WO2024094211A1 - Lipid composition - Google Patents

Abstract

The present invention relates to mucosal drug delivery systems, and specifically to a lipid composition; the lipid composition containing a therapeutic agent and/or prophylactic agent such as RNA can be used for mucosal delivery of the therapeutic agent and/or prophylactic agent to treat or prevent disease such as infectious disease.

Description

A lipid composition

CROSS-REFERENCE TO RELATED APPLICATIONS

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

The present invention relates to a drug delivery system, and in particular to a lipid composition for mucosal administration and related products and their application in treating or preventing infectious diseases.

Background technique

Nanoparticle compositions, liposomes and liposome complexes containing lipids can effectively deliver biologically active substances such as small molecule drugs, proteins and nucleic acids to cells and/or intracellular compartments as transport vehicles. These lipid compositions generally include cationic lipids, structural lipids, auxiliary lipids and/or surfactants.

Existing lipid-based drug delivery systems, such as liposomes, lipid nanoparticles (LNP) drug delivery systems, etc., have been widely used in recent years. However, in actual use, it is found that when these drug delivery systems are used for mucosal administration of bioactive substances such as nucleic acids, there are problems such as low expression of mRNA in the mucosa and weak immunogenicity caused by them.

At present, some researchers have found that the use of publicly available polymers, positively charged liposomes, viral vectors or some commercial transfection reagents for mucosal administration, such as nasal administration, will lead to a large amount of protein expression in the lungs. Some researchers have also compared three-component LNP drug delivery systems containing different lipid combinations such as cationic lipids, auxiliary phospholipids and PEG, and found that the same nucleic acid delivered through different routes of administration (intramuscular route, intradermal route, nasal administration) has different immunogenicity and expression in vivo. The immune effect of nasal administration is significantly weaker than that of intramuscular and subcutaneous administration. After nasal administration, the delivered nucleic acid will be distributed in large quantities in the throat and stomach of mice (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.).

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

Summary of the invention

In one aspect, the present invention provides a lipid composition for mucosal administration, comprising a therapeutic agent or a preventive agent and a lipid encapsulating the therapeutic agent or the preventive agent, wherein the lipid encapsulating the therapeutic agent or the preventive 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 preventive agent as a complex, and is co-encapsulated in the lipid to form a lipid polymer 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.

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

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.

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

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.

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.

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.

In one embodiment, the lipid composition 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 comprises 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.

In a preferred embodiment, the lipid composition comprises 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.

In a preferred embodiment, the lipid composition 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.

In a particularly 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.

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.

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.

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

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

In one embodiment, the nasal spray administration is performed by an aerosol administration device.

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

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

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 the preparation of a medicament for treating or preventing a disease or condition in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and Figure 1B show the in vivo expression of LPP preparations prepared by different prescriptions in the third round of screening. Figure 1A is a mouse live imaging diagram; Figure 1B is a statistical diagram of luciferase expression, represented by the area under the curve (AUC).

Figures 2A-2D show the immunization of LPP preparations containing COVID-19 mRNA using different immunization protocols Figure 2A shows the immunization scheme; Figure 2B shows the ELISpot test results of spleen cells in each group of mice immunized with different immunization schemes; Figure 2C shows the ELISpot test results of lung cells in each group of mice; Figure 2D shows the results of ELISA test of IgA antibody levels in nasal lavage fluid of each group of mice.

Figures 3A-3E show the results of immunogenicity detection of LPP preparations containing mRNA encoding influenza virus antigens using different immunization schemes. Figure 3A is the immunization scheme; Figure 3B is the ELISpot detection results of spleen cells in each group of mice immunized with different immunization schemes; Figure 3C is the ELISpot detection results of lung cells in each group of mice; Figure 3D is the result of ELISA detection of IgA antibody levels in nasal lavage fluid of each group of mice; Figure 3E is the result of ELISA detection of IgA antibody levels in lung lavage fluid of each group of mice.

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

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

Figures 6A-6C show the cell transfection efficiency of LPP preparations before and after nebulization. Figure 6A is a schematic diagram of the nebulization drug delivery device; Figure 6B shows the luciferase expression of B11/Luc LPP preparations containing luciferase mRNA in DC2.4 cells before and after nebulization; Figure 6C shows the eGFP expression of B11/eGFP LPP preparations containing eGFP mRNA in A549 cells before nebulization, in the front section of nebulization, in the middle section of nebulization, and in the rear section of nebulization.

Specific implementation plan

General Definitions and Terminology

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.

Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology related terms used herein are terms widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning 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."

As used herein, the singular forms "a", "an" or "the" include plural references 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.

Recitation of ranges of values herein is intended merely to serve as a shorthand method of referring individually to each different 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 indicated herein are modified by "about" to indicate that the recited or claimed value is about Or range ±20%, ±10%, ±5% or ±3%.

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

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

In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "Nucleotide" is usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylic acid 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.

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 should 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, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.

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 as "altered" groups in this article. Groups can be modified or altered chemically, structurally or functionally. For example, a modified nucleobase can include one or more non-naturally occurring substitutions.

As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may 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."

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

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

As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon including one or more carbon atoms. The term "C ₁ -C ₁₂ alkyl" or "C _1-12 alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon including 1-12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein, which is connected to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom. The term "C ₁ -C ₁₂ alkylene" or "C _1-12 alkylene" refers to an optionally substituted straight or branched chain alkylene group including 1-12 carbon atoms.

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. The term "C ₂ -C ₁₂ alkenyl" or "C _2-12 alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. An optionally substituted straight or branched chain hydrocarbon containing one or more carbon-carbon double bonds. The alkenyl group may include one, two, three, four or more carbon-carbon double bonds.

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

As used herein, the term "carbocycle" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings consisting of carbon atoms. The term "C _3-8 carbocycle" means a carbocycle comprising 3-8 carbon atoms. The carbocycle may include one or more carbon-carbon double bonds or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, etc. As used herein, when the carbocycle is saturated (i.e., without unsaturated bonds), it may also refer to the corresponding cycloalkyl. Unless otherwise specifically stated, the carbocycle described herein refers to unsubstituted and substituted, i.e., optionally substituted carbocycles.

As used herein, the term "heterocycle" refers to a monocyclic or polycyclic system including one or more rings and including at least one heteroatom. The heteroatom can be, for example, nitrogen, oxygen, phosphorus or sulfur atoms. 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 contain, for example, 3-10 atoms (non-hydrogen), i.e., 3-10 yuan heterocycles (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 yuan), 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, heterocycles described herein refer to two types of unsubstituted and substituted heterocyclic groups, i.e., optionally substituted heterocycles.

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

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, the remaining ring atoms being C, and having at least one aromatic ring. The heteroaryl group may have 5-10 ring atoms (5-10 membered heteroaryl), including 5, 6, 7, 8, 9 or 10 members, particularly 5 or 6 membered heteroaryl groups. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.

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

Unless otherwise specifically stated, the groups described herein (eg, any of R ₁ -R ₇ , such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. Optional substituents may be selected from the following, 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) ₂ R"", 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) ₂ OH), thialdehydes (e.g., -C(S)H), sulfates (e.g., S(O) ₄ ^2- ), sulfonyl (e.g., -S(O) ₂ -), amide (e.g., -C(O)NR ₂ or -N(R)C(O)R), azido (e.g., -N ₃ ), nitro (e.g., -NO ₂ ), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(O)R), amino (e.g., -NR ₂ , NRH, or -NH ₂ ), carbamoyl (e.g., -OC(O)NR ₂ , -OC(O)NRH, or -OC(O)NH ₂ ), sulfonamide (e.g., -S(O) ₂ NR ₂ , -S(O) ₂ NRH, -S(O) ₂ NH ₂ , -N(R)S(O) ₂ R, -N(H)S(O) ₂ R, -N(R)S(O) ₂ H, -N(H)S(O) ₂ H), C ₁ -C ₁₂ Alkyl, C ₂ -C ₁₂ alkenyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 3-10 membered heterocycle. In any of the foregoing, R can 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, alkyl can be further substituted with one, two, three, four, five or six substituents as described herein.

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 different numbers of neutrons in the nucleus, 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 in combination with solvents or water molecules to form solvates and hydrates by conventional methods.

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 group may or may not be substituted, and that the description includes substituted alkyl radicals and unsubstituted alkyl radicals.

It should be understood that when chemical groups are written in a particular order, the reverse order is also contemplated unless otherwise stated. For example, in the general formula -(R) _i- (M1) _k- (R) _m- where _M1 is defined as -C(O)NH- (i.e., -(R) _i -C(O)-NH-(R) _m- ), compounds where _M1 is -NHC(O)- (i.e., -(R) _i -NHC(O)-(R) _m- ) are also contemplated unless otherwise stated.

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 may involve administering a composition comprising the therapeutic or prophylactic agent to the subject.

As used herein, the term "subject" describes an organism to which the compositions of the present invention may be provided. 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.

As used herein, "encapsulation efficiency" refers to the ratio of the amount of therapeutic or preventive agent that becomes part of the composition to the initial total amount of the therapeutic or preventive agent used to prepare the composition. For example, if 97 mg of the total 100 mg of therapeutic or preventive agent initially provided to the composition is encapsulated in the composition, it can be concluded that the encapsulation efficiency is 97%. As used herein, "encapsulation" can refer to complete, major or partial encapsulation, sealing, surrounding or packaging.

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.

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.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds in which 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, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate The pharmaceutically acceptable salts of the present invention include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like. The pharmaceutically acceptable salts of the present invention include, but are not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed by non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from the parent compound containing a basic or acidic moiety. Generally speaking, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred.

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.

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

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

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 underlying disease or preventing worsening of symptoms or development of disease.

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 and severity of development 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 may be administered in one or more doses. The therapeutically or prophylactically effective amount may be achieved by continuous or intermittent administration.

Lipid composition

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

As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic portion and optionally also a hydrophilic portion. Lipids are generally insoluble 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, etc.

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

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 having a core-shell structure, wherein nucleic acids are contained in polyplexes, and the polyplexes themselves are encapsulated in a biocompatible lipid bilayer shell to constitute the lipid nanoparticles of the present invention. In some embodiments, the lipid composition of the invention is a lipid polyplex (LPP). In some embodiments, the lipid composition of the invention is a lipid polyplex (LPP) comprising RNA.

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.

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 encapsulating the therapeutic agent or the preventive agent. The lipid encapsulating the therapeutic agent or the preventive agent comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid.

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.

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 phospholipids as described herein, 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecene-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), dipalmitoyl phosphatidylglycerol (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, fecal sterol, 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 and a steroid as described herein. 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 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 cationic lipid 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, phospholipid DOPE and cholesterol. In one embodiment, the lipid composition of the present invention comprises cationic lipid 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, phospholipid DSPC and cholesterol.

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).

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

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

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

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

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.

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.

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.

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 %.

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%.

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

In one embodiment, the amount of cholesterol in lipid composition of the present invention is about 10-about 70 mol %, based on the total amount of all lipids in lipid composition.In one embodiment, the amount of cholesterol in 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 %.

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.

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.

For the application purposes of the lipid composition of the present invention, the above-mentioned content and the range thereof are advantageous.

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 as a complex. In one embodiment, the LPP comprises 2.5-20 mol% of a polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

In one embodiment, the lipid composition of the present invention comprises a therapeutic agent or a prophylactic agent and a lipid encapsulating the therapeutic agent or the prophylactic agent, wherein the lipid encapsulating 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 as 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 a polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid composition.

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.

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

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

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.

In a preferred embodiment, the lipid composition comprises 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.

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

In a preferred embodiment, the lipid composition comprises 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.

In a preferred embodiment, the lipid composition 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.

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.

In a particularly preferred embodiment, the lipids of the encapsulation complex comprise 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.

Cationic lipids

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.

Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (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), dioctadecyldimethyl ammonium chloride, 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 (DMRIE), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane ... -dimethylaminopropane, DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (1,2-dilinolenyloxy-N,N-dimethylaminopropane, DLenDMA), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutan-4-oxy)-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), dioctadecylamidolycyl 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.

In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. An ionizable cationic lipid has a net positive charge at, for example, acidic pH, but is 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), compounds of formula (I), (II), (III) or (IV) as described herein, or combinations thereof.

In one embodiment, 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 - _C12 alkyl group, and a _C2 - _C12 alkenyl group;

_R3 and _R4 are each independently selected from _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C6 - _C10 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;

R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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

R ₅ , one of M ₁ and M ₂ , together with the nitrogen atom to which they are attached, form a 3-10 membered heterocyclic ring, and the corresponding R ₁ /R ₃ or

or R ₂ /R ₄ is absent, the heterocyclic ring is optionally substituted by R ₇ ;

R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H, -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆ -C ₁₀ aryl, each of the above groups is optionally substituted with one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O);

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-, -NR ₇ -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C _3-8 carbocycle, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R ₇ ;

_R7 is each independently selected from H, _C1 - _C12 alkyl, C2- _C12 alkenyl, _C1 - _C12 alkoxy, carboxylic acid _, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, _C3-8 carbocycle, and each of the above groups is optionally substituted with one or more _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C1 - _{C12 alkoxy, C6-C10 aryl ,} 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O).

In one embodiment, _R1 and _R2 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl, such as _C1 - _C12 alkyl. In another embodiment, one of _R1 and _R2 is a bond and the other is independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl, such as _C1 - _C12 alkyl.

In one embodiment, _R3 and _R4 are each independently selected from _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C6 - _C10 aryl and 5-10 membered heteroaryl. In another embodiment, _R3 and _R4 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl.

R ₃ and R ₄ may each independently be optionally substituted by t R ₆ , t being 1, 2, 3, 4, or 5. In one embodiment, R ₆ is each independently selected from C ₁ -C ₁₂ alkyl.

In yet another embodiment, at least one of R ₃ and R ₄ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, for example C ₆ -C ₁₀ aryl.

In one embodiment, R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q. Q may be selected from H, -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , cyano, C _{3- 8} carbocycle, 3-10 membered heterocycle, C ₆ -C ₁₀ aryl. The above groups, including groups covering the options of Q, when appropriate, Each may be optionally substituted by one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (═O).

