schedules; Figure 2C shows the ELISpot test results of lung cells in each group of mice; and Figure 2D shows the results of ELISA testing of IgA antibody levels in nasal lavage fluid from each group of mice.

[0031] Figures 3A-3E show the immunogenicity testing results of LPP preparations containing mRNA encoding influenza virus antigens using different immunization schedules. Figure 3A shows the immunization schedule; Figure 3B shows the ELISpot results of splenocytes from each group of mice immunized using different immunization schedules; Figure 3C shows the ELISpot results of lung cells from each group of mice; Figure 3D shows the results of ELISA testing of IgA antibody levels in nasal lavage fluid from each group of mice; and Figure 3E shows the results of ELISA testing of IgA antibody levels in lung lavage fluid from each group of mice.

[0032] Figures 4A and 4B show the cell transfection efficiency of LPP preparations for mucosal administration prepared with different cations. Figure 4A shows the results of measuring cell transfection efficiency in A549 cells; Figure 4B shows the results of measuring cell transfection efficiency in DC2.4 cells.

[0033] Figures 5A and 5B show the in vivo expression of LPP formulations for mucosal administration prepared with different cations. Figure 5A is an in vivo imaging image of mice; Figure 5B is a statistical graph of luciferase expression, represented by total flux.

[0034] Figures 6A-6C show the cell transfection efficiency of LPP formulations before and after nebulization. Figure 6A is a schematic diagram of the nebulization drug delivery device; Figure 6B shows the luciferase expression of a B11 / Luc LPP formulation containing luciferase mRNA in DC2.4 cells before and after nebulization; Figure 6C shows the eGFP expression of a B11 / eGFP LPP formulation containing eGFP mRNA in A549 cells before, during, and after nebulization. Specific implementation plan

[0035] General Definitions and Terminology

[0036] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0037] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are those widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0038] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0039] As used herein, the singular forms "a," "an," or "the" include plural referents unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0040] Recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless expressly indicated to the contrary, values ​​or ranges recited herein are modified by "about" to mean ±20%, ±10%, ±5%, or ±3% of the recited or claimed value or range.

[0041] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0042] In lipid compositions of the present invention, unless otherwise indicated, the amount of various lipids is calculated with molar percentage (mol %). Said percentage can be calculated based on the total amount of all lipids in the lipid compositions. It will be appreciated by those skilled in the art that the content of each lipid can be suitably selected so that the total amount is 100%.

[0043] As used herein, "nucleotides" include deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotides" are constituents of ribonucleic acid (RNA), consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotides" are constituents of deoxyribonucleic acid (DNA), also consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and are the main chemical components of chromosomes. "Nucleotides" are usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylate containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridine containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0044] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes.

[0045] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linking groups. "Modified" groups can also be referred to herein as "altered" groups. Groups can be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase can include one or more non-naturally occurring substitutions.

[0046] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0047] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, or express a polynucleotide or vector.

[0048] As used herein, an "aliphatic" group is a non-aromatic group in which the carbon atoms are linked in a chain, and can be saturated or unsaturated.

[0049] As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms. 12 Alkyl" or "C 1-12 "Alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising 1 to 12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein, which is attached to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group having lost one hydrogen atom. The term "C1-C 12 "Alkylene" or "C 1-12 "Alkylene" refers to an optionally substituted straight or branched chain alkylene group comprising 1 to 12 carbon atoms.

[0050] As used herein, the term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. 12 Alkenyl" or "C 2-12 "Alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds.

[0051] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0052] As used herein, the term "carbocycle" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings consisting of carbon atoms. 3-8"Carbocycle" means a carbon ring comprising 3-8 carbon atoms. The carbocycle may include one or more carbon-carbon double or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like. As used herein, when a carbocycle is saturated (i.e., does not contain unsaturated bonds), it may also refer to the corresponding cycloalkyl group. Unless otherwise specifically stated, the carbocycles described herein refer to unsubstituted and substituted, i.e., optionally substituted, carbocycles.

[0053] As used herein, the term "heterocycle" refers to a monocyclic or polycyclic ring system comprising one or more rings and comprising at least one heteroatom. The heteroatom can be, for example, a nitrogen, oxygen, phosphorus or sulfur atom. The heterocycle can include one or more double bonds or triple bonds and can be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl and piperidinyl. The heterocycle can comprise, for example, 3-10 atoms (non-hydrogen), i.e., 3-10 membered heterocycles (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 members), wherein one or more atoms are heteroatoms (e.g., N, O, S or P). When the heterocycle is saturated (i.e., without unsaturated bonds), it can also refer to the corresponding heterocycloalkyl. Unless otherwise specifically stated, the heterocycles described herein refer to both unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

[0054] As used herein, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. 10 The alkylaryl group can have 6 to 10 carbon atoms, for example 6, 7, 8, 9, 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

[0055] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, with the remaining ring atoms being C, and having at least one aromatic ring. A heteroaryl group can have 5-10 ring atoms (5-10 membered heteroaryl), including 5-, 6-, 7-, 8-, 9-, or 10-membered, particularly 5- or 6-membered heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

[0056] As used herein, the term "interrupted by one or more groups" means that the one or more groups are present on the carbon chain, and the rest of the carbon chain is connected to both ends of the one or more groups.

[0057] Unless otherwise specifically stated, the groups described herein (e.g., any one of R1-R7, such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. Optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, or iodo), carboxylic acids (e.g., -C(O)OH), alcohols (e.g., hydroxy, -OH), esters (e.g., -C(O)OR or -OC(O)R), aldehydes (e.g., -C(O)H), carbonyls (e.g., -C(O)R, or represented by C=O), acyl halides (e.g., -C(O)X, wherein X is a halide selected from bromo, fluoro, chloro, and iodo), carbonates (e.g., -OC(O)OR), alkoxy groups (e.g., -OR), acetals (e.g., -C(OR)2R"", wherein each OR is the same or different alkoxy group and R"" is an alkyl or alkenyl group), phosphates (e.g., P(O)4 3- ), thiols (e.g., -SH), sulfoxides (e.g., -S(O)R), sulfinic acids (e.g., -S(O)OH), sulfonic acids (e.g., -S(O)2OH), thialdehydes (e.g., -C(S)H), sulfates (e.g., S(O)4 2- ), sulfonyl (e.g., -S(O)2-), amide (e.g., -C(O)NR2 or -N(R)C(O)R), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR2, NRH, or -NH2), carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, -N(H)S(O)2H), C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 aryl, 5-10 membered heteroaryl or 3-10 membered heterocycle. In any of the foregoing, each R can independently be a substituent as defined herein, such as alkyl, alkoxy, alkylene, halogen, carbocycle, heterocycle, aryl, heteroaryl, alkenyl. In some embodiments, the substituent itself can be further substituted with, for example, one, two, three, four, five or six substituents as defined herein. For example, an alkyl group can be further substituted with one, two, three, four, five or six substituents as described herein.

[0058] As used herein, the term "compound" is intended to include isotopic compounds of the depicted structure. "Isotopes" refer to atoms having the same atomic number but different mass numbers due to the number of neutrons in their nuclei, such as deuterium isotopes. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present invention can be prepared by combining with solvents or water molecules to form solvates and hydrates by conventional methods.

[0059] The term "optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted and unsubstituted alkyl radicals.

[0060] It should be understood that when chemical groups are written in a particular order, the reverse order is also encompassed unless otherwise stated. For example, in the formula -(R) where M1 is defined as -C(O)NH- i -(M1) k -(R) m -(i.e., -(R) i -C(O)-NH-(R) m -), unless otherwise stated, also encompasses compounds where M1 is -NHC(O)- (i.e., -(R) i -NHC(O)-(R) m -).

[0061] As used herein, the term "delivery" refers to providing an entity to a target. For example, delivering a therapeutic or prophylactic agent to a subject can involve administering a composition comprising the therapeutic or prophylactic agent to the subject.

[0062] As used herein, the term "subject" describes an organism to which the compositions of the present invention may be administered. Subjects to which these compositions may be administered include, but are not limited to, humans, other primates, and other mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, or rats. Preferably, the subject may be a mammal, particularly a human.

[0063] As used herein, "encapsulation efficiency" refers to the ratio of the amount of therapeutic or prophylactic agent that becomes part of the composition to the initial total amount of therapeutic or prophylactic agent used to prepare the composition. For example, if 97 mg of the therapeutic or prophylactic agent is encapsulated in the composition out of a total of 100 mg of therapeutic or prophylactic agent initially provided to the composition, then the encapsulation efficiency can be determined to be 97%. As used herein, "encapsulation" can refer to complete, substantial, or partial encapsulation, sealing, surrounding, or packaging.

[0064] As used herein, a "lipid component" is a component of a composition that includes one or more lipids. For example, the lipid component can include one or more cationic lipids, PEGylated lipids, structural lipids, or helper lipids.

[0065] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, salts, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are consistent with a reasonable benefit / risk ratio.

[0066] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety into its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like; and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally speaking, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent, or in a mixture of the two; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0067] As used herein, "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system. Smaller values, such as less than 0.3, indicate a narrower particle size distribution.

[0068] As used herein, "zeta potential" refers to the electrokinetic potential of lipids in a lipid composition, and is an important indicator for characterizing the stability of a dispersion.

[0069] As used herein, "size" or "average size" in the context of a composition refers to the average diameter of the composition.

[0070] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, or reducing the occurrence of one or more symptoms or features of a particular infection, disease, disorder, or condition. "Preventing" refers to guarding against an underlying disease or preventing the worsening of symptoms or the development of a disease.

[0071] The term "therapeutically or prophylactically effective amount" refers to an amount of an agent (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) sufficient to prevent or inhibit the occurrence of a disease or symptom and / or slow down, alleviate, or delay the development or severity of a disease or symptom. The therapeutically or prophylactically effective amount is affected by factors including, but not limited to, the rate of development and severity of the disease or symptom, the age, sex, weight, and physiological condition of the subject, the duration of treatment, and the specific route of administration. The therapeutically or prophylactically effective amount can be administered in one or more doses. The therapeutically or prophylactically effective amount can be achieved by continuous or intermittent administration.

[0072] Lipid composition

[0073] The present invention provides a lipid composition for mucosal administration. The lipid composition is a lipid delivery vehicle, and the lipid can encapsulate nucleotides to form nanoparticles, thereby delivering them into the body.

[0074] As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic portion and optionally a hydrophilic portion. Lipids are generally poorly soluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters.

[0075] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle with a uniform lipid core, which is a particle formed by lipids, the lipid components of which undergo intermolecular interactions to form a nanostructured entity. Nucleic acids (eg, mRNA) are encapsulated in the lipids.

[0076] Particularly preferred lipid compositions can be, for example, lipid polyplexes (LPPs) as described herein. Methods for preparing such compositions can be as described herein. LPPs are particles with a core-shell structure, wherein nucleic acids are contained in polyplexes, and the polyplexes themselves are encapsulated in a biocompatible lipid bilayer shell to form the lipid nanoparticles of the present invention. In some embodiments, the lipid composition of the present invention is a lipid polyplex (LPP). In some embodiments, the lipid composition of the present invention is a lipid polyplex (LPP) comprising RNA.

[0077] In some embodiments, the lipid encapsulating the polynucleotide is selected from one or more of the following lipids: cationic lipids, phospholipids, steroids and / or polyethylene glycol-modified lipids. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.

[0078] The lipid composition of the present invention can be used for administration through mucosa, and it comprises a therapeutic agent or a preventive agent and a lipid that encapsulates the therapeutic agent or the preventive agent. The lipid that encapsulates the therapeutic agent or the preventive agent comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid.

[0079] In one embodiment, the lipid composition of the present invention comprises a cationic lipid, wherein the cationic lipid comprises DOTMA, DOTAP, DDAB, DOSPA, DODAC, DODAP, DC-Chol, DMRIE, DMOBA, DLinDMA, DLenDMA, CLinDMA, DMORIE, DLDMA, DMDMA, DOGS, N4-cholesteryl-spermine, DLin-KC2-DMA, DLin-MC3-DMA, a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof. In a preferred embodiment, the cationic lipid comprises M5, MC3, ALC-0315, SM-102. In a preferred embodiment, the cationic lipid comprises SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1, or SW-II-140-2.

[0080] In one embodiment, the lipid composition of the present invention comprises phospholipids and / or steroids. In one embodiment, the lipid composition of the present invention comprises a phospholipid as described herein, wherein the phospholipid comprises 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oil Acylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) or a combination thereof. In one embodiment, the lipid composition of the present invention comprises a steroid as described herein, wherein the steroid comprises cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof.In one embodiment, the lipid composition of the present invention comprises a phospholipid as described herein and a steroid. In one embodiment, the lipid composition comprises DOPE. In one embodiment, the lipid composition of the present invention comprises DSPC. In one embodiment, the lipid composition of the present invention comprises cholesterol. In one embodiment, the lipid composition of the present invention comprises DOPE and cholesterol. In one embodiment, the lipid composition of the present invention comprises DSPC and cholesterol.

[0081] In one embodiment, the lipid composition of the present invention comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2, phospholipids DOPE and cholesterol. In one embodiment, the lipid composition of the present invention comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2, phospholipids DSPC and cholesterol.

[0082] In some embodiments, the lipid encapsulating the polynucleotide further comprises a polyethylene glycol-modified lipid. In one embodiment, the polyethylene glycol-modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000), DOG-PEG, DSPE-PEG, ALC-0159, or a combination thereof. In one embodiment, the polyethylene glycol-modified lipid is ALC-0159. In one embodiment, the polyethylene glycol-modified lipid is DSPE-PEG. In one embodiment, the polyethylene glycol-modified lipid is DMG-PEG (e.g., DMG-PEG 2000).

[0083] In one embodiment, the lipid composition of the invention comprises a cationic lipid, DOPE, cholesterol, and DSPE-PEG.

[0084] In one embodiment, the lipid composition of the present invention comprises a cationic lipid, DSPC, cholesterol, and DSPE-PEG.

[0085] In one embodiment, the lipid composition of the present invention comprises a cationic lipid, DSPC, cholesterol, and DMG-PEG.

[0086] In a preferred embodiment, the lipid composition of the present invention comprises a cationic lipid, DOPE, cholesterol and DMG-PEG.

[0087] In a preferred embodiment, the lipid composition of the present invention comprises cationic lipids M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.

[0088] In some embodiments, the lipid composition of the present invention further comprises a cationic polymer, which is associated with the polynucleotide as a complex and is co-encapsulated in the lipid.

[0089] In one embodiment, the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine. In one embodiment, the cationic polymer is polyethyleneimine.

[0090] In one embodiment, the amount of lipid in the lipid composition of the present invention is calculated as molar percentage (mol %), which is determined based on the total mole of all lipids in the lipid composition. Unless otherwise specified, the sum of the amount (mol %) of each lipid in the composition is 100 mol %, i.e., the sum of the amount (mol %) of cationic lipids, phospholipids, steroids and polyethylene glycol-modified lipids is 100 mol %.

[0091] In one embodiment, the amount of cationic lipid in the lipid composition of the present invention is about 30-about 60 mol%, based on the total amount of all lipids in the lipid composition. In some embodiments, the amount of cationic lipid in the lipid composition of the present invention is about 35-about 60 mol%, about 30-about 50 mol%, about 35-about 50 mol%, about 35-about 45 mol%, about 35-about 42.5 mol%, about 37.5-about 45 mol%, about 37.5%-about 42.5 mol%, about 40-about 45 mol% or about 40-about 50 mol%. For example, the amount of cationic lipid can be about 30,32.5,35,37.5,40,42.5,45,47.5,50,52.5,55,57.5 or 60 mol%.

[0092] In one embodiment, the amount of phospholipid in lipid composition of the present invention is the about 40 mol %, based on the total amount of all lipids in lipid composition.In one embodiment, the amount of phospholipid in lipid composition of the present invention is the about 35 mol %, about 5 mol %, about 5 mol %, about 25 mol %, about 5 mol %, about 20 mol %, about 10 mol %, about 10 mol %, about 30 mol %, about 15 mol %, about 20 mol %, about 35 mol %, about 25 mol %, about 10 mol % or about 25 mol %.For example, the amount of phospholipid can be about 5,10,15,20,25,30,35 or 40 mol %.

[0093] In one embodiment, the amount of cholesterol in the lipid composition of the present invention is about 10-about 70 mol %, based on the total amount of all lipids in the lipid composition. In one embodiment, the amount of cholesterol in the lipid composition of the present invention is about 10-about 65 mol %, about 10-about 60 mol %, about 15-about 50 mol %, about 15-about 30 mol %, about 20-about 60 mol %, about 30-about 50 mol %, about 35-about 40 mol %, about 35-about 45 mol %, about 40-about 45 mol % or about 45-about 50 mol %. For example, the amount of cholesterol can be about 10, 15, 17.5, 18.75, 20, 22.5, 25, 27.5, 28.75, 30, 32.5, 33.75, 35, 40, 42.5, 45, 46.25, 47.5, 48.75, 50, 52.5, 53.75, 55, 60, 62.5, 63.75, 65, or 70 mole percent.

[0094] In one embodiment, the amount of the polyethylene glycol-modified lipid in the lipid composition of the present invention is about 2.5-about 20 mol%, based on the total amount of all lipids in the lipid composition. In one embodiment, the amount of the polyethylene glycol-modified lipid in the lipid composition of the present invention is about 2.5-about 10 mol%, about 3-about 10 mol%, about 3.5-about 10 mol%, about 3.75-about 10 mol%, about 3.75-about 7.5 mol%, about 3.75-about 5 mol%, about 4-about 10 mol%, about 5-about 10 mol%, about 7.5-about 10 mol%, about 5-about 7.5 mol%, about 10-about 20 mol%, about 10-about 15 mol% or about 15-about 20 mol%. For example, the amount of polyethylene glycol-modified lipid can be about 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 15, or 20 mole percent.

[0095] In one embodiment, the lipid composition of the present invention comprises 30-60 mol% of cationic lipids, 5-40 mol% of phospholipids, 10-70 mol% of steroids and 2.5-20 mol% of polyethylene glycol modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 30-60 mol% of cationic lipids, 5-40 mol% of phospholipids, 10-60 mol% of steroids and 2.5-20 mol% of polyethylene glycol modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 35-50 mol% of cationic lipids, 10-35 mol% of phospholipids, 15-50 mol% of steroids and 2.5-20 mol% of polyethylene glycol modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 2.5-20 mol% of polyethylene glycol modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 2.5-10 mol% of polyethylene glycol-modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 3.75-10 mol% of polyethylene glycol-modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 3.75-7.5 mol% of polyethylene glycol-modified lipids. In a preferred embodiment, the lipid composition of the present invention comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 3.75-5 mol% of polyethylene glycol-modified lipids.

[0096] For the purpose of application of the lipid composition of the present invention, the above-mentioned content and range thereof are advantageous.

[0097] In one embodiment, the LPP comprises a therapeutic or prophylactic agent and a lipid encapsulating the therapeutic or prophylactic agent, wherein the lipid encapsulating the therapeutic or prophylactic agent comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid; the LPP further comprises a cationic polymer, wherein the cationic polymer is associated with the therapeutic or prophylactic agent to form a complex. In one embodiment, the LPP comprises 2.5-20 mol% of the polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

[0098] In one embodiment, the lipid composition of the present invention comprises a therapeutic agent or prophylactic agent and a lipid encapsulating the therapeutic agent or prophylactic agent, wherein the lipid encapsulating the therapeutic agent or prophylactic agent comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer associates with the therapeutic agent or prophylactic agent to form a complex and is co-encapsulated in the lipid to form a lipid multimer complex. In one embodiment, the lipid composition comprises 2.5-20 mol% of polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

[0099] In one embodiment, the phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the cationic polymer is protamine. In one embodiment, the polyethylene glycol-modified lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosane (ALC-0159), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), or a combination thereof. In one embodiment, the cationic lipid is selected from M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2.

[0100] In a preferred embodiment, the lipid composition comprises 30-60 mol% of a cationic lipid, 5-40 mol% of DOPE, 10-70 mol% of cholesterol, and 2.5-20 mol% of DMG-PEG.

[0101] In a preferred embodiment, the lipid composition comprises 30-60 mol% of a cationic lipid, 5-40 mol% of DOPE, 10-60 mol% of cholesterol, and 2.5-20 mol% of DMG-PEG.

[0102] In a preferred embodiment, the lipid composition comprises 35-50 mol% of a cationic lipid, 10-35 mol% of DOPE, 15-50 mol% of cholesterol, and 2.5-20 mol% of DMG-PEG.

[0103] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 2.5-20 mol% of DMG-PEG.

[0104] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 2.5-10 mol% of DMG-PEG.

[0105] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 3.75-10 mol% of DMG-PEG.

[0106] In a preferred embodiment, the lipid composition comprises 37.5-42.5 mol% of a cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol, and 3.75-5 mol% of DMG-PEG.

[0107] In a preferred embodiment, the lipid composition comprises 42.5 mol% of cationic lipid, 35 mol% of DOPE, 18.75 mol% of cholesterol and 3.75 mol% of DMG-PEG.

[0108] In a particularly preferred embodiment, the lipid encapsulation complex comprises 42.5 mol% of M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2, 35 mol% of DOPE, 18.75 mol% of cholesterol and 3.75 mol% of DMG-PEG.

[0109] Cationic lipids

[0110] Cationic lipids are lipids that can have a net positive charge at a given pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.

[0111] Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), didecyldimethylammonium bromide (DDAB), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), dioctadecyldimethylammonium chloride ... dioctadecyldimethylammonium chloride (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethylammonium bromide (DDAB), dioctadecyldimethyl chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (D MRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis, cis-9,1-(cis,cis-9,12-octadecadienoxy)propane (3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane, CLinDMA), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide bromide, DMORIE), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), dioctadecylamidoglycyl spermine spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate, DLin-MC3-DMA, a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof.

[0112] In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. Ionizable cationic lipids carry a net positive charge at, for example, acidic pH, but are neutral at higher pH (eg, physiological pH). Examples of ionizable cationic lipids include, but are not limited to, dioctadecylamidoglycyl spermine (DOGS), N4-cholesteryl-spermine, 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), a compound of formula (I), (II), (III) or (IV) as described herein, or a combination thereof.

[0113] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0114] in,

[0115] R1 and R2 are each independently selected from a bond, a C1-C 12 Alkyl and C2-C 12 alkenyl;

[0116] R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 aryl and 5-10 membered heteroaryl; and R3 and R4 are each independently optionally substituted by t R6, t being an integer selected from 1-5;

[0117] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0118] M1 and M2 are each independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR7-, or

[0119] R5, one of M1 and M2, together with the nitrogen atom to which they are connected, form a 3-10 membered heterocyclic ring, and the corresponding R1 / R3 or

[0120] or R2 / R4 are absent, the heterocyclic ring is optionally substituted by R7;

[0121] R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 Aryl, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution;

[0122] m and n are each independently an integer selected from 0-12;

[0123] The alkyl, alkenyl and alkylene groups are each optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle, and said alkyl, alkenyl and alkylene are each optionally substituted with one or more R7;

[0124] R7 are each independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0125] In one embodiment, R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C12 Alkenyl, such as C1-C 12 In another embodiment, one of R1 and R2 is a bond, and the other is independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, such as C1-C 12 alkyl.

[0126] In one embodiment, R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 In another embodiment, R3 and R4 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl.