In yet another embodiment, R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H, -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , cyano, C _3-8 carbocycle, _3-10 membered heterocycle, C ₆ -C ₁₀ aryl. The above groups, including groups covering the options of Q, may each be optionally substituted with one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (═O), where appropriate.

In the compounds of formula (I), _R7 can be each independently selected from H, _C1 - _C12 alkyl, C2- _C12 alkenyl, _{C1 - C12} alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, _C3-8 carbocycle, preferably selected from H, _C1 - _C12 alkyl, C2- _C12 alkenyl, _{C1 - C12} alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl and 5-10 membered heteroaryl. The above groups (when appropriate, for example H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, C _3-8 carbocycle) are each optionally substituted by one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (═O).

In one embodiment, each group described above, such as C _3-8 carbocycle, -C _1-12 alkylene-Q, includes -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , C _3-8 carbocycle, 3-10 membered heterocycle, C ₆ -C ₁₀ aryl, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _{The 3-8} carbon ring and the like may each be optionally substituted by one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (═O).

In one embodiment, the alkyl, alkenyl and alkylene groups (e.g., those mentioned in R ₁ -R ₇ ) in the compounds of formula (I) may each be optionally and independently interrupted by one or more groups selected from: -O-, -S-, -NR ₇ -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C _3-8 carbocycle, and the alkyl, alkenyl and alkylene groups may each be optionally substituted by one or more R _7. That is, the chains (straight or branched) of the alkyl, alkenyl and alkylene groups may each optionally contain one or more groups selected from: -O-, -S-, -NR ₇ -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C _3-8 carbocycle.

_R7 is each independently selected from H, _C1 - _C12 alkyl, C2- _C12 alkenyl, _C1 - _C12 alkoxy, carboxylic acid _, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, _C3-8 carbocycle; preferably, _R7 is independently selected from H, _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C1 - _C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 - _C10 aryl and 5-10 membered heteroaryl. The above groups (when appropriate, for example H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, C _3-8 carbocycle) are each optionally substituted by one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (═O).

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.

In one embodiment, _M1 or _M2 is a bond, the corresponding m or n is not 0, and the carbon chain before _M1 or _M2 is _R1 or _R2 connection.

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 .

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 .

In one embodiment, M ₁ and M ₂ are each independently selected from -C(O)-, -OC(O)- and -C(O)O-. In another embodiment, M ₁ and M ₂ are each independently selected from -NR ₇ -, and R ₇ is as described above.

In another embodiment, R ₅ and one of M ₁ and M ₂ together with the attached N atom form a 3-10 membered heterocyclic ring, and the corresponding R ₁ /R ₃ or R ₂ /R ₄ are absent, and the heterocyclic ring is optionally substituted with R ₇ , and R ₇ is as described above.

In one embodiment, R ₅ is selected from -C _1-12 alkylene-Q, Q is selected from H, -OR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ ) ₂ , cyano, and R ₇ is as described above.

In a preferred embodiment, R ₁ and R ₂ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

wherein R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; and R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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-;

R ₅ is selected from -C _{1 -12} alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl;

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

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

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

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

In a preferred embodiment, the cationic lipid comprises MC3.

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

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

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

_R1 and _R2 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl;

R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl;

Provided that at least one of R ₃ and R ₄ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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-;

R ₅ is selected from -C _1-12 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl;

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

In one embodiment, R ₂ is selected from C ₁ -C ₁₂ alkyl. In another embodiment, R ₂ is selected from C ₁ -C ₆ alkyl.

In one embodiment, one of R ₃ and R ₄ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, and the other is C ₁ -C ₁₂ alkyl or C ₂ -C ₁₂ alkenyl.

In one embodiment, _R3 and _R4 are each independently selected from _C1 - _C12 alkyl and phenyl, provided that at least one of _R3 and _R4 is phenyl. In another embodiment, one of _R3 and _R4 is phenyl and the other is _C1 - _C12 alkyl.

In yet another embodiment, _R3 and _R4 are each independently substituted by 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.

In one embodiment, each R ₆ is independently selected from C ₁ -C ₁₂ alkyl, such as C ₁ -C ₁₀ alkyl.

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring at the meta or para position relative to R ₁ or R ₂ .

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring at the meta and para positions relative to R ₁ or R ₂ .

In one embodiment, R ₄ is substituted at the 1st position or the last position of R _2. The 1st position refers to the position of the C atom in R ₂ that is directly connected to M _2. The last position refers to the position of the C atom in R ₂ that is farthest from M _2. In a specific embodiment, R ₄ is selected from C ₁ -C ₁₂ alkyl, and R ₃ is phenyl.

In one embodiment, R ₃ is substituted at the 1st position or the last position of R _1. The 1st position refers to the position of the C atom in R ₁ that is directly connected to M _1. The last position refers to the position of the C atom in R ₁ that is farthest from M _1. In a specific embodiment, R ₃ is selected from C ₁ -C ₁₂ alkyl, and R ₄ is phenyl.

In one embodiment, _M1 and _M2 are each independently selected from -OC(O)-, -C(O)O- and -OC(O)O-.

In one embodiment, R ₅ is selected from -C _1-5 alkylene-Q, such as C ₁ , C ₂ , C ₃ , C ₄ or C ₅ alkylene-Q. In an exemplary embodiment, R ₅ is selected from -C _1-3 alkylene-Q, such as C ₁ , C ₂ or C ₃ alkylene-Q.

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

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.

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

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

In one embodiment,

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

_M1 and _M2 are each independently selected from -OC(O)-, -C(O)O- and -OC(O)O-;

R ₅ is selected from -C _1-5 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl;

R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl, in particular C ₁ -C ₁₂ alkyl;

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

t is an integer selected from 1-3.

In one embodiment, R ₅ is selected from -C _1-3 alkylene-Q, Q is selected from -OH and -SH, especially -OH.

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

In some embodiments, t is 1 or 2.

In one embodiment, R ₄ is substituted at the 1st position or the last position of R _2. The 1st position refers to the position of the C atom in R ₂ that is directly connected to M _2. The last position refers to the position of the C atom in R ₂ that is farthest from M ₂ .

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring at the meta or para position relative to R ₁ .

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring at the meta and para positions relative to R ₁ .

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

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

In one embodiment,

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

R ₅ is selected from -C _1-3 alkylene-Q, Q is selected from -OH and -SH, especially -OH;

t is 1 or 2;

R ₆ is selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl, in particular C ₁ -C ₁₂ alkyl;

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

In one embodiment, R ₄ is substituted at the 1st or last position of R _2. The 1st position refers to the position of R ₂ that is The last position refers to the position of the C atom in R ₂ that is directly connected to the The position of the C atom that is farthest from the others.

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring at the meta or para position relative to R ₁ .

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring at the meta and para positions relative to R ₁ .

In certain embodiments, the compound of formula (I) includes a compound of formula (IV):

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

In one embodiment,

R ₁ is selected from C ₁ -C ₆ alkyl;

R ₂ is selected from C ₁ -C ₁₀ alkyl;

R ₄ is selected from C ₁ -C ₁₀ alkyl;

t is 1 or 2;

R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl, in particular C ₁ -C ₁₂ alkyl;

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

In one embodiment, R ₄ is substituted at the 1st or last position of R _2. The 1st position refers to the position of R ₂ that is The last position refers to the position of the C atom in R ₂ that is directly connected to the The position of the C atom that is farthest from the others.

In one embodiment, t is 1 and R ₆ is substituted on the phenyl ring at the meta or para position relative to R ₁ .

In another embodiment, t is 2 and R ₆ is substituted on the phenyl ring at the meta and para positions relative to R ₁ .

In a specific embodiment, the substituents (eg, R ₁ -R ₇ ) in the lipid compounds of the present invention do not include alkenyl groups.

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

In a preferred embodiment, the cationic lipid comprises the following lipid compound: 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 does not comprise T5',

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.

Phospholipids

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

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), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC). ), 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-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine Ethanolamine (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), dipalmitoyl Acylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (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.

Steroid

The lipid composition of the present invention comprises a steroid, which can serve as a structural component of the lipid composition.

Examples of steroids include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and derivatives thereof.

PEGylated lipids

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 The following are the main ingredients of the peptide mixture: 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.

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:

The average value of n is 44.

Cationic polymers

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 polyethyleneimine.

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 binds to DNA instead of histones. In a preferred embodiment, the cationic polymer is protamine (eg, protamine sulfate).

Physical and chemical properties

The physicochemical property of lipid composition can depend on its component.For example, the composition comprising cholesterol as structured lipid can have the physicochemical property different from the composition comprising different structured lipid.Similarly, the physicochemical property of composition can depend on the absolute or relative amount of its component.For example, the composition comprising higher mole fraction phospholipid can have the physicochemical property different from the composition comprising lower mole fraction phospholipid.Physicochemical property can also depend on the method and condition of preparing composition and change.

The physicochemical properties of the lipid composition can be characterized by a variety of methods. For example, the morphology and size distribution of the composition can be examined 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 the zeta potential. Dynamic light scattering can also be used to determine particle size. Multiple characteristics 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).

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 about 40nm to about 250nm, such as about 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm or 300nm. 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, In some 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.

The lipid composition can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the lipid composition, such as the particle size distribution of the lipid composition. Smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of the composition can be about 0 to about 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 the lipid composition can be about 0.10 to about 0.20.

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 charge, 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.

The encapsulation efficiency of therapeutic or preventive agents describes the ratio of the amount of therapeutic or preventive agents that are 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 therapeutic or preventive agents 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 therapeutic or preventive agents (e.g., RNA) in the solution. For compositions as described herein, the encapsulation efficiency of therapeutic or preventive agents can be at least 50%, such as 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%.

Pharmaceutical composition

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

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), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffers, histidine buffers and acetate buffers), surfactants (e.g., nonionic surfactants such as polysorbate 60, For example, the carrier 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 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.

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 various 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 (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches) for surface and/or transdermal administration. Suspensions, powders and other forms.

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.

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

Nasal drug delivery system refers to a type of preparation that is administered through the nasal cavity to exert local or systemic therapeutic or preventive effects. It is particularly suitable for drugs that need to be highly expressed locally in the nasal cavity or drugs that avoid the first-pass effect of the liver. The advantages of nasal drug delivery are that the nasal mucosa has a large area and rich blood vessels, so the drug is absorbed quickly and takes effect quickly after administration. In addition, the first-pass effect of the liver is low, the systemic toxicity is low, and the bioavailability is high.

Common dosage forms for nasal administration include nasal drops, nasal sprays, powders, gel preparations and emulsions. Among them, the diffusion degree and dispersion area of nasal spray drugs in the nasal mucosa are relatively wide. Currently, the commonly used atomization devices are quantitative 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 residence time of the drug in the nasal cavity, which is beneficial to absorption and improves bioavailability.

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.

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.

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

In a preferred embodiment, the nasal administration includes nasal instillation or nasal spray administration.

In one embodiment, the nasal spray administration is performed by an aerosol administration device.

In one embodiment, the aerosol drug delivery device comprises a syringe, a plastic needle, a nasal spray device and a dose stopper.

In one embodiment, the atomization drug delivery device can convert liquid medicine into mist particles (jet atomization) for delivery. Umbrella spray.

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. 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 particles and spraying out from the air outlet.

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 body tissues (or organs).

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.

Therapeutic/preventive agents

The lipid composition may contain 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 a mammalian mucosa, and treating a disease or condition in a mammal in need thereof, the methods comprising administering a lipid composition or pharmaceutical composition containing a therapeutic or prophylactic agent to the mammalian mucosa.

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.

In some embodiments, the disease is an infectious disease.

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

In some embodiments, the 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 associated with infectious diseases such as new 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 mRNA encoding infectious disease-derived antigens and/or epitopes. In some embodiments, vaccines and/or compounds that can cause immune responses are administered by mucosal administration of lipid compositions including compounds according to formula (I), (II), (III) or (IV).

Polynucleotide

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

In some embodiments, the therapeutic agent or preventive agent of the present invention is 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 RNA produced by chemical synthesis, recombinant production and in vitro transcription. In one embodiment, the RNA of the present invention is used to express a polypeptide in a host cell.

In one embodiment, the therapeutic agent 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 by RNA polymerase.

In some embodiments, the therapeutic or preventive agent of the present invention is a messenger RNA (mRNA). Generally speaking, the mRNA may include 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.

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.

As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence comprising continuous or discontinuous adenylic acid. The poly(A) sequence is typically located at the 3' end of the RNA, such as the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not contain nucleotides other than adenylic acid 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 linked to the free 3' end of the IVT-RNA, such as the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

As used herein, the term "5' cap" generally refers to an N7-methylguanosine structure (also known as "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 can be attached to the RNA after transcription using a capping enzyme.

In some embodiments, the therapeutic agent 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. DNA can be double-stranded, single-stranded, linear, and circular DNA.

The DNA template can be provided in a suitable transcription vector. In general, the DNA template can be a double-stranded complex comprising 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, a coding sequence, 3'-UTR, and an optional poly (A) sequence. The promoter can be a promoter available 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) sequences in the DNA template are the corresponding sequences contained in the RNA described herein or are complementary thereto. As a polynucleotide of a DNA vaccine, it can be provided in a plasmid vector (e.g., a circular plasmid vector).

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

Modified nucleotides

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- 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.

Application of lipid composition and pharmaceutical composition

The lipid composition of the present invention, pharmaceutical composition, nasal drops and nasal spray can be used to treat diseases, illnesses or the patient's condition. Specifically, these lipid compositions, pharmaceutical compositions, nasal drops and nasal sprays can be used to treat diseases, illnesses 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. The mRNA can be translated subsequently to produce the polypeptide, thus reducing or eliminating the problems caused by the absence or abnormal activity of the polypeptide.

There are a variety of diseases, disorders or conditions that can be characterized by the loss of protein activity (or substantially reduced 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 conditions of a subject by administering a lipid composition, pharmaceutical composition, nasal drops or nasal spray of the present invention, wherein the lipid composition includes RNA and a lipid component, the lipid component includes 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.

The methods provided by the present invention relate to the administration of lipid compositions containing one or more therapeutic or preventive agents, pharmaceutical compositions, nasal drops or nasal sprays comprising these compositions. For features and embodiments of the present invention, the terms therapeutic agent and preventive agent can be used interchangeably herein. Lipid compositions and pharmaceutical compositions can be administered to subjects using any reasonable amount and any route of administration, which can effectively achieve the prevention, treatment, diagnosis of 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, etc.

Diseases, disorders or conditions characterized by malfunction or abnormal protein or polypeptide activity to which lipid compositions, pharmaceutical compositions, nasal drops and nasal sprays may be administered include, but are not limited to, rare diseases, infectious diseases (in the form of vaccines and therapeutic agents), cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. The cancers include, 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's lymphoma, non-Hodgkin's 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 disease includes, for example, type II diabetes, scurvy, hypoglycemia, hyperlipidemia or osteoporosis.

In some embodiments, the disease is an infectious disease.

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

In one aspect, the present invention also provides the 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.

In one embodiment, the disease or condition is characterized by malfunction or abnormal 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.

On the other hand, the present invention also provides a method for treating or preventing a disease or condition, comprising administering a therapeutic agent or a 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.

In one embodiment, at least one additional dose in 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.

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

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 by a mucosal route, for example, with a lipid composition or pharmaceutical composition of the present invention; and at least one other dose is administered by 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 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 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.

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.

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

In one embodiment, a dose of a therapeutic agent or prophylactic agent as described herein is administered first by an intramuscular route, and then a dose of a lipid composition or pharmaceutical composition of the present invention is administered by a mucosal route.

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 via a mucosal route.

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

The present invention also relates to the lipid composition of the present invention or the pharmaceutical composition of the present invention in the preparation for the treatment or prevention of a disease or condition, wherein a therapeutic agent or a preventive agent is administered to a subject in a multiple dose regimen, wherein at least one dose is administered via a mucosal route to the lipid composition of the present invention or the pharmaceutical composition of the present invention. The multiple dose regimen is as described above. The disease or condition is as described above.

Beneficial Effects

The lipid composition, pharmaceutical composition, nasal drops or nasal spray provided by the present invention can show 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) good targeting and low systemic toxicity; (3) no obvious changes in the physicochemical properties and expression efficiency before and after atomization; (4) can induce mucosal immune response; (5) can be combined with other routes such as intramuscular administration in a "primary immunization + boost" mode (systemic immunity + mucosal immunity), which can induce a higher level of humoral immunity and cellular immunity, and can also induce mucosal immune response.

Example

The present invention is further described by 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.

Unless otherwise specified, all percentages in the following examples are mole percentages (mol %).

Experimental Materials

The cationic lipid according to formula (I) is synthesized by Silicomai or prepared by reference, such as CN110520409A, WO2018081480A1 or US11,246,933B1; phospholipid (DOPE) is 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.

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

General considerations

Unless otherwise noted, all solvents and reagents used were commercially available and used as received. ¹ H NMR spectra were recorded at 300 K using a Bruker Ultrashield 300 MHz instrument in CDCl _3. Chemical shifts are reported in parts per million (ppm) relative to TMS (0.00) for ¹ H. Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using an ISCO RediSep Rf Gold fast column (particle size: 20-40 microns).

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

This article uses the following abbreviations:
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

A. Compound SW-II-115

1. Synthesis of intermediate 3

EDCI (17.3 g, 90 mmol, 2 eq.) and DMAP (2.2 g, 18 mmol, 0.4 eq.) were added 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.), followed by DIEA (23.2 g, 180 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N ₂ 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 H ₂ O (40 mL), dried over anhydrous Na ₂ SO ₄ , 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.

2. Synthesis of Intermediate 5

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 _N2 protection. TLC (DCM: MeOH = 10: 1) showed that compound 3 was consumed, and TLC (DCM / MeOH = 10 / 1) showed that a new major 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 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 a yellow oily compound 5 (264 mg, 56%).

3. Synthesis of Intermediate 8

Pd(dppf)Cl ₂ (112 mg, 0.171 mmol, 0.1 eq.) and potassium carbonate (709 mg, 5.136 mmol, 3 eq.) were added to compound 6 (500 mg, 1.712 mmol, 1 eq.) and compound 7 (1.113 g, 8.562 mmol, 5 eq.) in a mixed solvent of dioxane/water (5 mL/0.5 mL). The mixture was stirred at 100° C. overnight under N _2. TLC (PE:EA=15:1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water, and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluted with PE:EA (1:0-10:1) to give compound 8 (455 mg, 88%) as a colorless oil.

4. Synthesis of Intermediate 9

To a solution of compound 8 (455 mg, 1.497 mmol, 1 eq.) in THF (5 mL) was added LiAlH ₄ (1.5 mL, 1.497 mmol, 1 M in THF, 1 eq.) at 0°C under N ₂ protection. The mixture was stirred at room temperature under N ₂ for 2 hours. TLC (PE: EtOAc = 5: 1) showed that 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 Na ₂ SO ₄ , filtered and concentrated in vacuo to give crude compound 9 (419 mg, >100%) as a colorless oil without further purification.

5. Synthesis of Intermediate 10

EDCI (583 mg, 3.036 mmol, 2 eq.) and DMAP (74 mg, 0.607 mmol, 0.4 eq.) were added to a DCM (4 mL) solution containing compound 1 (339 mg, 1.518 mmol, 1 eq.) and compound 9 (419 mg, 1.518 mmol, 1 eq.), followed by DIEA (783 mg, 6.072 mmol, 4 eq.). The reaction mixture was stirred at room temperature under N ₂ protection for 16 hours. TLC (petroleum ether: ethyl acetate = 10: 1) showed that the desired product was formed. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , 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 (443 mg, 60.7%) as a colorless oil.

6. Synthesis of the final product SW-II-115

To a mixture containing compound 10 (307 mg, 0.64 mmol, 1 eq.) and compound 5 (210 mg, 0.64 mmol, 1 eq.) K ₂ CO ₃ (530 mg, 3.84 mmol, 6 eq.) and KI (212 mg, 1.28 mmol, 2 eq.) were added to the solvent CPME/CH ₃ CN (3 mL/3 mL). After the addition was complete, the mixture was stirred at 90°C overnight under N _2. TLC (DCM:MeOH=10:1) showed that 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 Na ₂ SO ₄ , 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-115 (266 mg, 57%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.21–6.99 (m, 3H), 5.05 (s, 2H), 4.05 (t, J=6.8 Hz, 2H), 3.58 (t, J=5.3 Hz, 2H), 2.69–2.46 (m, 10H), 2.31 (dt, J=20.0, 7.5 Hz, 4H), 1.69–1.18 (m, 51H), 0.89 (dt, J=12.4, 6.3 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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),5 3.90(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).

B. Compound SW-II-118

1. Synthesis of intermediate 3

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(PPh ₃ ) ₄ (tetrakistriphenylphosphine palladium, 289 mg, 0.25 mmol, 0.05 eq.) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 2.0 eq.) in toluene (10 ml) and H ₂ O (1 ml) was stirred at 110° C. under N ₂ 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 H ₂ O (40 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.5 g, 45%) as a colorless oil.

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

2. Synthesis of intermediate 4

LiAlH ₄ (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 then the reaction was allowed to react at 10°C for 3 hours. TLC showed that the reaction was good, and the reaction was concentrated and diluted with Na ₂ SO ₄ (20 mL) and extracted with EA (30 mL×2), and the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain yellow oily compound 4 (3.12 g, crude product).

3. Synthesis of Intermediate 6

A DCM (5 mL) solution containing 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.) was stirred at 10 ° C for 16 h under N ₂ protection. TLC (DCM: MeOH = 10: 1) showed that the reaction was complete and a new main spot was observed. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography, eluted with PE: EA (1: 0-10: 1, v / v) to give a colorless oil compound 6 (145 mg, 31%).

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

4. Synthesis of the final product SW-II-118

A mixture containing compound 6 (140 mg, 0.37 mmol, 1.0 eq.), compound 7 (243 mg, 0.55 mmol, 1.5 eq.), K ₂ CO ₃ (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 CH ₃ CN (1 mL) under N ₂ 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 NaHCO ₃ (30 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ , 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.

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.11 (s, 4H), 4.91–4.79 (m, 1H), 4.26 (t, J=7.2 Hz, 2H), 3.80–3.68 (m, 2H), 2.90 (t, J=7.1 Hz, 4H), 2.81–2.67 (m, 4H), 2.62–2.52 (m, 2H), 2.28 (td, J=7.5, 2.6 Hz, 4H), 1.64–1.51 (m, 11H), 1.38–1.17 (m, 42H), 0.93–0.82 (m, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.61(d, J＝11.7 Hz),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).

C. Compound SW-II-120

1. Synthesis of intermediate 3

A mixed 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(PPh ₃ ) ₄ (289 mg, 0.25 mmol, 0.05 eq.) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 2.0 eq.) in toluene (10 ml) and H ₂ O (1 ml) was stirred at 110° C. under N ₂ 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 H ₂ O (40 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (1:0-10:1) to give compound 3 (0.78 g, 62%) as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ7.19 (d, J=8.1 Hz, 2H), 7.13 (d, J=8.1 Hz, 2H), 4.14 (q, J=7.1 Hz, 2H), 3.57 (s, 2H), 2.62-2.51 (m, 2H), 1.58 (d, J=11.1 Hz, 2H), 1.35-1.21 (m, 9H), 0.88 (t, J=6.7 Hz, 3H).

2. Synthesis of intermediate 4

LiAlH ₄ (477 mg, 12.56 mmol, 4.0 eq.) was added to a THF (10 mL) solution containing compound 3 (780 mg, 3.14 mmol, 1.0 eq.) at -78°C, and the reaction was stirred at 10°C for 3 hours. Thin layer chromatography showed that the reaction was proceeding well. The reaction was concentrated and diluted with Na ₂ SO ₄ (20 mL) and extracted with EA (30 mL*2), and the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give a colorless oily compound 4 (640 mg, crude product).

3. Synthesis of Intermediate 6

Contains compound 4 (640 mg, 3.10 mmol, 1.0 eq.), compound 5 (1.06 g, 4.70 mmol, 1.5 eq.), EDCI A solution of 4-(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 N ₂ protection. TLC (DCM: MeOH = 10: 1) showed that the reaction was complete and a new main point was observed. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography, eluted with PE: EA (1: 0-10: 1, v / v) to give a colorless oily compound 6 (465 mg, 36%).

4. Synthesis of the final product SW-II-120

A mixture containing compound 6 (100 mg, 0.25 mmol, 1.0 eq.), compound 7 (161 mg, 0.36 mmol, 1.5 eq.), K ₂ CO ₃ (104 mg, 0.75 mmol, 3.0 eq.) and KI (83 mg, 0.50 mmol, 2.0 eq.) was stirred in CPME (1 mL) and CH ₃ CN (1 mL) under N ₂ 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 NaHCO ₃ (30 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ , 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.

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.07 (d, J=8.9 Hz, 4H), 4.89–4.73 (m, 1H), 4.23 (t, J=7.2 Hz, 2H), 3.83–3.65 (m, 2H), 2.87 (t, J=7.2 Hz, 4H), 2.82–2.67 (m, 4H), 2.61–2.45 (m, 2H), 2.25 (td, J=7.5, 2.5 Hz, 4H), 1.65–1.44 (m, 15H), 1.27 (dd, J=13.2, 11.3 Hz, 42H), 0.85 (t, J=6.8 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.57 (d, J＝11.5 Hz), 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＝16.1Hz),34.14 (d, J = 3.3 Hz), 31.79 (d, J = 13.4 Hz), 31.49 (s), 29.50 (d, J = 2.2 Hz), 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).

D. Compound SW-II-121

1. Synthesis of intermediate 3

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 h. TLC (Stone The reaction mixture was diluted with DCM (20 mL) and washed with H ₂ O (40 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether: ethyl acetate (1:0-10:1) to give yellow oily compound 3 (1.2 g, 66.9%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.92–4.82 (m, 1H), 3.42 (t, J=6.8 Hz, 2H), 2.31 (t, J=7.5 Hz, 2H), 1.95–1.82 (m, 2H), 1.70–1.19 (m, 36H), 0.90 (t, J=6.8 Hz, 6H).

2. Synthesis of Intermediate 5

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 ₂ protection. TLC (petroleum ether: ethyl acetate = 19: 1) showed that compound 3 was consumed and TLC (DCM / MeOH = 10/1) showed that a new major spot was observed. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with EtOAc (50 mL) and washed with H ₂ O (3×50 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, and 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 5 (3 g, 60%).

3. Synthesis of Intermediate 8

Pd(pph ₃ ) ₄ (238 mg, 0.206 mmol, 0.05 eq.) and K 2 CO 3 (1.7 g, 12.35 mmol, 3 eq.) were added to a mixed solution of toluene/water (10 mL/1 mL) containing compound _{6 (1 g, 4.115 mmol, 1 eq.) and compound 7 (} 889 mg, 6.173 mmol, 1.5 eq.). The mixture was stirred under N ₂ and 110° C. for 2 hours. TLC (PE:EA=10:1) showed that 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 Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography eluted with PE:EA (1:0-10:1) to give compound 8 (714 mg, 66%) as a colorless oil.

4. Synthesis of Intermediate 9

Under _N2 protection, _LiAlH4 (2.7mL, 2.725mmol, 1M, in THF, 1eq.) was added to a mixture of compound 8 (714mg, 2.725mmol, 1eq.) in THF (7mL) 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 main spot was observed. The mixture was quenched with water (2.7mL) 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. The residue was purified by silica gel column chromatography, eluted with PE:EA (1:0-10:1) to give compound 9 (103mg, 63%) as a colorless oil.

5. Synthesis of Intermediate 11

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.) were added 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.). The reaction mixture was stirred at room temperature under N ₂ for 16 hours. TLC (petroleum ether: ethyl acetate = 10: 1) showed that the desired product was formed. The reaction mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , 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 11 (169 mg, 29%) as a colorless oil.