[0127] R3 and R4 can each be independently optionally substituted by t R6, where t is 1, 2, 3, 4, or 5. In one embodiment, R6 is each independently selected from C1-C 12 alkyl.

[0128] In yet another embodiment, at least one of R3 and R4 is C6-C 10 Aryl or 5-10 membered heteroaryl, such as C6-C 10 Aryl.

[0129] In one embodiment, R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q. Q can be selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3- 8-carbon ring, 3-10-membered heterocyclic ring, C6-C 10 The above groups, including those covering the options of Q, may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0130] In yet another embodiment, R5 is selected from C 3-8 Carbocyclic ring, -C 1-12Alkylene-Q, Q is selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3- 8-carbon ring, 3-10-membered heterocyclic ring, C6-C 10 The above groups, including those encompassing the options of Q, may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0131] In the compound of formula (I), R7 can be independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring, preferably selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl. The above groups (when appropriate, such as H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, C 3-8 carbocycle) are each optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0132] In one embodiment, each group described above, such as C 3-8 Carbocyclic ring, -C 1-12Alkylene-Q, including -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 Aryl, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 The carbocyclic rings etc. may each be optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0133] In one embodiment, the alkyl, alkenyl and alkylene groups (e.g., those mentioned in R1-R7) in the compounds of formula (I) may each be optionally and independently interrupted by one or more groups selected from the group consisting of: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 The alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R7. That is, the chains (straight or branched) of the alkyl, alkenyl and alkylene groups may each optionally contain one or more groups selected from the following: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 Carbon ring.

[0134] R7 are each independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring; preferably, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl. The above groups (when appropriate, such as H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic ring, C 3-8 carbocycle) are each optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

[0135] In the compound of formula (I), m and n can each independently be an integer selected from 0 to 12, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. When 0 is taken, it means that the corresponding group does not exist.

[0136] In one embodiment, M1 or M2 is a bond, the corresponding m or n is not 0, and the carbon chain preceding M1 or M2 is connected to the corresponding R1 or R2.

[0137] In one embodiment, m or n is 0, the corresponding M1 or M2 is not a bond, and the N atom is directly connected to M1 or M2.

[0138] In one embodiment, M1 or M2 is a bond, the corresponding m or n is 0, and the N atom is directly connected to the corresponding R1 or R2.

[0139] In one embodiment, M1 and M2 are each independently selected from -C(O)-, -OC(O)- and -C(O)O-. In another embodiment, M1 and M2 are each independently selected from -NR7-, and R7 is as described above.

[0140] In another embodiment, R5 and one of M1 and M2 together with the nitrogen atom to which they are attached form a 3-10 membered heterocyclic ring, and the corresponding R1 / R3 or R2 / R4 are absent, said heterocyclic ring being optionally substituted with R7, R7 being as described above.

[0141] In one embodiment, R5 is selected from -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)2, cyano, and R7 is as described above.

[0142] In a preferred embodiment, R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0143] wherein R3 and R4 are each independently selected from C1-C 12 Alkyl and C2-C 12 and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl.

[0144] M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, and -C(S)S-;

[0145] R5 is selected from -C1- 12 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl;

[0146] m and n are each independently an integer selected from 1-12.

[0147] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0148] In a preferred embodiment, the cationic lipid comprises M5 or SM-102.

[0149] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0150] In a preferred embodiment, the cationic lipid comprises MC3.

[0151] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0152] In a preferred embodiment, the cationic lipid comprises ALC-0315.

[0153] In one embodiment, the cationic lipid comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0154] R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0155] R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 Aryl and 5-10 membered heteroaryl;

[0156] Provided that at least one of R3 and R4 is C6-C 10 aryl or 5-10 membered heteroaryl, and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl;

[0157] M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, and -C(S)S-;

[0158] R5 is selected from -C 1-12 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl;

[0159] m and n are each independently an integer selected from 1-12.

[0160] In one embodiment, R2 is selected from C1-C 12 In another embodiment, R2 is selected from C1-C6 alkyl.

[0161] In one embodiment, one of R3 and R4 is C6-C 10 Aryl or 5-10 membered heteroaryl, the other is C1-C 12 Alkyl or C2-C 12 Alkenyl.

[0162] In one embodiment, R3 and R4 are each independently selected from C1-C 12In another embodiment, one of R3 and R4 is phenyl and the other is C1-C 12 alkyl.

[0163] In another embodiment, R3 and R4 are each independently substituted with t R6, t being an integer selected from 1-5; for example 1, 2, 3, 4 or 5. Preferably, t is an integer from 1-3, for example 1, 2 or 3, in particular 1 or 2.

[0164] In one embodiment, R6 is independently selected from C1-C 12 Alkyl groups, such as C1-C 10 alkyl.

[0165] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 or R2 on the phenyl ring.

[0166] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1 or R2.

[0167] In one embodiment, R4 is substituted at the 1st or last position of R2. The 1st position refers to the position of the carbon atom in R2 that is directly connected to M2. The last position refers to the position of the carbon atom in R2 that is farthest from M2. In a specific embodiment, R4 is selected from C1-C 12 Alkyl, R3 is phenyl.

[0168] In one embodiment, R3 is substituted at the 1st position or the last position of R1. The 1st position refers to the position of the carbon atom in R1 that is directly connected to M1. The last position refers to the position of the carbon atom in R1 that is farthest from M1. In a specific embodiment, R3 is selected from C1-C 12 Alkyl, R4 is phenyl.

[0169] In one embodiment, M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, and -OC(O)O-.

[0170] In one embodiment, R5 is selected from -C 1-5 Alkylene-Q, such as C1, C2, C3, C4 or C5 alkylene-Q. In exemplary embodiments, R5 is selected from -C 1-3 Alkylene-Q, for example C1, C2 or C3 alkylene-Q.

[0171] In another embodiment, Q is selected from -OH and -SH, in particular -OH.

[0172] In some embodiments, m and n are each independently an integer selected from 2-9, such as 2, 3, 4, 5, 6, 7, 8 or 9. Preferably, m and n are each independently an integer selected from 2-7, such as 2, 3, 4, 5, 6 or 7, more preferably, m and n are each independently an integer selected from 5-7, such as 5, 6 or 7.

[0173] In certain embodiments, the compound of formula (I) includes a compound represented by formula (II):

[0174] or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein.

[0175] In one embodiment,

[0176] R1 is selected from C1-C6 alkyl;

[0177] R2 is selected from C1-C 10 alkyl;

[0178] R4 is selected from C1-C 10 alkyl;

[0179] M1 and M2 are each independently selected from -OC(O)-, -C(O)O- and -OC(O)O-;

[0180] R5 is selected from -C 1-5 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl and C2-C 12 alkenyl;

[0181] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0182] m and n are each independently an integer selected from 2-9, such as 2, 3, 4, 5, 6, 7, 8 or 9;

[0183] t is an integer selected from 1-3.

[0184] In one embodiment, R5 is selected from -C 1-3 Alkylene-Q, Q is selected from -OH and -SH, in particular -OH.

[0185] In one embodiment, m and n are each independently an integer selected from 2-7, such as 2, 3, 4, 5, 6 or 7.

[0186] In some embodiments, t is 1 or 2.

[0187] In one embodiment, R4 is substituted at position 1 or the last position of R2. Position 1 refers to the position of the carbon atom in R2 that is directly connected to M2. The last position refers to the position of the carbon atom in R2 that is farthest from M2.

[0188] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 on the phenyl ring.

[0189] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1.

[0190] In certain embodiments, the compound of formula (I) includes a compound represented by formula (III):

[0191] or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein.

[0192] In one embodiment,

[0193] R1 is selected from C1-C6 alkyl;

[0194] R2 is selected from C1-C 10 alkyl;

[0195] R4 is selected from C1-C 10 alkyl;

[0196] R5 is selected from -C 1-3 Alkylene-Q, Q is selected from -OH and -SH, in particular -OH;

[0197] t is 1 or 2;

[0198] R6 is selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0199] m and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.

[0200] In one embodiment, R4 is substituted at the 1st or last position of R2. The last position refers to the position of the C atom in R2 that is directly connected to the The position of the furthest C atom in the part.

[0201] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 on the phenyl ring.

[0202] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1.

[0203] In certain embodiments, the compound of formula (I) includes a compound represented by formula (IV):

[0204] or a pharmaceutically acceptable salt thereof, wherein each group is as defined herein.

[0205] In one embodiment,

[0206] R1 is selected from C1-C6 alkyl;

[0207] R2 is selected from C1-C 10 alkyl;

[0208] R4 is selected from C1-C 10 alkyl;

[0209] t is 1 or 2;

[0210] R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl, especially C1-C 12 alkyl;

[0211] m and n are each independently an integer selected from 2-7, for example 2, 3, 4, 5, 6 or 7.

[0212] In one embodiment, R4 is substituted at the 1st or last position of R2. The last position refers to the position of the C atom in R2 that is directly connected to the The position of the furthest C atom in the part.

[0213] In one embodiment, t is 1 and R6 is substituted at the meta or para position relative to R1 on the phenyl ring.

[0214] In another embodiment, t is 2 and R6 is substituted at the meta and para positions on the phenyl ring relative to R1.

[0215] In a specific embodiment, the substituents (eg, R1-R7) in the lipid compounds of the present invention do not include alkenyl groups.

[0216] In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

[0217] In a preferred embodiment, the cationic lipid comprises the following lipid compounds: SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1, or SW-II-140-2.

[0218] In a preferred embodiment, the cationic lipid does not comprise T5',

[0219] In a preferred embodiment, the cationic lipid comprises the following lipid compound: M5, MC3, ALC-0315, SM-102, SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2.

[0220] phospholipids

[0221] The lipid composition of the present invention contains phospholipids, which can assist the cell penetration of the lipid composition.

[0222] Examples of phospholipids include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl oil Acylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof.

[0223] steroids

[0224] The lipid compositions of the present invention include steroids, which may serve as structural components of the lipid compositions.

[0225] Examples of steroids include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and derivatives thereof.

[0226] PEG-modified lipids

[0227] As used herein, the term "polyethylene glycol-modified lipid" or "PEG-modified lipid" or "PEG lipid" refers to a molecule comprising a polyethylene glycol portion and a lipid portion, which is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG-modified dialkylglycerol, or a combination thereof. For example, examples of polyethylene glycol-modified lipids include, but are not limited to, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (also known as ALC-0159), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol (DOGPEG), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG).

[0228] In one embodiment, the polyethylene glycol-modified lipid is DMG-PEG, such as DMG-PEG 2000. In one embodiment, DMG-PEG 2000 has the following structure:

[0229] The average value of n is 44.

[0230] Cationic polymers

[0231] As used herein, the term "cationic polymer" refers to any ionic polymer that can carry a net positive charge at a specified pH, thereby electrostatically binding to nucleic acids. Examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, polyethyleneimine (PEI), or a combination thereof. The polyethyleneimine can be linear or branched.

[0232] The term "protamine" refers to a low molecular weight basic protein rich in arginine, which exists in sperm cells of various animals (especially fish) and replaces histones to bind to DNA. In a preferred embodiment, the cationic polymer is protamine (e.g., protamine sulfate).

[0233] Physical and chemical properties

[0234] The physicochemical properties of lipid compositions can depend on their components. For example, a composition comprising cholesterol as a structural lipid can have physicochemical properties different from compositions comprising different structural lipids. Similarly, the physicochemical properties of a composition can depend on the absolute or relative amounts of its components. For example, a composition comprising a higher mole fraction phospholipid can have physicochemical properties different from compositions comprising a lower mole fraction phospholipid. Physicochemical properties can also vary depending on the method and conditions for preparing the composition.

[0235] The physicochemical properties of lipid compositions can be characterized by a variety of methods. For example, the morphology and size distribution of the composition can be checked using microscopy (e.g., transmission electron microscopy or scanning electron microscopy). Dynamic light scattering or potentiometric analysis (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to measure particle size. Multiple features of the composition, such as particle size, polydispersity index, and zeta potential, can also be measured using instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0236] For example, as measured by dynamic light scattering (DLS), the average size of the composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be from about 40 nm to about 250 nm, such as about 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm. In some embodiments, the average size of the composition can be from about 50 nm to about 300 nm, from about 50 nm to about 290 nm, from about 50 nm to about 280 nm, from about 50 nm to about 270 nm, from about 50 nm to about 260 nm, from about 60 nm to about 300 nm, from about 60 nm to about 290 nm, from about 60 nm to about 280 nm, from about 60 nm to about 270 nm, from about 70 nm to about 300 nm, from about 70 nm to about 290 nm, from about 70 nm to about 280 nm, from about 70 nm to about 270 nm, from about 70 nm to about 260 nm, from about 80 nm to about 280 nm, from about 80 nm to about 270 nm, from about 80 nm to about 260 nm, from about 80 nm to about 250 nm, from about 90 nm to about 280 nm, from about 90 nm to about 270 nm, or from about 90 nm to about 260 nm. In certain embodiments, the average size of the lipid composition can be about 90nm to about 290nm or about 100nm to about 250nm. In a specific embodiment, the average size can be about 100nm. In other embodiments, the average size can be about 150nm. In other embodiments, the average size can be about 200nm.

[0237] In some embodiments, the lipid composition can be relatively homogeneous. Polydispersity index can be used to indicate the homogeneity of lipid composition, such as the particle size distribution of lipid composition. Smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of composition can be approximately 0 to approximately 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polydispersity index of lipid composition can be approximately 0.10 to approximately 0.20.

[0238] The zeta potential of a composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a composition. Compositions with relatively low charges, i.e., positively or negatively charged, are generally desirable because compositions with higher charges may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0239] The encapsulation efficiency of therapeutic or preventive agent describes the ratio of the amount of the therapeutic or preventive agent that is encapsulated in the composition or otherwise combined with the composition after preparation relative to the initial amount provided. Higher encapsulation efficiency is ideal (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic or preventive agent in the solution containing the composition before and after splitting the composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of the therapeutic or preventive agent (e.g., RNA) in the solution. For compositions as described herein, the encapsulation efficiency of the therapeutic or preventive agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0240] Pharmaceutical composition

[0241] The present invention also provides a pharmaceutical composition comprising the lipid composition of the present invention and a pharmaceutically acceptable carrier.

[0242] Pharmaceutically acceptable carriers may include, but are not limited to, propellants, diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA),

[0014] In some embodiments, the carrier may be a solubilizing agent, a gelling agent, a softening agent, a solvent (e.g., water, alcohol, acetic acid, and syrup), a buffer (e.g., phosphate buffer, histidine buffer, and acetate buffer), a surfactant (e.g., a nonionic surfactant, such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), an antibacterial agent, an antifungal agent, an isotonic agent (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), an absorption delaying agent, a chelating agent, and an emulsifier. For pharmaceutical compositions, suitable carriers may be selected from a buffer (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), an isotonic agent (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), a nonionic surfactant (e.g., polysorbate 80, polysorbate 20, poloxamer), or a combination thereof.

[0243] The pharmaceutical compositions provided herein can be in various dosage forms, including but not limited to solid, semisolid, liquid, powder or lyophilized forms. For pharmaceutical compositions, preferred dosage forms can generally be, for example, solutions and lyophilized powders. Pharmaceutical compositions can be prepared into various forms suitable for multiple routes of administration and methods. For example, pharmaceutical compositions can be prepared into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), dosage forms for surface and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

[0244] In another aspect, the lipid composition or pharmaceutical composition of the present invention is used for administration to the nasal cavity, oral cavity, conjunctiva, rectum, or vaginal mucosa.

[0245] In a preferred embodiment, the lipid composition or pharmaceutical composition is for nasal administration.

[0246] Intranasal drug delivery systems are a class of preparations that deliver drugs through the nasal cavity to exert local or systemic therapeutic or preventive effects. They are particularly suitable for drugs that require high local nasal expression or that avoid first-pass effects in the liver. The advantages of intranasal drug delivery are that the nasal mucosa is large and rich in blood vessels, resulting in rapid drug absorption and a rapid onset of action after administration. Furthermore, they offer minimal first-pass effects in the liver, low systemic toxicity, and high bioavailability.

[0247] Common dosage forms for nasal administration include nasal drops, nasal sprays, powders, gel preparations and emulsions. Among them, nasal spray drugs have a wider diffusion rate and dispersion area in the nasal mucosa. Currently, the commonly used atomization devices are metered dose constant pressure inhalers (MDI), dry powder inhalers (DPI) and nebulizers. After nasal spraying, the drug is deposited in the front of the nasal cavity, and only a small part is slowly cleared into the throat, which prolongs the retention time of the drug in the nasal cavity, which is beneficial to absorption and improves bioavailability.

[0248] The pharmaceutical composition of the present invention can be a nasal drop or a nasal spray, which comprises the lipid composition of the present invention and a pharmaceutically acceptable excipient. Therefore, the present invention also provides a nasal drop or a nasal spray, which comprises the lipid composition of the present invention and a pharmaceutically acceptable excipient.

[0249] The excipient may include a propellant, such as trichloromonofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane, etc. The excipient may also include one or more of water, a sugar solution, an electrolyte solution, and an amino acid solution. For example, the excipient may include one or more of water, Ringer's solution, glucose solution, glucose-sodium chloride solution, isotonic sodium chloride solution, fructose solution, dextran, amino acid solution, heparin solution, mannitol solution, and sodium bicarbonate solution.

[0250] In some embodiments, the nasal administration comprises nasal instillation, nasal spray administration, or nasal inhalation.

[0251] In a preferred embodiment, the nasal administration includes nasal drip or nasal spray administration.

[0252] In one embodiment, the nasal spray administration is performed via an aerosol delivery device.

[0253] In one embodiment, the aerosol drug delivery device comprises a syringe, a plastic needle, a nasal spray device, and a dose limiter.

[0254] In one embodiment, the atomization drug delivery device can convert liquid medicament into mist particles (jet atomization) for umbrella-shaped spraying.

[0255] As used in this article, "jet atomization" is based on the Venturi injection principle, using compressed air or high-flow medical oxygen to form a high-speed airflow through a small tube orifice. The negative pressure generated drives the liquid or other fluid to be sprayed onto the obstruction, and the liquid droplets are splashed around under high-speed impact, turning into mist-like particles and spraying out from the air outlet.

[0256] In one embodiment, the liquid medicine is atomized into fine mist particles with a size of 10-70 μm and sprayed on the surface of human tissues (or organs).

[0257] In one embodiment, the aerosol drug delivery device has a built-in self-destruction design, and the push rod self-destructs after use, ensuring single-use.

[0258] Therapeutic / preventive agents

[0259] The lipid composition may comprise one or more therapeutic or prophylactic agents. The present invention provides methods for delivering a therapeutic or prophylactic agent to a mammalian mucosa, producing a target polypeptide in the mammalian mucosa, and treating a disease or condition in a mammal in need thereof, comprising administering a lipid composition or pharmaceutical composition comprising a therapeutic or prophylactic agent to the mammalian mucosa.

[0260] In some embodiments, the disease or condition is selected from rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0261] In some embodiments, the disease is an infectious disease.

[0262] In some embodiments, the infectious diseases include novel coronavirus pneumonia, influenza, acute upper respiratory tract infection, pneumococcal disease, Hemophilus influenzae meningitis, epidemic cerebrospinal meningitis, diphtheria, pertussis, measles, human papillomavirus (HPV), rabies, tetanus, plague, hepatitis and tuberculosis.

[0263] In some embodiments, therapeutic agent or preventive agent is a vaccine or a compound causing an immune response. Vaccines include compounds and preparations that can provide immunity for one or more conditions related to infectious diseases such as novel coronavirus pneumonia, influenza, acute upper respiratory tract infection, pneumococcal disease, hemophilic influenza bacillus meningitis, epidemic cerebrospinal meningitis, diphtheria, pertussis, measles, human papillomavirus (HPV), rabies, tetanus, plague, hepatitis and tuberculosis and can include encoding infectious disease-derived antigens and / or epitopes. In some embodiments, vaccines and / or compounds causing an immune response can be administered by including a lipid composition mucosal administration of a compound according to formula (I), (II), (III) or (IV).

[0264] polynucleotides

[0265] In some embodiments, the therapeutic or prophylactic agent is a polynucleotide or nucleic acid (eg, ribonucleic acid or deoxyribonucleic acid).

[0266] In some embodiments, the therapeutic or preventive agents of the present invention are RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear and circular RNA. The RNA of the present invention can be chemically synthesized, recombinantly produced and in vitro transcribed RNA. In one embodiment, the RNA of the present invention is used to express a polypeptide in a host cell.

[0267] In one embodiment, the therapeutic or preventive agent of the present invention is a single-stranded RNA. In one embodiment, the RNA of the present invention is an in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription using a DNA template using RNA polymerase.

[0268] In some embodiments, the therapeutic or preventive agent of the present invention is a messenger RNA (mRNA). Generally speaking, an mRNA may comprise a 5'-UTR sequence, a coding sequence for a polypeptide, a 3'-UTR sequence, and an optional poly(A) sequence. The mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the present invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3'-UTR, and (4) an optional poly(A) sequence. In one embodiment, the mRNA of the present invention is a nucleoside-modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.

[0269] As used herein, the term "untranslated region (UTR)" generally refers to a region (non-coding region) in RNA (such as mRNA) that is not translated into an amino acid sequence, or a corresponding region in DNA. Generally, the UTR located at the 5' end (upstream) of the open reading frame (start codon) can be referred to as the 5' untranslated region 5'-UTR; the UTR located at the 3' end (downstream) of the open reading frame (stop codon) can be referred to as the 3'-UTR. In the presence of a 5' cap, the 5'-UTR is located downstream of the 5' cap, for example, directly adjacent to the 5' cap. In a specific embodiment, an optimized "Kozak sequence" can be included in the 5'-UTR, for example, near the start codon, to improve translation efficiency. In the presence of a poly (A) sequence, the 3'-UTR is located upstream of the poly (A) sequence, for example, directly adjacent to the poly (A) sequence.

[0270] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence containing continuous or discontinuous adenylate nucleotides. The poly(A) sequence is typically located at the 3' end of the RNA, for example, at the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylate nucleotides at its 3' end. The poly(A) sequence can be transcribed from the coding sequence of the DNA template by a DNA-dependent RNA polymerase during the preparation of the IVT-RNA, or can be attached to the free 3' end of the IVT-RNA, for example, at the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

[0271] As used herein, the term "5' cap" generally refers to an N7-methylguanosine structure (also known as an "m7G cap," "m7Gppp-") attached to the 5' end of an mRNA via a 5' to 5' triphosphate bond. The 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or it can be attached to the RNA post-transcriptionally using a capping enzyme.

[0272] In some embodiments, the therapeutic or preventive agent of the present invention is DNA. Such DNA can be, for example, a DNA template for in vitro transcription of the RNA of the present invention or a DNA vaccine for expressing a polypeptide antigen in a host cell. The DNA can be double-stranded, single-stranded, linear, or circular.

[0273] The DNA template can be provided in a suitable transcription vector. Generally speaking, the DNA template can be a double-stranded complex, which comprises a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand). As known to those skilled in the art, the DNA template can include a promoter, 5'-UTR, coding sequence, 3'-UTR and an optional poly (A) sequence. The promoter can be an available promoter for a suitable RNA polymerase (particularly DNA-dependent RNA polymerase) known to those skilled in the art, including but not limited to promoters of SP6, T3 and T7 RNA polymerases. The 5'-UTR, coding sequence, 3'-UTR and poly (A) sequence in the DNA template are the corresponding sequences included in the RNA described herein or are complementary thereto. As the polynucleotide of a DNA vaccine, it can be provided in a plasmid vector (e.g., a circular plasmid vector).