6. Synthesis of the final product SW-II-121

K ₂ CO ₃ (330 mg, 2.394 mmol, 6 eq.) and KI (132 mg, 0.798 mmol, 2 eq.) were added to a CPME/CH ₃ CN (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.). After the addition was complete, the mixture was stirred at 90° C. under N ₂ overnight. TLC (DCM:MeOH=10:1) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with EA and washed with water, and the organic layer was dried over anhydrous Na ₂ SO ₄ , 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 yellow oily compound SW-II-121 (145 mg, 46%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.11 (s, 4H), 4.92–4.80 (m, 1H), 4.26 (t, J=7.2 Hz, 2H), 3.80 (s, 2H), 2.87 (dd, J=26.6, 19.4 Hz, 7H), 2.62–2.51 (m, 2H), 2.28 (td, J=7.2, 3.6 Hz, 4H), 1.75–1.45 (m, 14H), 1.42–1.09 (m, 45H), 0.88 (t, J=6.8 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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).

E. Compound SW-II-122

1. Synthesis of compound 3

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 then K ₂ CO ₃ (1.92 g, 13.9 mmol, 3 eq.) and Pd(pph ₃ ) ₄ (269 mg, 0.23 mmol, 0.05 eq.) were added to the mixture. The reaction mixture was placed in N ₂ and heated to 110° C. 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 H ₂ O (80 mL) and extracted with ethyl acetate (60 mL×3), and 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 petroleum ether/ethyl acetate (1/0-10/1) to obtain yellow oily compound 3 (800 mg, 78%).

2. Synthesis of Compound 4

Under nitrogen protection at 0°C, LiAlH ₄ (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 reactants were heated 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 ethyl acetate (60 mL×3) was extracted. 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, eluted with ethyl acetate/petroleum ether=1/10, to give yellow oily compound 4 (600 mg, 98%).

3. Synthesis of Compound 6

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 protection. TLC (DCM/MeOH=30/1) showed that the starting material was consumed and a new spot was formed. The mixture was quenched with water (70 mL) and extracted with DCM (80 mL×3). The combined organic layer was washed with brine (2×20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether = 3/97 solution as eluent to give yellow oily compound 6 (680 mg, 48.5%).

4. Synthesis of SW-II-122

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 CH ₃ CN (2 mL), and 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. for 16 hours under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the reaction was complete. The reaction mixture was 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 SW-II-122 (68 mg, 40%) as a colorless oil.

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.32 (d, J=26.4 Hz, 1H), 7.17 (dd, J=27.2, 21.1 Hz, 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=7.6 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 1.74–1.57 (m, 9H), 1.50 (d, J=5.6 Hz, 4H), 1.37–1.15 (m, 43H), 0.94–0.80 (m, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.55 (d, J＝2.4 Hz),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 = 4.8 Hz), 25.33 (s), 24.92 (s), 24.71 (s), 24.48 (s), 22.64 (d, J = 6.8 Hz), 14.12 (s).

F. Compound SW-II-127

1. Synthesis of compound 3

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), and 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 protection. 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 H ₂ O (40 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (1/0-10/1) to give yellow oily compound 3 (1.2 g, 66.9%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.92–4.82 (m, 1H), 3.42 (t, J=6.8 Hz, 2H), 2.31 (t, J=7.5 Hz, 2H), 1.95–1.82 (m, 2H), 1.70–1.19 (m, 36H), 0.90 (t, J=6.8 Hz, 6H).

2. Synthesis of Compound 5

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 major spot was observed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with EtOAc (50 mL) and washed with H ₂ O (3X 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.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.95–4.75 (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=7.5 Hz, 2H), 1.65–1.42 (m, 8H), 1.38–1.17 (m, 30H), 0.88 (t, J=6.8 Hz, 6H).

3. Synthesis of Compound 8

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), and 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 protection. TLC (PE:EA=10:1) showed that the starting material was consumed and a new spot was formed. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1/0-10/1) to obtain compound 8 (454 mg, 57%) as a colorless oil.

4. Synthesis of SW-II-127

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), and 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 overnight at 90 ° C under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the reaction was complete and the desired product was 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 and eluted with DCM/MeOH (1/0-10:1, v/v) to obtain a yellow oily compound SW-II-127 (42.49 mg, 22%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.25 (s, 2H), 7.17 (d, J=8.0 Hz, 2H), 5.07 (s, 2H), 4.91-4.82 (m, 1H), 3.83 (s, 2H), 2.90 (d, J=44.8 Hz, 5H), 2.64-2.55 (m, 2H), 2.35 (t, J=7.4 Hz, 2H), 2.28 (t, J=7.5 Hz, 2H), 1.76-1.46 (m, 14H), 1.42-1.19 (m, 41H), 0.88 (t, J=6.8 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.50 (d, J＝8.5 Hz),133.17 (s),128.61 (s),128.34 (s),77.29 (d, J＝11.4 Hz),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 = 8.8 Hz),31.80(d, J = 15.4 Hz),31.43(s),29.53(d, J = 2.5 Hz),29.25(s),28.95(d, J = 10.5 Hz),28.63(s),26.71(d, J = 18.2 Hz),25.33(s),24.93(s),24.62(s),22.65(d, J = 6.6 Hz),14.13(s).

G. Compound SW-II-134-1

1. Synthesis of compound 3

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) was 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) 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, eluted with PE/EA (1/0-20/1) to give compound 3 (723 mg, 99.6%) as a colorless oil.

2. Synthesis of Compound 4

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 and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed 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.

3. Synthesis of Compound 6

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 was 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 and purified by sodium sulfate column chromatography. Elution with oily ether/ethyl acetate (1/0-20/1) gave compound 6 (272 mg, 44%) as a colorless oil.

4. Synthesis of SW-II-134-1

Potassium carbonate (251 mg, 1.818 mmol, 6 eq.) and potassium iodide (101 mg, 0.61 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). After addition, the mixture was stirred at 90° C. overnight under nitrogen. 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-134-1 (168 mg, 75%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.06 (d, J=7.6 Hz, 1H), 7.01-6.93 (m, 2H), 4.25 (t, J=7.3 Hz, 2H), 4.05 (t, J=6.8 Hz, 2H), 3.85-3.72 (m, 2H), 2.98-2.69 (m, 8H), 2.62-2.48 (m, 4H), 2.29 (t, J=7.5 Hz, 4H), 1.72-1.48 (m, 14H), 1.45-1.17 (m, 36H), 0.89 (dt, J=11.9, 6.0 Hz, 9H).

¹³ CNMR (101MHz,CDCl ₃ )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), 34.22(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).

H. Compound SW-II-134-2

1. Synthesis of compound 3

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) was 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) showed that the reaction was complete and a new major spot was observed. The mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography using PE/EA (1/0-20/1) as eluent to give compound 3 (854 mg, 100%) as a colorless oil.

2. Synthesis of Compound 4

To a mixture of compound 3 (854 mg, 2.28 mmol, 1 eq.) in THF (9 mL) was added lithium aluminum hydride (2.3 mL, 2.28 mmol, 1 M, in THF, 1 eq.) at 0 ° C and 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 (724 mg, 92%) as a colorless oil without further purification.

3. Synthesis of Compound 6

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) was 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 that the desired product was 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 (473 mg, 41%) as a colorless oil.

4. Synthesis of SW-II-134-2

Potassium carbonate (225 mg, 1.63 mmol, 6 eq.) and potassium iodide (90 mg, 0.54 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). After addition, the mixture was stirred at 90° C. overnight under nitrogen. 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%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.06 (d, J=7.6 Hz, 1H), 6.96 (d, J=9.6 Hz, 2H), 4.25 (t, J=7.3 Hz, 2H), 4.05 (t, J=6.8 Hz, 2H), 3.80–3.66 (m, 2H), 2.86 (dd, J=12.8, 5.6 Hz, 4H), 2.78–2.67 (m, 4H), 2.60–2.52 (m, 4H), 2.29 (t, J=7.5 Hz, 4H), 1.57 (dt, J=15.8, 7.3 Hz, 14H), 1.30 (d, J=20.3 Hz, 45H), 0.88 (t, J=6.7 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.82(d,J＝16.9 Hz),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),28.65(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).

I. Compound SW-II-134-3

1. Synthesis of compound 3

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) 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 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 a colorless oily compound 3 (4.365 g, 28%).

2. Synthesis of Compound 5

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 main 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 a yellow oily compound 5 (1.008 g, 21%).

3. Synthesis of Compound 8

Compound 6 (500 mg, 2.283 mmol, 1 eq.) and compound 7 (699 mg, 6.849 mmol, 3 eq.) were dissolved in toluene/water. To the mixture in (5mL/1mL) was added palladium acetate (51mg, 0.228mmol, 0.1eq.), Ruphos (213mg, 0.457mmol, 0.2eq.) and potassium carbonate (945mg, 6.849mmol, 3eq.). The mixture was stirred at 110°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, eluted with PE/EA (1/0-20/1) to give compound 8 (507mg, 85%) as a colorless oil.

4. Synthesis of Compound 9

To a mixture of compound 8 (507 mg, 1.935 mmol, 1 eq.) in THF (5 mL) was added lithium aluminum hydride (2 mL, 1.935 mmol, 1 M, in THF, 1 eq.) at 0 ° C and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed that the reaction was complete 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 give compound 9 (492 mg,>100%) as a colorless oil without further purification.

5. Synthesis of Compound 10

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) was 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 that the desired product was 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 (1/0-10/1) to give compound 10 (329 mg, 36%) as a colorless oil.

6. Synthesis of SW-II-134-3

Potassium carbonate (282 mg, 2.04 mmol, 6 eq.) and potassium iodide (113 mg, 0.68 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-134-3 (63.59 mg, 25%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.07 (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).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.86 (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＝3.9Hz),33.54(d, J＝4.5Hz),32.41(s),31.95(d, J＝16.5Hz),29.49(s),29.15(dd, J＝21.1,2.4Hz),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).

J.SW-II-135-1

1. Synthesis of compound 3

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/H ₂ O (5 mL/1 mL), and Ruphos (201 mg, 0.43 mmol, 0.2 eq), Pd(OAc) ₂ (48.5 mg, 0.22 mmol, 0.1 eq) and Cs ₂ CO ₃ (2.10 g, 6.46 mmol, 3.0 eq.) were added to the mixture. The reaction mixture was heated to reflux at 110° C. for 16 hours under nitrogen protection. TLC (petroleum ether/ethyl acetate=10/1) showed that the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (40 mL) and extracted 3 times with EA (50 mL), and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , 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.

2. Synthesis of Compound 4

Under nitrogen protection at 0°C, LiAlH ₄ (3.55 mL, 3.55 mmol, 2 eq. in 1M THF) was added to compound 3 (540 mg, 1.78 mmol, 1.0 eq.) dissolved in THF (5 mL). The reactants were warmed 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 (10 mL), then adjusted to pH=6-7 with 1M hydrochloric acid, and extracted 3 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, eluted with PE/EA (1/0-10/1) to give compound 4 (442 mg, 90.2%) as a colorless oil.

3. Synthesis of Compound 6

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), 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, and then DIEA (826 mg, 6.4 mmol, 4.0 eq.) was added. 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 H ₂ O (40 mL) and extracted with EA (50 mL) 3 times, and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , 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.

4. Synthesis of SW-II-135-1

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/CH ₃ CN (1 mL/1 mL), and 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 protection. TLC (DCM/MeOH=10/1) showed that the reaction was complete and the desired product was 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, eluted with DCM/MeOH (1/0-10:1, v/v) to obtain yellow oily compound SW-II-135-1 (83.89 mg, 55.6%).

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

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

¹ H NMR (400 MHz, CDCl3) δ 6.97 (d, J = 7.6 Hz, 1H), 6.91–6.74 (m, 2H), 4.76 (s, 1H), 3.99 (dt, J = 13.6, 6.4 Hz, 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, 7.2 Hz, 4H), 1.93–1.79 (m, 2H), 1.62–1.41 (m, 14H), 1.23 (d, J = 24.4 Hz, 32H), 0.82 (ddd, J = 13.6, 8.0, 5.6 Hz, 9H).

¹³ C NMR (101 MHz, CDCl3) δ 172.81 (d, J = 6.4 Hz), 139.55 (s), 137.38 (s), 137.14 (s), 128.16 (d, J = 2.4 Hz), 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＝4.7 Hz).

K. Compound SW-II-135-2

SW-II-135-2

1. Synthesis of compound 3

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/H ₂ O (5 mL/1 mL), and Ruphos (201 mg, 0.43 mmol, 0.2 eq), Pd(OAc) ₂ (48.5 mg, 0.22 mmol, 0.1 eq) and Cs ₂ CO ₃ (2.10 g, 6.46 mmol, 3.0 eq.) were added to the mixture. The reaction mixture was heated to reflux at 110° C. for 16 hours under nitrogen protection. TLC (petroleum ether/ethyl acetate=10/1) showed that the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (40 mL) and extracted 3 times with EA (50 mL), and the resulting organic phase was washed twice with brine (20 mL), dried over anhydrous Na ₂ SO ₄ , 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.

2. Synthesis of Compound 4

Under nitrogen protection at 0°C, LiAlH ₄ (3.62 mL, 3.62 mmol, 1 M, in THF, 2 eq.) was added to compound 3 (651 mg, 1.81 mmol, 1.0 eq.) dissolved in THF (7 mL). The reactants were warmed 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 (10 mL), then adjusted to pH=6-7 with 1 M hydrochloric acid, and extracted 3 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, eluted with PE/EA (1/0-10/1) to give compound 4 (571 mg, 95.2%) as a colorless oil.

3. Synthesis of Compound 6

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), EDCI (657 mg, 3.44 mmol, 2.0 eq.) and DMAP (84 mg, 0.68 mmol, 0.4 eq.) were added to the mixture, and then DIEA (887.5 mg, 6.88 mmol, 4.0 eq.) was added. 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 H ₂ O (50 mL) and extracted with EA (60 mL) 3 times, and the resulting organic phase was washed twice with brine (25 mL), dried over anhydrous Na ₂ SO ₄ , 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.