[0274] In a preferred embodiment, the therapeutic or prophylactic agent of the present invention is an mRNA encoding a SARS-CoV-2 spike protein variant, an exemplary coding sequence of which can be found in SEQ ID NO: 2. In another preferred embodiment, the therapeutic or prophylactic agent of the present invention is an mRNA encoding an influenza virus antigen such as NP protein, an exemplary coding sequence of which can be found in SEQ ID NO: 3.

[0275] Modified nucleotides

[0276] In some embodiments, the mRNA herein comprises modified nucleotides, wherein the modified nucleotides are selected from one or more of the following nucleotides: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5- The present invention also includes methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, N-1-methyl-pseudouridine, 2-thiouridine and 2-thiocytidine; methylated bases; inserted bases; 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose; thiophosphate and 5'-N-phosphoramidite bond. And the modified nucleotides described in PCT / CN2020 / 074825 and PCT / CN2020 / 106696 are modified.

[0277] Application of lipid compositions and pharmaceutical compositions

[0278] Lipid compositions, pharmaceutical compositions, nasal drops and nasal sprays of the present invention can be used for treating disease, disease or the patient's condition. Specifically, these lipid compositions, pharmaceutical compositions, nasal drops and nasal sprays can be used for treating disease, disease or the patient's condition characterized by loss or abnormal protein or polypeptide activity. For example, lipid compositions and pharmaceutical compositions comprising mRNA encoding loss or abnormal polypeptide can be applied or delivered to mucosa. This mRNA can be translated subsequently to produce the polypeptide, thus reducing or eliminating the problem caused by the absence or abnormal activity of the polypeptide.

[0279] There are various diseases, disorders or conditions that can be characterized by loss of protein activity (or generally reduction so that appropriate protein function cannot occur). These proteins may not exist, or they may be substantially non-functional. The invention provides a method for treating such diseases, disorders or the patient's condition of a subject by administering a lipid composition, pharmaceutical composition, nasal drops or nasal spray of the present invention, wherein the lipid composition comprises RNA and a lipid component, the lipid component comprising a cationic lipid, a phospholipid, a PEG lipid and a structural lipid, wherein the RNA can be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the abnormal protein activity present in the subject's cell.

[0280] The methods provided herein relate to administering lipid compositions containing one or more therapeutic or prophylactic agents, pharmaceutical compositions, nasal drops or nasal sprays comprising these compositions. For features and embodiments of the present invention, the terms therapeutic agent and prophylactic agent can be used interchangeably herein. Lipid compositions and pharmaceutical compositions can be administered to a subject using any reasonable amount and any route of administration that is effective for preventing, treating, diagnosing, or treating a disease, disorder, or condition, or for any other purpose. The specific amount administered to a given subject can vary depending on the species, age, and general condition of the subject; the purpose of administration; the specific composition; the mode of administration, and the like.

[0281] Diseases, disorders or conditions characterized by malfunction or aberrant protein or polypeptide activity for which the lipid compositions, pharmaceutical compositions, nasal drops and nasal sprays can be administered include, but are not limited to, rare diseases, infectious diseases (in the form of vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. The cancer includes, for example, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, nasopharyngeal carcinoma, laryngeal cancer, pharyngeal cancer, tracheal cancer, melanoma, thyroid cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, brain cancer, colon cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, leukemia, lymphoma, myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma or multiple myeloma. The autoimmune disease includes, for example, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis or type I diabetes. The neurodegenerative disease includes, for example, Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degeneration, stroke, Huntington's disease or amyotrophic lateral sclerosis. The metabolic diseases include, for example, type II diabetes, scurvy, hypoglycemia, hyperlipidemia or osteoporosis.

[0282] In some embodiments, the disease is an infectious disease.

[0283] In some embodiments, the infectious diseases include novel coronavirus pneumonia, influenza, acute upper respiratory tract infection, pneumococcal disease, Hemophilus influenzae meningitis, epidemic cerebrospinal meningitis, diphtheria, pertussis, measles, human papillomavirus (HPV), rabies, tetanus, plague, hepatitis and tuberculosis.

[0284] In one aspect, the present invention further provides use of the lipid composition, pharmaceutical composition, nasal drops, or nasal spray of the present invention in the preparation of a medicament for treating or preventing a disease or condition in a subject in need thereof. The disease or condition is as described above.

[0285] In one embodiment, the disease or condition is characterized by malfunction or aberrant protein or polypeptide activity. In one embodiment, the disease or condition is selected from rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. In a preferred embodiment, the disease is an infectious disease.

[0286] In another aspect, the present invention also provides a method for treating or preventing a disease or condition, comprising administering a therapeutic or preventive agent to a subject in a multiple-dose regimen, wherein at least one dose is a lipid composition of the present invention or a pharmaceutical composition of the present invention administered via a mucosal route. The disease or condition is as described above.

[0287] In one embodiment, at least one additional dose of the multiple dose regimen is administered by a route selected from the group consisting of intramuscular, intratumoral, transdermal, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, or a combination thereof.

[0288] In a preferred embodiment, the at least one additional dose is administered via the intramuscular route of administration.

[0289] As described above, the therapeutic or prophylactic agent as described herein can be administered by two or more modes of administration, in other words, the therapeutic or prophylactic agent is administered in a multi-dose regimen. At least one dose is administered via a mucosal route, for example, with a lipid composition or pharmaceutical composition of the present invention; and at least one other dose is administered via a route of administration different from the mucosal route. In a particularly preferred embodiment, the therapeutic or prophylactic agent administered by different routes of administration is the same. In a preferred embodiment, the therapeutic or prophylactic agent is an mRNA encoding a variant of the SARS-CoV-2 spike protein, an exemplary coding sequence of which can be found in SEQ ID NO: 2. In another preferred embodiment, the therapeutic or prophylactic agent is an mRNA encoding an influenza virus antigen such as an NP protein, an exemplary coding sequence of which can be found in SEQ ID NO: 3.

[0290] Different administration modes can be carried out sequentially or simultaneously. In appropriate cases, a multiple-dose regimen can achieve better therapeutic effects, such as eliciting a better immune response.

[0291] In one embodiment, at least one dose of a therapeutic or prophylactic agent as described herein is administered intramuscularly prior to administering at least one dose of a lipid composition or pharmaceutical composition of the invention via a mucosal route.

[0292] In one embodiment, a dose of a therapeutic or prophylactic agent as described herein is administered intramuscularly first, followed by administration of a dose of a lipid composition or pharmaceutical composition of the invention via a mucosal route.

[0293] In one embodiment, at least two doses of a therapeutic or prophylactic agent as described herein are administered intramuscularly, followed by at least one dose of a lipid composition or pharmaceutical composition of the invention being administered mucosally.

[0294] In one embodiment, two doses of a therapeutic or prophylactic agent as described herein are administered intramuscularly followed by one dose of a lipid composition or pharmaceutical composition of the invention via a mucosal route.

[0295] The present invention also relates to the use of the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation for treating or preventing a disease or condition, wherein the therapeutic agent or preventive agent is administered to a subject in a multiple dose regimen, wherein at least one dose is administered via a mucosal route. The multiple dose regimen is as described above. The disease or condition is as described above. Beneficial effects

[0296] The lipid composition, pharmaceutical composition, or nasal drops or nasal spray provided by the present invention can exhibit excellent effects, for example, at least one of the following beneficial effects: (1) improving the expression efficiency of the contained mRNA in the nasal cavity; (2) having good targeting and low systemic toxicity; (3) having no significant changes in physicochemical properties and expression efficiency before and after atomization; (4) being able to induce mucosal immune response; (5) being able to be administered in combination with other routes such as intramuscular administration in a "primary immunization + boost" manner (systemic immunization + mucosal immunization), which can induce a higher level of humoral immunity and cellular immunity, and at the same time induce a mucosal immune response.

[0297] Example

[0298] The present invention is further described with reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0299] Unless otherwise specified, all percentages in the following examples are molar percentages (mol %).

[0300] Experimental Materials

[0301] The cationic lipid according to formula (I) is synthesized by Si microorganisms or prepared by reference, such as CN110520409A, WO2018081480A1 or US11,246,933B1; phospholipids (DOPE) are purchased from CordenPharma; cholesterol is purchased from Sigma-Aldrich; mPEG2000-DMG (i.e., DMG-PEG 2000) is purchased from Avanti Polar Lipids, Inc.; PBS is purchased from Invitrogen; protamine sulfate is purchased from Beijing Silian Pharmaceutical Co., Ltd.; mPEG2000-DSPE is purchased from lipoid GmbH; DSPC is purchased from Avanti Polar Lipids, Inc.

[0302] Example 1 Synthesis of the compound according to formula (I)

[0303] General considerations

[0304] Unless otherwise noted, all solvents and reagents used were commercially available and used as received. 1 H NMR spectra were recorded in CDCl3 at 300 K using a Bruker Ultrashield 300 MHz instrument. Chemical shifts are relative to 1 H is reported as parts per million (ppm) relative to TMS (0.00). Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using ISCO RediSep Rf Gold flash columns (particle size: 20-40 microns).

[0305] The procedure described below can be used to synthesize compounds SW-II-115 to SW-II-140-2.

[0306] The following abbreviations are used herein: THF: tetrahydrofuran MeCN: acetonitrile LAH: lithium aluminum hydride DCM: dichloromethane DMAP: 4-dimethylaminopyridine LDA: lithium diisopropylamide rt: room temperature DME: 1,2-dimethoxyethane n-BuLi: n-butyllithium CPME: cyclopentyl methyl ether EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide DIEA: N,N-diisopropylethylamine PE: petroleum ether EA: ethyl acetate

[0307] A. Compound SW-II-115

[0308] 1. Synthesis of Intermediate 3

[0309] To a DCM solution (100 mL) containing compound 1 (10 g, 45 mmol, 1 eq.) and compound 2 (7.8 g, 54 mmol, 1.2 eq.) was added EDCI (17.3 g, 90 mmol, 2 eq.) and DMAP (2.2 g, 18 mmol, 0.4 eq.), followed by DIEA (23.2 g, 180 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N protection for 16 hours. TLC (petroleum ether: ethyl acetate = 30: 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether: ethyl acetate (1: 0-20: 1) to give compound 3 (4.365 g, 28%) as a colorless oil.

[0310] 2. Synthesis of Intermediate 5

[0311] A solution of compound 3 (500 mg, 1.437 mmol, 1 eq.) and compound 4 (2.63 g, 43.103 mmol, 30 eq.) in EtOH was stirred at 60 ° C for 16 hours under N protection. TLC (DCM: MeOH = 10: 1) showed that compound 3 was consumed, and TLC (DCM / MeOH = 10 / 1) showed that a new main point was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (1: 0-10: 1, v / v) to give compound 5 (264 mg, 56%) as a yellow oil.

[0312] 3. Synthesis of Intermediate 8

[0313] To compound 6 (500 mg, 1.712 mmol, 1 eq.) and compound 7 (1.113 g, 8.562 mmol, 5 eq.) in a dioxane / water (5 mL / 0.5 mL) solvent mixture were added Pd(dppf)Cl2 (112 mg, 0.171 mmol, 0.1 eq.) and potassium carbonate (709 mg, 5.136 mmol, 3 eq.). The mixture was stirred at 100°C overnight under N2. TLC (PE:EA = 15:1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to give compound 8 (455 mg, 88%) as a colorless oil.

[0314] 4. Synthesis of Intermediate 9

[0315] To a solution of compound 8 (455 mg, 1.497 mmol, 1 eq.) in THF (5 mL) was added LiAlH₄ (1.5 mL, 1.497 mmol, 1 M in THF, 1 eq.) at 0°C under N₂ protection. The mixture was stirred at room temperature under N₂ for 2 hours. TLC (PE:EtOAc=5:1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (1.5 mL) and treated with 2N HCl to adjust the pH between 6 and 7, extracted with EA, and washed with brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford crude compound 9 (419 mg, >100%) as a colorless oil without further purification.

[0316] 5. Synthesis of Intermediate 10

[0317] To a solution of compound 1 (339 mg, 1.518 mmol, 1 eq.) and compound 9 (419 mg, 1.518 mmol, 1 eq.) in DCM (4 mL) were added EDCI (583 mg, 3.036 mmol, 2 eq.) and DMAP (74 mg, 0.607 mmol, 0.4 eq.), followed by DIEA (783 mg, 6.072 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N protection for 16 hours. TLC (petroleum ether:ethyl acetate = 10:1) showed the formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to obtain compound 10 (443 mg, 60.7%) as a colorless oil.

[0318] 6. Synthesis of the final product SW-II-115

[0319] To a mixed solvent of CPME / CH3CN (3 mL / 3 mL) containing compound 10 (307 mg, 0.64 mmol, 1 eq.) and compound 5 (210 mg, 0.64 mmol, 1 eq.) were added K2CO3 (530 mg, 3.84 mmol, 6 eq.) and KI (212 mg, 1.28 mmol, 2 eq.). After the addition was complete, the mixture was stirred at 90°C overnight under N2. TLC (DCM:MeOH = 10:1) indicated the reaction was complete, with a new major spot observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM:MeOH (1:0-10:1, v / v) to afford compound SW-II-115 (266 mg, 57%) as a yellow oil.

[0320] LCMS:Rt:1.293min;MS m / z(ELSD):730.5[M+H] + ;

[0321] HPLC: 99.472% purity, ELSD; RT = 4.895 min.

[0322] 1 H NMR (400MHz, CDCl3) δ7.21–6.99(m,3H),5.05(s,2H),4.05(t,J=6.8Hz,2H),3.58(t,J=5.3Hz,2H), 2.69–2.46(m,10H),2.31(dt,J=20.0,7.5Hz,4H),1.69–1.18(m,51H),0.89(dt,J=12.4,6.3Hz,9H).

[0323] 13C NMR (101MHz, CDCl3) δ173.90 (s), 173.68 (s), 140.80 (d, J = 13.0Hz), 133.31 (s), 129.25 (d, J = 16.2Hz) ,128.30(s),125.75(s),77.30(d,J=11.5Hz),77.04(s),76.72(s),66.22(s),64.43(s),58.12(s),55 .72(s),53.90(s),34.32(d,J=1.9Hz),32.69(s),32.48(s),31.81(d,J=11.2Hz),31.25(s),29.59–28 .91(m),28.66(s),27.17(s),26.64(s),25.94(s),24.91(d,J=5.1Hz),22.65(d,J=3.3Hz),14.10(s).

[0324] B. Compound SW-II-118

[0325] 1. Synthesis of Intermediate 3

[0326] A solution of compound 1 (1.22 g, 5.0 mmol, 1.0 eq.) and compound 2 (765 mg, 7.5 mmol, 1.5 eq.), Pd(PPh3)4 (tetrakistriphenylphosphine palladium, 289 mg, 0.25 mmol, 0.05 eq.), and K2CO3 (1.38 g, 10.0 mmol, 2.0 eq.) in toluene (10 ml) and H2O (1 ml) was stirred at 110°C under N2 protection for 1 hour. TLC (petroleum ether:ethyl acetate = 19:1) showed that compound 1 was consumed and a new spot was observed. The reaction mixture was diluted with DCM (50 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.5 g, 45%) as a colorless oil.

[0327] 1 H NMR (400MHz, CDCl3) δ7.16 (dd, J=23.5, 8.1Hz, 4H), 4.14 (q, J=7.1Hz, 2H), 3.57 (s, 2H), 2.64–2.48 (m ,2H),1.66–1.51(m,2H),1.35(dd,J=15.0,7.4Hz,2H),1.25(t,J=7.1Hz,3H),0.92(t,J=7.3Hz,3H).

[0328] 2. Synthesis of Intermediate 4

[0329] LiAlH4 (193 mg, 5.09 mmol, 4.0 eq.) was added to a THF (10 mL) solution containing compound 3 (280 mg, 1.27 mmol, 1.0 eq.) at -78°C, and the reaction was allowed to react at 10°C for 3 hours. TLC showed that the reaction was well, and the reaction was concentrated and diluted with Na2SO4 (20 mL) and extracted with EA (30 mL×2). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound 4 (3.12 g, crude product) as a yellow oil.

[0330] 3. Synthesis of Intermediate 6

[0331] A solution of compound 4 (215 mg, 1.2 mmol, 1.0 eq.), compound 5 (404 mg, 1.8 mmol, 1.5 eq.), EDCI (1.15 g, 6.0 mmol, 5.0 eq.), DMAP (732 mg, 1.8 eq.), DIEA (1.29 g, 12.0 mmol, 10.0 eq.), and DIEA (1.29 g, 12.0 mmol, 10.0 eq.) in DCM (5 mL) was stirred at 10°C for 16 h under N₂ protection. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v / v) to afford compound 6 (145 mg, 31%) as a colorless oil.

[0332] 1 H NMR (400MHz, CDCl3) δ7.12(s,4H),4.27(t,J=7.1Hz,2H),3.52(t,J=6.7Hz,1H),3.40(t,J=6.8Hz,1H),2.90(t,J=7.1Hz,2H ),2.65–2.50(m,2H),2.28(t,J=7.5Hz,2H),1.93–1.70(m,2H),1.64–1.56(m,4H),1.44–1.27(m,8H),0.92(t,J=7.3Hz,3H).

[0333] 4. Synthesis of the final product SW-II-118

[0334] A mixture containing compound 6 (140 mg, 0.37 mmol, 1.0 eq.), compound 7 (243 mg, 0.55 mmol, 1.5 eq.), K2CO3 (153 mg, 1.11 mmol, 3.0 eq.) and KI (123 mg, 0.74 mmol, 2.0 eq.) was stirred in a mixed solvent of CPME (1 mL) and CH3CN (1 mL) under N2 at 90 ° C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO3 (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with DCM:MeOH (1: 0-10: 1, v / v) to give SW-II-118 (105 mg, 61%) as a yellow oil.

[0335] LCMS:Rt:1.946min; MS m / z(ELSD):744.4[M+H] + ;

[0336] HPLC: 99.64% purity, ELSD; RT = 5.875 min.

[0337] 1 H NMR (400MHz, CDCl3) δ7.11(s,4H),4.91–4.79(m,1H),4.26(t,J=7.2Hz,2H),3.80–3.68(m,2H),2.90(t,J=7.1Hz,4H),2.8 1–2.67(m,4H),2.62–2.52(m,2H),2.28(td,J=7.5,2.6Hz,4H),1.64–1.51(m,11H),1.38–1.17(m,42H),0.93–0.82(m,9H).

[0338] 13C NMR (101MHz, CDCl3) δ173.61 (d, J = 11.7Hz), 141.11 (s), 134.90 (s), 128.74 (s), 128.51 ( s),77.40(s),77.08(s),76.77(s),74.17(s),64.90(s),57.48(s),56.24(s),53.98(s), 35.25(s),34.66(d,J=14.4Hz),34.16(d,J=5.1Hz),33.67(s),31.86(s),29.52(d,J=2.4Hz),29.24(s),29.21–28. 74(m),26.90(d,J=4.9Hz),25.42–24.92(m),24.92–24.88(m),24.74(s),22.67(s),22.37(s),14.04(d,J=15.7Hz).

[0339] C. Compound SW-II-120

[0340] 1. Synthesis of Intermediate 3

[0341] A solution containing compound 1 (1.22 g, 5.0 mmol, 1.0 eq.), compound 2 (1.30 mg, 10.0 mmol, 2.0 eq.), Pd(PPh3)4 (289 mg, 0.25 mmol, 0.05 eq.), and K2CO3 (1.38 g, 10.0 mmol, 2.0 eq.) in a mixture of toluene (10 ml) and H2O (1 ml) was stirred at 110°C under N2 protection for 1 hour. TLC (petroleum ether:ethyl acetate = 19:1) showed that compound 1 was consumed and a new spot was observed. The reaction mixture was diluted with DCM (50 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.78 g, 62%) as a colorless oil.

[0342] 1 H NMR (400MHz, CDCl3) δ7.19(d,J=8.1Hz,2H),7.13(d,J=8.1Hz,2H),4.14(q,J=7.1Hz,2H),3.5 7(s,2H),2.62–2.51(m,2H),1.58(d,J=11.1Hz,2H),1.35–1.21(m,9H),0.88(t,J=6.7Hz,3H).

[0343] 2. Synthesis of Intermediate 4

[0344] LiAlH4 (477 mg, 12.56 mmol, 4.0 eq.) was added to a solution of compound 3 (780 mg, 3.14 mmol, 1.0 eq.) in THF (10 mL) at -78°C, and the reaction was stirred at 10°C for 3 hours. Thin-layer chromatography indicated that the reaction proceeded well. The reaction was concentrated, diluted with Na2SO4 (20 mL), and extracted with EA (30 mL x 2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford compound 4 (640 mg, crude) as a colorless oil.

[0345] 3. Synthesis of Intermediate 6

[0346] A solution of compound 4 (640 mg, 3.10 mmol, 1.0 eq.), compound 5 (1.06 g, 4.70 mmol, 1.5 eq.), EDCI (2.98 g, 15.5 mmol, 5.0 eq.), DMAP (1.85 g, 15.0 eq.), and DIEA (4.0 g, 31.0 mmol, 10.0 eq.) in DCM (10 mL) was stirred at 10°C for 16 h under N₂ protection. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1, v / v) to afford compound 6 (465 mg, 36%) as a colorless oil.

[0347] 4. Synthesis of the final product SW-II-120

[0348] A mixture containing compound 6 (100 mg, 0.25 mmol, 1.0 eq.), compound 7 (161 mg, 0.36 mmol, 1.5 eq.), K2CO3 (104 mg, 0.75 mmol, 3.0 eq.) and KI (83 mg, 0.50 mmol, 2.0 eq.) was stirred at 90 ° C for 16 hours in CPME (1 mL) and CH3CN (1 mL) under N2. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with NaHCO3 (30 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM:MeOH (1: 0-10: 1, v / v) to give SW-II-120 (100 mg, 52%) as a yellow oil.

[0349] LCMS:Rt:2.500min; MS m / z(ELSD):772.4[M+H] + ;

[0350] HPLC: 99.70% purity, ELSD; RT = 8.675 min.

[0351] 1 H NMR(400MHz, CDCl3) δ7.07(d,J=8.9Hz,4H),4.89–4.73(m,1H),4.23(t,J=7.2Hz,2H),3.83–3.65(m,2H),2.87(t,J=7.2Hz,4H),2.82–2 .67(m,4H),2.61–2.45(m,2H),2.25(td,J=7.5,2.5Hz,4H),1.65–1.44(m,15H),1.27(dd,J=13.2,11.3Hz,42H),0.85(t,J=6.8Hz,9H).

[0352] 13 C NMR (101MHz, CDCl3) δ173.57 (d, J = 11.5Hz), 141.13 (s), 134.88 (s), 128.73 (s), 128.48 (s), 77.45 (s), 77.13(s),76.81(s),74.14(s),64.89(s),57.34(s),56.17(s),53.92(s),35.57(s),34.64(d,J=16.1H z),34.14(d,J=3.3Hz),31.79(d,J=13.4Hz),31.49(s),29.50(d,J=2.2Hz),29.23(s),29.10–28.71(m ),26.85(d,J=5.0Hz),25.49–25.38(m),25.13(d,J=35.4Hz),24.72(s),22.63(d,J=5.8Hz),14.11(s).

[0353] D. Compound SW-II-121

[0354] 1. Synthesis of Intermediate 3

[0355] To a DCM (20 mL) solution containing compound 1 (1.3 g, 5.86 mmol, 1.5 eq.) and compound 2 (1 g, 3.9 mmol, 1.0 eq.) was added EDCI (1.495 g, 7.8 mmol, 2.0 eq.), DMAP (0.19 g, 1.56 mmol, 0.4 eq.), and DIEA (2.57 mL, 15.6 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature under N2 for 16 hours. TLC (petroleum ether: ethyl acetate = 19:1) showed that compound 2 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

[0356] 1 H NMR (400MHz, CDCl3) δ4.92–4.82(m,1H),3.42(t,J=6.8Hz,2H),2.31(t,J=7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J=6.8Hz,6H).

[0357] 2. Synthesis of Intermediate 5

[0358] A solution of EtOH (5 mL) containing compound 3 (5.2 g, 11.30 mmol, 1.0 eq.) and compound 4 (20.6 g, 339 mmol, 30 eq.) was stirred at 60 ° C for 16 hours under N protection. TLC (petroleum ether: ethyl acetate = 19: 1) showed that compound 3 was consumed and TLC (DCM / MeOH = 10 / 1) showed that a new main point was observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM: MeOH (1: 0-10: 1, v / v) to give compound 5 (3 g, 60%) as a yellow oil.

[0359] 3. Synthesis of Intermediate 8

[0360] To a mixed solution of compound 6 (1 g, 4.115 mmol, 1 eq.) and compound 7 (889 mg, 6.173 mmol, 1.5 eq.) in toluene / water (10 mL / 1 mL) were added Pd(pph) (238 mg, 0.206 mmol, 0.05 eq.) and KCO (1.7 g, 12.35 mmol, 3 eq.). The mixture was stirred at 110°C under N2 for 2 hours. TLC (PE:EA = 10:1) indicated the reaction was complete, with a new major spot observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE:EA (1:0-10:1) to afford compound 8 (714 mg, 66%) as a colorless oil.

[0361] 4. Synthesis of Intermediate 9

[0362] Under N protection, LiAlH (2.7 mL, 2.725 mmol, 1 M in THF, 1 eq.) was added to a mixture of compound 8 (714 mg, 2.725 mmol, 1 eq.) in THF (7 mL) at 0°C, and the mixture was stirred at room temperature for 2 hours. TLC (PE:EtOAc=10:1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.7 mL) and treated with 2N HCl to adjust the pH between 6 and 7, extracted with EA and washed with brine. The organic layer was dried over anhydrous NaSO, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with PE:EA (1:0-10:1) to give compound 9 (103 mg, 63%) as a colorless oil.

[0363] 5. Synthesis of Intermediate 11

[0364] To DCM (3 mL) containing compound 9 (300 mg, 1.364 mmol, 1 eq.) and compound 10 (363 mg, 1.64 mmol, 1.2 eq.) was added EDCI (524 mg, 2.728 mmol, 2 eq.), DMAP (67 mg, 0.546 mmol, 0.4 eq.), and DIEA (704 mg, 5.456 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N2 for 16 hours. TLC (petroleum ether: ethyl acetate = 10:1) showed the formation of the desired product. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether: ethyl acetate (1:0-10:1) to give compound 11 (169 mg, 29%) as a colorless oil.

[0365] 6. Synthesis of the final product SW-II-121

[0366] To a CPME / CH3CN (2 mL / 2 mL) mixed solvent containing compound 11 (169 mg, 0.399 mmol, 1 eq.) and compound 5 (176 mg, 0.399 mmol, 1 eq.) was added K2CO3 (330 mg, 2.394 mmol, 6 eq.) and KI (132 mg, 0.798 mmol, 2 eq.). After the addition was complete, the mixture was stirred at 90°C overnight under N2. TLC (DCM:MeOH = 10:1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM:MeOH (1:0-10:1, v / v) to afford compound SW-II-121 (145 mg, 46%) as a yellow oil.

[0367] LCMS:Rt:1.493min;MS m / z(ELSD):786.5[M+H] + ;

[0368] HPLC: 99.869% purity, ELSD; RT = 10.655 min.

[0369] 1 H NMR (400MHz, CDCl3) δ7.11 (s, 4H), 4.92–4.80 (m, 1H), 4.26 (t, J = 7.2Hz, 2H), 3.80 (s, 2H), 2.87 (dd, J = 26.6, 19.4Hz, 7H),2.62–2.51(m,2H),2.28(td,J=7.2,3.6Hz,4H),1.75–1.45(m,14H),1.42–1.09(m,45H),0.88(t,J=6.8Hz,9H).

[0370] 13C NMR (101MHz, CDCl3) δ173.61 (d, J = 12.3Hz), 141.20 (s), 134.90 (s), 128.75 (s), 128.51 (s),77.35(s),77.03(s),76.72(s),74.21(s),64.93(s),54.15(s),35.59(s),34.66( d,J=16.6Hz),34.16(d,J=3.0Hz),31.85(d,J=4.4Hz),31.55(s),29.64–29.15(m),29. 15–28.78(m),26.85(d,J=4.5Hz),25.33(s),24.95(s),24.72(s),22.68(s),14.12(s).

[0371] E. Compound SW-II-122

[0372] 1. Synthesis of compound 3

[0373] Compound 1 (1 g, 4.65 mmol, 1 eq.) and compound 2 (726 mg, 5.58 mmol, 1.2 eq.) were dissolved in toluene / water (10 / 1, 20 mL), and KCO (1.92 g, 13.9 mmol, 3 eq.) and Pd(pph) (269 mg, 0.23 mmol, 0.05 eq.) were added to the mixture. The reaction mixture was heated to 110°C under N2 and stirred for 2 hours. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 1 was consumed and a new major spot was observed. The reaction mixture was quenched with H2O (80 mL) and extracted with ethyl acetate (60 mL x 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (800 mg, 78%) as a yellow oil.

[0374] 2. Synthesis of compound 4

[0375] Under nitrogen protection at 0°C, LiAlH4 (3.2 mL, 3.18 mmol, 1 eq.) was added to compound 3 (700 mg, 3.18 mmol, 1.0 eq.) dissolved in THF (14 mL). The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen protection. TLC (PE / EtOAc = 10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was quenched with water (3.2 mL) and 1M HCl (3.2 mL), respectively. Water (6 mL) was added to the mixture and extracted with ethyl acetate (60 mL × 3). The organic layer was washed with brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with ethyl acetate / petroleum ether = 1 / 10 to give compound 4 (600 mg, 98%) as a yellow oil.

[0376] 3. Synthesis of Compound 6

[0377] Compound 4 (680 mg, 3.5 mmol, 1.0 eq.) and compound 5 (1.13 g, 5.1 mmol, 1.5 eq.) were dissolved in DCM (10 mL), and EDCI (1.20 g, 6.25 mmol, 2.0 eq.), DMAP (166 mg, 1.36 mmol, 0.4 eq.), and DIEA (1.78 g, 13.8 mmol, 4.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at room temperature overnight under nitrogen. TLC (DCM / MeOH = 30 / 1) showed that the starting material was consumed and a new spot had formed. The mixture was quenched with water (70 mL) and extracted with DCM (80 mL x 3). The combined organic layers were washed with brine (2 x 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with ethyl acetate / petroleum ether = 3 / 97 solution to give compound 6 (680 mg, 48.5%) as a yellow oil.

[0378] 4. Synthesis of SW-II-122

[0379] Compound 6 (108 mg, 0.27 mmol, 1.2 eq) and compound 7 (100 mg, 0.23 mmol, 1 eq.) were dissolved in CPME (2 mL) and CHCN (2 mL). Potassium carbonate (157 mg, 1.14 mmol, 5.0 eq) and potassium iodide (75 mg, 0.45 mmol, 2.0 eq) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C under nitrogen for 16 hours. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain SW-II-122 (68 mg, 40%) as a colorless oil.

[0380] LCMS:Rt:1.487min;MS m / z(ELSD):758.5[M+H] + ;

[0381] HPLC: 97.3% purity, ELSD; RT = 7.622 min.

[0382] 1 H NMR (400MHz, CDCl3) δ7.32(d,J=26.4Hz,1H),7.17(dd,J=27.2,21.1Hz,3H),5.09(s,2H),4.91–4.79(m,1H),3.85(s,2H),2.98(s,2H),2.87(s,4H) ,2.65–2.54(m,2H),2.35(t,J=7.6Hz,2H),2.28(t,J=7.6Hz,2H),1.74–1 .57(m,9H),1.50(d,J=5.6Hz,4H),1.37–1.15(m,43H),0.94–0.80(m,9H).

[0383] 13C NMR (101MHz, CDCl3) δ173.55 (d, J = 2.4Hz), 143.35 (s), 135.92 (s), 128.67–128.19 (m), 125.47 (s), 77. 36(s),77.04(s),76.73(s),74.22(s),66.27(s),57.15(s),56.74(s),54.14(s),35.88(s),34.55(s) ,34.15(d,J=3.6Hz),31.79(d,J=15.2Hz),31.43(s),29.52(d,J=2.8Hz),29.25(s),28.92(dd,J=14.2 ,5.8Hz),26.77(d,J=4.8Hz),25.33(s),24.92(s),24.71(s),24.48(s),22.64(d,J=6.8Hz),14.12(s).

[0384] F. Compound SW-II-127

[0385] 1. Synthesis of compound 3

[0386] Compound 1 (1.3 g, 5.86 mmol, 1.5 eq.) and compound 2 (1 g, 3.9 mmol, 1.0 eq.) were dissolved in DCM (20 mL). EDCI (1.495 g, 7.8 mmol, 2.0 eq.) and DMAP (0.19 g, 1.56 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (2.57 mL, 15.6 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 2 was consumed and the desired product was formed. The reaction mixture was diluted with DCM (20 mL) and washed with H2O (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (1.2 g, 66.9%) as a yellow oil.

[0387] 1 H NMR (400MHz, CDCl3) δ4.92–4.82(m,1H),3.42(t,J=6.8Hz,2H),2.31(t,J=7.5Hz,2H),1.95–1.82(m,2H),1.70–1.19(m,36H),0.90(t,J=6.8Hz,6H).

[0388] 2. Synthesis of compound 5

[0389] Compound 3 (5.2 g, 11.30 mmol, 1.0 eq.) and compound 4 (20.6 g, 339 mmol, 30 eq.) were added to EtOH (5 mL), and the mixture was stirred at 60 ° C for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 19 / 1) showed that compound 3 was consumed and TLC (DCM / MeOH = 10 / 1) showed that a new main spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H2O (3 × 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM / MeOH (1 / 0-10: 1, v / v) to obtain compound 5 (3 g, 60%) as a yellow oil.

[0390] 1 H NMR (400MHz, CDCl3) δ4.95–4.75(m,1H),3.74–3.58(m,2H),2.87–2.74(m,2H),2.69–2.56(m,2H), 2.36(s,2H),2.28(t,J=7.5Hz,2H),1.65–1.42(m,8H),1.38–1.17(m,30H),0.88(t,J=6.8Hz,6H).

[0391] 3. Synthesis of Compound 8

[0392] Compound 7 (522 mg, 2.5 mmol, 1.2 eq.) and compound 6 (400 mg, 2.083 mmol, 1 eq.) were dissolved in DCM (4 mL). EDCI (800 mg, 4.166 mmol, 2 eq.), DMAP (102 mg, 0.833 mmol, 0.4 eq.), and DIEA (1.075 mg, 8.332 mmol, 4 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at room temperature overnight under nitrogen. TLC (PE:EA = 10:1) showed that the starting material was consumed and a new spot had formed. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1), to afford compound 8 (454 mg, 57%) as a colorless oil.

[0393] 4. Synthesis of SW-II-127

[0394] Compound 8 (100 mg, 0.262 mmol, 1 eq.) and compound 5 (139 mg, 0.314 mmol, 1.2 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (217 mg, 1.572 mmol, 6 eq.) and potassium iodide (87 mg, 0.524 mmol, 2 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and the desired product had formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain compound SW-II-127 (42.49 mg, 22%) as a yellow oil.

[0395] LCMS:Rt:1.323min; MS m / z(ELSD):744.5[M+H] + ;

[0396] HPLC: 99.742% purity, ELSD; RT = 7.339 min.

[0397] 1 H NMR(400MHz, CDCl3)δ7.25(s,2H),7.17(d,J=8.0Hz,2H),5.07(s,2H),4.91–4.82(m,1H),3.83(s,2H),2.90(d,J=44.8Hz,5H), 2.64–2.55(m,2H),2.35(t,J=7.4Hz,2H),2.28(t,J=7.5Hz,2H),1.76–1.46(m,14H),1.42–1.19(m,41H),0.88(t,J=6.8Hz,9H).

[0398] 13C NMR (101MHz, CDCl3) δ173.50 (d, J = 8.5Hz), 133.17 (s), 128.61 (s), 128.34 (s), 77.29 (d ,J=11.4Hz),77.03(s),76.71(s),74.23(s),66.19(s),54.20(s),35.71(s),34.56(s), 34.10(d,J=8.8Hz),31.80(d,J=15.4Hz),31.43(s),29.53(d,J=2.5Hz),29.25(s),28.95(d,J=10.5 Hz), 28.63 (s), 26.71 (d, J = 18.2Hz), 25.33 (s), 24.93 (s), 24.62 (s), 22.65 (d, J = 6.6Hz), 14.13 (s).

[0399] G. Compound SW-II-134-1

[0400] 1. Synthesis of compound 3

[0401] To a mixture of compound 1 (500 mg, 2.283 mmol, 1 eq.) and compound 2 (890 mg, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (723 mg, 99.6%) as a colorless oil.

[0402] 2. Synthesis of compound 4

[0403] To a mixture of compound 3 (723 mg, 2.27 mmol, 1 eq.) in THF (8 mL) was added lithium aluminum hydride (2.3 mL, 2.27 mmol, 1 M in THF, 1 eq.) at 0°C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated that the reaction was complete and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give compound 4 (381 mg, 58%) as a colorless oil without further purification.

[0404] 3. Synthesis of Compound 6

[0405] To a mixture of compound 4 (381 mg, 1.3 mmol, 1 eq.) and compound 5 (352 mg, 1.6 mmol, 1.2 eq.) in DCM (4 mL) was added EDCI (499 mg, 2.6 mmol, 2 eq.) and DMAP (63 mg, 0.52 mmol, 0.4 eq.), followed by DIEA (671 mg, 5.2 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 20 / 1) showed that the desired product had formed. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (272 mg, 44%) as a colorless oil.

[0406] 4. Synthesis of SW-II-134-1

[0407] To a mixture of compound 6 (150 mg, 0.303 mmol, 1 eq.) and compound 7 (110 mg, 0.333 mmol, 1.1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (251 mg, 1.818 mmol, 6 eq.) and potassium iodide (101 mg, 0.61 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-134-1 (168 mg, 75%) as a yellow oil.

[0408] LCMS:Rt:1.276min;MS m / z(ELSD):744.4[M+H] + ;

[0409] HPLC: 98.481% purity, ELSD; RT = 10.724 min.

[0410] 1 H NMR(400MHz, CDCl3) δ7.06(d,J=7.6Hz,1H),7.01–6.93(m,2H),4.25(t,J=7.3Hz,2H),4.05(t,J=6.8Hz,2H),3.85–3.72(m,2H),2 .98–2.69(m,8H),2.62–2.48(m,4H),2.29(t,J=7.5Hz,4H),1.72–1.48(m,14H),1.45–1.17(m,36H),0.89(dt,J=11.9,6.0Hz,9H).

[0411] 13 CNMR (101MHz, CDCl3) δ173.78 (d, J = 16.7Hz), 140.72 (s), 138.81 (s), 134.91 (s), 129.70 (s), 129.22 (s), 126.19 (s),77.30(d,J=11.4Hz),77.03(s),76.72(s),65.02(s),64.49(s),57.42(s),56.36(s),54.08(s),34.76(s),3 4.22(d,J=4.2Hz),32.74(s),32.36(s),31.81(d,J=9.1Hz),31.35(d,J=5.3Hz),29.49(d,J=2.8Hz),29.24(d,J= 2.2Hz), 28.92(s), 28.66(s), 26.86(s), 25.93(s), 25.04(s), 24.78(d,J=6.6Hz), 22.65(d,J=2.6Hz), 14.10(s).

[0412] H. Compound SW-II-134-2

[0413] 1. Synthesis of compound 3

[0414] To a mixture of compound 1 (500 mg, 2.283 mmol, 1 eq.) and compound 2 (1.08 g, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (854 mg, 100%) as a colorless oil.

[0415] 2. Synthesis of compound 4

[0416] To a mixture of compound 3 (854 mg, 2.28 mmol, 1 eq.) in THF (9 mL) at 0°C under nitrogen was added lithium aluminum hydride (2.3 mL, 2.28 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction, with a new major spot observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (724 mg, 92%) as a colorless oil without further purification.

[0417] 3. Synthesis of Compound 6

[0418] To a mixture of compound 4 (724 mg, 2.09 mmol, 1 eq.) and compound 5 (560 mg, 2.51 mmol, 1.2 eq.) in DCM (8 mL) were added EDCI (803 mg, 4.18 mmol, 2 eq.) and DMAP (102 mg, 0.84 mmol, 0.4 eq.), followed by DIEA (1.078 g, 8.36 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 20 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (473 mg, 41%) as a colorless oil.

[0419] 4. Synthesis of SW-II-134-2

[0420] To a mixture of compound 6 (150 mg, 0.27 mmol, 1 eq.) and compound 7 (108 mg, 0.33 mmol, 1.1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (225 mg, 1.63 mmol, 6 eq.) and potassium iodide (90 mg, 0.54 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=15 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-134-2 (71.77 mg, 33%).

[0421] LCMS:Rt:1.527min;MS m / z(ELSD):800.4[M+H] + ;

[0422] HPLC: 97.311% purity, ELSD; RT = 9.025 min.

[0423] 1 H NMR (400MHz, CDCl3) δ7.06(d,J=7.6Hz,1H),6.96(d,J=9.6Hz,2H),4.25(t,J=7.3Hz,2H),4.05(t,J=6.8Hz,2H),3.80–3.66(m,2H),2.86(dd,J=12.8,5 .6Hz,4H),2.78–2.67(m,4H),2.60–2.52(m,4H),2.29(t,J=7.5Hz,4H),1.5 7(dt,J=15.8,7.3Hz,14H),1.30(d,J=20.3Hz,45H),0.88(t,J=6.7Hz,9H).

[0424] 13C NMR (101MHz, CDCl3) δ173.82 (d, J = 16.9Hz), 140.73 (s), 138.82 (s), 134.91 (s), 129.71(s),129.23(s),126.19(s),77.36(s),77.14(d,J=20.4Hz),76.72(s),65.03(s),64 .49(s),57.57(s),56.13(s),54.02(s),34.76(s),34.25(d,J=4.2Hz),32.76(s),32.37(s) ,31.89(d,J=5.3Hz),31.40(d,J=6.0Hz),29.84(d,J=3.7Hz),29.63–29.14(m),28.97(s),2 8.65(s), 26.93(s), 25.66(d,J=54.4Hz), 24.80(d,J=6.6Hz), 22.68(d,J=1.8Hz), 14.12(s).

[0425] I. Compound SW-II-134-3

[0426] 1. Synthesis of compound 3

[0427] To a mixture of compound 1 (10 g, 45 mmol, 1 eq.) and compound 2 (7.8 g, 54 mmol, 1.2 eq.) in DCM (100 mL) were added EDCI (17.3 g, 90 mmol, 2 eq.) and DMAP (2.2 g, 18 mmol, 0.4 eq.), followed by DIEA (23.2 g, 180 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 30 / 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (20 mL) and washed with water (40 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 3 (4.365 g, 28%) as a colorless oil.

[0428] 2. Synthesis of compound 5

[0429] A mixture of compound 3 (5 g, 14.38 mmol, 1 eq.) and compound 4 (8.8 g, 143.7 mmol, 10 eq.) in ethanol (2 mL) was stirred at 55 ° C under nitrogen for 16 hours. TLC (DCM / MeOH=10 / 1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (50 mL) and washed with water (3×50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give compound 5 (1.008 g, 21%) as a yellow oil.

[0430] 3. Synthesis of Compound 8

[0431] To a mixture of compound 6 (500 mg, 2.283 mmol, 1 eq.) and compound 7 (699 mg, 6.849 mmol, 3 eq.) in toluene / water (5 mL / 1 mL) were added palladium acetate (51 mg, 0.228 mmol, 0.1 eq.), Ruphos (213 mg, 0.457 mmol, 0.2 eq.), and potassium carbonate (945 mg, 6.849 mmol, 3 eq.). The mixture was stirred at 110°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 8 (507 mg, 85%) as a colorless oil.

[0432] 4. Synthesis of Compound 9

[0433] To a mixture of compound 8 (507 mg, 1.935 mmol, 1 eq.) in THF (5 mL) at 0°C under nitrogen was added lithium aluminum hydride (2 mL, 1.935 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 9 (492 mg, >100%) as a colorless oil without further purification.

[0434] 5. Synthesis of Compound 10

[0435] To a mixture of compound 9 (492 mg, 2.103 mmol, 1 eq.) and compound 1 (563 mg, 2.523 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (808 mg, 4.206 mmol, 2 eq.) and DMAP (103 mg, 0.84 mmol, 0.4 eq.), followed by DIEA (1.085 g, 8.412 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 10 (329 mg, 36%) as a colorless oil.

[0436] 6. Synthesis of SW-II-134-3

[0437] To a mixture of compound 10 (150 mg, 0.34 mmol, 1 eq.) and compound 5 (134 mg, 0.41 mmol, 1.2 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (282 mg, 2.04 mmol, 6 eq.) and potassium iodide (113 mg, 0.68 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give compound SW-II-134-3 (63.59 mg, 25%) as a yellow oil.

[0438] LCMS:Rt:1.247min;MS m / z(ELSD):688.3[M+H] + ;

[0439] HPLC: 95.945% purity, ELSD; RT = 6.186 min.

[0440] 11H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 7.6 Hz, 1H), 6.97 (dd, J = 9.9, 2.2 Hz, 2H), 4.26 (t, J = 7.2 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 2.88 (dd, J = 14.8, 7.6 Hz, 4H), 2.78–2.74 (m, 2H), 2.67–2.54 (m, 8H), 2.29 (t, J = 7.5 Hz, 4H), 1.68–1.47 (m, 15H), 1.37–1.22 (m, 27H), 0.98–0.86 (m, 9H).

[0441] 13 13C NMR (101 MHz, CDCl3) δ 173.86 (d, J = 17.1 Hz), 140.66 (s), 138.76 (s), 134.93 (s), 129.74 (s), 129.24 (s), 126.19 (s), 77.36 (s), 77.04 (s), 76.72 (s), 65.01 (s), 64.48 (s), 57.73 (s), 55.73 (s), 53.93 (s), 34.76 (s), 34.28 (d, J = 3.9 Hz), 33.54 (d, J = 4.5 Hz), 32.41 (s), 31.95 (d, J = 16.5 Hz), 29.49 (s), 29.15 (dd, J = 21.1, 2.4 Hz), 28.66 (s), 27.04 (s), 25.95 (d, J = 3.3 Hz), 24.85 (d, J = 6.6 Hz), 22.98–22.58 (m), 14.08 (d, J = 7.5 Hz).