4. Synthesis of SW-II-135-2

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/CH ₃ CN (1 mL/1 mL), and 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 protection. TLC (DCM/MeOH=10/1) showed that the reaction was complete and the desired product was 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 and eluted with DCM/MeOH (1/0-10:1, v/v) to give a yellow oily compound SW-II-135-2 (31.41 mg, 21.9%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.98 (d, J = 7.6 Hz, 1H), 6.87 (d, J = 2.4 Hz, 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, 7.2 Hz, 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).

¹³ C NMR (100 MHz, CDCl ₃ )δ172.82 (d, J＝6.8 Hz), 139.61 (s), 137.29 (d, J＝16.4 Hz), 128.15 (s), 124.67 (s), 76.41 (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).

L. Compound SW-II-136-2

SW-II-136-2

1. Synthesis of compound 3

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/H ₂ O (30 mL/3 mL), and Ruphos (1.28 g, 2.74 mmol, 0.2 eq), Pd(OAc) ₂ (308.3 mg, 1.37 mmol, 0.1 eq) and K ₂ CO ₃ (5.67 g, 41.10 mmol, 3.0 eq.) were added to the mixture. The reaction mixture was heated to reflux at 110° C. for 16 hours under nitrogen protection. TLC (PE/EA=10/1) showed that the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (90 mL) and extracted with EA (110 mL) three times, and the organic phase was washed twice with brine (40 mL), dried over anhydrous Na ₂ SO ₄ , 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.

2. Synthesis of Compound 4

Under nitrogen protection at 0°C, LiAlH ₄ (1M, 12.45 mL, 2.0 eq.) was added to compound 3 (1.98 g, 6.23 mmol, 1.0 eq.) dissolved in THF (20 mL). The reactants were warmed 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 H ₂ O (70 mL), Then, the pH was adjusted to 6-7 with 1M hydrochloric acid and extracted with EA (80 mL) for 3 times. The organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1/0-10/1) to obtain compound 4 (1.28 g, 71.1%) as a colorless oil.

3. Synthesis of Compound 7

Under nitrogen protection at 0°C, DMSO (3.63 g, 51.72 mmol, 15 eq.), TEA (1.25 g, 12.4 mmol, 4.0 eq.) and PySO ₃ (1.27 g, 7.97 mmol, 2.57 eq.) were added to compound 4 (900 g, 3.1 mmol, 1.0 eq.) dissolved in DCM (9 mL). The mixture was stirred at 0°C for 30 minutes, then warmed to room temperature and stirred for 90 minutes under nitrogen protection. Compound 6 (4.74 g, 13.62 mmol, 3.0 eq.) was then added to the mixture, and the reaction mixture was reacted at 25°C for 2 hours under nitrogen protection. TLC (PE/EA=10/1) showed that the reaction was complete and the desired product was formed. The reaction mixture was washed with H ₂ O (60 mL) and extracted 3 times with EA (70 mL), and the resulting organic phase was washed twice with brine (40 mL), dried over anhydrous Na ₂ SO ₄ , 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.

4. Synthesis of Compound 8

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 hydrogen protection for 16 h. TLC (PE/EA=10/1) showed that the starting material was completely consumed and the desired product was generated. The reaction mixture was filtered through celite and washed with MeOH (40 mL×2), dried over anhydrous Na ₂ SO ₄ and the filtrate was concentrated under reduced pressure to obtain a light yellow oily compound 8 (298 mg, 88.2%).

5. Synthesis of Compound 9

Under nitrogen protection at 0°C, LiAlH ₄ (1M, 1.66mL, 2.0eq.) was added to compound 8 (298mg, 0.83mmol, 1.0eq.) dissolved in THF (3mL). The reactants were warmed 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 H ₂ O (20mL), then adjusted to pH=6-7 with 1M hydrochloric acid, and extracted 3 times with EA (30mL). The organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1/0-10/1) to give compound 9 (254mg, 98.3%) as a colorless oil.

6. Synthesis of Compound 11

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 for 16 hours under nitrogen protection. 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 1M hydrochloric acid and extracted 3 times with EA (30 mL), and the resulting organic phase was washed twice with brine (15 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE/EA (1/0-7/1) to give compound 11 (210 mg, 50.5%) as a yellow oil.

7. Synthesis of SW-II-136-2

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/CH ₃ CN (1.5 mL/1.5 mL), and K ₂ CO ₃ (220.2 mg, 1.60 mmol, 5.0 eq) and KI (106 mg, 0.64 mmol, 2.0 eq) were added to the mixture. After the addition, the reaction mixture was stirred at 90° C. overnight under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the reaction was complete and the desired product was formed. The mixture was extracted with EA and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ , 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 yellow oily compound SW-II-136-2 (208 mg, 90.4%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.04 (d, J=7.6 Hz, 1H), 6.92 (d, J=9.6 Hz, 2H), 4.45 (s, 1H), 4.06 (dd, J=12.0, 5.2 Hz, 4H), 3.64 (t, J=5.2 Hz, 2H), 2.72 (t, J=5.2 Hz, 2H), 2.65–2.50 (m, 10H), 2.29 (t, J=7.6 Hz, 4H), 1.69–1.48 (m, 18H), 1.41–1.24 (m, 36H), 0.95–0.78 (m, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.86 (d, J＝2.8 Hz), 140.48 (s), 139.24 (s), 138.01 (s), 129.13 (d, J＝14.8 Hz), 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＝18.0,2.0Hz),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).

M. Compound SW-II-137-1

1. Synthesis of compound 3

Compound 1 (500 mg, 1.95 mmol, 1.0 eq.) was dissolved in toluene (5.0 mL), and then compound 2 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh ₃ ) ₄ (225 mg, 0.19 mmol, 0.1 eq.), water (1 mL) and K ₂ CO ₃ (808 g, 5.85 mmol, 3.0 eq.) were added. The mixture was reacted at 110°C for 3 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the raw material had reacted completely and the desired product was formed. H ₂ O (70 mL) was added to the reaction, and EA (80 mL×3) was extracted. The combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, eluted with PE/EA (1/0-5:1, v/v) to obtain a colorless oily compound (320 mg, 70%).

2. Synthesis of Compound 4

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 protection. Then the reaction was carried out at room temperature for 2 hours. TLC (PE/EA=10/1) showed that the raw material was completely reacted and the desired product was generated. HCl (1M, 4 mL) solution and H ₂ O (10 mL) were added to quench, and EA (50 mL×3) was used for extraction. The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, eluted with PE/EA (1/0-10/1, v/v) to obtain yellow oily compound 4 (224 mg, 84.8%).

3. Synthesis of Compound 6

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. Then, the mixture was reacted at room temperature overnight under nitrogen protection. TLC (PE/EA=20/1) showed that the raw material had reacted completely and the desired product was formed. The reaction was quenched with HCl (1 M) solution, the pH was adjusted to 4-6, and the mixture was extracted with EA (40 mL×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified using a silica gel column and eluted with PE/EA (1/0-20/1, v/v) to give compound 6 (90 mg, 48.6%) as a colorless oil.

4. Synthesis of SW-II-137-1

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 K ₂ CO ₃ (157 mg, 1.25 mmol, 5.0 eq.) was added. The mixture was reacted overnight at 90°C under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the raw material was completely reacted and the desired product was formed. The mixture was quenched with water (50 mL) and extracted with EA (40 mL×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with DCM/MeOH (1/0-10/1, v/v) to obtain a yellow oily compound (98 mg, 52.12%, SW-II-137-1).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.02 (d, J=8.8 Hz, 4H), 4.92-4.71 (m, 1H), 4.01 (t, J=6.4 Hz, 2H), 3.78 (s, 1H), 3.55 (t, J=5.2 Hz, 2H), 2.76-2.40 (m, 10H), 2.21 (dd, J=15.6, 7.7 Hz, 4H), 1.95-1.80 (m, 2H), 1.49 (ddd, J=24.4, 15.8, 6.2 Hz, 15H), 1.34-1.13 (m, 37H), 0.82 (dt, J=13.6, 7.2 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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＝11.6 Hz),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) ^.

N. Compound SW-II-137-2

1. Synthesis of compound 3

Compound 1 (500 mg, 2.06 mmol, 1.0 eq.), compound 2 (286 mg, 2.47 mmol, 1.2 eq.), Pd(PPh ₃ ) ₄ (119 mg, 0.1 mmol, 0.1 eq.) and K ₂ CO ₃ (851 mg, 6.21 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL), and water (0.5 mL) was added. Then, the mixture was reacted at 110° C. for 3 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the raw materials were completely reacted and the desired compound was formed. The reaction was quenched with H ₂ O (70 mL) and extracted with EA (80 mL×3). The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with (PE/EA=5/1, v/v) to obtain colorless oily compound 3 (420 mg, 87.5%).

2. Synthesis of Compound 4

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 protection. Then, the reaction was allowed to proceed at room temperature for 2 hours. TLC (PE/EA = 5/1) showed that the raw material had reacted completely and the desired product had been formed. The mixture was quenched with HCl (1 M, 4 mL) solution and H ₂ O (10 mL), and extracted with EA (50 mL × 3). The organic phase was washed with saturated brine (2 × 30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, and eluted with (PE/EA = 5/1, v/v) to obtain compound 4 (320 mg, 94%) as a colorless oil.

3. Synthesis of Compound 6

Compound 4 (320 mg, 1.55 mmol, 1.0 eq.) was dissolved in DCM (4.0 mL), and compound 5 (416 mg, 1.86mmol, 1.2eq.), EDCI (594mg, 3.11mmol, 2.0eq.), DIEA (802mg, 6.21mmol, 4.0eq.) and DMAP (76mg, 0.62mmol, 0.4eq.). Then, the reaction was allowed to proceed overnight at room temperature under nitrogen protection. TLC (PE/EA=20/1) showed that the raw materials had reacted completely and the desired product was formed. The reaction was quenched with HCl (1M) solution and the pH was adjusted to 4-6, and extracted with DCM (60mL×3). The organic phase was washed with saturated brine (2×35mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with (PE/EA=5/1, v/v) to obtain colorless oily compound 6 (300mg, 47.17%).

4. Synthesis of SW-II-137-2

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 K ₂ CO ₃ (235 mg, 1.70 mmol, 5.0 eq) was added. The mixture was reacted at 90°C overnight under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the raw materials reacted completely and the desired product was generated. The reaction was quenched with water (50 mL) and extracted with EA (60 mlx3). The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with DCM/MeOH (1/0-10/1, v/v) to obtain a light yellow oily compound (105 mg, 40.3%, SW-II-137-2).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.10 (d, J=8.8 Hz, 4H), 5.04–4.74 (m, 1H), 4.08 (t, J=6.4 Hz, 2H), 3.58 (t, J=5.2 Hz, 2H), 2.65 (dd, J=9.6, 5.6 Hz, 4H), 2.60–2.44 (m, 6H), 2.29 (dd, J=16.4, 7.6 Hz, 4H), 2.01–1.88 (m, 2H), 1.59 (dt, J=9.2, 7.2 Hz, 6H), 1.54–1.42 (m, 8H), 1.39–1.11 (m, 41H), 0.88 (dt, J=11.8, 6.0 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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＝14.8Hz) ,3 1.82(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 = 2.4 Hz), 26.64 (s), 25.33 (s), 25.02 (d, J = 15.6 Hz), 22.62 (d, J = 11.6 Hz), 14.08 (d, J = 8.0 Hz).

O. Compound SW-II-137-3

1. Synthesis of compound 3

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) was 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 a colorless oily compound 3 (7.391 g, 37%).

2. Synthesis of Compound 5

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 main 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 and eluted with DCM/MeOH (1/0-10:1, v/v) to give a yellow oily compound 5 (3.695 g, 52%).

3. Synthesis of Compound 8

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) was added Pd(dtbpf)Cl ₂ (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.

4. Synthesis of Compound 9

To a mixture of compound 8 (568 mg, 2.29 mmol, 1 eq.) in THF (6 mL) was added lithium aluminum hydride (2.3 mL, 2.29 mmol, 1 M, in THF, 1 eq.) at 0 ° C and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed 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 9 (541 mg,>100%) as a colorless oil without further purification.

5. Synthesis of Compound 10

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) was 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 that the desired product was 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 (1/0-10/1) to give compound 10 (372 mg, 44%) as a colorless oil.

6. Synthesis of SW-II-137-3

Potassium carbonate (244 mg, 1.765 mmol, 6 eq.) and potassium iodide (117 mg, 0.706 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-137-3 (56.17 mg, 20%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.09 (s, 4H), 4.92–4.78 (m, 1H), 4.08 (t, J=6.6 Hz, 2H), 3.62 (t, J=5.2 Hz, 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, 4.5 Hz, 14H), 1.38–1.20 (m, 42H), 0.88 (t, J=6.8 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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 = 13.0 Hz), 31.81 (d, J = 12.3 Hz), 31.54 (s), 30.32 (s), 29.52 (d, J = 3.1 Hz), 29.34–28.94 (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).

P. Compound SW-II-138-1

1. Synthesis of Compound 2

Compound 1 (4 g, 16.46 mmol, 1.0 eq.) was dissolved in MeOH (40 mL), cooled to 0 °C and added dropwise with SOCl ₂ (3.9 g, 32.92 mmol, 2.0 eq.). Then the mixture was reacted at room temperature for 1 hour. TLC (PE/EA=5/1) showed that the starting material was consumed and the desired product was formed. The system was directly dried under reduced pressure, and NaHCO ₃ (70 mL) solution was added to the residue, and extracted with EA (80 mL×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, eluted with PE/EA (1/0-5:1, v/v) to obtain yellow oily compound 2 (4.1 mg, 95%).