[0442] J.SW-II-135-1

[0443] 1. Synthesis of Compound 3

[0444] Compound 1 (500 mg, 2.16 mmol, 1.0 eq.) and compound 2 (750 mg, 6.46 mmol, 3.0 eq.) were dissolved in toluene / H₂O (5 mL / 1 mL). To this mixture were added Ruphos (201 mg, 0.43 mmol, 0.2 eq.), Pd(OAc)₂ (48.5 mg, 0.22 mmol, 0.1 eq.), and Cs₂CO₃ (2.10 g, 6.46 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (40 mL) and extracted three times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-30 / 1) to give compound 3 (540 mg, 82.44%) as a yellow oil.

[0445] 2. Synthesis of compound 4

[0446] To compound 3 (540 mg, 1.78 mmol, 1.0 eq.) dissolved in THF (5 mL) was added LiAlH4 (3.55 mL, 3.55 mmol, 2 eq. in 1 M THF) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen. TLC (PE / EtOAc = 10 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1) to give compound 4 (442 mg, 90.2%) as a colorless oil.

[0447] 3. Synthesis of Compound 6

[0448] Compound 4 (442 mg, 1.60 mmol, 1.0 eq.) and compound 5 (428.5 mg, 1.92 mmol, 1.2 eq.) were dissolved in DCM (5 mL), and EDCI (612 mg, 3.2 mmol, 2.0 eq.) and DMAP (78.2 mg, 0.64 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (826 mg, 6.4 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4 was consumed and the desired product was formed. The reaction mixture was washed with H2O (40 mL) and extracted 3 times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (342 mg, 44.5%) as a yellow oil.

[0449] 4. Synthesis of SW-II-135-1

[0450] Compound 6 (175 mg, 0.365 mmol, 1.2 eq.) and compound 7 (100 mg, 0.304 mmol, 1.0 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (210 mg, 1.52 mmol, 5.0 eq.) and potassium iodide (101 mg, 0.61 mmol, 2.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) showed that the reaction was complete and the desired product had formed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to obtain compound SW-II-135-1 (83.89 mg, 55.6%) as a yellow oil.

[0451] LCMS:Rt:1.356min; MS m / z(ELSD):730.5[M+H] + ;

[0452] HPLC: 100% purity at ELSD; RT=12.614min.

[0453] 1H NMR(400MHz, CDCl3)δ6.97(d,J=7.6Hz,1H),6.91–6.74(m,2H),4.76(s,1H),3 .99(dt,J=13.6,6.4Hz,4H),3.72–3.58(m,2H),2.85–2.73(m,2H),2.72–2.61( m,4H),2.59–2.41(m,6H),2.22(dd,J=13.2,7.2Hz,4H),1.93–1.79(m,2H),1. 62–1.41(m,14H),1.23(d,J=24.4Hz,32H),0.82(ddd,J=13.6,8.0,5.6Hz,9H).

[0454] 13 C NMR (101MHz, CDCl3) δ172.81 (d, J = 6.4Hz), 139.55 (s), 137.38 (s), 137.14 (s), 128.16 (d, J = 2.4Hz), 124.69 ( s),76.51(s),76.19(s),75.88(s),63.43(s),62.78(s),56.53(s),54.90(s),52.84(s),33.23(d,J=2.4Hz) ,31.73(s),31.28(s),30.91(dd,J=20.0,6.4Hz),30.10(d,J=3.2Hz),29.29(s),28.36(d,J=22.8Hz),28.23 (s),27.97(s),27.64(s),25.92(s),24.92(s),24.34(s),23.84(s),21.62(d,J=7.6Hz),13.08(d,J=4.7Hz).

[0455] K. Compound SW-II-135-2

[0456] SW-II-135-2

[0457] 1. Synthesis of compound 3

[0458] Compound 1 (500 mg, 2.16 mmol, 1.0 eq.) and compound 2 (931 mg, 6.46 mmol, 3.0 eq.) were dissolved in toluene / H₂O (5 mL / 1 mL). To this mixture were added Ruphos (201 mg, 0.43 mmol, 0.2 eq.), Pd(OAc)₂ (48.5 mg, 0.22 mmol, 0.1 eq.), and Cs₂CO₃ (2.10 g, 6.46 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (40 mL) and extracted three times with EA (50 mL). The resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-30 / 1) to give compound 3 (651 mg, 84%) as a yellow oil.

[0459] 2. Synthesis of compound 4

[0460] To compound 3 (651 mg, 1.81 mmol, 1.0 eq.) dissolved in THF (7 mL) was added LiAlH4 (3.62 mL, 3.62 mmol, 1 M in THF, 2 eq.) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred for 2 hours under nitrogen. TLC (PE / EtOAc = 10 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (10 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1) to give compound 4 (571 mg, 95.2%) as a colorless oil.

[0461] 3. Synthesis of Compound 6

[0462] Compound 4 (571 mg, 1.72 mmol, 1.0 eq.) and compound 5 (459 mg, 2.06 mmol, 1.2 eq.) were dissolved in DCM (6 mL), and EDCI (657 mg, 3.44 mmol, 2.0 eq.) and DMAP (84 mg, 0.68 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (887.5 mg, 6.88 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature for 16 hours under nitrogen protection. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 4 was consumed and the desired product was formed. The reaction mixture was washed with H2O (50 mL) and extracted 3 times with EA (60 mL). The resulting organic phase was washed twice with brine (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 3 (245 mg, 26.5%) as a yellow oil.

[0463] 4. Synthesis of SW-II-135-2

[0464] Compound 6 (245 mg, 0.456 mmol, 1.5 eq.) and compound 7 (100 mg, 0.3 mmol, 1.0 eq.) were dissolved in CPME / CH3CN (1 mL / 1 mL). Potassium carbonate (210 mg, 1.52 mmol, 5.0 eq.) and potassium iodide (101 mg, 0.61 mmol, 2.0 eq.) were added to the mixture. After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated completion of the reaction and formation of the desired product. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to afford SW-II-135-2 (31.41 mg, 21.9%) as a yellow oil.

[0465] LCMS:Rt:1.608min; MS m / z(ELSD):786.4[M+H] + ;

[0466] HPLC: 95.16% purity, ELSD; RT = 7.919 min.

[0467] 1H NMR(400MHz, CDCl3)δ6.98(d,J=7.6Hz,1H),6.87(d,J=2.4Hz,2H),4.28–4.13(m,1H),4.04–3.95(m,4H),3.94–3.84(m,2H),3.14–2.89(m,6H) ,2.59–2.43(m,6H),2.23(dd,J=13.8,7.2Hz,4H),1.88–1.82(m,2H),1 .70(s,4H),1.57–1.46(m,10H),1.33–1.16(m,40H),0.90–0.72(m,9H).

[0468] 13 C NMR(100MHz, CDCl3)δ172.82(d,J=6.8Hz),139.61(s),137.29(d,J=16.4Hz),128.15(s),124.67(s),7 6.41(s),76.09(s),75.77(s),63.50(s),62.87(s),55.49(s),54.92(s),52.98(s),33.16(d,J=2.4Hz ),31.77(s),31.33(s),30.80(d,J=6.5Hz),30.42(d,J=3.6Hz),29.29(s),28.99–28.66(m),28.47(s) ,28.23(d,J=2.8Hz),28.06–27.45(m),25.58(s),24.91(s),23.71(s),22.79(s),21.66(s),13.10(s).

[0469] L. Compound SW-II-136-2

[0470] SW-II-136-2

[0471] 1. Synthesis of compound 3

[0472] Compound 1 (3 g, 13.70 mmol, 1.0 eq.) and compound 2 (5.34 g, 41.09 mmol, 3.0 eq.) were dissolved in toluene / H₂O (30 mL / 3 mL). To this mixture were added Ruphos (1.28 g, 2.74 mmol, 0.2 eq.), Pd(OAc)₂ (308.3 mg, 1.37 mmol, 0.1 eq.), and K₂CO₃ (5.67 g, 41.10 mmol, 3.0 eq.). The reaction mixture was heated at reflux at 110°C under nitrogen for 16 hours. TLC (PE / EA = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H₂O (90 mL) and extracted three times with EA (110 mL). The resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-30 / 1) to give compound 3 (1.98 g, 45.5%) as a yellow oil.

[0473] 2. Synthesis of compound 4

[0474] To compound 3 (1.98 g, 6.23 mmol, 1.0 eq.) dissolved in THF (20 mL) was added LiAlH₄ (1 M, 12.45 mL, 2.0 eq.) at 0°C under nitrogen. The reaction was allowed to warm to room temperature and stirred under nitrogen for 2 hours. TLC (PE / EtOAc = 10 / 1) indicated completion of the reaction, with a new major spot observed. The mixture was quenched with H₂O (70 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with EA (80 mL). The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-10 / 1), to afford compound 4 (1.28 g, 71.1%) as a colorless oil.

[0475] 3. Synthesis of Compound 7

[0476] To compound 4 (900 g, 3.1 mmol, 1.0 eq.) dissolved in DCM (9 mL) under nitrogen at 0°C were added DMSO (3.63 g, 51.72 mmol, 15 eq.), TEA (1.25 g, 12.4 mmol, 4.0 eq.), and PySO (1.27 g, 7.97 mmol, 2.57 eq.). The mixture was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred under nitrogen for 90 minutes. Compound 6 (4.74 g, 13.62 mmol, 3.0 eq.) was then added to the mixture, and the reaction mixture was allowed to react at 25°C under nitrogen for 2 hours. TLC (PE / EA = 10 / 1) indicated the reaction was complete and the desired product had formed. The reaction mixture was washed with H2O (60 mL) and extracted three times with EA (70 mL). The resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-10 / 1) to give compound 7 (345 mg, 27.9%) as a yellow oil.

[0477] 4. Synthesis of Compound 8

[0478] Compound 7 (340 mg, 0.95 mmol, 1.0 eq.) and Pd / C (100 mg) were added to MeOH (4 ml), and the reaction mixture was stirred at room temperature under a hydrogen atmosphere for 16 h. TLC (PE / EA = 10 / 1) showed complete consumption of the starting material and the formation of the desired product. The reaction mixture was filtered through Celite and washed with MeOH (40 mL x 2), dried over anhydrous NaSO, and the filtrate was concentrated under reduced pressure to obtain Compound 8 (298 mg, 88.2%) as a pale yellow oil.

[0479] 5. Synthesis of Compound 9

[0480] To compound 8 (298 mg, 0.83 mmol, 1.0 eq.) dissolved in THF (3 mL) was added LiAlH₄ (1 M, 1.66 mL, 2.0 eq.) under nitrogen at 0°C. The reaction was allowed to warm to room temperature and stirred under nitrogen for 2 hours. TLC (PE / EtOAc = 10 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with H₂O (20 mL), then adjusted to pH 6-7 with 1 M hydrochloric acid and extracted three times with EA (30 mL). The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-10 / 1) to afford compound 9 (254 mg, 98.3%) as a colorless oil.

[0481] 6. Synthesis of Compound 11

[0482] Compound 9 (254 mg, 0.80 mmol, 1.0 eq.) and compound 10 (214 mg, 0.96 mmol, 1.2 eq.) were dissolved in DCM (3 mL). EDCI (305.6 mg, 1.6 mmol, 2.0 eq.) and DMAP (39 mg, 0.32 mmol, 0.4 eq.) were added to the mixture, followed by DIEA (412.8 mg, 3.2 mmol, 4.0 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (PE / EA = 10 / 1) showed that compound 9 was consumed and the desired product was formed. The reaction mixture was adjusted to pH 4-6 with 1 M hydrochloric acid and extracted three times with EA (30 mL). The resulting organic phase was washed twice with brine (15 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using PE / EA (1 / 0-7 / 1) as eluent to give compound 11 (210 mg, 50.5%) as a yellow oil.

[0483] 7. Synthesis of SW-II-136-2

[0484] Compound 11 (200 mg, 0.38 mmol, 1.2 eq.) and compound 12 (105 mg, 0.32 mmol, 1.0 eq.) were dissolved in CPME / CHCN (1.5 mL / 1.5 mL). To this mixture were added KCO (220.2 mg, 1.60 mmol, 5.0 eq.) and KI (106 mg, 0.64 mmol, 2.0 eq.). After the addition, the reaction mixture was stirred at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated completion of the reaction and formation of the desired product. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with DCM / MeOH (1 / 0-10:1, v / v) to afford SW-II-136-2 (208 mg, 90.4%) as a yellow oil.

[0485] LCMS:Rt:2.146min; MS m / z(ELSD):773.3[M+H] + ;

[0486] HPLC: 99.49% purity, ELSD; RT = 8.055 min.

[0487] 1H NMR (400MHz, CDCl3) δ7.04(d,J=7.6Hz,1H),6.92(d,J=9.6Hz,2H),4.45(s,1H),4.06(dd,J=12.0,5.2Hz,4H),3.64(t,J=5.2Hz,2 H),2.72(t,J=5.2Hz,2H),2.65–2.50(m,10H),2.29(t,J=7.6Hz,4H),1.69–1.48(m,18H),1.41–1.24(m,36H),0.95–0.78(m,9H).

[0488] 13 C NMR (101MHz, CDCl3) δ173.86 (d, J = 2.8Hz), 140.48 (s), 139.24 (s), 138.01 (s), 129.13 (d, J = 14.8Hz), 125.67 (s),77.37(s),77.05(s),76.73(s),64.45(s),64.23(s),57.88(s),55.91(s),53.94(s),35.07(s),34.29(d ,J=3.2Hz),32.79(s),32.35(s),31.82(d,J=8.4Hz),31.38(s),29.50(d,J=2.4Hz),29.16(dd,J=18.0,2.0H z),28.66(s),28.35(s),27.78(s),27.08(s),26.02(d,J=17.2Hz),24.89(d,J=1.6Hz),22.65(s),14.10(s).

[0489] M. Compound SW-II-137-1

[0490] 1. Synthesis of compound 3

[0491] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.) was dissolved in toluene (5.0 mL), followed by the addition of compound 2 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.19 mmol, 0.1 eq.), water (1 mL), and K2CO3 (808 g, 5.85 mmol, 3.0 eq.). The reaction was allowed to proceed at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and the formation of the desired product. H2O (70 mL) was added to the reaction mixture, and the mixture was extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford the compound (320 mg, 70%) as a colorless oil.

[0492] 2. Synthesis of compound 4

[0493] Compound 3 (300 mg, 1.28 mmol, 1.0 eq.) was dissolved in THF (4.0 mL) and LAH (97 mg, 2.56 mmol, 2.0 eq.) was added at 0°C under nitrogen. The reaction was then allowed to react at room temperature for 2 hours. TLC (PE / EA = 10 / 1) indicated complete reaction of the starting material and the formation of the desired product. The mixture was quenched with 1 M HCl (4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-10 / 1, v / v) as the eluent to afford Compound 4 (224 mg, 84.8%) as a yellow oil.

[0494] 3. Synthesis of Compound 6

[0495] Compound 4 (90 mg, 0.47 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL), and compound 5 (127 mg, 0.56 mmol, 1.2 eq.), EDCI (180 mg, 0.94 mmol, 2.0 eq.), DIEA (242 mg, 1.88 mmol, 4.0 eq.), and DMAP (23 mg, 0.18 mmol, 0.4 eq.) were added. The mixture was then allowed to react overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete reaction of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution, the pH adjusted to 4-6, and extracted with EA (40 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-20 / 1, v / v) as eluent to give compound 6 (90 mg, 48.6%) as a colorless oil.

[0496] 4. Synthesis of SW-II-137-1

[0497] Compound 6 (90 mg, 0.25 mmol, 1.0 eq.) was dissolved in MeCN (2 mL) and compound 7 (110 mg, 0.25 mmol, 1.0 eq.), KI (76 mg, 0.50 mmol, 2.0 eq.), CPME (2 mL), and KCO (157 mg, 1.25 mmol, 5.0 eq.) were added. The reaction was allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete reaction of the starting material and formation of the desired product. The mixture was quenched with water (50 mL) and extracted with EA (40 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous NaSO, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10 / 1, v / v) as the eluent to afford the compound (98 mg, 52.12%, SW-II-137-1) as a yellow oil.

[0498] LCMS:Rt:1.596min; MS m / z(ELSD):758.4[M+H] + ;

[0499] HPLC: 98.02% purity, ELSD; RT = 5.993 min.

[0500] 1H NMR(400MHz, CDCl3) δ7.02(d,J=8.8Hz,4H),4.92–4.71(m,1H),4.01(t,J=6.4Hz,2H),3.78(s,1H),3.55(t,J=5.2Hz,2H),2.76–2.40(m,10H ),2.21(dd,J=15.6,7.7Hz,4H),1.95–1.80(m,2H),1.49(ddd,J=24.4,15.8,6.2Hz,15H),1.34–1.13(m,37H),0.82(dt,J=13.6,7.2Hz,9H).

[0501] 13 C NMR(101MHz, CDCl3)δ173.79(s),173.57(s),140.49(s),138.30(s),128.43(s),128.22(s),77.43(s), 77.11(s),76.79(s),74.11(s),63.66(s),57.96(s),55.75(s),53.90(s),35.22(s),34.63(s),34.20( d,J=11.6Hz),33.70(s),31.80(d,J=11.2Hz),30.30(s),29.51(d,J=2.8Hz),29.13(dd,J=9.6,6.8Hz), 27.12(d,J=2.8Hz),26.29(s),25.31(s),24.97(d,J=15.6Hz),22.66(s),22.37(s),14.02(d,J=15.2Hz) .

[0502] N. Compound SW-II-137-2

[0503] 1. Synthesis of compound 3

[0504] Compound 1 (500 mg, 2.06 mmol, 1.0 eq.), compound 2 (286 mg, 2.47 mmol, 1.2 eq.), Pd(PPh3)4 (119 mg, 0.1 mmol, 0.1 eq.), and K2CO3 (851 mg, 6.21 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL), and water (0.5 mL) was added. The mixture was then reacted at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting materials and the formation of the desired compound. The reaction was quenched with H2O (70 mL) and extracted with EA (80 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA = 5 / 1, v / v as the eluent to afford Compound 3 (420 mg, 87.5%) as a colorless oil.

[0505] 2. Synthesis of compound 4

[0506] Compound 3 (420 mg, 1.78 mmol, 1.0 eq.) was dissolved in THF (3.0 mL), and LAH (1 M, 7 mL, 2.0 eq.) was added dropwise at 0°C under nitrogen. The reaction was then allowed to react at room temperature for 2 hours. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and formation of the desired product. The reaction was quenched with HCl (1 M, 4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA = 5 / 1, v / v as the eluent to afford compound 4 (320 mg, 94%) as a colorless oil.

[0507] 3. Synthesis of Compound 6

[0508] Compound 4 (320 mg, 1.55 mmol, 1.0 eq.) was dissolved in DCM (4.0 mL), and compound 5 (416 mg, 1.86 mmol, 1.2 eq.), EDCI (594 mg, 3.11 mmol, 2.0 eq.), DIEA (802 mg, 6.21 mmol, 4.0 eq.), and DMAP (76 mg, 0.62 mmol, 0.4 eq.) were added. The reaction was then allowed to proceed overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete reaction of the starting materials and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 4-6. The product was then extracted with DCM (60 mL x 3). The organic phase was washed with saturated brine (2 x 35 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA=5 / 1, v / v) to give compound 6 (300 mg, 47.17%) as a colorless oil.

[0509] 4. Synthesis of SW-II-137-2

[0510] Compound 6 (167 mg, 0.41 mmol, 1.2 eq.), compound 7 (150 mg, 0.34 mmol, 1.0 eq.), KI (113 mg, 0.68 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL), and KCO (235 mg, 1.70 mmol, 5.0 eq.) was added. The reaction was allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete reaction of the starting materials and the formation of the desired product. The reaction was quenched with water (50 mL) and extracted with EA (60 mL x 3). The organic phase was dried over anhydrous NaSO, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10 / 1, v / v) as the eluent to afford the compound (105 mg, 40.3%, SW-II-137-2) as a pale yellow oil.

[0511] LCMS:Rt:1.660min;MS m / z(ELSD):772.4[M+H] + ;

[0512] HPLC: 98.38% purity, ELSD; RT = 8.743 min.

[0513] 1H NMR (400MHz, CDCl3) δ7.10 (d, J=8.8Hz, 4H), 5.04–4.74 (m, 1H), 4.08 (t, J=6 .4Hz,2H),3.58(t,J=5.2Hz,2H),2.65(dd,J=9.6,5.6Hz,4H),2.60–2.44(m, 6H),2.29(dd,J=16.4,7.6Hz,4H),2.01–1.88(m,2H),1.59(dt,J=9.2,7.2H z,6H),1.54–1.42(m,8H),1.39–1.11(m,41H),0.88(dt,J=11.8,6.0Hz,9H).

[0514] 13 C NMR(101MHz, CDCl3)δ173.86(s),173.63(s),140.59(s),138.34(s),128.45(s),128.24(s),77.36(s),77 .04(s),76.72(s),74.14(s),63.69(s),58.11(s),55.71(s),53.90(s),35.53(s),34.68(s),34.23(d,J=1 4.8Hz),31.82(d,J=11.6Hz),31.56(s),31.26(s),30.32(s),29.53(d,J=2.8Hz),29.19(dd,J=8.0,4.4Hz ), 27.20 (d, J = 2.4Hz), 26.64 (s), 25.33 (s), 25.02 (d, J = 15.6Hz), 22.62 (d, J = 11.6Hz), 14.08 (d, J = 8.0Hz).

[0515] O. Compound SW-II-137-3

[0516] 1. Synthesis of compound 3

[0517] To a mixture of compound 1 (11.8 g, 53 mmol, 1.2 eq.) and compound 2 (11.2 g, 44 mmol, 1 eq.) in DCM (110 mL) were added EDCI (16.9 g, 88 mmol, 2 eq.) and DMAP (2.1 g, 18 mmol, 0.4 eq.), followed by DIEA (22.7 g, 176 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 30 / 1) showed that compound 1 was consumed and the desired product was formed. The reaction mixture was extracted with ethyl acetate (200 mL) and washed with water (200 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 3 (7.391 g, 37%) as a colorless oil.

[0518] 2. Synthesis of compound 5

[0519] A mixture of compound 3 (7.391 mg, 16.07 mmol, 1 eq.) and compound 4 (29.4 g, 482.02 mmol, 30 eq.) in ethanol (2 mL) was stirred at 55 ° C under nitrogen for 16 hours. TLC (DCM / MeOH=10 / 1) showed that a new major spot was observed. The reaction mixture was extracted with ethyl acetate (100 mL) and washed with water (3×100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give compound 5 (3.695 g, 52%) as a yellow oil.

[0520] 3. Synthesis of Compound 8

[0521] To a mixture of compound 6 (1 g, 4.12 mmol, 1 eq.) and compound 7 (803 g, 6.17 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (269 mg, 0.41 mmol, 0.1 eq.) and potassium carbonate (1.7 g, 12.36 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 8 (568 mg, 56%) as a colorless oil.

[0522] 4. Synthesis of Compound 9

[0523] To a mixture of compound 8 (568 mg, 2.29 mmol, 1 eq.) in THF (6 mL) at 0°C under nitrogen was added lithium aluminum hydride (2.3 mL, 2.29 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2.3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 9 (541 mg, >100%) as a colorless oil without further purification.

[0524] 5. Synthesis of Compound 10

[0525] To a mixture of compound 9 (441 mg, 2 mmol, 1 eq.) and compound 1 (536 mg, 2.4 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (768 mg, 4 mmol, 2 eq.) and DMAP (98 mg, 0.8 mmol, 0.4 eq.), followed by DIEA (1.032 g, 8 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-10 / 1) to give compound 10 (372 mg, 44%) as a colorless oil.

[0526] 6. Synthesis of SW-II-137-3

[0527] To a mixture of compound 10 (150 mg, 0.353 mmol, 1 eq.) and compound 5 (156 mg, 0.353 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (244 mg, 1.765 mmol, 6 eq.) and potassium iodide (117 mg, 0.706 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-137-3 (56.17 mg, 20%) as a yellow oil.

[0528] LCMS:Rt:1.550min;MS m / z(ELSD):786.4[M+H] + ;

[0529] HPLC: 98.597% purity, ELSD; RT = 13.153 min.

[0530] 1 H NMR(400MHz, CDCl3)δ7.09(s,4H),4.92–4.78(m,1H),4.08(t,J=6.6Hz,2H),3.62(t,J=5.2Hz,2H),2.78–2.50(m,10H), 2.35–2.22(m,4H),2.00–1.88(m,2H),1.57(ddd,J=28.9,13.5,4.5Hz,14H),1.38–1.20(m,42H),0.88(t,J=6.8Hz,9H).

[0531] 13C NMR(101MHz, CDCl3)δ173.83(s),173.60(s),140.60(s),138.33(s),128.44(s),128.24(s),77.36 (s),77.04(s),76.72(s),74.15(s),63.69(s),57.95(s),55.83(s),53.95(s),35.56(s),34.65(s) ),34.21(d,J=13.0Hz),31.81(d,J=12.3Hz),31.54(s),30.32(s),29.52(d,J=3.1Hz),29.34–28.9 4(m),27.13(d,J=2.5Hz),26.31(s),25.33(s),24.99(d,J=15.7Hz),22.64(d,J=5.7Hz),14.11(s).

[0532] P. Compound SW-II-138-1

[0533] 1. Synthesis of Compound 2

[0534] Compound 1 (4 g, 16.46 mmol, 1.0 eq.) was dissolved in MeOH (40 mL), cooled to 0°C, and SOCl2 (3.9 g, 32.92 mmol, 2.0 eq.) was added dropwise. The reaction was then allowed to react at room temperature for 1 hour. TLC (PE / EA = 5 / 1) showed that the starting material had been consumed and the desired product had formed. The system was directly evaporated to dryness under reduced pressure. NaHCO3 solution (70 mL) was added to the residue, and the mixture was extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford Compound 2 (4.1 mg, 95%) as a yellow oil.

[0535] 2. Synthesis of compound 4

[0536] Compound 2 (500 mg, 1.95 mmol, 1.0 eq.), compound 3 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.19 mmol, 0.1 eq.), and K2CO3 (808 g, 5.85 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. The mixture was then reacted at 110°C under nitrogen for 3 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with H2O (70 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford compound 4 (320 mg, 70%) as a yellow oil.

[0537] 3. Synthesis of compound 5

[0538] Compound 4 (300 mg, 1.28 mmol, 1.0 eq.) was dissolved in THF (4.0 mL), and LAH (97 mg, 2.56 mmol, 2.0 eq.) was added at 0°C. The reaction was then allowed to react at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and the formation of the desired product. The reaction was quenched with 1 M HCl (4 mL) and H₂O (10 mL), and extracted with EA (50 mL x 3). The organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford Compound 5 (224 mg, 84.8%) as a yellow oil.

[0539] 4. Synthesis of Compound 7

[0540] Compound 7 (224 mg, 1.09 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL), and compound 6 (290 mg, 1.30 mmol, 1.2 eq.), EDCI (415 mg, 2.17 mmol, 2.0 eq.), DIEA (561 mg, 4.35 mmol, 4.0 eq.), and DMAP (53 mg, 0.43 mmol, 0.4 eq.) were added. The reaction was then allowed to proceed overnight at room temperature under nitrogen. TLC (PE / EA = 30 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 4-6, followed by extraction with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as eluent to give compound 7 (208 mg, 46.7%) as a colorless oil.

[0541] 5. Synthesis of SW-II-138-1

[0542] Compound 10 (110 mg, 0.25 mmol, 1 eq.), compound 7 (153 mg, 0.37 mmol, 1.5 eq.), KI (83 mg, 0.50 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL) and KCO (172 mg, 1.25 mmol, 5.0 eq.) was added. The reaction was allowed to proceed overnight at 90°C under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then dried under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the compound (65 mg, 32%, SW-II-138-1) as a light yellow oil.

[0543] LCMS:Rt:1.684min;MS m / z(ELSD):772.4[M+H] + ;

[0544] HPLC: 96.56% purity, ELSD; RT = 6.346 min.

[0545] 11H NMR (400 MHz, CDCl3) δ 7.09 (s, 4H), 4.86 (s, 1H), 4.09 (d, J = 6.0 Hz, 2H), 3.97 (s, 2H), 3.07 (d, J = 38.8 Hz, 6H), 2.69–2.51 (m, 4H), 2.28 (td, J = 7.3, 3.6 Hz, 4H), 1.79 (s, 4H), 1.70–1.46 (m, 16H), 1.42–1.17 (m, 37H), 0.90 (dt, J = 13.6, 7.2 Hz, 9H).

[0546] 13 13C NMR (101 MHz, CDCl3) δ 173.80 (s), 173.53 (s), 140.32 (s), 139.13 (s), 128.28 (d, J = 13.6 Hz), 77.43 (s), 77.11 (s), 76.80 (s), 74.21 (s), 64.22 (s), 56.85 (s), 55.98 (s), 53.93 (s), 35.22 (s), 35.01 (s), 34.54 (s), 34.14 (d, J = 5.6 Hz), 33.71 (s), 31.85 (s), 29.50 (d, J = 2.8 Hz), 29.22 (s), 29.12–28.60 (m), 28.26 (s), 27.78 (s), 26.70 (d, J = 4.4 Hz), 25.31 (s), 24.82 (d, J = 17.6 Hz), 24.28 (s), 22.65 (s), 22.37 (s), 14.03 (d, J = 15.2 Hz).

[0547] Q.SW-II-138-2

[0548] 1. Synthesis of Compound

[0549] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (271 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.20 mmol, 0.1 eq.), and K2CO3 (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL). The mixture was then reacted at 110°C under nitrogen for 3 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with water (70 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as the eluent to afford compound 3 (320 mg, 70%) as a colorless oil.

[0550] 2. Synthesis of compound 4

[0551] Compound 3 (320 mg, 1.29 mmol, 1.0 eq.) was dissolved in THF (3.0 mL) and LAH (67 mg, 1.77 mmol, 2.0 eq.) was added at 0°C. The mixture was then reacted at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl (2 mL) and H₂O (10 mL) and extracted with EA (50 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as the eluent to afford compound 4 (180 mg, 64%) as a colorless oil.

[0552] 3. Synthesis of Compound 6

[0553] Compound 4 (180 mg, 0.82 mmol, 1.0 eq.) was dissolved in DCM (3.0 mL) and compound 5 (245 mg, 1.10 mmol, 1.2 eq.), EDCI (347 mg, 1.82 mmol, 2.0 eq.), DIEA (470 mg, 3.63 mmol, 4.0 eq.), and DMAP (45 mg, 0.36 mmol, 0.4 eq.) were added. The reaction was then allowed to proceed under nitrogen at room temperature overnight. TLC (PE / EA = 30 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction was quenched with 1 M HCl solution and adjusted to pH 5-6, followed by extraction with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-30:1, v / v) as eluent to give compound 6 (220 mg, 63.6%) as a colorless oil.

[0554] 4. Synthesis of SW-II-138-2

[0555] Compound 6 (158 mg, 0.37 mmol, 1.5 eq.) and compound 7 (110 mg, 0.25 mmol, 1.0 eq.), KI (83 mg, 0.50 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL), and KCO (172 mg, 1.25 mmol, 5.0 eq.) was added. The reaction was then allowed to proceed at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then evaporated to dryness under reduced pressure, and the residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the desired product (100 mg, 51%, SW-II-138-2) as a colorless oil.

[0556] LCMS:Rt:1.834min; MS m / z(ELSD):786.4[M+H] + ;

[0557] HPLC: 99.20% purity, ELSD; RT = 7.990 min.

[0558] 11H NMR (400 MHz, CDCl3) δ 7.00 (s, 4H), 4.88–4.73 (m, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.81–3.54 (m, 2H), 3.00–2.81 (m, 2H), 2.81–2.65 (m, 4H), 2.50 (dd, J = 16.4, 8.4 Hz, 4H), 2.20 (td, J = 7.6, 3.2 Hz, 4H), 1.56 (ddd, J = 18.4, 10.4, 5.2 Hz, 13H), 1.43 (d, J = 5.6 Hz, 4H), 1.34–1.07 (m, 40H), 0.81 (dt, J = 11.2, 5.6 Hz, 9H).

[0559] 13 13C NMR (101 MHz, CDCl3) δ 173.78 (s), 173.52 (s), 140.32 (s), 139.11 (s), 128.25 (d, J = 11.6 Hz), 77.49 (s), 77.17 (s), 76.85 (s), 74.14 (s), 64.17 (s), 57.25 (s), 55.82 (s), 53.85 (s), 35.50 (s), 35.01 (s), 34.56 (s), 34.14 (d, J = 7.2 Hz), 31.84 (s), 31.53 (s), 31.23 (s), 29.49 (d, J = 2.8 Hz), 29.21 (s), 28.94 (dd, J = 6.4, 4.4 Hz), 28.25 (s), 27.77 (s), 26.84 (d, J = 4.4 Hz), 25.30 (s), 25.25–24.59 (m), 22.59 (d, J = 11.2 Hz), 14.05 (d, J = 7.6 Hz).

[0560] R.SW-II-138-3

[0561] 1. Synthesis of Compound 3

[0562] Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (305 mg, 2.34 mmol, 1.2 eq.), Pd(PPh3)4 (225 mg, 0.20 mmol, 0.1 eq.), and K2CO3 (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. The reaction was then incubated at 110°C for 3 hours under nitrogen. TLC (PE / EA = 5:1) indicated complete consumption of the starting material and formation of the desired compound. The reaction was quenched with water (80 mL) and extracted with EA (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 40 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford compound 3 (260 mg, 51.3%) as a colorless oil.

[0563] 2. Synthesis of compound 4

[0564] Compound 3 (260 mg, 0.99 mmol, 1.0 eq.) was dissolved in THF (4.0 mL), and LAH (75 mg, 1.98 mmol, 2.0 eq.) was added at 0°C. The reaction was then allowed to proceed at room temperature under nitrogen for 2 hours. TLC (PE / EA = 5 / 1) indicated complete reaction of the starting material and the formation of the desired compound. The reaction was quenched with 1 M HCl (4 mL) and H₂O (20 mL), and extracted with EA (50 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-5:1, v / v) as the eluent to afford compound 4 (230 mg, 98%) as a colorless oil.

[0565] 3. Synthesis of Compound 6

[0566] Compound 4 (240 mg, 1.03 mmol, 1.0 eq.) was dissolved in DCM (4.0 mL), and compound 5 (275 mg, 1.23 mmol, 1.2 eq.), EDCI (392 mg, 2.07 mmol, 2.0 eq.), DIEA (530 mg, 4.10 mmol, 4.0 eq.), and DMAP (50 mg, 0.41 mmol, 0.4 eq.) were added sequentially. The reaction was then allowed to proceed overnight at room temperature under nitrogen. TLC (PE / EA = 20 / 1) indicated complete consumption of the starting material and formation of the desired compound. The reaction was quenched with HCl (1 M) and the pH was adjusted to 5-6. The mixture was then extracted with DCM (80 mL x 3). The combined organic phases were washed with saturated brine (2 x 30 mL), dried over anhydrous Na2SO4, filtered, and dried under reduced pressure. The residue was purified on a silica gel column using PE / EA (1 / 0-20:1, v / v) as eluent to give compound 6 (180 mg, 40.9%) as a colorless oil.

[0567] 4. Synthesis of SW-II-138-3

[0568] Compound 6 (164 mg, 0.37 mmol, 1 eq.), compound 7 (110 mg, 0.24 mmol, 1.0 eq.), KI (83 mg, 0.49 mmol, 2.0 eq.), and CPME (2 mL) were dissolved in MeCN (2 mL) and KCO (172 mg, 1.24 mmol, 5.0 eq.) was added. The mixture was then reacted at 90°C overnight under nitrogen. TLC (DCM / MeOH = 10 / 1) indicated complete consumption of the starting material and formation of the desired product. The reaction mixture was then dried under reduced pressure. The residue was purified on a silica gel column using DCM / MeOH (1 / 0-10:1, v / v) as the eluent to afford the desired product (108 mg, 52.76%, SW-II-138-3) as a colorless oil.

[0569] LCMS:Rt:2.007min;MS m / z(ELSD):800.4[M+H] + ;

[0570] HPLC: 97.95% purity, ELSD; RT = 9.455 min.

[0571] 1H NMR (400MHz, CDCl3) δ7.08(s,4H),4.86(p,J=6.4Hz,1H),4.08(s,2H),3.60(t,J=5.2Hz,3H),2.76–2.42( m,10H),2.28(td,J=7.6,2.8Hz,4H),1.70–1.42(m,18H),1.28(d,J=20.0Hz,41H),0.88(t,J=6.8Hz,9H).

[0572] 13 C NMR(101MHz, CDCl3)δ173.85(s),173.59(s),140.39(s),139.15(s),128.27(d,J=12.0Hz),77.38(s),7 7.07(s),76.75(s),74.13(s),64.17(s),58.06(s),55.75(s),53.92(s),35.57(s),35.03(s),34.66(s) ,34.22(d,J=13.2Hz),31.81(d,J=12.4Hz),31.54(s),29.52(d,J=2.9Hz),29.34–28.95(m),28.29(s), 27.79 (s), 27.16 (d, J = 3.6Hz), 26.50 (s), 25.32 (s), 24.99 (d, J = 17.6Hz), 22.64 (d, J = 5.6Hz), 14.10 (s).

[0573] S. Compound SW-II-139-1

[0574] 1. Synthesis of compound 3

[0575] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (852 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (691 mg, 68%) as a colorless oil.

[0576] 2. Synthesis of compound 4

[0577] To a mixture of compound 3 (691 mg, 2.95 mmol, 1 eq.) in THF (7 mL) at 0°C under nitrogen was added lithium aluminum hydride (3 mL, 2.95 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (547 mg, 90%) as a colorless oil without further purification.

[0578] 3. Synthesis of Compound 6

[0579] To a mixture of compound 4 (447 mg, 2.17 mmol, 1 eq.) and compound 5 (581 mg, 2.6 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (833 mg, 4.34 mmol, 2 eq.) and DMAP (106 mg, 0.87 mmol, 0.4 eq.), followed by DIEA (1.12 g, 8.68 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (455 mg, 51%) as a colorless oil.

[0580] 4. Synthesis of SW-II-139-1

[0581] To a mixture of compound 6 (150 mg, 0.365 mmol, 1 eq.) and compound 7 (161 mg, 0.365 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (252 mg, 1.825 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-139-1 (54.53 mg, 19%).

[0582] LCMS:Rt:1.521min;MS m / z(ELSD):772.4[M+H] + ;

[0583] HPLC: 99.637% purity, ELSD; RT = 12.347 min.

[0584] 1 H NMR (400MHz, CDCl3) δ7.20(t,J=7.7Hz,1H),7.03(t,J=6.8Hz,3H),4.94–4.78(m,1H),4.27(t,J=7.2Hz,2H),3.65(t,J=5.1Hz,2H),2.90(t,J=7.2H z,2H),2.73(t,J=4.9Hz,2H),2.67–2.41(m,6H),2.28(td,J=7.5,2.7Hz, 4H),1.67–1.45(m,14H),1.41–1.19(m,42H),0.88(dd,J=7.9,5.7Hz,9H).

[0585] 13C NMR(101MHz, CDCl3)δ173.65(d,J=11.3Hz),143.17(s),137.67(s),129.04(s),128.34(s),126.61(s),126 .11(s),77.30(d,J=11.6Hz),77.04(s),76.72(s),74.16(s),64.85(s),57.88(s),55.93(s),53.97(s),35 .94(s),35.13(s),34.64(s),34.20(d,J=10.5Hz),31.80(d,J=13.7Hz),31.50(s),29.52(d,J=2.9Hz),29. 34–28.92(m),27.08(d,J=3.9Hz),26.10(s),25.33(s),25.05(s),24.82(s),22.64(d,J=6.5Hz),14.11(s).

[0586] T. Compound SW-II-139-2

[0587] 1. Synthesis of compound 3

[0588] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (668 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dtbpf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (605 mg, 67%) as a colorless oil.

[0589] 2. Synthesis of compound 4

[0590] To a mixture of compound 3 (605 mg, 2.94 mmol, 1 eq.) in THF (7 mL) was added lithium aluminum hydride (3 mL, 2.94 mmol, 1 M in THF, 1 eq.) at 0°C under nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (534 mg, >100%) as a colorless oil without further purification.

[0591] 3. Synthesis of Compound 6

[0592] To a mixture of compound 4 (434 mg, 2.44 mmol, 1 eq.) and compound 5 (652 mg, 2.93 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (937 mg, 4.88 mmol, 2 eq.) and DMAP (119 mg, 0.976 mmol, 0.4 eq.), followed by DIEA (1.259 g, 9.76 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (355 mg, 38%) as a colorless oil.

[0593] 4. Synthesis of SW-II-139-2

[0594] To a mixture of compound 6 (122 mg, 0.319 mmol, 1 eq.) and compound 7 (140 mg, 0.319 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (220 mg, 1.595 mmol, 5 eq.) and potassium iodide (106 mg, 0.638 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-139-2 (45.48 mg, 19%) as a yellow oil.

[0595] LCMS:Rt:1.346min; MS m / z(ELSD):744.3[M+H] + ;

[0596] HPLC: 97.994% purity, ELSD; RT = 11.235 min.

[0597] 1 H NMR (400MHz, CDCl3) δ7.20(t,J=7.8Hz,1H),7.03(t,J=7.6Hz,3H),4.91–4.81(m,1H),4.27(t,J=7.2Hz,2H),3.89–3.75(m ,2H),2.99–2.79(m,7H),2.64–2.48(m,2H),2.28(td,J=7.5,3.1Hz,4H),1.74–1.08(m,53H),0.90(dt,J=13.6,7.2Hz,9H).

[0598] 13 C NMR (101MHz, CDCl3) δ173.60 (d, J = 11.7Hz), 143.13 (s), 137.65 (s), 129.06 (s), 128.34(s),126.64(s),126.11(s),77.30(d,J=11.4Hz),77.04(s),76.72(s),74.22(s),64.8 8(s),57.28(s),56.55(s),54.11(s),35.60(s),35.12(s),34.56(s),34.15(d,J=4.0Hz),33. 68(s),31.86(s),29.52(d,J=2.8Hz),29.24(s),28.91(dd,J=7.0,4.2Hz),26.81(d,J=3.9Hz) ,25.33(s),25.12–24.98(m),24.83(d,J=22.2Hz),22.67(s),22.40(s),14.04(d,J=14.4Hz).

[0599] U. Compound SW-II-140-1

[0600] 1. Synthesis of compound 3

[0601] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (852 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dppf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (748 mg, 73%) as a colorless oil.

[0602] 2. Synthesis of compound 4

[0603] To a mixture of compound 3 (748 mg, 3.2 mmol, 1 eq.) in THF (8 mL) at 0°C under nitrogen was added lithium aluminum hydride (3.2 mL, 3.2 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (3 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (493 mg, 75%) as a colorless oil without further purification.

[0604] 3. Synthesis of Compound 6

[0605] To a mixture of compound 4 (393 mg, 1.91 mmol, 1 eq.) and compound 5 (511 mg, 2.29 mmol, 1.2 eq.) in DCM (5 mL) were added EDCI (733 mg, 3.82 mmol, 2 eq.) and DMAP (93 mg, 0.76 mmol, 0.4 eq.), followed by DIEA (986 mg, 7.64 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (327 mg, 42%) as a colorless oil.

[0606] 4. Synthesis of SW-II-140-1

[0607] To a mixture of compound 6 (150 mg, 0.365 mmol, 1 eq.) and compound 7 (161 mg, 0.365 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (302 mg, 2.19 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C under nitrogen overnight. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give a yellow oily compound SW-II-140-1 (180 mg, 64%).

[0608] LCMS:Rt:1.568min; MS m / z(ELSD):772.4[M+H] + ;

[0609] HPLC: 98.053% purity, ELSD; RT = 8.702 min.

[0610] 1 H NMR (400MHz, CDCl3) δ7.23–7.05(m,4H),4.95–4.79(m,1H),4.25(t,J=7.4Hz,2H),3.62(t,J=4.8Hz,2H),2.96(dd,J=15.4,8.0 Hz,2H),2.74–2.49(m,8H),2.28(dd,J=14.2,7.2Hz,4H),1.67–1.44(m,14H),1.41–1.20(m,42H),0.90(dt,J=13.2,7.1Hz,9H).

[0611] 13C NMR (101MHz, CDCl3) δ173.68 (d, J = 10.2Hz), 141.26 (s), 135.23 (s), 129.73 (s), 129.37 (s), 126. 72(s),125.92(s),77.35(s),77.03(s),76.71(s),74.17(s),64.52(s),57.99(s),55.87(s),53 .94(s),34.66(s),34.21(d,J=11.5Hz),32.75(s),31.83(d,J=9.8Hz),31.32(s),29.65–28.88( m), 27.15 (d, J = 3.7Hz), 26.35 (s), 25.33 (s), 25.07 (s), 24.83 (s), 22.66 (d, J = 3.4Hz), 14.12 (s).

[0612] V.SW-II-140-2

[0613] 1. Synthesis of compound 3

[0614] To a mixture of compound 1 (1 g, 4.37 mmol, 1 eq.) and compound 2 (668 g, 6.55 mmol, 1.5 eq.) in 1,4-dioxane / water (10 mL / 1 mL) were added Pd(dppf)Cl2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.). The mixture was stirred at 100°C overnight under nitrogen. TLC (PE / EA = 20 / 1) indicated the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography eluting with PE / EA (1 / 0-20 / 1) to give compound 3 (406 mg, 45%) as a colorless oil.

[0615] 2. Synthesis of compound 4

[0616] To a mixture of compound 3 (406 mg, 1.97 mmol, 1 eq.) in THF (5 mL) at 0°C under nitrogen was added lithium aluminum hydride (2 mL, 1.97 mmol, 1 M in THF, 1 eq.). The mixture was stirred at room temperature for 3 hours. TLC (PE / EA = 5 / 1) indicated completion of the reaction and a new major spot was observed. The mixture was quenched with water (2 mL) and treated with 2N hydrochloric acid to adjust the pH between 6 and 7, extracted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford compound 4 (341 mg, 97%) as a colorless oil without further purification.