2. Synthesis of Compound 4

Compound 2 (500 mg, 1.95 mmol, 1.0 eq.), compound 3 (239 mg, 2.34 mmol, 1.2 eq.), Pd(PPh ₃ ) ₄ (225 mg, 0.19 mmol, 0.1 eq.) and K ₂ CO ₃ (808 g, 5.85 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. Then the mixture was reacted at 110°C for 3 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the starting material was consumed and the desired product was formed. The reaction was quenched with H ₂ O (70 mL) and extracted with EA (80 mL×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with PE/EA (1/0-5:1, v/v) to obtain yellow oily compound 4 (320 mg, 70%).

3. Synthesis of Compound 5

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. Then the mixture was reacted at room temperature for 2 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the starting material was consumed and the desired product was formed. HCl (1M, 4 mL) solution and H ₂ O (10 mL) were added to quench the reaction, and the mixture was extracted with EA (50 mL×3). The organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, and eluted with PE/EA (1/0-5:1, v/v) to obtain yellow oily compound 5 (224 mg, 84.8%).

4. Synthesis of Compound 7

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. Then, the reaction was carried out at room temperature overnight under nitrogen protection. TLC (PE/EA=30/1) showed that the starting material was consumed and the desired product was formed. The reaction was quenched with HCl (1 M) solution and the pH was adjusted to 4-6, and extracted with DCM (80 mL×3). The combined organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column eluting with PE/EA (1/0-30:1, v/v) to give compound 7 (208 mg, 46.7%) as a colorless oil.

5. Synthesis of SW-II-138-1

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 K ₂ CO ₃ (172 mg, 1.25 mmol, 5.0 eq.) was added. The reaction was carried out at 90°C overnight under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the starting material was consumed and the desired product was formed. The reaction was directly decompressed and dried. The residue was purified by silica gel column and eluted with DCM/MeOH (1/0-10:1, v/v) to obtain a light yellow oily compound (65 mg, 32%, SW-II-138-1).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.09 (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).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.80(s),173.53(s),140.32(s),139.13(s),128.28(d,J＝13.6Hz),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 .6Hz),33.71(s),31.85(s),29.50(d,J＝2.8Hz),29.22(s),29.12–28.60(m),28.26(s),27.78(s),26.70(d, J＝4.4Hz),25.31(s),24.82(d,J＝17.6Hz),24.28(s),22.65(s),22.37(s),14.03(d,J＝15.2Hz).

Q.SW-II-138-2

1. Synthesis of compound 3

Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (271 mg, 2.34 mmol, 1.2 eq.), Pd(PPh ₃ ) ₄ (225 mg, 0.20 mmol, 0.1 eq.) and K ₂ CO ₃ (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added, and then the mixture was reacted at 110°C for 3 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the starting material was consumed and the desired product was formed. The reaction was quenched by adding water (70 mL) and extracted with EA (80 mL×3). The organic phases were combined and washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with PE/EA (1/0-30:1, v/v) to obtain colorless oily compound 3 (320 mg, 70%).

2. Synthesis of Compound 4

Compound 3 (320 mg, 1.29 mmol, 1.0 eq.) was dissolved in THF (3.0 mL), LAH (67 mg, 1.77 mmol, 2.0 eq.) was added at 0°C, and then reacted at room temperature for 2 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the starting material was consumed and the desired product was formed. The reaction was quenched with HCl (1M, 2 mL) solution and H ₂ O (10 mL), and extracted with EA (50 mL×3). The combined organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with PE/EA (1/0-30:1, v/v) to obtain colorless oily compound 4 (180 mg, 64%).

3. Synthesis of Compound 6

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. Then, the reaction was carried out at room temperature overnight under nitrogen protection. TLC (PE/EA=30/1) showed that the starting material was completely consumed and the desired product was formed. The reaction was quenched with HCl (1 M) solution and the pH was adjusted to 5-6, and extracted with DCM (80 mL×3). The combined organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column eluting with PE/EA (1/0-30:1, v/v) to give compound 6 (220 mg, 63.6%) as a colorless oil.

4. Synthesis of SW-II-138-2

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 K ₂ CO ₃ (172 mg, 1.25 mmol, 5.0 eq.) was added. Then, the reaction was carried out at 90°C overnight under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the starting material was consumed and the desired product was formed. The reaction was directly dried under reduced pressure, and the residue was purified by silica gel column, eluted with DCM/MeOH (1/0-10:1, v/v) to obtain the target product (100 mg, 51%, SW-II-138-2) as a colorless oil.

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ7.00 (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).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.78(s),173.52(s),140.32(s),139.11(s),128.25(d,J＝11.6Hz),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＝2.8Hz),29.21(s),28.94(dd,J＝6.4,4.4Hz),28.25(s), 27.77(s),26.84(d,J＝4.4Hz),25.30(s),25.25–24.59(m),22.59(d,J＝11.2Hz),14.05(d,J＝7.6Hz).

R.SW-II-138-3

1. Synthesis of compound 3

Compound 1 (500 mg, 1.95 mmol, 1.0 eq.), compound 2 (305 mg, 2.34 mmol, 1.2 eq.), Pd(PPh ₃ ) ₄ (225 mg, 0.20 mmol, 0.1 eq.) and K ₂ CO ₃ (809 g, 5.86 mmol, 3.0 eq.) were dissolved in toluene (5.0 mL) and water (1 mL) was added. Then, the reaction was carried out at 110° C. for 3 hours under nitrogen protection. TLC (PE/EA=5/1) showed that the starting material was completely consumed and the desired compound was formed. The reaction was quenched with water (80 mL) and extracted with EA (80 mL×3). The organic phases were combined and washed with saturated brine (2×40 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column and eluted with PE/EA (1/0-5:1, v/v) to obtain colorless oily compound 3 (260 mg, 51.3%).

2. Synthesis of Compound 4

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. Then, the reaction was carried out at room temperature under nitrogen protection for 2 hours. TLC (PE/EA = 5/1) showed that the raw material had reacted completely and the desired compound was formed. The reaction was quenched with HCl (1M, 4 mL) solution and H ₂ O (20 mL), and extracted with EA (50 mL × 3). The combined organic phases were washed with saturated brine (2 × 30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column, eluted with PE/EA (1/0-5:1, v/v) to obtain colorless oily compound 4 (230 mg, 98%).

3. Synthesis of Compound 6

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 in sequence. Then the reaction was allowed to proceed overnight at room temperature under nitrogen protection. TLC (PE/EA=20/1) showed that the starting material was consumed and the desired compound was formed. The reaction was quenched with HCl (1 M) and the pH was adjusted to 5-6, and extracted with DCM (80 mL×3). The combined organic phase was washed with saturated brine (2×30 mL), dried over anhydrous Na ₂ SO ₄ , filtered and dried under reduced pressure. The residue was purified by silica gel column eluting with PE/EA (1/0-20:1, v/v) to give compound 6 (180 mg, 40.9%) as a colorless oil.

4. Synthesis of SW-II-138-3

Compound 6 (164 mg, 0.37 mmol, 1 eq.) and 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 K ₂ CO ₃ (172 mg, 1.24 mmol, 5.0 eq.) was added. Then, the mixture was reacted at 90°C overnight under nitrogen protection. TLC (DCM/MeOH=10/1) showed that the starting material was consumed and the desired product was formed. The reaction was directly dried under reduced pressure. The residue was purified by silica gel column and eluted with DCM/MeOH (1/0-10:1, v/v) to obtain the desired product (108 mg, 52.76%, SW-II-138-3) as a colorless oil.

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

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

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

¹³ C NMR (101 MHz, CDCl ₃ )δ173.85(s),173.59(s),140.39(s),139.15(s),128.27(d,J＝12.0Hz),77.38(s),77.07(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 = 3.6 Hz), 26.50 (s), 25.32 (s), 24.99 (d, J = 17.6 Hz), 22.64 (d, J = 5.6 Hz), 14.10 (s).

S. Compound SW-II-139-1

1. Synthesis of compound 3

Pd(dtbpf)Cl _{2 (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11 mmol, 3 eq.) were added 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). 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.

2. Synthesis of Compound 4

To a mixture of compound 3 (691 mg, 2.95 mmol, 1 eq.) in THF (7 mL) was added lithium aluminum hydride (3 mL, 2.95 mmol, 1 M, in THF, 1 eq.) at 0 ° C and 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 (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 (547 mg, 90%) as a colorless oil without further purification.

3. Synthesis of Compound 6

Compound 4 (447 mg, 2.17 mmol, 1 eq.) and compound 5 (581 mg, 2.6 mmol, 1.2 eq.) were added in DCM. EDCI (833 mg, 4.34 mmol, 2 eq.) and DMAP (106 mg, 0.87 mmol, 0.4 eq.) were added to the mixture in (5 mL), and then DIEA (1.12 g, 8.68 mmol, 4 eq.) was added. The reaction mixture was stirred at room temperature under nitrogen for 16 hours. TLC (petroleum ether/ethyl acetate = 15/1) showed that the desired product was 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 (455 mg, 51%) as a colorless oil.

4. Synthesis of SW-II-139-1

Potassium carbonate (252 mg, 1.825 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-139-1 (54.53 mg, 19%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.20 (t, J = 7.7 Hz, 1H), 7.03 (t, J = 6.8 Hz, 3H), 4.94-4.78 (m, 1H), 4.27 (t, J = 7.2 Hz, 2H), 3.65 (t, J = 5.1 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 2.73 (t, J = 4.9 Hz, 2H), 2.67-2.41 (m, 6H), 2.28 (td, J = 7.5, 2.7 Hz, 4H), 1.67-1.45 (m, 14H), 1.41-1.19 (m, 42H), 0.88 (dd, J = 7.9, 5.7 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.65 (d, J＝11.3 Hz), 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) ,3 5.13(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).

T. Compound SW-II-139-2

1. Synthesis of compound 3

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)Cl ₂ (286 mg, 0.437 mmol, 0.1 eq.) and potassium carbonate (1.8 g, 13.11mmol, 3eq). 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 (605mg, 67%) as a colorless oil.

2. Synthesis of Compound 4

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 and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed that 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 give compound 4 (534 mg,>100%) as a colorless oil without further purification.

3. Synthesis of Compound 6

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) was 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 that the desired product was 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 (355 mg, 38%) as a colorless oil.

4. Synthesis of SW-II-139-2

Potassium carbonate (220 mg, 1.595 mmol, 5 eq.) and potassium iodide (106 mg, 0.638 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-139-2 (45.48 mg, 19%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.20 (t, J = 7.8 Hz, 1H), 7.03 (t, J = 7.6 Hz, 3H), 4.91-4.81 (m, 1H), 4.27 (t, J = 7.2 Hz, 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, 3.1 Hz, 4H), 1.74-1.08 (m, 53H), 0.90 (dt, J = 13.6, 7.2 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )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),64.88(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).

U. Compound SW-II-140-1

1. Synthesis of compound 3

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) was added Pd(dppf)Cl ₂ (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 (748 mg, 73%) as a colorless oil.

2. Synthesis of Compound 4

To a mixture of compound 3 (748 mg, 3.2 mmol, 1 eq.) in THF (8 mL) was added lithium aluminum hydride (3.2 mL, 3.2 mmol, 1 M, in THF, 1 eq.) at 0 ° C and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed that 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 give compound 4 (493 mg, 75%) as a colorless oil without further purification.

3. Synthesis of Compound 6

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) was 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 that the desired product was 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 (327 mg, 42%) as a colorless oil.

4. Synthesis of SW-II-140-1

Potassium carbonate (302 mg, 2.19 mmol, 6 eq.) and potassium iodide (121 mg, 0.73 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-140-1 (180 mg, 64%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.23–7.05 (m, 4H), 4.95–4.79 (m, 1H), 4.25 (t, J=7.4 Hz, 2H), 3.62 (t, J=4.8 Hz, 2H), 2.96 (dd, J=15.4, 8.0 Hz, 2H), 2.74–2.49 (m, 8H), 2.28 (dd, J=14.2, 7.2 Hz, 4H), 1.67–1.44 (m, 14H), 1.41–1.20 (m, 42H), 0.90 (dt, J=13.2, 7.1 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.68 (d, J＝10.2 Hz),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),3 4.66(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).

V.SW-II-140-2

1. Synthesis of compound 3

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) was added Pd(dppf)Cl ₂ (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 (406 mg, 45%) as a colorless oil.

2. Synthesis of Compound 4

To a mixture of compound 3 (406 mg, 1.97 mmol, 1 eq.) in THF (5 mL) was added lithium aluminum hydride (2 mL, 1.97 mmol, 1 M, in THF, 1 eq.) at 0 ° C and nitrogen. The mixture was stirred at room temperature for 3 hours. TLC (PE/EA=5/1) showed that the reaction was complete 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 give compound 4 (341 mg, 97%) as a colorless oil without further purification.

3. Synthesis of Compound 6

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) was 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 that the desired product was 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 (185 mg, 32%) as a colorless oil.

4. Synthesis of SW-II-140-2

Potassium carbonate (400 mg, 2.898 mmol, 6 eq.) and potassium iodide (160 mg, 0.966 mmol, 2 eq.) were added 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/CH ₃ CN (2 mL/2 mL). 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 a yellow oily compound SW-II-140-2 (161 mg, 45%).

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

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

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22–7.03 (m, 4H), 4.94–4.78 (m, 1H), 4.25 (t, J=7.3 Hz, 2H), 3.70–3.54 (m, 2H), 2.96 (t, J=7.4 Hz, 2H), 2.77–2.41 (m, 8H), 2.28 (dd, J=14.3, 7.1 Hz, 4H), 1.65–1.18 (m, 52H), 0.91 (dt, J=13.3, 7.1 Hz, 9H).

¹³ C NMR (101 MHz, CDCl ₃ )δ173.67 (d, J＝10.8 Hz),141.22 (s),135.23 (s),129.73 (s),129.39 (s),126.72 (s),125.92 (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),34.21(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=11.3,8.5Hz),27.12(d , J = 4.1 Hz), 26.23 (s), 25.33 (s), 25.06 (s), 24.82 (s), 22.73 (d, J = 9.9 Hz), 14.08 (d, J = 8.8 Hz).

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

In this example, the four-lipid LPP preparation containing luciferase mRNA (coding sequence as shown in SEQ ID NO: 1) for mucosal administration was prepared using the prescriptions in Tables 1, 2 and 3, and three rounds of comparison of physicochemical properties and in vivo expression levels of the prepared LPP preparation were performed to screen the lipid-lipid ratio suitable for mucosal administration. The MC3 LNP preparation was used as a positive control.

2.1 Preparation of lipid nanoparticle (LNP-mRNA) formulation

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

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

Preparation of LNP: Using microfluidic technology (Inano (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 LNP-mRNA solution.

Centrifugal ultrafiltration: Add the LNP-mRNA solution to an ultrafiltration tube for centrifugal ultrafiltration concentration (centrifugal force 3000 rpm, centrifugal time 60 min, temperature 4°C), ultrafiltration to ethanol content <0.5%, and set the LNP mRNA concentration to 0.2 mg/mL. Obtain the LNP-mRNA preparation numbered MC3 LNP.

2.2 Preparation of lipid polyplex (LPP-mRNA) preparation

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.

Preparation of mRNA aqueous solution: Dilute luciferase mRNA (coding sequence as shown in SEQ ID NO: 1) with 10 mM sodium citrate (pH = 4.0) buffer solution to get 0.1 mg/mL mRNA aqueous solution.

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.

Preparation of core nanoparticle solution: Using microfluidics technology, the protamine sulfate solution and the 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.

Preparation of LPP: The core nanoparticle solution and 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.

Centrifugal ultrafiltration: Remove ethanol from the LPP-mRNA solution by ultrafiltration centrifugation (centrifugal force 3000rpm, centrifugal time 60min, temperature 4°C), ultrafilter until the ethanol content is <0.5%, and adjust the LPP mRNA concentration to 0.2mg/mL. LPP preparations of 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.

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

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

Particle size detection: Take 50 μL of the LPP sample prepared in Example 2.2 and dilute it with 950 mL of purified water to obtain a diluted LPP sample, and place the sample in a dynamic light scattering laser particle size analyzer (Malvern, ZS-90) for detection.

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). 100μL of each diluted sample was transferred to a 96-well plate, and 100μL 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 way. A calibration curve of fluorescence and mRNA concentration was drawn by linear regression, from which the mRNA content of the sample free outside the LPP particles was calculated.

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 of the above samples 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 well 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 way. 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.

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.

Polydispersity index (PDI) detection: The polydispersity index (PDI) of LPP solution was determined using Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

Purity detection: Use Q-Analyzer and RNA Carisge Kit (RNA Low Marker (5x); Dilution Buffer (10x); Separation Buffer (10x); Mininer oil) to detect the purity of mRNA in the LPP solution prepared in Example 2.2. 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 heparin sodium solution in sequence, vortex to mix, and let stand for 5 minutes. After standing, place the sample in a 70°C water bath for 20 minutes, and then quickly transfer to a 4°C refrigerator to cool for 5-10 minutes; take out 50 μL of sample and add it to the eight-tube strip, and add 20 μL Mininer oil on top. The purity of the sample was detected by capillary electrophoresis Qsep100 method.

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, encapsulation efficiency, etc. 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%, it had better and more stable particle size, polydispersity index and encapsulation efficiency.

Table 1. First round of mucosal drug delivery prescription screening

Table 2. Second round of mucosal drug delivery prescription screening

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

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

While the prepared LPP preparation was subjected to three rounds of physical and chemical property tests as in Example 2.3, the prepared LPP preparation was tested for in vivo expression to screen lipids and lipid ratios suitable for mucosal administration. The specific testing method is as follows:

During the three rounds of in vivo expression detection, 6-8 week old female BALB/c mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were anesthetized with sodium pentobarbital (70 mg/kg) and then the prepared LPP solution (3 mice per group), and during each round of screening, 2 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). 3 mg of D-luciferin substrate (Mao Kang Biotechnology) was injected intraperitoneally into the mice 6 hours and 24 hours after administration. Ten minutes after substrate injection, the mice were imaged in vivo using the Xenogen IVIS-200 imaging system to detect the expression of luciferase in vivo.

The experimental results showed that all LPP preparations were expressed only in the nasal cavity, and the highest luciferase expression was achieved when M5: 37.5-42.5%; phospholipids (DOPE): 25-35%; PEG (DMG-PEG): 3.75-5%; and cholesterol: 15-30%. The AUC of IN-3-11 LPP preparation, which expressed the most luciferase in the third round, was about 35 times that of IN-1-4, which expressed the most luciferase in the first round, and about 100 times that of MC3 LNP preparation.

Among them, the luciferase expression in mice 6 hours after administration in the third round is shown in Figure 1A and Figure 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.

According to the test results of the physicochemical properties of Example 2.3 and the results of the in vivo expression in this example, the optimized lipid-lipid ratio range suitable for mucosal administration was confirmed, namely, cationic lipid (M5 in this example): 37.5-42.5%; phospholipid (DOPE): 25-35%; PEG (DMG-PEG): 3.75-5%; cholesterol: 15-30%. And based on the above results, the prescriptions of IN-2-6, IN-3-4, IN-3-5, IN-3-11 and IN-3-12 LPP preparations with higher luciferase expression levels in the nasal cavity were selected for further testing.

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

In this example, mice were immunized using different immunization schemes to detect the application of the preferred LPP preparation prescription in Example 2 in the treatment of the new coronavirus vaccine.

3.1 Preparation of lipid polyplex (LPP-mRNA) preparation

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

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 0.1 mg/mL mRNA aqueous solution.

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.

Preparation of core nanoparticle solution: Using microfluidics technology, the protamine sulfate solution and the 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.

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 mixed solution), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL/min, room temperature, to obtain LPP-mRNA solution.

Centrifugal ultrafiltration: The LPP-mRNA solution was centrifuged by ultrafiltration to remove ethanol (centrifugal force 3000 rpm, centrifugation time 60 min, temperature 4°C), ultrafiltered to an ethanol content of <0.5%, and the LPP mRNA concentration was fixed to 1 mg/mL. LPP preparations numbered IN-2-6/Covid-19, IN-3-11/Covid-19, and IN-3-12/Covid-19 were prepared.

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

3.2 Immune effects of different immunization regimens

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

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

ELISpot assay

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, placed in a cell culture incubator, and left to stand for more than 1 h. Lung cells or spleen cells were plated at 3×10 ⁶ cells/well and stimulated in vitro with 100 μL/well S protein peptide, 100 μL of medium was added to the negative control, and 100 μL of PMA+Ionomycine was added to the positive control, and incubated at 37°C, 5% CO2 for 20 hours. Afterwards, biotinylated IFN-γ-detection antibody and streptavidin-alkaline phosphatase (ALP) were used for detection, and BCIP/NBT-plus (5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium-plus) substrate was added for color development and counted using an ELISpot plate reader (ImmunoSpot S6 Core Analyzer (CTL)).

Enzyme-linked immunosorbent assay (ELISA)

When performing ELISA testing of animal serum/lavage fluid, the S protein antigen (SARS-CoV-2 (2019-nCoV) Spike Protein, purchased from Beijing Sino Biological Technology Co., Ltd.) was diluted to 2μg/mL using ELISA coating buffer, and 96-well plates were coated with 100μL per well, 200ng/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 100μL of the diluted serum or lavage fluid was added to the plate and incubated at 37°C for 1h. Then, the liquid in the plate was discarded, the plate was washed 3 times in PBST, and the secondary antibody (Goat pAb to Mouse IgG-HRP) was diluted with sample diluent at a ratio of 1:10000, and 100 μL was added to each well and incubated at 37°C for 0.5 h. After incubation, the plate was washed 3 times with PBST, 100 μL 1x TMB substrate solution was added to each well for color development for 3 minutes, and 50 μL TMB stop solution was added to stop the color development after color development. The absorbance was recorded at 450nm and 610nm using Synegry H1 microplate reader. For result analysis, the OD450 value of each well was first subtracted from the OD610 value, and then the average value of the blank well was deducted to obtain the actual sample absorbance value. When the absorbance value corresponding to the highest dilution factor is greater than 0.21, the highest dilution is used as the titer of the sample.

For IgA, 100 μL of diluted lavage fluid was added to the plate and incubated at 37°C for 1 hour. Then the liquid in the plate was aspirated and the plate was washed 3 times in PBST. The secondary antibody (Goat pAb to Mouse IgA-biotin) was diluted with sample diluent at a ratio of 1:10000 and 100 μL was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed with Wash 3 times with PBST, dilute the antibody Streptavidin-HRP with sample diluent at a ratio of 1:5000, add 100μL to each well and incubate at 37℃ for 1h. After incubation, wash the plate 3 times with PBST, add 100μL 1x TMB substrate solution to each well for color development for 10 minutes, add 100μL TMB stop solution to stop color development after color development, and record the absorbance at 450nm using Synegry H1 microplate reader. Result analysis: First, deduct the average value of the blank well from the OD450 of each well to obtain the actual sample absorbance value. When the absorbance value corresponding to the highest dilution factor is greater than 0.21, the highest dilution is used as the titer of the sample.

The results of ELISpot detection are shown in Table 5 and Figures 2B and 2C. The third booster immunization resulted in better immune effects. During the third immunization, the cellular immune responses after nasal administration of IN-3-11/Covid-19 LPP preparations and IN-3-12/Covid-19 LPP preparations were equivalent to or higher than those after intramuscular administration of B11/Covid-19 LPP preparations.

The results of ELISA 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, and there will be better immune effects. Compared with the administration of B11/Covid-19 LPP preparation by intramuscular injection at 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 in 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.

The above results indicate that the “primary immunization + boosting” approach (systemic immunization + mucosal immunization) can combine the advantages of both immunization approaches and induce a higher level of immune effect.

Table 4. Immunization schedule

Table 5. Immune effects of different immunization regimens

Example 4 Application of LPP preparation for mucosal administration in influenza vaccine treatment

In this example, mice were immunized by different immunization schemes to detect the application of the preferred LPP preparation in Example 2 in influenza vaccine treatment.

4.1 Preparation of lipid polyplex (LPP-mRNA) preparation

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

Preparation of mRNA aqueous solution: Dilute the mRNA encoding influenza virus antigen (the coding sequence is shown in SEQ ID NO: 3) into 0.1 mg/mL mRNA aqueous solution with 10 mM sodium citrate (pH = 4.0) buffer solution.

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.

Preparation of core nanoparticle solution: Using microfluidics technology, the protamine sulfate solution and the 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.

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 mixed solution), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL/min, room temperature, to obtain LPP-mRNA solution.

Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3000 rpm, centrifugal time 60 min, temperature 4°C), ultrafiltered to an ethanol content of <0.5%, and the LPP mRNA concentration was fixed 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.

And the B11/Flu LPP preparation with a lipid ratio of M5: phospholipid: cholesterol: PEG of 40:15:43.5:1.5 was prepared by the above method, and the concentration of B11/Flu LPP mRNA was fixed to 0.1 mg/mL.

4.2 Immune effects of different immunization regimens

Mice were immunized by the immunization scheme shown in FIG3A and Table 6. Female BALB/c mice aged 6-8 weeks (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were divided into 8 groups (n=5), and the mice were immunized twice on day 0 and day 14 (2 weeks after the primary immunization). In the two immunizations, 10% sucrose solution was administered to group 1 mice by nasal instillation; IN-2-6/Flu LPP preparation, IN-3-4/Flu LPP preparation, IN-3-5/Flu LPP preparation, IN-3-11/Flu LPP preparation and IN-3-12/Flu LPP preparation were administered to groups 2, 3, 4, 5 and 6 respectively by nasal instillation; B11/Flu LPP preparation was administered to group 7 mice by intramuscular route. At the time of primary immunization, mice in group 8 were given B11/Flu LPP preparations by intramuscular route, and at the time of secondary immunization, mice in group 8 were given IN-3-11/Flu LPP preparations by nasal instillation. At the fourth week after the primary immunization, mice were sacrificed, and lungs, spleens, and lung/nasal lavage fluids (obtained by lavage with 0.7 mL PBS) were obtained for subsequent testing.

ELISpot was used to detect the immune cell response in spleen cells and lung cells, and ELISA was used to detect the antibody levels in serum, nasal lavage fluid, and lung lavage fluid. The specific detection method of ELISpot is shown in Example 3.2. The specific detection method of ELISA is as follows:

When performing ELISA testing of animal serum, use ELISA coating buffer to dilute influenza virus antigen (NP protein, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) to 1μg/mL, coat 96-well plates with 100μL per well, 100ng/well of antigen, and incubate overnight at 4°C. After incubation overnight at 4°C, wash the plates three times with phosphate-buffered saline PBS-Tween (PBST) (0.5% tween-20, w/v) and block in 10% FCS-PBST at 37°C for 2 hours. Wash the plates three times in PBST, then dilute the serum in 10% FCS-PBST. For IgG detection, dilute the samples in multiples, add 100μL of diluted serum to the plates, and incubate at 37°C for 1h. Then aspirate and discard the plates. The plate was washed 3 times in PBST, and the secondary antibody (Goat pAb to Mouse IgG-HRP) was diluted with sample diluent at a ratio of 1:50000, and 100 μL was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST, and 100 μL 1x TMB substrate solution was added to each well for color development for 10 minutes. After color development, 50 μL TMB stop solution was added to stop color development, and the absorbance was recorded at 450 nm using a Synegry H1 microplate reader. For result analysis, the actual sample absorbance value was obtained by first subtracting the average value of the blank well from the OD450 value of each well. When the absorbance value corresponding to the highest dilution factor was greater than 0.21, the highest dilution was used as the titer of the sample.