[0617] 3. Synthesis of Compound 6

[0618] To a mixture of compound 4 (241 mg, 1.35 mmol, 1 eq.) and compound 5 (361 mg, 1.62 mmol, 1.2 eq.) in DCM (3 mL) were added EDCI (518 mg, 2.7 mmol, 2 eq.) and DMAP (66 mg, 0.54 mmol, 0.4 eq.), followed by DIEA (697 mg, 5.4 mmol, 4 eq.). The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether / ethyl acetate = 15 / 1) showed the formation of the desired product. The reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluted with petroleum ether / ethyl acetate (1 / 0-20 / 1) to give compound 6 (185 mg, 32%) as a colorless oil.

[0619] 4. Synthesis of SW-II-140-2

[0620] To a mixture of compound 6 (185 mg, 0.483 mmol, 1 eq.) and compound 7 (213 mg, 0.483 mmol, 1 eq.) in CPME / CH3CN (2 mL / 2 mL) were added potassium carbonate (400 mg, 2.898 mmol, 6 eq.) and potassium iodide (160 mg, 0.966 mmol, 2 eq.). After addition, the mixture was stirred at 90 ° C overnight under nitrogen. TLC (DCM / MeOH=10 / 1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with DCM / MeOH (1 / 0-10:1, v / v) to give SW-II-140-2 (161 mg, 45%) as a yellow oil.

[0621] LCMS:Rt:1.696min;MS m / z(ELSD):744.3[M+H] + ;

[0622] HPLC: 94.658% purity, ELSD; RT = 5.938 min.

[0623] 1 H NMR (400MHz, CDCl3) δ7.22–7.03(m,4H),4.94–4.78(m,1H),4.25(t,J=7.3Hz,2H),3.70–3.54(m,2H),2.96(t,J =7.4Hz,2H),2.77–2.41(m,8H),2.28(dd,J=14.3,7.1Hz,4H),1.65–1.18(m,52H),0.91(dt,J=13.3,7.1Hz,9H).

[0624] 13 C NMR (101MHz, CDCl3) δ173.67 (d, J = 10.8Hz), 141.22 (s), 135.23 (s), 129.73 (s), 129.39 (s), 126.72 (s), 12 5.92(s),77.36(s),77.04(s),76.72(s),74.17(s),64.52(s),57.92(s),55.92(s),53.96(s),34.66(s),3 4.21(d,J=11.2Hz),33.51(s),32.44(s),31.83(d,J=9.3Hz),29.53(d,J=2.9Hz),29.14(dd,J=11.3,8.5H z), 27.12 (d, J = 4.1Hz), 26.23 (s), 25.33 (s), 25.06 (s), 24.82 (s), 22.73 (d, J = 9.9Hz), 14.08 (d, J = 8.8Hz).

[0625] Example 2 Comparison of physicochemical properties and expression levels of LPP preparations with different lipid to lipid ratios for mucosal administration

[0626] In this example, a four-lipid LPP formulation containing luciferase mRNA (encoding sequence as shown in SEQ ID NO: 1) was prepared using the formulations in Tables 1, 2, and 3 for mucosal administration. Three rounds of comparisons of physicochemical properties and in vivo expression levels were performed on the prepared LPP formulations to screen for lipid-to-lipid ratios suitable for mucosal administration. An MC3 LNP formulation was used as a positive control.

[0627] 2.1 Preparation of lipid nanoparticle (LNP-mRNA) formulations

[0628] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0629] Preparation of lipid solution: MC3:phospholipid:cholesterol:PEG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a 6 mg / mL lipid solution.

[0630] Preparation of LNPs: Using microfluidic technology (Maianna (Shanghai) Technology Co., Ltd., model: Inano D), the lipid solution and the mRNA aqueous solution were mixed under the following conditions: volume = 4.0 mL; flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, to obtain an LNP-mRNA solution.

[0631] Centrifugal Ultrafiltration: The LNP-mRNA solution was added to an ultrafiltration tube and concentrated by centrifugation (3000 rpm, 60 min, 4°C) until the ethanol content was <0.5%. The LNP mRNA concentration was then adjusted to 0.2 mg / mL. This resulted in the LNP-mRNA formulation designated MC3 LNP.

[0632] 2.2 Preparation of lipid polyplex (LPP-mRNA) preparation

[0633] Preparation of lipid mixture: M5, phospholipids, cholesterol and PEG lipids were dissolved in ethanol solution according to the lipids and lipid ratios shown in Table 1, Table 2 and Table 3 to prepare a 6 mg / mL lipid mixture.

[0634] Preparation of mRNA aqueous solution: Luciferase mRNA (coding sequence shown in SEQ ID NO: 1) was diluted with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0635] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.125 mg / mL.

[0636] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: mass ratio = 4 (mRNA solution): 1 (protamine solution), flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, room temperature.

[0637] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: mass ratio = 20 (core nanoparticle solution): 1 (lipid mixture), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, room temperature, to obtain LPP-mRNA solution.

[0638] Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration to remove ethanol (centrifugal force 3000 rpm, centrifugation time 60 min, temperature 4°C), ultrafiltration was performed until the ethanol content was <0.5%, and the LPP mRNA concentration was adjusted to 0.2 mg / mL. The prepared samples were numbered IN-1-1, IN-1-2, IN-1-3, IN-1-4, IN-1-5, IN-1-6, IN-1-7, IN-1-8, IN-1-9, IN-1-10, IN-1-11, IN-1-12, IN-1-13, IN-1-14, IN-1-15, IN-1-16, IN-2-1, IN-2-2, IN-2-3, IN-2-4, IN-1-5 N-2-5, IN-2-6, IN-2-7, IN-2-8, IN-2-9, IN-2-10, IN-2-11, IN-2-12, IN-2-13, IN-2-14, IN- 2-15, IN-2-16, IN-3-1, IN-3-4, IN-3-5, IN-3-6, IN-3-7, IN-3-9, IN-3-11 and IN-3-12 LPP formulations.

[0639] 2.3 Examination of the physicochemical properties of LPP preparations with different lipid to lipid ratios

[0640] The prepared LPP preparations were tested for their physical and chemical properties to screen for lipid-to-lipid ratios suitable for mucosal administration. The specific testing methods are as follows:

[0641] Particle size detection: 50 μL of the LPP sample prepared in Example 2.2 was taken and diluted with 950 mL of purified water to obtain a diluted LPP sample, which was then placed in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

[0642] Encapsulation efficiency detection: The encapsulation efficiency of mRNA in the LPP solution prepared in Example 2.2 was detected using Quant-iT RiboGreen RNA reagent (Thermo Scientific). First, the amount of mRNA free outside the LPP particles in the LPP solution was detected, and the LPP solution was diluted with nuclease-free water and 1xTE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.5). 100 μL of each diluted sample was transferred to a 96-well plate, and 100 μL of 200-fold diluted RiboGreen RNA reagent was added thereto. After the 96-well plate was placed on a plate mixer and mixed at room temperature for 10 minutes, the fluorescence value was read by a Bio-Tek Synergy I plate reader (BioTek). The standard sample was treated in the same manner. A calibration curve of fluorescence and mRNA concentration was drawn by linear regression, from which the mRNA content of the sample free from the LPP particles was calculated.

[0643] To detect the total mRNA content inside and outside the LPP sample particles, the LPP solution was diluted with nuclease-free water, then vortexed with an equal volume of 2% Triton X-100 and incubated at room temperature for 10 minutes to destroy the LPP structure and release the mRNA encapsulated in the LPP particles. 100 μL of each sample was transferred to a 96-well plate, and 100 μL of 200-fold diluted RiboGreen RNA reagent was added thereto. After the 96-well plate was placed on a plate mixer and mixed at room temperature for 5 minutes, the fluorescence value was read by a Bio-Tek Synergy I plate reader (BioTek). The standard sample was treated in the same manner. A calibration curve of fluorescence and mRNA concentration was drawn by linear regression, from which the total mRNA content inside and outside the LPP sample particles was calculated.

[0644] The encapsulation efficiency of the LPP solution was defined as the percentage of mRNA encapsulated inside the LPP particles to the total mRNA in the test sample.

[0645] Polydispersity index (PDI) measurement: The polydispersity index (PDI) of the LPP solution was determined using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0646] Purity detection: The mRNA purity in the LPP solution prepared in Example 2.2 was detected using a Q-Analyzer and RNA Carisge Kit (RNA Low Marker (5x); Dilution Buffer (10x); Separation Buffer (10x); Mininer oil). Take 50 μL of sample, first dilute the LPP solution with an equal volume of 1x Dilution Buffer, and then add an equal volume of 2% Triton; add RNase inhibitor (Invitrogen) and 0.1 mg / mL sodium heparin solution in sequence, vortex mix, and let stand for 5 minutes. After standing, place the sample in a 70°C water bath for 20 minutes, then quickly transfer to a 4°C refrigerator to cool for 5-10 minutes; take out 50 μL of sample and add it to eight tube strips, and add 20 μL Mininer oil on top of each tube. The sample purity was detected by capillary electrophoresis Qsep100 method.

[0647] The results of the physicochemical property tests are shown in Tables 1, 2, and 3. Different lipids and lipid ratios will affect the physicochemical properties of the lipid composition, such as polydispersity index, particle size, and encapsulation efficiency. In the first round of screening, the lipid ratio and lipids are shown in Table 1, and the screening range is M5: 30-60%; phospholipids (DOPE, DSPC): 5-20%; PEG (DMG-PEG, DSPE-PEG, ACL-0159): 1.25-10%; cholesterol: 15-65%; in the second round of screening, the lipid ratio and lipids are shown in Table 2, and the screening range is M5: 35-50%; phospholipids (DOPE): 10-30%; PEG (DMG-PEG, DSPE-PEG): 1.25-10%; cholesterol: 15-55%; in the third round of screening, the lipid ratio and lipids are shown in Table 3, and the screening range is M5: 37.5-42.5%; phospholipids (DOPE): 25-35%; PEG (DMG-PEG): 3.75-7.5%; cholesterol: 15-30%. It was observed that when M5: 37.5-42.5%; phospholipid (DOPE): 25-35%; PEG (DMG-PEG): 3.75-7.5%; cholesterol: 15-30%, the particle size, polydispersity index and encapsulation efficiency were excellent and stable.

[0648] Table 1. First round of mucosal drug delivery prescription screening

[0649] Table 2. Second round of mucosal drug delivery prescription screening

[0650] Table 3. The third round of mucosal drug delivery prescription screening

[0651] 2.4 In vivo expression of LPP preparations with different lipid to lipid ratios

[0652] While the prepared LPP preparation was subjected to three rounds of physicochemical property testing as in Example 2.3, the prepared LPP preparation was also tested for in vivo expression to screen for lipids and lipid ratios suitable for mucosal administration. The specific testing methods are as follows:

[0653] During the three rounds of in vivo expression detection, 6-8 week old female BALB / c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were anesthetized with sodium pentobarbital (70 mg / kg) and then administered the prepared LPP solution by nasal instillation (3 mice per group). During each round of screening, two groups of mice were administered MC3 LNP solution and PBS solution (negative control) to the nasal mucosa. Each LPP solution or LNP solution administered contained 2 μg of luciferase mRNA (10 μL). 6 hours and 24 hours after administration, the mice were intraperitoneally injected with 3 mg of D-luciferin substrate (Mao Kang Biotechnology). Ten minutes after substrate injection, the mice were imaged in vivo using a Xenogen IVIS-200 imaging system to detect luciferase expression in vivo.

[0654] The results showed that all LPP formulations expressed luciferase exclusively in the nasal cavity, with the highest luciferase expression observed when the M5 content was 37.5-42.5%, the phospholipid (DOPE) content was 25-35%, the PEG (DMG-PEG) content was 3.75-5%, and the cholesterol content was 15-30%. The AUC of the IN-3-11 LPP formulation, which showed the highest luciferase expression in the third round, was approximately 35 times that of the IN-1-4 formulation, which showed the highest luciferase expression in the first round, and approximately 100 times that of the MC3 LNP formulation.

[0655] Among them, the luciferase expression in mice 6 hours after administration in the third round is shown in Figures 1A and 1B. All LPP preparations were expressed only in the nasal cavity, indicating low systemic toxicity, and among them, the IN-3-11 LPP preparation had the highest luciferase expression.

[0656] Based on the physicochemical property test results in Example 2.3 and the in vivo expression results in this example, the optimized lipid-to-lipid ratio range suitable for mucosal administration was confirmed to be: cationic lipid (M5 in this example): 37.5-42.5%; phospholipid (DOPE): 25-35%; PEG (DMG-PEG): 3.75-5%; and cholesterol: 15-30%. Based on these results, the formulations IN-2-6, IN-3-4, IN-3-5, IN-3-11, and IN-3-12 LPP formulations, which showed high luciferase expression levels in the nasal cavity, were selected for further testing.

[0657] Example 3 Application of LPP preparations for mucosal administration in COVID-19 vaccine treatment

[0658] In this example, mice were immunized using different immunization schemes to test the application of the preferred LPP preparation in Example 2 in the treatment of COVID-19 vaccines.

[0659] 3.1 Preparation of Lipid Polyplex (LPP-mRNA) Preparation

[0660] Preparation of lipid mixture: M5, phospholipids, cholesterol and PEG lipids were dissolved in ethanol solution according to the recipe of IN-2-6, IN-3-11 and IN-3-12 LPP preparations to prepare a 6 mg / mL lipid mixture.

[0661] Preparation of mRNA aqueous solution: Dilute COVID-19 mRNA (coding sequence as shown in SEQ ID NO: 2) with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0662] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.125 mg / mL.

[0663] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: mass ratio = 4 (mRNA solution): 1 (protamine solution), flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, room temperature.

[0664] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: mass ratio = 20 (core nanoparticle solution): 1 (lipid mixture), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, room temperature, to obtain LPP-mRNA solution.

[0665] Centrifugal Ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration to remove ethanol (3000 rpm, 60 min, 4°C). Ultrafiltration was performed to a concentration of <0.5% ethanol, and the LPP mRNA concentration was adjusted to 1 mg / mL. These LPP preparations were designated IN-2-6 / Covid-19, IN-3-11 / Covid-19, and IN-3-12 / Covid-19.

[0666] The B11 / Covid-19 LPP preparation was prepared by the above method with a lipid ratio of M5:phospholipid:cholesterol:PEG of 40:15:43.5:1.5, and the B11 / Covid-19 LPP mRNA concentration was adjusted to 0.1 mg / mL.

[0667] 3.2 Immune effects of different immunization regimens

[0668] Mice were immunized using the immunization protocol shown in Figure 2A and Table 4. Female BALB / c mice aged 6-8 weeks (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were divided into 6 groups (n=5). On days 0 and 14 (2 weeks after the initial immunization), mice in groups 2, 3, 4, 5, and 6 were immunized with a B11 / Covid-19 LPP formulation containing 3 μg of mRNA by intramuscular injection. Six weeks after the initial immunization, the third immunization was performed, and mice in groups 2, 3, 4, 5, and 6 were administered PBS, B11 / Covid-19 LPP formulation (intramuscular route), IN-2-6 / Covid-19 LPP formulation (nasal instillation), IN-3-11 / Covid-19 LPP formulation (nasal instillation), and IN-3-12 / Covid-19 LPP formulation (nasal instillation), respectively; Group 1 mice did not undergo the first two immunizations and were administered PBS six weeks after the initial immunization as a control. At 10 weeks after the initial immunization, the mice were euthanized, and the lung, spleen, and nasal lavage fluid (obtained by lavage with 0.7 mL of PBS) were collected for subsequent testing.

[0669] ELISpot was used to detect immune cell responses in splenocytes and lung cells, and ELISA was used to detect antibody levels in serum and nasal lavage fluid. The specific detection methods are as follows:

[0670] Enzyme-linked immunospot (ELISpot) assay

[0671] Mouse IFN-γ ELISpot assay was performed using the IFN-γ ELISpotPLUS kit (Mabtech, 3321-4APT-10) according to the manufacturer's instructions. Briefly, the plate was blocked with 1640 medium containing 10% FBS, 200 μL / well, and placed in a cell culture incubator for more than 1 hour. Lung cells or spleen cells were plated at 3×10 6 Cells were plated / well and stimulated in vitro with 100 μL / well of S protein peptide. 100 μL of culture medium was added as a negative control, and 100 μL of PMA + Ionomycine was added as a positive control. The cells were incubated at 37°C, 5% CO2 for 20 hours. Afterwards, cells were detected using a biotinylated IFN-γ detection antibody and streptavidin-alkaline phosphatase (ALP). Color was developed using BCIP / NBT-plus (5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium-plus) substrate, and cells were counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).

[0672] Enzyme-linked immunosorbent assay (ELISA)

[0673] When performing ELISA testing of animal serum / lavage fluid, the S protein antigen (SARS-CoV-2 (2019-nCoV) Spike Protein, purchased from Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.) was diluted to 2 μg / mL using ELISA coating buffer, and 96-well plates were coated with 100 μL per well, 200 ng / well of antigen, and incubated overnight at 4°C. After incubation at 4°C overnight, the plates were washed three times with phosphate-buffered saline PBS-Tween (PBST) (0.5% tween-20, w / v) and blocked in 10% FCS-PBST at 37°C for 2 hours. The serum was then diluted in 10% FCS-PBST, and 100 μL of diluted serum or lavage fluid was added to the plate and incubated at 37°C for 1 hour. The plate was then aspirated and washed three times in PBST. The secondary antibody (Goat pAb to Mouse IgG-HRP) was diluted 1:10,000 in sample diluent and 100 μL was added to each well for incubation at 37°C for 0.5 h. Following incubation, the plate was washed three times with PBST. 100 μL of 1x TMB substrate solution was added to each well for color development for 3 minutes. After color development, 50 μL of TMB stop solution was added, and absorbance was recorded at 450 nm and 610 nm using a Synegry H1 microplate reader. For analysis, the OD610 value was subtracted from the OD450 value for each well, followed by subtracting the average of the blank wells to obtain the actual sample absorbance. If the absorbance corresponding to the highest dilution factor was greater than 0.21, the highest dilution was used as the sample titer.

[0674] For IgA, 100 μL of diluted lavage fluid was added to the plate and incubated at 37°C for 1 hour. The plate was then aspirated and washed three times in PBST. A secondary antibody (Goat pAb to Mouse IgA-biotin) was diluted 1:10,000 in sample diluent and 100 μL was added to each well and incubated at 37°C for 1 hour. Following incubation, the plate was washed three times with PBST. Streptavidin-HRP antibody was diluted 1:5,000 in sample diluent and 100 μL was added to each well and incubated at 37°C for 1 hour. Following incubation, the plate was washed three times with PBST. 100 μL of 1x TMB substrate solution was added to each well and the reaction was developed for 10 minutes. After development, 100 μL of TMB stop solution was added and absorbance was recorded at 450 nm using a Synegry H1 microplate reader. To analyze the results, first deduct the average value of the blank wells from the OD450 of each well to obtain the actual sample absorbance value. If the absorbance value corresponding to the highest dilution factor is greater than 0.21, the highest dilution factor is used as the sample titer.

[0675] The ELISpot test results are shown in Table 5 and Figures 2B and 2C. The third booster immunization resulted in a better immune effect. During the third immunization, the cellular immune response after nasal administration of the IN-3-11 / Covid-19 LPP preparation and the IN-3-12 / Covid-19 LPP preparation was equivalent to or higher than that after intramuscular administration of the B11 / Covid-19 LPP preparation.

[0676] The ELISA test results are shown in Table 5 and Figure 2D. After the third booster immunization, there will be higher antibody levels in the serum and nasal lavage fluid, indicating a better immune effect. Compared with the administration of B11 / Covid-19 LPP preparation by intramuscular injection during the third immunization, the administration of IN-2-6 / Covid-19 LPP preparation, IN-3-11 / Covid-19 LPP preparation or IN-3-12 / Covid-19 LPP preparation by intranasal instillation during the third immunization can produce equivalent or higher cellular immune responses and activate mucosal immunity, resulting in significantly higher IgA antibody levels in the nasal lavage fluid of mice.

[0677] The above results show that the "primary immunization + boost" approach (systemic immunization + mucosal immunization) can combine the advantages of the two immunization methods and induce a higher level of immune effect.

[0678] Table 4. Immunization schedule

[0679] Table 5. Immune effects of different immunization regimens

[0680] Example 4 Application of LPP Preparation for Mucosal Administration in Influenza Vaccine Treatment

[0681] In this example, mice were immunized using different immunization protocols to test the application of the preferred LPP formulation in Example 2 in influenza vaccine treatment.

[0682] 4.1 Preparation of Lipid Polyplex (LPP-mRNA) Preparation

[0683] Preparation of lipid mixture: M5, phospholipids, cholesterol and PEG lipids were dissolved in ethanol solution according to the recipe of IN-2-6, IN-3-4, IN-3-5, IN-3-11 and IN-3-12 LPP preparations to prepare a 6 mg / mL lipid mixture.

[0684] Preparation of mRNA aqueous solution: mRNA encoding influenza virus antigen (coding sequence as shown in SEQ ID NO: 3) was diluted with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0685] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.125 mg / mL.

[0686] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: mass ratio = 4 (mRNA solution): 1 (protamine solution), flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, room temperature.

[0687] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: mass ratio = 20 (core nanoparticle solution): 1 (lipid mixture), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, room temperature, to obtain LPP-mRNA solution.

[0688] Centrifugal Ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration to remove ethanol (3000 rpm, 60 min, 4°C). Ultrafiltration was performed to a concentration of <0.5% ethanol, and the LPP mRNA concentration was adjusted to 1 mg / mL. LPP preparations numbered IN-2-6 / Flu, IN-3-4 / Flu, IN-3-5 / Flu, IN-3-11 / Flu, and IN-3-12 / Flu were prepared.

[0689] A B11 / Flu LPP preparation was prepared by the above method with a lipid ratio of M5:phospholipid:cholesterol:PEG of 40:15:43.5:1.5 in molar ratio, and the B11 / Flu LPP mRNA concentration was adjusted to 0.1 mg / mL.

[0690] 4.2 Immune effects of different immunization regimens

[0691] Mice were immunized using the immunization protocol shown in Figure 3A and Table 6. Female BALB / c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were divided into 8 groups (n=5) and immunized twice, on day 0 and day 14 (2 weeks after the initial immunization). In both immunizations, mice in Group 1 were administered a 10% sucrose solution via nasal instillation; mice in Groups 2, 3, 4, 5, and 6 were administered the IN-2-6 / Flu LPP formulation, the IN-3-4 / Flu LPP formulation, the IN-3-5 / Flu LPP formulation, the IN-3-11 / Flu LPP formulation, and the IN-3-12 / Flu LPP formulation, respectively, via nasal instillation; and mice in Group 7 were administered the B11 / Flu LPP formulation via intramuscular route. At the time of the primary immunization, the B11 / Flu LPP formulation was administered intramuscularly to Group 8 mice, and at the time of the secondary immunization, the IN-3-11 / Flu LPP formulation was administered intranasally to Group 8 mice. Four weeks after the primary immunization, the mice were sacrificed, and the lungs, spleens, and lung / nasal lavage fluid (obtained by lavage with 0.7 mL of PBS) were collected for subsequent testing.