For IgA, for ELISA of animal serum/lavage fluid, dilute influenza virus antigen to 2 μg/mL with ELISA coating buffer, coat 96-well plates with 100 μL per well, 200 ng/well antigen, and incubate overnight at 4°C. Dilute samples 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 plates three times with PBST, dilute the secondary antibody (Goat pAb to Mouse IgA-biotin) at a ratio of 1:10000 with sample diluent, add 100 μL per well, and incubate at 37°C for 1 hour. After incubation, wash the plates three times with PBST, dilute the antibody Streptavidin-HRP at a ratio of 1:5000 with sample diluent, add 100 μL per well, and incubate at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST, and 100 μL 1x TMB substrate solution was added to each well for color development for 10 minutes. After color development, 100 μL TMB stop solution was added to stop color development, and the absorbance was recorded at 450 nm using a Synegry H1 microplate reader. For result analysis, the actual sample absorbance value was obtained by first deducting the average value of the blank well from the OD450 of each well. When the absorbance value corresponding to the highest dilution factor was greater than 0.21, the highest dilution was used as the titer of the sample.

The ELISpot test results and ELISA test results are shown in Table 7 and Figures 3B, 3C, and 3D. In the primary immunization, the B11/Flu LPP preparation was administered by intramuscular route, and in the second immunization, the mice were administered IN-3-11/Flu LPP preparation by nasal instillation. The mice had excellent cellular immune response and had high antibody levels in their serum, nasal lavage fluid, and alveolar lavage fluid.

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.

Table 6. Immunization schedule

Table 7. Immune effects of different immunization regimens

Example 5 Effects of different cationic lipids on the physicochemical properties and expression levels of LPP preparations for mucosal administration

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

5.1 Preparation of lipid nanoparticle (LNP-mRNA) formulations

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

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

Preparation of LNP: Using microfluidic technology (Inano (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 LNP-mRNA solution.

Centrifugal ultrafiltration: Add the LNP-mRNA solution to an ultrafiltration tube for centrifugal ultrafiltration concentration (centrifugal force 3000 rpm, centrifugal time 60 min, temperature 4°C), ultrafiltration to ethanol content <0.5%, and set the LNP mRNA concentration to 0.2 mg/mL. Obtain the LNP-mRNA preparation numbered MC3 LNP.

5.2 Preparation of lipid polyplex (LPP-mRNA) preparation

Preparation of lipid mixture: According to the lipid ratio in the formulation of IN-3-11 and the cationic lipids shown in Table 8, cationic lipids, phospholipids, cholesterol and PEG lipids were dissolved in ethanol solution to prepare a 6 mg/mL lipid mixture.

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

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.

Preparation of core nanoparticle solution: Using microfluidics technology, the protamine sulfate solution and the 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.

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 mixed solution), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL/min, room temperature, to obtain LPP-mRNA solution.

Centrifugal ultrafiltration: Remove ethanol from the LPP-mRNA solution by ultrafiltration centrifugation (centrifugal force 3000 rpm, centrifugation time 60 min, temperature 4°C), ultrafilter until the ethanol content is <0.5%, and adjust the LPP mRNA concentration 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.

And the B11 LPP preparation with 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.

5.3 Testing of the physicochemical properties of LPP preparations with different cationic lipids

The prepared LPP preparation and LNP preparation were tested for physical and chemical properties. The specific testing methods for particle size testing, encapsulation efficiency testing, polydispersity index (PDI) testing and purity testing are shown in Example 2.3.

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

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

5.4 In vitro expression of LPP preparations containing different cationic lipids

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. LPP solutions of B11, 121 LPP, 138-1 LPP, 139-1 LPP, 140-1 LPP and 140-2 LPP and LNP solution of MC3 LNP containing 100 ng of luciferase mRNA were taken respectively, and PBS was used as a control to transfect A549 cells and dendritic cells DC2.4 cells. The cells were lysed 24 hours after administration 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.

5.5 In vivo expression of LPP preparations containing different cationic lipids

The in vivo expression of the LPP preparation prepared in Example 5.2 was detected. The specific detection method is as follows:

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 in each 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 (Maokang Biotechnology). Ten minutes after the substrate injection, the mice were stained with Xenogen IVIS-200. The imaging system was used to perform in vivo imaging of mice to detect the expression of luciferase in vivo.

The experimental results are shown in Figures 5A and 5B. All LPP preparations were expressed only in the nasal cavity, with high local expression and low systemic toxicity. Among them, the luciferase expression levels of LPP preparations containing different cationic lipids prepared with the lipid ratio in the IN-3-11 prescription were similar, but the luciferase expression in the nasal cavity was higher than that of the B11 LPP preparation.

The above results indicate that the lipid ratio screened by the present invention is particularly suitable for intranasal administration. LPP preparations containing self-made 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.

Example 6 Effect of atomization on the physicochemical properties and transfection efficiency of LPP preparations

In this example, a four-lipid LPP preparation containing luciferase mRNA (coding sequence as shown in SEQ ID NO: 1) or eGFP protein mRNA (coding sequence as shown in SEQ ID NO: 4) was prepared using the B11 formulation, and atomized by the atomization drug delivery device as shown in FIG6A . The physicochemical properties and transfection efficiency of the LPP preparation before and after atomization were measured to determine the effect of atomization on it.

6.1 Preparation of lipid polyplex (LPP-mRNA) preparation

Preparation of lipid mixture: M5, phospholipids, cholesterol and PEG lipids were dissolved in ethanol solution according to the lipid ratio in the B11 prescription to prepare a 6 mg/mL lipid mixture.

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

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.

Preparation of core nanoparticle solution: Using microfluidics technology, the protamine sulfate solution and the 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.

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 mixed solution), flow rate ratio = 3 (core nanoparticle solution): 1 (lipid solution), total flow rate = 12 mL/min, room temperature, to obtain LPP-mRNA solution.

Centrifugal ultrafiltration: The LPP-mRNA solution was subjected to ultrafiltration centrifugation to remove ethanol (centrifugal force 3000 rpm, centrifugal time 60 min, temperature 4°C), ultrafiltration was performed until the ethanol content was <0.5%, and the LPP mRNA concentration was fixed to 0.1 mg/mL. LPP preparations numbered B11/Luc and B11/eGFP were obtained.

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

The LPP preparations of B11/Luc and B11/eGFP were aerosolized using the aerosol drug delivery device as shown in FIG6A , and the physical and chemical properties of the preparations before and after aerosolization were tested. For specific detection methods of particle size detection, encapsulation efficiency detection, polydispersity index (PDI) detection and purity detection, see Example 2.3.

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.

Table 9. Physicochemical properties before and after atomization

6.3 Detection of transfection efficiency of LPP preparations before and after atomization

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.

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

B11/eGFP LPP preparations containing 12.5, 25, 50, and 100 ng of luciferase mRNA eGFP mRNA were taken before nebulization, in the front section of nebulization, in the middle section of nebulization, and in the rear section of nebulization, respectively, and A549 cells were transfected. The expression of green fluorescent protein was observed under a fluorescence microscope 24 hours after administration, and photos were taken and compared.

The test results are shown in FIG6B and FIG6C , 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 atomized sample compared with the sample before atomization.

Although the present invention has been disclosed as above in terms of preferred embodiments, it 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.

Sequence Listing

Claims (35)

A lipid composition for mucosal administration, comprising a therapeutic agent or a preventive agent and a lipid encapsulating the therapeutic agent or the preventive agent, wherein the lipid encapsulating the therapeutic agent or the preventive agent comprises a cationic lipid, a phospholipid, a steroid and a lipid modified with polyethylene glycol; the lipid composition further comprises a cationic polymer, wherein the cationic polymer is associated with the therapeutic agent or the preventive agent to form a complex and is co-encapsulated in the lipid to form a lipid polymer complex; the lipid composition comprises 2.5-20 mol % of the lipid modified with polyethylene glycol, based on the total amount of all lipids in the lipid composition. The lipid composition of claim 1, wherein the therapeutic or prophylactic agent is a nucleic acid, such as RNA, particularly mRNA. 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 - _C12 alkyl group, and a _C2 - _C12 alkenyl group; _R3 and _R4 are each independently selected from _C1 - _C12 alkyl, _C2 - _C12 alkenyl, _C6 - _C10 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; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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 R _5, together with one of M ₁ and M ₂ and the N atom to which they are attached, form a 3-10 membered heterocyclic ring, and the corresponding R ₁ /R ₃ or R ₂ /R ₄ do not exist, and the heterocyclic ring is optionally substituted by R ₇ ; R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H, -OR ₇ , -SR ₇ , -OC(O)R ₇ , -C(O)OR ₇ , -N(R ₇ )C(O)R ₇ , -N(R ₇ )S(O) ₂ R ₇ , -N(R ₇ )C(S)R ₇ , -N(R ₇ ) ₂ , cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆ -C ₁₀ aryl, each of the above groups is optionally substituted with one or more C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, oxo (=O); 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-, -NR ₇ -, -C(O)-, -OC(O)-, -C(O)O-, -SC(S)-, -C(S)S-, C _3-8 carbocycle, and the alkyl, alkenyl and alkylene groups are each optionally substituted by one or more R ₇ ; _R7 is each independently selected from H, _C1 - _C12 alkyl, C2- _C12 alkenyl, _C1 - _C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, _C6 -C10 aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C3-8 carbocycle, each of the above groups is optionally substituted with one or more _{C1 - C12} alkyl, C2-C12 alkenyl, C1- _C12 alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C6 _{- C10} aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, _C3-8 carbocycle, The invention may be substituted with alkyl, C ₁ -C ₁₂ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxyl, or oxo (═O). The lipid composition of claim 3, wherein _R1 and _R2 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl; wherein R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; and R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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-; R ₅ is selected from -C _1-12 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl; m and n are each independently an integer selected from 1-12. 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:

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:

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

The lipid composition of claim 3, wherein _R1 and _R2 are each independently selected from _C1 - _C12 alkyl and _C2 - _C12 alkenyl; R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl; Provided that at least one of R ₃ and R ₄ is C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted by t R ₆ , t being an integer selected from 1-5; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -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-; R ₅ is selected from -C _1-12 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆ -C ₁₀ aryl and 5-10 membered heteroaryl; m and n are each independently an integer selected from 1-12. The lipid composition of claim 8, wherein _R1 and _R2 are each independently selected from _C1 - _C12 alkyl groups. The lipid composition of claim 8 or 9, wherein R ₃ and R ₄ are each independently selected from C ₁ -C ₁₂ alkyl and C ₆ -C ₁₀ aryl; Provided that one of R ₃ and R ₄ is a C ₆ -C ₁₀ aryl group and the other is a C ₁ -C ₁₂ alkyl group; R ₃ and R ₄ are each independently substituted by t R ₆ , where t is an integer selected from 1-3; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl groups. 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-. The lipid composition according to any one of claims 8 to 11, wherein _R5 is selected from _-C1-5 alkylene-Q, where Q is -OH. 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. The lipid composition according to any one of claims 8 to 13, wherein R ₄ is substituted at the 1st or last position of R ₂ ; and/or R ₃ is substituted at the 1st position or the last position of R ₁ . The lipid composition according to any one of claims 8 to 14, wherein t is 1 or 2, and R ₆ is substituted at the meta and/or para position relative to R ₁ or R ₂ on the benzene ring. The lipid composition according to any one of claims 8 to 15, wherein t is 1 or 2, and R ₆ is each independently selected from a C ₁ -C ₁₀ alkyl group. 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 R ₁ , R ₂ , R ₄ , R ₅ , R ₆ , M ₁ , M ₂ , t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (II) R ₁ is selected from C ₁ -C ₆ alkyl; R ₂ is selected from C ₁ -C ₁₀ alkyl; R ₄ is selected from C ₁ -C ₁₀ alkyl; _M1 and _M2 are each independently selected from: -OC(O)-, -C(O)O- and -OC(O)O-; R ₅ is selected from -C _1-5 alkylene-Q, Q is selected from -OR ₇ and -SR ₇ , R ₇ is independently selected from H, C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; m and n are each independently an integer selected from 2-9; t is an integer selected from 1-3; R ₆ is each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl. 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 R ₁ , R ₂ , R ₄ , R ₅ , R ₆ , t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (III), R ₁ is selected from C ₁ -C ₆ alkyl; R ₂ is selected from C ₁ -C ₁₀ alkyl; R ₄ is selected from C ₁ -C ₁₀ alkyl; _R5 is selected from _-C1-3 alkylene-Q, Q is selected from -OH and -SH; t is 1 or 2; R ₆ is selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; m and n are each independently an integer selected from 2-7. 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 R ₁ , R ₂ , R ₄ , R ₆ , t, m and n are as defined in any one of claims 8 to 16; Preferably, in formula (IV), R ₁ is selected from C ₁ -C ₆ alkyl; R ₂ is selected from C ₁ -C ₁₀ alkyl; R ₄ is selected from C ₁ -C ₁₀ alkyl; t is 1 or 2; R ₆ are each independently selected from C ₁ -C ₁₂ alkyl and C ₂ -C ₁₂ alkenyl; m and n are each independently an integer selected from 2-7. 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. 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), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmit ...diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DP 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. The lipid composition of claim 21, wherein the steroid comprises cholesterol, coproposterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and derivatives thereof; preferably, the steroid is cholesterol. 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-glycerol, 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. The lipid composition of claim 23, comprising 30-60 mol% cationic lipids, 5-40 mol% phospholipids, 10-70 mol% steroids, and 2.5-20 mol% polyethylene glycol-modified lipids; Preferably comprising 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% of cationic lipid, 35 mol% of DOPE, 18.75 mol% of cholesterol and 3.75 mol% of DMG-PEG. The lipid composition of claim 24, wherein the cationic polymer comprises poly-L-lysine, protamine White, polyethyleneimine (PEI) or a combination thereof; preferably, the cationic polymer is protamine. A pharmaceutical composition comprising the lipid composition according to any one of claims 1 to 25, and optionally a pharmaceutically acceptable excipient. The lipid composition of any one of claims 1 to 25 or the pharmaceutical composition of claim 26, which is used 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. The lipid composition or pharmaceutical composition of claim 27, wherein the nasal spray administration is performed by an atomization administration device. 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 limiter. 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 as a lipid composition of any one of claims 1 to 25 or a pharmaceutical composition of claim 26. The method of claim 30, wherein the additional at least one dose 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 additional at least one dose is administered by an intramuscular route of administration. 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. 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. 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. A nasal drop or nasal spray comprising the lipid composition according to any one of claims 1 to 25 and a pharmaceutically acceptable excipient.