[0692] ELISpot was used to detect immune cell responses in splenocytes and lung cells, and ELISA was used to detect antibody levels in serum, nasal lavage fluid, and lung lavage fluid. The specific ELISpot detection method is shown in Example 3.2. The specific ELISA detection method is as follows:

[0693] When performing ELISA testing of animal serum, influenza virus antigen (NP protein, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) was diluted to 1 μg / mL using ELISA coating buffer, and 96-well plates were coated with 100 μL of 100 ng / well antigen per well and incubated overnight at 4°C. After incubation overnight at 4°C, the plates were washed 3 times with phosphate-buffered saline PBS-Tween (PBST) (0.5% tween-20, w / v) and blocked in 10% FCS-PBST at 37°C for 2 hours. The plates were washed 3 times in PBST, and then the serum was diluted in 10% FCS-PBST. For IgG detection, the samples were diluted in series, 100 μL of diluted serum was added to the plates, and incubated at 37°C for 1 hour. The plate was then aspirated and washed three times in PBST. The secondary antibody (Goat pAb to Mouse IgG-HRP) was diluted 1:50,000 in sample diluent and 100 μL was added to each well for incubation at 37°C for 1 hour. After incubation, the plate was washed three times with PBST and 100 μL of 1x TMB substrate solution was added to each well for color development for 10 minutes. After color development, 50 μL of TMB stop solution was added to terminate the color development. The absorbance was recorded at 450 nm using a Synegry H1 microplate reader. For analysis, the OD450 value for each well was first subtracted from the average of the blank wells to determine the actual sample absorbance. If the absorbance corresponding to the highest dilution was greater than 0.21, the highest dilution was used as the sample titer.

[0694] For IgA ELISA testing of animal serum / lavage fluid, dilute influenza virus antigen to 2 μg / mL in ELISA coating buffer, coat a 96-well plate with 100 μL of antigen per well (200 ng / well), and incubate overnight at 4°C. Dilute the sample in 10% FCS-PBST, add 100 μL of diluted sample to each well, and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST, dilute the secondary antibody (Goat pAb to Mouse IgA-biotin) at a ratio of 1:10,000 in sample diluent, add 100 μL to each well, and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST, dilute the Streptavidin-HRP antibody at a ratio of 1:5,000 in sample diluent, and add 100 μL to each well at 37°C for 1 hour. After incubation, the plate was washed three times with PBST. 100 μL of 1x TMB substrate solution was added to each well and the reaction was allowed to develop for 10 minutes. After color development, 100 μL of TMB stop solution was added to terminate the color development. The absorbance was recorded at 450 nm using a Synegry H1 microplate reader. For analysis, the OD450 value of each well was first calculated, followed by subtracting the average of the blank wells to obtain the actual sample absorbance. If the absorbance value corresponding to the highest dilution factor was greater than 0.21, the highest dilution was used as the sample titer.

[0695] The ELISpot test results and ELISA test results are shown in Table 7 and Figures 3B, 3C, and 3D. Mice administered with the B11 / Flu LPP preparation via intramuscular administration during the primary immunization and with the IN-3-11 / Flu LPP preparation via nasal instillation during the secondary immunization had excellent cellular immune responses and high antibody levels in their serum, nasal lavage fluid, and bronchoalveolar lavage fluid.

[0696] The above results once again demonstrate that the "primary immunization + boost" approach (systemic immunization + mucosal immunization) can combine the advantages of the two immunization methods, induce a higher level of humoral immunity and cellular immunity, and at the same time induce mucosal immune responses.

[0697] Table 6. Immunization schedule

[0698] Table 7. Immune effects of different immunization schemes

[0699] Example 5 Effects of Different Cationic Lipids on the Physicochemical Properties and Expression of LPP Preparations for Mucosal Administration

[0700] In this example, a four-lipid LPP formulation containing luciferase mRNA (coding sequence as shown in SEQ ID NO: 1) for mucosal administration was prepared using the formulation in Table 8, and the physicochemical properties and in vivo expression levels of the prepared LPP formulations were compared.

[0701] 5.1 Preparation of lipid nanoparticle (LNP-mRNA) formulations

[0702] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0703] Preparation of lipid solution: MC3:phospholipid:cholesterol:PEG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a 6 mg / mL lipid solution.

[0704] Preparation of LNPs: Using microfluidic technology (Maianna (Shanghai) Technology Co., Ltd., model: Inano D), the lipid solution and the mRNA aqueous solution were mixed under the following conditions: volume = 4.0 mL; flow rate ratio = 3 (mRNA aqueous solution): 1 (lipid solution), total flow rate = 12 mL / min, to obtain an LNP-mRNA solution.

[0705] Centrifugal Ultrafiltration: The LNP-mRNA solution was added to an ultrafiltration tube and concentrated by centrifugation (3000 rpm, 60 min, 4°C) until the ethanol content was <0.5%. The LNP mRNA concentration was then adjusted to 0.2 mg / mL. This resulted in the LNP-mRNA formulation designated MC3 LNP.

[0706] 5.2 Preparation of Lipid Polyplex (LPP-mRNA) Preparation

[0707] Preparation of lipid mixture: Cationic lipid, phospholipid, cholesterol and PEG lipid were dissolved in ethanol solution according to the lipid ratio in the formulation of IN-3-11 and the cationic lipid shown in Table 8 to prepare a 6 mg / mL lipid mixture.

[0708] Preparation of mRNA aqueous solution: Dilute luciferase mRNA with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0709] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.125 mg / mL.

[0710] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: mass ratio = 4 (mRNA solution): 1 (protamine solution), flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, room temperature.

[0711] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: mass ratio = 20 (core nanoparticle solution): 1 (lipid mixture), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, room temperature, to obtain LPP-mRNA solution.

[0712] Centrifugal Ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration to remove ethanol (3000 rpm, 60 min, 4°C). Ultrafiltration was performed to a concentration of <0.5% ethanol, and the LPP mRNA concentration was adjusted to 0.2 mg / mL. LPP preparations numbered 121 LPP, 138-1 LPP, 139-1 LPP, 140-1 LPP, and 140-2 LPP were prepared.

[0713] Furthermore, a B11 LPP preparation having a lipid ratio of M5:phospholipid:cholesterol:PEG of 40:15:43.5:1.5 was prepared by the above method, and the LPP mRNA concentration was fixed to 0.2 mg / mL.

[0714] 5.3 Testing of the physicochemical properties of LPP preparations containing different cationic lipids

[0715] The prepared LPP and LNP preparations were tested for physical and chemical properties. Specific testing methods for particle size testing, encapsulation efficiency testing, polydispersity index (PDI) testing, and purity testing are described in Example 2.3.

[0716] The results of physicochemical property tests are shown in Table 8. LPP preparations prepared with different cationic lipids have higher encapsulation efficiency and purity and lower polydispersity index.

[0717] Table 8. Formulations and physicochemical properties of LPP preparations with different cationic lipids

[0718] 5.4 In vitro expression of LPP preparations containing different cationic lipids

[0719] Non-small cell lung cancer cells (A549 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, 10566-016) supplemented with 10% FBS (Hyclone, 35-081-CV) and 1% penicillin-streptomycin (GIBCO, 15140-122) at 37°C and 5% CO. Dendritic cells (DC2.4 cells) were cultured in Gibco RPMI 1640 medium supplemented with 10% FBS (Hyclone, 35-081-CV) and 1% penicillin-streptomycin (GIBCO, 15140-122) at 37°C and 5% CO. LPP solutions of B11, 121 LPP, 138-1 LPP, 139-1 LPP, 140-1 LPP, and 140-2 LPP, as well as an LNP solution of MC3 LNP containing 100 ng of luciferase mRNA, were taken, and PBS was used as a control to transfect A549 cells and dendritic cells DC2.4 cells. 24 hours after administration, the cells were lysed and the expression of luciferase protein was detected. The results are shown in Figures 4A and 4B. Both B11 LPP and 140-2 LPP showed good in vitro transfection effects and had high expression levels.

[0720] 5.5 In vivo expression of LPP preparations containing different cationic lipids

[0721] The LPP preparation prepared in Example 5.2 was tested for in vivo expression. The specific detection method is as follows:

[0722] Female BALB / c mice aged 6-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were anesthetized with sodium pentobarbital (70 mg / kg) and then administered the prepared LPP solution by nasal instillation (3 mice per group). Two groups of mice were administered MC3 LNP solution and PBS solution (negative control) through the nasal mucosa. Each LPP solution or LNP solution administered contained 2 μg of luciferase mRNA (10 μL). Six hours after administration, the mice were intraperitoneally injected with 3 mg of D-luciferin substrate (Mao Kang Biotechnology). Ten minutes after substrate injection, the mice were imaged in vivo using a Xenogen IVIS-200 imaging system to detect luciferase expression in vivo.

[0723] The experimental results, as shown in Figures 5A and 5B, show that all LPP preparations were expressed exclusively in the nasal cavity, demonstrating high local expression and minimal systemic toxicity. LPP preparations containing different cationic lipids, prepared using the lipid ratios used in the IN-3-11 formulation, showed similar luciferase expression levels, but exhibited higher luciferase expression in the nasal cavity than the B11 LPP preparation.

[0724] The above results show that the lipid ratio screened by the present invention is particularly suitable for intranasal administration. LPP preparations containing the homemade novel cationic lipids have good expression in the nasal cavity at the same lipid ratio, among which SW-II-140-2 cationic lipid is particularly preferred.

[0725] Example 6 Effects of atomization on the physicochemical properties and transfection efficiency of LPP preparations

[0726] In this example, a four-lipid LPP formulation containing luciferase mRNA (coding sequence shown in SEQ ID NO: 1) or eGFP protein mRNA (coding sequence shown in SEQ ID NO: 4) was prepared using a B11 formulation and aerosolized using the aerosol delivery device shown in Figure 6A. The physicochemical properties and transfection efficiency of the LPP formulation were measured before and after aerosolization to determine the effects of aerosolization on the LPP formulation.

[0727] 6.1 Preparation of Lipid Polyplex (LPP-mRNA) Preparation

[0728] Preparation of lipid mixture: M5, phospholipid, cholesterol and PEG lipid were dissolved in ethanol solution according to the lipid ratio in the B11 recipe to prepare a 6 mg / mL lipid mixture.

[0729] Preparation of mRNA aqueous solution: dilute luciferase mRNA or eGFP mRNA with 10 mM sodium citrate (pH = 4.0) buffer solution to a 0.1 mg / mL mRNA aqueous solution.

[0730] Preparation of protamine sulfate solution: Dissolve protamine sulfate in nuclease-free water to prepare a protamine sulfate solution with a working concentration of 0.125 mg / mL.

[0731] Preparation of core nanoparticle solution: Using microfluidics technology, a protamine sulfate solution and an mRNA solution were mixed under the following conditions to obtain a core nanoparticle solution formed by protamine and mRNA: mass ratio = 4 (mRNA solution): 1 (protamine solution), flow rate ratio = 5 (mRNA): 1 (protamine solution), total flow rate = 12 mL / min, room temperature.

[0732] Preparation of LPP: The core nanoparticle solution and the lipid solution were mixed twice under the following conditions: mass ratio = 20 (core nanoparticle solution): 1 (lipid mixture), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL / min, room temperature, to obtain LPP-mRNA solution.

[0733] Ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration to remove ethanol (3000 rpm, 60 min, 4°C). Ultrafiltration was performed to a concentration of <0.5% ethanol, and the LPP mRNA concentration was adjusted to 0.1 mg / mL. These LPP preparations were designated B11 / Luc and B11 / eGFP.

[0734] 6.2 Testing the physical and chemical properties of LPP preparations before and after atomization

[0735] LPP formulations of B11 / Luc and B11 / eGFP were aerosolized using the aerosol delivery device shown in Figure 6A , and the physical and chemical properties of the formulations were tested before and after aerosolization. Specific methods for particle size, encapsulation efficiency, polydispersity index (PDI), and purity testing are described in Example 2.3.

[0736] The results of the physical and chemical property tests are shown in Table 9. After the LPP sample was atomized by the nasal spray device, there was no significant change in the physical and chemical properties of the sample before and after atomization.

[0737] Table 9. Physicochemical properties before and after atomization

[0738] 6.3 Detection of transfection efficiency of LPP preparations before and after atomization

[0739] Mouse myoblasts (C2C12 cells) and non-small cell lung cancer cells (A549 cells) were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO, 10566-016) supplemented with 10% FBS (Hyclone, 35-081-CV) and 1% penicillin-streptomycin (GIBCO, 15140-122) at 37°C and 5% CO2. Dendritic cells (DC2.4 cells) were cultured in Gibco RPMI 1640 medium supplemented with 10% FBS (Hyclone, 35-081-CV) and 1% penicillin-streptomycin (GIBCO, 15140-122) at 37°C and 5% CO2.

[0740] B11 / Luc LPP preparations containing 12.5, 25, 50, and 100 ng of luciferase mRNA were taken before and after nebulization, and C2C12 cells, A549 cells, and dendritic cells DC2.4 cells were transfected. The cells were lysed 24 hours after administration and the expression of luciferase protein was detected.

[0741] B11 / eGFP LPP preparations containing 12.5, 25, 50, and 100 ng of luciferase mRNA and eGFP mRNA were taken before, during, and after atomization, respectively, and transfected into A549 cells. The expression of green fluorescent protein was observed under a fluorescence microscope 24 hours after administration, and photographs were taken and compared.

[0742] The test results are shown in Figures 6B and 6C, respectively. After the LPP sample was atomized by the nasal spray device, there was no significant change in the cell transfection efficiency and in vitro expression of the sample after atomization compared with the sample before atomization.

[0743] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0744] Sequence Listing

Claims

1. A lipid composition for mucosal administration, comprising a therapeutic agent or a prophylactic agent and a lipid that encapsulates the therapeutic agent or the prophylactic agent, wherein the lipid that encapsulates the therapeutic agent or the prophylactic agent comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; the lipid composition further comprises a cationic polymer, wherein the cationic polymer is associated with the therapeutic agent or the prophylactic agent to form a complex and is co-encapsulated in the lipid to form a lipid multimer complex; the lipid composition comprises 2.5-20 mol% of the polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

2. The lipid composition of claim 1, wherein the therapeutic or prophylactic agent is a nucleic acid, such as RNA, particularly mRNA.

3. The lipid composition of claim 1 or 2, wherein the cationic lipid comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof in, R1 and R2 are each independently selected from a bond, a C1-C 12 Alkyl and C2-C 12 alkenyl; R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 aryl and 5-10 membered heteroaryl; and R3 and R4 are each independently optionally substituted by t R6, where t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl; M1 and M2 are each independently selected from a bond, H, -O-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, -C(S)S-, a 3-10 membered heterocycle, -NR7-, or R5 together with one of M1 and M2 together with the nitrogen atom to which they are attached forms a 3-10 membered heterocyclic ring, and the corresponding R1 / R3 or R2 / R4 are absent, said heterocyclic ring being optionally substituted by R7; R5 is selected from C 3-8 Carbocyclic ring, -C 1-12 Alkylene-Q, Q is selected from H, -OR7, -SR7, -OC(O)R7, -C(O)OR7, -N(R7)C(O)R7, -N(R7)S(O)2R7, -N(R7)C(S)R7, -N(R7)2, cyano, C 3-8 Carbocyclic ring, 3-10 membered heterocyclic ring, C6-C 10 Aryl, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O) substitution; m and n are each independently an integer selected from 0-12; The alkyl, alkenyl and alkylene groups are each optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR7-, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C 3-8 carbocycle, and said alkyl, alkenyl and alkylene are each optionally substituted with one or more R7; R7 are each independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C 3-8 Carbocyclic ring, each of the above groups is optionally replaced by one or more C1-C 12 Alkyl, C2-C 12 ene Base, C1-C 12 Alkoxy, C6-C 10 Aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O) substitution.

4. The lipid composition of claim 3, wherein R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl; wherein R3 and R4 are each independently selected from C1-C 12 Alkyl and C2-C 12 and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl. M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, and -C(S)S-; R5 is selected from -C 1-12 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl; m and n are each independently an integer selected from 1-12.

5. The lipid composition of claim 3, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

6. The lipid composition of claim 3, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof:

7. The lipid composition of claim 3, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof 8. The lipid composition of claim 3, wherein R1 and R2 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl; R3 and R4 are each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C6-C 10 Aryl and 5-10 membered heteroaryl; Provided that at least one of R3 and R4 is C6-C 10 aryl or 5-10 membered heteroaryl, and R3 and R4 are each independently optionally substituted by t R6, t is an integer selected from 1-5; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl; M1 and M2 are each independently selected from -OC(O)-, -C(O)O-, -OC(O)O-, -SC(S)-, and -C(S)S-; R5 is selected from -C 1-12 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6-C 10 Aryl and 5-10 membered heteroaryl; m and n are each independently an integer selected from 1-12.

9. The lipid composition of claim 8, wherein R1 and R2 are each independently selected from C1-C 12 alkyl.

10. The lipid composition according to claim 8 or 9, wherein R3 and R4 are each independently selected from C1-C 12 Alkyl and C6-C 10 aryl; The condition is that one of R3 and R4 is C6-C 10 Aryl, the other is C1-C 12 alkyl; R3 and R4 are each independently substituted by t R6, where t is an integer selected from 1-3; R6 are each independently selected from C1-C 12 alkyl.

11. The lipid composition according to any one of claims 8 to 10, wherein M1 and M2 are each independently selected from: -OC(O)-, -C(O)O- and -OC(O)O-.

12. The lipid composition according to any one of claims 8 to 11, wherein R5 is selected from -C 1-5 Alkylene-Q, Q is -OH.

13. The lipid composition according to any one of claims 8 to 12, wherein m and n are each independently an integer selected from 2-7.

14. The lipid composition according to any one of claims 8 to 13, wherein R4 is substituted at the first or last position of R2; and / or R3 is substituted at the first or last position of R1.

15. The lipid composition according to any one of claims 8 to 14, wherein t is 1 or 2, and R6 is substituted at the meta and / or para position relative to R1 or R2 on the benzene ring.

16. The lipid composition according to any one of claims 8 to 15, wherein t is 1 or 2, and R6 is independently selected from C1-C 10 alkyl.

17. The lipid composition of any one of claims 8 to 16, wherein the cationic lipid comprises a compound of formula (II), or a pharmaceutically acceptable salt thereof: wherein R1, R2, R4, R5, R6, M1, M2, t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (II) R1 is selected from C1-C6 alkyl; R2 is selected from C1-C 10 alkyl; R4 is selected from C1-C 10 alkyl; M1 and M2 are each independently selected from: -OC(O)-, -C(O)O- and -OC(O)O-; R5 is selected from -C 1-5 Alkylene-Q, Q is selected from -OR7 and -SR7, R7 is independently selected from H, C1-C 12 Alkyl and C2-C 12 alkenyl; m and n are each independently an integer selected from 2-9; t is an integer selected from 1-3; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 Alkenyl.

18. The lipid composition of any one of claims 8 to 16, wherein the cationic lipid comprises a compound of formula (III), or a pharmaceutically acceptable salt thereof: wherein R1, R2, R4, R5, R6, t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (III), R1 is selected from C1-C6 alkyl; R2 is selected from C1-C 10 alkyl; R4 is selected from C1-C 10 alkyl; R5 is selected from -C 1-3 Alkylene-Q, Q is selected from -OH and -SH; t is 1 or 2; R6 is selected from C1-C 12 Alkyl and C2-C 12 alkenyl; m and n are each independently an integer selected from 2-7.

19. The lipid composition of any one of claims 8 to 16, wherein the cationic lipid comprises a compound of formula (IV), or a pharmaceutically acceptable salt thereof: wherein R1, R2, R4, R6, t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (IV), R1 is selected from C1-C6 alkyl; R2 is selected from C1-C 10 alkyl; R4 is selected from C1-C 10 alkyl; t is 1 or 2; R6 are each independently selected from C1-C 12 Alkyl and C2-C 12 alkenyl; m and n are each independently an integer selected from 2-7.

20. The lipid composition of claim 8, wherein the cationic lipid comprises a lipid compound having the structure shown below or a pharmaceutically acceptable salt thereof: Preferably, the cationic lipid is SW-II-121, SW-II-138-1, SW-II-139-1, SW-II-140-1 or SW-II-140-2.

21. The lipid composition of any one of claims 3 to 20, wherein the phospholipids comprise 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof; DSPC, DOPE or a combination thereof is preferred.

22. The lipid composition of claim 21, wherein the steroid comprises cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof; preferably, the steroid is cholesterol.

23. The lipid composition of claim 22, wherein the polyethylene glycol-modified lipid comprises 2-[(polyethylene glycol)-2000]-N,N-tetracosane, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-dioleoyl-rac-glycero, methoxy-polyethylene glycol (DOGPEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG); preferably DSPE-PEG, DMG-PEG or a combination thereof.

24. The lipid composition of claim 23, comprising 30-60 mol% cationic lipid, 5-40 mol% phospholipid, 10-70 mol% steroid and 2.5-20 mol% polyethylene glycol-modified lipid; Preferably, it comprises 37.5-42.5 mol% of cationic lipids, 25-35 mol% of phospholipids, 15-30 mol% of steroids and 2.5-20 mol% of polyethylene glycol-modified lipids; Preferably comprising 37.5-42.5 mol% of cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol and 2.5-20 mol% of DMG-PEG; Preferably comprising 37.5-42.5 mol% of cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol and 3.75-10 mol% of DMG-PEG; More preferably, it comprises 37.5-42.5 mol% of cationic lipid, 25-35 mol% of DOPE, 15-30 mol% of cholesterol and 3.75-5 mol% of DMG-PEG; Most preferably, it comprises 42.5 mol% cationic lipid, 35 mol% DOPE, 18.75 mol% cholesterol, and 3.75 mol% DMG-PEG.

25. The lipid composition of claim 24, wherein the cationic polymer comprises poly-L-lysine, protamine, polyethyleneimine (PEI) or a combination thereof; preferably, the cationic polymer is protamine.

26. A pharmaceutical composition comprising the lipid composition of any one of claims 1 to 25, and optionally a pharmaceutically acceptable excipient.

27. The lipid composition according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26, for administration to the nasal cavity, oral cavity, conjunctiva, rectum, or vaginal mucosa; Nasal administration is preferred, and the nasal administration preferably includes nasal drip, nasal spray administration or nasal inhalation; most preferably, the nasal administration includes nasal drip or nasal spray administration.

28. The lipid composition or pharmaceutical composition of claim 27, wherein the nasal spray administration is performed by an aerosol administration device.

29. The lipid composition or pharmaceutical composition of claim 28, wherein the aerosol drug delivery device comprises a syringe, a plastic needle, a nasal spray device, and a dose stopper.

30. A method for treating or preventing a disease or condition comprising administering to a subject a therapeutic or prophylactic agent in a multiple dose regimen, wherein at least one dose is administered by a mucosal route as a lipid composition according to any one of claims 1 to 25 or a pharmaceutical composition according to claim 26.

31. The method of claim 30, wherein the at least one additional agent is administered by a route selected from the group consisting of intramuscular, intratumoral, transdermal, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, or a combination thereof; preferably, the at least one additional agent is administered by an intramuscular route of administration.

32. Use of the lipid composition of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 in the preparation of a medicament for treating or preventing a disease or condition in a subject in need thereof.

33. The method of claim 30 or 31 or the use of claim 32, wherein the disease or disorder is characterized by a malfunction or abnormal protein or polypeptide activity.

34. The method or use of claim 33, wherein the disease or condition is selected from rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases; preferably, the disease is an infectious disease.

35. A nasal drop or nasal spray comprising the lipid composition according to any one of claims 1 to 25 and a pharmaceutically acceptable excipient.

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